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1
Schmid, Christina, Najah Abi-Gerges, Michael Leitner, Dietmar Zellner, and Georg Rast. "Ion Channel Expression and Electrophysiology of Singular Human (Primary and Induced Pluripotent Stem Cell-Derived) Cardiomyocytes." Cells 10, no. 12 (November 30, 2021): 3370. http://dx.doi.org/10.3390/cells10123370.
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Subtype-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are promising tools, e.g., to assess the potential of drugs to cause chronotropic effects (nodal hiPSC-CMs), atrial fibrillation (atrial hiPSC-CMs), or ventricular arrhythmias (ventricular hiPSC-CMs). We used single-cell patch-clamp reverse transcriptase-quantitative polymerase chain reaction to clarify the composition of the iCell cardiomyocyte population (Fujifilm Cellular Dynamics, Madison, WI, USA) and to compare it with atrial and ventricular Pluricytes (Ncardia, Charleroi, Belgium) and primary human atrial and ventricular cardiomyocytes. The comparison of beating and non-beating iCell cardiomyocytes did not support the presence of true nodal, atrial, and ventricular cells in this hiPSC-CM population. The comparison of atrial and ventricular Pluricytes with primary human cardiomyocytes showed trends, indicating the potential to derive more subtype-specific hiPSC-CM models using appropriate differentiation protocols. Nevertheless, the single-cell phenotypes of the majority of the hiPSC-CMs showed a combination of attributes which may be interpreted as a mixture of traits of adult cardiomyocyte subtypes: (i) nodal: spontaneous action potentials and high HCN4 expression and (ii) non-nodal: prominent INa-driven fast inward current and high expression of SCN5A. This may hamper the interpretation of the drug effects on parameters depending on a combination of ionic currents, such as beat rate. However, the proven expression of specific ion channels supports the evaluation of the drug effects on ionic currents in a more realistic cardiomyocyte environment than in recombinant non-cardiomyocyte systems.
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Xie, Duanyang, Ke Xiong, Xuling Su, Guanghua Wang, Qiang Ji, Qicheng Zou, Lingling Wang, et al. "Identification of an endogenous glutamatergic transmitter system controlling excitability and conductivity of atrial cardiomyocytes." Cell Research 31, no. 9 (April 6, 2021): 951–64. http://dx.doi.org/10.1038/s41422-021-00499-5.
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AbstractAs an excitatory transmitter system, the glutamatergic transmitter system controls excitability and conductivity of neurons. Since both cardiomyocytes and neurons are excitable cells, we hypothesized that cardiomyocytes may also be regulated by a similar system. Here, we have demonstrated that atrial cardiomyocytes have an intrinsic glutamatergic transmitter system, which regulates the generation and propagation of action potentials. First, there are abundant vesicles containing glutamate beneath the plasma membrane of rat atrial cardiomyocytes. Second, rat atrial cardiomyocytes express key elements of the glutamatergic transmitter system, such as the glutamate metabolic enzyme, ionotropic glutamate receptors (iGluRs), and glutamate transporters. Third, iGluR agonists evoke iGluR-gated currents and decrease the threshold of electrical excitability in rat atrial cardiomyocytes. Fourth, iGluR antagonists strikingly attenuate the conduction velocity of electrical impulses in rat atrial myocardium both in vitro and in vivo. Knockdown of GRIA3 or GRIN1, two highly expressed iGluR subtypes in atria, drastically decreased the excitatory firing rate and slowed down the electrical conduction velocity in cultured human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocyte monolayers. Finally, iGluR antagonists effectively prevent and terminate atrial fibrillation in a rat isolated heart model. In addition, the key elements of the glutamatergic transmitter system are also present and show electrophysiological functions in human atrial cardiomyocytes. In conclusion, our data reveal an intrinsic glutamatergic transmitter system directly modulating excitability and conductivity of atrial cardiomyocytes through controlling iGluR-gated currents. Manipulation of this system may open potential new avenues for therapeutic intervention of cardiac arrhythmias.
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Freundt, Johanna K., Gerrit Frommeyer, Fabian Wötzel, Andreas Huge, Andreas Hoffmeier, Sven Martens, Lars Eckardt, and Philipp S. Lange. "The Transcription Factor ATF4 Promotes Expression of Cell Stress Genes and Cardiomyocyte Death in a Cellular Model of Atrial Fibrillation." BioMed Research International 2018 (May 29, 2018): 1–15. http://dx.doi.org/10.1155/2018/3694362.
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Introduction. Cardiomyocyte remodelling in atrial fibrillation (AF) has been associated with both oxidative stress and endoplasmic reticulum (ER) stress and is accompanied by a complex transcriptional regulation. Here, we investigated the role the oxidative stress and ER stress responsive bZIP transcription factor ATF4 plays in atrial cardiomyocyte viability and AF induced gene expression. Methods. HL-1 cardiomyocytes were subjected to rapid field stimulation. Forced expression of ATF4 was achieved by adenoviral gene transfer. Using global gene expression analysis and chromatin immunoprecipitation, ATF4 dependent transcriptional regulation was studied, and tissue specimen of AF patients was analysed by immunohistochemistry. Results. Oxidative stress and ER stress caused a significant reduction in cardiomyocyte viability and were associated with an induction of ATF4. Accordingly, ATF4 was also induced by rapid field stimulation mimicking AF. Forced expression of wild type ATF4 promoted cardiomyocyte death. ATF4 was demonstrated to bind to the promoters of several cell stress genes and to induce the expression of a number of ATF4 dependent stress responsive genes. Moreover, immunohistochemical analyses showed that ATF4 is expressed in the nuclei of cardiomyocytes of tissue specimen obtained from AF patients. Conclusion. ATF4 is expressed in human atrial cardiomyocytes and is induced in response to different types of cell stress. High rate electrical field stimulation seems to result in ATF4 induction, and forced expression of ATF4 reduces cardiomyocyte viability.
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Nesterova, Tatyana, Dmitry Shmarko, Konstantin Ushenin, and Olga Solovyova. "In-silico analysis of aging mechanisms of action potential remodeling in human atrial cardiomyocites." BIO Web of Conferences 22 (2020): 01025. http://dx.doi.org/10.1051/bioconf/20202201025.
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Electrophysiology of cardiomyocytes changes with aging. Agerelated ionic remodeling in cardiomyocytes may increase the incidence and prevalence of atrial fibrillation (AF) in the elderly and affect the efficiency of antiarrhythmic drugs. There is the deep lack of experimental data on an action potential and transmembrane currents recorded in the healthy human cardiomyocytes of different age. Experimental data in mammals is also incomplete and often contradicting depending on the experimental conditions. In this in-silico study, we used a population of ionic models of human atrial cardiomyocytes to transfer data on the age- related ionic remodeling in atrial cardiomyocytes from canines and mice to predict possible consequences for human cardiomyocyte activity. Based on experimental data, we analyzes two hypotheses on the aging effect on the ionic currents using two age-related sets of varied model parameters and evaluated corresponding changes in action potential morphology with aging. Using the two populations of aging models, we analyzed the agedependent sensitivity of atrial cardiomyocytes to Dofetilide which is one of the antiarrhythmic drugs widely used in patients with atrial fibrillation.
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Li, Jiuru, Alexandra Wiesinger, Lianne Fokkert, Bastiaan J. Boukens, Arie O. Verkerk, Vincent M. Christoffels, Gerard J. J. Boink, and Harsha D. Devalla. "Molecular and electrophysiological evaluation of human cardiomyocyte subtypes to facilitate generation of composite cardiac models." Journal of Tissue Engineering 13 (January 2022): 204173142211279. http://dx.doi.org/10.1177/20417314221127908.
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Paucity of physiologically relevant cardiac models has limited the widespread application of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in drug development. Here, we performed comprehensive characterization of hiPSC-derived cardiomyocyte subtypes from 2D and 3D cultures and established a novel 3D model to study impulse initiation and propagation. Directed differentiation approaches were used to generate sinoatrial nodal (SANCM), atrial (ACM) and ventricular cardiomyocytes (VCM). Single cell RNA sequencing established that the protocols yield distinct cell populations in line with expected identities, which was also confirmed by electrophysiological characterization. In 3D EHT cultures of all subtypes, we observed prominent expression of stretch-responsive genes such as NPPA. Response to rate modulating drugs noradrenaline, carbachol and ivabradine were comparable in single cells and EHTs. Differences in the speed of impulse propagation between the subtypes were more pronounced in EHTs compared with 2D monolayers owing to a progressive increase in conduction velocities in atrial and ventricular cardiomyocytes, in line with a more mature phenotype. In a novel binary EHT model of pacemaker-atrial interface, the SANCM end of the tissue consistently paced the EHTs under baseline conditions, which was inhibited by ivabradine. Taken together, our data provide comprehensive insights into molecular and electrophysiological properties of hiPSC-derived cardiomyocyte subtypes, facilitating the creation of next generation composite cardiac models for drug discovery, disease modeling and cell-based regenerative therapies.
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Rajala, Kristiina, Mari Pekkanen-Mattila, and Katriina Aalto-Setälä. "Cardiac Differentiation of Pluripotent Stem Cells." Stem Cells International 2011 (2011): 1–12. http://dx.doi.org/10.4061/2011/383709.
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The ability of human pluripotent stem cells to differentiate towards the cardiac lineage has attracted significant interest, initially with a strong focus on regenerative medicine. The ultimate goal to repair the heart by cardiomyocyte replacement has, however, proven challenging. Human cardiac differentiation has been difficult to control, but methods are improving, and the process, to a certain extent, can be manipulated and directed. The stem cell-derived cardiomyocytes described to date exhibit rather immature functional and structural characteristics compared to adult cardiomyocytes. Thus, a future challenge will be to develop strategies to reach a higher degree of cardiomyocyte maturationin vitro, to isolate cardiomyocytes from the heterogeneous pool of differentiating cells, as well as to guide the differentiation into the desired subtype, that is, ventricular, atrial, and pacemaker cells. In this paper, we will discuss the strategies for the generation of cardiomyocytes from pluripotent stem cells and their characteristics, as well as highlight some applications for the cells.
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Wells, Simon P., Helen M. Waddell, Choon Boon Sim, Shiang Y. Lim, Gabriel B. Bernasochi, Davor Pavlovic, Paulus Kirchhof, Enzo R. Porrello, Lea M. D. Delbridge, and James R. Bell. "Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems." American Journal of Physiology-Cell Physiology 317, no. 6 (December 1, 2019): C1256—C1267. http://dx.doi.org/10.1152/ajpcell.00306.2019.
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Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48–120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the β-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.
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Hochman-Mendez, Camila, Dilza Balteiro Pereira de Campos, Rafael Serafim Pinto, Bernardo Jorge da Silva Mendes, Gustavo Miranda Rocha, Gustavo Monnerat, Gilberto Weissmuller, et al. "Tissue-engineered human embryonic stem cell-containing cardiac patches: evaluating recellularization of decellularized matrix." Journal of Tissue Engineering 11 (January 2020): 204173142092148. http://dx.doi.org/10.1177/2041731420921482.
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Decellularized cardiac extracellular matrix scaffolds with preserved composition and architecture can be used in tissue engineering to reproduce the complex cardiac extracellular matrix. However, evaluating the extent of cardiomyocyte repopulation of decellularized cardiac extracellular matrix scaffolds after recellularization attempts is challenging. Here, we describe a unique combination of biochemical, biomechanical, histological, and physiological parameters for quantifying recellularization efficiency of tissue-engineered cardiac patches compared with native cardiac tissue. Human embryonic stem cell-derived cardiomyocytes were seeded into rat heart atrial and ventricular decellularized cardiac extracellular matrix patches. Confocal and atomic force microscopy showed cell integration within the extracellular matrix basement membrane that was accompanied by restoration of native cardiac tissue passive mechanical properties. Multi-electrode array and immunostaining (connexin 43) were used to determine synchronous field potentials with electrical coupling. Myoglobin content (~60%) and sarcomere length measurement (>45% vs 2D culture) were used to evaluate cardiomyocyte maturation of integrated cells. The combination of these techniques allowed us to demonstrate that as cellularization efficiency improves, cardiomyocytes mature and synchronize electrical activity, and tissue mechanical/biochemical properties improve toward those of native tissue.
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Dobrev, Dobromir, and Ursula Ravens. "Remodeling of cardiomyocyte ion channels in human atrial fibrillation." Basic Research in Cardiology 98, no. 3 (May 2003): 137–48. http://dx.doi.org/10.1007/s00395-003-0409-8.
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Baena-Montes, Jara M., Tony O’Halloran, Cormac Clarke, Kevin Donaghey, Eoghan Dunne, Martin O’Halloran, and Leo R. Quinlan. "Electroporation Parameters for Human Cardiomyocyte Ablation In Vitro." Journal of Cardiovascular Development and Disease 9, no. 8 (July 28, 2022): 240. http://dx.doi.org/10.3390/jcdd9080240.
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Cardiac ablation with irreversible electroporation (IRE) is quickly being established as a modality of choice for atrial fibrillation treatment. While it has not yet been optimised, IRE has the potential to significantly limit collateral damage and improve cell-specific targeting associated with other energy sources. However, more tissue and cell-specific evidence is required to demonstrate the selective threshold parameters for human cells. The aim here is to determine the optimal ablation threshold parameters related to lesion size for human cardiomyocytes in 2D culture. Conventional biphasic pulses of different field strengths and on-times were delivered in a monolayer culture system of human AC16 cardiomyocytes. The dynamics of cell death and lesion dimensions were examined at different time points. Human cardiomyocytes are susceptible to significant electroporation and cell death at a field strength of 750 V/cm or higher with 100 μs pulses. Increasing the IRE on-time from 3 ms to 60 ms reduces the effective field threshold to 250 V/cm. Using very short pulses of 2 μs and 5 μs also causes significant cell death, but only at fields higher than 1000 V/cm. A longer on-time results in more cell death and induced greater lesion area in 2D models. In addition, different forms of cell death are predicted based on the evolution of cell death over time. This study presents important findings on the ability of different IRE parameters to induce human cardiomyocyte cell death. Lesion size can be tuned by appropriate choice of IRE parameters and cardiomyocytes display an upregulation of delayed cell death 24 h after electroporation, which is an important consideration for clinical practice.
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Tsai, Su-Yi, Zaniar Ghazizadeh, Hou-Jun Wang, Sadaf Amin, Francis A. Ortega, Zohreh Sadat Badieyan, Zi-Ting Hsu, et al. "A human embryonic stem cell reporter line for monitoring chemical-induced cardiotoxicity." Cardiovascular Research 116, no. 3 (June 7, 2019): 658–70. http://dx.doi.org/10.1093/cvr/cvz148.
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Abstract Aims Human embryonic stem cells (hESCs) can be used to generate scalable numbers of cardiomyocytes (CMs) for studying cardiac biology, disease modelling, drug screens, and potentially for regenerative therapies. A fluorescence-based reporter line will significantly enhance our capacities to visualize the derivation, survival, and function of hESC-derived CMs. Our goal was to develop a reporter cell line for real-time monitoring of live hESC-derived CMs. Methods and results We used CRISPR/Cas9 to knock a mCherry reporter gene into the MYH6 locus of hESC lines, H1 and H9, enabling real-time monitoring of the generation of CMs. MYH6:mCherry+ cells express atrial or ventricular markers and display a range of cardiomyocyte action potential morphologies. At 20 days of differentiation, MYH6:mCherry+ cells show features characteristic of human CMs and can be used successfully to monitor drug-induced cardiotoxicity and oleic acid-induced cardiac arrhythmia. Conclusion We created two MYH6:mCherry hESC reporter lines and documented the application of these lines for disease modelling relevant to cardiomyocyte biology.
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Eisenberg, Leonard M., Keerat Kaur, John M. Castillo, John G. Edwards, and Carol A. Eisenberg. "Dexamethasone Treatment Preserves the Structure of Adult Cardiac Explants and Supports Their Long-Term Contractility In Vitro." International Journal of Translational Medicine 3, no. 3 (September 5, 2023): 360–73. http://dx.doi.org/10.3390/ijtm3030025.
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Normal contractile function of the myocardium is essential for optimal cardiovascular health. Evaluating drug effects on cardiomyocyte function at the cellular level is difficult for long-term studies. Present culture systems rely on isolated, cardiomyocyte preparations or cardiomyocytes derived from pluripotent stem cells (PSCs), all of which have limitations. Isolated, endogenous cardiomyocytes do not remain contractile in culture long term. While PSC-derived cardiomyocytes show contractile activity for longer periods of time, their phenotype is more embryonic than adult. Here we report that dexamethasone (DEX) treatment of adult mouse atrial tissue can extend its functionality in culture. Normally, cardiac explants cease their capacity as a contractile tissue within the first month, as the tissue flattens and spreads out on the culture substrate, while the cells dedifferentiate and lose their myocardial phenotype. However, with DEX treatment, cardiac explants maintain their contractile function, 3D morphology, and myocyte phenotype for up to 6 months. Moreover, DEX also preserved the contractile phenotype of isolated rat cardiomyocytes. These data with DEX suggest that simple modifications in culture conditions can greatly improve the long-term utility of in vitro model systems for screening drugs and agents that could be employed to alleviate human cardiac disease.
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Martin, Kendall E., and Joshua S. Waxman. "Atrial and Sinoatrial Node Development in the Zebrafish Heart." Journal of Cardiovascular Development and Disease 8, no. 2 (February 9, 2021): 15. http://dx.doi.org/10.3390/jcdd8020015.
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Proper development and function of the vertebrate heart is vital for embryonic and postnatal life. Many congenital heart defects in humans are associated with disruption of genes that direct the formation or maintenance of atrial and pacemaker cardiomyocytes at the venous pole of the heart. Zebrafish are an outstanding model for studying vertebrate cardiogenesis, due to the conservation of molecular mechanisms underlying early heart development, external development, and ease of genetic manipulation. Here, we discuss early developmental mechanisms that instruct appropriate formation of the venous pole in zebrafish embryos. We primarily focus on signals that determine atrial chamber size and the specialized pacemaker cells of the sinoatrial node through directing proper specification and differentiation, as well as contemporary insights into the plasticity and maintenance of cardiomyocyte identity in embryonic zebrafish hearts. Finally, we integrate how these insights into zebrafish cardiogenesis can serve as models for human atrial defects and arrhythmias.
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Liu, Yang, Shuang Li, Zhanqun Gao, Shuangjia Li, Qingyun Tan, Yanmei Li, Dongwei Wang, and Qingdong Wang. "Indoleamine 2,3-Dioxygenase 1 (IDO1) Promotes Cardiac Hypertrophy via a PI3K-AKT-mTOR-Dependent Mechanism." Cardiovascular Toxicology 21, no. 8 (May 21, 2021): 655–68. http://dx.doi.org/10.1007/s12012-021-09657-y.
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AbstractIndoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme for tryptophan metabolism, involved in immune cell differentiation/maturation and cancer biology. IDO1 is also expressed in cardiomyocytes, but its roles in the cardiovascular system are not fully understood. Here, we reported the functions of IDO1 during cardiac hypertrophy. Quantitative real-time PCR and Western blot experiments demonstrated the upregulation of IDO1 mRNA and protein levels in human and hypertrophic mouse hearts, as well as in angiotensin II (Ang II)-induced hypertrophic rat cardiomyocytes. IDO1 activity and metabolite product kynurenine were upregulated in rodent hypertrophic hearts and cardiomyocytes. Inhibition of IDO1 activity with PF-06840003 reduced Ang II-induced cardiac hypertrophy and rescued cardiac function in mice. siRNA-mediated knockdown of Ido1 repressed Ang II-induced growth in cardiomyocyte size and overexpression of hypertrophy-associated genes atrial natriuretic peptide (Anp or Nppa), brain natriuretic peptide (Bnp or Nppb), β-myosin heavy chain (β-Mhc or Myh7). By contrast, adenovirus-mediated rat Ido1 overexpression in cardiomyocytes promoted hypertrophic growth induced by Ang II. Mechanism analysis showed that IDO1 overexpression was associated with PI3K-AKT-mTOR signaling to activate the ribosomal protein S6 kinase 1 (S6K1), which promoted protein synthesis in Ang II-induced hypertrophy of rat cardiomyocytes. Finally, we provided evidence that inhibition of PI3K with pictilisib, AKT with perifosine, or mTOR with rapamycin, blocked the effects of IDO1 on protein synthesis and cardiomyocyte hypertrophy in Ang II-treated cells. Collectively, our findings identify that IDO1 promotes cardiomyocyte hypertrophy partially via PI3K-AKT-mTOR-S6K1 signaling.
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Zang, Rongjia, Qingyun Tan, Fanrong Zeng, Dongwei Wang, Shuang Yu, and Qingdong Wang. "JMJD1A Represses the Development of Cardiomyocyte Hypertrophy by Regulating the Expression of Catalase." BioMed Research International 2020 (May 13, 2020): 1–14. http://dx.doi.org/10.1155/2020/5081323.
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The histone demethylase JMJD family is involved in various physiological and pathological functions. However, the roles of JMJD1A in the cardiovascular system remain unknown. Here, we studied the function of JMJD1A in cardiac hypertrophy. The mRNA and protein levels of JMJD1A were significantly downregulated in the hearts of human patients with hypertrophic cardiomyopathy and the hearts of C57BL/6 mice underwent cardiac hypertrophy induced by transverse aortic constriction (TAC) surgery or isoproterenol (ISO) infusion. In neonatal rat cardiomyocytes (NRCMs), siRNA-mediated JMJD1A knockdown facilitated ISO or angiotensin II-induced increase in cardiomyocyte size, protein synthesis, and expression of hypertrophic fetal genes, including atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), and Myh7. By contrast, overexpression of JMJD1A with adenovirus repressed the development of ISO-induced cardiomyocyte hypertrophy. We observed that JMJD1A reduced the production of total cellular and mitochondrial levels of reactive oxygen species (ROS), which was critically involved in the effects of JMJD1A because either N-acetylcysteine or MitoTEMPO treatment blocked the effects of JMJD1A deficiency on cardiomyocyte hypertrophy. Mechanism study demonstrated that JMJD1A promoted the expression and activity of Catalase under basal condition or oxidative stress. siRNA-mediated loss of Catalase blocked the protection of JMJD1A overexpression against ISO-induced cardiomyocyte hypertrophy. These findings demonstrated that JMJD1A loss promoted cardiomyocyte hypertrophy in a Catalase and ROS-dependent manner.
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van Ouwerkerk, Antoinette F., Fernanda M. Bosada, Karel van Duijvenboden, Arjan C. Houweling, Koen T. Scholman, Vincent Wakker, Cornelis P. Allaart, et al. "Patient-Specific TBX5-G125R Variant Induces Profound Transcriptional Deregulation and Atrial Dysfunction." Circulation 145, no. 8 (February 22, 2022): 606–19. http://dx.doi.org/10.1161/circulationaha.121.054347.
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Background: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt–Oram syndrome (also known as hand–heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. Methods: We analyzed ECGs of an extended family pedigree of Holt–Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome ( Tbx5 G125R ) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). Results: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt–Oram syndrome patients. Tbx5 G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5 G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. Conclusions: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.
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Madsen, Alexandra, Grit Höppner, Julia Krause, Marc N. Hirt, Sandra D. Laufer, Michaela Schweizer, Wilson Lek Wen Tan, et al. "An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility." Circulation 142, no. 16 (October 20, 2020): 1562–78. http://dx.doi.org/10.1161/circulationaha.119.044444.
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Background: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell–derived cardiomyocytes. Methods: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell–derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. Results: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. Conclusion: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.
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Liu, Chuyu, and Ning-Yi Shao. "The Differences in the Developmental Stages of the Cardiomyocytes and Endothelial Cells in Human and Mouse Embryos at the Single-Cell Level." International Journal of Molecular Sciences 25, no. 6 (March 13, 2024): 3240. http://dx.doi.org/10.3390/ijms25063240.
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Our research focuses on expression patterns in human and mouse embryonic cardiomyocytes and endothelial cells at the single-cell level. We analyzed single-cell datasets containing different species, cardiac chambers, and cell types. We identified developmentally dynamic genes associated with different cellular lineages in the heart and explored their expression and possible roles during cardiac development. We used dynamic time warping, a method that aligns temporal sequences, to compare these developmental stages across two species. Our results indicated that atrial cardiomyocytes from E9.5 to E13.5 in mice corresponded to a human embryo age of approximately 5–6 weeks, whereas in ventricular cardiomyocytes, they corresponded to a human embryo age of 13–15 weeks. The endothelial cells in mouse hearts corresponded to 6–7-week-old human embryos. Next, we focused on expression changes in cardiac transcription factors over time in different species and chambers, and found that Prdm16 might be related to interspecies cardiomyocyte differences. Moreover, we compared the developmental trajectories of cardiomyocytes differentiated from human pluripotent stem cells and embryonic cells. This analysis explored the relationship between their respective developments and provided compelling evidence supporting the relevance of our dynamic time-warping results. These significant findings contribute to a deeper understanding of cardiac development across different species.
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Wang, Xiaoyu, Susan V. McLennan, Terri J. Allen, Tatiana Tsoutsman, Christopher Semsarian, and Stephen M. Twigg. "Adverse effects of high glucose and free fatty acid on cardiomyocytes are mediated by connective tissue growth factor." American Journal of Physiology-Cell Physiology 297, no. 6 (December 2009): C1490—C1500. http://dx.doi.org/10.1152/ajpcell.00049.2009.
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Diabetic cardiomyopathy is characterized by interstitial fibrosis and cardiomyocyte hypertrophy and apoptosis. Also known as CCN2, connective tissue growth factor (CTGF) is implicated in the fibrosis; however, whether it contributes to cardiomyocytes changes and adverse effects of high glucose and lipids on these cells remains unknown. Hearts from streptozotocin-induced diabetic rats had elevated CTGF and changes of pathological myocardial hypertrophy, fibrosis, and cardiomyocyte apoptosis. Rat H9c2 cardiomyocytes were then treated with recombinant human (rh)CTGF, high glucose, or the saturated free fatty acid palmitate. Each reagent induced cell hypertrophy, as indicated by the ratio of total protein to cell number, cell size, and gene expression of cardiac hypertrophy marker genes atrial natriuretic peptide (ANP), and α-skeletal actin. Each treatment also caused apoptosis measured by increased caspase3/7 activity, apoptotic cells by transferase-mediated dUTP nick end labeling (TUNEL) assay, and lower viable cell number. Further studies showed CTGF mRNA was rapidly induced by high glucose and palmitate in H9c2 cells and in mouse neonatal cardiomyocyte primary cultures. small interfering RNA against CTGF blocked the high glucose and palmitate induction of hypertrophy and apoptosis. In addition, these CTGF effects were through the tyrosine kinase A (TrkA) receptor with tyrosine kinase activity, which has previously been implicated in CTGF signaling: TrkA was phosphorylated by CTGF, and a specific TrkA blocker abrogated CTGF-induced effects on hypertrophy and apoptosis. For the first time in any system, fatty acid is newly identified as a regulator of CTGF, and this work implicates autocrine CTGF as a mediator of adverse effects of high glucose and fatty acids in cardiomyocytes.
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Abi-Gerges, Najah, Paul E. Miller, and Andre Ghetti. "Human Heart Cardiomyocytes in Drug Discovery and Research: New Opportunities in Translational Sciences." Current Pharmaceutical Biotechnology 21, no. 9 (June 9, 2020): 787–806. http://dx.doi.org/10.2174/1389201021666191210142023.
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In preclinical drug development, accurate prediction of drug effects on the human heart is critically important, whether in the context of cardiovascular safety or for the purpose of modulating cardiac function to treat heart disease. Current strategies have significant limitations, whereby, cardiotoxic drugs can escape detection or potential life-saving therapies are abandoned due to false positive toxicity signals. Thus, new and more reliable translational approaches are urgently needed to help accelerate the rate of new therapy development. Renewed efforts in the recovery of human donor hearts for research and in cardiomyocyte isolation methods, are providing new opportunities for preclinical studies in adult primary cardiomyocytes. These cells exhibit the native physiological and pharmacological properties, overcoming the limitations presented by artificial cellular models, animal models and have great potential for providing an excellent tool for preclinical drug testing. Adult human primary cardiomyocytes have already shown utility in assessing drug-induced cardiotoxicity risk and helping in the identification of new treatments for cardiac diseases, such as heart failure and atrial fibrillation. Finally, strategies with actionable decision-making trees that rely on data derived from adult human primary cardiomyocytes will provide the holistic insights necessary to accurately predict human heart effects of drugs.
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Duelen, Robin, Guillaume Gilbert, Abdulsamie Patel, Nathalie de Schaetzen, Liesbeth De Waele, Llewelyn Roderick, Karin R. Sipido, et al. "Activin A Modulates CRIPTO-1/HNF4α+ Cells to Guide Cardiac Differentiation from Human Embryonic Stem Cells." Stem Cells International 2017 (2017): 1–17. http://dx.doi.org/10.1155/2017/4651238.
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The use of human pluripotent stem cells in basic and translational cardiac research requires efficient differentiation protocols towards cardiomyocytes. In vitro differentiation yields heterogeneous populations of ventricular-, atrial-, and nodal-like cells hindering their potential applications in regenerative therapies. We described the effect of the growth factor Activin A during early human embryonic stem cell fate determination in cardiac differentiation. Addition of high levels of Activin A during embryoid body cardiac differentiation augmented the generation of endoderm derivatives, which in turn promoted cardiomyocyte differentiation. Moreover, a dose-dependent increase in the coreceptor expression of the TGF-β superfamily member CRIPTO-1 was observed in response to Activin A. We hypothesized that interactions between cells derived from meso- and endodermal lineages in embryoid bodies contributed to improved cell maturation in early stages of cardiac differentiation, improving the beating frequency and the percentage of contracting embryoid bodies. Activin A did not seem to affect the properties of cardiomyocytes at later stages of differentiation, measuring action potentials, and intracellular Ca2+ dynamics. These findings are relevant for improving our understanding on human heart development, and the proposed protocol could be further explored to obtain cardiomyocytes with functional phenotypes, similar to those observed in adult cardiac myocytes.
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Lavall, Daniel, Pia Schuster, Nadine Jacobs, Andrey Kazakov, Michael Böhm, and Ulrich Laufs. "Rac1 GTPase regulates 11β hydroxysteroid dehydrogenase type 2 and fibrotic remodeling." Journal of Biological Chemistry 292, no. 18 (March 20, 2017): 7542–53. http://dx.doi.org/10.1074/jbc.m116.764449.
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The aim of the study was to characterize the role of Rac1 GTPase for the mineralocorticoid receptor (MR)-mediated pro-fibrotic remodeling. Transgenic mice with cardiac overexpression of constitutively active Rac1 (RacET) develop an age-dependent phenotype with atrial dilatation, fibrosis, and atrial fibrillation. Expression of MR was similar in RacET and WT mice. The expression of 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) was age-dependently up-regulated in the atria and the left ventricles of RacET mice on mRNA and protein levels. Statin treatment inhibiting Rac1 geranylgeranylation reduced 11β-HSD2 up-regulation. Samples of human left atrial myocardium showed a positive correlation between Rac1 activity and 11β-HSD2 expression (r = 0.7169). Immunoprecipitation showed enhanced Rac1-bound 11β-HSD2 relative to Rac1 expression in RacET mice that was diminished with statin treatment. Both basal and phorbol 12-myristate 13-acetate (PMA)-induced NADPH oxidase activity were increased in RacET and correlated positively with 11β-HSD2 expression (r = 0.788 and r = 0.843, respectively). In cultured H9c2 cardiomyocytes, Rac1 activation with l-buthionine sulfoximine increased; Rac1 inhibition with NSC23766 decreased 11β-HSD2 mRNA and protein expression. Connective tissue growth factor (CTGF) up-regulation induced by aldosterone was prevented with NSC23766. Cardiomyocyte transfection with 11β-HSD2 siRNA abolished the aldosterone-induced CTGF up-regulation. Aldosterone-stimulated MR nuclear translocation was blocked by the 11β-HSD2 inhibitor carbenoxolone. In cardiac fibroblasts, nuclear MR translocation induced by aldosterone was inhibited with NSC23766 and spironolactone. NSC23766 prevented the aldosterone-induced proliferation and migration of cardiac fibroblasts and the up-regulation of CTGF and fibronectin. In conclusion, Rac1 GTPase regulates 11β-HSD2 expression, MR activation, and MR-mediated pro-fibrotic signaling.
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Çubukçuoğlu Deniz, Günseli, Serkan Durdu, Yeşim Doğan, Esra Erdemli, Hilal Özdağ, and Ahmet Ruchan Akar. "Molecular Signatures of Human Chronic Atrial Fibrillation in Primary Mitral Regurgitation." Cardiovascular Therapeutics 2021 (October 15, 2021): 1–12. http://dx.doi.org/10.1155/2021/5516185.
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Objectives. Transcriptomics of atrial fibrillation (AFib) in the setting of chronic primary mitral regurgitation (MR) remains to be characterized. We aimed to compare the gene expression profiles of patients with degenerative MR in AFib and sinus rhythm (SR) for a clearer picture of AFib pathophysiology. Methods. After transcriptomic analysis and bioinformatics ( n = 59 ), differentially expressed genes were defined using 1.5-fold change as the threshold. Additionally, independent datasets from GEO were included as meta-analyses. Results. QRT-PCR analysis confirmed that AFib persistence was associated with increased expression molecular changes underlying a transition to heart failure (NPPB, P = 0.002 ; ANGPTL2, P = 0.002 ; IGFBP2, P = 0.010 ), structural remodeling including changes in the extracellular matrix and cellular stress response (COLQ, P = 0.003 ; COMP, P = 0.028 ; DHRS9, P = 0.038 ; CHGB, P = 0.038 ), and cellular stress response (DNAJA4, P = 0.038 ). Furthermore, AFib persistence was associated with decreased expression of the targets of structural remodeling (BMP7, P = 0.021 ) and electrical remodeling (CACNB2, P = 0.035 ; MCOLN3, P = 0.035 ) in both left and right atrial samples. The transmission electron microscopic analysis confirmed ultrastructural atrial remodeling and autophagy in human AFib atrial samples. Conclusions. Atrial cardiomyocyte remodeling in persistent AFib is closely linked to alterations in gene expression profiles compared to SR in patients with primary MR. Study findings may lead to novel therapeutic targets. This trial is registered with ClinicalTrials.gov identifier: NCT00970034.
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Goruppi, Sandro, Richard D. Patten, Thomas Force, and John M. Kyriakis. "Helix-Loop-Helix Protein p8, a Transcriptional Regulator Required for Cardiomyocyte Hypertrophy and Cardiac Fibroblast Matrix Metalloprotease Induction." Molecular and Cellular Biology 27, no. 3 (November 20, 2006): 993–1006. http://dx.doi.org/10.1128/mcb.00996-06.
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ABSTRACT Cardiomyocyte hypertrophy and extracellular matrix remodeling, primarily mediated by inflammatory cytokine-stimulated cardiac fibroblasts, are critical cellular events in cardiac pathology. The molecular components governing these processes remain nebulous, and few genes have been linked to both hypertrophy and matrix remodeling. Here we show that p8, a small stress-inducible basic helix-loop-helix protein, is required for endothelin- and α-adrenergic agonist-induced cardiomyocyte hypertrophy and for tumor necrosis factor-stimulated induction, in cardiac fibroblasts, of matrix metalloproteases (MMPs) 9 and 13—MMPs linked to general inflammation and to adverse ventricular remodeling in heart failure. In a stimulus-dependent manner, p8 associates with chromatin containing c-Jun and with the cardiomyocyte atrial natriuretic factor (anf) promoter and the cardiac fibroblast mmp9 and mmp13 promoters, established activator protein 1 effectors. p8 is also induced strongly in the failing human heart by a process reversed upon therapeutic intervention. Our results identify an unexpectedly broad involvement for p8 in key cellular events linked to cardiomyocyte hypertrophy and cardiac fibroblast MMP production, both of which occur in heart failure.
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Moulin, Sophie, Amandine Thomas, Stefan Wagner, Michael Arzt, Hervé Dubouchaud, Frédéric Lamarche, Sophie Bouyon, et al. "Intermittent Hypoxia-Induced Cardiomyocyte Death Is Mediated by HIF-1 Dependent MAM Disruption." Antioxidants 11, no. 8 (July 27, 2022): 1462. http://dx.doi.org/10.3390/antiox11081462.
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Rationale: Intermittent hypoxia (IH) is one of the main features of sleep-disordered breathing (SDB). Recent findings indicate that hypoxia inducible factor-1 (HIF-1) promotes cardiomyocytes apoptosis during chronic IH, but the mechanisms involved remain to be elucidated. Here, we hypothesize that IH-induced ER stress is associated with mitochondria-associated ER membrane (MAM) alteration and mitochondrial dysfunction, through HIF-1 activation. Methods: Right atrial appendage biopsies from patients with and without SDB were used to determine HIF-1α, Grp78 and CHOP expressions. Wild-type and HIF-1α+/− mice were exposed to normoxia (N) or IH (21–5% O2, 60 cycles/h, 8 h/day) for 21 days. Expressions of HIF-1α, Grp78 and CHOP, and apoptosis, were measured by Western blot and immunochemistry. In isolated cardiomyocytes, we examined structural integrity of MAM by proximity ligation assay and their function by measuring ER-to-mitochondria Ca2+ transfer by confocal microscopy. Finally, we measured mitochondrial respiration using oxygraphy and calcium retention capacity (CRC) by spectrofluorometry. MAM structure was also investigated in H9C2 cells incubated with 1 mM CoCl2, a potent HIF-1α inducer. Results: In human atrial biopsies and mice, IH induced HIF-1 activation, ER stress and apoptosis. IH disrupted MAM, altered Ca2+ homeostasis, mitochondrial respiration and CRC. Importantly, IH had no effect in HIF-1α+/− mice. Similar to what observed under IH, HIF-1α overexpression was associated with MAM alteration in H9C2. Conclusion: IH-induced ER stress, MAM alterations and mitochondrial dysfunction were mediated by HIF-1; all these intermediate mechanisms ultimately inducing cardiomyocyte apoptosis. This suggests that HIF-1 modulation might limit the deleterious cardiac effects of SDB.
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Mazhar, Fazeelat, Chiara Bartolucci, Francesco Regazzoni, Michelangelo Paci, Luca Dedè, Alfio Quarteroni, Cristiana Corsi, and Stefano Severi. "A detailed mathematical model of the human atrial cardiomyocyte: Integration of electrophysiology and cardiomechanics." Vascular Pharmacology 155 (June 2024): 107330. http://dx.doi.org/10.1016/j.vph.2024.107330.
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Ragulya, M. R., L. P. Goralskyi, I. M. Sokulskyi, and N. L. Kolesnik. "Morphometric parameters of the heart of domestic sheep Ovis aries L., 1758." Ukrainian Journal of Veterinary and Agricultural Sciences 7, no. 1 (March 26, 2024): 94–101. http://dx.doi.org/10.32718/ujvas7-1.15.
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The cardiovascular system is one of the most critical animal systems. Its main functions are to supply organs and tissues with oxygen and nutrients and to remove metabolic products from tissues. Diseases of the cardiovascular system of animals cause significant economic damage, including reduced productivity, reproductive qualities, and the development of concomitant diseases. The prevention, diagnosis, surgery, and treatment of such pathologies are only possible by knowing the morphofunctional parameters of comparative anatomy, histology, and physiology. The heart plays a vital role in blood circulation and regulates the proper functioning and development of all organs of animal and human organisms. The study of the heart structure reveals topography features, age-related morphology, and development of this organ in domestic animals and remains relevant. The article is a fragment of the scientific developments of the Department of Normal and Pathological Morphology, Hygiene and Forensics of Polissia National University on the following topics: “Features of the morphology of the heart of domestic mammals” (state registration number 0121U108884); “Development, morphology and histochemistry of animal organs in normal and pathological conditions”, state registration number 0113U000900. In the study, sexually mature clinically healthy animals (n = 5) belonging to the class Mammalia – Mammals, species Ovis aries L., 1758 – domestic sheep (ram) were investigated. The study aimed to evaluate the morphological structures of the heart of mature sheep using macro- and microscopic, morphometric, and statistical research methods. The heart of a sexually mature domestic sheep was subjected to anatomical dissection. Sections were stained with hematoxylin and eosin to study the cyto- and histoarchitectonics of the heart for microscopic examination of transverse striated myocardial muscle tissue, detection of cardiomyocyte cytostructure, and Heidenhain staining of histological specimens were used. The morphology of the heart in sheep has a similar organization plan, topographic location of the organ, and anatomical and histological structure. However, there are some striking species and morphological features. The heart of the domestic sheep belongs to the expanded-shortened anatomical type (according to its development index – 145.5 ± 4.02 %). According to the studies, the absolute and relative weight of the heart of mature sheep is, accordingly, 208.4 ± 9.82 g and 0.44 ± 0.007 %, and the weight without epicardial fat is 175.0 ± 8.17 g. It has been shown that the microscopic structure of the ventricles and atria of the sheep heart differ in cytometric parameters. Cardiomyocytes of the left ventricle have the most significant volume (3982.99 ± 423.96 μm3), the smaller – of the right ventricle (2463.02 ± 318.04 μm3). The lowest index was observed in atrial cardiomyocytes (1215.93 ± 176.94 μm3). The volumes of cardiomyocyte nuclei in the left ventricle were (53.42 ± 5.18 μm3) and in the right ventricle (52.85 ± 4.33 μm3). The volume of atrial nuclei (50.16 ± 4.57 μm3) is almost the same. Such ambiguous morphometric parameters of cardiomyocytes and their nuclei volumes are directly reflected in their nuclear-cytoplasmic ratio, which is the smallest in cardiomyocytes of the left ventricle (0.0136 ± 0.0062), larger in cardiomyocytes of the right ventricle (0.0219 ± 0.0079) and the largest (0.0430 ± 0.0096) in atrial cardiomyocytes. The obtained results of the study of the macro- and microscopic structure of the heart of domestic sheep significantly supplement the information on heart morphology in the relevant sections of comparative anatomy and histology and are a significant contribution to clinical cardiology.
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Kuwana, Masataka, Hiroaki Kodama, Yuka Okazaki, Takashi Satoh, Takafumi Inoue, Yutaka Kawakami, and Yasuo Ikeda. "Multi-Lineage Potential of Human Monocyte-Derived Mesenchymal Progenitors (MOMPs)." Blood 104, no. 11 (November 16, 2004): 3595. http://dx.doi.org/10.1182/blood.v104.11.3595.3595.
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Abstract Circulating CD14+ monocytes are known to be precursors of phagocytes, such as macrophages and dendritic cells. We have recently identified a novel CD14+CD45+CD34+type I collagen+ cell fraction derived from human circulating CD14+ monocytes, monocyte-derived mesenchymal progenitor (MOMP), which contains progenitors capable of differentiating into a variety of mesenchymal cells, including bone, cartilage, fat and skeletal muscle (J Leukoc Biol2003;74:833). Here, we investigated a differentiation potential of human MOMPs along endothelial, cardiomyocytic, and neuronal lineages. MOMPs treated with angiogenic factors for 7 days underwent a change in their morphology from spindle-shaped to caudated. Transmission electron microscopic analysis revealed that these cells displayed rod-shaped microtubulated structures corresponding to Weibel-Palade bodies. Almost every cell expressed CD31, VE-vadherin, VEGFR2, Tie-2, von Willeband factor (vWF), eNOS and CD146, but CD14/CD45 expression was markedly down-regulated. Functional characteristics, including vWF release upon histamine stimulation, acetylated LDL uptake, and up-regulated expression of VEGFR1 in response to hypoxia, were indistinguishable between MOMP-derived endothelial-like cells and human umbilical vein endothelial cells. We further performed xenogenic transplantation studies using a SCID mouse model, in which syngeneic colon carcinoma cells were injected subcutaneously with or without human MOMPs. Tumors generated from carcinoma cells alone showed central necrosis and less blood vessel formation, but co-transplantation with MOMPs resulted in promotion of blood vessel formation and no areas of necrosis. Immunohistochemical analysis using human specific antibodies to CD31 and vWF demonstrated that >50% of blood vessels incorporated MOMP-derived endothelial cells. To investigate whether MOMPs were able to differentiate along cardiomyocytic and neuronal lineages, pre-labeled human MOMPs were co-cultivated with primary cultures of rat cardiomyocytes or neurons. Shortly after co-cultivation with rat cardiomyocytes, the majority of MOMPs expressed cardiomyocyte-specific transcription factors, Nkx2.5, GATA-4, eHAND and MEF2, together with CD14/CD45. Subsequently, a subpopulation of MOMPs expressed troponin I and atrial natriuretic peptide and lost CD14/CD45 expression. Spontaneously beating cells formed gap junctions with adjacent rat cardiomyocytes and exhibited electrophysiological properties of ventricular myocytes. MOMPs co-cultured with rat neurons for 3 days expressed neuron-specific transcription factors, Ngn-2, NeuroD, Mash1 and nestin. At day 7, these cells expressed neuron-specific markers, NeuN and Hu. At day 18, a subpopulation of the cells exhibited a neuron-like morphology, including characteristic axons and a refractile round cell body, and expressed MAP2 and β3-tubulin. Co-cultivation of MOMPs with rat cells induced to express GFP by adenoviral gene transfer resulted in appearance of human cardiomyoocytes and neurons without GFP staining, suggesting that our observations are not solely explained by cell fusion. In summary, human MOMPs are capable of differentiating along endothelial and cardiomyocytic lineages as well as a neuronal lineage of an ectoderm-origin. Circulating CD14+ monocytes can be an abundant and easily accessible source for autologous cell transplantation for tissue regeneration.
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Sutanto, Henry. "Individual Contributions of Cardiac Ion Channels on Atrial Repolarization and Reentrant Waves: A Multiscale In-Silico Study." Journal of Cardiovascular Development and Disease 9, no. 1 (January 14, 2022): 28. http://dx.doi.org/10.3390/jcdd9010028.
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The excitation, contraction, and relaxation of an atrial cardiomyocyte are maintained by the activation and inactivation of numerous cardiac ion channels. Their collaborative efforts cause time-dependent changes of membrane potential, generating an action potential (AP), which is a surrogate marker of atrial arrhythmias. Recently, computational models of atrial electrophysiology emerged as a modality to investigate arrhythmia mechanisms and to predict the outcome of antiarrhythmic therapies. However, the individual contribution of atrial ion channels on atrial action potential and reentrant arrhythmia is not yet fully understood. Thus, in this multiscale in-silico study, perturbations of individual atrial ionic currents (INa, Ito, ICaL, IKur, IKr, IKs, IK1, INCX and INaK) in two in-silico models of human atrial cardiomyocyte (i.e., Courtemanche-1998 and Grandi-2011) were performed at both cellular and tissue levels. The results show that the inhibition of ICaL and INCX resulted in AP shortening, while the inhibition of IKur, IKr, IKs, IK1 and INaK prolonged AP duration (APD). Particularly, in-silico perturbations (inhibition and upregulation) of IKr and IKs only minorly affected atrial repolarization in the Grandi model. In contrast, in the Courtemanche model, the inhibition of IKr and IKs significantly prolonged APD and vice versa. Additionally, a 50% reduction of Ito density abbreviated APD in the Courtemanche model, while the same perturbation prolonged APD in the Grandi model. Similarly, a strong model dependence was also observed at tissue scale, with an observable IK1-mediated reentry stabilizing effect in the Courtemanche model but not in the Grandi atrial model. Moreover, the Grandi model was highly sensitive to a change on intracellular Ca2+ concentration, promoting a repolarization failure in ICaL upregulation above 150% and facilitating reentrant spiral waves stabilization by ICaL inhibition. Finally, by incorporating the previously published atrial fibrillation (AF)-associated ionic remodeling in the Courtemanche atrial model, in-silico modeling revealed the antiarrhythmic effect of IKr inhibition in both acute and chronic settings. Overall, our multiscale computational study highlights the strong model-dependent effects of ionic perturbations which could affect the model’s accuracy, interpretability, and prediction. This observation also suggests the need for a careful selection of in-silico models of atrial electrophysiology to achieve specific research aims.
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Wei, Chi-Ming, Margarita Bracamonte, Shi-Wen Jiang, Richard C. Daly, Christopher G. A. McGregor, Shaobo Zhang, and Charles Y. F. Young. "Localization of endothelial nitric oxide synthase in the normal and failing human atrial myocardium." Proceedings, annual meeting, Electron Microscopy Society of America 54 (August 11, 1996): 786–87. http://dx.doi.org/10.1017/s0424820100166397.
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Nitric oxide (NO) is a potent endothelium-derived relaxing factor which also may modulate cardiomyocyte inotropism and growth via increasing cGMP. While endothelial nitric oxide synthase (eNOS) isoforms have been detected in non-human mammalian tissues, expression and localization of eNOS in the normal and failing human myocardium are poorly defined. Therefore, the present study was designed to investigate eNOS in human cardiac tissues in the presence and absence of congestive heart failure (CHF).Normal and failing atrial tissue were obtained from six cardiac donors and six end-stage heart failure patients undergoing primary cardiac transplantation. ENOS protein expression and localization was investigated utilizing Western blot analysis and immunohistochemical staining with the polyclonal rabbit antibody to eNOS (Transduction Laboratories, Lexington, Kentucky).
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S. Ramos, Kennedy, Lisa Pool, Mathijs S. van Schie, Leonoor F. J. M. Wijdeveld, Willemijn F. B. van der Does, Luciënne Baks, H. M. Danish Sultan, et al. "Degree of Fibrosis in Human Atrial Tissue Is Not the Hallmark Driving AF." Cells 11, no. 3 (January 26, 2022): 427. http://dx.doi.org/10.3390/cells11030427.
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Background: The current paradigm is that fibrosis promotes electrophysiological disorders and drives atrial fibrillation (AF). In this current study, we investigated the relation between the degree of fibrosis in human atrial tissue samples of controls and patients in various stages of AF and the degree of electrophysiological abnormalities. Methods: The degree of fibrosis was measured in the atrial tissue and serum of patients in various stages of AF and the controls. Hereto, picrosirius and H&E staining were performed to quantify degree of total, endo-perimysial fibrosis, and cardiomyocyte diameter. Western blot quantified fibrosis markers: neural cell adhesion molecule, tissue inhibitor of metalloproteinase, lysyl oxidase, and α-smooth muscle actin. In serum, the ratio carboxyl-terminal telopeptide of collagen/matrix-metalloproteinase1 was determined. High-resolution epicardial mapping evaluated low-voltage areas and conduction abnormalities. Results: No significant differences were observed in the degree of fibrosis between the groups. Finally, no significant correlation—absolute nor spatial—was observed between all electrophysiological parameters and histological fibrosis markers. Conclusions: No differences in the degree of fibrosis were observed in patients from various stages of AF compared to the controls. Moreover, electrophysiological abnormalities did not correlate with any of the fibrosis markers. The findings indicate that fibrosis is not the hallmark of structural remodeling in AF.
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Godoy-Marín, Héctor, Verónica Jiménez-Sábado, Carmen Tarifa, Antonino Ginel, Joana Larupa Dos Santos, Bo Hjorth Bentzen, Leif Hove-Madsen, and Francisco Ciruela. "Increased Density of Endogenous Adenosine A2A Receptors in Atrial Fibrillation: From Cellular and Porcine Models to Human Patients." International Journal of Molecular Sciences 24, no. 4 (February 11, 2023): 3668. http://dx.doi.org/10.3390/ijms24043668.
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Adenosine, an endogenous nucleoside, plays a critical role in maintaining homeostasis during stressful situations, such as energy deprivation or cellular damage. Therefore, extracellular adenosine is generated locally in tissues under conditions such as hypoxia, ischemia, or inflammation. In fact, plasma levels of adenosine in patients with atrial fibrillation (AF) are elevated, which also correlates with an increased density of adenosine A2A receptors (A2ARs) both in the right atrium and in peripheral blood mononuclear cells (PBMCs). The complexity of adenosine-mediated effects in health and disease requires simple and reproducible experimental models of AF. Here, we generate two AF models, namely the cardiomyocyte cell line HL-1 submitted to Anemonia toxin II (ATX-II) and a large animal model of AF, the right atrium tachypaced pig (A-TP). We evaluated the density of endogenous A2AR in those AF models. Treatment of HL-1 cells with ATX-II reduced cell viability, while the density of A2AR increased significantly, as previously observed in cardiomyocytes with AF. Next, we generated the animal model of AF based on tachypacing pigs. In particular, the density of the key calcium regulatory protein calsequestrin-2 was reduced in A-TP animals, which is consistent with the atrial remodelling shown in humans suffering from AF. Likewise, the density of A2AR in the atrium of the AF pig model increased significantly, as also shown in the biopsies of the right atrium of subjects with AF. Overall, our findings revealed that these two experimental models of AF mimicked the alterations in A2AR density observed in patients with AF, making them attractive models for studying the adenosinergic system in AF.
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Anderson, Ethan J., Evelio Rodriguez, Curtis A. Anderson, Kathleen Thayne, W. Randolph Chitwood, and Alan P. Kypson. "Increased propensity for cell death in diabetic human heart is mediated by mitochondrial-dependent pathways." American Journal of Physiology-Heart and Circulatory Physiology 300, no. 1 (January 2011): H118—H124. http://dx.doi.org/10.1152/ajpheart.00932.2010.
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Progressive energy deficiency and loss of cardiomyocyte numbers are two prominent factors that lead to heart failure in experimental models. Signals that mediate cardiomyocyte cell death have been suggested to come from both extrinsic (e.g., cytokines) and intrinsic (e.g., mitochondria) sources, but the evidence supporting these mechanisms remains unclear, and virtually nonexistent in humans. In this study, we investigated the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium (Ca2+) using permeabilized myofibers of right atrium obtained from diabetic ( n = 9) and nondiabetic ( n = 12) patients with coronary artery disease undergoing nonemergent coronary revascularization surgery. Under conditions that mimic the energetic state of the heart in vivo (pyruvate, glutamate, malate, and 100 μM ADP), cardiac mitochondria from diabetic patients show an increased sensitivity to Ca2+-induced mPTP opening compared with nondiabetic patients. This increased mPTP Ca2+ sensitivity in diabetic heart mitochondria is accompanied by a substantially greater rate of mitochondrial H2O2 emission under identical conditions, despite no differences in respiratory capacity under these conditions or mitochondrial enzyme content. Activity of the intrinsic apoptosis pathway mediator caspase-9 was greater in diabetic atrial tissue, whereas activity of the extrinsic pathway mediator caspase-8 was unchanged between groups. Furthermore, caspase-3 activity was not significantly increased in diabetic atrial tissue. These data collectively suggest that the myocardium in diabetic patients has a greater overall propensity for mitochondrial-dependent cell death, possibly as a result of metabolic stress-imposed changes that have occurred within the mitochondria, rendering them more susceptible to insults such as Ca2+ overload. In addition, they lend further support to the notion that mitochondria represent a viable target for future therapies directed at ameliorating heart failure and other comorbidities that come with diabetes.
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Amin, Mohamed, Yoshihiro Kushida, Shohei Wakao, Masaaki Kitada, Kazuki Tatsumi, and Mari Dezawa. "Cardiotrophic Growth Factor–Driven Induction of Human Muse Cells Into Cardiomyocyte-Like Phenotype." Cell Transplantation 27, no. 2 (February 2018): 285–98. http://dx.doi.org/10.1177/0963689717721514.
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Multilineage-differentiating stress-enduring (Muse) cells are endogenous nontumorigenic stem cells collectable as stage-specific embryonic antigen 3 (SSEA-3) + from various organs including the bone marrow and are pluripotent-like. The potential of human bone marrow-derived Muse cells to commit to cardiac lineage cells was evaluated. We found that (1) initial treatment of Muse cells with 5′-azacytidine in suspension culture successfully accelerated demethylation of cardiac marker Nkx2.5 promoter; (2) then transferring the cells onto adherent culture and treatment with early cardiac differentiation factors including wingless-int (Wnt)-3a, bone morphogenetic proteins (BMP)-2/4, and transforming growth factor (TGF) β1; and (3) further treatment with late cardiac differentiation cytokines including cardiotrophin-1 converted Muse cells into cardiomyocyte-like cells that expressed α-actinin and troponin-I with a striation-like pattern. MLC2a expression in the final step suggested differentiation of the cells into an atrial subtype. MLC2v, a marker for a mature ventricular subtype, was expressed when cells were treated with Dickkopf-related protein 1 (DKK-1) and Noggin, inhibitors of Wnt3a and BMP-4, respectively, between steps (2) and (3). None of the steps included exogenous gene transfection, making induced cells feasible for future clinical application.
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Ivashchenko, Christine Y., Gordon C. Pipes, Irina M. Lozinskaya, Zuojun Lin, Xu Xiaoping, Saul Needle, Eugene T. Grygielko, et al. "Human-induced pluripotent stem cell-derived cardiomyocytes exhibit temporal changes in phenotype." American Journal of Physiology-Heart and Circulatory Physiology 305, no. 6 (September 15, 2013): H913—H922. http://dx.doi.org/10.1152/ajpheart.00819.2012.
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Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been recently derived and are used for basic research, cardiotoxicity assessment, and phenotypic screening. However, the hiPS-CM phenotype is dependent on their derivation, age, and culture conditions, and there is disagreement as to what constitutes a functional hiPS-CM. The aim of the present study is to characterize the temporal changes in hiPS-CM phenotype by examining five determinants of cardiomyocyte function: gene expression, ion channel functionality, calcium cycling, metabolic activity, and responsiveness to cardioactive compounds. Based on both gene expression and electrophysiological properties, at day 30 of differentiation, hiPS-CMs are immature cells that, with time in culture, progressively develop a more mature phenotype without signs of dedifferentiation. This phenotype is characterized by adult-like gene expression patterns, action potentials exhibiting ventricular atrial and nodal properties, coordinated calcium cycling and beating, suggesting the formation of a functional syncytium. Pharmacological responses to pathological (endothelin-1), physiological (IGF-1), and autonomic (isoproterenol) stimuli similar to those characteristic of isolated adult cardiac myocytes are present in maturing hiPS-CMs. In addition, thyroid hormone treatment of hiPS-CMs attenuated the fetal gene expression in favor of a more adult-like pattern. Overall, hiPS-CMs progressively acquire functionality when maintained in culture for a prolonged period of time. The description of this evolving phenotype helps to identify optimal use of hiPS-CMs for a range of research applications.
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Han, Wei, Songbin Fu, Na Wei, Baodong Xie, Weimin Li, Shusen Yang, Yue Li, Zijun Liang, and Hong Huo. "Nitric oxide overproduction derived from inducible nitric oxide synthase increases cardiomyocyte apoptosis in human atrial fibrillation." International Journal of Cardiology 130, no. 2 (November 2008): 165–73. http://dx.doi.org/10.1016/j.ijcard.2008.02.026.
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Zhao, Guo-Jun, Chang-Ling Zhao, Shan Ouyang, Ke-Qiong Deng, Lihua Zhu, Augusto C. Montezano, Changjiang Zhang, et al. "Ca 2+ -Dependent NOX5 (NADPH Oxidase 5) Exaggerates Cardiac Hypertrophy Through Reactive Oxygen Species Production." Hypertension 76, no. 3 (September 2020): 827–38. http://dx.doi.org/10.1161/hypertensionaha.120.15558.
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NOX5 (NADPH oxidase 5) is a homolog of the gp91 phox subunit of the phagocyte NOX, which generates reactive oxygen species. NOX5 is involved in sperm motility and vascular contraction and has been implicated in diabetic nephropathy, atherosclerosis, and stroke. The function of NOX5 in the cardiac hypertrophy is unknown. Because NOX5 is a Ca 2+ -sensitive, procontractile NOX isoform, we questioned whether it plays a role in cardiac hypertrophy. Studies were performed in (1) cardiac tissue from patients undergoing heart transplant for cardiomyopathy and heart failure, (2) NOX5-expressing rat cardiomyocytes, and (3) mice expressing human NOX5 in a cardiomyocyte-specific manner. Cardiac hypertrophy was induced in mice by transverse aorta coarctation and Ang II (angiotensin II) infusion. NOX5 expression was increased in human failing hearts. Rat cardiomyocytes infected with adenoviral vector encoding human NOX5 cDNA exhibited elevated reactive oxygen species levels with significant enlargement and associated increased expression of ANP (atrial natriuretic peptides) and β-MHC (β-myosin heavy chain) and prohypertrophic genes ( Nppa , Nppb , and Myh7 ) under Ang II stimulation. These effects were reduced by N-acetylcysteine and diltiazem. Pressure overload and Ang II infusion induced left ventricular hypertrophy, interstitial fibrosis, and contractile dysfunction, responses that were exaggerated in cardiac-specific NOX5 trangenic mice. These phenomena were associated with increased reactive oxygen species levels and activation of redox-sensitive MAPK (mitogen-activated protein kinase). N-acetylcysteine treatment reduced cardiac oxidative stress and attenuated cardiac hypertrophy in NOX5 trangenic. Our study defines Ca 2+ -regulated NOX5 as an important NOX isoform involved in oxidative stress- and MAPK-mediated cardiac hypertrophy and contractile dysfunction.
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Richer, Romain, Sandrine Lemoine, Jean-Luc Hanouz, Benoît Bernay, Marie Nowoczyn, Stéphane Allouche, and Laurent Coulbault. "0485 : Subsarcolemmal and interfibrillar mitochondria in human atrial cardiomyocyte: an enzymatic and proteomic study in diabetic patients." Archives of Cardiovascular Diseases Supplements 8, no. 3 (April 2016): 208. http://dx.doi.org/10.1016/s1878-6480(16)30377-9.
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Gündel, Daniel, Thu Hang Lai, Sladjana Dukic-Stefanovic, Rodrigo Teodoro, Winnie Deuther-Conrad, Magali Toussaint, Klaus Kopka, et al. "Non-Invasive Assessment of Locally Overexpressed Human Adenosine 2A Receptors in the Heart of Transgenic Mice." International Journal of Molecular Sciences 23, no. 3 (January 18, 2022): 1025. http://dx.doi.org/10.3390/ijms23031025.
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A2A adenosine receptors (A2A-AR) have a cardio-protective function upon ischemia and reperfusion, but on the other hand, their stimulation could lead to arrhythmias. Our aim was to investigate the potential use of the PET radiotracer [18F]FLUDA to non-invasively determine the A2A-AR availability for diagnosis of the A2AR status. Therefore, we compared mice with cardiomyocyte-specific overexpression of the human A2A-AR (A2A-AR TG) with the respective wild type (WT). We determined: (1) the functional impact of the selective A2AR ligand FLUDA on the contractile function of atrial mouse samples, (2) the binding parameters (Bmax and KD) of [18F]FLUDA on mouse and human atrial tissue samples by autoradiographic studies, and (3) investigated the in vivo uptake of the radiotracer by dynamic PET imaging in A2A-AR TG and WT. After A2A-AR stimulation by the A2A-AR agonist CGS 21680 in isolated atrial preparations, antagonistic effects of FLUDA were found in A2A-AR-TG animals but not in WT. Radiolabelled [18F]FLUDA exhibited a KD of 5.9 ± 1.6 nM and a Bmax of 455 ± 78 fmol/mg protein in cardiac samples of A2A-AR TG, whereas in WT, as well as in human atrial preparations, only low specific binding was found. Dynamic PET studies revealed a significantly higher initial uptake of [18F]FLUDA into the myocardium of A2A-AR TG compared to WT. The hA2A-AR-specific binding of [18F]FLUDA in vivo was verified by pre-administration of the highly affine A2AAR-specific antagonist istradefylline. Conclusion: [18F]FLUDA is a promising PET probe for the non-invasive assessment of the A2A-AR as a marker for pathologies linked to an increased A2A-AR density in the heart, as shown in patients with heart failure.
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Nakano, Austin, Yasuhiro Nakashima, Diana A. Yanez, Marlin Touma, Haruko Nakano, Artur Jarodzewicz, Maria C. Jordan, Matteo Pellegrini, Kenneth P. Roos, and Kenneth P. Roos. "Abstract 13: Nkx2-5-notch Signaling Axis Regulates The Proliferation Of The Atrial Myocytes And Conduction System." Circulation Research 115, suppl_1 (July 18, 2014). http://dx.doi.org/10.1161/res.115.suppl_1.13.
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Rationale: Tight control of cardiomyocyte proliferation is essential for the formation of four-chambered heart. Although human mutation of NKX2-5 is linked to septal defects and atrioventricular conduction abnormalities, early lethality and hemodynamic alteration in the mutant models have caused controversy as to whether Nkx2-5 regulates cardiomyocyte proliferation. Objective: In this study, we circumvented these limitations by atrial-restricted deletion of Nkx2-5. Method and Results: Atrial-specific Nkx2-5 mutants died shortly after birth with hyperplastic working myocytes and conduction system including two nodes and internodal tracts. Multicolor reporter analysis revealed that Nkx2-5-null cardiomyocytes displayed clonal proliferative activity throughout the atria, indicating the suppressive role of Nkx2-5 in the cardiomyocyte proliferation after chamber ballooning stages. Transcriptome analysis revealed that aberrant activation of Notch signaling underlies hyperproliferation of mutant cardiomyocytes, and forced activation of Notch signaling recapitulates hyperproliferation of working myocytes but not conduction system. Conclusion: Collectively, these data suggest that Nkx2-5 regulates proliferation of atrial working and conduction myocardium in coordination with Notch pathway.
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Guo, Huixin, Chengwen Hang, Bowen Lin, Zheyi Lin, Hui Xiong, Mingshuai Zhang, Renhong Lu, et al. "HAND factors regulate cardiac lineage commitment and differentiation from human pluripotent stem cells." Stem Cell Research & Therapy 15, no. 1 (February 5, 2024). http://dx.doi.org/10.1186/s13287-024-03649-9.
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Abstract Background Transcription factors HAND1 and HAND2 (HAND1/2) play significant roles in cardiac organogenesis. Abnormal expression and deficiency of HAND1/2 result in severe cardiac defects. However, the function and mechanism of HAND1/2 in regulating human early cardiac lineage commitment and differentiation are still unclear. Methods With NKX2.5eGFP H9 human embryonic stem cells (hESCs), we established single and double knockout cell lines for HAND1 and HAND2, respectively, whose cardiomyocyte differentiation efficiency could be monitored by assessing NKX2.5-eGFP+ cells with flow cytometry. The expression of specific markers for heart fields and cardiomyocyte subtypes was examined by quantitative PCR, western blot and immunofluorescence staining. Microelectrode array and whole-cell patch clamp were performed to determine the electrophysiological characteristics of differentiated cardiomyocytes. The transcriptomic changes of HAND knockout cells were revealed by RNA sequencing. The HAND1/2 target genes were identified and validated experimentally by integrating with HAND1/2 chromatin immunoprecipitation sequencing data. Results Either HAND1 or HAND2 knockout did not affect the cardiomyocyte differentiation kinetics, whereas depletion of HAND1/2 resulted in delayed differentiation onset. HAND1 knockout biased cardiac mesoderm toward second heart field progenitors at the expense of first heart field progenitors, leading to increased expression of atrial and outflow tract cardiomyocyte markers, which was further confirmed by the appearance of atrial-like action potentials. By contrast, HAND2 knockout cardiomyocytes had reduced expression of atrial cardiomyocyte markers and displayed ventricular-like action potentials. HAND1/2-deficient hESCs were more inclined to second heart field lineage and its derived cardiomyocytes with atrial-like action potentials than HAND1 single knockout during differentiation. Further mechanistic investigations suggested TBX5 as one of the downstream targets of HAND1/2, whose overexpression partially restored the abnormal cardiomyocyte differentiation in HAND1/2-deficient hESCs. Conclusions HAND1/2 have specific and redundant roles in cardiac lineage commitment and differentiation. These findings not only reveal the essential function of HAND1/2 in cardiac organogenesis, but also provide important information on the pathogenesis of HAND1/2 deficiency-related congenital heart diseases, which could potentially lead to new therapeutic strategies.
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Quaranta, Roberto, Jakob Fell, Frank Rühle, Jyoti Rao, Ilaria Piccini, Marcos J. Araúzo-Bravo, Arie O. Verkerk, Monika Stoll, and Boris Greber. "Revised roles of ISL1 in a hES cell-based model of human heart chamber specification." eLife 7 (January 16, 2018). http://dx.doi.org/10.7554/elife.31706.
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The transcription factor ISL1 is thought to be key for conveying the multipotent and proliferative properties of cardiac precursor cells. Here, we investigate its function upon cardiac induction of human embryonic stem cells. We find that ISL1 does not stabilize the transient cardiac precursor cell state but rather serves to accelerate cardiomyocyte differentiation. Conversely, ISL1 depletion delays cardiac differentiation and respecifies nascent cardiomyocytes from a ventricular to an atrial identity. Mechanistic analyses integrate this unrecognized anti-atrial function of ISL1 with known and newly identified atrial inducers. In this revised view, ISL1 is antagonized by retinoic acid signaling via a novel player, MEIS2. Conversely, ISL1 competes with the retinoic acid pathway for prospective cardiomyocyte fate, which converges on the atrial specifier NR2F1. This study reveals a core regulatory network putatively controlling human heart chamber formation and also bears implications for the subtype-specific production of human cardiomyocytes with enhanced functional properties.
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Churko, Jared, Barbara Treutlein, Priyanka Garg, Meenakshi Venkatasubramanian, Haodi Wu, Shih-Yu Chen, Wen-Yi Chen, et al. "Abstract 13841: Transcriptomic Signatures of Atrial- And Ventricular-like Human Induced Pluripotent Stem Cell Derived Cardiomyocytes." Circulation 134, suppl_1 (November 11, 2016). http://dx.doi.org/10.1161/circ.134.suppl_1.13841.
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Differentiation of cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) can be used to gain insight into the signaling pathways that regulate human heart development. The resulting hiPSC-CMs formed have been reported to possess either atrial- or ventricular-like characteristics and uncovering the molecular mechanisms which regulate the development of these cardiomyocyte subtypes will be instrumental in drug screening and disease modelling research efforts. To investigate the molecular signatures of atrial- and/or ventricular-like hiPSC-CMs formed after differentiation, we profiled the transcriptomic signatures of day 30 hiPSC-CMs using single-cell RNA-seq. Using unsupervised and supervised clustering approaches, we defined distinct subpopulations of hiPSC-CMs representing different stages of maturity as well as expressing atrial or ventricular markers. Expression of NR2F2 was found to be specifically enriched within the atrial defined subpopulation while HEY2 expression was enriched within the ventricular defined subpopulation. To further investigate the role that these transcription factors play in defining each cardiomyocyte subpopulation, we created cell lines deficient in either HEY2 or NR2F2 using CRISPR technology. Patch-clamp analysis revealed an increase in atrial-like action potentials within HEY2 gene-edited cardiomyocytes and single cell Westerns revealed a decrease in the expression of the ventricular marker MYL2 within these cells. Finally, by assessing the temporal expression of transcription factors during differentiation by RNA-seq (along with CyTOF analysis), we observed that the expression of atrial markers preceded the expression of ventricular markers. By defining the transcriptomic signatures of cardiomyocyte subpopulations using single-cell RNA-seq and additional novel supporting methods, we provide a platform for understanding human heart developmental pathways in vitro, hiPSC-CM heterogeneity, and cardiomyocyte subtype specificity.
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E Molina, C., L. Sommerfeld, T. Zeller, J. Obergassel, H. Wieboldt, L. Conradi, H. Reichenspurner, V. O. Nikolaev, P. Kirchhof, and L. Fabritz. "From cells to circulating biomarker: BMP10 is a myocyte-secreted peptide with potential to detect atrial fibrillation." European Heart Journal 44, Supplement_2 (November 2023). http://dx.doi.org/10.1093/eurheartj/ehad655.3140.
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Abstract Background Early onset detection and recurrence prediction of atrial fibrillation (AF) may improve therapeutic decision-making, prognosis and long-term outcome. Plasma proteome analyses and mechanistic studies identified bone morphogenetic protein 10 (BMP10) as an atrial-specific secreted biomolecule for AF associated with reduced paired-like homeodomain transcription factor 2 (PITX2) expression. Purpose Our aim is 1) to confirm BMP10 synthesis and secretion by human atrial isolated myocytes, 2) to compare BMP10 secreted levels between right atrial, left atrial and left ventricular myocytes, and 3) to assess if BMP10 secretion is indeed associated with AF in human myocytes. Methods Right atrial (RA), left atrial (LA) and left ventricular (LV) myocytes were isolated from 16 tissues samples of patients undergoing open heart surgery which were currently in sinus rhythm (SR) or AF. Freshly isolated myocytes were resuspended in minimal essential medium (MEM, M1) containing fetal bovine serum (FBS) and plated in laminin coated dishes. M1 was collected after 3 hours of culture and substituted by FBS-free medium (M2), which was collected after 48 hours of culture. Cardiomyocyte-released BMP10 levels were measured in M1 and M2 by ELISA. Results Cardiomyocytes from twelve patients (68±2.7 years, 52±2.1 % ejection fraction, 17 % women, 50 % with AF, 50 % valvular surgery and 75 % bypass) were cultured and BMP10 secreted levels analyzed. BMP10 concentrations were high in medium from atrial cardiomyocytes (2.76±1.8 ng/µl) and negligible in medium from ventricular cardiomyocytes (0.01±0.005 ng/µl). Left atrial cardiomyocytes released less BMP10 than right atrial cardiomyocytes (LA 0.02±0.007 ng/µl; RA 3.50±2.3 ng/µl). In addition, BMP10 concentrations were higher in right atrial cardiomyocytes from patients with AF (3.76±3.6 ng/µl) than in the right atrial cardiomyocytes from patients in SR (0.97±0.9 ng/µl). Conclusion Our results provide a direct proof that BMP10 is secreted by human atrial myocytes, mainly right atrial myocytes. Furthermore, atrial myocytes from patients with AF secreted significantly more BMP10 than myocytes from patients in sinus rhythm. These observations validate the atrial cardiomyocyte origin of circulating BMP10, making BMP10 a potentially attractive atrial-specific cardiac biomarker.
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Ye, Shiqiao, Cankun Wang, Zhaohui Xu, Hui Lin, Xiaoping Wan, Yang Yu, Subhodip Adhicary, et al. "Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency." Circulation Research, December 30, 2022. http://dx.doi.org/10.1161/circresaha.122.321398.
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BACKGROUND: NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive. METHODS: CRISPR/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq. RESULTS: Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations. CONCLUSIONS: NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.
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Broman, Michael T., Rangarajan D. Nadadur, Carlos Perez-Cervantes, Ozanna Burnicka-Turek, Sonja Lazarevic, Anna Gams, Brigitte Laforest, et al. "A Genomic Link From Heart Failure to Atrial Fibrillation Risk: FOG2 Modulates a TBX5/GATA4-Dependent Atrial Gene Regulatory Network." Circulation, January 8, 2024. http://dx.doi.org/10.1161/circulationaha.123.066804.
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BACKGROUND: The relationship between heart failure (HF) and atrial fibrillation (AF) is clear, with up to half of patients with HF progressing to AF. The pathophysiological basis of AF in the context of HF is presumed to result from atrial remodeling. Upregulation of the transcription factor FOG2 (friend of GATA2; encoded by ZFPM2 ) is observed in human ventricles during HF and causes HF in mice. METHODS: FOG2 expression was assessed in human atria. The effect of adult-specific FOG2 overexpression in the mouse heart was evaluated by whole animal electrophysiology, in vivo organ electrophysiology, cellular electrophysiology, calcium flux, mouse genetic interactions, gene expression, and genomic function, including a novel approach for defining functional transcription factor interactions based on overlapping effects on enhancer noncoding transcription. RESULTS: FOG2 is significantly upregulated in the human atria during HF. Adult cardiomyocyte-specific FOG2 overexpression in mice caused primary spontaneous AF before the development of HF or atrial remodeling. FOG2 overexpression generated arrhythmia substrate and trigger in cardiomyocytes, including calcium cycling defects. We found that FOG2 repressed atrial gene expression promoted by TBX5 . FOG2 bound a subset of GATA4 and TBX5 co-bound genomic locations, defining a shared atrial gene regulatory network. FOG2 repressed TBX5-dependent transcription from a subset of co-bound enhancers, including a conserved enhancer at the Atp2a2 locus. Atrial rhythm abnormalities in mice caused by Tbx5 haploinsufficiency were rescued by Fog2 haploinsufficiency. CONCLUSIONS: Transcriptional changes in the atria observed in human HF directly antagonize the atrial rhythm gene regulatory network, providing a genomic link between HF and AF risk independent of atrial remodeling.
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Hendrickson, Troy, William Perez, Abigail Giese, and Francisco Altamirano. "Abstract P1088: Polycystin-1 Protects Against Experimental Atrial Fibrillation." Circulation Research 131, Suppl_1 (August 5, 2022). http://dx.doi.org/10.1161/res.131.suppl_1.p1088.
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Atrial fibrillation (AF) is the most common cardiac arrhythmia with a 1 in 4 lifetime risk. AF impairs cardiac function and increases the risk of stroke and heart attack. Although this is a common and harmful disease, little is known about the molecular mechanisms driving this disease. We have previously demonstrated that the protein polycystin-1 (PC1) plays a role in atrial cardiomyocyte excitation-contraction coupling in ventricular cardiomyocytes. Interestingly, patients with mutations in PC1 encoding gene have increased rates of atrial arrhythmias, including atrial fibrillation. Here, we sought to study the role of PC1 in atrial cardiomyocyte function and atrial fibrillation inducibility. We performed intracardiac electrophysiology with burst pacing in two mouse models with impaired PC1 function: 1) a mouse model harboring R3277C mutation (RC/RC) known to cause autosomal polycystic kidney disease, and 2) cardiomyocyte-specific PC1 KO (CKO). We observed increased rates of atrial fibrillation inducibility in both RC/RC and PC1 CKO mice compared to their respective control littermates (76.9% in RC/RC and 81.8% in CKO vs 20.0% and 11.1% in their respective WT controls). To elucidate molecular mechanisms driving increased susceptibility to atrial fibrillation, we determined whether PC1 affects excitation-contraction coupling in atrial cardiomyocytes. Human induced pluripotent stem cells-derived atrial cardiomyocytes (hiPSC-aCM) were treated with control or PC1 siRNA. Action potentials and intracellular calcium were measured using Fluovolt and Fluo-4, respectively. PC1 ablation decreased action potential duration and calcium transient peak amplitude elicited by electrical stimulation in hiPSC-CM. Decreased calcium transient peaks were also observed in HL-1 atrial cell line treated with siRNA and adult atrial cardiomyocytes isolated from RC/RC mice compared to their control counterparts. Unexpectedly, RNA-sequencing revealed altered DNA Damage Response gene expression. DNA damage has been implicated in the pathogenesis of atrial fibrillation. Therefore, we studied whether tachypacing elicited DNA damage in hiPSC-aCM. We observed increased levels of H2AX phosphorylation (gamma H2AX) in hiPSC-aCM with reduced PC1 expression. Our data show that PC1 mutations increase atrial fibrillation inducibility in multiple mouse models, likely due to impaired DNA damage response and altered action potential and calcium handling.
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Dai, Wenli, Brigitte Laforest, Leonid Tyan, Kaitlyn M. Shen, Rangarajan D. Nadadur, Francisco J. Alvarado, Stefan R. Mazurek, et al. "A calcium transport mechanism for atrial fibrillation in Tbx5-mutant mice." eLife 8 (March 21, 2019). http://dx.doi.org/10.7554/elife.41814.
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Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adult-specific Tbx5-mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5-deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2)-mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca2+ sequestration, enhanced trans-sarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca2+-dependent pathway for AF risk.
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Munro, Michelle L., Isabelle van Hout, Hamish M. Aitken-Buck, Ramanen Sugunesegran, Krishna Bhagwat, Philip J. Davis, Regis R. Lamberts, Sean Coffey, Christian Soeller, and Peter P. Jones. "Human Atrial Fibrillation Is Not Associated With Remodeling of Ryanodine Receptor Clusters." Frontiers in Cell and Developmental Biology 9 (February 25, 2021). http://dx.doi.org/10.3389/fcell.2021.633704.
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The release of Ca2+ by ryanodine receptor (RyR2) channels is critical for cardiac function. However, abnormal RyR2 activity has been linked to the development of arrhythmias, including increased spontaneous Ca2+ release in human atrial fibrillation (AF). Clustering properties of RyR2 have been suggested to alter the activity of the channel, with remodeling of RyR2 clusters identified in pre-clinical models of AF and heart failure. Whether such remodeling occurs in human cardiac disease remains unclear. This study aimed to investigate the nanoscale organization of RyR2 clusters in AF patients – the first known study to examine this potential remodeling in diseased human cardiomyocytes. Right atrial appendage from cardiac surgery patients with paroxysmal or persistent AF, or without AF (non-AF) were examined using super-resolution (dSTORM) imaging. Significant atrial dilation and cardiomyocyte hypertrophy was observed in persistent AF patients compared to non-AF, with these two parameters significantly correlated. Interestingly, the clustering properties of RyR2 were remarkably unaltered in the AF patients. No significant differences were identified in cluster size (mean ∼18 RyR2 channels), density or channel packing within clusters between patient groups. The spatial organization of clusters throughout the cardiomyocyte was also unchanged across the groups. RyR2 clustering properties did not significantly correlate with patient characteristics. In this first study to examine nanoscale RyR2 organization in human cardiac disease, these findings indicate that RyR2 cluster remodeling is not an underlying mechanism contributing to altered channel function and subsequent arrhythmogenesis in human AF.
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Thorpe, Jordan, Matthew D. Perry, Osvaldo Contreras, Emily Hurley, George Parker, Richard P. Harvey, Adam P. Hill, and Jamie I. Vandenberg. "Development of a robust induced pluripotent stem cell atrial cardiomyocyte differentiation protocol to model atrial arrhythmia." Stem Cell Research & Therapy 14, no. 1 (July 27, 2023). http://dx.doi.org/10.1186/s13287-023-03405-5.
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Abstract Background Atrial fibrillation is the most common arrhythmia syndrome and causes significant morbidity and mortality. Current therapeutics, however, have limited efficacy. Notably, many therapeutics shown to be efficacious in animal models have not proved effective in humans. Thus, there is a need for a drug screening platform based on human tissue. The aim of this study was to develop a robust protocol for generating atrial cardiomyocytes from human-induced pluripotent stem cells. Methods A novel protocol for atrial differentiation, with optimized timing of retinoic acid during mesoderm formation, was compared to two previously published methods. Each differentiation method was assessed for successful formation of a contractile syncytium, electrical properties assayed by optical action potential recordings and multi-electrode array electrophysiology, and response to the G-protein-gated potassium channel activator, carbamylcholine. Atrial myocyte monolayers, derived using the new differentiation protocol, were further assessed for cardiomyocyte purity, gene expression, and the ability to form arrhythmic rotors in response to burst pacing. Results Application of retinoic acid at day 1 of mesoderm formation resulted in a robust differentiation of atrial myocytes with contractile syncytium forming in 16/18 differentiations across two cell lines. Atrial-like myocytes produced have shortened action potentials and field potentials, when compared to standard application of retinoic acid at the cardiac mesoderm stage. Day 1 retinoic acid produced atrial cardiomyocytes are also carbamylcholine sensitive, indicative of active Ikach currents, which was distinct from ventricular myocytes and standard retinoic addition in matched differentiations. A current protocol utilizing reduced Activin A and BMP4 can produce atrial cardiomyocytes with equivalent functionality but with reduced robustness of differentiation; only 8/17 differentiations produced a contractile syncytium. The day 1 retinoic acid protocol was successfully applied to 6 iPSC lines (3 male and 3 female) without additional optimization or modification. Atrial myocytes produced could also generate syncytia with rapid conduction velocities, > 40 cm s−1, and form rotor style arrhythmia in response to burst pacing. Conclusions This method combines an enhanced atrial-like phenotype with robustness of differentiation, which will facilitate further research in human atrial arrhythmia and myopathies, while being economically viable for larger anti-arrhythmic drug screens.