Auswahl der wissenschaftlichen Literatur zum Thema „Mitochondrial adaptation“

Mitochondria are descended from free-living bacteria that were engulfed by another cell between one and a half to two billion years ago. A redistribution of DNA led to most genetic information being lost or transferred to a large central genome in the nucleus, leaving a residual genome in each mitochondrion. Oxidative phosphorylation, the most critical function of mitochondria, depends on the functional compatibility of proteins encoded by both the nucleus and mitochondria. We investigate whether selection for adaptation between the nuclear and mitochondrial genomes (mitonuclear co-adaptation) could, in principle, have promoted uniparental inheritance of mitochondria and thereby the evolution of two mating types or sexes. Using a mathematical model, we explore the importance of the radical differences in ploidy levels, sexual and asexual modes of inheritance, and mutation rates of the nucleus and mitochondria. We show that the major features of mitochondrial inheritance, notably uniparental inheritance and bottlenecking, enhance the co-adaptation of mitochondrial and nuclear genes and therefore improve fitness. We conclude that, under a wide range of conditions, selection for mitonuclear co-adaptation favours the evolution of two distinct mating types or sexes in sexual species.

After billions of years of evolution, mitochondrion retains its own genome, which gets expressed in mitochondrial matrix. Mitochondrial translation machinery rather differs from modern bacterial and eukaryotic cytosolic systems. Any disturbance in mitochondrial translation drastically impairs mitochondrial function. In budding yeast Saccharomyces cerevisiae, deletion of the gene coding for mitochondrial translation initiation factor 3 - AIM23, leads to an imbalance in mitochondrial protein synthesis and significantly delays growth after shifting from fermentable to non-fermentable carbon sources. Molecular mechanism underlying this adaptation to respiratory growth was unknown. Here, we demonstrate that slow adaptation from glycolysis to respiration in the absence of Aim23p is accompanied by a gradual increase of cytochrome c oxidase activity and by increased levels of Tma19p protein, which protects mitochondria from oxidative stress.

Mitochondria play crucial roles in programmed cell death and aging. Different stimuli activate distinct mitochondrion-dependent cell death pathways, and aging is associated with a progressive increase in mitochondrial damage, culminating in oxidative stress and cellular dysfunction. Mitochondria are highly dynamic organelles that constantly fuse and divide, forming either interconnected mitochondrial networks or separated fragmented mitochondria. These processes are believed to provide a mitochondrial quality control system and enable an effective adaptation of the mitochondrial compartment to the metabolic needs of the cell. The baker's yeast, Saccharomyces cerevisiae, is an established model for programmed cell death and aging research. The present review summarizes how mitochondrial morphology is altered on induction of cell death or on aging and how this correlates with the induction of different cell death pathways in yeast. We highlight the roles of the components of the mitochondrial fusion and fission machinery that affect and regulate cell death and aging.

Mitochondrial evolution has been examined on the basis of properties of mitochondria from representatives of key adaptive stages. The major step in the evolution of mitochondria was the transfer of mitochondrial genes to the nucleus to take advantage of recombination during meiosis. The ensuing increase in variability facilitated adaptation to environmental stress. The role of environmental factors such as atmospheric oxygen levels in the evolution of mitochondria is discussed on the basis of evidence obtained from mitochondria of living representatives of important groups and the fossil record. Rate enhancement has been a central theme in the evolution of animal mitochondria. Optimization of mitochondrial oxidation rates occurred through adjustments in intracellular solute systems. This took place in several stages, including (i) a reduction of intracellular inorganic ion levels by substitution of a variety of compatible solutes, (ii) a counteracting solute system (urea and methylamines), and (iii) osmoregulation.

Thermal tolerance and the respiratory properties of isolated red muscle mitochondria were investigated in Oreochromis alcalicus grahami from the alkaline hot-springs, Lake Magadi, Kenya. Populations of O. a. grahami were resident in pools at 42.8 °C and migrated into water reaching temperatures of 44.8 °C for short periods. The maximum respiration rates of mitochondria with pyruvate as substrate were 217 and 284 natom O mg-1 mitochondrial protein min-1 at 37 °C and 42 °C, respectively (Q10=1.71). Fatty acyl carnitines (chain lengths C8, C12 and C16), malate and glutamate were oxidised at 70­80 % of the rate for pyruvate. In order to assess evolutionary temperature adaptation of maximum mitochondrial oxidative capacities, the rates of pyruvate and palmitoyl carnitine utilisation in red muscle mitochondria were measured from species living at other temperatures: Notothenia coriiceps from Antarctica (-1.5 to +1 °C); summer-caught Myoxocephalus scorpius from the North Sea (10­15 °C); and Oreochromis andersoni from African lakes and rivers (22­30 °C). State 3 respiration rates had Q10 values in the range 1.8­2.7. At the lower lethal temperature of O. andersoni (12.5 °C), isolated mitochondria utilised pyruvate at a similar rate to mitochondria from N. coriiceps at 2.5 °C (30 natom O mg-1 mitochondrial protein min-1). Rates of pyruvate oxidation by mitochondria from M. scorpius and N. coriiceps were similar and were higher at a given temperature than for O. andersoni. At their normal body temperature (-1.2 °C), mitochondria from the Antarctic fish oxidised pyruvate at 5.5 % and palmitoyl-dl-carnitine at 8.8 % of the rates of mitochondria from the hot-spring species at 42 °C. The results indicate only modest evolutionary adjustments in the maximal rates of mitochondrial respiration in fish living at different temperatures.

Variable durations of food restriction (FR; lasting weeks to years) and variable FR intensities are applied to animals in life span-prolonging studies. A reduction in mitochondrial proton leak is suggested as a putative mechanism linking such diet interventions and aging retardation. Early mechanisms of mitochondrial metabolic adaptation induced by FR remain unclear. We investigated the influence of different degrees of FR over 3 days on mitochondrial proton leak and mitochondrial energy metabolism in rat hindlimb skeletal muscle. Animals underwent 25, 50, and 75% and total FR compared with control rats. Proton leak kinetics and mitochondrial functions were investigated in two mitochondrial subpopulations, intermyofibrillar (IMF) and subsarcolemmal (SSM) mitochondria. Regardless of the degree of restriction, skeletal muscle mass was not affected by 3 days of FR. Mitochondrial basal proton conductance was significantly decreased in 50% restricted rats in both mitochondrial subpopulations (46 and 40% for IMF and SSM, respectively) but was unaffected in other groups compared with controls. State 3 and uncoupled state 3 respiration rates were decreased in SSM mitochondria only for 50% restricted rats when pyruvate + malate was used as substrate (−34.5 and −38.9% compared with controls, P < 0.05). IMF mitochondria respiratory rates remained unchanged. Three days of FR, particularly at 50% FR, were sufficient to lower mitochondria energetic metabolism in both mitochondrial populations. Our study highlights an early step in mitochondrial adaptation to FR and the influence of the severity of restriction on this adaptation. This step may be involved in an aging-retardation process.

The interaction of mitochondria with cellular components evolved differently in plants and mammals; in plants, the organelle contains proteins such as ALTERNATIVE OXIDASES (AOXs), which, in conjunction with internal and external ALTERNATIVE NAD(P)H DEHYDROGENASES, allow canonical oxidative phosphorylation (OXPHOS) to be bypassed. Plant mitochondria also contain UNCOUPLING PROTEINS (UCPs) that bypass OXPHOS. Recent work revealed that OXPHOS bypass performed by AOXs and UCPs is linked with new mechanisms of mitochondrial retrograde signaling. AOX is functionally associated with the NO APICAL MERISTEM transcription factors, which mediate mitochondrial retrograde signaling, while UCP1 can regulate the plant oxygen-sensing mechanism via the PRT6 N-Degron. Here, we discuss the crosstalk or the independent action of AOXs and UCPs on mitochondrial retrograde signaling associated with abiotic stress responses. We also discuss how mitochondrial function and retrograde signaling mechanisms affect chloroplast function. Additionally, we discuss how mitochondrial inner membrane transporters can mediate mitochondrial communication with other organelles. Lastly, we review how mitochondrial metabolism can be used to improve crop resilience to environmental stresses. In this respect, we particularly focus on the contribution of Brazilian research groups to advances in the topic of mitochondrial metabolism and signaling.

Abstract Mitochondria have been known to be involved in speciation through the generation of Dobzhansky–Muller incompatibilities, where functionally neutral co-evolution between mitochondrial and nuclear genomes can cause dysfunction when alleles are recombined in hybrids. We propose that adaptive mitochondrial divergence between populations can not only produce intrinsic (Dobzhansky–Muller) incompatibilities, but could also contribute to reproductive isolation through natural and sexual selection against migrants, post-mating prezygotic isolation, as well as by causing extrinsic reductions in hybrid fitness. We describe how these reproductive isolating barriers can potentially arise through adaptive divergence of mitochondrial function in the absence of mito-nuclear coevolution, a departure from more established views. While a role for mitochondria in the speciation process appears promising, we also highlight critical gaps of knowledge: (1) many systems with a potential for mitochondrially-mediated reproductive isolation lack crucial evidence directly linking reproductive isolation and mitochondrial function; (2) it often remains to be seen if mitochondrial barriers are a driver or a consequence of reproductive isolation; (3) the presence of substantial gene flow in the presence of mito-nuclear incompatibilities raises questions whether such incompatibilities are strong enough to drive speciation to completion; and (4) it remains to be tested how mitochondrial effects on reproductive isolation compare when multiple mechanisms of reproductive isolation coincide. We hope this perspective and the proposed research plans help to inform future studies of mitochondrial adaptation in a manner that links genotypic changes to phenotypic adaptations, fitness, and reproductive isolation in natural systems, helping to clarify the importance of mitochondria in the formation and maintenance of biological diversity.

Long-term heat acclimation (LTHA; 30 days, 34°C) causes phenotypic adaptations that render protection against ischemic/reperfusion insult (I/R, 30 min global ischemia and 40 min reperfusion) via heat acclimation-mediated cross-tolerance (HACT) mechanisms. Short-term acclimation (STHA, 2 days, 34°C), in contrast, is characterized by cellular perturbations, leading to increased susceptibility to insults. Here, we tested the hypothesis that enhanced mitochondrial respiratory function is part of the acclimatory repertoire and that the 30-day regimen is required for protection via HACT. We subjected isolated hearts and mitochondria from controls (C), STHA, or LTHA rats to I/R, hypoxia/reoxygenation, or Ca2+ overload insults. Mitochondrial function was assessed by measuring O2 consumption, membrane potential (ΔΨm), mitochondrial Ca2+ ([Ca2+]m), ATP production, respiratory chain complex activities, and molecular markers of mitochondrial biogenesis. Our results, combining physiological and biochemical parameters, confirmed that mitochondria from LTHA rats subjected to insults, in contrast to C, preserve respiratory functions (e.g., upon I/R, C mitochondria fueled by glutamate-malate, demonstrated decreases of 81%, 13%, 25%, and 50% in O2/P ratio, ATP production, ΔΨm, and complex I activity, respectively, whereas the corresponding LTHA parameters remained unchanged). STHA mitochondria maintained ΔΨm but did not preserve ATP production. LTHA [Ca2+]m was significantly higher than that of C and STHA and was not affected by the hypoxia/reoxygenation protocol compared with C. Enhanced mitochondrial biogenesis markers, switched-on during STHA coincidentally with enhanced membrane integrity (ΔΨm), were insufficient to confer intact respiratory function upon insult. LTHA was required for respiratory complex I adaptation and HACT. Stabilized higher basal [Ca2+]m and attenuated Ca2+ overload are likely connected to this adaptation.

Rapid adaptation to extreme hypoxia is a challenging problem, and there is no effective scheme to achieve rapid adaptation to extreme hypoxia. In this study, we found that withaferin A (WA) can significantly reduce myocardial damage, maintain cardiac function, and improve survival in rats in extremely hypoxic environments. Mechanistically, WA protects against extreme hypoxia by affecting BCL2-interacting protein 3 (BNIP3)-mediated mitophagy and the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α)-mediated mitochondrial biogenesis pathway among mitochondrial quality control mechanisms. On the one hand, enhanced mitophagy eliminates hypoxia-damaged mitochondria and prevents the induction of apoptosis; on the other hand, enhanced mitochondrial biogenesis can supplement functional mitochondria and maintain mitochondrial respiration to ensure mitochondrial ATP production under acute extreme hypoxia. Our study shows that WA can be used as an effective drug to improve tolerance to extreme hypoxia.

Mitochondrial Ca2+ homeostasis plays a fundamental role in the regulation of several biological processes, ranging from the regulation of ATP production to the control of cell death. Recent studies have identified the multimolecular complex responsible for Ca2+ entry into mitochondria: the mitochondrial calcium uniporter (MCU) complex [1]. It is widely accepted that mitochondria actively participate in the regulation of cellular Ca2+ homeostasis by dictating the spatio-temporal properties of [Ca2+]cyt rises [2]. Mitochondrial calcium uptake, in specific cells, contributes to regulate cellular Ca2+ homeostasis acting as high-capacity fixed buffer, sequestering large amounts of Ca2+ from a subcellular domain[2]. Furthermore, one of the most important roles of mitochondrial Ca2+ uptake is the mitochondrial Ca2+-dependent control of the rate of mitochondrial adenosine triphosphate (ATP) production, the main fuel for sustaining cellular functions [3], [4]. This general picture is particularly relevant in skeletal muscle, a tissue where mitochondria produce most of the ATP required to sustain muscle contraction [3], [5]. It is thus not surprising that skeletal muscle mitochondria display the highest mitochondrial Ca2+ transients, as demonstrated by the measurement of the MCU current by patch-clamp from IMM-derived mitoplasts from different tissues [6]. Moreover, pivotal findings have highlighted the role of mitochondria as key players in the dynamic regulation of crucial signalling pathways in skeletal muscle [7], [8], involved not only in muscle contraction but also in skeletal muscle homeostasis [9], [10]. However, whether skeletal muscle mitochondria act also as a possible high capacity Ca2+ buffer remains a fundamental question on muscle physiology and diseases. Our research investigated the regulatory processes that modulate mitochondrial Ca2+ signalling in skeletal muscle. In detail, to understand the impact of changes in cytosolic Ca2+ concentrations ([Ca2+]cyt) on mitochondrial Ca2+ uptake and muscle physiology, we explored a condition where intra-fiber Ca2+ kinetics have been profoundly altered by removing parvalbumin (PV), one of the crucial cytosolic Ca2+ buffers in skeletal muscle, specifically expressed in fast twitch muscle fibers [11], [12]. To this end, as study tool, we used a PV knockout (KO) mouse model obtained from the laboratory of Prof. Beat Schwaller (Dept. of Medicine, University of Fribourg, Switzerland) [13]. PV plays an important role in skeletal muscle, acting as a temporary Ca2+ buffer (e.g. increasing the relaxation rate of fast twitch muscle contraction) [14]. To investigate the physiological role of PV in muscle fibers and in Ca2+ homeostasis, we investigated cytosolic and mitochondrial Ca2+ transients in PV KO mice compared. We observed that basal [Ca2+]cyt was not affected in PV knockout fibers, but kinetics of Ca2+ transients and Ca2+ clearance were altered. In detail, consistently with the role of PV in buffering cytosolic Ca2+, the time-to-peak and the half-relaxation time was increased in PV KO FDB fibers. Unexpectedly, however, under tetanic stimulation, PV KO FDB muscle fibers showed a decrease in [Ca2+]cyt. To explain this result we asked whether the lack of PV could induce rearrangements of one of the two main Ca2+ stores, the sarcoplasmic reticulum (SR) and mitochondria. SR Ca2+ measurements demonstrated that lack of PV increases SR Ca2+ release during stimulation. Therefore, we concluded that SR is not causative of the effect of PV removal on cytosolic Ca2+ transients. Consistently, we found no difference in the mRNA levels of RyR1, the main Ca2+ releasing channel in muscle, and on the expression of two different isoforms of SERCA in PV KO muscles compared to WT. We then focused our attention on mitochondrial Ca2+ homeostasis. The data obtained demonstrated that the lack of PV induces an increase of mitochondrial Ca2+ uptake and this is accompanied by the induction of the expression of MCU complex components, the channel responsible for Ca2+ entry in mitochondria [1], [2], [15], [16]. In addition, electron microscopy analysis demonstrated that the volume of PV KO mitochondria was doubled compared to WT with an increase of mitochondria associated to the Ca2+ release units (CRUs), suggesting a tight connection of PV expression with mitochondrial morphology and function in muscle cells. Furthermore, to further prove that mitochondria are responsible for cytosolic Ca2+ buffering in fibers lacking PV, we silenced MCU on WT and PV KO FDB fibers and we measured [Ca2+]cyt.. In WT animals, [Ca2+]cyt was not affected by the absence of MCU, while MCU silencing in PV KO fibers resulted in a significant higher [Ca2+]cyt, reinforcing the hypothesis that, while in WT animals mitochondria do not significantly buffer [Ca2+]cyt, mitochondria of fibers lacking PV adapt to buffer [Ca2+]cyt increases. Moreover, since PV is one of the most downregulated “atrogenes”, the genes commonly up- and down-regulated during both disuse and systemic types of atrophy [17], [18] and that mitochondrial Ca2+ controls skeletal muscle trophism [10], the role of PV in the regulation of muscle mass was investigated through denervation experiments. In PV KO muscles, loss of muscle mass caused by denervation is reduced compared to WT fibers, demonstrating that the lack of PV can partially protect muscles from denervation-induced atrophy. Since the effect of PV ablation on denervated muscle was modest and the effect on innervated muscles was negligible, we decided to perform PV acute silencing and overexpression in adult WT tibialis anterior (TA) muscles and we monitored fiber size. We demonstrated that the acute modulation of PV protein controls skeletal muscle size. In detail, we observed an increase of fiber size in PV silenced muscles and coherently, PV overexpressing muscles displayed an atrophic phenotype. Since the regulation of muscle size involves a precise transcriptional program [18], [19], we focused our attention on PGC-1α4, a splicing variant of the PGC-1α gene, that plays a key role in triggering muscle hypertrophy as adaptive response to exercise [20]. Intriguingly, we found an up-regulation of PGC-1α4 mRNA in PV KO skeletal muscles, suggesting the activation of this hypertrophic pathway. Of note, our data are in accordance with previous studies showing that mitochondrial Ca2+ positively regulates skeletal muscle mass by impinging also on PGC-1α4 pathway [10]. Our results show that the lack of PV in skeletal muscle leads to morphological and functional adaptations of mitochondria. In particular, mitochondria of fibers lacking PV, either constitutively or transiently, adapt to take up more Ca2+ to control [Ca2+]cyt increases. Furthermore, we demonstrated that the absence of PV partially counteracts denervation atrophy by triggering the expression of PGC-1α4. Our hypothesis is that PV ablation, leading to an increase of mitochondrial Ca2+ uptake, activates mitochondrial Ca2+-dependent pathways to control skeletal muscle trophism.

The New World Junonia butterflies include well-studied model organisms yet their phylogeny remains unresolved by traditional cox1 DNA barcodes. Sixteen Junonia mitochondrial genomes were sequenced using next generation MiSeq technology. Junonia lemonias, an Old World species, has mitochondrial genome features typical of Ditrysian Lepidoptera, and synteny is maintained throughout Junonia. Analysis of Junonia mitogenomes produced a robust phylogeny that was used with biogeographic information to infer that Junonia crossed the Pacific Ocean to invade the New World on 3 separate occasions. Junonia vestina, a high elevation species from the Andes Mountains, shows high altitude adaptation in the mitochondrial protein coding loci atp6, atp8, cox1, cob, nad1, and nad2, with the strongest effects seen in cox1 and nad1. There is some overlap between these genes with human loci that have disease associations with the same amino acid positions which could help elucidate the function of high elevation mutations in J. vestina.
February 2016

From birth, throughout the entire lifespan, the myocardium is constituted by an almost fixed number of cardiomyocytes (CMs). Post-natal heart growth occurs through CM hypertrophy, and the cell achieves the adult phenot ype through profound structural, functional and metabolic maturation. Once fully developed, the heart continuously adapts its performance and structure in response to the varying requests of the organism, elicited by changes in intrinsic and environmental conditions. While acute stresses operate through reversible modulation of contractility, CMs subjected to prolonged increase in workload undergo complex structural remodelling, mostly occurring through further growth necessary to sustain the chronic elevation of mechanical load. Depending on the nature, intensity and duration of the hypertrophying stimuli, cardiac remodelling may lead to either the so-called "physiologic" (i.e., in the athletic heart), or "pathologic" hypertrophy (i.e., in pressure overload), the latter resulting, with time, in cardiac dysfunction, heart failure and death. Although the cellular and clinical phenotypes of the two conditions are different, the common tenet is that, in the initial phases, they share the same adaptive mechanisms, including increased sarcomeric deposition and enhanced/preserved contractility, both of which require increased ATP supply. Unsurprisingly, the regulation of mitochondrial function is a critical process for ATP production to match energetic demand during cell growth. Mitochondria are the CM powerhouse, and [Ca2+] operates as a primary dynamic regulator of ATP production. In CMs, Ca2+ influx into the mitochondrial matrix occurs during the systolic elevation in cytosolic Ca2+, and is mediated by the recently identified mitochondrial Ca2+ uniporter complex (MCUC). Mitochondrial Ca2+ uptake is fundamental in the acute modulation of contractility during the fight-or-flight response triggered by β-adrenergic receptor (β-AR) activation. Consistently, deletion of MCU in mice impairs exercise capacity, and reduction of functioning MCU channels in sino-atrial node cells or ventricular CMs, blunts the chronotropic or contractile response, respectively, to β-AR stimulation. Whether changes in the expression of MCUC proteins take place during cardiac diseases and, conversely, the effect of modulating mitochondrial Ca2+ uptake on myocardial remodelling is, at present not known. The aims of this thesis were to: Aim 1) identify the molecular mechanisms involved in the endogenous regulation of MCU; Aim 2) determine whether MCU has a role in physiologic and pathologic cardiac remodelling and Aim 3) develop an experimental model of cultured cardiomyocytes suited for the in vitro characterization of mitochondrial Ca2+ dynamics in prolonged observations. Results. 1. Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy. In our preliminary experiments, we compared the protein levels of MCU in normal and hypertrophied hearts, and observed that changes in mitochondrial MCU protein density were not accompanied by parallel alteration in its transcriptional levels. This prompted us to investigate whether post-translational regulation of MCU might occur in myocardial remodelling. We thus focused on microRNAs (miRs), which are small, non-coding RNA sequences (18-25 nt) capable of finely tuning the expression of a variety of genes by interfering with either the stability or the translation of mRNA. By bioinformatics analysis that identified several microRNAs predicted to target MCU 3’UTR (untranslated region). Among these, we focused on miR-1 for its muscle specific expression, the critical role in the activation in cardiac hypertrophy, its conserved homology among species, and its specificity for MCU among MCUC members. Luciferase assay confirmed the prediction and identified the specific seed sequences on the MCU gene. Consistently, CMs expressing miR-1 showed decreased MCU protein content, with no alterations in mRNA expression, which resulted in significant reduction in mitochondrial Ca2+ uptake. We thus investigated whether MCU content was modulated in hypertrophic conditions associated to changes in miR-1, including: i) post-natal development, ii) moderate exercise and iii) pressure overload. By comparing neonatal and adult mouse hearts we observed that, in line with its role of repressor of fetal gene program, miR-1 expression increased during postnatal development and, coherently, MCU protein content decreased without alterations at transcriptional level. Moreover, this modulation was specific for MCU among the molecular components of MCUC, with the exceptions of mitochondrial calcium uniporter b (MCUb) mRNA, which increased. We then investigated the miR-1/MCU axis, in murine and human heart models of physiologic and pathologic hypertrophy. Physiologic hypertrophy was obtained in mice with chronic exercise protocol, which caused enlargement in cardiac size, CM cross-sectional area, and a slight increase in contractility. As compared to sedentary littermates, miR-1 expression level decreased and, consistently, MCU protein content increased. Analysis of the uniporter complex biochemistry in hearts undergone pressure-overload through transverse aortic constriction (TAC) surgery demonstrated that during the initial, compensatory hypertrophy, characterized by modest CM growth with no contractile failure, changes in miR-1 and MCU were similar to those observed in hearts from exercised mice. Remarkably, the reciprocal miR-1 and MCU modulation occurred in a clinically relevant model of cardiac hypertrophy, as shown by the analysis of human heart biopsies obtained from healthy subjects and patients with aortic stenosis-induced hypertrophy. These results suggest that, regardless of the nature of the hypertrophic stimulus (physiologic or pathologic), the initial CM adaptation to increased heart work is characterized by similar enhancement in the availability of uniporter-forming MCU molecules. Given that similar changes in the miR1/MCU axis were detected both upon exercise and compensated pathologic hypertrophy, we made the hypothesis that a common regulatory mechanism may exist. We thus focused on the β-AR system, the primary physiologic mechanism engaged in response to increased heart load, a condition in common between exercise and TAC-induced pressure-overload. Activation of β-AR signalling leads to enhancement of cytosolic Ca2+ oscillations and mitochondrial Ca2+ uptake, and is involved in the parallel activation of hypertrophic pathways, such as the Akt-FOXO cascade. Interestingly, miR-1 expression has been shown to depend on FOXO3a, suggesting that in conditions of chronic β-AR activation, the blockade of FOXO3a nuclear translocation may inhibit miR-1 increase. In support of this hypothesis, treatment of mice undergone TAC with the β1-blocker metoprolol ablated miR-1 repression and prevented accordingly the increase in MCU protein content. Altogether, our data identifies miR-1 as a novel post-translational regulator of MCU, and supports that the miR-1/MCU axis is involved in physiologic and pathologic myocardial remodelling. Future experiments will be aimed at exploiting the mechanicism of miR-1 action on MCU, as well as understanding the complete signalling pathway involved in MCU modulation. Given that miRs are well-suited therapeutic targets, as they can easily be mimicked or antagonized pharmacologically (miR mimics or antagomiRs, respectively), even with target selectivity, our study of the miR-1/MCU axis may open to the refinement of the current therapeutic approaches to treat myocardial hypertrophy. 2. MCU participates to the myocardial adaptation to hypertrophic stimuli. The observation that MCU protein content drops during long-term TAC, in which maladaptation occurs, suggested that MCU protein content fluctuates during pathologic hypertrophy. This led us to investigate whether MCU may have a role in myocardial remodelling caused by chronic increase in cardiac workload. To test this hypothesis, we sought to characterize functionally, biochemically, and morphologically the effect of modulating MCU expression level prior to exposing hearts to pressure overload through TAC. To increase the insight on cellular signalling, we used adrenergic receptor agonists to study the effect of prolonged adrenergic stimulation in cultured CMs. To study the role of MCU in cardiac adaptation to hypertrophy in vivo, we efficiently overexpressed or downregulated MCU, via AAV9 injection. Altering MCU expression did not affect cardiac structure and performance at baseline. However, in mice undergone TAC, MCU overexpression resulted in enhanced hypertrophy, as demonstrated by higher increase in cardiac mass, as compared to TAC-operated WT TAC (injected with AAV9-Empty vector). Interestingly, hypertrophic remodelling had characteristics similar to that of physiologic hypertrophy (i.e. increased capillary density, reduced fibrotic remodelling, and preserved cardiac contractility) also in the advanced stages of hypertrophy (i.e. 8 weeks). On the contrary, silenced mice subjected to TAC displayed a dramatic phenotype caused by the rapid appearance of severe maladaptive remodelling with typical hallmarks of dilated cardiomyopathy, including reduced capillary density, massive replacement fibrosis and decreased cardiac function. Altogether, these processes result in HF and increased susceptibility to sudden cardiac death already four weeks after TAC. To gain insight on the molecular mechanism whereby changes in MCU impact on stress-induced CM growth, we used neonatal rat CMs in which MCU overexpression or downregulation were obtained with adenoviral vectors. Consistently, MCU overexpression and downregulation resulted in enhanced and reduced mitochondrial calcium uptake, respectively. Interestingly, while MCU overexpression did not affect CM size and morphology at baseline, MCU KD cells displayed a significant increased area and disarranged sarcomeres. To mimic the increased sympathetic tone that characterises both physiologic and pathologic hypertrophy, we treated CMs with the onset of both adrenergic agonist norepinephrine (NE). Interestingly, MCU OE cells had a significantly enhanced increase in cell size growth. Conversely, MCU KD cells had a remarkably divergent phenotype, characterized by sarcomere disarray and activation of apoptosis. These data were intriguingly similar to the phenotype observed in MCU KD hearts developing dilated cardiomyopathy after TAC. The following analyses regarded the activation state of several pro-hypertrophic pathways. Interestingly, MCU overexpression determined faster activation of calcineurin/NFAT pathway upon adrenergic stimulation. Our data point at the participation of Akt/GSK3axis in NFAT enhanced nuclear translocation, presumably downstream of CaMKII-mediated Akt phosphorylation. Indeed, inhibition of CaMKII in MCU OE cardiomyocytes resulted in hypertrophic growth comparable to control cells. To conclude, our studies show that increased heart workload, as achieved in vivo by TAC and mimicked in cells by NE treatment, is well tolerated when MCU levels are augmented by overexpression. Conversely, MCU downregulation leads, in the same conditions, to cell death and consistently faster maladaptive cellular and tissue remodelling. These data are well in accord with our preliminary observation that MCU content, increased in the compensated hypertrophy, decreases in the advanced remodelling associated to HF. Second, we have identified the AR/CaMKII/Akt cascade as a key signalling pathway involved in myocardial hypertrophy and dependent on MCU modulation. 3. In vitro maturation of cultured neonatal cardiomyocytes. Primary neonatal CMs are a widely used cellular model in molecular cardiology, which can be maintained in culture for several days and is easily amenable to genetic manipulation. However, this cell type has important functional and structural differences with the mature CMs. These differences range from the expression of different myosin isoforms to maximize contractile performance, to changes in metabolism allowing increased ATP production to sustain higher consumption. Importantly, postnatal cellular maturation involves structures that regulate Ca2+ dynamics. In particular, in neonatal cells contraction is mostly due to Ca2+ entering through the plasmalemmal L-type Ca2+ channels (LTCC), directly triggering the activation of the sarcomeres, with little contribution from intracellular Ca2+ release from the immature SR stores. In contrast, in adult cells the plasma membrane has fully developed invaginations known as T-tubules which face the terminal SR cisternae, so that LTCC are in close juxtaposition to the Ca2+ Release Units (CRU) formed by the intracellular Ca2+ release channel, ryanodine receptor (RyR). Such arrangement allows few Ca2+ ions entering the cell to trigger release of further Ca2+ from the SR, in a process known as Ca2+-Induced-Ca2+-Release (CICR), which drives contraction. In parallel with the development of SR, the mitochondrial population enriches and interfibrillary mitochondria tether to the SR, in proximity to the CRUs, a condition in which the organelle is found within the confines of a high Ca2+ microdomain, fundamental to drive the ion into the mitochondrial matrix. With these notions in mind, we sought to develop a protocol promoting maturation of neonatal CMs, thus obtaining a cellular model better suited to the study of subcellular Ca2+ handling in order to identify the mechanisms linking mitochondrial Ca2+ dynamics to hypertrophic remodelling. To induce maturation of neonatal CMs, we modified the composition of the media traditionally used to maintain cells in culture. By removing serum from the culture medium, we could avoid cell proliferation and de-differentiation. In addition, we reduced glucose content and added vitamin co-factors and trophic hormones, such as insulin, to compensate the absence of mammalian serum. Furthermore, we improved the preparation purity by eliminating contaminating cardiac fibroblasts, which secrete growth factors and matrix components, promoting cell de-differentiation and hyperplastic growth. With these changes in the isolation conditions, we obtained a pure population of CMs that can be maintained in culture for several weeks, and after few days already acquired a different morphology, compared to those obtained with the more commonly used protocol. Indeed, microscopy imaging showed that the cells were larger, rectangular-shaped, with a regular perimeter, lacking the typical ramifications of neonatal CMs, and a higher long/short axis ratio. Moreover, we observed an increased area occupied by the contractile apparatus, which appeared more regularly displaced. Mitochondria appeared longitudinally displaced along and between the sarcomeres, similarly to adult cells. In addition, immunostaining of RyRs revealed that the protein appeared in clusters more regularly distributed, thus mimicking the phenotype observed in fully differentiated CMs and suggesting increased maturation of the SR. In line with this, we observed shorter and smaller Ca2+ sparks, which are elementary Ca2+ signalling events depending on RyR opening, thus supporting that the more organized RyR clusters formed functionally active CRU, alike those of more mature cells. Interestingly, cells were more receptive to adrenergic agonists, displaying a more pronounced growth by hypertrophy as compared to traditional neonatal CMs. All the aforementioned aspects demonstrate that these cells may represent an in vitro model system well-suited to the study of Ca2+ dynamics and its relation with hypertrophic growth. Remarkably, these properties did not compromise the amenability for genetic manipulation, either via viral infection or transient plasmid transfection. Future experiment will aim at fully characterizing the Ca2+-related structures, such as T-Tubules, as well as formation of dyads.
Dal momento della nascita, per tutta la durata della vita, il miocardio è costituito da un numero pressoché fisso di cardiomiociti (CM). Infatti, la crescita postnatale del cuore è di tipo ipertrofico, per cui lo sviluppo del cardiomiocita, anch’esso di tipo ipertrofico, avviene attraverso un profondo rimodellamento strutturale, funzionale e metabolico. Una volta raggiunto un completo sviluppo, il cuore adatta continuamente la sua contrattilità e struttura in base alle richieste perfusionali dell’organismo, che variano in base a fattori intrinseci ed ambientali. Stimoli acuti determinano la modulazione della contrattilità, mentre stimoli cronici, che richiedono una performance elevata nel tempo, fanno sì che i cardiomiociti rimodellino la loro struttura, crescendo ulteriormente per sostenere l’aumentato carico meccanico. In base a tipo, intensità e durata dello stimolo ipertrofico, il rimodellamento cardiaco può portare ad ipertrofia fisiologica (come nel caso del “cuore d’atleta”) o patologica (ad esempio nel sovraccarico pressorio): in quest’ultimo caso, la crescita ipertrofica risulterà nel tempo in scompenso cardiaco, insufficienza cardiaca e morte. Nonostante i fenotipi cellulare e clinico siano distinti, il comune denominatore di queste condizioni è che, nelle fasi iniziali, i processi sono di tipo adattativo e comprendono la deposizione di nuovi sarcomeri nei cardiomiociti, per garantire una contrattilità migliorata o quanto meno preservata. Queste proprietà richiedono entrambe una maggiore produzione di ATP. Non sorprende quindi il fatto che la regolazione della funzione mitocondriale sia un processo critico per la produzione di ATP, per soddisfare il fabbisogno energetico durante la crescita ipertrofica. I mitocondri sono la “centrale energetica” della cellula e la concentrazione di Ca2+ opera come un regolatore dinamico primario della produzione di ATP. Nei cardiomiociti, l’influsso di Ca2+ nella matrice mitocondriale avviene durante l’aumento di Ca2+ sistolico ed è mediato dal complesso dell’uniporto mitocondriale per il calcio (MCUC), recentemente identificato. L’uptake di Ca2+ mitocondriale è un processo fondamentale nella modulazione acuta della contrattilità durante la risposta “fight-or-flight” attivata dall’attivazione dei recettori ß-adrenergici. A prova di ciò, la delezione di MCU nel modello murino diminuisce la capacità d’esercizio7, mentre la riduzione di MCU nelle cellule del nodo senoatriale o dei ventricoli riduce le risposte cronotropiche o contrattili, rispettivamente, indotte dalla stimolazione ß-adrenergica. Al momento non è noto se avvengano cambi nell’espressione delle proteine formanti MCUC in diverse situazioni fisiopatologiche, così come non è noto l’effetto della modulazione dell’uptake di Ca2+ mitocondriale durante il rimodellamento cardiaco. Su queste basi, gli obiettivi del mio progetto di dottorato sono: 1) Identificare i meccanismi molecolari coinvolti nella regolazione endogena di MCU; 2) Determinare se MCU ha un ruolo nel rimodellamento fisiologico e patologico del cuore; 3) Sviluppare un modello sperimentale di cardiomiociti isolati da cuori neonati per la caratterizzazione in vitro delle dinamiche del Ca2+mitocondriale su tempi prolungati. Risultati. 1. Il contenuto dell’uniporto mitocondriale per il calcio (MCU) nei cardiomiociti è dinamicamente regolato da miR-1 nell’ipertrofia fisiologica e patologica. In esperimenti preliminari condotti nel nostro laboratorio, abbiamo confrontato i livelli proteici di MCU in cuori normali ed ipertrofici, ed abbiamo osservato che le variazioni nel contenuto proteico di MCU non erano accompagnate da variazioni in parallelo del suo trascritto. Ciò ci ha portato ad investigare se, nel rimodellamento cardiaco, potesse avvenire una regolazione post-trascrizionale di MCU. Ci siamo così focalizzati sui microRNA (miR), piccole sequenze non codificanti di RNA (18-25 nucleotidi) capaci di modulare finemente l’espressione di svariati geni, grazie all’interferenza con la stabilità o la traduzione dell’mRNA target. Un numero crescente di evidenze rivela il ruolo fondamentale dei miRs nell’ipertrofia cardiaca e, in altri tessuti, è stato dimostrato come certi miR regolino il contenuto di MCU. Tramite ricerca bioinformatica, abbiamo identificato diversi microRNA che potrebbero appaiarsi alla regione 3’UTR di MCU. Tra questi, ci siamo focalizzati su miR-1 per la sua espressione muscolo-specifica, il suo ruolo critico nell’ipertrofia cardiaca, la sua omologia conservata tra diverse specie e la specificità per MCU tra i membri del complesso MCUC. Il saggio di luciferasi ha confermato quanto predetto dalla bioinformatica ed ha permesso di identificare specifiche sequenze complementari sul gene di MCU. Consistentemente, cardiomiociti over-esprimenti miR-1 hanno mostrato un diminuito contenuto proteico di MCU senza alterazioni nel suo mRNA, risultando in una riduzione significativa nella capacità di importare Ca2+ nella matrice mitocondriale. Quindi, abbiamo testato l’ipotesi che il contenuto di MCU fosse modulato in condizioni di ipertrofia associate a variazioni nell’espressione di miR-1, quali: i) lo sviluppo postnatale, ii) l’esercizio moderato, iii) il sovraccarico pressorio. Confrontando cuori neonati ed adulti abbiamo osservato che l’espressione di miR-1 aumenta, in linea col suo ruolo di repressore del programma genico fetale. Questo calo di miR-1 è accompagnato da un aumento nel contenuto proteico di MCU senza che ne aumentasse il trascritto. Inoltre, abbiamo osservato come solo il contenuto di MCU vari, tra i vari membri del complesso, eccezion fatta per l’mRNA di MCUb, che aumenta. Quindi, abbiamo analizzato l’asse miR-1/MCU in cuori ipertrofici murini e umani, con rimodellamenti sia fisiologici che patologici. Nei topi, l’ipertrofia fisiologica è stata indotta tramite protocollo di esercizio cronico, efficace nel determinare ingrandimento cardiaco, dei singoli cardiomiociti ed un aumento della contrattilità35. Il confronto coi cuori di topi sedentari ho dimostrato come il livello di miR-1 scenda nell’esercizio e, consistentemente, quello proteico di MCU salga. L’analisi del complesso in cuori sottoposti a costrizione aortica ha dimostrato come, durante l’iniziale fase compensata, caratterizzata da crescita dei cardiomiociti senza scompenso, le variazioni di miR-1 e MCU rispecchino quelle osservate nei topi esercitati. Inoltre, le reciproche variazioni di miR-1 e MCU accadono anche in un modello di ipertrofia di rilevanza clinica, come dimostrato dalle analisi di biopsie cardiache umane provenienti da donatori sani e pazienti con ipertrofia causata da stenosi aortica. Questi risultati indicano che, indipendentemente dalla natura dello stimolo ipertrofico (fisiologico o ipertrofico), l’iniziale adattamento cardiaco all’aumentata richiesta contrattile è caratterizzato da analoghi aumenti nella disponibilità cellulare di MCU. Viste le variazioni analoghe dell’asse miR-1/MCU riscontrate sia in ipertrofia indotta da esercizio che in quella compensata patologica, abbiamo ipotizzato che ci sia un meccanismo regolatorio comune. Ci siamo così focalizzati sul sistema ß-adrenergico, il primo meccanismo fisiologico coinvolto nella risposta all’aumentato carico di lavoro, condizione che accomuna sia ipertrofia da esercizio che da costrizione aortica. L’attivazione del signalling ß-adrenergico, infatti, determina aumento delle oscillazioni di Ca2+ citosolico e conseguentemente dell’uptake mitocondriale. In parallelo, l’attivazione di queste cascate di segnale è coinvolta nell’attivazione di vie di segnale di ipertrofia come Akt-FOXO. È interessante notare che l’espressione di miR-1, come è stato dimostrato, dipende da FOXO3a, indicando che, in condizioni di attivazione cronica dei recettori ß-adrenergici, il blocco della traslocazione nucleare di FOXO3a potrebbe inibire l’aumento di miR-1. Per supportare questa ipotesi, abbiamo trattato topi sottoposti a costrizione aortica col ß-bloccante metoprololo che, in linea con quanto ipotizzato, è stato in grado di abolire la repressione di miR-1 e di conseguenza l’aumento di MCU. Conclusioni e prospettive future. Complessivamente, i nostri dati identificano miR-1 come un nuovo regolatore post-trascrizionale di MCU e supportano l’idea che l’asse miR-1/MCU sia coinvolto nel rimodellamento ipertrofico fisiologico e patologico. Esperimenti futuri mireranno ad approfondire il ruolo causale di miR-1 nella modulazione di MCU, ed a identificare la via molecolare coinvolta nel processo. Attualmente esistono tools farmacologici (quali miR-mimics o antagomiRs) in grado di interagire coi miR endogeni, antagonizzandoli o sostituendoli, modulando con efficacia e selettività l’espressione degli mRNA target. Su queste basi, il nostro studio sull’asse miR-1/MCU può aprire a nuove prospettive terapeutiche per trattare l’ipertrofia cardiaca. 2. MCU partecipa all’adattamento cardiaco a stimoli ipertrofici. L’osservazione di come il contenuto di MCU cali durante la fase maladattativa dell’ipertrofia patologica, suggerisce che esso fluttui nelle varie fasi dell’ipertrofia. Questa osservazione ci ha indotto a cercare di determinare se MCU potesse avere un ruolo attivo nel rimodellamento cardiaco. Per testare quest’ipotesi, abbiamo modulato il livello di MCU in topi successivamente sottoposti a sovraccarico pressorio. Inoltre, per avere dettagli meccanicistici sul signalling cellulare, abbiamo modulato l’espressione di MCU in vitro, e abbiamo studiato l’effetto della sua overespressione o silenziamento nella risposta ad incubazione cronica con agonisti adrenergici. Per studiare il ruolo di MCU nell’adattamento cardiaco in vivo, abbiamo overespresso o silenziato l’uniporto mediante l’uso di vettori virali (AAV9). La modulazione di MCU, per sé, non ha alterato la struttura e la performance cardiaca. Tuttavia, quando abbiamo sottoposto gli animali a TAC, abbiamo osservato come l’overespressione di MCU comporti aumentata crescita ipertrofica, confrontando con animali WT allo stesso tempo dopo l’inizio della costrizione aortica. Inoltre, il rimodellamento nei topi overesprimenti ha caratteristiche simili a quello dell’ipertrofia fisiologica, quali aumentata densità capillare, scarsa fibrosi, funzionalità cardiaca preservata anche dopo 8 settimane di sovraccarico pressorio. Al contrario, il silenziamento di MCU ostacola l’adattamento cardiaco all’aumentata pressione, determinando un maladattamento prematuro, con caratteristiche tipiche della cardiomiopatia dilatativa, quali ridotta densità capillare, fibrosi diffusa ed inadeguata contrattilità. Queste caratteristiche hanno portato i topi MCU silenziati a sviluppare scompenso ed insufficienza cardiaca, ed a morire dopo solo 4 settimane dalla TAC. Per approfondire i meccanismi molecolari mediante i quali MCU impatta nella crescita ipertrofica dei cardiomiociti, abbiamo overespresso o silenziato MCU in cardiomiociti neonatali di ratto. Eseguendo esperimenti di live imaging delle dinamiche di Ca2+ mitocondriali con la sonda “mito-CaMeleon”, abbiamo appurato come la modulazione di MCU risulti in aumentato o diminuito uptake di Ca2+ mitocondriale. Se da un lato l’over-espressione di MCU non determina alterazioni morfologiche in condizioni basali, cellule silenziate dimostrano dimensioni maggiori rispetto a cellule di controllo, con evidente alterazioni nella struttura sarcomerica. Per mimare l’iperattivazione del sistema nervoso simpatico che si riscontra nell’ipertrofia sia fisiologica che patologica, abbiamo incubato le cellule con norepinefrina. Anche in questo caso, l’overespressione di MCU aumenta la crescita ipertrofica, mentre il suo silenziamento ha un effetto opposto, contraddistinto da compromissione dei sarcomeri ad attivazione di apoptosi, in evidente analogia ai dati ottenuti in vivo. Le successive analisi sono state mirate per approfondire lo stato di attivazione di divere vie di segnale medianti ipertrofia. Abbiamo rilevato come l’overespressione di MCU, in cardiomiociti sottoposti a stimolazione adrenergica, acceleri l’attivazione dell’asse calcineurina/NFAT. Inoltre, i nostri dati suggeriscono la partecipazione dell’asse Akt/ GSK3ß all’aumentata attivazione di NFAT, in una cascata presumibilmente a valle di CaMKII che fosforila Akt. Infatti, l’inibizione di CaMKII in cardiomiociti MCU overesprimenti determina una crescita ipertrofica comparabile a cellule di controllo. Per concludere, i nostri risultati dimostrano come l’aumento del carico cardiaco, indotto in vivo da TAC ed in vitro da trattamento con noradrenalina, sia ben tollerato quando i livelli di MCU sono aumentati dall’overespressione. Al contrario, il silenziamento di MCU induce, nelle stesse condizioni, morte cellulare e prematuro rimodellamento maladattativo. Questi dati sono in accordo con le nostre osservazioni preliminari che indicano come il contenuto proteico di MCU, che aumenta nell’ipertrofia compensata, diminuisca nel successivo rimodellamento patologico che determina scompenso cardiaco. Inoltre, abbiamo identificato l’asse ß-AR/CaMKII/Akt come cruciale nell’ipertrofia cardiaca e dipendente dalla modulazione di MCU. 3. Sviluppo di un protocollo di coltura che induca la maturazione di cardiomiociti neonatali in vitro Le colture primarie di cardiomiociti neonatali sono un modello cellulare ampiamente utilizzato nella cardiologia molecolare, in quanto possono esser mantenuti in coltura per più giorni e sono facilmente manipolabili geneticamente28. Tuttavia, questo tipo cellulare ha importanti differenze funzionali e strutturali rispetto ai cardiomiociti adulti. Queste differenze vanno dall’espressione di diverse isoforme di miosina (nel topo, dalla ß alla α), necessario per ottimizzare la performance contrattile, a cambi nel metabolismo (che passa da glucidico ad ossidativo), in modo da garantire maggior apporto di ATP in vista di un maggior consumo29. Inoltre, il processo di maturazione postnatale delle cellule comprende alterazioni nelle strutture coinvolte nelle dinamiche di Ca2+ 30. In particolare, nelle cellule neonatali, la contrazione avviene principalmente grazie al Ca2+ che entra dai canali del Ca2+ di tipo L situati nella membrana citoplasmatica. Il Ca2+ che entra attiva direttamente i sarcomeri, con un minimo contributo del Ca2+ contenuto nelle vescicole che costituiscono un immaturo reticolo sarcoplasmatico. Al contrario, nelle cellule adulte la membrana plasmatica ha sviluppato una serie di invaginazioni note come tubuli T che penetrano nella cellula e giungono all’estremità del reticolo sarcoplasmatico, ora costituito dal tipico sistema di cisterne, cosicché i canali del Ca2+ di tipo L siano a stretto contatto coi RyR2, formando cosi le Unità deputate al Rilascio del Ca2+ (CRUs). Questa sofisticata struttura fa sì che le poche molecole di Ca2+ che entrano dai canali nei tubuli T possano scatenare il Rilascio di Ca2+ indotto dal Ca2+ (CICR), determinando l’uscita di un’ingente quantità di ione dal reticolo sarcoplasmatico. Un altro importante cambiamento interessa i mitocondri che, se nel cardiomiocita neonatale occupano principalmente la zona perinucleare, in quello adulto si dispongono anche negli spazi sub-sarcolemmali ed inter-miofibrillari. In questi distretti, i mitocondri sono in prossimità del reticolo sarcoplasmatico, al quale possono ancorarsi fisicamente, trovandosi così in distretti cellulari caratterizzati da elevate concentrazioni di Ca2+. Tenendo a mente questi fattori, il nostro obiettivo è stato quello di sviluppare un protocollo che promuovesse la maturazione di cardiomiociti neonatali verso un fenotipo adulto, ottenendo così un modello sperimentale ottimale per lo studio delle dinamiche del Ca2+ cellulare, ed identificare così i meccanismi che connettono il Ca2+ mitocondriale al rimodellamento ipertrofico. Per indurre la maturazione dei cardiomiociti neonatali abbiamo modificato la composizione dei terreni di coltura tradizionalmente usati. Per mantenere le cellule ad una concentrazione di glucosio simile a quella fisiologica, abbiamo cambiato il costituente principale del terreno, passando da DMEM (Dulbecco’s modified eagle medium) a MEM (minimum essential medium) e riducendo così la concentrazione da 25 mM a 5 mM, valore, quest’ultimo, paragonabile alla concentrazione fisiologica in vivo. Per ridurre la proliferazione dei fibroblasti, che tramite secrezione di fattori di crescita e componenti della matrice extracellulare determinerebbero de-differenziamento dei cardiomiociti, abbiamo fortemente ridotto il quantitativo di siero ed aggiunto un agente proliferativo (BrdU). Per compensare la rimozione del siero, abbiamo aggiunto co-fattori vitaminici ed ormoni trofici, come l’insulina. In tal modo abbiamo ottenuto una popolazione pura di cardiomiociti che può essere tenuta in coltura per più settimane, e che già dopo pochi giorni mostrano una morfologia diversa dalle cellule ottenute col protocollo tradizionale. Analisi alla microscopia hanno evidenziato come queste cellule siano più grandi, rettangolari con un asse maggiore ben distinto da un asse minore, ed un perimetro regolare senza le tipiche ramificazioni dei cardiomiociti immaturi neonatali. A livello subcellulare, abbiamo osservato una maggiore estensione dell’apparato contrattile, rivelatosi disposto in maniera più regolare. I mitocondri appaiono disposti longitudinalmente accanto e tra i sarcomeri, come nelle cellule adulte. Inoltre, l’immunofluorescenza per il recettore rianodinico ne ha evidenziato la presenza in clusters, distribuiti in maniera regolare, in analogia alla loro distribuzione in cellule mature, suggerendo così la presenza di un reticolo sarcoplasmatico maggiorente formato. Consistentemente con ciò, abbiamo osservato minori e più rapidi Ca2+ sparks, eventi elementari di dinamiche di calcio, determinati dall’apertura transiente di RyR. La minore frequenza ed entità di questi sparks suggerisce che i RyR disposti in maniera regolare in clusters determini la formazione di vere e proprie unità deputate al rilascio di calcio (Calcium Release Units, CRUs), strutture fondamentali nei cardiomiociti adulti. Infine, queste cellule han risposto maggiormente al trattamento con agonisti adrenergici, riportando una crescita ipertrofica maggiore rispetto a cellule neonatali tradizionali sottoposte allo stesso trattamento. Tutte queste caratteristiche sopracitate indicano come queste cellule possano rappresentare un modello in vitro adatto allo studio delle dinamiche di Ca2+ intracellulare, specialmente nel rimodellamento ipertrofico. È importante sottolineare come questo maggior grado di maturazione dei cardiomiociti neonatali non sia a discapito della capacità di manipolarli geneticamente, con tecniche di trasfezione od infezione. Esperimenti futuri cercheranno di caratterizzare a fondo le strutture coinvolte nelle dinamiche di calcio intracellulari, come ad esempio la formazione di Tubuli T ed il rapporto di questi con il reticolo sarcoplasmatico ed i mitocondri.

Abstract The impact of hand-rearing on the morphology and physiology of captive and wild grey partridges (Perdix perdix) and capercaillies (Tetrao urogallus) was studied in three feeding trials conducted under laboratory conditions, and two comparative studies between wild and captive birds. Finally, wild and hand-reared grey partridges from several localities in Europe were sampled and the control region 1 of mitochondrial DNA was sequenced to reveal genetic variation between populations, as well as to compare wild and captive stocks. Wild capercaillies had heavier pectoral muscles, hearts, livers and gizzards, longer small intestines than hand-reared ones, and a higher cytochrome-c oxidase activity in muscle and heart. Invertebrates were essential to the growth, primary and temperature regulation development in grey partridge chicks. Fish was not sufficient to replace invertebrates in the diet. A change in diet from commercial to natural decreased the assimilation efficiency in the grey partridge. It also increased the mass of gizzard reflecting the need for greater grinding ability. Of hepatic P450 enzymes used in this study 7-ethoxyresorufin-0-deethylase and 7-pentoxyresorufin-0-deethylase differed between wild and hand-reared birds. Coumarin-7-hydroxylase activity was higher in grey partridges than capercaillies. Diet differences may have caused these differences. Quebracho tannin added to the diet lowered nitrogen concentration in caecal feces, and elevated the level of excreted tannin. Otherwise its effects were slight. Mitochondrial control region revealed 14 variable sites between two main lineages detected. Nucleotide and haplotype diversities varied greatly between populations. The markedly deep divergence between the two lineages indicated most probably post-glacial recolonisations from geographically isolated refuges. In Finland, wild birds represented the eastern lineage, while the farmstock represented the western lineage. Surprisingly little trace, contrary to expectations, from the large-scale releasing of imported partridges could be seen in the European populations.

Le cancer du sein triple négatif (TNBC) est un sous-type particulièrement agressif du cancer du sein, traité principalement par chimiothérapie. Cependant, environ 50% des patients connaissent une rechute avec métastases dans les 3 à 5 ans suivant le traitement. Afin de mieux comprendre l'évasion post-chimiothérapie et la formation de métastases des cellules cancéreuses TNBC, nous avons établi des modèles de cellules TNBC en traitant les cellules SUM159-PT et MDA-MB-231 avec de l'épirubicine, du cyclophosphamide et du paclitaxel, simulant des protocoles cliniques. Nous nous sommes initialement concentrés sur l'adaptation mitochondriale de ces cellules persistantes. Les cellules MDA-MB-231 ont montré une sensibilité réduite à la chimiothérapie, associée à une phosphorylation oxydative accrue et à des intermédiaires du cycle de l'acide tricarboxylique modifiés. En revanche, les cellules SUM159-PT ont conservé leur sensibilité. Le ciblage du métabolisme mitochondrial du pyruvate avec le UK-5099 a resensibilisé les cellules persistantes MDA-MB-231 aux agents thérapeutiques. Les cellules persistantes ont montré une migration, une invasion et une survie accrues en culture en suspension, les cellules SUM159-PT présentant une adhésion accrue aux cellules endothéliales. Des études de xénogreffe in vivo ont confirmé ces observations, mettant l'accent sur une croissance cellulaire accrue et une colonisation métastatique dans des organes vitaux, en particulier le cerveau. Le tropisme accru pour le cerveau pourrait s'expliquer par le fait que les cellules TNBC persistantes présentaient une capacité accrue de traverser la barrière hémato-encéphalique, d'envahir le parenchyme cérébral et de croître dans une matrice 3D similaire au cerveau. Dans une deuxième phase de notre étude, nous avons étudié les mécanismes moléculaires facilitant la formation de métastases cérébrales de ces cellules persistantes. L'analyse protéomique a identifié des protéines surexprimées, notamment le COL1A1, fréquemment élevé chez les patients atteints de TNBC. Une augmentation de COL1A1 était corrélée à un mauvais pronostic et à une augmentation de la formation de métastase. L'inhibition de COL1A1 a réduit le potentiel métastatique à la fois in vitro et in vivo, soulignant son potentiel en tant que cible thérapeutique pour prévenir les métastases cérébrales après un traitement par chimiothérapie.L'ensemble de ces résultats offre un aperçu des mécanismes d'adaptation mis en place par les cellules cancéreuses en réponse à la chimiothérapie, et suggère que cibler le métabolisme du pyruvate mitochondrial pourrait contribuer à surmonter les adaptations mitochondriales des cellules de cancer du sein triple négatif. De plus, nos résultats mettent en lumière la manière dont une chimiothérapie combinée et séquentielle peut accroître le potentiel métastatique des cellules TNBC, en particulier vers le cerveau. Nous avons identifié la protéine COL1A1 comme un élément clé favorisant les différentes étapes de formation des métastases cérébrales dans les cellules TNBC résistantes à la chimiothérapie. Des recherches complémentaires sont nécessaires pour élucider les mécanismes détaillés de la surexpression de COL1A1.En utilisant le même schéma thérapeutique, nous avons mis en œuvre un traitement court de 48h, combiné et séquentiel pour évaluer les modifications protéomique précoces dans les vésicules extracellulaires libérées par les cellules TNBC persistantes. Avec cette approche, on a également exploré l'impact de la chimiothérapie sur les facteurs angiocrines des cellules endothéliales, suggérant le rôle du sécrétome induit par la chimiothérapie dans la facilitation des métastases post-chimiothérapie. Bien que ce volet de notre étude en soit à un stade préliminaire, les résultats encouragent à approfondir davantage l'investigation expérimentale
Triple negative breast cancer (TNBC) is a highly aggressive breast cancer subtype, primarily treated with chemotherapy. However, approximately 50% of patients experience relapse with metastasis within 3 to 5 years post-treatment. To gain insight into the post-chemotherapy escape and metastasis formation of TNBC cancer cells, we established TNBC cell models by treating SUM159-PT and MDA-MB-231 cells with epirubicin, cyclophosphamide, and paclitaxel. simulating clinical protocols. We initially focused on the mitochondrial adaptation of these chemo-persistent cells. MDA-MB-231 cells showed reduced chemosensitivity, associated with increased oxidative phosphorylation and altered tricarboxylic acid cycle intermediates. In contrast, SUM159-PT cells retained sensitivity. Targeting mitochondrial pyruvate metabolism with UK-5099 re-sensitized persistent cells to therapeutic agents, suggesting a potential strategy to overcome mitochondrial adaptation. Persistent cells exhibited increased migration, invasion, survival in suspension culture, with SUM159-PT cells displaying increased adhesion to endothelial cells. In vivo xenograft studies confirmed these observations, emphasizing increased cell growth and metastatic colonization in vital organs, particularly the brain. The enhanced trophism for brain could be explained by the fact that persistent TNBC cells exhibited increased abilities to transmigrate through BBB, to invade the brain parenchyma and to grow in a brain-like 3D matrix. In a second phase of our study, we investigated the molecular mechanisms facilitating brain metastasis of these persistent cells. proteomic analysis identified upregulated proteins, notably COL1A1, frequently elevated in TNBC patients. Increased COL1A1 correlated with poor prognosis and enhanced metastasis. Inhibition of COL1A1 reduced metastatic potential both in vitro and in vivo, highlighting its potential as a therapeutic target in preventing brain metastasis post chemotherapy treatment.Collectively, these findings provide insight into the adaptive mechanisms employed by cancer cells in response to chemotherapy, and suggest that targeting mitochondrial pyruvate metabolism may help to overcome the mitochondrial adaptations in TNBC cells. Furthermore, our data illuminate how combined and sequential chemotherapy may increase the metastatic potential of TNBC cells, particularly towards the brain. We have pinpointed COL1A1 as a key factor promoting various stages of brain metastasis formation in chemotherapy-resistant TNBC cells. Additional research is required to elucidate the detailed mechanisms behind COL1A1 overexpression.Using the identical drug regimen, we implemented a short, combined, and sequential treatment to replicate initial proteomic alterations in extracellular vesicles released by persistent TNBC cells. This approach also explored the impact of chemotherapy on angiocrine factors from endothelial cells, suggesting the role of the chemo-induced secretome in evading treatment and facilitating metastasis post-chemotherapy. Although this aspect of our study is currently in its early phases, the findings underscore the necessity for further experimental validation

Collembola (=springtails) is one of the most abundant, widespread and ancient lineages of basal hexapods. During their long evolutionary history, springtails have adapted to most damp environments on Earth, including those of South Pole. Antarctic springtails are endemic to the frozen Continent and among the few invertebrate taxa adapted to its strictly terrestrial ecosystem. These species have evolved when Antarctica was still linked to the Gondwanaland at lower latitudes and have adapted and survived to the cooling, isolation and southwards migration of the landmass. Antarctic springtails’ habitats are restricted to the few coastal areas, seasonally ice-free and accounting for less than the 0.5% of the entire continental area and off-shore islands. The niche fragmentation, together with springtails poor dispersal capability (due to the primary absence of wings), entail a severe degree of isolation among populations, with very low levels of gene flow. The Antarctic springtail species composition is limited without overlap among the two main Antarctic bioregions (i.e., the maritime and the continental Antarctica), with Friesea antarctica being the only species found both in the Antarctic Peninsula and Victoria Land (continental Antarctica). The high levels of endemism and fragmentation among populations, as well as the low invertebrate biodiversity and the complex and delicate array of physiological adaptation these species evolved, make Antarctic taxa particularly susceptible to anthropogenic climate changes, that we are all experiencing since the second industrial revolution in the XIXth century. In this respect, studying the molecular mechanisms underlying springtail adaptation to such a harsh environment, as well as the genetic structure of the populations and the way in which specimens may have been and can be influenced by the Antarctic terrestrial environment, may greatly assist the development of adequate and biogeographically-specific (thus, effective) conservational plans. In order to address these issues, different studies have been carried out during the current PhD project. A genetics of population study was performed to investigate the genetic structure of the Antarctic springtail species Cryptopygus terranovus. As previously observed in other Antarctic species (i.e, F. antarctica), high levels of genetic divergence were detected, with very few haplotypes shared among populations nearly suggesting the absence of gene flow, as well as the presence of cryptic species. One way to address this issue is the integration of morphological, molecular and biogeographic data to assess whether the detection of cryptic species is due to an ongoing phylogenetic niche conservatism process or to overlooked morphological differences. In this respect, an integrative taxonomic analysis has been carried out on the Antarctic springtail F. antarctica. Nuclear and mitochondrial markers were used in bioinformatic analyses of species delimitation. Although applied tools rely on different algorithms and biological assumptions, and the chosen molecular markers are generally subject to different evolutionary pressures, the results obtained in this study would suggest that at least two more species may be hidden within the F. antarctica complex. Specimens from the same localities were also morphologically re-described so that new species could be possibly established. These analyses would suggest an even higher species richness of the Antarctic terrestrial ecosystem, that should be taken into account when developing conservational plans. Our ability to safeguard Antarctica relies also on our in-depth understanding of the terrestrial ecosystem functioning and dynamics. In this perspective, an initial descriptive analysis of the microbial communities associated to four Antarctic springtail species was performed (specifically on: Cryptopygus antarcticus antarcticus and F. antarctica from the Antarctic Peninsula and C. terranovus and F. antarctica collected along Victoria Land). The results obtained are in line with previous studies on Collembola microbiomes. In addition, the occurrence of particularly interesting OTUs, such as those of the genera Streptomyces and Bacillus, was, to my knowledge, firstly detected among springtails and may break new grounds for biotechnology development, especially starting from such an unspoiled ecosystem, like the Antarctic one. Finally, the mitochondrial genomes from 13 springtail species, both living at low and high latitudes, have been applied to maximum likelihood analyses of positive selection in order to investigate whether or not the organelle chromosome may have been involved in Antarctic springtails adaptation to such an extreme environment. The results pointed out that some mitochondrial genes involved in the oxidative phosphorylation process may have been under positive selection, thus suggesting the development of additional thermoregulatory mechanism within the mitochondrion, complementary to the well-known cold hardiness strategies.

L’anorexie mentale (AN), un trouble du comportement alimentaire multifactoriel, se traduit par une perte de poids. La sévère dénutrition retrouvée dans l’AN est associée à des altérations métaboliques induisant une dérégulation de l’axe intestin cerveau. Les mécanismes physiopathologiques sont encore mal connus. Le travail de cette thèse était de mieux appréhender les dysfonctions de l’axe intestin cerveau en évaluant le métabolisme protéique de divers tissus (hypothalamus, côlon et estomac) dans un modèle murin d’anorexie par une approche protéomique. Le premier travail a permis de mieux caractériser le modèle d’anorexie nommé activity-based anorexia (ABA) en fonction du sexe. Puis les différentes analyses protéomiques ont permis de constater une adaptation tissu dépendant des mécanismes régulant l’équilibre énergétique, avec une activité cérébrale potentiellement augmentée au détriment des fonctions digestives. Chez les souris femelles ABA, il a été constaté une augmentation d’expression de protéines mitochondriales au niveau de l’hypothalamus et à l’inverse, une diminution du métabolisme protéino-énergétique au niveau colique avec un rôle de la voie de signalisation mTOR. L’autophagie était augmentée dans ces deux tissus. Ensuite, nous avons démontré un ralentissement de la vidange gastrique secondaire à la dénutrition, et l’analyse protéomique a permis de constater une augmentation du stress oxydant au niveau de l’antre des souris ABA femelles. Ces altérations peuvent contribuer aux troubles fonctionnels gastro intestinaux. En conclusion, nos études soulignent des mécanismes d’adaptation tissu dépendants dans l’anorexie, qui devront être ultérieurement approfondis
Anorexia nervosa, a multifactorial eating disorder, is a major public health problem and results in a severe body weight loss. The severe malnutrition observed in anorectic patients is associated with metabolic alterations inducing disturbance of the gut-brain axis. However, involved mechanisms remained poorly understood. The aim of the present thesis was to better understand the alterations of the gut-brain axis in the activity-based anorexia (ABA) model by evaluating the protein metabolism of various tissues (hypothalamus, colon and stomach) by proteomic approach. Firstly, we have better characterized the response to ABA model according to sex. Then, different proteomic analyses were performed using female C57BL/6 mice. Our results revealed a tissue-dependent adaptation of protein and energy metabolism with an increased hypothalamic activity and a decrease in the gastrointestinal tract. Indeed, ABA mice exhibited an increased expression of proteins involved in mitochondrial metabolism at the level of the hypothalamus, and conversely a decrease of proteins involved in protein and energy metabolism in colonic mucosa with a key role of the mTOR signaling pathway. Both in hypothalamus and colon, autophagy was increased. We were also able to show that gastric emptying was delayed in ABA mice that is mainly due to malnutrition. In addition, proteomic analysis revealed an increase in gastric oxidative stress in female ABA mice. These alterations may contribute to the gastrointestinal functional disorders frequently described in anorexia nervosa. In conclusions, our study underlined tissue-dependent adaptive metabolic process during anorexia that should be further explored

Bien que les statines forment la classe d'hypolipidémiants la plus utilisée, une toxicité musculaire a été reportée, pouvant ainsi provoquer l’apparition d’une myopathie. Dans la première partie, nous avons montré chez l’Homme et l’animal que les statines inhibent directement la chaine respiratoire mitochondriale, et induisent la production de radicaux libres dérivés de l’oxygène (RLO), qui active les voies apoptotiques dans les muscles glycolytiques, alors que les muscles oxydatifs ne sont pas atteints. Nous avons ensuite montré in vitro que le stress réducteur peut engendrer une oxydation mitochondriale, pouvant conduire à une activation de la voie de biogenèse mitochondriale. De plus l’augmentation du contenu mitochondrial induite a permis de protéger les cellules contre l’apoptose induite par les statines. Enfin, nous avons montré in vivo que l’induction des voies de biogenèse mitochondriale est nécessaire à la tolérance des statines dans les muscles oxydatifs. En conclusion, le phénotype mitochondrial, tant au niveau quantitatif que qualitatif, semble être un facteur clé dans l’apparition de la myopathie aux statines
Although statins are the most prescribed class of lipid-lowering agents, adverse muscular toxicity has been reported, which can lead to the appearance of a myopathy. In the first part, we showed in Humans and animals that statins inhibit directly the mitochondrial respiratory chain, and induce the production of reactive oxygen species (ROS), that trigger apoptotic pathways in glycolytic skeletal muscles, whereas oxidative muscles are not impaired. We then showed in vitro that reductive stress can provoke mitochondrial oxidation, that could lead to an activation of mitochondrial biogenesis pathways. Moreover, the consequent increase in mitochondrial content enabled to protect cells against statin-induced apoptosis. Finally, we showed in vivo that the induction of mitochondrial biogenesis is necessary for statin tolerance in oxidative skeletal muscles. In conclusion, mitochondrial phenotype, both quantitatively and qualitatively, seems to be a key factor in the appearance of statin myopathy

An important goal of biology is to understand the key mechanisms of evolution underlying the diversity of living organisms on Earth. In that respect, the recent innovations in the field of new generation sequencing technologies (NGS) are bringing new and exciting opportunities. This thesis presents results obtained with these tools in the specific context of the study of two sister species of cold-adapted leaf beetles, Gonioctena intermedia and G. quinquepunctata. More specifically, this work is focused around four research directions: the two first explore methods of statistical inference using a spatially explicit model of coalescence, by (1) evaluating the potential of various summary statistics to discriminate phylogeographic hypotheses, and (2) investigating the dispersal abilities of a montane leaf beetle, G. quinquepunctata, using an original method that avoids using summary statistics. The third research direction focuses on the adaptation to cold conditions in this montane leaf beetle, by testing the association between genetic polymorphism across tens of thousands of genetic markers and altitude in samples collected at various elevation levels in the Vosges (France). Finally, the fourth, and last, research axis presents the discovery of mitochondrial heteroplasmy, i.e. the presence in an individual of multiple copies of the mitochondrial genome, in natural populations of G. intermedia. We illustrate, here, how NGS technologies could help identify this phenomenon, probably underestimated in animals, on a large scale.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Eukaryotes were born of a chimeric union of two prokaryotes. The legacy of this fusion is organisms with both a nuclear and mitochondrial genome that must work in a coordinated fashion to enable cellular respiration. The coexistence of two genomes in a single organism requires tight coadaptation to enable function. The need for coadaptation, the challenge of co-transmission, and the possibility of genomic conflict between mitochondrial and nuclear genes have profound consequences for the ecology and evolution of eukaryotic life. This book defines mitonuclear ecology as an emerging field that reassesses core concepts in evolutionary ecology in light of the necessity of mitonuclear coadaptation. I discuss and summarize research that tests new mitonuclear-based theories for the evolution of sex, two sexes, senescence, a sequestered germ line, speciation, sexual selection, and adaptation. The ideas presented in this book represent a paradigm shift for evolutionary ecology. Through the twentieth century, mitochondrial genomes were dismissed as unimportant to the evolution of complex life because variation within mitochondrial genomes was proposed to be functionally neutral. These conceptions about mitochondrial genomes and mitonuclear genomic interactions have been changing rapidly, and a growing literature in top journals is making it increasingly clear that the interactions of the mitochondrial and nuclear genomes over the past 2 billion years have played a major role in shaping the evolution of eukaryotes. These new hypotheses for the evolution of quintessential characteristics of complex life hold the potential to fundamentally reshape the field of evolutionary ecology and to inform the emerging fields of mitochondrial medicine and mitochondrial-based reproductive therapies.

The molecular basis of ageing is reviewed. This includes the concept of a summation of DNA damage over a lifetime causing genome instability. Epigenetic alterations, telomeric shortening, and the possibility of their modification are discussed. Oxidative and mitochondrial DNA damage and the resulting dysfunction leading to senescence are briefly described. Systemic problems and resultant behavioural adaptation may mask the decline in functional reserve and cause some of the difficulties in identifying its presence in ill elderly patients. Specific organ system changes are then described in some detail. These include the major concerns with the cardiovascular, respiratory, renal, hepatic, neurologic, endocrine, and musculoskeletal systems. The effect of ageing on the special senses of vision and hearing are covered, with emphasis on issues of informed consent.

Abstract The plastome is usually considered a highly conserved genome. Compared with the nuclear genome, it is small and has different genetic rules. Through different molecular methods (TILLING, candidate gene sequencing, amplicon massive sequencing and plastome re-sequencing) applied to barley chloroplast mutator (cpm) seedlings, we detected more than 60 polymorphisms affecting a wide variety of plastid genes and several intergenic regions. The genes affected belonged mostly to the plastid genetic machinery and the photosynthetic apparatus, but there were also genes like matK, whose functions are so far not clearly established. Among the isolated mutants, we found the first infA gene mutant in higher plants, two mutants in ycf3 locus and the first psbA gene mutant in barley. The latter is used in breeding barley cultivars where PSII is tolerant to toxic herbicides. Most of the molecular changes were substitutions, and small indels located in microsatellites. However, particular combinations of polymorphisms observed in the rpl23 gene and pseudogene suggest that, besides an increased rate of mutations, an augmented rate of illegitimate recombination also occurred. Although a few substitutions were observed in the mitochondria of cpm plants, we have not yet determined the implications of the cpm for mitochondrial stability. The spectrum of plastome polymorphisms highly suggests that the cpm gene is involved in plastid DNA repair, more precisely taking part in the mismatch repair system. All results show that the cpm mutant is an extraordinary source of plastome variability for plant research and/or plant breeding. This mutant also provides an interesting experimental system in which to investigate the mechanisms responsible for maintaining plastid stability.

Knockout of the HSP90AA1 gene encoding Hsp90α in HT1080 human fibrosarcoma cells was accompanied by adaptation of the cellular chaperone machine to Hsp90α loss. Adaptation included increased expression of Hsp90β, key Hsp90 co-chaperones, chaperones and co-chaperones of the Hsp70/Hsp40 complex, components of the TRiC/CCT complex, prefoldins, prefoldin-like PFDL, R2TP, and R2SP chaperone complexes, as well as key mitochondrial chaperones and chaperonins.

Биогенез митохондрий регулируется организованной индукции нескольких транскрипционных факторов. Утрата митохондриальной адаптации способствует сахарному диабету 2 типа. Высокий уровень IL-6 в плазме крови пациентов с ожирением взаимосвязан со снижением экспрессии гена TFAM в биоптатах печени. Уровень экспрессии гена TFAM в биоптатах печени у больных с СД 2 типа снижался относительно контрольной группы. Mitochondrial biogenesis is regulated by the organized induction of several transcription factors. Loss of mitochondrial adaptation contributes to type 2 diabetes. A high level of IL-6 in the blood plasma of obese patients is associated with a decrease in TFAM gene expression in liver biopsies. The expression level of the TFAM gene in liver biopsies of patients with type 2 diabetes decreased relative to the control group.

This paper presents the adaptation of a diffusion neural network to generate a labeled synthetic dataset of electron microscopy of the brain. A model was trained can generate images and markup for them at the same time, which is an undoubted advantage of the chosen approach. Using the trained model, a set of labeled images was generated. The synthetic images are visually very similar to the original ones, the FID similarity metric between the synthetic and original datasets is 27.1. A simplified U-Net segmentation model trained on a mixed data set (original data + synthetic data) obtained a Dice score of 0.856 versus 0.858 on the original training set. Despite the good quality of synthetic data, their use in training the segmentation network does not improve the segmentation results.

New potential for engineering chloroplasts to express novel traits has stimulated research into relevant techniques and genetic processes, including plastid transformation and gene regulation. This proposal continued our long time BARD-funded collaboration research into mechanisms that influence chloroplast RNA accumulation, and thus gene expression. Previous work on cpRNA catabolism has elucidated a pathway initiated by endonucleolytic cleavage, followed by polyadenylation and exonucleolytic degradation. A major player in this process is the nucleus-encoded exoribonuclease/polymerasepolynucleotidephoshorylase (PNPase). Biochemical characterization of PNPase has revealed a modular structure that controls its RNA synthesis and degradation activities, which in turn are responsive to the phosphate (P) concentration. However, the in vivo roles and regulation of these opposing activities are poorly understood. The objectives of this project were to define how PNPase is controlled by P and nucleotides, using in vitro assays; To make use of both null and site-directed mutations in the PNPgene to study why PNPase appears to be required for photosynthesis; and to analyze plants defective in P sensing for effects on chloroplast gene expression, to address one aspect of how adaptation is integrated throughout the organism. Our new data show that P deprivation reduces cpRNA decay rates in vivo in a PNPasedependent manner, suggesting that PNPase is part of an organismal P limitation response chain that includes the chloroplast. As an essential component of macromolecules, P availability often limits plant growth, and particularly impacts photosynthesis. Although plants have evolved sophisticated scavenging mechanisms these have yet to be exploited, hence P is the most important fertilizer input for crop plants. cpRNA metabolism was found to be regulated by P concentrations through a global sensing pathway in which PNPase is a central player. In addition several additional discoveries were revealed during the course of this research program. The human mitochondria PNPase was explored and a possible role in maintaining mitochondria homeostasis was outlined. As polyadenylation was found to be a common mechanism that is present in almost all organisms, the few examples of organisms that metabolize RNA with no polyadenylation were analyzed and described. Our experiment shaded new insights into how nutrient stress signals affect yield by influencing photosynthesis and other chloroplast processes, suggesting strategies for improving agriculturally-important plants or plants with novel introduced traits. Our studies illuminated the poorly understood linkage of chloroplast gene expression to environmental influences other than light quality and quantity. Finely, our finding significantly advanced the knowledge about polyadenylation of RNA, the evolution of this process and its function in different organisms including bacteria, archaea, chloroplasts, mitochondria and the eukaryotic cell. These new insights into chloroplast gene regulation will ultimately support plant improvement for agriculture