Littérature scientifique sur le sujet « Sarcomere mechanics »

AbstractThe sarcomere is the fundamental structural and functional unit of striated muscle and is directly responsible for most of its mechanical properties. The sarcomere generates active or contractile forces and determines the passive or elastic properties of striated muscle. In the heart, mutations in sarcomeric proteins are responsible for the majority of genetically inherited cardiomyopathies. Here, we review the major determinants of cardiac sarcomere mechanics including the key structural components that contribute to active and passive tension. We dissect the molecular and structural basis of active force generation, including sarcomere composition, structure, activation, and relaxation. We then explore the giant sarcomere-resident protein titin, the major contributor to cardiac passive tension. We discuss sarcomere dynamics exemplified by the regulation of titin-based stiffness and the titin life cycle. Finally, we provide an overview of therapeutic strategies that target the sarcomere to improve cardiac contraction and filling.

Muscle contraction is commonly associated with the cross-bridge and sliding filament theories, which have received strong support from experiments conducted over the years in different laboratories. However, there are studies that cannot be readily explained by the theories, showing 1) a plateau of the force-length relation extended beyond optimal filament overlap, and forces produced at long sarcomere lengths that are higher than those predicted by the sliding filament theory; 2) passive forces at long sarcomere lengths that can be modulated by activation and Ca2+, which changes the force-length relation; and 3) an unexplained high force produced during and after stretch of activated muscle fibers. Some of these studies even propose “new theories of contraction.” While some of these observations deserve evaluation, many of these studies present data that lack a rigorous control and experiments that cannot be repeated in other laboratories. This article reviews these issues, looking into studies that have used intact and permeabilized fibers, myofibrils, isolated sarcomeres, and half-sarcomeres. A common mechanism associated with sarcomere and half-sarcomere length nonuniformities and a Ca2+-induced increase in the stiffness of titin is proposed to explain observations that derive from these studies.

Whereas it is evident that a well aligned and regular sarcomeric structure in cardiomyocytes is vital for heart function, considerably less is known about the contribution of individual elements to the mechanics of the entire cell. For instance, it is unclear whether altered Z-disc elements are the reason or the outcome of related cardiomyopathies. Therefore, it is crucial to gain more insight into this cellular organization. This study utilizes femtosecond laser-based nanosurgery to better understand sarcomeres and their repair upon damage. We investigated the influence of the extent and the location of the Z-disc damage. A single, three, five or ten Z-disc ablations were performed in neonatal rat cardiomyocytes. We employed image-based analysis using a self-written software together with different already published algorithms. We observed that cardiomyocyte survival associated with the damage extent, but not with the cell area or the total number of Z-discs per cell. The cell survival is independent of the damage position and can be compensated. However, the sarcomere alignment/orientation is changing over time after ablation. The contraction time is also independent of the extent of damage for the tested parameters. Additionally, we observed shortening rates between 6–7% of the initial sarcomere length in laser treated cardiomyocytes. This rate is an important indicator for force generation in myocytes. In conclusion, femtosecond laser-based nanosurgery together with image-based sarcomere tracking is a powerful tool to better understand the Z-disc complex and its force propagation function and role in cellular mechanisms.

Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment.

Cette thèse est consacrée à la modélisation de la réponse transitoire d'une fibre musculaire squelettique soumise à des sollicitations mécaniques rapides. A l'échelle du nanomètre, la fibre musculaire contient des filaments d'actine et de myosine regroupés en unités contractiles appelées "sarcomères". Le filament de myosine est un assemblage de moteurs mol ́eculaires qui, en présence d'ATP, s'attachent et se d ́etachent p ́eriodiquement au filament d'actine. Au cours de ce processus d'attachement-détachement, la myosine génère une force lors d'un changement de conformation appelé "power-stroke". Ses caractéristiques peuvent être étudiées lors de la réponse transitoire de la fibre soumise à des sollicitations mécaniques rapides. Nous proposons un modèle mécanique innovant du demi-sarcomere permettant de relier les caractéristiques de la myosine à la réponse de la fibre complète. A la différence des modèles existants, privilégiant une approche discrète, ce modèle s'appuie sur la définition d'un potentiel d'énergie continu qui prend en compte une interaction de champ moyen entre les moteurs moléculaires. Ce système présente des réponses radicallement différentes à longueur imposée et à force imposée. Nous proposons en particulier une explication à la différence de cinétique observée expérimentalement. Nous montrons également que le demi-sarcomere est m ́ecaniquement instable ce qui explique les inhomogénéités de longueurs observées dans une myofibrille.

Tendon transfer surgery is used to improve the hand function of patients with nerve injuries, spinal cord lesions, cerebral palsy (CP), stroke, or muscle injuries. The tendon of a muscle, usually with function opposite that of the lost muscle function, is transferred to the tendon of the deficient muscle. The aim is to balance the wrist and fingers to achieve better hand function. The position, function, and length at which the donor muscle is sutured is essential for the outcome for the procedure. In these studies the significance of the transferred muscle’s morphology, length and apillarization was investigated using both animal and human models. Immunohistochemical, biochemical, and laser diffraction techniques were used to examine muscle structure. In animal studies (rabbit), the effects of immobilization and of tendon transfers at different muscle lengths were analyzed. Immobilization of highly stretched muscles resulted in fibrosis and aberrant regeneration. A greater pull on the tendon while suturing a tendon transfer resulted in larger sarcomere lengths as measured in vivo. On examination of the number of sarcomeres per muscle fiber and the sarcomere lengths after 3 weeks of immobilization and healing time, we found a cut-off point up to which the sarcomerogenesis was optimal. Transfer at too long sarcomere lengths inhibited adaptation of the muscle to its new length, probably resulting in diminished function. In human studies we defined the sarcomere lengths of a normal human flexor carpi ulnaris muscle through the range of motion, and then again after a routinely performed tendon transfer to the finger extensor. A calculated model illustrated that after a transfer the largest force was predicted to occur with the wrist in extension. Morphological studies of spastic biceps brachii muscle showed, compared with control muscle, smaller fiber areas and higher variability in fiber size. Similar changes were also found in the more spastic wrist flexors comparing with wrist extensors in children with CP. In flexors, more type 2B fibers were found. These observations could all be due to the decreased use in the spastic limb, but might also represent a specific effect of the spasticity. In children and adults with spasticity very small fibers containing developmental myosin were present in all specimens, while none were found in controls. These fibers probably represent newly formed fibers originating from activated satellite cells. Impaired supraspinal control of active motion as well as of spinal reflexes, both typical of upper motor syndrome, could result in minor eccentric injuries of the muscle, causing activation of satellite cells. Spastic biceps muscles had fewer capillaries per cross-sectional area compared to age-matched controls, and also a smaller number of capillaries around each fiber. Nevertheless, the number of capillaries related to the specific fiber area was normal, and hence the spastic fibers are sufficiently supplied with capillaries. This study shows that the length of the muscle during tendon transfer is crucial for optimization of force output. Laser diffraction can be used for accurate measurement of sarcomere length during tendon transfer surgery. Wrist flexor muscles have more morphological alterations typical of spasticity compared to extensors.

Thesis advisor: Eric S. Folker
Thesis advisor: David R. Burgess
During muscle development, myonuclei undergo a complex set of movements that result in evenly spaced nuclei throughout the muscle cell. In many muscle diseases mispositioned myonuclei have been used as a hallmark phenotype of disease. A number of studies over the last decade have started to piece together the cytoskeletal elements that govern these movements. In Drosophila, two separate pools of Kinesin and Dynein work in synchrony to drive nuclear movement. However, it is still not clear how these two pools of microtubule motors become specified. In addition, it is not clear how nuclear position impacts the other defining feature of the muscle cell, which is the highly organized contractile network of sarcomeres. Previously, mispositioned myonuclei have been correlated with improper muscle function, yet no direct link between nuclear position and sarcomere development or function has been demonstrated. In this thesis, we show a role for Aplip1 (the Drosophila homolog of JIP1), a known regulator of both Kinesin and Dynein, in myonuclear positioning. Aplip1 localizes to the myotendinous junction and has genetically separable roles in myonuclear positioning and muscle stability. Furthermore, we show that a number of sarcomeric proteins, including ZASP, Actin and β-integrin localize to the nucleus prior to being incorporated into the sarcomere, regardless of nuclear position. Finally, we show that the LINC complex is required for nuclear dependent sarcomere assembly and that disruption of nuclear dependent sarcomere assembly or nuclear position resulted in a compromised sarcomeric network. Together, this thesis adds to the mechanisms that are important in positioning nuclei and shows the first direct link between the nucleus and sarcomere assembly
Thesis (PhD) — Boston College, 2018
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Proper contraction of striated muscle requires the assembly of actin filaments with precise spacing, polarity and lengths, however the mechanisms by which the cell accomplishes this remain unclear. In one model, the giant protein nebulin is proposed to function as a "molecular ruler" specifying the final lengths of the actin filaments. This dissertation focuses on determining the mechanisms by which nebulin regulates thin filament assembly. We found that nebulin physically interacts with CapZ, a protein that caps the barbed end of the actin filament within the Z-disc. Reduction of nebulin levels in chick skeletal myocytes via siRNA results in a reduction of assembled CapZ, and a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin-filament barbed ends to the Z-disc via a direct interaction with CapZ. Unexpectedly, the CapZ binding site was mapped to a site on nebulin that was previously predicted to localize outside of the Z-disc. Thus, we also propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres. To determine the mechanism by which nebulin regulates thin filament length and directly test the molecular ruler hypothesis, a unique small nebulin molecule ("mini-nebulin") was constructed. The introduction of mini-nebulin into chick skeletal myocytes, with endogenous nebulin knocked down, does not result in corresponding shorter actin filaments; an observation that is inconsistent with a strict ruler function. Treatment of these cells, however, with the actin depolymerizing agent Latrunculin A produces filaments that match the length of the mini-nebulin molecule, indicating mini-nebulin stabilizes the actin filaments. Furthermore, knockdown of nebulin results in more dynamic populations of the thin filament components actin, tropomyosin and tropomodulin. Strikingly, introduction of mini-nebulin is able to restore the normal stability of the actin filaments. Taken together, these data indicate that nebulin is responsible for proper actin organization within the Z-disc and contributes to actin filament length regulation by stabilizing the filament, preventing actin depolymerization.

Hypertrophic cardiomyopathy is a heart disease that is characterized by an enlarged heart muscle. Mutations to sarcomere proteins in the muscle fibers give rise to the disease, and this review aims to compile the mechanisms by which the mutations cause the disease phenotype. β-myosin heavy chain mutants affect the thick filament structure and contraction velocity of the muscle. Mutations to the myosin-binding protein C produces truncated proteins with decreased expression in the cells. Troponin T mutants cause myofibrillar disarray, alters affinity to α-tropomyosin, and are linked to a higher risk of sudden death. Troponin I is an unpredictable mutant that needs to be further researched but is thought to cause regulatory problems. Mutations to α-tropomyosin and the regulatory myosin light chain both affect the Ca2+-affinity of the proteins and leads to contractile problems. Hypercontractility as a result of the mutations seems to be the primary cause of the disease. Hypertrophic cardiomyopathy is linked to sudden death, and factors such as a family history of sudden death, multiple simultaneous mutations, unexplained syncope, non-sustained ventricular tachycardia, abnormal blood pressure response and extreme hypertrophy (>30 mm) heightens the risk of a sudden death. An increased knowledge about the disease will aid in the mission to better the treatments for the affected, but further investigation of pathological pathways needs to be performed.

The assembly, form, and function of the heart is regulated by complex mechanical signals originating from intrinsic and extrinsic sources, such as the cytoskeleton and the extracellular matrix. During development, mechanical forces influence the self-assembly of highly organized ventricular myocardium. However, mechanical overload induces maladaptive remodeling of tissue structure and eventual failure. Thus, mechanical forces potentiate physiological or pathological remodeling, depending on factors such as frequency and magnitude. We hypothesized that mechanical stimuli in the form of microenvironmental stiffness, cytoskeletal architecture, or cyclic stretch regulate cell-cell junction formation and cytoskeletal remodeling during development and disease. To test this, we engineered cardiac tissues in vitro and quantified structural and functional remodeling over multiple spatial scales in response to diverse mechanical perturbations mimicking development and disease. We first asked if the mechanical microenvironment impacts tissue assembly. To investigate this, we cultured two-cell cardiac µtissues on flexible substrates with tunable stiffness and monitored cell-cell junction formation over time. As myocytes transitioned from isolated cells to interconnected µtissues, focal adhesions disassembled near cell-cell interfaces and mechanical forces were transmitted almost completely through cell-cell junctions. However, µtissues cultured on stiff substrates mimicking fibrotic microenvironments retained focal adhesions near the cell-cell interface, potentially to reinforce the cell-cell junction in response to excessive forces generated by myofibrils in stiff microenvironments. Intercellular electrical conductance between myocytes was measured as a function of connexin 43 immunosignal and the length-to-width ratio of cell pairs. We observed that conductance was correlated to connexin 43 immunosignal and cell pair length-to-width ratio, indicating that tissue architecture can affect electrical coupling. The impact of mechanical overload was also determined by applying chronic cyclic stretch to engineered cardiac tissues. Stretch activated gene expression patterns characteristic of pathological remodeling, including up-regulation of focal adhesion genes, and impacted sarcomere alignment and myocyte shape. Furthermore, chronic cyclic stretch altered intracellular calcium cycling in a manner similar to heart failure and decreased contractile stress generation, suggestive of maladaptive remodeling. In summary, we show that the assembly, form, and function of cardiac tissue is sensitive to a wide range of mechanical cues that emerge during physiological and pathological growth.
Engineering and Applied Sciences

A literatura refere que os estímulos produzidos pelos diferentes tipos de contração no sistema músculo-esquelético geram adaptações específicas. A locomoção em aclive e declive vem sendo utilizada como modelo de treinamento concêntrico e excêntrico para estudos dessas adaptações em animais. Sendo assim este estudo teve o objetivo de avaliar características mecânicas e histológicas do músculo sóleo de ratos submetidos a treinamento em aclive ou declive. Foram avaliados 36 ratos Wistar machos (90 dias de idade no início do treinamento) divididos igualmente em três grupos: aclive (A), declive (D) e controle (C). Os treinamentos foram realizados em uma esteira adaptada com raias individuais e inclinação de + 16° (aclive) ou – 16° (declive). Os animais passaram por um período de adaptação de uma semana na esteira com a inclinação específica e, em seguida, foram avaliados quanto à velocidade máxima suportada. Os treinamentos aconteceram com velocidade (relativa à máxima avaliada) e tempo progressivos ao longo de oito semanas (29 sessões). Dois dias após o último treino os animais foram anestesiados com Tiopental Sódico (± 5 ml), os músculos sóleos direitos e esquerdos foram removidos e os animais decapitados. Os músculos sóleos da pata direita (n = 7C, 8A, 9D) foram submetidos a ensaios de tração em uma máquina EMIC DL2000, com célula de carga de 50 N a velocidade de 1,66 m·s-1. Durante o ensaio, as amostras foram borrifadas com solução salina a cada um minuto. Os músculos sóleos esquerdos foram retirados para contagem de sarcômeros (n = 4A, 4C e 5D) e análise na proteína titina por Western Blot (n = 4 por grupo). Foi verificada a normalidade e homogeneidade dos dados e a confiabilidade das medidas foi avaliada pelo coeficiente Alpha de Cronbach. Comportamentos individuais foram analisados pelo Coeficiente de Correlação Intraclasse (ICC); ANOVA one way e post hoc de Bonferroni e teste não paramétrico Kruskal-Walls foram aplicados para identificação das diferenças entre os grupos. Dos 36 animais, três morreram antes do final do experimento. Os coeficientes Alpha de Cronbach indicaram confiabilidade das medidas com valores próximos a um para todas as variáveis. Os dados mecânicos dos animais que treinaram em declive apresentaram menores valores de ICC, mesmo assim significativos. As comparações entre os grupos indicaram aumento significativo da rigidez da curva tensão X deformação para os animais treinados em aclive e declive, e maior deformação da curva para os animais controle. Foi encontrado aumento da média da tensão passiva dos músculos dos animais treinados (57 ± 5 e 56 ± 11 para A e D; 51,6 ± 7,7 para C), contudo essa diferença não foi estatisticamente significativa, possivelmente devido a grande variabilidade dos dados mecânicos. Em relação ao número de sarcômeros em série os animais que treinaram em declive apresentaram menores comprimentos de sarcômero (2,806 ± 0,059 μm) e maior número de sarcômeros em série (8170 ± 510) quando comparados aos grupos controle (3,06 ± 0,054 μm e 7510 ± 240) e aclive (2,990 ± 0,023 μm e 7390 ± 270). As demais variáveis analisadas não apresentaram diferenças entre os grupos. É possível concluir com este estudo que adaptações estruturais nem sempre acontecem em paralelo às adaptações funcionais, que o treinamento em declive parece produzir resultados com maiores variações e que os diferentes estímulos provocam diferentes adaptações no músculo sóleo de ratos.
The literature states that the stimulus produced by different types of contraction in skeletal muscle generates specific adaptations. Downhill and uphill locomotion have been used as a model of concentric and eccentric training in studies that verify the different adaptations in animals. Therefore this study intended to evaluate mechanical and histological properties of rats’ soleus muscle submitted to uphill or downhill running. We evaluated 36 male Wistar rats (90 days old at the start of training). They were divided equally into three groups: uphill (A), downhill (D) and control (C). Training was performed on a treadmill adapted to individual lanes and +16º (uphill) or -16º (downhill) incline. The animals went through a one week adjustment period on the treadmill with the specific inclines, and then there were evaluated the maximal speeds they reached. The training took place with progressive speed (relative to the maximum tested) and time over 8 weeks (29 sessions). Two days after the last training the animals were anesthetized with sodium thiopental (± 5 ml), the right and left soleus muscles were removed and the animals were decapitated. The right soleus muscles (n = 7C, 8A, 9D) were subjected to tensile tests on one machine EMIC DL2000, with a load cell of 50 N, speed of 1,66 m.s-1. During the test, the samples were sprayed with saline solution every one minute. The left soleus muscles were removed for counting of sarcomeres (n = 4A, 4C, 5D) and the titin Western blot analysis (n = 4 per group). Data normality and homogeneity were verified and reliability of the measures was assessed by Cronbach's Alpha coefficient. Individual behaviors were analyzed by Intraclass Correlation Coefficient (ICC); one-way ANOVA and post hoc Bonferroni test and nonparametric Kruskal-Wallis were applied to identify the differences between the groups. Three out of the 36 animals died before the end of the experiment. The Cronbach's Alpha coefficients indicated reliability of the measures with values close to one for all the analyzed variables. The mechanical data of downhill trained animals had significantly lower ICC, even though significant. Comparisons between groups showed increased rigidity of the muscles of trained animals and greater deformation of the muscles of control animals. It has been found greater passive tension mean for both training groups (57 ± 5 and 56 ± 11 for A and D; 51.6 ± 7.7 for C), but it was not statistically significant probably due to the high mechanical data variability. Regarding the number of sarcomeres in series the downhill trained animals showed smaller sarcomere lengths (2.806 ± 0.059 μm) and greater serial sarcomere number (8170 ± 510) when compared to control (3.06 ± 0.054 μm and 7510 ± 240) and uphill (2.990 ± 0.023 μm e 7390 ± 270) groups. The remaining variables did not differ between groups. It can be concluded from this study that structural adaptations do not happen in parallel with functional adaptations, downhill running appear to produce more variable results and that different stimuli generate different adaptations in rats’ soleus muscle.

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Left ventricular hypertrophy (LVH) is a major risk factor for cardiovascular morbidity and mortality, and is a feature of common diseases, such as hypertension and diabetes. It is therefore vital to understand the underlying mechanisms influencing its development. However, investigating the mechanisms underlying LVH in such complex disorders can be challenging. For this reason, many researchers have focused their attention on the autosomal dominant cardiac muscle disorder, hypertrophic cardiomyopathy (HCM), since it is considered a model disease in which to study the causal molecular factors underlying isolated cardiac hypertrophy. HCM is a heterogeneous disease that manifests with various phenotypes and clinical symptoms, even in families with the same genetic defects, suggesting that additional factors contribute to the disease phenotype. Despite the identification of several HCM-causing genes, the genetic factors that modify the extent of hypertrophy in HCM patients remain relatively unknown. The gene encoding the sarcomeric protein, cardiac myosin binding protein C, cMyBPC (MyBPC3) is one of the most frequently implicated genes in HCM. Identification of proteins that interact with cMyBPC has led to improved insights into the function of this protein and its role in cardiac hypertrophy. However, very little is known about another member of the myosin binding protein family, myosin binding protein H (MyBPH). Given the sequence homology and similarity in structure between cMyBPC and MyBPH, we propose that MyBPH, like cMyBPC, may play a critical role in the structure and functionality of the cardiac sarcomere and could therefore be involved in HCM pathogenesis. The present study aimed to identify MyBPH-interacting proteins by using yeast two-hybrid (Y2H) analysis and to verify these interactions using three-dimensional (3D) co-localisation and co-immunoprecipitation (Co-IP) analyses. We further hypothesized that both MyBPH and cMyBPC may be involved in autophagy. To test this hypothesis, both MyBPH and cMyBPC were analysed for co-localisation with a marker for autophagy, LC3b-II. The role of MyBPH and cMyBPC in cardiac cell contractility were analysed by measuring the planar cell surface area of differentiated H9c2 rat cardiomyocytes in response to β-adrenergic stress after individual and concurrent siRNA-mediated knockdown of MyBPH and cMyBPC. In the present study we employed a family-based genetic association analysis approach to investigate the contribution of genes encoding the novel MyBPH-interacting proteins in modifying the hypertrophy phenotype. This study investigated the hypertrophy modifying effects of 38 SNPs and haplotypes in four candidate HCM modifier genes, in 388 individuals from 27 HCM families, in which three unique South African HCM-causing founder mutations segregate. Yeast two-hybrid analysis identified three putative MyBPH-interacting proteins namely, cardiac β-myosin heavy chain (MYH7), cardiac α-actin (ACTC1) and the SUMO-conjugating enzyme UBC9 (UBC9). These interactions were verified using both 3D co-localisation and Co-IP analyses. Furthermore, MyBPH and cMyBPC were implicated in autophagy, since both these proteins were being recruited to the membrane of autophagosomes. In addition, a cardiac contractility assay demonstrated that the concurrent siRNA-mediated knockdown of MyBPH and cMyBPC resulted in a significant reduction in cardiomyocyte contractility, compared to individual protein and control knockdowns under conditions of β-adrenergic stress. These results indicated that MyBPH could compensate for cMyBPC, and vice versa, further confirming that both these proteins are required for efficient sarcomere contraction. Results from genetic association analyses found a number of SNPs and haplotypes that had a significant effect on HCM hypertrophy. Single SNP and haplotype analyses identified SNPs and haplotypes within genes encoding MyBPH, MYH7, ACTC1 and UBC9, which contribute to the extent of hypertrophy in HCM. In addition, we found that several variants and haplotypes had markedly different statistical significant effects in the presence of each of the three HCM founder mutations. The results of this study ascribe novel functions to MyBPH. Cardiac MyBPC and MyBPH play a critical role in sarcomere contraction and have been implicated in autophagy. This has further implications for understanding the patho-etiology of HCM-causing mutations in the gene encoding MyBPH and its interacting proteins. This is to our knowledge the first genetic association analysis to investigate the modifying effect of interactors of MyBPH, as indication of the risk for developing LVH in the context of HCM. Our findings suggest that the hypertrophic phenotype of HCM is modulated by the compound effect of a number of variants and haplotypes in MyBPH, and genes encoding protein interactors of MyBPH. These results provide a basis for future studies to investigate the risk profile of hypertrophy development in the context of HCM, which could consequently lead to improved risk stratification and patient management.
AFRIKAANSE OPSOMMING: Linker ventrikulêre hipertrofie (LVH) is 'n primêre risikofaktor vir kardiovaskulêre morbiditeit en mortaliteit asook 'n kenmerk van algemene siektes soos hipertensie en diabetes. Daarom is dit van kardinale belang om te verstaan wat die onderliggende meganismes is wat die ontwikkeling van LVH beïnvloed. Die ondersoek na die onderliggende meganismes wat lei tot LVH in sulke komplekse siektes is ‟n uitdaging. Om hierdie rede fokus baie navorsers hul aandag op die autosomaal dominante hartspier siekte, hipertrofiese kardiomiopatie (HKM), wat beskou word as 'n model siekte om die molekulêre oorsake onderliggend tot geïsoleerde kardiovaskulêre hipertrofie te ondersoek. HKM is 'n heterogene siekte wat manifesteer met verskeie fenotipes en kliniese simptome, selfs in families met dieselfde genetiese defekte, wat impliseer dat addisionele faktore bydra tot die modifisering van die siekte fenotipe. Ten spyte van die identifisering van verskeie HKM-versoorsakende gene, bly die genetiese faktore wat die mate van hipertrofie in HKM pasiente modifiseer relatief onbekend. Die geen wat kodeer vir die sarkomeriese proteïen, kardiale miosien-bindingsproteïen C (kMyBPC) is die algemeenste betrokke in HKM. Die identifisering van proteïene wat bind met kMyBPC het gelei tot verbeterde insigte tot die funksie van hierdie proteïen en die rol wat hierdie proteïen in hipertrofie speel. Ten spyte hiervan, is daar baie min inligting beskikbaar oor 'n ander lid van die miosien-bindingsproteïen families, miosien-bindingsproteïen H (MyBPH). Gegewe die ooreenstemming tussen die DNA basispaar-volgorde en struktuur tussen hierdie twee proteïene, stel ons voor dat MyBPH, net soos kMyBPC, 'n kritiese rol in die struktuur en funksie van die kardiale sarkomeer speel en kan daarom betrokke wees in die patogenese van HKM. Die huidige studie het beoog om proteïene wat met MyBPH bind te identifiseer deur die gebruik van gis-twee-hibried (G2H) kardiale biblioteek sifting en om hierdie interaksies te verifieer met behulp van drie-dimensionele (3D) ko-lokalisering en ko-immunopresipitasie eksperimente. Ons het verder gehipotiseer dat beide MyBPH and kMyBPC betrokke kan wees in outofagie. Om hierdie hipotese te toets is beide MyBPH en kMyBPC geanaliseer vir ko-lokalisering met 'n merker vir outofagie, LC3b-II. Verder het ons beplan om die rol van MyBPH en kMyBPC in kardiale spiersel-sametrekking te ondersoek deur die oppervlak van gedifferensieerde H9c2 rot kardiomiosiete in reaksie op β-adrenergiese stres te meet, na individuele en gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC. In hierdie studie het ons 'n familie-gebaseerde genetiese assosiasie analise benadering gevolg om vas te stel of MyBPH en gene wat kodeer vir die geverifieerde bindingsgenote van MyBPH bydra tot die modifisering van die hipertrofiese fenotipe. Die doel van hierdie studie was om die hipertrofiese effek van 38 enkel nukleotied polimorfismes (SNPs) en haplotipes in vier kandidaat HKM modifiserende gene in 388 individue van 27 HCM families te toets, waarin drie unieke Suid-Afrikaanse HKM-stigters mutasies segregeer. G2H analise het drie verneemde MyBPH bindingsgenote geidentifiseer, naamlik miosien (MYH7), alfa kardiale aktien (ACTC1) en die SUMO-konjugerende ensiem UBC9 (UBC9). Hierdie interaksies is geverifieer deur middel van 3D ko-lokalisering en ko-immunopresipitasie analises. Verder is bewys dat MyBPH en kMyBPC betrokke is in outofagie, siende dat beide proteïene gewerf is tot die membraan van die outofagosoom. 'n Kardiale sametrekkings eksperiment het gevind dat die gesamentlike siRNA-bemiddelde uitklopping van MyBPH en kMyBPC 'n merkwaardige vermindering in die kardiomiosiet sametrekking veroorsaak het in reaksie op β-adrenergiese stres kondisies, in vergelyking met die individuele proteïen en kontrole uitkloppings eksperimente. Hierdie resultate bevestig dat MyBPH vir kMyBPC kan instaan en ook andersom, wat verder bevestig dat beide proteïene benodig word vir effektiewe sarkomeer sametrekking. Resultate van die genetiese assosiasie studie het gevind dat 'n aantal SNPs en haplotipes 'n beduidende effek of HKM hipertrofie het. Enkel SNP en haplotipe analises in gene wat kodeer vir MyBPH, MYH7, ACTC1 en UBC9 het SNPs en haplotipes geidentifiseer wat bydra tot die omvang van hipertrofie in HKM. Verder het ons gevind dat sekere SNPs en haplotipes kenmerkend verskillende statisties beduidende effekte in die teenwoordigheid van elk van die drie HKM-stigter mutasies gehad het. Die resultate van hierdie studie skryf twee nuwe funksies aan MyBPH toe. Kardiale MyBPC en MyBPH speel 'n kritiese rol in sarkomeer sametrekking en is betrokke in outofagie. Hierdie resultate het verdere implikasies vir die verstaan van die pato-etiologie van die HKM-veroorsakende mutasies in die MyBPH, MYH7, ACTC1 en UBC9 gene. So vêr dit ons kennis strek is dit die eerste genetiese assosiasie studie wat die modifiserende effek van bindingsgenote van MyBPH ondersoek as risiko aanduiding vir die ontwikkeling van LVH in die konteks van HKM. Ons bevindinge bewys dat die hipertrofiese fenotipe van HKM gemoduleer word deur die komplekse effekte van SNPs en haplotipes in die MyBPH geen en gene wat MyBPH proteïen-bindingsgenote enkodeer. Hierdie resultate verskaf dus 'n basis vir toekomstige studies om die risiko profiel van hipertrofie ontwikkeling met betrekking tot HKM te ondersoek, wat gevolglik kan bydra tot die verbeterde risiko stratifikasie en pasiënte bestuur.

The aim of this work is to define the function of I-band titin as a dynamic element in parallel whit the array of myosin motors using fast half-sarcomere level mechanics on intact fibres of frog muscle in the presence of 20 uM para-nitro-blebbistatin (PNB)

While atrial fibrillation (AF) is common and has serious consequences, a lot is yet unknown about the causative factors underlying this arrhythmia. The role of genetics in the development of AF has become more evident in the past decade. Family history is now a firmly established risk factor and many common and rare sequence variants linked to AF have been identified. Genome-wide association studies have identified common sequence variants that associate with AF, including variants on chromosomes 4q25, 16q22, and 1q22. Nevertheless, it has become apparent that despite these findings, a substantial fraction of heritability of most complex traits remained unaccounted for. This raises the possibility that development of AF is determined by the combination of common and rare susceptibility variants. Whole genome sequencing is the most comprehensive method to analyse individual genetic variation. A paradigm shift from microarray-based genotyping studies to whole exome and whole genome sequencing is ongoing. Whole genome sequencing studies have shown mutations in myosin genes may be associated with AF, implying that variants encoding sarcomere genes may be involved in the development of this arrhythmia. While some of the sequence variants discovered suggest novel mechanisms in the pathophysiology of this complex arrhythmia, much work is still needed to fully understand the mechanisms linking many of these loci to AF. Likewise, the current clinical applicability of this information is still unclear. However, further developments in this field are expected to add to our understanding of this complex arrhythmia and hopefully lead to new therapeutic possibilities.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder of cardiac myocytes that is characterized by cardiac hypertrophy, unexplained by the loading conditions, a non-dilated left ventricle and a normal or increased left ventricular ejection fraction (LV-EF). Prevalence of HCM has been estimated at 0.16% to 0.29% (≈ 1:625–1:344 individuals) in the general adult population. HCM represents the most common genetic heart disease and represent an archetypical single gene disorder with an autosomal dominant pattern of inheritance and historically termed a “disease of the sarcomere”. The precise mechanisms by which sarcomere variants result in the clinical phenotype have not been fully understood. Mutant sarcomere genes trigger several myocardial changes, leading to hypertrophy and fibrosis, which ultimately result in a small, stiff ventricle with impaired systolic and diastolic performance despite a preserved LV-EF. The most common differential diagnosis challenges in the presence of hypertrophic heart disease are represented by: athlete’s heart, hypertensive heart and other cardiomyopathies mimicking HCM. A multimodality approach using ECG, echocardiography, CMR, cardiac computed tomography (CCT) and cardiac nuclear imaging provides unique information about diagnosis, staging and clinical profiles, anatomical and functional assessment, metabolic evaluation, monitoring of treatment, follow-up, prognosis and risk stratification, as well as preclinical screening and differential diagnosis. HCM may be associated with a normal life expectancy and a very stable clinical course. However, about a third of patients develop heart failure (HF); in addition, 5–15% of cases show progression to either the restrictive or the dilated hypokinetic evolution of HCM, both of which may require evaluation for cardiac transplantation. The clinical course of HCM has been classified into four clinical stages: non-hypertrophic, classic, adverse remodeling and overt dysfunction phenotype. No evidence-based treatments are available for non-hypertrophic HCM patients (pre-hypertrophic stage), on the other hand in classic HCM, adverse remodeling and overt dysfunction phenotype, pharmacological or interventional strategies have the target to improve functional capacity, reduce symptoms, prevent disease progression. Therapeutic approach mainly differs on the basis of the presence or absence of significant obstructive HCM. Adult patients with HCM report an annual incidence for cardiovascular death of 1–2%, with sudden cardiac death (SCD), HF and thromboembolism being the main causes of death; the most commonly recorded fatal arrhythmic event is spontaneous ventricular fibrillation. For this reason, SCD risk estimation is an integral part of clinical management of HCM. International guidelines suggest the evaluation of several risk factor for SCD based on personal and family history, non-invasive testing including echocardiography, ambulatory electrocardiographic 24 hours monitoring and CMR imaging in order to identity those patients most likely to benefit implantable cardioverter-defibrillator (ICD) implantation. The present chapter summarize genetics, pathogenesis, diagnosis, clinical course and therapy of HCM as well as novel therapeutic options.

Actin stress fibers (SFs), bundles of actin filaments crosslinked by α-actinin and myosin II in non-muscle cells, are mechanosensitive structural elements that respond to applied stress and strain to regulate cell morphology, signal transduction and cell function. Results from various studies indicate that myosin-generated contraction extends SFs beyond their unloaded lengths and cells maintain fiber strain at an optimal level that depends on actomyosin activity (Lu et al., 2008). Stretching the matrix upon which cells adhere perturbs the cell-matrix traction forces and cells respond by actively re-establishing the preexisting level of force (Brown et al., 1998; Gavara et al., 2008). We have developed a sarcomeric model of SF networks (Kaunas et al., 2011) to predict the effects of stretch on SF reorganization depending on the rates of matrix stretching, SF turnover, and SF stress relaxation.

Tropomodulin is an actin-capping protein in cardiac muscle, and is associated with both sarcomeric and cytoskeletal actin filaments. Homozygous knockout of erythrocyte tropomodulin (E-Tmod) is embryonically lethal, but heterozygous knockout (+/-) mice survive. Heterozygous E-Tmod knockout resulted in smaller right ventricle (RV) cavities and free walls compared to wild type. To investigate the effect of heterozygous tropomodulin knockout on mouse cardiac function and remodeling, mice (n=6 to 9) were subjected to 5 weeks of hypoxia to increase loading conditions on the RV via pulmonary hypertension. The effect of loading was determined by measuring the volume of RV anatomical features, and surface strain during the cardiac cycle. Although there was no significant change due to loading on RV cavity or free wall volume for wild type, geometrical measurements suggest that tissue had been redistributed. Under equal loading conditions, knockout mice exhibited a significant increase in volume for both RV features. RV epicardial function showed an increase in surface area strain at peak systole for hypoxic knockout mice. Thus it appears that heterozygous knockout of E-Tmod affects RV volume under normal and adverse loading conditions, as well as RV function.

The remarkable energetic versatility and adaptability of skeletal muscle provides great inspiration to develop advanced adaptive structures and materials. These notable properties may arise from the assembly of skeletal muscle’s nanoscale cross-bridges into microscale structures known as sarcomeres. Essential understanding of muscle energetics has been developed from models of the micro/nanoscale constituents which incorporate an intriguing, asymmetric, bistable potential energy landscape to capture trends in the experimental data on cross-bridge power stroke motions. Inspired by the capability of cross-bridges to strategically absorb and store elastic energy for achieving high energetic efficiency of sudden motions, this research studies the assembly of modular mechanical structures from asymmetrically bistable constituents to absorb and trap energy in higher-energy stable configurations. Specifically, energy features of a module with an asymmetrically bistable element are first studied; these modules are then assembled in series and parallel to generate structures with multiple stable configurations having different quantities of stored energy. Dynamic analyses are performed to illustrate the ability of these structures to trap a portion of the kinetic energy due to an impulsive excitation as recoverable and reusable strain energy, and insight is gained into how asymmetries and damping influence the energy trapping performance.