Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20(Delta RRm)) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20(Delta RRm) animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20(Delta RRm) on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca2+-activated, skinned papillary muscle strips from Rbrn20<^>R'Rm and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca2+-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20(Delta RRm) vs. WT mice. As sarcomere length increased from 1.9 to 2.2 mu m, length-dependent activation of contraction was eliminated in the Rbrn20<^>R'Rm myocardium, even though myosin MgADP release rate decreased similar to 20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.

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Missense single-nucleotide polymorphisms (mSNPs) in titin are emerging as a main causative factor of heart failure. However, distinguishing between benign and disease-causing mSNPs is a substantial challenge. Here, we research the question of whether a single mSNP in a generic domain of titin can affect heart function as a whole and, if so, how. For this, we studied the mSNP T2850I, seemingly linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). We used structural biology, computational simulations and transgenic muscle in vivo methods to track the effect of the mutation from the molecular to the organismal level. The data show that the T2850I exchange is compatible with the domain three-dimensional fold, but that it strongly destabilizes it. Further, it induces a change in the conformational dynamics of the titin chain that alters its reactivity, causing the formation of aberrant interactions in the sarcomere. Echocardiography of knock-in mice indicated a mild diastolic dysfunction arising from increased myocardial stiffness. In conclusion, our data provide evidence that single mSNPs in titin's I-band can alter overall muscle behaviour. Our suggested mechanisms of disease are the development of non-native sarcomeric interactions and titin instability leading to a reduced I-band compliance. However, understanding the T2850I-induced ARVC pathology mechanistically remains a complex problem and will require a deeper understanding of the sarcomeric context of the titin region affected.

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Thèse réalisée en cotutelle avec l'Université Pierre et Marie Curie, Paris 6(UPMC, Paris, France). / Bien que les bases physiopathologiques de beaucoup de maladies musculaires soient dorénavant connues, les myopathies congénitales à cores (MCs), maladies génétiques qui se présentent dès la naissance avec un retard du développement moteur, une faiblesse musculaire et des complications respiratoires et/ou cardiaques parfois mortelles, demeurent mal comprises. Des mutations dans RYR1, SEPN1, TTN, ACTA1, CFL2 et MEGF10 ont été associées aux MCs, pourtant, dans plus de 50% des cas, le gène responsable reste à identifier. L’objectif de ma thèse a été de clarifier les mécanismes physiopathologiques des MCs par l’identification de nouveaux gènes ou de nouvelles mutations. Cette thèse a eu une dimension internationale concrétisée par la mise en place d’une cotutelle UPMC (France) et UdeM (Québec). J’ai développé deux axes de recherche complémentaires. D’une part j’ai étudié 21 familles informatives avec MC récessive, scoliose et atteinte respiratoire, en combinant clonage positionnel et étude de gènes candidats et en utilisant des outils variés allant du génotypage au séquençage de nouvelle génération (NGS). En parallèle, j’ai étudié 24 familles avec une MC autosomique récessive affectant les muscles cardiaque et squelettiques et dont le phénotype était semblable à celui observé chez des patients avec des délétions dans les 6 derniers exons de TTN. Ainsi pour l'analyse de cette deuxième cohorte, nous avons appliqué une stratégie de séquençage de gène candidat ciblée sur ces exons et de NGS pour le reste du gène. Pendant mon doctorat j'ai identifié les défauts génétiques de 8 des 45 familles étudiées (18 %), et caractérisé 3 nouvelles entités médicales, dont deux MCs dues à des nouvelles mutations de TTN. Ces résultats ont servi à l’identification de nouvelles interactions protéiques de la titine et contribuent à définir TTN comme une cause majeure de pathologies musculaires cardiaques et/ou squelettiques. Une troisième nouvelle forme de MC est provoquée par une mutation d'un coactivateur transcriptionnel peu connu et jamais associé à une maladie. Ces résultats ont révélé un nouvel acteur clef et une nouvelle voie de signalisation dans la physiopathologie du muscle, ont eu un bénéfice direct en termes de conseil génétique et ouvrent la voie pour le développement de thérapies. / While the pathophysiological bases of many muscular diseases are nowadays well known, congenital core myopathies (CMs) remain poorly understood. CMs are genetic diseases which generally present at birth with delayed motor development, muscle weakness, and sometimes fatal respiratory or cardiological complications. Mutations in RYR1, SEPN1, ACTA1, TTN and MEGF10 have been associated with various CMs, yet for about 50% of CM cases the responsible gene has not been identified. The objective of my thesis was to clarify the pathophysiological mechanisms of new forms of CM through the identification of new genes or new mutations in known genes. This thesis had an international dimension as manifested by a UPMC (France) and UdeM (Québec) joint direction. I developed two complementary axes of research. First, I studied 21 informative families with a recessive CM with scoliosis and respiratory failure, for which I combined positional cloning and candidate gene studies, using various tools from genotyping to next generation sequencing (NGS). The second part of this work consisted on the analysis of 24 families with recessive CM affecting both cardiac and skeletal muscles. Their phenotype was similar to that previously observed in cases with deletions in the last 6 exons of the giant gene TTN. Thus we applied a candidate gene strategy through direct Sanger sequencing coupled with NGS for the analysis of this second cohort. During my PhD work I identified the molecular defect in 8 out of the 45 families included (18%), which led to the identification and characterization of 3 novel medical entities, including two new CMs due to novel defects of TTN. These results served to identify new titin protein interactions, and participate in the definition of TTN defects as a major cause of both cardiac and skeletal muscle conditions. A third new form of CM is due to mutations of a poorly-known transcriptional coactivator whose role in striated muscle physiology was unknown and which had never been associated to a human condition. Globally, these results unveiled a novel important protein and pathway in muscle pathophysiology, have direct health benefits (molecular diagnosis) and open the way for therapeutic investigations.

The aim of this study was to investigate the mode of action of pavetamine on rat cardiomyocytes. Pavetamine is the causative agent of gousiekte (“quick-disease”), a disease of ruminants characterized by acute heart failure following ingestion of certain rubiaceous plants. Two in vitro rat cardiomyocyte models were utilized in this study, namely the rat embryonic cardiac cell line, H9c2, and primary neonatal rat cardiomyocytes. Cytotoxicity of pavetamine was evaluated in H9c2 cells using the MTT and LDH release assays. The eventual cell death of H9c2 cells was due to necrosis, with LDH release into the culture medium after exposure to pavetamine for 72 h. Pavetamine did not induce apoptosis, as the typical features of apoptosis were not observed. Electron microscopy was employed to study ultrastructural alterations caused by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormalities after 48 h exposure of the cells to pavetamine. Abundant secondary lysosomes with electron dense material were present in treated cells. Numerous vacuoles were also present in treated cells, indicative of autophagy. During this exposure time, the nuclei appeared normal, with no chromatin condensation as would be expected for apoptosis. Abnormalities in the morphology of the nuclei were only evident after 72 h exposure. The nuclei became fragmented and plasma membrane blebbing occurred. The mitochondrial membrane potential was investigated with a fluorescent probe, which demonstrated that pavetamine caused significant hyperpolarization of the mitochondrial membrane, in contrast to the depolarization caused by apoptotic inducers. Pavetamine did not cause opening of the mitochondrial permeability transition pore, because cyclosporine A, which is an inhibitor of the mitochondrial permeability transition pore, did not reduce the cytotoxicity of pavetamine significantly. Fluorescent probes were used to investigate subcellular changes induced by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormal features compared to the control cells, which is consistent with the electron microscopy studies. The lysosomes of treated cells were more abundant and enlarged. The activity of cytosolic hexosaminidase was nearly three times higher in the treated cells than in the control cells, which suggested increased lysosomal membrane permeability. The activity of acid phosphatase was also increased in comparison to the control cells. In addition, the organization of the cytoskeletal F-actin of treated cells was severely affected by pavetamine. Rat neonatal cardiomyocytes were labelled with antibodies to detect the three major contractile proteins (titin, actin and myosin) and cytoskeletal proteins (F-actin, desmin and β-tubulin). Cells treated with pavetamine had degraded myosin and titin, with altered morphology of sarcomeric actin. Vacuoles appeared in the β-tubulin network, but the appearance of desmin was normal. F-actin was severely disrupted in cardiomyocytes treated with pavetamine and was degraded or even absent in treated cells. Ultrastructurally, the sarcomeres of rat neonatal cardiomyocytes exposed to pavetamine were disorganized and disengaged from the Z-lines, which can also be observed in the hearts of ruminants that have died of gousiekte. It is concluded that the pathological alteration to the major contractile and cytoskeleton proteins caused by pavetamine could explain the cardiac dysfunction that characterizes gousiekte. F-actin is involved in protein synthesis and therefore can play a role in the inhibition of protein synthesis in the myocardium of ruminants suffering from gousiekte. Apart from inhibition of protein synthesis in the heart, there is also increased degradation of cardiac proteins in an animal with gousiekte. The mitochondrial damage will lead to an energy deficiency and possibly to generation of reactive oxygen species. The sarcoplasmic reticula are involved in protein synthesis and any damage to them will affect protein synthesis, folding and post-translational modifications. This will activate the unfolded protein response (UPR) and sarcoplasmic reticula-associated protein degradation (ERAD). If the oxidizing environment of the sarcoplasmic reticula is disturbed, it will activate the ubiquitin-proteasome pathway (UPP) to clear aggregated and misfolded proteins. Lastly, the mitochondria, sarcoplasmic reticula and F-actin are involved in calcium homeostasis. Any damage to these organelles will have a profound influence on calcium flux in the heart and will further contribute to the contractile dysfunction that characterizes gousiekte. / Thesis (PhD)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted