L’insuffisance cardiaque (IC) est une cause majeure de mortalité dans les pays industrialisés. Ce syndrome est le résultat de nombreuses maladies cardiaques qui induisent dans un premier temps un remodelage adaptatif du myocarde : l’hypertrophie du ventricule gauche (HVG). Dans le cœur, le calcium libéré à partir du réticulum sarcoplasmique (RS) est à l’origine de la contractilité. Ce mécanisme est contrôlé par un macro-complexe moléculaire, composé du récepteur de la ryanodine (RyR2), et de protéines stabilisatrices associées dont la junctine (JCN), la calséquestrine (CSQ2), et la triadine (Trd). Ces dernières années, des dysfonctionnements de ce complexe, par des relâchements aberrants de Ca2+ du RS (vu comme des fuites de Ca2+ hors du RS) ont été remarqué au cours de l’IC, conduisant à une HVG associée à une dysfonction contractile et à la survenue d’arythmies cardiaques létales. De très nombreuses études se sont intéressées aux protéines principales du RS, telles que RyR2 et CSQ2, mais peu de données sont disponibles sur le rôle de Trd, protéine considérée comme mineure en physiopathologie cardiaque. Afin d’étudier son rôle dans le cœur, notre travail s’est articulé autour de trois modèles de pathologie cardiaque : 1-une surcharge de pression par une sténose de l’aorte transverse (TAC), 2-une diffusion de catécholamines (isoprotérénol, Iso) par mini-pompe osmotique et 3-une IC chronique par un infarctus du myocarde (IM), chez des souris dont le gène de la triadine a été invalidé (KO Trd). En réponse à une TAC ou à l’ISO, les animaux développent une HVG plus importante que les souris WT. Suite à une TAC, cette HVG est supérieure et excentrique et s’accompagne d’une dysfonction cardiaque comparativement aux animaux sauvages. Suite à un IM, les souris KO Trd présentent une mortalité accrue post-chirurgie. L’accroissement de cette mortalité accrue résiderait dans l’augmentation significative d’arythmies ventriculaires sévères (tachycardies ventriculaires, TV) chez ces souris suite à une stimulation catécholaminergique, pouvant être la conséquence d’une augmentation des fuites de Ca2+ hors du RS. Nous avons également observé qu’en réponse à la TAC la réexpression du gène TRDN avec un adénovirus AAV9 dans notre modèle KO Trd permet le maintien de la fonction cardiaque et de prévenir le développement de l’HVG. Au final, ces travaux montrent que l’absence de la triadine accélère la transition vers l’IC en modulant à la fois l’HVG et la dysfonction contractile associée mais également la survenue d’arythmies ventriculaires létales. / Heart failure (HF) is a serious public health issue with a growing prevalence in industrialized countries. This syndrome results from several cardiac diseases which begin with an adaptative myocardial remodeling: left ventricular hypertrophy (LVH). In heart, contractility depends on calcium release from sarcoplasmic reticulum (SR). This release is controlled by a macro-molecular complex, composed by ryanodine receptor (RyR2) and its associated regulatory protein junctin (JCN), calsequestrin (CSQ2) and triadin (Trd). During the past years, alterations of this complex by disturbed calcium release outside SR (as « sparks ») was often observed during the development of HF, being associated with LVH, dysfunction and fatal ventricular arrhythmias. Most studies were focused on RyR2 and CSQ2 function but few data are available regarding the role of Trd, considered until now having minor role in cardiac physiopathology. To elucidate its role, we realized 3 cardiac pathological experimental models on mice with triadin gene invalidation (KO Trd): 1- a pressure overload with transversal aorta constriction (TAC) 2-a chronic infusion of catecholamines (Isoproterenol, Iso) with osmotic minipumps and 3- a chronic HF with myocardial infarction (MI). In response to TAC or Iso, KO mice developed a greater LVH compared to wild-type mice. Also, with TAC, KO mice show an eccentric LVH associated with a severe cardiac dysfunction, as compared to wild-type mice. After MI, we observed a greater mortality post-surgery in KO Trd mice. This prevalence may be due to increasing of severe ventricular arrhythmias (ventricular tachycardia, VT) after catecholaminergic stimulation. This observation could be a consequence of increasing number of « sparks », and thus an increased calcium release during diastole. More interestingly, delivery of TRDN gene using AAV9 in KO mice, prevent adverse remodeling and the associated cardiac dysfunction following 28 days TAC surgery. To conclude, this work shows that the lack of triadin accelerate the transition towards heart failure, acting on LVH , contractile dysfunction, and the occurrence of lethal ventricular arrhythmias.

Abstract During cardiac hypertrophy individual cardiac myocytes increase in size, which is accompanied by augmented protein synthesis and selective induction of a subset of genes. These phenotypic changes of myocytes are a result from altered intracellular signaling mechanisms and molecules. B-type natriuretic peptide (BNP) gene was selected as a target gene for the study of cardiac signaling mechanisms, since it is activated by mechanical, neural and humoral stimuli during myocyte hypertrophy. To generate hypertrophy of cardiac myocytes, neonatal rat cardiac myocytes were subjected to exogenous hypertrophic agonists such as endothelin-1 (ET-1) or to cyclic mechanical stretch. The role and regulation of transcription factors were studied by utilizing promoter analysis together with site-specific mutations and measurement of DNA binding activity and phosphorylation. GATA-4 mediated signaling was inhibited by blocking DNA binding with decoy oligonucleotides or by decreasing GATA-4 synthesis via adenoviral antisense delivery. ET-1 activated GATA-4 via serine residue phosphorylation, and this effect was mediated via p38 kinase. Similarly, GATA-4 binding activity was increased by ET-1 and mechanical stretch, but it was essential for activation of BNP gene only in the latter stimulation. Importantly, downregulation of GATA-4 protein levels prevented mechanical stretch induced hypertrophy of cardiac myocytes. In contrast, separate mechanism for an ET-1 specific signaling was composed of p38 kinase regulated ETS-like transcription factor-1 (Elk-1). Finally, the effect of mechanical stretch on endogenous endothelin-1 (ET-1) synthesis in cardiac cells was studied. Intrinsic ET-1 synthesis was activated in stretched cardiac myocytes, yet the levels of ET-1 were relatively low. This work suggests that GATA-4 transcription factor is required for mechanical stretch mediated hypertrophic program, and Elk-1 may act as a downstream effector of ET-1 in cardiac myocytes. Taken together, induction of ET-1 and BNP genes as well as activation of GATA-4 and Elk-1 transcription factors are regulated via a network of mitogen activated protein kinase pathways.

Abstract Cardiac myocytes react to increased workload and hypertrophic neurohumoral stimuli by increasing protein synthesis, reinitiating expression of fetal forms of structural genes, α-skeletal actin (α-SkA) and β-myosin heavy chain (β-MHC), and by increasing expression and secretion of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP). Initially, the response is beneficial, but when prolonged, it leads to pathological cardiomyocyte hypertrophy. In this study, cardiomyocyte hypertrophy was initiated by hypertrophic agonists, endothelin-1 (ET-1) and phenylephrine (PE), and by increased stretching of atrial wall. Transcription factor GATA4 was studied to identify the mechanism leading to increased gene expression of BNP. In BNP promoter, GATA4 binds to cis elements mediating hypertrophic response. Eliminating GATA4 binding by using the decoy approach, basal BNP gene expression was reduced. To identify mechanisms regulating GATA4, the roles of mitogen-activated protein kinases (MAPKs) were studied. Activation of p38 MAPK increased GATA4 binding to BNP gene and led to increased GATA4 dependent BNP gene expression. p38 MAPK was required for ET-1 induced GATA4 binding, whereas extracellular signal-regulated kinase (ERK) was required for maintaining basal GATA4 binding activity. PE and ET-1 activated protein kinase C (PKC) signaling in cardiac myocytes. Antisense oligonucleotide inhibition of PKCα markedly reduced PE induced ANP secretion and ET-1 induced BNP secretion, whereas gene expression of natriuretic peptides was not affected. Antisense PKCα treatment inhibited PE induced expression of α-SkA, while increased protein synthesis or β-MHC gene expression were not affected. Sretching of the perfused rat atria increased BNP, c-fos and BNP gene expression via mechanism involving p38 MAP kinase activation of transcription factor Elk-1. In cultured neonatal rat atrial myocytes stretch induced BNP gene expression was dependent upon transcription factor Elk-1 binding sites within the BNP gene promoter. In conclusion, hypertrophic signaling in cardiac myocytes involves multiple signaling cascades. Activation of p38 MAPK is required for the development of ET-1 induced hypertrophic phenotype and GATA4 mediated BNP gene expression in cultured ventricular myocytes, and for stretch induced Elk-1 dependent BNP gene expression in atrial myocytes. PKCα is involved in PE induced hypertrophic response and PE induced switch in gene programming inducing expression of α-SkA, the fetal form of cardiac α-actin.

Abstract Cardiac hypertrophy is an adaptive response to increased cardiac workload. It is initially beneficial, since it helps to maintain cardiac output, but ultimately it is considered as an independent predictor for heart failure and sudden cardiac death. The cardiac hypertrophic response is triggered by mechanical and neurohumoral stimuli and is associated with the activation of complex changes in gene programming and intracellular signaling pathways. The purpose of this study was to investigate the expression of some novel load-induced factors i.e. melusin, thrombospondin (TSP)-1 and -4 and LIM and cysteine-rich domains protein 1 (LMCD1)/dyxin during the hypertrophic response. Melusin was expressed in cardiac tissue both in the atria and ventricles, furthermore its expression was very rapidly activated in response to multiple hypertrophic stimuli predominantly in the left atria. Melusin gene expression was activated when cultured cardiac myocytes were subjected to mechanical stretch or hypertrophic agonists such as endothelin-1 or angiotensin (Ang II). TSP-1 and TSP-4 gene expressions were rapidly activated at an early stage of pressure overload. Myocardial infarction (MI) induced the expression of both TSP-4 and TSP-1 mRNA in the heart. TSP-4 may also be an endothelial cell-specific marker of pressure overload since its expression was limited to endothelial cells in the adult heart. The expression of LMCD1/dyxin was found to be induced during the cardiac hypertrophic response and after MI. By itself, mechanical load was a critical regulator of LMCD1/dyxin gene expression. LMCD1/dyxin is a putative novel p38 mitogen-activated protein kinase (MAPK) target since adenovirus-mediated overexpression of p38 MAPK upregulated LMCD1/dyxin expression. In addition, during the Ang II-induced pressure overload p38 MAPK phosphorylation levels correlated with the early induction of LMCD1/dyxin expression. In conclusion, this study provides new information on the expression of melusin, TSP-1 and -4 and LMCD1/dyxin in the cardiac hypertrophic response. Early induction of their gene expression may represent an initial step in the adaptive and protective remodeling processes following increased workload in the heart. / Tiivistelmä Sydänlihas mukautuu lisääntyneeseen kuormitukseen lihassolujen koon kasvun eli hypertrofian avulla. Pitkittyessään hypertrofinen kasvu on kuitenkin tärkeä sydämen vajaatoimintaa ja äkkikuolemaa ennakoiva riskitekijä. Hypertrofisessa vasteessa mekaaninen venytys sekä neurohumoraaliset tekijät saavat aikaan solunsisäisten signaalinvälitysreittien aktivoitumisen, mikä johtaa lisääntyneeseen geenien luentaan ja proteiinituotantoon. Väitöskirjassa tutkittiin uusien kuormitusaktivoituvien tekijöiden, melusiinin, trombospondiini (TSP) -1:n ja -4:n sekä dyksiinin ilmentymistä hypertrofisen vasteen aikana. Melusiinia ilmentyy sydämessä sekä kammioissa että eteisissä, mutta painekuormituksen myötä se aktivoituu nopeasti pääasiassa vasemmassa eteisessä. Sydänlihassolujen soluviljelymallissa melusiinin luenta lisääntyy suoraan mekaanisen venytyksen ja hypertrofisten agonistien vaikutuksesta. Painekuormitus aktivoi nopeasti myös TSP-1:n ja -4:n luentaa sydämessä. TSP-1:n ja -4:n geeniluenta lisääntyy myös kokeellisessa sydäninfarktimallissa. Lisäksi sydämessä TSP-4:ää havaittiin olevan ensisijaisesti endoteelisoluissa. Dyksiinin ilmentyminen lisääntyi sekä painekuormituksen että sydäninfarktin aiheuttaman sydänlihaksen uudelleenmuovautumisen aikana. Mekaaninen kuormitus riitti jo yksinään aktivoimaan dyksiinin geeniluentaa sydämessä. Lisäksi mitogeeni-aktivoituvan p38-proteiinikinaasin havaittiin säätelevän dyksiinin ilmentämistä. Väitöskirjatyössä saatiin uutta tietoa sydänlihaksen kuormituksen aikaisista muutoksista geenien luennassa sydänlihaksessa. Työssä osoitettiin, että painekuormitus aktivoi sydämessä aiemmin vähän tutkittujen geenien, melusiinin, TSP-1:n ja -4:n sekä dyksiinin, ilmentymistä. Näiden tekijöiden aktivoituminen hypertrofisen vasteen alkuvaiheessa antaa viitettä siitä, että tekijät osallistuvat kuormittuneen sydänlihaskudoksen uudelleenmuovautumiseen. Melusiini voi toimia erityisesti eteiskudosta kuormitukselta suojaavissa mekanismeissa, kun taas TSP-4 osoittautui aktivoituvan painekuormituksessa nimenomaan endoteelisoluissa.

Abstract Cardiac hypertrophy provides an adaptive mechanism to maintain cardiac output in response to increased workload, and although initially beneficial, hypertrophy eventually leads to heart failure, a major cause of morbidity and mortality in Western countries. The hypertrophic response in cardiac myocytes is accompanied by e.g. activation of signal transduction pathways, such as mitogen-activated protein kinases (MAPKs), and complex changes in gene programming. The purpose of this study was to characterize gene expression patterns in experimental models of cardiac load by using high-throughput DNA microarray technologies. In the present study, changes in gene expression were evaluated in response to acute pressure overload and prolonged hypertension as well as during the development of left ventricular hypertrophy (LVH) and the transition to diastolic heart failure in an animal model of genetic hypertension, the spontaneously hypertensive rat (SHR). Increased expression of several immediate early genes was seen in response to acute hemodynamic overload in vivo. The transition from LVH to diastolic hypertensive heart failure was almost exclusively associated with changes in genes encoding extracellular matrix proteins and their regulatory processes showing the importance of progressive extracellular matrix remodeling. The effect of p38 MAPK activation on gene expression patterns in vivo was elucidated. Cardiac-specific overexpression of p38 MAPK resulted in upregulation of genes controlling cell division and inflammation as well as cell signaling and adhesion. Accordingly, the functional role of p38 MAPK was related to myocardial cell proliferation, inflammation and fibrosis. Finally, temporal analysis of mechanical stretch induced gene expression changes in neonatal rat cardiomyocyte cultures in vitro indicated that mechanical stretch induced complex gene expression profiles, demonstrating that both positive and negative regulators are involved in the hypertrophic process. Many novel stretch responsive genes were identified, and a subset of them may be putative downstream targets of p38 MAPK. In conclusion, in the present study a number of well-established gene expression changes of cardiac hypertrophy were observed and novel modulators associated with increased cardiac load, such as thrombospondin-4, were identified. The study provides a better understanding of molecular mechanisms associated with increased cardiac load, and may indicate potential targets for novel therapeutic interventions.

Introduction : Le diagnostic de cardiomyopathie hypertrophique (CMH) est difficile chez l’athlète. En effet, le remodelage physiologique induit par l’entraînement physique intense entraîne des modifications électriques et morphologiques qui peuvent mimer une cardiomyopathie. Or il est indispensable de poser le diagnostic de cardiomyopathie avec certitude chez un athlète. Ne pas contre-indiquer un athlète avec une cardiomyopathie l’expose à un risque de mort subite, mais poser un diagnostic par excès l’expose à de lourdes répercussions tant professionnelles que sociales. Méthodes : (1) Nous avons cherché à améliorer les critères ECG actuels de détection de cardiomyopathie chez l’athlète à partir d’une cohorte multicentrique d’athlètes et de CMH. (2) Nous avons cherché à déterminer quel bilan complémentaire réaliser en cas d’anomalie ECG par un suivi longitudinal d’athlètes avec ondes T négatives. (3) Nous avons essayé de mieux caractériser le phénotype des athlètes atteints de CMH par rapport aux CMH sédentaires dans une cohorte multicentrique. (4) Nous avons tenté de déterminer si l’utilisation des nouvelles techniques d’imagerie de déformation myocardique permettait d’améliorer la pertinence diagnostique et pronostique en cas de CMH dans une cohorte de CMH et d’athlètes rennais. Résultats : Nous avons proposé une nouvelle classification ECG permettant de mieux identifier les athlètes avec modifications ECG non pathologiques sans diminuer pour autant la capacité à détecter les CMH. En cas d’ondes T négatives chez l’athlète, nous avons démontré qu’il était indispensable de réaliser une IRM myocardique. En effet l’échocardiographie peut être prise en défaut dans près de 35% des cas. Néanmoins, les critères diagnostiques actuels de CMH peuvent être pris en défaut; en effet les athlètes avec une CMH ont un phénotype différent des CMH sédentaires avec une meilleure fonction systolique, notamment longitudinale, et diastolique. L’évaluation de la fonction longitudinale à l’effort et l’évaluation de la dispersion mécanique sont des paramètres qui semblent prometteurs en terme de diagnostic. En effet l’altération la fonction longitudinale semble être en lien avec la fibrose myocardique. L’échocardiographie d’effort, notamment la présence d’une insuffisance mitrale à l’effort, semble être un facteur pronostic important dans les CMH. Conclusions : les travaux réalisés ont permis de développer des outils pour mieux différencier une hypertrophie ventriculaire gauche (HVG) pathologique d’une HVG physiologique mais également pour mieux caractériser cette HVG et déterminer avec plus de précision le pronostic des CMH . / Introduction: the diagnosis of hypertrophic cardiomyopathy (HCM) in athlete is difficult. Indeed, intense sports practice induces an electrical and morphological physiological remodeling which can be difficult to differentiate from the changes induced in pathology. However, it is essential to diagnose an athlete with a cardiomyopathy. Indeed, in case of underlying cardiomyopathy the athlete will be at risk of sudden cardiac death, but an excessive over diagnosis has strong professional and social consequences. Methods: (1) we have tried to improve the ECG criteria’s, which enable the differentiation between ECG changes induced by exercise and the ECG changes induced by an underlying cardiomyopathy. (2) We tried to define the best investigation algorithm in case of abnormal ECG changes in athletes. (3) We tried to improve the characterization of the phenotype of athletes with HCM as compared to sedentary HCM. (4) We tried to investigate if the use of new imaging technics, i.e. speckle tracking, might improve the diagnostic accuracy and enable a better prognostic evaluation in HCM. Results: We have proposed a new classification of ECG in athletes enabling to decrease the rate of false positive ECG in athletes without decreasing its diagnostic accuracy in HCM. In case of pathological T wave inversion (PTWI) in athletes, we demonstrated that a CMR is mandatory, as echocardiography missed a diagnosis of pathology in 35% of PTWI athletes. Nevertheless, the diagnosis of HCM with current criteria’s of HCM can be challenging. Indeed, HCM athletes have a different phenotype from HCM sedentary, with a better systolic and diastolic function; they also have a better longitudinal function. The assessment of longitudinal function during exercise and mechanical dispersion are promising tool for the diagnosis of HCM in athletes. Indeed, the alteration of longitudinal strain is related to myocardial fibrosis. Exercise echocardiography, i.e. exercise mitral insufficiency, seems to be a prognostic factor in HCM patients. Conclusions: Ours results enabled to develop tools which might help to better differentiate pathological and physiological left ventricular hypertrophy (LVH); but also to better characterize LVH and the prognosis in HCM patients.

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.

Cardiac hypertrophy is a compensatory response to increased work load or stress on the heart, but over time can lead to heart failure and death. The molecular mechanisms underlying this disease are still not completely understood, however the Rho/Rho kinase pathway has been shown to play a role. N-[11C]-methyl-hydroxyfasudil, a PET radiotracer, binds to active Rho kinase and could be a possible tracer for hypertrophy. Hypertrophy was induced in vitro using the β-adrenergic receptor agonist isoproterenol to evaluate optimal Rho kinase activity. Rho kinase activity data was correlated to N-[11C]-methyl-hydroxyfasudil binding. Cardiac hypertrophy was verified with an increase in nuclear size (1.74 fold) and cell size (~2 fold), activation of hypertrophic signalling pathways, and increased Rho kinase activity (1.64 fold). This correlated to a 10.3% increase in N-[11C]-methyl-hydroxyfasudil binding. This data suggests that N-[11C]-methyl-hydroxyfasudil may be useful as a radiotracer for detecting cardiac hypertrophy and merits further in vivo investigation.

Aim The main aim of this study was to investigate if a combination of classic resistance training and blood flow restricted resistance exercise (BFRE) training would result in greater increases in quadriceps muscle growth compared with other strength training studies. The second aim was to investigate if there would be any difference in muscle hypertrophy between men and women after the training intervention.   Method Twenty untrained subjects (10 males and 10 female) were recruited to participate in a 10-week unilateral resistance training intervention. Sixteen subjects completed the training intervention. After two familiarization sessions subjects performed three sessions per week in leg press and leg extension, except for week 4 and 8 were subjects performed five BFRE training sessions Monday to Friday. All subjects performed a one repetition maximum test in leg press and leg extension pre and post the training intervention. Ultrasound screening was performed pre and post training intervention to measure muscle thickness in m. vastus lateralis (VL).   Results The 10-week intervention resulted in a significant increase of VL muscle thickness by 15,1 % ± 7,6 (p ≤ 0,01). Both men and women increased in VL muscle thickness, men (n=7) by 15,4 % ± 9,3 (p ≤ 0,01) and women (n=9) by 14,8 % ± 6,0 (p ≤ 0,01), with no difference between genders. Maximal strength increased for the entire group in the leg press by 59,1 % ± 27,4 (p ≤ 0,01) and in the leg extension by 19,8 % ± 13,1 (p ≤ 0,01). Men had an increase of 58,1 % ± 18,0 (p ≤ 0,01) and women with 60,3 % ± 32,8 (p ≤ 0,01) in the leg press. In the leg extension women and men increased their maximal strength by 23,3 % ± 7,4 (p ≤ 0,01) respectively 17,0 % ± 14,4 (p = 0,051).   Conclusions Our unique training protocol resulted in a superior increase in muscle growth in comparison with most other strength training studies. Our result can be converted to an increase of 17,3 % (0,25 % per day) in VL muscle CSA, which is much greater than the mean increase of 0,11 % per day reported in a large meta-analysis (Wernbom, Augustsson &amp; Thomeé 2007).

Familial Hypertrophic cardiomyopathy (HCM) causes ventricle walls to thicken and often leads to sudden death especially in adults. Mutations in the subfragment 2 (S2) of &#946;-cardiac myosin are implicated in the genetic disorder. This S2 region is a coiled-coil rod region resulting from the dimeric form of myosin II. It has been proposed that an elastic quality allows normal S2 to absorb force during the powerstroke according to the sliding filament model. To test the flexibility of single molecules of S2 against levels of physiological force, the Gravitational Force Spectrometer (GFS) is being developed. This novel system employs a standard microscope on an equatorial mount that allows the spectrometer to be rotated freely in space. Stationary glass beads are attached to a microscope slide where the molecule is tethered between the stationary bead and a smaller mobile bead. The GFS is oriented so that the force of gravity can act on the mobile bead and so impart a small force to the tethered subfragment. Additionally, a video system in conjunction with ImageJ software makes a distance measurement of the molecule possible with a resolution of around 11 nm. The S2 can be stretched parallel or perpendicular to the coiled coil to elucidate different structural properties of the rod. This study is the first to show structural evidence that S2 in vertebrate skeletal myosin uncoils proportionally to physiological force loads. Because of this, the usefulness and promise of the novel GFS is highlighted, and the biological role of S2's flexibility can be directly commented on. If the dimer undergoes uncoiling at physiological force loads as shown, then it is reasonable to think that this might occur in nature in response to the stress of the powerstroke on a single molecule. This unwinding could be to absorb force as a mechanism to protect the muscle fiber.