Hypertrophic Cardiomyopathy (HCM) has been related to many different mutations in more than 20 different, mostly sarcomeric proteins. While development of the HCM-phenotype is thought to be triggered by the different mutations, a common mechanism remains elusive. Studying missense-mutations in the ventricular beta-myosin heavy chain (β-MyHC, MYH7) we hypothesized that significant contractile heterogeneity exists among individual cardiomyocytes of HCM-patients that results from cell-to-cell variation in relative expression of mutated vs. wildtype β-MyHC. To test this hypothesis, we measured force-calcium-relationships of cardiomyocytes isolated from myocardium of heterozygous HCM-patients with either β-MyHC-mutation Arg723Gly or Arg200Val, and from healthy controls. From the myocardial samples of the HCM-patients we also obtained cryo-sections, and laser-microdissected single cardiomyocytes for quantification of mutated vs. wildtype MYH7-mRNA using a single cell RT-qPCR and restriction digest approach. We characterized gene transcription by visualizing active transcription sites by fluorescence in situ hybridization of intronic and exonic sequences of MYH7-pre-mRNA. For both mutations, cardiomyocytes showed large cell-to-cell variation in Ca++-sensitivity. Interestingly, some cardiomyocytes were essentially indistinguishable from controls what might indicate that they had no mutant β-MyHC while others had highly reduced Ca++-sensitivity suggesting substantial fractions of mutant β-MyHC. Single-cell MYH7-mRNA-quantification in cardiomyocytes of the same patients revealed high cell-to-cell variability of mutated vs. wildtype mRNA, ranging from essentially pure mutant to essentially pure wildtype MYH7-mRNA. We found 27% of nuclei without active transcription sites which is inconsistent with continuous gene transcription but suggests burst-like transcription of MYH7. Model simulations indicated that burst-like, stochastic on/off-switching of MYH7 transcription, which is independent for mutant and wildtype alleles, could generate the observed cell-to-cell variation in the fraction of mutant vs. wildtype MYH7-mRNA, a similar variation in β-MyHC-protein, and highly heterogeneous Ca++-sensitivity of individual cardiomyocytes. In the long run, such contractile imbalance in the myocardium may well induce progressive structural distortions like cellular and myofibrillar disarray and interstitial fibrosis, as they are typically observed in HCM.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent an attractive model to investigate CM function and disease mechanisms. One characteristic marker of ventricular specificity of human CMs is expression of the ventricular, slow b-myosin heavy chain (MyHC), as opposed to the atrial, fast a-MyHC. The main aim of this study was to investigate at the single-cell level whether contraction kinetics and electrical activity of hESC-CMs are influenced by the relative expression of a-MyHC versus b-MyHC. For effective assignment of functional parameters to the expression of both MyHC isoforms at protein and mRNA levels in the very same hESC-CMs, we developed a single-cell mapping technique. Surprisingly, aversus b-MyHC was not related to specific contractile or electrophysiological properties of the same cells. The multiparametric cell-by-cell analysis suggests that in hESC-CMs the expression of genes associated with electrical activity, contraction, calcium handling, and MyHCs is independently regulated.

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Mutations throughout cardiac troponin T (cTnT), a cardiac thin filament (CTF) component cause changes in protein structure and dynamics leading to pathologic cardiac remodeling observed in patients with hypertrophic (HCM) and dilated (DCM) cardiomyopathies. Of note, mutations within the cTnT-linker region cause particularly severe and highly penetrant cardiomyopathies. Our understanding of the precise molecular mechanisms involved has been limited by the lack of a high-resolution structure in this highly flexible domain. We employed time-resolved fluorescence resonance energy transfer (TR-FRET) utilizing fully reconstituted CTFs with donor-labeled (IAEDANS) cTnT on one of 4 Cys-substituted residues (A168/177/192/198C) and acceptor-labeled (5-IAF) actin on Cys-374 to gain high-resolution insight into the cTnT-linker's positioning across the actin filament. Our data indicate the cTnT-linker is proximal to three adjacent actin monomers in both þ/-Ca 2þ conditions. To determine how mutations in the cTnT-linker region alter the native structure of this region, we investigated three cardiomyopathy-linked mutations: DCMassociated mutations R173W and R173Q and HCM-associated mutation D160E. We hypothesize that R173Q/W and D160E cause differential repositioning of the cTnT-linker in relationship to actin. Preliminary investigation of R173W and D160E effects on the positioning of the cTnT-linker þCa 2þ indicates a trending compaction of the linker towards actin. R173Q, however, exhibits both increases and decreases in the cTnT-linker to actin distances. Further TR-FRET studies are ongoing to confirm and extend these preliminary mutation-specific structural results. Lastly, in vivo studies functional studies are ongoing to compare our new cTnT R173W transgenic mouse model and previous D160E mouse model to begin to elucidate genotype-phenotype mechanisms of disease. Through this approach, we can craft a high-resolution structure of the flexible cTnT-linker region and gain an understanding of mutation-specific structural alterations in this region.

In this work, we developed a model to quantitatively access the influence of different types of interactions within the sarcomere on properties of cardiac muscle. We use the set of partial differentsial equations to describe the dynamics of ensembles consisting of cross-bridge groups connected by elastic tropomyosin. The mathematical model gives thermodynamically consistent description of isometric and shortening contractions allowing us to study biophysical principles behind the cooperativity.Through large scan in the free energy landscape, we demonstrate the different influence of RU-RU, XB-XB, and XB-RU interactions on cooperativity coefficient of calcium binding, developed maximal force, and calcium sensitivity. The model solution was fitted to reproduce experimental data on force development during isometric contraction, shortening in physiological contraction, and ATP consumption by actomyosin during different types of contractions. On the basis of the fits, we showed that RU-RU interaction leads to about 5 times larger change in the free energy profile of the reaction than XB-XB interaction. This predicted mechanism behind cooperativity of the muscle contraction is in quantitative agreement with the studies comparing the azimuthal tropomyosin movement induced by binding calcium or myosin. Due to the deterministic description of muscle contraction and its thermodynamic consistency for shortening contractions, we envision that the developed model can be used to study biophysics of heart muscle contraction, its energy cost, link between calcium release and force development not just on the tissue level, but on a level of the whole heart.