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Summary 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 β-myosin heavy chain (MyHC), as opposed to the atrial, fast α-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 α-MyHC versus β-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, α- versus β-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., Highlights • Single-cell mapping technique allows multiparametric characterization of hESC-CMs • Twitch and action potential kinetics are independent of MyHC isoform in hESC-CMs • Myosin-mRNA levels are only weakly correlated with respective protein levels, In this article, Natalie Weber and colleagues developed a single-cell mapping technique for multiparametric analysis of hESC cardiomyocytes (hESC-CMs). Characterization of function (twitch and action potential) in direct relation to the expressed myosin heavy chain isoform by multiparametric analysis suggests that in hESC-CMs the expression of genes associated with electrical activity, contraction, calcium handling, and myosin heavy chains is independently regulated.

Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disease with an incidence of about 1 in 500 individuals.1 It is characterized by asymmetrical hypertrophy of the left ventricle in the absence of other causes for hypertrophy. HCM can vary from essentially asymptomatic to highly malignant up to end-stage heart failure or cause life-threatening arrhythmias with sudden cardiac death particularly in young adults. In most familial HCM cases, heterozygous mutations in sarcomeric proteins have been identified as underlying cause. About one third of the patients are heterozygous for mutations in the β-cardiac myosin heavy chain gene MYH7 , another third in cardiac myosin-binding protein C (cMyBPC).2 Few mutations were found in nonsarcomeric proteins. Cardiomyocyte and myofibrillar disarray with interstitial fibrosis and hypertrophy are hallmarks of HCM.3 The degree of myocardial disarray correlates with risk factors for sudden cardiac death,4 and it was suggested that myocyte disarray directly results from functional changes induced by the HCM-related mutations at the sarcomeric level.5 From studies mostly based on animal models or recombinant contractile proteins, it was postulated that HCM-associated mutations generally enhance contractility of the myocardium, whereas mutations with reduced contractility would lead to dilated cardiomyopathy.6–8 Accordingly, the different HCM mutations are thought to lead to the HCM phenotype by increasing calcium sensitivity, contractility, and ATPase activity of the cardiomyocytes.5 Results from our group and others, however, were inconsistent with this hypothesis. Instead, contractility and calcium sensitivity can be enhanced or decreased in HCM.6,9–12 Thus, it is still unclear what triggers development of HCM. On the other hand, trials with new therapeutic approaches for HCM would greatly benefit from detailed knowledge of the disease-causing mechanisms. Here, we propose an entirely new concept of how different mutations in sarcomeric proteins may lead …

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.

Familial hypertrophic cardiomyopathy (HCM) has been related to many different mutations in at least 20 different sarcomeric and some non-sarcomeric proteins. While development of the HCM phenotype is thought to be triggered by the respective mutation, a common mechanism has not yet been identified.We hypothesized that functional imbalance among individual cardiomyocytes, seen in our previous work on M. soleus fibers of patients with different β-MyHC missense mutations, may be due to cell-to-cell variation in expression of mutated myosin and may trigger development of the HCM-phenotype.We tested this hypothesis by recording force-pCa relations of individual cardiomyocytes and quantified expression of mutant β-MyHC mRNA in individual cardiomyocytes of myocardium of two HCM-patients with β-MyHC mutations R723G and A200V. For both mutations, cardiomyocytes showed a large cell-to-cell variation in Ca++-sensitivity with some cells being essentially indistinguishable from controls as if they had not expressed mutant β-MyHC while other cells had highly reduced Ca++-sensitivity suggesting substantial levels of mutant β-MyHC. Quantification of the fraction of mutant β-MyHC-mRNA in individual cardiomyocytes of both patients showed a wide spectrum from cardiomyocytes with essentially pure mutant β-MyHC-mRNA to cells with essentially pure wildtype β-MyHC-mRNA.This behavior could be modelled on the basis of random, independent, burst-like transcription of the mutant and wildtype alleles using known turnover rates of the β-MyHC-protein and β-MyHC-mRNA. We therefore propose that random, independent, burst-like transcription of mutant and wildtype alleles, via large cell-to-cell variation in mutant mRNA and related cell-to-cell functional variation, results in structural distortions (cellular/myofibrillar disarray). This may trigger altered cell signaling leading to interstitial fibrosis, another hallmark of the HCM phenotype. Such a mechanism may well act for other mutations in other proteins as well.

Mechanical and two-dimensional (2D) x-ray diffraction studies suggest that during isometric steady-state contraction, strongly bound cross-bridges mostly occupy early states in the power stroke, whereas rigor or rigor-like cross-bridges could not be detected. However, it remained unclear whether cross-bridges accumulate, at least transiently, in rigor or rigor-like states in response to rapid-length releases. We addressed this question using time-resolved recording of 2D x-ray diffraction patterns of permeabilized fibers from rabbit psoas muscles during isometric contraction and when small, ramp-shaped length-releases were applied to these fibers. This maneuver allows a transient accumulation of cross-bridges in states near the end of their power stroke. By lowering the temperature to 5 degrees C, force transients were slowed sufficiently to record diffraction patterns in several 2-4-ms time frames before and during such releases, using the RAPID detector (Refined ADC Per Input Detector) at beam line ID02 of the European Synchrotron Radiation Facility (Grenoble, France). The same sequence of frames was recorded in relaxation and rigor. Comparisons of 2D patterns recorded during isometric contraction, with patterns recorded at different [MgATPgammaS] and at 1 degrees C, showed that changes in intensity profiles along the first and sixth actin layer lines (ALL1 and ALL6, respectively) allowed for discernment of the formation of rigor or rigor-like cross-bridges. During ramp-shaped releases of activated fibers, intensity profiles along ALL1 and ALL6 did not reveal evidence for the accumulation of rigor-like cross-bridges. Instead, changes in the ALL6-profile suggest that during ramp-shaped releases, cross-bridges transiently accumulate in a structural state that, to our knowledge, was not previously seen, but that could well be a strongly bound state with the light-chain binding domain in a conformation between a near prepower-stroke (isometric) orientation and the orientation in rigor.

Hypertrophic Cardiomyopathy (HCM) is an inherited disease mainly linked to mutations in genes coding for sarcomeric proteins. Most of these mutations result in a similar phenotype which is characterized by hypertrophy, cellular and myofibrillar disarray and interstitial fibrosis within the myocardium. The similar phenotype suggests an as yet unknown trigger for HCM-development common to different mutations.In our previous work on HCM-mutations in MYH7 we found a large cell-to-cell functional variability in HCM-tissue samples, presumably due to cell-to-cell variability in the expression of mutant protein. This was supported by a large cell-to-cell variability in mutant mRNA. We hypothesized that this observed functional imbalance among individual cardiomyocytes in the cellular network of the myocardium results in hypertrophy, disarray and interstitial fibrosis, i.e., triggers development of the HCM phenotype. Furthermore, FISH-based visualization of active transcription sites showed a substantial proportion of nuclei without active transcription, indicating discontinuous, burst-like transcription of the MYH7-alleles.To test whether independent, stochastic, burst-like transcription of the mutant and wildtype MYH7-alleles could indeed account for all these observations, we programmed a numerical model where the transcription of both alleles is switched on and off stochastically, resulting in sequential generation of pre-mRNA, mRNA and myosin molecules with defined rate constants. Rate constants for generation and decay of pre-mRNA, mRNA and myosin were taken from the literature. The model shows that independent, stochastic, burst-like transcription can indeed account for all of our above mentioned experimental results by adjusting the on and off rates of the transcription yielding the large cell-to-cell variability in mutant mRNA and protein. The fraction of nuclei without active transcription sites is determined by the on/off rates of transcription and modulated by the pre-mRNA to mRNA transition.

Hypertrophic Cardiomyopathy (HCM) has a prevalence of 1 in 500 individuals. More than 1000 mutations in >20 different sarcomeric and some non-sarcomeric proteins have been identified in HCM-patients. In 30-40% the HCM-mutation is found in the β-myosin heavy chain (β-MyHC) gene (MYH7). Until now, a common mechanism triggering HCM is unknown.From previous work of our group on MYH7-mutations we hypothesized that the characteristic HCM-phenotype with hypertrophy, myocyte disarray, and interstitial fibrosis could be a consequence of cell-to-cell functional imbalance due to cell-to-cell variation in the expression of mutated vs. wildtype β-MyHC. Such cell-to-cell variability could result from stochastic, independent burst-like transcription of mutant and wildtype MYH7-alleles.To investigate whether the MYH7-alleles are indeed transcribed in a discontinuous, burst-like manner, we visualized active transcription sites of MYH7 in cardiomyocytes of a patient with β-MyHC-mutation R723G and of a non-transplanted donor heart using fluorescence in situ hybridization. To visualize pre-mRNA in active transcription sites we used two probes sets, each containing 48 single-labeled 20-bases-long oligonucleotides to probe intronic and exonic sequences of MYH7-pre-mRNA, while cytoplasmic MYH7-mRNA was labelled by the exonic probe set. Both probe sets were hybridized to their target sequences in 16 µm thick sections of cardiac samples.Active transcription sites were identified as bright spots inside the nuclei of cardiomyocytes showing co-localization of both probe sets. 26% of the nuclei had no active transcription sites while cytoplasmic MYH7-mRNA was revealed by the exonic probes. Absence of active transcription sites is inconsistent with continuous transcription of the two MYH7-alleles but expected for random burst-like transcription.Model calculations showed that independent, stochastic, burst-like transcription of mutant and wildtype MYH7-alleles can account for the experimentally observed cell-to-cell variability in mutant MYH7-mRNA and functional imbalances.

Coupling of ATP hydrolysis to structural changes in the motor domain is fundamental to the driving of motile functions by myosins. Current understanding of this chemomechanical coupling is primarily based on ensemble average measurements in solution and muscle fibers. Although important, the averaging could potentially mask essential details of the chemomechanical coupling, particularly for mixed populations of molecules. Here, we demonstrate the potential of studying individual myosin molecules, one by one, for unique insights into established systems and to dissect mixed populations of molecules where separation can be particularly challenging. We measured ATP turnover by individual myosin molecules, monitoring appearance and disappearance of fluorescent spots upon binding/dissociation of a fluorescent nucleotide to/from the active site of myosin. Surprisingly, for all myosins tested, we found two populations of fluorescence lifetimes for individual myosin molecules, suggesting that termination of fluorescence occurred by two different paths, unexpected from standard kinetic schemes of myosin ATPase. In addition, molecules of the same myosin isoform showed substantial intermolecular variability in fluorescence lifetimes. From kinetic modeling of our two fluorescence lifetime populations and earlier solution data, we propose two conformers of the active site of myosin, one that allows the complete ATPase cycle and one that dissociates ATP uncleaved. Statistical analysis and Monte Carlo simulations showed that the intermolecular variability in our studies is essentially due to the stochastic behavior of enzyme kinetics and the limited number of ATP binding events detectable from an individual myosin molecule with little room for static variation among individual molecules, previously described for other enzymes.

Two-dimensional x-ray diffraction was used to investigate structural features of cross-bridges that generate force in isometrically contracting skeletal muscle. Diffraction patterns were recorded from arrays of single, chemically skinned rabbit psoas muscle fibers during isometric force generation, under relaxation, and in rigor. In isometric contraction, a rather prominent intensification of the actin layer lines at 5.9 and 5.1nm and of the first actin layer line at 37nm was found compared with those under relaxing conditions. Surprisingly, during isometric contraction, the intensity profile of the 5.9-nm actin layer line was shifted toward the meridian, but the resulting intensity profile was different from that observed in rigor. We particularly addressed the question whether the differences seen between rigor and active contraction might be due to a rigor-like configuration of both myosin heads in the absence of nucleotide (rigor), whereas during active contraction only one head of each myosin molecule is in a rigor-like configuration and the second head is weakly bound. To investigate this question, we created different mixtures of weak binding myosin heads and rigor-like actomyosin complexes by titrating MgATPγS at saturating [Ca2+] into arrays of single muscle fibers. The resulting diffraction patterns were different in several respects from patterns recorded under isometric contraction, particularly in the intensity distribution along the 5.9-nm actin layer line. This result indicates that cross-bridges present during isometric force generation are not simply a mixture of weakly bound and single-headed rigor-like complexes but are rather distinctly different from the rigor-like cross-bridge. Experiments with myosin-S1 and truncated S1 (motor domain) support the idea that for a force generating cross-bridge, disorder due to elastic distortion might involve a larger part of the myosin head than for a nucleotide free, rigor cross-bridge.