Revisiting Frank–Starling: regulatory light chain phosphorylation alters the rate of force redevelopment (k<inf>tr</inf>) in a length-dependent fashion

© 2016 Wellcome Trust. Key points: Regulatory light chain (RLC) phosphorylation has been shown to alter the ability of muscle to produce force and power during shortening and to alter the rate of force redevelopment (ktr) at submaximal [Ca2+]. Increasing RLC phosphorylation ∼50% from the in vivo level in maximally [Ca2+]-activated cardiac trabecula accelerates ktr. Decreasing RLC phosphorylation to ∼70% of the in vivo control level slows ktr and reduces force generation. ktr is dependent on sarcomere length in the physiological range 1.85–1.94 μm and RLC phosphorylation modulates this response. We demonstrate that Frank–Starling is evident at maximal [Ca2+] activation and therefore does not necessarily require length-dependent change in [Ca2+]-sensitivity of thin filament activation. The stretch response is modulated by changes in RLC phosphorylation, pinpointing RLC phosphorylation as a modulator of the Frank–Starling law in the heart. These data provide an explanation for slowed systolic function in the intact heart in response to RLC phosphorylation reduction. Abstract: Force and power in cardiac muscle have a known dependence on phosphorylation of the myosin-associated regulatory light chain (RLC). We explore the effect of RLC phosphorylation on the ability of cardiac preparations to redevelop force (ktr) in maximally activating [Ca2+]. Activation was achieved by rapidly increasing the temperature (temperature-jump of 0.5–20ºC) of permeabilized trabeculae over a physiological range of sarcomere lengths (1.85–1.94 μm). The trabeculae were subjected to shortening ramps over a range of velocities and the extent of RLC phosphorylation was varied. The latter was achieved using an RLC-exchange technique, which avoids changes in the phosphorylation level of other proteins. The results show that increasing RLC phosphorylation by 50% accelerates ktr by ∼50%, irrespective of the sarcomere length, whereas decreasing phosphorylation by 30% slows ktr by ∼50%, relative to the ktr obtained for in vivo phosphorylation. Clearly, phosphorylation affects the magnitude of ktr following step shortening or ramp shortening. Using a two-state model, we explore the effect of RLC phosphorylation on the kinetics of force development, which proposes that phosphorylation affects the kinetics of both attachment and detachment of cross-bridges. In summary, RLC phosphorylation affects the rate and extent of force redevelopment. These findings were obtained in maximally activated muscle at saturating [Ca2+] and are not explained by changes in the Ca2+-sensitivity of acto-myosin interactions. The length-dependence of the rate of force redevelopment, together with the modulation by the state of RLC phosphorylation, suggests that these effects play a role in the Frank–Starling law of the heart.Wellcome Trust Grant 091460/Z/10/Z

Genetic studies of hypertrophic cardiomyopathy in Singaporeans identify variants in TNNI3 and TNNT2 that are common in Chinese patients

Background - To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry. Methods - We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3,634), compared findings with additional populations and Caucasian HCM cohorts (n=6,179) and performed in vitro functional studies. Results - Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (Pathogenic (P)/Likely Pathogenic (LP):18%, p<0.0001) but an excess of variants of unknown significance (exVUS: 24%, p<0.0001), as compared to Caucasians (P/LP: 31%, exVUS: 7%). Two missense variants in thin filament encoding genes were commonly seen in Singaporean HCM (TNNI3:p.R79C, disease allele frequency (AF)=0.018; TNNT2:p.R286H, disease AF=0.022) and are enriched in Singaporean HCM when compared with Asian controls (TNNI3:p.R79C, Singaporean controls AF=0.0055, p=0.0057, gnomAD-East Asian (gnomAD-EA) AF=0.0062, p=0.0086; TNNT2:p.R286H, Singaporean controls AF=0.0017, p<0.0001, gnomAD-EA AF=0.0009, p<0.0001). Both these variants have conflicting annotations in ClinVar and are of low penetrance (TNNI3:p.R79C, 0.7%; TNNT2:p.R286H, 2.7%) but are predicted to be deleterious by computational tools. In population controls, TNNI3:p.R79C carriers had significantly thicker left ventricular walls compared to non-carriers while its etiological fraction is limited (0.70, 95% CI: 0.35-0.86) and thus TNNI3:p.R79C is considered a VUS. Mutant TNNT2:p.R286H iPSC-CMs show hypercontractility, increased metabolic requirements and cellular hypertrophy and the etiological fraction (0.93, 95% CI: 0.83-0.97) support the likely pathogenicity of TNNT2:p.R286H. Conclusions - As compared to Caucasians, Chinese HCM patients commonly have low penetrance risk alleles in TNNT2 or TNNI3 but exhibit few clinically actionable HCM variants overall. This highlights the need for greater study of HCM genetics in non-Caucasian populations

Cardiac myosin super relaxation (SRX): a perspective on fundamental biology, human disease and therapeutics

The fundamental basis of muscle contraction ‘the sliding filament model’ (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954) and the ‘swinging, tilting crossbridge-sliding filament mechanism’ (Huxley, 1969; Huxley and Brown, 1967) nucleated a field of research that has unearthed the complex and fascinating role of myosin structure in the regulation of contraction. A recently discovered energy conserving state of myosin termed the super relaxed state (SRX) has been observed in filamentous myosins and is central to modulating force production and energy use within the sarcomere. Modulation of myosin function through SRX is a rapidly developing theme in therapeutic development for both cardiovascular disease and infectious disease. Some 70 years after the first discoveries concerning muscular function, modulation of myosin SRX may bring the first myosin targeted small molecule to the clinic, for treating hypertrophic cardiomyopathy (Olivotto et al., 2020). An often monogenic disease HCM afflicts 1 in 500 individuals, and can cause heart failure and sudden cardiac death. Even as we near therapeutic translation, there remain many questions about the governance of muscle function in human health and disease. With this review, we provide a broad overview of contemporary understanding of myosin SRX, and explore the complexities of targeting this myosin state in human disease

A post-MI power struggle: adaptations in cardiac power occur at the sarcomere level alongside MyBP-C and RLC phosphorylation.

Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic compensation and congestive decompensation. Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae

Plakophilin-2 truncating variants impair cardiac contractility by disrupting sarcomere stability and organization

Progressive loss of cardiac systolic function in arrhythmogenic cardiomyopathy (ACM) has recently gained attention as an important clinical consideration in managing the disease. However, the mechanisms leading to reduction in cardiac contractility are poorly defined. Here, we use CRISPR gene editing to generate human induced pluripotent stem cells (iPSCs) that harbor plakophilin-2 truncating variants (PKP2tv), the most prevalent ACM-linked mutations. The PKP2tv iPSC–derived cardiomyocytes are shown to have aberrant action potentials and reduced systolic function in cardiac microtissues, recapitulating both the electrical and mechanical pathologies reported in ACM. By combining cell micropatterning with traction force microscopy and live imaging, we found that PKP2tvs impair cardiac tissue contractility by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. These findings highlight the interplay between cell-cell adhesions and sarcomeres required for stabilizing cardiomyocyte structure and function and suggest fundamental pathogenic mechanisms that may be shared among different types of cardiomyopathies

Plakophilin-2 truncating variants impair cardiac contractility by disrupting sarcomere stability and organization

Progressive loss of cardiac systolic function in arrhythmogenic cardiomyopathy (ACM) has recently gained attention as an important clinical consideration in managing the disease. However, the mechanisms leading to reduction in cardiac contractility are poorly defined. Here, we use CRISPR gene editing to generate human induced pluripotent stem cells (iPSCs) that harbor plakophilin-2 truncating variants (PKP2tv), the most prevalent ACM-linked mutations. The PKP2tv iPSC-derived cardiomyocytes are shown to have aberrant action potentials and reduced systolic function in cardiac microtissues, recapitulating both the electrical and mechanical pathologies reported in ACM. By combining cell micropatterning with traction force microscopy and live imaging, we found that PKP2tvs impair cardiac tissue contractility by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. These findings highlight the interplay between cell-cell adhesions and sarcomeres required for stabilizing cardiomyocyte structure and function and suggest fundamental pathogenic mechanisms that may be shared among different types of cardiomyopathies

CalTrack: high throughput automated calcium transient analysis in cardiomyocytes

Rationale: Calcium transient analysis is central to understanding inherited and acquired cardiac physiology and disease. While the development of novel calcium reporters enables assays of CRISPR/Cas-9 genome edited pluripotent stem cell derived cardiomyocytes (iPSC-CMs) and primary adult cardiomyocytes, existing calcium-detection technologies are often proprietary and require specialist equipment, while open source workflows necessitate considerable user expertise and manual input. Objective: We aimed to develop an easy to use open source, adaptable, and automated analysis pipeline for measuring cellular calcium transients, from image stack to data output, inclusive of cellular identification, background subtraction, photobleaching correction, calcium transient averaging, calcium transient fitting, data collation and aberrant behavior recognition. Methods and Results: We developed CalTrack, a MatLab based algorithm, to monitor fluorescent calcium transients in living cardiomyocytes, including isolated single cells or those contained in 3-dimensional tissues or organoids and to analyze data acquired using photomultiplier tubes or employing line scans. CalTrack uses masks to segment cells allowing multiple cardiomyocyte transients to be measured from a single field of view. After automatically correcting for photobleaching, CalTrack averages and fits a string of transients and provides parameters that measure time to peak, time of decay, tau, Fmax/F0 and others. We demonstrate the utility of CalTrack in primary and iPSC-derived cell lines in response to pharmacological compounds and in phenotyping cells carrying hypertrophic cardiomyopathy variants. Conclusions: CalTrack, an open source tool that runs on a local computer, provides automated high-throughput analysis of calcium transients in response to development, genetic or pharmacological manipulations, and pathological conditions. We expect that CalTrack analyses will accelerate insights into physiologic and abnormal calcium homeostasis that influence diverse aspects of cardiomyocyte biology

Hypertrophic cardiomyopathy mutations in MYBPC3 dysregulate myosin

The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin-binding protein C; cMyBPC) or myosin missense mutations cause hypercontractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches, we explored how depletion of cMyBPC altered sarcomere function. We demonstrated that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM), normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation, enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in patients with DCM, whereas inhibiting myosin by MYK-461 should benefit the substantial proportion of patients with HCM with MYBPC3 mutations

Myosin sequestration regulates sarcomere function, cardiomyocyte energetics, and metabolism, informing the pathogenesis of hypertrophic cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. Methods: We assayed myosin ATP binding to define the proportions of myosin in SRX or DRX conformations in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants with unknown clinical significance (VUS) that were identified in HCM patients, predicted functional consequences and associations with heart failure and arrhythmias. Results: Myosins undergo physiologic shifts between SRX conformations that maximized energy-conservation and active states (DRX) that enable cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacologic modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased SRX conformations while pathogenic variants destabilized these and increased the proportion of DRX myosins, which enhanced cardiomyocyte contractility but impaired relaxation, and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify VUS, we showed that variants that unbalanced myosin conformations were associated with higher rates of heart failure and arrhythmias in HCM patients. Conclusions: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy conserving states promotes contractile abnormalities, morphological and metabolic remodeling and adverse clinical outcomes in HCM patients. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in HCM patients.</p