Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.

Long-Term Prevalence of Systolic Dysfunction in MYBPC3 Versus MYH7-Related Hypertrophic Cardiomyopathy.

BACKGROUND: The 2 sarcomere genes most commonly associated with hypertrophic cardiomyopathy (HCM), MYBPC3 (myosin-binding protein C3) and MYH7 (ß-myosin heavy chain), are indistinguishable at presentation, and genotype-phenotype correlations have been elusive. Based on molecular and pathophysiological differences, however, it is plausible to hypothesize a different behavior in myocardial performance, impacting lifetime changes in left ventricular (LV) function. METHODS: We reviewed the initial and final echocardiograms of 402 consecutive HCM patients with pathogenic or likely pathogenic MYBPC3 (n=251) or MYH7 (n=151) mutations, followed over 9±8 years. RESULTS: At presentation, MYBPC3 patients were less frequently obstructive (15% versus 26%; P=0.005) and had lower LV ejection fraction compared with MYH7 (66±8% versus 68±8%, respectively; P=0.03). Both HCM patients harboring MYBPC3 and MYH7 mutations exhibited a small but significant decline in LV systolic function during follow-up; however, new onset of severe LV systolic dysfunction (LV ejection fraction, <50%) was greater among MYBPC3 patients (15% versus 5% among MYH7; P=0.013). Prevalence of grade II/III diastolic dysfunction at final evaluation was comparable between MYBPC3 and MYH7 patients (P=0.509). In a Cox multivariable analysis, MYBPC3-positive status (hazard ratio, 2.53 [95% CI, 1.09-5.82]; P=0.029), age (hazard ratio, 1.03 [95% CI, 1.00-1.06]; P=0.027), and atrial fibrillation (hazard ratio, 2.39 [95% CI, 1.14-5.05]; P=0.020) were independent predictors of severe systolic dysfunction. No statistically significant differences occurred with regard to incidence of atrial fibrillation, heart failure, appropriate implanted cardioverter defibrillator shock, or cardiovascular death. CONCLUSIONS: MYBPC3-related HCM showed increased long-term prevalence of systolic dysfunction compared with MYH7, in spite of similar outcome. Such observations suggest different pathophysiology of clinical progression in the 2 subsets and may prove relevant for understanding of genotype-phenotype correlations in HCM.

Corrigendum: Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: the impact of full-length dystrophin deficiency.

[This corrects the article DOI: 10.3389/fphys.2022.1030920.].

The Role of Crosslinker Molecular Structure on Mechanical and Light-Actuation Properties in Liquid Crystalline Networks.

Phase behavior modulation of liquid crystalline molecules can be addressed by structural modification at molecular level. Starting from a rigid rod-like core reduction of the symmetry or increase of the steric hindrance by different substituents generally reduces the clearing temperature. Similar approaches can be explored to modulate the properties of liquid crystalline networks (LCNs)-shape-changing materials employed as actuators in many fields. Depending on the application, the polymer properties have to be adjusted in terms of force developed under stimuli, kinetics of actuation, elasticity, and resistance to specific loads. In this work, the crosslinker modification at molecular level is explored towards the optimization of LCN properties as light-responsive artificial muscles. The synthesis and characterization of photopolymerizable crosslinkers, bearing different lateral groups on the aromatic core is reported. Such molecules are able to strongly modulate the material mechanical properties, such as kinetics and maximum tension under light actuation, opening up to interesting materials for biomedical applications.

Slower Calcium Handling Balances Faster Cross-Bridge Cycling in Human MYBPC3 HCM.

BACKGROUND: The pathogenesis of MYBPC3-associated hypertrophic cardiomyopathy (HCM) is still unresolved. In our HCM patient cohort, a large and well-characterized population carrying the MYBPC3:c772G>A variant (p.Glu258Lys, E258K) provides the unique opportunity to study the basic mechanisms of MYBPC3-HCM with a comprehensive translational approach. METHODS: We collected clinical and genetic data from 93 HCM patients carrying the MYBPC3:c772G>A variant. Functional perturbations were investigated using different biophysical techniques in left ventricular samples from 4 patients who underwent myectomy for refractory outflow obstruction, compared with samples from non-failing non-hypertrophic surgical patients and healthy donors. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and engineered heart tissues (EHTs) were also investigated. RESULTS: Haplotype analysis revealed MYBPC3:c772G>A as a founder mutation in Tuscany. In ventricular myocardium, the mutation leads to reduced cMyBP-C (cardiac myosin binding protein-C) expression, supporting haploinsufficiency as the main primary disease mechanism. Mechanical studies in single myofibrils and permeabilized muscle strips highlighted faster cross-bridge cycling, and higher energy cost of tension generation. A novel approach based on tissue clearing and advanced optical microscopy supported the idea that the sarcomere energetics dysfunction is intrinsically related with the reduction in cMyBP-C. Studies in single cardiomyocytes (native and hiPSC-derived), intact trabeculae and hiPSC-EHTs revealed prolonged action potentials, slower Ca2+ transients and preserved twitch duration, suggesting that the slower excitation-contraction coupling counterbalanced the faster sarcomere kinetics. This conclusion was strengthened by in silico simulations. CONCLUSIONS: HCM-related MYBPC3:c772G>A mutation invariably impairs sarcomere energetics and cross-bridge cycling. Compensatory electrophysiological changes (eg, reduced potassium channel expression) appear to preserve twitch contraction parameters, but may expose patients to greater arrhythmic propensity and disease progression. Therapeutic approaches correcting the primary sarcomeric defects may prevent secondary cardiomyocyte remodeling.

Clinical scenarios of hypertrophic cardiomyopathy-related mortality: Relevance of age and stage of disease at presentation.

The evolving epidemiology of hypertrophic cardiomyopathy (HCM) has progressively changed our perception of HCM-related mortality. However, recent studies detailing individual causes of death based on age and clinical setting are lacking. Thus, the present study aimed to describe the modes of death in a consecutive cohort of HCM patients based on presenting clinical features and stage of disease. METHODS: By retrospective analysis of a large HCM cohort, we identified 161 patients with >1 year follow-up who died between 2000 and 2020 and thoroughly investigated their modes of death. HCM stage at presentation was defined as "classic", "adverse remodeling" or "overt dysfunction". RESULTS: Of the 161 patients, 103 (64%) died of HCM-related causes, whereas 58 (36%) died of non-HCM-related causes. Patients who died of HCM-related causes were younger than those who died of non-HCM related causes. The most common cause of death was heart failure (HF). Sudden cardiac death (SCD) ranked third, after non cardiovascular death, and mostly occurred in young individuals. The proportion of HF related death and SCD per stage of disease was 14% and 27% in "classic", 38% and 21% in "adverse remodeling" and 74% and 10% in "overt dysfunction". CONCLUSIONS: Most HCM patients die due to complications of their own disease, mainly in the context of HF. While SCD tends to be juvenile, HF related deaths often occur in age groups no longer amenable to cardiac transplant. Modes of death vary with the stage of disease, with SCD becoming less prevalent in more advanced phases, when competitive risk of HF becomes overwhelming.

Safety and efficacy of ranolazine in hypertrophic cardiomyopathy: Real-world experience in a National Referral Center.

OBJECTIVES: We assessed the efficacy and safety of ranolazine in real-world patients with hypertrophic cardiomyopathy (HCM). BACKGROUND: Ranolazine is an anti-anginal drug that inhibits the late phase of the inward sodium current. In a small prospective trial, ranolazine reduced the arrhythmic burden and improved biomarker profile in HCM patients. However, systematic reports reflecting real-world use in this setting are lacking. METHODS: Changes in clinical and instrumental features, symptoms and arrhythmic burden were evaluated in 119 patients with HCM before and during treatment with ranolazine at a national referral centre for HCM. RESULTS: Patients were treated with ranolazine for 2 [1-4] years; 83 (70%) achieved a dosage ≥1000 mg per day. Treatment interruption was necessary in 24 patients (20%) due to side effects (n = 10, 8%) or disopyramide initiation (n = 8, 7%). Seventy patients (59%) were treated with ranolazine for relief of angina. Among them, 51 (73%) had total symptomatic relief and 47 patients (67%) showed ≥2 Canadian Cardiovascular society (CCS) angina grade improvement. Sixteen patients (13%) were treated for recurrent ventricular arrhythmias, including 4 with a clear ischemic trigger, who experienced no further arrhythmic episodes while on ranolazine. Finally, 33 patients (28%) were treated for heart failure associated with severe diastolic dysfunction: no symptomatic benefit could be observed in this group. CONCLUSION: Ranolazine was safe and well tolerated in patients with HCM. The use of ranolazine may be considered in patients with HCM and microvascular angina.

Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency.

Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis).

Paradoxical prolongation of QT interval during exercise in patients with hypertrophic cardiomyopathy: cellular mechanisms and implications for diastolic function.

Aims: Ventricular cardiomyocytes from hypertrophic cardiomyopathy (HCM) patient hearts show prolonged action potential duration (APD), impaired intracellular Ca2+ homeostasis and abnormal electrical response to beta -adrenergic stimulation. We sought to determine whether this behaviour is associated with abnormal changes of repolarization during exercise and worsening of diastolic function, ultimately explaining the intolerance to exercise experienced by some patients without obstruction. Methods and results: Non-obstructive HCM patients (178) and control subjects (81) underwent standard exercise testing, including exercise echocardiography. Ventricular myocytes were isolated from myocardial samples of 23 HCM and eight non-failing non-hypertrophic surgical patients. The APD shortening in response to high frequencies was maintained in HCM myocytes, while ß-adrenergic stimulation unexpectedly prolonged APDs, ultimately leading to a lesser shortening of APDs in response to exercise. In HCM vs. control subjects, we observed a lesser shortening of QT interval at peak exercise (QTc: +27 ± 52 ms in HCM, -4 ± 50 ms in controls, P < 0.0001). In patients showing a marked QTc prolongation (>30 ms), the excessive shortening of the electrical diastolic period was linked with a limited increase of heart-rate and deterioration of diastolic function at peak effort. Conclusions: Abnormal balance of Ca2+- and K+-currents in HCM cardiomyocytes determines insufficient APD and Ca2+-transient shortening with exercise. In HCM patients, exercise-induced QTc prolongation was associated with impaired diastolic reserve, contributing to the reduced exercise tolerance. Our results support the idea that severe electrical cardiomyocyte abnormalities underlie exercise intolerance in a subgroup of HCM patients without obstruction.

Genotype-Driven Pathogenesis of Atrial Fibrillation in Hypertrophic Cardiomyopathy: The Case of Different TNNT2 Mutations.

Atrial dilation and atrial fibrillation (AF) are common in Hypertrophic CardioMyopathy (HCM) patients and associated with a worsening of prognosis. The pathogenesis of atrial myopathy in HCM remains poorly investigated and no specific association with genotype has been identified. By re-analysis of our cohort of thin-filament HCM patients (Coppini et al. 2014) AF was identified in 10% of patients with sporadic mutations in the cardiac Troponin T gene (TNNT2), while AF occurrence was much higher (25-75%) in patients carrying specific "hot-spot" TNNT2 mutations. To determine the molecular basis of arrhythmia occurrence, two HCM mouse models expressing human TNNT2 variants (a "hot-spot" one, R92Q, and a "sporadic" one, E163R) were selected according to the different pathophysiological pathways previously demonstrated in ventricular tissue. Echocardiography studies showed a significant left atrial dilation in both models, but more pronounced in the R92Q. In E163R atrial trabeculae, in line with what previously observed in ventricular preparations, the energy cost of tension generation was markedly increased. However, no changes of twitch amplitude and kinetics were observed, and there was no atrial arrhythmic propensity. R92Q atrial trabeculae, instead, displayed normal ATP consumption but markedly increased myofilament calcium sensitivity, as previously observed in ventricular preparations. This was associated with reduced inotropic reserve and slower kinetics of twitch contractions and, importantly, with an increased occurrence of spontaneous beats and triggered contractions that represent an intrinsic arrhythmogenic mechanism promoting AF. The association of specific TNNT2 mutations with AF occurrence depends on the mutation-driven pathomechanism (i.e., increased atrial myofilament calcium sensitivity rather than increased myofilament tension cost) and may influence the individual response to treatment.

The harder the climb the better the view: The impact of substrate stiffness on cardiomyocyte fate.

The quest for novel methods to mature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for cardiac regeneration, modelling and drug testing has emphasized a need to create microenvironments with physiological features. Many studies have reported on how cardiomyocytes sense substrate stiffness and adapt their morphological and functional properties. However, these observations have raised new biological questions and a shared vision to translate it into a tissue or organ context is still elusive. In this review, we will focus on the relevance of substrates mimicking cardiac extracellular matrix (cECM) rigidity for the understanding of the biomechanical crosstalk between the extracellular and intracellular environment. The ability to opportunely modulate these pathways could be a key to regulate in vitro hiPSC-CM maturation. Therefore, both hiPSC-CM models and substrate stiffness appear as intriguing tools for the investigation of cECM-cell interactions. More understanding of these mechanisms may provide novel insights on how cECM affects cardiac cell function in the context of genetic cardiomyopathies.

Quantification of Myocyte Disarray in Human Cardiac Tissue.

Proper three-dimensional (3D)-cardiomyocyte orientation is important for an effective tension production in cardiac muscle. Cardiac diseases can cause severe remodeling processes in the heart, such as cellular misalignment, that can affect both the electrical and mechanical functions of the organ. To date, a proven methodology to map and quantify myocytes disarray in massive samples is missing. In this study, we present an experimental pipeline to reconstruct and analyze the 3D cardiomyocyte architecture in massive samples. We employed tissue clearing, staining, and advanced microscopy techniques to detect sarcomeres in relatively large human myocardial strips with micrometric resolution. Z-bands periodicity was exploited in a frequency analysis approach to extract the 3D myofilament orientation, providing an orientation map used to characterize the tissue organization at different spatial scales. As a proof-of-principle, we applied the proposed method to healthy and pathologically remodeled human cardiac tissue strips. Preliminary results suggest the reliability of the method: strips from a healthy donor are characterized by a well-organized tissue, where the local disarray is log-normally distributed and slightly depends on the spatial scale of analysis; on the contrary, pathological strips show pronounced tissue disorganization, characterized by local disarray significantly dependent on the spatial scale of analysis. A virtual sample generator is developed to link this multi-scale disarray analysis with the underlying cellular architecture. This approach allowed us to quantitatively assess tissue organization in terms of 3D myocyte angular dispersion and may pave the way for developing novel predictive models based on structural data at cellular resolution.

Novel Optics-Based Approaches for Cardiac Electrophysiology: A Review.

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 2018, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research.

Alpha and beta myosin isoforms and human atrial and ventricular contraction.

Human atrial and ventricular contractions have distinct mechanical characteristics including speed of contraction, volume of blood delivered and the range of pressure generated. Notably, the ventricle expresses predominantly ß-cardiac myosin while the atrium expresses mostly the α-isoform. In recent years exploration of the properties of pure α- & ß-myosin isoforms have been possible in solution, in isolated myocytes and myofibrils. This allows us to consider the extent to which the atrial vs ventricular mechanical characteristics are defined by the myosin isoform expressed, and how the isoform properties are matched to their physiological roles. To do this we Outline the essential feature of atrial and ventricular contraction; Explore the molecular structural and functional characteristics of the two myosin isoforms; Describe the contractile behaviour of myocytes and myofibrils expressing a single myosin isoform; Finally we outline the outstanding problems in defining the differences between the atria and ventricles. This allowed us consider what features of contraction can and cannot be ascribed to the myosin isoforms present in the atria and ventricles.

Pathophysiology and Treatment of Hypertrophic Cardiomyopathy: New Perspectives.

PURPOSE OF REVIEW: We provide a state of the art of therapeutic options in hypertrophic cardiomyopathy (HCM), focusing on recent advances in our understanding of the pathophysiology of sarcomeric disease. RECENT FINDINGS: A wealth of novel information regarding the molecular mechanisms associated with the clinical phenotype and natural history of HCM have been developed over the last two decades. Such advances have only recently led to a number of controlled randomized studies, often limited in size and fortune. Recently, however, the allosteric inhibitors of cardiac myosin adenosine triphosphatase, countering the main pathophysiological abnormality associated with HCM-causing mutations, i.e. hypercontractility, have opened new management perspectives. Mavacamten is the first drug specifically developed for HCM used in a successful phase 3 trial, with the promise to reach symptomatic obstructive patients in the near future. In addition, the fine characterization of cardiomyocyte electrophysiological remodelling has recently highlighted relevant therapeutic targets. Current therapies for HCM focus on late disease manifestations without addressing the intrinsic pathological mechanisms. However, novel evidence-based approaches have opened the way for agents targeting HCM molecular substrates. The impact of these targeted interventions will hopefully alter the natural history of the disease in the near future.

Mavacamten has a differential impact on force generation in myofibrils from rabbit psoas and human cardiac muscle.

Mavacamten (MYK-461) is a small-molecule allosteric inhibitor of sarcomeric myosins being used in preclinical/clinical trials for hypertrophic cardiomyopathy treatment. A better understanding of its impact on force generation in intact or skinned striated muscle preparations, especially for human cardiac muscle, has been hindered by diffusional barriers. These limitations have been overcome by mechanical experiments using myofibrils subject to perturbations of the contractile environment by sudden solution changes. Here, we characterize the action of mavacamten in human ventricular myofibrils compared with fast skeletal myofibrils from rabbit psoas. Mavacamten had a fast, fully reversible, and dose-dependent negative effect on maximal Ca2+-activated isometric force at 15°C, which can be explained by a sudden decrease in the number of heads functionally available for interaction with actin. It also decreased the kinetics of force development in fast skeletal myofibrils, while it had no effect in human ventricular myofibrils. For both myofibril types, the effects of mavacamten were independent from phosphate in the low-concentration range. Mavacamten did not alter force relaxation of fast skeletal myofibrils, but it significantly accelerated the relaxation of human ventricular myofibrils. Lastly, mavacamten had no effect on resting tension but inhibited the ADP-stimulated force in the absence of Ca2+. Altogether, these effects outline a motor isoform-specific dependence of the inhibitory effect of mavacamten on force generation, which is mediated by a reduction in the availability of strongly actin-binding heads. Mavacamten may thus alter the interplay between thick and thin filament regulation mechanisms of contraction in association with the widely documented drug effect of stabilizing myosin motor heads into autoinhibited states.

Arrhythmogenic potential of myocardial disarray in hypertrophic cardiomyopathy: genetic basis, functional consequences and relation to sudden cardiac death.

Myocardial disarray is defined as disorganized cardiomyocyte spatial distribution, with loss of physiological fibre alignment and orientation. Since the first pathological descriptions of hypertrophic cardiomyopathy (HCM), disarray appeared as a typical feature of this condition and sparked vivid debate regarding its specificity to the disease and clinical significance as a diagnostic marker and a risk factor for sudden death. Although much of the controversy surrounding its diagnostic value in HCM persists, it is increasingly recognized that myocardial disarray may be found in physiological contexts and in cardiac conditions different from HCM, raising the possibility that central focus should be placed on its quantity and distribution, rather than a mere presence. While further studies are needed to establish what amount of disarray should be considered as a hallmark of the disease, novel experimental approaches and emerging imaging techniques for the first time allow ex vivo and in vivo characterization of the myocardium to a molecular level. Such advances hold the promise of filling major gaps in our understanding of the functional consequences of myocardial disarray in HCM and specifically on arrhythmogenic propensity and as a risk factor for sudden death. Ultimately, these studies will clarify whether disarray represents a major determinant of the HCM clinical profile, and a potential therapeutic target, as opposed to an intriguing but largely innocent bystander.

The relation between sarcomere energetics and the rate of isometric tension relaxation in healthy and diseased cardiac muscle.

Full muscle relaxation happens when [Ca2+] falls below the threshold for force activation. Several experimental models, from whole muscle organs and intact muscle down to skinned fibers, have been used to explore the cascade of kinetic events leading to mechanical relaxation. The use of single myofibrils together with fast solution switching techniques, has provided new information about the role of cross-bridge (CB) dissociation in the time course of isometric force decay. Myofibril's relaxation is biphasic starting with a slow seemingly linear phase, with a rate constant, slow kREL, followed by a fast mono-exponential phase. Sarcomeres remain isometric during the slow force decay that reflects CB detachment under isometric conditions while the final fast relaxation phase begins with a sudden give of few sarcomeres and is then dominated by intersarcomere dynamics. Based on a simple two-state model of the CB cycle, myofibril slow kREL represents the apparent forward rate with which CBs leave force generating states (gapp) under isometric conditions and correlates with the energy cost of tension generation (ATPase/tension ratio); in short slow kREL ~ gapp ~ tension cost. The validation of this relationship is obtained by simultaneously measuring maximal isometric force and ATP consumption in skinned myocardial strips that provide an unambiguous determination of the relation between contractile and energetic properties of the sarcomere. Thus, combining kinetic experiments in isolated myofibrils and mechanical and energetic measurements in multicellular cardiac strips, we are able to provide direct evidence for a positive linear correlation between myofibril isometric relaxation kinetics (slow kREL) and the energy cost of force production both measured in preparations from the same cardiac sample. This correlation remains true among different types of muscles with different ATPase activities and also when CB kinetics are altered by cardiomyopathy-related mutations. Sarcomeric mutations associated to hypertrophic cardiomyopathy (HCM), a primary cardiac disorder caused by mutations in genes encoding sarcomeric proteins, have been often found to accelerate CB turnover rate and increase the energy cost of myocardial contraction. Here we review data showing that faster CB detachment results in a proportional increase in the energetic cost of tension generation in heart samples from both HCM patients and mouse models of the disease.

T-tubule remodeling in human hypertrophic cardiomyopathy.

The highly organized transverse T-tubule membrane system represents the ultrastructural substrate for excitation-contraction coupling in ventricular myocytes. While the architecture and function of T-tubules have been well described in animal models, there is limited morpho-functional data on T-tubules in human myocardium. Hypertrophic cardiomyopathy (HCM) is a primary disease of the heart muscle, characterized by different clinical presentations at the various stages of its progression. Most HCM patients, indeed, show a compensated hypertrophic disease ("non-failing hypertrophic phase"), with preserved left ventricular function, and only a small subset of individuals evolves into heart failure ("end stage HCM"). In terms of T-tubule remodeling, the "end-stage" disease does not differ from other forms of heart failure. In this review we aim to recapitulate the main structural features of T-tubules during the "non-failing hypertrophic stage" of human HCM by revisiting data obtained from human myectomy samples. Moreover, by comparing pathological changes observed in myectomy samples with those introduced by acute (experimentally induced) detubulation, we discuss the role of T-tubular disruption as a part of the complex excitation-contraction coupling remodeling process that occurs during disease progression. Lastly, we highlight how T-tubule morpho-functional changes may be related to patient genotype and we discuss the possibility of a primitive remodeling of the T-tubule system in rare HCM forms associated with genes coding for proteins implicated in T-tubule structural integrity, formation and maintenance.