Sarcomere Contraction Research Articles

Differentiating the effects of β-adrenergic stimulation and stretch on calcium and force dynamics using a novel electromechanical cardiomyocyte model.

Cardiac electrophysiology and mechanics are strongly interconnected. Calcium is crucial in this complex interplay through its role in cellular electrophysiology and sarcomere contraction. We aim to differentiate the effects of acute β-adrenergic stimulation (β-ARS) and cardiomyocyte stretch (increased sarcomere length) on calcium-transient dynamics and force generation, using a novel computational model of cardiac electromechanics. We implemented a bidirectional coupling between the O'Hara-Rudy model of human ventricular electrophysiology and the MechChem model of sarcomere mechanics through the buffering of calcium by troponin. The coupled model was validated using experimental data from large mammals or human samples. Calcium transient and force were simulated for various degrees of β-ARS and initial sarcomere lengths. The model reproduced force-frequency, quick-release, and isotonic contraction experiments, validating the bidirectional electromechanical interactions. An increase in β-ARS increased the amplitudes of force (augmented inotropy) and calcium transient, and shortened both force and calcium-transient duration (lusitropy). An increase in sarcomere length increased force amplitude even more, but decreased calcium-transient amplitude and increased both force and calcium-transient duration. Finally, a gradient in relaxation along the thin filament may explain the nonmonotonic decay in cytosolic calcium observed with high tension. Using a novel coupled human electromechanical model, we identified differential effects of β-ARS and stretch on calcium and force. Stretch mostly contributed to increased force amplitude and β-ARS to the reduction of calcium and force duration. We showed that their combination, rather than individual contributions, is key to ensure force generation, rapid relaxation, and low diastolic calcium levels.NEW & NOTEWORTHY This work identifies the contribution of electrical and mechanical alterations to regulation of calcium and force under exercise-like conditions using a novel human electromechanical model integrating ventricular electrophysiology and sarcomere mechanics. By better understanding their individual and combined effects, this can uncover arrhythmogenic mechanisms in exercise-like situations. This publicly available model is a crucial step toward understanding the complex interplay between cardiac electrophysiology and mechanics to improve arrhythmia risk prediction and treatment.

Abstract MP103: Methylglyoxal Modifications on Myofilament Proteins Caused by Diabetes Block Heart Failure Drug Targets Through Changing Tropomyosin Position

We previously discovered methylglyoxal (MG), a glycolysis byproduct, modifies arginine and lysine residues on myofilament proteins to a greater extent in patients with diabetes and heart failure than non-failing controls. These modifications reduced myofilament calcium sensitivity (pCa 50 ), maximal calcium activated force (F max ) and cross-bridge cycling dynamics. We hypothesized that compensating for these modifications might slow or halt the development of diabetic cardiomyopathy. We attempted to pharmacologically compensate for the negative functional effects of MG using omecamtiv mecarbil (OM), a myosin activator currently in clinical trials. As reported previously, we found that OM increased pCa 50 and decreased F max in normal myocytes. However, in myocytes pretreated with MG, OM had no effect of pCa 50 but still decreased F max . These data suggest OM may have less, or potentially detrimental effects in diabetic patients.To understand why MG might block the actions of sarcomere drug targets, we aimed to further elucidate the molecular mechanisms of MG. We utilized a computer model of sarcomere contraction to predict what molecular effects of MG would explain the functional data we collected. The model predicted a simultaneous increase in cross-bridge detachment rate and a decrease in the closed-to-blocked tropomyosin transition rate could explain the data. Stopped flow experiments measuring myofilament kinetics indicated a decreased transition rate between “closed” and “blocked” tropomyosin state but did not indicate a difference in the cross-bridge detachment rate upon treatment with MG. This finding further explained the observation that MG blocks OM’s effects - since a myosin activator would not necessarily be effective if MG affected tropomyosin position on actin. We hypothesize that a residue we found to be modified by MG on actin, K291 would be important for the molecular mechanism observed, since it is located in a critical position for the interaction between actin and tropomyosin. In this study, we have elucidated the mechanism of MG’s detrimental myofilament impact and found that that these modifications reduce the efficacy of OM. This mechanistic understanding will help drive future identification of therapeutic targets.

The architecture and function of cardiac dyads.

Cardiac excitation-contraction (EC) coupling, which links plasma membrane depolarization to activation of cardiomyocyte contraction, occurs at dyads, the nanoscopic microdomains formed by apposition of transverse (T)-tubules and junctional sarcoplasmic reticulum (jSR). In a dyadic junction, EC coupling occurs through Ca2+-induced Ca2+ release. Membrane depolarization opens voltage-gated L-type Ca2+ channels (LTCCs) in the T-tubule. The resulting influx of extracellular Ca2+ into the dyadic cleft opens Ca2+ release channels known as ryanodine receptors (RYRs) in the jSR, leading to the rapid increase in cytosolic Ca2+ that triggers sarcomere contraction. The efficacy of LTCC-RYR communication greatly affects a myriad of downstream intracellular signaling events, and it is controlled by many factors, including T-tubule and jSR structure, spatial distribution of ion channels, and regulatory proteins that closely regulate the activities of channels within dyads. Alterations in dyad architecture and/or channel activity are seen in many types of heart disease. This review will focus on the current knowledge regarding cardiac dyad structure and function, their alterations in heart failure, and new approaches to study the composition and function of dyads.

Sarcomere Lengths Become More Uniform Over Time in Intact Muscle-Tendon Unit During Isometric Contractions

The seemingly uniform striation pattern of skeletal muscles, quantified in terms of sarcomere lengths (SLs), is inherently non-uniform across all hierarchical levels. The SL non-uniformity theory has been used to explain the force creep in isometric contractions, force depression following shortening of activated muscle, and residual force enhancement following lengthening of activated muscle. Our understanding of sarcomere contraction dynamics has been derived primarily from in vitro experiments using regular bright-field light microscopy or laser diffraction techniques to measure striation/diffraction patterns in isolated muscle fibers or myofibrils. However, the collagenous extracellular matrices present around the muscle fibers, as well as the complex architecture in the whole muscles may lead to different contraction dynamics of sarcomeres than seen in the in vitro studies. Here, we used multi-photon excitation microscopy to visualize in situ individual sarcomeres in intact muscle tendon units (MTUs) of mouse tibialis anterior (TA), and quantified the temporal changes of SL distribution as a function of SLs in relaxed and maximally activated muscles for quasi-steady state, fixed-end isometric conditions. The corresponding muscle forces were simultaneously measured using a force transducer. We found that SL non-uniformity, quantified by the coefficient of variation (CV) of SLs, decreased at a rate of 1.9–3.1%/s in the activated muscles, but remained constant in the relaxed muscles. The force loss during the quasi-steady state likely did not play a role in the decrease of SL non-uniformity, as similar force losses were found in the activated and relaxed muscles, but the CV of SLs in the relaxed muscles underwent negligible change over time. We conclude that sarcomeres in the mid-belly of maximally contracting whole muscles constantly re-organize their lengths into a more uniform pattern over time. The molecular mechanisms accounting for SL non-uniformity appear to differ in active and passive muscles, and need further elucidation, as do the functional implications of the SL non-uniformity.

Foci of Segmentally Contracted Sarcomeres in Trapezius Muscle Biopsy Specimens in Myalgic and Nonmyalgic Human Subjects: Preliminary Results.

The myofascial trigger point hypothesis postulates that there are small foci of contracted sarcomeres in resting skeletal muscle. Only one example, in canine muscle, has been published previously. This study evaluated human muscle biopsies for foci of contracted sarcomeres. The Departments of Rehabilitation Sciences and Physiotherapy at Ghent University, Ghent, Belgium. Biopsies from 28 women with or without trapezius myalgia were evaluated, 14 in each group. Muscle biopsies were obtained from regions of taut bands in the trapezius muscle and processed for light and electron microscopy and for histochemical analysis. Examination of the biopsies was blinded as to group. A small number of foci of segmentally contracted sarcomeres were identified. One fusiform segmental locus involved the entire muscle fiber in tissue from a myalgic subject. Several transition zones from normal to contracted sarcomeres were found in both myalgic and nonmyalgic subjects. The distance between Z-lines in contracted sarcomeres was about 25-45% of the same distance in normal sarcomeres. Z-lines were disrupted and smeared in the contracted sarcomeres. A small number of foci of segmentally contracted sarcomeres were found in relaxed trapezius muscle in human subjects, a confirmation of the only other example of spontaneous segmental contraction of sarcomeres (in a canine muscle specimen), consistent with the hypothesis of trigger point formation and with the presence of trigger point end plate noise.

A cardiomyocyte show of force: A fluorescent alpha-actinin reporter line sheds light on human cardiomyocyte contractility versus substrate stiffness

Cardiovascular disease is often associated with cardiac remodeling, including cardiac fibrosis, which may lead to increased stiffness of the heart wall. This stiffness in turn may cause subsequent failure of cardiac myocytes, however the response of these cells to increased substrate stiffness is largely unknown. To investigate the contractile response of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to increased substrate stiffness, we generated a stable transgenic human pluripotent stem cell line expressing a fusion protein of α-Actinin and fluorescent mRubyII in a previously characterized NKX2.5-GFP reporter line. Cardiomyocytes differentiated from this line were subjected to a substrate with stiffness ranging from 4 kPa to 101 kPa, while contraction of sarcomeres and bead displacement in the substrate were measured for each single cardiomyocyte. We found that sarcomere dynamics in hPSC-CMs on polyacrylamide gels of increasing stiffness are not affected above physiological levels (21 kPa), but that contractile force increases up to a stiffness of 90 kPa, at which cell shortening, deducted from bead displacement, is significantly reduced compared to physiological stiffness. We therefore hypothesize that this discrepancy may be the cause of intracellular stress that leads to hypertrophy and consequent heart failure in vivo.

Variant R94C in TNNT2-Encoded Troponin T Predisposes to Pediatric Restrictive Cardiomyopathy and Sudden Death Through Impaired Thin Filament Relaxation Resulting in Myocardial Diastolic Dysfunction.

BackgroundPediatric‐onset restrictive cardiomyopathy (RCM) is associated with high mortality, but underlying mechanisms of disease are under investigated. RCM‐associated diastolic dysfunction secondary to variants in TNNT2‐encoded cardiac troponin T (TNNT2) is poorly described.Methods and ResultsGenetic analysis of a proband and kindred with RCM identified TNNT2‐R94C, which cosegregated in a family with 2 generations of RCM, ventricular arrhythmias, and sudden death. TNNT2‐R94C was absent among large, population‐based cohorts Genome Aggregation Database (gnomAD) and predicted to be pathologic by in silico modeling. Biophysical experiments using recombinant human TNNT2‐R94C demonstrated impaired cardiac regulation at the molecular level attributed to reduced calcium‐dependent blocking of myosin's interaction with the thin filament. Computational modeling predicted a shift in the force‐calcium curve for the R94C mutant toward submaximal calcium activation compared within the wild type, suggesting low levels of muscle activation even at resting calcium concentrations and hypercontractility following activation by calcium.ConclusionsThe pathogenic TNNT2‐R94C variant activates thin‐filament–mediated sarcomeric contraction at submaximal calcium concentrations, likely resulting in increased muscle tension during diastole and hypercontractility during systole. This describes the proximal biophysical mechanism for development of RCM in this family.

Quantifying Contributions of Cellular Mechanical Myocardial Properties on Left Ventricular Contractile Function in Aortic Banded Rats

Left ventricular (LV) contractile dysfunction is known to be associated with altered cellular myocardial properties. Quantitative translation from cellular mechanical properties to whole-organ cardiomyopathy phenotype, however, is an ongoing challenge. To quantitatively map cell-level pathophysiology to whole-organ contractile function in aortic banded (AB) 6-weeks post-surgery rats (Røe et al. 2017), we consider an in silicomodel of LV hypertrophy which combines mathematical models of cellular electrophysiology and calcium dynamics, sarcomere contraction and whole-organ mechanics solved over geometries derived from control and AB rat hearts. The resulting multi-scale computationally expensive model is regulated by >100 parameters. To assess parameters impact on the model outputs we developed a computationally effective approach for global sensitivity analysis (GSA) of rat cardiac mechanics based on Gaussian process (GP) emulation. We identified eight key parameters regulating ventricular mechanics such as myofilament calcium sensitivity (Ca50), and described the LV contractile function using eleven measurements including ejection fraction (EF) and pressure-volume loop. We found Ca50 to be the most significant parameter in explaining the total variance in both the control and AB rat (56%, 53%, 56%, 57% of the total variance of EF, isovolumetric relaxation time, peak pressure and maximum pressure rise rate, respectively, in control, and 65%, 47%, 58%, 56%, respectively, in AB rat). Calcium unbinding rate from Troponin C and maximal cellular tension yielded, respectively, the second and the third highest impact on the above LV features' total variances. Tissue stiffness and cross-bridge cycling rate impacted EF, ejection time, diastolic time and peak and end-systolic pressure features in the AB rat only. Our GP-based GSA approach enables computationally efficient identification of key components in 3D ventricular mechanics models, a potential path towards discovering new targets for heart failure treatment.

Why Myocardial Relaxation Always Slows at Cardiac Pathology?

Chronic heart failure (CHF) in most cases is due to a decrease in myocardial contractility. In particular, this results in a reduction in the maximum rate of the pressure development in the left ventricle. At the same time the maximal rate of pressure fall at relaxation is also reduced. This is not surprising, since both depend on Ca ++ myoplasmic concentration. But most of cardiac pathologies have been associated with the impairement of myocardial relaxation to a greater extent than the contraction. In the review a new view has been proposed according to which this phenomenon is attributable to restructuring of titin, the sarcomeric protein that connects the ends of myosin filaments with the sarcomeric board, lines Z. A spring-like molecule of titin shrinks at sarcomeric contraction and straightens in parallel with removing of Ca ++ from myofibrils. A reduction of its stiffness, facilitating the filling of the left ventricle, can reduce restoring force of titin and thereby slow relaxation. The survey provides information about the functions of the calcium transport system and titin in the normal heart and in CHF observed both in experimental models and in patients.

Spatiotemporal Characterizations of Spontaneously Beating Cardiomyocytes with Adaptive Reference Digital Image Correlation

We developed an Adaptive Reference-Digital Image Correlation (AR-DIC) method that enables unbiased and accurate mechanics measurements of moving biological tissue samples. We applied the AR-DIC analysis to a spontaneously beating cardiomyocyte (CM) tissue, and could provide correct quantifications of tissue displacement and strain for the beating CMs utilizing physiologically-relevant, sarcomere displacement length-based contraction criteria. The data were further synthesized into novel spatiotemporal parameters of CM contraction to account for the CM beating homogeneity, synchronicity, and propagation as holistic measures of functional myocardial tissue development. Our AR-DIC analyses may thus provide advanced non-invasive characterization tools for assessing the development of spontaneously contracting CMs, suggesting an applicability in myocardial regenerative medicine.

P.112Two murine models for tubular aggregate myopathy with mutations in Stim1 and Orai1

Calcium (Ca2+) is a key mediator in a wide range of cellular functions. Ca2+ released from the sarcoplasmic reticulum (SR) induces sarcomeric contraction of myofibrils, which is a primary mechanism mediating Ca2+ homeostasis in skeletal muscle. At the depletion of Ca2+ in the SR, store-operated Ca2+ entry (SOCE) is induced to obtain Ca2+ from the extracellular area. SR protein, STIM1 and Ca2+ channel protein on the plasma membrane, ORAI1 work on SOCE in skeletal myofibers. Tubular aggregate myopathy (TAM) has been known to be caused by constitutively activated SOCE owing to the dominant mutations in ORAI1 or STIM1 consequently.

Abstract 860: Functional Characterization of a Novel Human Heart-specific Microprotein With a Potential Mitochondrial Localization and Role in Sarcomere Dynamics

Cardiac microproteins are an emerging class of important regulators of cellular homeostasis, often encoded by presumed long noncoding RNAs. With only few described so far, their prevalence and biological significance largely remains unclear. To create an atlas of cardiac-expressed microproteins, we performed ribosome profiling on 80 human heart samples. This revealed the translation of hundreds of previously undetected microproteins, which we validated in vivo by targeted mass spectrometry. A single heart-specific microprotein caught our attention as it showed strong expression coregulation with components of mitochondrial OXPHOS pathway. We next deleted this microprotein's lncRNA host gene in hiPSCs using CRISPR/Cas9, and, upon differentiation into 30-d old cardiomyocytes, find a strong expression upregulation of genes involved in oxidative phosphorylation and calcium handling. To investigate the impact of the microprotein KO on cardiomyocyte mechanical function, we assessed sarcomere dynamics using an endogenous TTN-GFP tag, enabling live cell imaging analysis with the SarcTrack algorithm. Preliminary analysis sowes a putative increase in sarcomere contraction and relaxation times, suggesting a repressive regulatory function of the eliminated microprotein encoding lncRNA.

Abstract 341: Efficient Large-scale Sarcomere Tracking (sarctrack) to Assess HCM Variants in iPSC-CMs

Variants that drive HCM and associated adverse patient outcomes are found in the cardiac sarcomere. These variants range from those that are known to be pathogenic to those that are likely pathogenic or even variants of unknown significance (VUS). CRISPR/Cas-9 engineering has accelerated our ability to generate variants in iPSC to probe changes in cellular function and assess cellular pathogenicity in VUSs. However, iPSC-CMs are not as functionally mature as adult cardiomyocytes. For this reason we have developed a platform to assess contractile function directly at the level of the sarcomere. We use a custom built MatLab algorithm to assess sarcomere length, contraction time, relaxation time, and beat rate of individual sarcomeres within iPSC-CMs. Sarcomeres are visualised using reporter lines that have been engineered with an N-terminal TTN-GFP. We assess the contractile function of thick filament variants in MYH7 and MYBPC3. We show the ability to detect changes in key contractile parameters. This platform allows the screening of pharmacological compounds against these reporter lines with engineered variants.

SarcTrack.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues. We aimed to develop an analysis platform that directly, rapidly, and automatically tracks sarcomeres in beating cardiomyocytes. The platform should assess sarcomere content, contraction and relaxation parameters, and beat rate. We developed SarcTrack, a MatLab software that monitors fluorescently tagged sarcomeres in hiPSC-CMs. The algorithm determines sarcomere content, sarcomere length, and returns rates of sarcomere contraction and relaxation. By rapid measurement of hundreds of sarcomeres in each hiPSC-CM, SarcTrack provides large data sets for robust statistical analyses of multiple contractile parameters. We validated SarcTrack by analyzing drug-treated hiPSC-CMs, confirming the contractility effects of compounds that directly activate (CK-1827452) or inhibit (MYK-461) myosin molecules or indirectly alter contractility (verapamil and propranolol). SarcTrack analysis of hiPSC-CMs carrying a heterozygous truncation variant in the myosin-binding protein C ( MYBPC3) gene, which causes hypertrophic cardiomyopathy, recapitulated seminal disease phenotypes including cardiac hypercontractility and diminished relaxation, abnormalities that normalized with MYK-461 treatment. SarcTrack provides a direct and efficient method to quantitatively assess sarcomere function. By improving existing contractility analysis methods and overcoming technical challenges associated with functional evaluation of hiPSC-CMs, SarcTrack enhances translational prospects for sarcomere-regulating therapeutics and accelerates interrogation of human cardiac genetic variants.

Molecular Mechanism of a Mutation Implicated in Pediatric-Onset Heart Disesase

Familial cardiomyopathies are the leading cause of sudden cardiac death among young people, and pediatric-onset disease is particularly devastating. Dilated cardiomyopathy (DCM) is a familial cardiomyopathy characterized by dilation of the left ventricular chamber of the heart. DCM can be caused by mutations in proteins involved in cardiac muscle contraction, including troponin T; however, it is not well understood how these mutations affect cardiac contraction and contribute to the disease phenotype. To address this critical gap in our knowledge, we examined the molecular-level impact of a troponin T mutation implicated in pediatric-onset DCM, R134G. In vitro motility assays, in which troponin/tropomyosin-regulated thin filaments are propelled across a myosin-coated surface, were carried out over a range of physiologically relevant calcium concentrations. These measurements revealed decreased calcium sensitivity in the troponin complex containing the R134G mutant. To elucidate the molecular mechanism underlying the altered calcium sensitivity conferred by the R134G mutation, we used stopped-flow and steady-state fluorescence measurements to determine the equilibrium constants that define binding of myosin to regulated thin filaments. By inputting these equilibrium constants into a computational model of the sarcomeric contraction, we were able to explain the observed changes in calcium-based myosin regulation and make testable predictions about the effect of the mutation on cardiac contractility. Taken together, our results provide new insights into the mechanism of pediatric heart disease.

Regulation of Myofilament Force and Loaded Shortening by Skeletal Myosin Binding Protein-C

Myosin binding protein C (MyBP-C) is a 125-140-kD protein located in the C-zone of each half-thick filament. It is thought to be an important regulator of contraction, but its precise role is unclear. Here we investigate mechanisms by which skeletal MyBP-C regulates myofilament function using rat permeabilized skeletal muscle fibers. We mount either slow-twitch or fast-twitch skeletal muscle fibers between a force transducer and motor, use Ca2+ to activate a range of forces, and measure contractile properties including transient force overshoot, rate of force development, and loaded sarcomere shortening. The transient force overshoot is greater in slow-twitch than fast-twitch fibers at all Ca2+ activation levels. In slow-twitch fibers, protein kinase A (PKA) treatment (a) augments phosphorylation of slow skeletal MyBP-C (sMyBP-C), (b) doubles the magnitude of the relative transient force overshoot at low Ca2+ activation levels, and (c) increases force development rates at all Ca2+ activation levels. We also investigate the role that phosphorylated and dephosphorylated sMyBP-C plays in loaded sarcomere shortening. We test the hypothesis that MyBP-C acts as a brake to filament sliding within the myofilament lattice by measuring sarcomere shortening as thin filaments traverse into the C-zone during lightly loaded slow-twitch fiber contractions. Before PKA treatment, shortening velocity decelerates as sarcomeres traverse from ∼3.10 to ∼3.00 µm. After PKA treatment, sarcomeres shorten a greater distance and exhibit less deceleration during similar force clamps. After sMyBP-C dephosphorylation, sarcomere length traces display a brief recoil (i.e., bump) that initiates at ∼3.06 µm during loaded shortening. Interestingly, the timing of the bump shifts with changes in load but manifests at the same sarcomere length. Our results suggest that sMyBP-C and its phosphorylation state regulate sarcomere contraction by a combination of cross-bridge recruitment, modification of cross-bridge cycling kinetics, and alteration of drag forces that originate in the C-zone.

Regulation of myofilament force and loaded shortening by skeletal myosin binding protein C.

Myosin binding protein C (MyBP-C) is a 125-140-kD protein located in the C-zone of each half-thick filament. It is thought to be an important regulator of contraction, but its precise role is unclear. Here we investigate mechanisms by which skeletal MyBP-C regulates myofilament function using rat permeabilized skeletal muscle fibers. We mount either slow-twitch or fast-twitch skeletal muscle fibers between a force transducer and motor, use Ca2+ to activate a range of forces, and measure contractile properties including transient force overshoot, rate of force development, and loaded sarcomere shortening. The transient force overshoot is greater in slow-twitch than fast-twitch fibers at all Ca2+ activation levels. In slow-twitch fibers, protein kinase A (PKA) treatment (a) augments phosphorylation of slow skeletal MyBP-C (sMyBP-C), (b) doubles the magnitude of the relative transient force overshoot at low Ca2+ activation levels, and (c) increases force development rates at all Ca2+ activation levels. We also investigate the role that phosphorylated and dephosphorylated sMyBP-C plays in loaded sarcomere shortening. We test the hypothesis that MyBP-C acts as a brake to filament sliding within the myofilament lattice by measuring sarcomere shortening as thin filaments traverse into the C-zone during lightly loaded slow-twitch fiber contractions. Before PKA treatment, shortening velocity decelerates as sarcomeres traverse from ∼3.10 to ∼3.00 µm. After PKA treatment, sarcomeres shorten a greater distance and exhibit less deceleration during similar force clamps. After sMyBP-C dephosphorylation, sarcomere length traces display a brief recoil (i.e., "bump") that initiates at ∼3.06 µm during loaded shortening. Interestingly, the timing of the bump shifts with changes in load but manifests at the same sarcomere length. Our results suggest that sMyBP-C and its phosphorylation state regulate sarcomere contraction by a combination of cross-bridge recruitment, modification of cross-bridge cycling kinetics, and alteration of drag forces that originate in the C-zone.

Role of intrinsic disorder in muscle sarcomeres.

The role and utility of intrinsically disordered regions (IDRs) is reviewed for two groups of sarcomeric proteins, such as members of tropomodulin/leiomodin (Tmod/Lmod) protein homology group and myosin binding protein C (MyBP-C). These two types of sarcomeric proteins represent very different but strongly interdependent functions, being responsible for maintaining structure and operation of the muscle sarcomere. The role of IDRs in the formation of complexes between thin filaments and Tmods/Lmods is discussed within the framework of current understanding of the thin filament length regulation. For MyBP-C, the function of IDRs is discussed in the context of MYBP-C-dependent sarcomere contraction and actomyosin activation.

Linking cross-bridge cycling kinetics to response to cardiac resynchronization therapy: a multiscale modelling study.

Cardiac resynchronization therapy (CRT) is currently the most widely used treatment for heart failure patients with left bundle branch block (LBBB). In recent years, the presence of septal rebound stretch (SRS) has been found to be a positive indicator for CRT response although the mechanism is unknown. In an attempt to understand the relation between cellular mechanics and global pump function in CRT patients, we utilize the CircAdapt closed-loop cardiovascular system model in combination with the MechChem model of cardiac sarcomere contraction. Left bundle branch block has been simulated with increasing delay in left ventricular free wall and septal wall activation. In addition to the electrical dyssynchrony, myocardial mechanical function was diminished by decreasing the cross-bridge cycling rate. Our results have shown that a decrease in the cross-bridge cycling rate in addition to LBBB resulted in a decrease in SRS with a concomitant decreased response to resynchronization. The results of our multiscale modelling study suggest that, while greater SRS during systole clearly indicates electrical dyssynchrony, it also predicts mechanical viability and healthy cross-bridge cycling rates in the myocardium. Hence, SRS positively indicates response to CRT.

Genetic Basis of Severe Childhood-Onset Cardiomyopathies

BackgroundChildhood cardiomyopathies are progressive and often lethal disorders, forming the most common cause of heart failure in children. Despite severe outcomes, their genetic background is still poorly characterized. ObjectivesThe purpose of this study was to characterize the genetics of severe childhood cardiomyopathies in a countrywide cohort. MethodsThe authors collected a countrywide cohort, KidCMP, of 66 severe childhood cardiomyopathies from the sole center in Finland performing cardiac transplantation. For genetic diagnosis, next-generation sequencing and subsequent validation using genetic, cell biology, and computational approaches were used. ResultsThe KidCMP cohort presents remarkable early-onset and severe disorders: the median age of diagnosis was 0.33 years, and 17 patients underwent cardiac transplantation. The authors identified the pathogenic variants in 39% of patients: 46% de novo, 34% recessive, and 20% dominantly-inherited. The authors report NRAP underlying childhood dilated cardiomyopathy, as well as novel phenotypes for known heart disease genes. Some genetic diagnoses have immediate implications for treatment: CALM1 with life-threatening arrhythmias, and TAZ with good cardiac prognosis. The disease genes converge on metabolic causes (PRKAG2, MRPL44, AARS2, HADHB, DNAJC19, PPA2, TAZ, BAG3), MAPK pathways (HRAS, PTPN11, RAF1, TAB2), development (NEK8 and TBX20), calcium signaling (JPH2, CALM1, CACNA1C), and the sarcomeric contraction cycle (TNNC1, TNNI3, ACTC1, MYH7, NRAP). ConclusionsChildhood cardiomyopathies are typically caused by rare, family-specific mutations, most commonly de novo, indicating that next-generation sequencing of trios is the approach of choice in their diagnosis. Genetic diagnoses may suggest intervention strategies and predict prognosis, offering valuable tools for prioritization of patients for transplantation versus conservative treatment.