The effect of muscle ultrastructure on the force, displacement and work capacity of skeletal muscle.

Skeletal muscle powers animal movement through interactions between the contractile proteins, actin and myosin. Structural variation contributes greatly to the variation in mechanical performance observed across muscles. In vertebrates, gross structural variation occurs in the form of changes in the muscle cross-sectional area : fibre length ratio. This results in a trade-off between force and displacement capacity, leaving work capacity unaltered. Consequently, the maximum work per unit volume-the work density-is considered constant. Invertebrate muscle also varies in muscle ultrastructure, i.e. actin and myosin filament lengths. Increasing actin and myosin filament lengths increases force capacity, but the effect on muscle fibre displacement, and thus work, capacity is unclear. We use a sliding-filament muscle model to predict the effect of actin and myosin filament lengths on these mechanical parameters for both idealized sarcomeres with fixed actin : myosin length ratios, and for real sarcomeres with known filament lengths. Increasing actin and myosin filament lengths increases stress without reducing strain capacity. A muscle with longer actin and myosin filaments can generate larger force over the same displacement and has a higher work density, so seemingly bypassing an established trade-off. However, real sarcomeres deviate from the idealized length ratio suggesting unidentified constraints or selective pressures.

Myosin inhibitor reverses hypertrophic cardiomyopathy in genotypically diverse pediatric iPSC-cardiomyocytes to mirror variant correction.

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 (V606M; R453C), MYBPC3 (G148R) or digenic variants (MYBPC3 P955fs, TNNI3 A157V). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls, variant-positive CMs show sarcomere disorganization, higher contractility, calcium transients, and ATPase activity. However, only MYH7 and biallelic MYBPC3 variant-positive CMs show stronger myosin-actin binding. Targeted myosin ATPase inhibitors show complete rescue of the phenotype in variant-positive CMs and in cardiac Biowires to mirror isogenic controls. The response is superior to verapamil or metoprolol. Myosin inhibitors can be effective in genotypically diverse HCM highlighting the need for myosin inhibitor drug trials in pediatric HCM.

Myosin folding boosts solubility in cardiac muscle sarcomeres.

The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates that the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected > 5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may affect cardiac contractility.

Myosin-binding protein C regulates the sarcomere lattice and stabilizes the OFF states of myosin heads.

Muscle contraction is produced via the interaction of myofilaments and is regulated so that muscle performance matches demand. Myosin-binding protein C (MyBP-C) is a long and flexible protein that is tightly bound to the thick filament at its C-terminal end (MyBP-CC8C10), but may be loosely bound at its middle- and N-terminal end (MyBP-CC1C7) to myosin heads and/or the thin filament. MyBP-C is thought to control muscle contraction via the regulation of myosin motors, as mutations lead to debilitating disease. We use a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-CC1C7 domains of fast MyBP-C in permeabilized skeletal muscle. We show that cleavage leads to alterations in crossbridge kinetics and passive structural signatures of myofilaments that are indicative of a shift of myosin heads towards the ON state, highlighting the importance of MyBP-CC1C7 to myofilament force production and regulation.

Force and kinetics of fast and slow muscle myosin determined with a synthetic sarcomere-like nanomachine.

Myosin II is the muscle molecular motor that works in two bipolar arrays in each thick filament of the striated (skeletal and cardiac) muscle, converting the chemical energy into steady force and shortening by cyclic ATP-driven interactions with the nearby actin filaments. Different isoforms of the myosin motor in the skeletal muscles account for the different functional requirements of the slow muscles (primarily responsible for the posture) and fast muscles (responsible for voluntary movements). To clarify the molecular basis of the differences, here the isoform-dependent mechanokinetic parameters underpinning the force of slow and fast muscles are defined with a unidimensional synthetic nanomachine powered by pure myosin isoforms from either slow or fast rabbit skeletal muscle. Data fitting with a stochastic model provides a self-consistent estimate of all the mechanokinetic properties of the motor ensemble including the motor force, the fraction of actin-attached motors and the rate of transition through the attachment-detachment cycle. The achievements in this paper set the stage for any future study on the emergent mechanokinetic properties of an ensemble of myosin molecules either engineered or purified from mutant animal models or human biopsies.

Lmod2 is necessary for effective skeletal muscle contraction.

Muscle contraction is a regulated process driven by the sliding of actin-thin filaments over myosin-thick filaments. Lmod2 is an actin filament length regulator and essential for life since human mutations and complete loss of Lmod2 in mice lead to dilated cardiomyopathy and death. To study the little-known role of Lmod2 in skeletal muscle, we created a mouse model with Lmod2 expressed exclusively in the heart but absent in skeletal muscle. Loss of Lmod2 in skeletal muscle results in decreased force production in fast- and slow-twitch muscles. Soleus muscle from rescued Lmod2 knockout mice have shorter thin filaments, increased Lmod3 levels, and present with a myosin fiber type switch from fast myosin heavy chain (MHC) IIA to the slower MHC I isoform. Since Lmod2 regulates thin-filament length in slow-twitch but not fast-twitch skeletal muscle and force deficits were observed in both muscle types, this work demonstrates that Lmod2 regulates skeletal muscle contraction, independent of its role in thin-filament length regulation.

Structure of mavacamten-free human cardiac thick filaments within the sarcomere by cryoelectron tomography.

Heart muscle has the unique property that it can never rest; all cardiomyocytes contract with each heartbeat which requires a complex control mechanism to regulate cardiac output to physiological requirements. Changes in calcium concentration regulate the thin filament activation. A separate but linked mechanism regulates the thick filament activation, which frees sufficient myosin heads to bind the thin filament, thereby producing the required force. Thick filaments contain additional nonmyosin proteins, myosin-binding protein C and titin, the latter being the protein that transmits applied tension to the thick filament. How these three proteins interact to control thick filament activation is poorly understood. Here, we show using 3-D image reconstruction of frozen-hydrated human cardiac muscle myofibrils lacking exogenous drugs that the thick filament is structured to provide three levels of myosin activation corresponding to the three crowns of myosin heads in each 429Å repeat. In one crown, the myosin heads are almost completely activated and disordered. In another crown, many myosin heads are inactive, ordered into a structure called the interacting heads motif. At the third crown, the myosin heads are ordered into the interacting heads motif, but the stability of that motif is affected by myosin-binding protein C. We think that this hierarchy of control explains many of the effects of length-dependent activation as well as stretch activation in cardiac muscle control.

Modeling thick filament activation suggests a molecular basis for force depression.

Multiscale models aiming to connect muscle's molecular and cellular function have been difficult to develop, in part due to a lack of self-consistent multiscale data. To address this gap, we measured the force response from single, skinned rabbit psoas muscle fibers to ramp shortenings and step stretches performed on the plateau region of the force-length relationship. We isolated myosin from the same muscles and, under similar conditions, performed single-molecule and ensemble measurements of myosin's ATP-dependent interaction with actin using laser trapping and in vitro motility assays. We fit the fiber data by developing a partial differential equation model that includes thick filament activation, whereby an increase in force on the thick filament pulls myosin out of an inhibited state. The model also includes a series elastic element and a parallel elastic element. This parallel elastic element models a titin-actin interaction proposed to account for the increase in isometric force after stretch (residual force enhancement). By optimizing the model fit to a subset of our fiber measurements, we specified seven unknown parameters. The model then successfully predicted the remainder of our fiber measurements and also our molecular measurements from the laser trap and in vitro motility. The success of the model suggests that our multiscale data are self-consistent and can serve as a testbed for other multiscale models. Moreover, the model captures the decrease in isometric force observed in our muscle fibers after active shortening (force depression), suggesting a molecular mechanism for force depression, whereby a parallel elastic element combines with thick filament activation to decrease the number of cycling cross-bridges.

Intrafusal cross-bridge dynamics shape history-dependent muscle spindle responses to stretch.

Computational models can be critical to linking complex properties of muscle spindle organs to the sensory information that they encode during behaviours such as postural sway and locomotion where few muscle spindle recordings exist. Here, we augment a biophysical muscle spindle model to predict the muscle spindle sensory signal. Muscle spindles comprise several intrafusal muscle fibres with varied myosin expression and are innervated by sensory neurons that fire during muscle stretch. We demonstrate how cross-bridge dynamics from thick and thin filament interactions affect the sensory receptor potential at the spike initiating region. Equivalent to the Ia afferent's instantaneous firing rate, the receptor potential is modelled as a linear sum of the force and rate change of force (yank) of a dynamic bag1 fibre and the force of a static bag2/chain fibre. We show the importance of inter-filament interactions in (i) generating large changes in force at stretch onset that drive initial bursts and (ii) faster recovery of bag fibre force and receptor potential following a shortening. We show how myosin attachment and detachment rates qualitatively alter the receptor potential. Finally, we show the effect of faster recovery of receptor potential on cyclic stretch-shorten cycles. Specifically, the model predicts history-dependence in muscle spindle receptor potentials as a function of inter-stretch interval (ISI), pre-stretch amplitude and the amplitude of sinusoidal stretches. This model provides a computational platform for predicting muscle spindle response in behaviourally relevant stretches and can link myosin expression seen in healthy and diseased intrafusal muscle fibres to muscle spindle function.

Association between left atrial myopathy and sarcomere mutation in patients with hypertrophic cardiomyopathy: insights into left atrial strain by MRI feature tracking.

OBJECTIVES: Left atrial (LA) myopathy, characterized by LA enlargement and mechanical dysfunction, is associated with worse prognosis in hypertrophic cardiomyopathy (HCM) while the impact of sarcomere mutation on LA myopathy remains unclear. We aimed to assess the association between LA myopathy and sarcomere mutation and to explore the incremental utility of LA strain in mutation prediction. METHODS: A total of 105 consecutive HCM patients (mean age 47.8 ± 11.9 years, 71% male) who underwent HCM-related gene screening and cardiac MRI were retrospectively enrolled. LA volume, ejection fraction and strain indices in reservoir, conduit, and booster-pump phases were investigated respectively. RESULTS: Fifty mutation-positive patients showed higher LA maximal volume index (59.4 ± 28.2 vs 43.8 ± 18.1 mL/m2, p = 0.001), lower reservoir (21.3 ± 7.9 vs 26.2 ± 6.6%, p < 0.001), and booster-pump strain (12.1 ± 5.4 vs 17.1 ± 5.0%, p < 0.001) but similar conduit strain (9.2 ± 4.5 vs 9.1 ± 4.5%, p = 0.909) compared with mutation-negative patients. In multivariate logistic regression, LA booster-pump strain was associated with sarcomere mutation (odds ratio = 0.86, 95% confidence interval: 0.77-0.96, p = 0.010) independent of maximal wall thickness, late gadolinium enhancement, and LA volume. Furthermore, LA booster-pump strain showed incremental value for mutation prediction added to Mayo II score (AUC 0.798 vs 0.709, p = 0.024). CONCLUSIONS: In HCM, mutation-positive patients suffered worse LA enlargement and worse reservoir and booster-pump functions. LA booster-pump strain was a strong factor for sarcomere mutation prediction added to Mayo II score. CLINICAL RELEVANCE STATEMENT: The independent association between sarcomere mutation and left atrial mechanical dysfunction provide new insights into the pathogenesis of atrial myopathy and is helpful to understand the adverse prognosis regarding atrial fibrillation and stroke in mutation-positive patients. KEY POINTS: • In patients with hypertrophic cardiomyopathy, left atrial (LA) reservoir and booster-pump function, but not conduit function, were significantly impaired in mutation-positive patients compared with mutation-negative patients. • LA booster-pump strain measured by MRI-derived feature tracking is feasible to predict sarcomere mutation with high incremental value added to Mayo II score.

Sarcomere level mechanics of the fast skeletal muscle of the medaka fish larva.

The medaka fish (Oryzias latipes) is a vertebrate model used in developmental biology and genetics. Here we explore its suitability as a model for investigating the molecular mechanisms of human myopathies caused by mutations in sarcomeric proteins. To this end, the relevant mechanical parameters of the intact skeletal muscle of wild-type medaka are determined using the transparent tail at larval stage 40. Tails were mounted at sarcomere length of 2.1 µm in a thermoregulated trough containing physiological solution. Tetanic contractions were elicited at physiological temperature (10°C-30°C) by electrical stimulation, and sarcomere length changes were recorded with nanometer-microsecond resolution during both isometric and isotonic contractions with a striation follower. The force output has been normalized for the actual fraction of the cross section of the tail occupied by the myofilament lattice, as established with transmission electron microscopy (TEM), and then for the actual density of myofilaments, as established with X-ray diffraction. Under these conditions, the mechanical performance of the contracting muscle of the wild-type larva can be defined at the level of the half-thick filament, where ∼300 myosin motors work in parallel as a collective motor, allowing a detailed comparison with the established performance of the skeletal muscle of different vertebrates. The results of this study point out that the medaka fish larva is a suitable model for the investigation of the genotype/phenotype correlations and therapeutic possibilities in skeletal muscle diseases caused by mutations in sarcomeric proteins.NEW & NOTEWORTHY The suitability of the medaka fish as a model for investigating the molecular mechanisms of human myopathies caused by mutations of sarcomeric proteins is tested by combining structural analysis and sarcomere-level mechanics of the skeletal muscle of the tail of medaka larva. The mechanical performance of the medaka muscle, scaled at the level of the myosin-containing thick filament, together with its reduced genome duplication makes this model unique for investigations of the genotype/phenotype correlations in human myopathies.

Myosin-binding protein H-like regulates myosin-binding protein distribution and function in atrial cardiomyocytes.

Mutations in atrial-enriched genes can cause a primary atrial myopathy that can contribute to overall cardiovascular dysfunction. MYBPHL encodes myosin-binding protein H-like (MyBP-HL), an atrial sarcomere protein that shares domain homology with the carboxy-terminus of cardiac myosin-binding protein-C (cMyBP-C). The function of MyBP-HL and the relationship between MyBP-HL and cMyBP-C is unknown. To decipher the roles of MyBP-HL, we used structured illumination microscopy, immuno-electron microscopy, and mass spectrometry to establish the localization and stoichiometry of MyBP-HL. We found levels of cMyBP-C, a major regulator of myosin function, were half as abundant compared to levels in the ventricle. In genetic mouse models, loss of MyBP-HL doubled cMyBP-C abundance in the atria, and loss of cMyBP-C doubled MyBP-HL abundance in the atria. Structured illumination microscopy showed that both proteins colocalize in the C-zone of the A-band, with MyBP-HL enriched closer to the M-line. Immuno-electron microscopy of mouse atria showed MyBP-HL strongly localized 161 nm from the M-line, consistent with localization to the third 43 nm repeat of myosin heads. Both cMyBP-C and MyBP-HL had less-defined sarcomere localization in the atria compared to ventricle, yet areas with the expected 43 nm repeat distance were observed for both proteins. Isometric force measurements taken from control and Mybphl null single atrial myofibrils revealed that loss of Mybphl accelerated the linear phase of relaxation. These findings support a mechanism where MyBP-HL regulates cMyBP-C abundance to alter the kinetics of sarcomere relaxation in atrial sarcomeres.

Dysregulated Autophagy and Sarcomere Dysfunction in Patients With Heart Failure With Co-Occurrence of P63A and P380S BAG3 Variants.

BACKGROUND: Mutations to the co-chaperone protein BAG3 (B-cell lymphoma-2-associated athanogene-3) are a leading cause of dilated cardiomyopathy (DCM). These mutations often impact the C-terminal BAG domain (residues 420-499), which regulates heat shock protein 70-dependent protein turnover via autophagy. While mutations in other regions are less common, previous studies in patients with DCM found that co-occurrence of 2 BAG3 variants (P63A, P380S) led to worse prognosis. However, the underlying mechanism for dysfunction is not fully understood. METHODS AND RESULTS: In this study, we used proteomics, Western blots, and myofilament functional assays on left ventricular tissue from patients with nonfailing, DCM, and DCM with BAG363/380 to determine how these mutations impact protein quality control and cardiomyocyte contractile function. We found dysregulated autophagy and increased protein ubiquitination in patients with BAG363/380 compared with nonfailing and DCM, suggesting impaired protein turnover. Expression and myofilament localization of BAG3-binding proteins were also uniquely altered in the BAG3,63/380 including abolished localization of the small heat shock protein CRYAB (alpha-crystallin B chain) to the sarcomere. To determine whether these variants impacted sarcomere function, we used cardiomyocyte force-calcium assays and found reduced maximal calcium-activated force in DCM and BAG363/380. Interestingly, myofilament calcium sensitivity was increased in DCM but not with BAG363/380, which was not explained by differences in troponin I phosphorylation. CONCLUSIONS: Together, our data support that the disease-enhancing mechanism for BAG3 variants outside of the BAG domain is through disrupted protein turnover leading to compromised sarcomere function. These findings suggest a shared mechanism of disease among pathogenic BAG3 variants, regardless of location.

Sorafenib induces cardiotoxicity through RBM20-mediated alternative splicing of sarcomeric and mitochondrial genes.

Sorafenib, a multi-targeted tyrosine kinase inhibitor, is a first-line treatment for advanced solid tumors, but it induces many adverse cardiovascular events, including myocardial infarction and heart failure. These cardiac defects can be mediated by alternative splicing of genes critical for heart function. Whether alternative splicing plays a role in sorafenib-induced cardiotoxicity remains unclear. Transcriptome of rat hearts or human cardiomyocytes treated with sorafenib was analyzed and validated to define alternatively spliced genes and their impact on cardiotoxicity. In rats, sorafenib caused severe cardiotoxicity with decreased left ventricular systolic pressure, elongated sarcomere, enlarged mitochondria and decreased ATP. This was associated with alternative splicing of hundreds of genes in the hearts, many of which were targets of a cardiac specific splicing factor, RBM20. Sorafenib inhibited RBM20 expression in both rat hearts and human cardiomyocytes. The splicing of RBM20's targets, SLC25A3 and FHOD3, was altered into fetal isoforms with decreased function. Upregulation of RBM20 during sorafenib treatment reversed the pathogenic splicing of SLC25A3 and FHOD3, and enhanced the phosphate transport into mitochondria by SLC25A3, ATP synthesis and cell survival.We envision this regulation may happen in many drug-induced cardiotoxicity, and represent a potential druggable pathway for mitigating sorafenib-induced cardiotoxicity.

Visualization of cardiac thick filament dynamics in ex vivo heart preparations.

RATIONALE: Cardiac muscle cells are terminally differentiated after birth and must beat continually throughout one's lifetime. This mechanical process is driven by the sliding of actin-based thin filaments along myosin-based thick filaments, organized within sarcomeres. Despite costly energetic demand, the half-life of the proteins that comprise the cardiac thick filaments is ∼10 days, with individual molecules being replaced stochastically, by unknown mechanisms. OBJECTIVES: To allow for the stochastic replacement of molecules, we hypothesized that the structure of thick filaments must be highly dynamic in vivo. METHODS AND RESULTS: To test this hypothesis in adult mouse hearts, we replaced a fraction of the endogenous myosin regulatory light chain (RLC), a component of thick filaments, with GFP-labeled RLC by adeno-associated viral (AAV) transduction. The RLC-GFP was properly localized to the heads of the myosin molecules within thick filaments in ex vivo heart preparations and had no effect on heart size or actin filament siding in vitro. However, the localization of the RLC-GFP molecules was highly mobile, changing its position within the sarcomere on the minute timescale, when quantified by fluorescence recovery after photobleaching (FRAP) using multiphoton microscopy. Interestingly, RLC-GFP mobility was restricted to within the boundaries of single sarcomeres. When cardiomyocytes were lysed, the RLC-GFP remained strongly bound to myosin heavy chain, and the intact myosin molecules adopted a folded, compact configuration, when disassociated from the filaments at physiological ionic conditions. CONCLUSIONS: These data demonstrate that the structure of the thick filament is highly dynamic in the intact heart, with a rate of molecular exchange into and out of thick filaments that is ∼1500 times faster than that required for the replacement of molecules through protein synthesis or degradation.

From amino-acid to disease: the effects of oxidation on actin-myosin interactions in muscle.

Actin-myosin interactions form the basis of the force-producing contraction cycle within the sarcomere, serving as the primary mechanism for muscle contraction. Post-translational modifications, such as oxidation, have a considerable impact on the mechanics of these interactions. Considering their widespread occurrence, the explicit contributions of these modifications to muscle function remain an active field of research. In this review, we aim to provide a comprehensive overview of the basic mechanics of the actin-myosin complex and elucidate the extent to which oxidation influences the contractile cycle and various mechanical characteristics of this complex at the single-molecule, myofibrillar and whole-muscle levels. We place particular focus on amino acids shown to be vulnerable to oxidation in actin, myosin, and some of their binding partners. Additionally, we highlight the differences between in vitro environments, where oxidation is controlled and limited to actin and myosin and myofibrillar or whole muscle environments, to foster a better understanding of oxidative modification in muscle. Thus, this review seeks to encompass a broad range of studies, aiming to lay out the multi layered effects of oxidation in in vitro and in vivo environments, with brief mention of clinical muscular disorders associated with oxidative stress.

MDM2 Regulation of HIF Signaling Causes Microvascular Dysfunction in Hypertrophic Cardiomyopathy.

BACKGROUND: Microvasculature dysfunction is a common finding in pathologic remodeling of the heart and is thought to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM), a disease caused by sarcomere gene mutations. We hypothesized that microvascular dysfunction in HCM was secondary to abnormal microvascular growth and could occur independent of ventricular hypertrophy. METHODS: We used multimodality imaging methods to track the temporality of microvascular dysfunction in HCM mouse models harboring mutations in the sarcomere genes Mybpc3 (cardiac myosin binding protein C3) or Myh6 (myosin heavy chain 6). We performed complementary molecular methods to assess protein quantity, interactions, and post-translational modifications to identify mechanisms regulating this response. We manipulated select molecular pathways in vivo using both genetic and pharmacological methods to validate these mechanisms. RESULTS: We found that microvascular dysfunction in our HCM models occurred secondary to reduced myocardial capillary growth during the early postnatal time period and could occur before the onset of myocardial hypertrophy. We discovered that the E3 ubiquitin protein ligase MDM2 (murine double minute 2) dynamically regulates the protein stability of both HIF1α (hypoxia-inducible factor 1 alpha) and HIF2α (hypoxia-inducible factor 2 alpha)/EPAS1 (endothelial PAS domain protein 1) through canonical and noncanonical mechanisms. The resulting HIF imbalance leads to reduced proangiogenic gene expression during a key period of myocardial capillary growth. Reducing MDM2 protein levels by genetic or pharmacological methods normalized HIF protein levels and prevented the development of microvascular dysfunction in both HCM models. CONCLUSIONS: Our results show that sarcomere mutations induce cardiomyocyte MDM2 signaling during the earliest stages of disease, and this leads to long-term changes in the myocardial microenvironment.

Sarcomeric network analysis of ex vivo cultivated human atrial appendage tissue using super-resolution microscopy.

Investigating native human cardiac tissue with preserved 3D macro- and microarchitecture is fundamental for clinical and basic research. Unfortunately, the low accessibility of the human myocardium continues to limit scientific progress. To overcome this issue, utilizing atrial appendages of the human heart may become highly beneficial. Atrial appendages are often removed during open-heart surgery and can be preserved ex vivo as living tissue with varying durability depending on the culture method. In this study, we prepared living thin myocardial slices from left atrial appendages that were cultured using an air-liquid interface system for overall 10 days. Metabolic activity of the cultured slices was assessed using a conventional methyl thiazolyl tetrazolium (MTT) assay. To monitor the structural integrity of cardiomyocytes within the tissue, we implemented our recently described super-resolution microscopy approach that allows both qualitative and quantitative in-depth evaluation of sarcomere network based on parameters such as overall sarcomere content, filament size and orientation. Additionally, expression of mRNAs coding for key structural and functional proteins was analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR). Our findings demonstrate highly significant disassembly of contractile apparatus represented by degradation of [Formula: see text]-actinin filaments detected after three days in culture, while metabolic activity was constantly rising and remained high for up to seven days. However, gene expression of crucial cardiac markers strongly decreased after the first day in culture indicating an early destructive response to ex vivo conditions. Therefore, we suggest static cultivation of living myocardial slices derived from left atrial appendage and prepared according to our protocol only for short-termed experiments (e.g. medicinal drug testing), while introduction of electro-mechanical stimulation protocols may offer the possibility for long-term integrity of such constructs.

Cardiomyocyte external mechanical unloading activates modifications of α-actinin differently from sarcomere-originated unloading.

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano-signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano-signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1 µm mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α-actinin by 6 h. In myosin inhibition, this was accompanied by redistribution of intracellular poly-ubiquitin K48 to the cellular periphery relative to the poly-ubiquitin K48 reservoir at the I-band. Moreover, loading and unloading of the cellular substrate led to a three-fold increase in post-translational modifications (PTMs) when compared to the myosin-specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve extracellular signal-regulated kinase 1/2 and focal adhesion kinase activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α-actinin to increase its propensity to bind F-actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α-actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α-actinin are discussed with respect to cardiac atrophy and heart failure.

The Definition of Sarcomeric and Non-Sarcomeric Gene Mutations in Hypertrophic Cardiomyopathy Patients: A Multicenter Diagnostic Study Across Türkiye.

BACKGROUND: Hypertrophic cardiomyopathy is a common genetic heart disease and up to 40%-60% of patients have mutations in cardiac sarcomere protein genes. This genetic diagnosis study aimed to detect pathogenic or likely pathogenic sarcomeric and non-sarcomeric gene mutations and to confirm a final molecular diagnosis in patients diagnosed with hypertrophic cardiomyopathy. METHODS: A total of 392 patients with hypertrophic cardiomyopathy were included in this nationwide multicenter study conducted at 23 centers across Türkiye. All samples were analyzed with a 17-gene hypertrophic cardiomyopathy panel using next-generation sequencing technology. The gene panel includes ACTC1, DES, FLNC, GLA, LAMP2, MYBPC3, MYH7, MYL2, MYL3, PLN, PRKAG2, PTPN11, TNNC1, TNNI3, TNNT2, TPM1, and TTR genes. RESULTS: The next-generation sequencing panel identified positive genetic variants (variants of unknown significance, likely pathogenic or pathogenic) in 12 genes for 121 of 392 samples, including sarcomeric gene mutations in 30.4% (119/392) of samples tested, galactosidase alpha variants in 0.5% (2/392) of samples and TTR variant in 0.025% (1/392). The likely pathogenic or pathogenic variants identified in 69 (57.0%) of 121 positive samples yielded a confirmed molecular diagnosis. The diagnostic yield was 17.1% (15.8% for hypertrophic cardiomyopathy variants) for hypertrophic cardiomyopathy and hypertrophic cardiomyopathy phenocopies and 0.5% for Fabry disease. CONCLUSIONS: Our study showed that the distribution of genetic mutations, the prevalence of Fabry disease, and TTR amyloidosis in the Turkish population diagnosed with hypertrophic cardiomyopathy were similar to the other populations, but the percentage of sarcomeric gene mutations was slightly lower.