Baby, A. | bioRxiv Science

bioRxiv2024

Fast Myosin Binding Protein-C is a Vital Regulator in Young and Aged Fast Skeletal Muscle Homeostasis

BackgroundSkeletal muscle plays a vital role in voluntary motion and locomotion. Fast-twitch muscle fibers are characterized by their rapid contraction kinetics, high force generation capacity, and a distinct gene expression profile compared to slow-twitch fibers. Skeletal myosin binding protein-C (MyBP-C) paralogs, slow (sMyBP-C) and fast (fMyBP-C), interact with myosin and actin filaments within sarcomeres to modulate force development during contraction. These paralogs are differentially expressed in muscle fibers, with fMyBP-C predominantly expressed in the fast-twitch fibers. However, the role of fMyBP-C in skeletal muscle disease states and aging remains poorly understood. This study employs mouse models with fMyBP-C ablation to investigate its significance in skeletal muscle physiology. MethodsAdult skeletal muscle samples aged 2[~]7 months from male and female wild-type, db/db, MDX, ECC injury model, were used to determine the differential expression of fMyBP-C. Next, Mybpc2 knockout (C2-/-) young (3[~]5 months) and old (22 months) male mice were used to define the role of fMyBP-C in aging. Western immunoblotting was employed to analyze the expression of fMyBP-C and sMyBP-C and the phosphorylation status of sMyBP-C. The impact of C2-/- and aging on the fiber type, size, and number as well as general muscle structure was assessed by immunohistochemistry and electron microscopy. The functional effect of C2-/- and aging was measured in terms of in vivo and ex vivo muscle force generation. Lastly, RNA sequencing was performed to identify the molecular pathways dysregulated in the C2-/- mediated muscle dysfunction in young and old mice. ResultsfMyBP-C was significantly reduced with a modest compensatory upregulation of sMyBP-C in the diseased fast-twitch muscles. fMyBP-C has a significantly higher expression in the male skeletal muscles compared to females. Further studies using young male C2-/- mice showed a significant reduction in isometric tetanic force generation and relaxation rate, fiber type switching, atrophy, and altered gene expressions related to muscle function and metabolism compared to wild-type mice. Similarly, compared to their wild-type counterparts, aged male C2-/- mice display significant deficits in muscle strength, endurance, and survival rate, accompanied by changes in muscle fiber size and molecular signaling pathways critical for muscle homeostasis. ConclusionfMyBP-C is an important regulator of muscle function and homeostasis in young and aged male fast-twitch muscle fibers. The absence of fMyBP-C aggravates the effect of aging on muscle structure and function. fMyBP-C has the potential to be a therapeutic target to modulate muscle wasting caused by aging and disease.

physiology↗

bioRxiv2024

MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids

BACKGROUNDMYBPC3, encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). METHODSThe hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro. RESULTSConfocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS(R)) in MYBPC3D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONSConceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIMYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. C_LIO_LID389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. C_LIO_LIThe molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined. C_LI What New Information Does This Article Contribute?O_LIThe authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. C_LIO_LIThe MYBPC3D389V-mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. C_LIO_LIOur data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS(R)), a myosin inhibitor. C_LIO_LIIn addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m), highlighting a possible early adaptive response to primary sarcomeric changes. C_LIO_LIEarly treatment of MYBPC3D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. C_LI GRAPHICAL ABSTRACTA graphical abstract is available for this article. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/596463v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1489301org.highwire.dtl.DTLVardef@1ab60c1org.highwire.dtl.DTLVardef@5d49d1org.highwire.dtl.DTLVardef@9944d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Unlocking the Role of sMyBP-C: A Key Player in Skeletal Muscle Development and Growth

Skeletal muscle is the largest organ in the body, responsible for gross movement and metabolic regulation. Recently, variants in the MYBPC1 gene have been implicated in a variety of developmental muscle diseases, such as distal arthrogryposis. How MYBPC1 variants cause disease is not well understood. Here, through a collection of novel gene-edited mouse models, we define a critical role for slow myosin binding protein-C (sMyBP-C), encoded by MYBPC1, across muscle development, growth, and maintenance during prenatal, perinatal, postnatal and adult stages. Specifically, Mybpc1 knockout mice exhibited early postnatal lethality and impaired skeletal muscle formation and structure, skeletal deformity, and respiratory failure. Moreover, a conditional knockout of Mybpc1 in perinatal, postnatal and adult stages demonstrates impaired postnatal muscle growth and function secondary to disrupted actomyosin interaction and sarcomere structural integrity. These findings confirm the essential role of sMyBP-C in skeletal muscle and reveal specific functions in both prenatal embryonic musculoskeletal development and postnatal muscle growth and function.

physiology↗