Jul 2026· Cellular and Molecular Life Sciences (CMLS)· 0 citations
Medicine
TL;DR
In the presence of Fhod3, sarcomere reassembly after mitosis was successful, whereas it failed in the absence of Fhod3, indicating the indispensable role of Fhod3 in sarcomere reorganization after mitosis.
Abstract
Fhod3, a member of the formin family of proteins that regulate actin polymerization, is essential for the organization of cardiac sarcomeres during embryonic cardiogenesis. We previously demonstrated that the perinatal deletion of Fhod3 in mice results in lethality with disorganized sarcomeres, whereas the deletion in the adult period is non-lethal and does not affect sarcomere morphology. To clarify the underlying mechanism of this developmental stage-dependent outcome, we examined the effects of Fhod3 depletion in primary cultured cardiomyocytes derived from Fhod3flox neonates. Long-range time-lapse imaging throughout the entire culture period revealed that Fhod3 depletion around birth using muscle creatine kinase-Cre caused sarcomere disruption only in cells that had undergone mitosis and not in cells that had not undergone mitosis, suggesting that sarcomere breakdown by Fhod3 deletion occurs after mitosis. To verify this, we varied the timing of Fhod3 deletion using a tamoxifen-inducible αMHC-MerCreMer line. Cells treated with tamoxifen from day 1 in vitro (DIV1) exhibited a disruption of sarcomeres, similar to that observed in the Fhod3-depleted cells by MCK-Cre around birth. In contrast, cells treated from DIV9, when postnatal mitosis was almost complete, showed organized sarcomere despite Fhod3 depletion. Thus, in the presence of Fhod3, sarcomere reassembly after mitosis was successful, whereas it failed in the absence of Fhod3, indicating the indispensable role of Fhod3 in sarcomere reorganization after mitosis.
It is shown that microtubule expression and network density decline with cardiac maturation, which identifies microtubule networks as an essential regulator modulating CM dedifferentiation and sarcomere reorganization, which is critical for CM cytokinesis and cardiac regenerative repair.
Feng Zhang, Jenna Cusick, Jie Liang et al.· Circulation· 0 citations
The simple nematode Caenorhabditis elegans has long served as a powerful genetic model system for studying muscle structure and function, with sarcomeres of the striated body wall muscle (BWM) that are considered largely homologous to those in vertebrates. However, we have observed a population of F-actin in BWM separate from I-bands and centered at the sarcomere M-line, something not seen in vertebrate sarcomeres. Here, we investigate whether M-line-associated F-actin is a true feature of worm muscle, or an artifact of sample preparation. Using thin filament components tagged with fluorescent proteins, we confirm that living C. elegans BWM has a traditional sarcomere organization with actin restricted to I-bands, whereas two methods of fixation resulted in varying degrees of mislocalization of actin as well as barbed end capping protein/CapZ and tropomodulin. Additionally, we observe in live animals that α-actinin-rich dense bodies are flanked by discrete patches of CapZ, something we cannot observe in fixed animals, but which provides some support for a model in which dense bodies anchor thin filaments in a manner similar to vertebrate Z-discs. Overall, our work highlights the susceptibility of thin filaments in worm BWM to fixation artifacts and the potential utility in studying sarcomere structure in live animals.
Michael J. Kimmich, Pu Zhang, Bob Goldstein et al.· Cytoskeleton· 0 citations
Background: Understanding the mechanisms of cardiomyocyte development is critical for fulfilling the potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Although myocyte development is known to depend on internal and external mechanical cues, further investigation is required to understand the contributions of different signals and how they are integrated together to generate an adult cardiomyocyte. Here, we address this gap by examining the role of calcium-activated contractility in sarcomere formation and maturation and its influence on the iPSC-CM response to nanopatterns. Methods: We generated iPSCs with homozygous D65A cardiac troponin C (cTnC) substitutions. This engineered cTnC cannot bind to calcium at site II, resulting in tropomyosin blocking strong myosin binding to the thin filament and inhibiting sarcomere contraction. The iPSCs were differentiated into cardiomyocytes and matured in culture over 60 days. Cells were characterized via imaging, metabolic assays, and calcium transient analysis. Proteomes were examined using mass spectrometry throughout differentiation and maturation. We also replated partially matured cardiomyocytes onto nanopatterned surfaces to investigate how external mechanical signals affect maturation in contractile versus non-contractile cells. Results: Surprisingly, we found that sarcomeres formed in the D65A cTnC cardiomyocytes, though these sarcomeres were underdeveloped and disorganized. The D65A cardiomyocytes also exhibited significant proteomic maturation defects and abnormal calcium transients. Replating the non-contractile cardiomyocytes onto nanopatterns improved several structural and proteomic maturation metrics. In contrast, WT maturation did not benefit from the introduction of nanopatterns. Conclusions: Calcium-activated contractility is dispensable for sarcomerogenesis but critical for cardiomyocyte maturation. In non-contractile, D65A cTnC cardiomyocytes, nanopatterns enhanced maturation, suggesting that external mechanical cues may partially compensate for defective contractility. However, nanopatterns did not facilitate WT maturation, suggesting that maturity may reduce the efficacy of nanopatterns. In addition to these novel findings, these mass spectrometry datasets cataloging iPSC-CM maturation represent a useful resource for the cardiovascular research community.
Laura A. Sherer, Abigail Nagle, M. Papadaki et al.· Circulation Research· 0 citations
Patterning is fundamental to the development and maintenance of organisms, ensuring functional and structural organization. While patterning is well studied at the level of multicellular organisms, single cells also form spatial patterns. Stentor coeruleus is a ciliate that has been a classical system for studying patterning and morphogenesis due to its distinctive shape and organization of easily visible cortical structures. The cortex of Stentor contains two cytoskeletal layers: microtubules and a network of centrin-family EF-hand proteins, which form a branched network in the anterior half of the cell and long, thick bundles known as myonemes in the posterior half. Sfi1 family proteins scaffold the assembly of centrin filaments throughout the eukaryotes and support contractile myonemal systems in some ciliates. In Stentor, a set of Sfi1 proteins upregulated during regeneration maintains anterior/posterior differences in centrin patterning, and this loss disrupts both regeneration and contraction. RNAi-mediated knockdown revealed that temporally expressed Sfi1 genes correlate with distinct stages of oral primordium growth. Additionally, the knockdown of Sfi1 isoforms that results in reduced myoneme cables impairs contraction, such that cells fail to contract in response to a stimulus or do not fully contract. These findings suggest a model in which regionalized differences in the patterning of cytoskeletal assemblies can be modulated by the regionalized localization of scaffolding proteins.
Connie Yan, N. Steube, G. Dey et al.· Current Biology· 0 citations
The actin cytoskeleton of the cardiomyocyte is organized into two structurally and functionally distinct filament networks. The sarcomeric thin filaments, built primarily from cardiac α-actin (αCA), generate contractile force, whereas a separate subsarcolemmal cytoplasmic network, built from β-actin (Actb gene) and γ-actin (Actg1 gene), lies beneath the sarcolemma and is comparatively understudied in the mature heart. We hypothesized that this cytoplasmic actin network is required for sarcolemmal membrane integrity, signal transduction, and mechanosensing in the adult cardiomyocyte, and we generated cardiomyocyte-specific Actb and Actg1 double-gene deleted mice, using loxP (fl)-targeted Actbfl/fl and Actg1fl/fl alleles combined with an αMHC (Myh6) promoter-driven Cre-recombinase transgene, to test this directly. Deletion of cytoplasmic β-actin and γ-actin from cardiomyocytes (Actb/g1fl/fl-Myh6-Cre mice) drove compensatory upregulation of a γ-interferon like stress response with compensatory upregulation of skeletal α-actin (αSKA) and smooth muscle α-actin (αSMA) protein in adult cardiomyocytes, without altering baseline cardiac structure or function. To test if lost β-actin and γ-actin in the mouse heart impacts sarcolemmal stability, we crossed Actb/g1fl/fl-Myh6-Cremice onto the dystrophin-deficient mdx background, which is characterized by a fragile sarcolemma. Unexpectedly, hearts from Actb/g1fl/fl-Myh6- Cre; mdx mice showed greater membrane stability than mdx hearts alone with intact Actb/Actg1. We also observed that Actb/g1fl/fl-Myh6-Cremice subjected to chronic pressure overload by transverse aortic constriction (TAC) were protected and developed less cardiac hypertrophy, had better preserved systolic function, and improved survival. Together, these data indicate induction of the cytoplasmic β-actin/γ-actin network in the heart during disease stimulation is maladaptive and weakens the sarcolemma, and 2 downstream mechanisms are considered that could mediate this effect.
Background Rare heterozygous loss-of-function (LoF) variants in SVIL, encoding the Z-disk and costameric protein supervillin, have recently been identified as a cause of hypertrophic cardiomyopathy (HCM). Although supervillin is implicated in actin-dependent mechanotransduction, the mechanisms linking SVIL deficiency to cardiomyopathy remain poorly understood. Homozygous LoF cause a novel skeletal Myofibrillar Myopathy-10 (MFM-10) while heterozygous LoF cause HCM without skeletal myopathy. In this study we use a human model system to disentangle the LoF pathomechanism of the scaffolding protein supervillin in cardiomyocytes and its clinical implications. Methods Using CRISPR/Cas-9 we engineered a representative pathogenic LoF variant Q255X into an isogenic induced pluripotent stem cell (iPSC) line creating the heterozygous SVILQ255X/+ and homozygous SVILQ255X/Q255X cell lines. These lines were differentiated into iPSC-derived cardiomyocytes (iPSC-CMs) and cellular phenotypes were assessed using bulk RNA-sequencing, LC-MS proteomics, electrophysiological and calcium handling analyses, contractility measurements, sarcomere organization analysis, Seahorse metabolic flux assay, and pharmacological intervention with mavacamten. Results The Q255X variant resulted in SVIL haploinsufficiency at both RNA and protein levels with no evidence of a truncated protein. Compared with isogenic controls, SVILQ255X/+ iPSC-CMs demonstrated action potential shortening, calcium transient elongation, sarcomeric disorganization and hypertrophy, and impaired mitochondrial respiration. Multi-omic analyses of SVILQ255X/+ iPSC-CMs showed a profile of cellular stress and inflammation, hypertrophic and pro-fibrotic signalling, and a pseudohypoxic state driven by decreased respiration and a HIF-induced glycolytic shift. These abnormalities were not present in SVILQ255X/Q255X cardiomyocytes, consistent with a relatively limited cardiac phenotype reported in homozygous variant carriers. Mavacamten improved sarcomeric disorganization and hypertrophy in SVILQ255X/+ cells but did not rescue energetic compromise. Conclusions Pathogenic heterozygous SVIL LoF produces a distinct cellular phenotype characterized by impaired mechanotransduction, mitochondrial dysfunction, and maladaptive metabolic remodelling that promotes hypertrophic and pro-fibrotic signalling. These findings define a mechanistic basis for SVIL-associated cardiomyopathy and identify metabolic dysfunction as a potential therapeutic target beyond sarcomere-directed therapy. Clinical Perspective What Is New? SVIL haploinsufficiency causes HCM through a mechanism distinct from canonical sarcomeric disease, characterized by impaired mechanotransduction, mitochondrial dysfunction, and pseudohypoxia-driven metabolic remodeling. Heterozygous SVIL loss of function produces a substantially more severe cardiomyocyte phenotype than homozygous loss of function, providing a mechanistic explanation for the predominance of cardiac disease in heterozygous variant carriers. Mavacamten improves sarcomeric organization but does not restore impaired mitochondrial respiration, demonstrating that energetic dysfunction persists despite sarcomere-directed therapy. What Are the Clinical Implications? Our findings give functional evidence to support SVIL as a clinically relevant HCM disease gene and its inclusion in clinical genetic testing panels. These findings establish SVIL-associated cardiomyopathy as a mechanistically distinct form of HCM and offer insight into the pathomechanism of Z-disk and costameric HCM The persistence of mitochondrial dysfunction despite myosin inhibition suggests that drugs targeting mitochondrial bioenergetics may be a therapeutic strategy in patients with SVIL-associated cardiomyopathy.
Yifan J. Li, BMedSci Yiangos Psaras MBChB, V. Steeples et al.· bioRxiv· 0 citations