YAP Promotes Microtubule Growth to Facilitate Sarcomere Disassembly in Adult Cardiomyocytes

The formin Fhod3 is required for sarcomere reassembly after mitosis in postnatal cardiomyocytes.

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.

Contractility Drives Cardiomyocyte Maturation and the Response to Nanopatterns

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.

Proteome dynamics reveal Leiomodin 1 as a key regulator of myogenic differentiation

During myogenic differentiation, the cellular architecture and proteome of muscle stem cells and myoblasts undergo extensive remodeling. These processes are partially understood and display alterations in disease and aging, resulting in impaired regeneration. Here, we used mass spectrometry to quantify the temporal dynamics of over 6000 proteins during myogenic differentiation. We identified the actin nucleator leiomodin 1 (LMOD1) among a restricted subset of cytoskeletal proteins increasing in abundance during early myogenic differentiation. LMOD1 is expressed by muscle stem cells in vivo and displays increased abundance during skeletal muscle regeneration in mice, particularly during early stages, suggesting its importance in myotube formation. Notably, LMOD1 knockdown in primary myoblasts and during regeneration severely affects differentiation, while its overexpression accelerates and improves myotube initiation. This suggests LMOD1 is a critical component regulating myogenic differentiation. Mechanistically, we show that LMOD1 physically and functionally interacts with the deacetylase sirtuin1 (SIRT1), a regulator of myogenic differentiation. We demonstrate that LMOD1 influences SIRT1 localization and the expression of its target genes. Consistently, depletion or pharmacological inhibition of SIRT1 partially rescues the differentiation impairment observed after LMOD1 knockdown. Our work identifies LMOD1 as a new regulator that might be targeted to improve muscle regeneration in aging and disease.

Piezo1 Mechanotransduction in Skeletal Muscle: Convergence with Noncoding RNA Regulation in Myogenesis, Regeneration, and Sarcopenia

Skeletal muscle is a continuously load-bearing tissue whose growth, repair, and age-related decline are governed by mechanical signals; failure of this mechano-regulation underlies disuse atrophy and sarcopenia. Piezo1, a mechanically activated cation channel, has emerged as a tractable transducer of these signals in muscle, contributing to satellite-cell quiescence and senescence, regenerative division, myoblast fusion, and the response to loading and unloading. In parallel, the myogenic noncoding RNA program is among the best defined in any lineage, with myomiRs miR-1/133/206, the long noncoding RNA LINC-MD1, and the circular RNA circ-ZNF609 being established regulators of the proliferation-to-differentiation transition. These layers are linked because Piezo1-evoked calcium influx feeds the RhoA/ROCK-actin-MRTFA-SRF and YAP/TAZ axis that drives myogenic transcription, yet no direct coupling between Piezo1 and noncoding RNAs has been demonstrated in skeletal myocytes. Drawing on validated precedents from vascular, cardiac, and tendon tissues, this review consolidates the two pillars, frames their convergence as a testable question, distinguishes validated relationships from hypotheses, and proposes three falsifiable predictions using an unbiased candidate selection strategy. The contribution of this review is this testable framework rather than any specific candidate list. Mechanically tunable noncoding RNAs may thus represent an underexplored node for counteracting disuse atrophy and sarcopenia.

Mdfi Regulates Myoblast Proliferation and Cell Cycle Progression through Cyclins

: Background: The inhibitor of MyoD family ( Mdfi ) has been characterized as a myogenic repressor that regulates transcription factor activity through cytoplasmic retention; however, its specific function in myoblast proliferation remains poorly understood. This study aimed to elucidate the precise role of Mdfi in regulating myoblast cell cycle progression and proliferation using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing. Methods: We employed the CRISPR/Cas9 system to construct Mdfi-knockout ( Mdfi − / − ) C2C12 cell lines. Cell cycle distribution was analyzed by flow cytometry, proliferation was assessed by EdU incorporation assays, and molecular mechanisms were investigated through quantitative RT-PCR, Western blotting, and RNA sequencing (RNA-Seq). Differentially expressed genes (DEGs) between wild-type (WT) and Mdfi -overexpressing ( Mdfi -OE) were identified and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Results: Mdfi overexpression significantly increased the proportion of cells in G1 ( p < 0.001) phase while reducing S and G2 phase populations ( p < 0.001), accompanied by decreased EdU-positive cells. Conversely, Mdfi knockout promoted cell cycle progression into S phase ( p < 0.05) and enhanced proliferation. Mechanistically, Mdfi overexpression downregulated cyclin B1 ( Ccnb1 ) ( p < 0.01), cyclin D1 ( Ccnd1 ) ( p < 0.05), and proliferating cell nuclear antigen ( Pcna ) ( p < 0.05), while upregulating the cyclin-dependent kinase inhibitor P21 ( p < 0.01). RNA-Seq analysis identified 889 DEGs (FDR < 0.05 and |log 2 FC| > 1), with enrichment in cell cycle and calcium signaling pathways. Conclusions: Mdfi functions as a negative regulator of myoblast proliferation by inducing G1 phase arrest, potentially through modulation of cyclin D within the cyclin-CDK-P21 pathway. These findings advance our understanding of the molecular mechanisms governing muscle cell proliferation and identify Mdfi as a potential therapeutic target for muscle regeneration and repair.

Role of semaphorin 4f in cardiac fibroblasts to regulate matrix production through actin remodeling and YAP/TAZ activation