Capsaicin, a naturally occurring polyphenol, is known to affect energy expenditure and muscle fatigue and modulate contractions in skeletal muscle. The L-type Ca2+ channels are known to be an important ion channel involved in the various muscle functions and the effect of capsaicin on the skeletal L-type Ca2+ channels is currently unknown. In this study, the effects of capsaicin and capsaicin analogs on depolarization-induced Ca2+ effluxes through L-type Ca2+ channels in transverse tubule membranes from rabbit skeletal muscle and L-type Ca2+ currents recorded using the whole-cell patch clamp technique in rat myotubes were examined. Capsaicin, in the concentration range of 3–100 µM, inhibited depolarization-induced Ca2+ effluxes. The effect of capsaicin was not reversed by TRPV1 antagonist SB-366791 (10 µM). While vanilloids (30 µM) including vanillin, vanillyl alcohol, and vanillylamine were ineffective, other capsaicinoids (30 µM) including dihydrocapsaicin, nonivamide, and nordihydrocapsaicin significantly inhibited Ca2+ effluxes, suggesting that hydrocarbon chains are required for inhibition. In rat myotubes, capsaicin inhibited L-type Ca2+ currents with an IC50 value of 27.2 μM in the presence of SB-366791. Furthermore, in docking studies and molecular dynamic simulations, capsaicinoids with an aliphatic tail showed stronger binding and stable bent conformations in CaV1.1, forming hydrogen bonds with Ser1011 and Thr935 and hydrophobic/π–alkyl contacts with Phe1008, Ile1052, Met1366, and Ala1369, resembling the binding mode of amlodipine. In conclusion, the results indicate that the function of L-type Ca2+ channels in mammalian skeletal muscle was inhibited by capsaicin and capsaicin analogs in a TRPV1-independent manner.

Model of muscle contraction is used to describe a muscle single twitch. An approach based on motion separation method is used to build reduced models of skeletal muscle contraction based on the model of sarcomere contraction. These models have different accuracy of modeling fast transient process. The basic sarcomere contraction model is a system of 7-th nonlinear differential equations. The integration of this system allowed to construct a simplified model of the 2-nd order, describing the behavior of an assembly of active motor units. The advantage of this model is the good validity of various parame-ters of the model, including those reflecting the processes occurring at the cellular level in the muscle. This advantage is due to the choice of a basic sarcomere contraction model, the parameters of which are properly validated. The input signal for reduced models is the average rate of influx of calcium ions in the muscle, which is assumed to be linearly related to the action potential that occurs on motor units in the muscle. Two re-gimes of muscle contraction are used to simulate skeletal muscle contraction: isometric and isotonic regimes. Muscle contraction due to short stimulation (5 ms) numerically calculated using reduced muscle models. Estimates of the contraction time, time of re-laxation phase, and the characteristic values of the concentration of calcium ions in the muscle are calculated. For the frog's sartorius muscle, these characteristics calculated from the models turn out to be close to those observed in experiments. Thus, suggested reduced muscle models allow to describe muscle contraction due to single nerve impulse from muscle motor unit.

Unconventional myosin VI (MVI) is an ATP-dependent actin-binding molecular motor that participates in numerous cellular and tissue functions, including striated muscle physiology. Lack of MVI expression significantly aberrates myogenesis and skeletal muscle metabolism, and alters myoblast adhesion, fusion, and cytoskeletal organisation. Concomitantly, MVI knockout mice display functional and structural cardiac defects. Here, for the first time, we investigate the impact of MVI on neuromuscular junctions (NMJs), the peripheral synapses crucial for skeletal muscle contraction. We show that MVI is enriched at the postsynaptic machinery of developing and adult NMJs. We analyse the morphology of NMJs of MVI knockout mice (Snell’s waltzer, SV) during early developmental remodelling and show that MVI deficiency delays NMJ maturation in fast- and slow-twitch muscles. It also reduces the NMJ size of the soleus muscle, as demonstrated by the decreased morphological parameters of both presynaptic and postsynaptic compartments. Simultaneously, synaptic elimination remains unaffected after MVI knockout, suggesting that the observed phenotypes are innervation-independent. Lastly, depletion of MVI impairs the grip strength of both female and male SV/SV mice. In summary, our studies show that MVI is an important regulator of NMJ size and maturation, controls muscle performance, and its impact is independent of innervation and sex.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-28650-x.

Microvascular control mechanisms involved in the blood flow response to muscle contractions have been well documented in males but remain poorly understood in females. Therefore, we characterized arteriolar vasodilation in situ using intravital microscopy of the retractor muscle of anesthetized female and male hamsters (8–13 weeks). Arterioles were stimulated physiologically by contracting 3–5 skeletal muscle fibers overlying the arteriole for 2 min using a range of twitch and tetanic stimulation parameters: 6, 15, and 60 contractions per minute (cpm) at 20 Hz, or 4, 20, and 70 Hz at 15 cpm (250 ms train duration) and pharmacologically via 2 min micropipette application of nitric oxide (NO, 10−5 M), adenosine (ADO, 10−6 M) and potassium (K+, 20 mM) as well as acetylcholine (ACh, 10−6 M) to assess local and conducted responses. Sex differences were not observed in the magnitude or rate of arteriolar vasodilation under any physiological or pharmacological condition. Collectively, these data demonstrate that arteriolar reactivity to muscle contractions and to pharmacological stimuli relevant to muscle contractions, were not different between females and males. These data suggest that the integrated vascular response during active hyperemia may not be sexually dimorphic.

Accounting for isoforms is likely key to understanding muscle transcriptomic divergence. Muscles offer classic examples of tissues where changes in isoforms alter function and structure in response to stimuli like increased exercise or novel nutrient regimes. To determine how an essential micronutrient alters muscle isoform production, we examined how vitamin D supplementation influences transcription at multiple hierarchical levels across four Atlantic salmon (Salmo salar) muscle tissues. Specifically, we investigated whether analyses of differential transcript expression (DTE), differential transcript usage (DTU), and alternative splicing (DAS) recovered different responses to vitamin D compared to differential gene expression (DGE) alone. Vitamin D modulates salmon muscle transcription at the level of the gene, transcript, and splice junctions in all four muscle tissues. However, the strongest effects at all levels were found in the heart. There was little overlap among significant genes found at the gene, transcript, and splicing levels and the distribution of isoforms per significant gene varied across DGE, DTE, and DTU, indicating that each method tends to identify unique sets of genes. For example, we found that several myosin light chain kinase isoforms were particularly impacted by vitamin D in the heart, but many of the genes exhibited isoforms that were differentially expressed in opposing directions and were thus masked in DGE analysis. We also identified myofibrillar genes that are impacted by vitamin D at all levels of gene regulation, demonstrating that vitamin D impacts many structural proteins that are directly involved in striated muscle contractions. Vitamin D influences several muscle tissue types across multiple levels of transcriptomic divergence. Further, the limited overlap among significant genes found at the gene, transcript, and splicing levels suggests examining only DGE can mask important genes that show differential effects at other levels. Myofibrillar gene isoforms that directly influence muscle contractions in critical organs like the heart could provide an especially fruitful avenue of additional investigation into the transcriptional impacts of vitamin D. Together, these findings clarify how vitamin D influences muscle differentiation, health, and function.

Precise remote control of skeletal muscle contraction could be beneficial to the study and treatment of muscular dysfunction. Recently, we reported a method regulating intracellular calcium signaling using molecular motors (MMs), molecules that rotate submolecular components unidirectionally upon absorption of light. Here, we explore the application of this methodology to skeletal muscle tissue. Our results demonstrate that MMs induce intracellular calcium release in C2C12 myoblasts and differentiated myotubes via IP3-mediated signaling in a fashion that depends on their fast unidirectional rotation. Inhibition of proteins involved in the cAMP pathway such as adenylyl cyclase and protein kinase A also reduced the magnitude of the elicited calcium responses. We further show that, in differentiated C2C12 myotubes, the calcium signaling events driven by MM activation cause localized myotube contraction. This work demonstrates the use of a molecular mechanical technique to directly control skeletal muscle contraction, expanding the scope of available tools to study muscle contraction in a single-cell regime and treat a range of myopathies.

Voiding dysfunction, characterized by abnormally slow and/or incomplete micturition, is a clinical challenge that significantly impacts quality of life among women. Dysfunctional voiding, a subtype of voiding dysfunction is identified by intermittent and/or fluctuating urine flow caused by involuntary periurethral striated muscle contractions during voiding in individuals without neurological abnormalities. Interferential stimulation is a non-invasive surface electrical stimulation modality widely employed in physiotherapy. The current evidence supports its clinical efficacy in managing both urinary and fecal disorders. However, its role in treating dysfunctional voiding in women remains understudied. Thus, we present a study protocol to investigate the effects of interferential electrical stimulation on clinical symptoms and urodynamic findings in women with dysfunctional voiding. This double-blind, randomized controlled trial will employ a parallel-group design and will be conducted at the Physiotherapy Clinic of Iran University of Rehabilitation Sciences, Tehran, Iran. The study population will comprise women aged 18-50 years diagnosed with dysfunctional voiding. This trial will enroll 28 participants, equally allocated to two parallel groups (n = 14 per group). Both groups will receive standard urotherapy and pelvic floor exercises as baseline interventions. The experimental group will additionally undergo 20-minute interferential stimulation twice weekly for 10 sessions under clinical supervision. Primary outcomes include maximum urine flow rate (Qmax) and severity of lower urinary tract symptoms, assessed at three times: before treatment, after treatment, and after a three-month follow-up. All statistical analyses will be conducted using SPSS software (version 26), with statistical significance set at p <0.05. It is hypothesized that using interferential stimulation will have a positive effect on clinical symptoms and urodynamic parameters in women with dysfunctional voiding. This trial is registered in the Iranian Registry of Clinical Trials (IRCT) under the registration number IRCT20180611040061N2.

BackgroundVoiding dysfunction, characterized by abnormally slow and/or incomplete micturition, is a clinical challenge that significantly impacts quality of life among women. Dysfunctional voiding, a subtype of voiding dysfunction is identified by intermittent and/or fluctuating urine flow caused by involuntary periurethral striated muscle contractions during voiding in individuals without neurological abnormalities. Interferential stimulation is a non-invasive surface electrical stimulation modality widely employed in physiotherapy. The current evidence supports its clinical efficacy in managing both urinary and fecal disorders. However, its role in treating dysfunctional voiding in women remains understudied. Thus, we present a study protocol to investigate the effects of interferential electrical stimulation on clinical symptoms and urodynamic findings in women with dysfunctional voiding.MethodsThis double-blind, randomized controlled trial will employ a parallel-group design and will be conducted at the Physiotherapy Clinic of Iran University of Rehabilitation Sciences, Tehran, Iran. The study population will comprise women aged 18–50 years diagnosed with dysfunctional voiding. This trial will enroll 28 participants, equally allocated to two parallel groups (n = 14 per group). Both groups will receive standard urotherapy and pelvic floor exercises as baseline interventions. The experimental group will additionally undergo 20-minute interferential stimulation twice weekly for 10 sessions under clinical supervision. Primary outcomes include maximum urine flow rate (Qmax) and severity of lower urinary tract symptoms, assessed at three times: before treatment, after treatment, and after a three-month follow-up. All statistical analyses will be conducted using SPSS software (version 26), with statistical significance set at p <0.05,Expected resultsIt is hypothesized that using interferential stimulation will have a positive effect on clinical symptoms and urodynamic parameters in women with dysfunctional voiding.Trial registrationThis trial is registered in the Iranian Registry of Clinical Trials (IRCT) under the registration number IRCT20180611040061N2.

The ryanodine receptor isoform-1 (RyR1) is a large intracellular calcium release channel essential for skeletal muscle contraction. While cryo-electron microscopy has revealed structural snapshots of RyR1 in closed and open states, the dynamic features associated with calcium-dependent gating remain incompletely understood. In this study, we integrated all-atom molecular dynamics (MD) simulations with domain-level bioinformatics analyses to characterize and compare the structural dynamics of RyR1 in its closed and open conformations. Our simulations revealed distinct structural differences, including domain flexibility patterns, solvent accessibility, and hydrogen bonding networks, between the closed and open states. The closed state exhibited more extensive inter-subunit contacts and stable hydrogen-bonding networks, supporting a compact architecture characterized by inter-subunit domain engagement and intra-subunit domain loosening. In contrast, the open state showed increased solvent exposure and reduced inter-subunit interactions, reflecting inter-subunit domain loosening coupled with intra-subunit domain engagement, particularly in regions connecting the cytoplasmic and pore-forming domains. The comparative approach provides structural perspectives on how calcium binding may contribute to RyR1's conformational organization relevant to gating function. Our findings highlight the utility of integrating MD simulations with domain-scale analyses to investigate large protein complexes and generate hypotheses for future experimental validation.

Dynamic Evaluation of Skeletal Muscle Voluntary Contraction Function Using Pulsed Wave Doppler Imaging: An Exploratory Study Based on Wearable Ultrasound.

RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Several dozen variants associated with DCM have been reported so far. Variants in the arginine/serine-rich domain and the RNA recognition motif domain have been well studied, but the pathogenicity of variants outside of these areas remains unknown. A patient with the Q373fs-RBM20 variant without a typical DCM phenotype was identified in a sudden death cohort. The Q374fs-Rbm20 mouse model was generated to determine the significance of this variant. In mouse experiments, cardiac dysfunction, such as reduced fractional shortening and an extended duration of QRS and the corrected QT interval, were observed in Q374fs-Rbm20 mice by ultrasound echocardiography and electrocardiography. RNA sequencing analysis showed that Q374fs-Rbm20 mice had different splicing patterns, such as Ttn, Ldb3, Camk2d, Obscn, and Ryr2. Casq1, Mybpc2, and Myot expression was also upregulated in Q374fs-Rbm20 mice. A pathway analysis indicated the involvement of some of the 1770 differentially expressed genes in cytoplasmic ribosomal proteins, calcium regulation in cardiac cells, and striated muscle contraction. Our findings suggest that the Q374fs-Rbm20 variant changes gene splicing, affects genes involved in sarcomere structure and calcium handling genes, and presents with cardiac dysfunction.

Non-neonatal tetanus is an acute, specific and toxic disease characterized by persistent tonic contraction and paroxysmal spasms of skeletal muscles caused by Clostridium tetani invading the human body through skin or mucosal breaks, reproducing in an anaerobic environment and producing tetanospasmin. After tetanus exposure, debridement, active immunization and passive immunization are mainly used for prevention, but there were still cases of incidence after prevention. With the development of passive immune agents, new research evidence has emerged. On the basis of debridement and active immunization, rational use of passive immune agents may further reduce the risk of disease and the occurrence of adverse reactions. Based on the Standard for Diagnosis and Treatment of Non-neonatal Tetanus (2024 edition) issued by National Health Commission of the People's Republic of China, experts in the field were organized by Animal Injury Treatment Branch of China Medical Rescue Association, and Animal Injury and Acute Infectious Disease Prevention Branch of Beijing Society of Integrative Medicine. This consensus was developed by referring to the guidelines and consensus, combined with the clinical experience of experts.

Adolescent idiopathic scoliosis (AIS) is a complex genetic disorder. This study used whole-genome sequencing (WGS) to investigate the genetic basis of AIS in 119 patients from 103 families. Our WGS analysis identified known pathogenic or protein-truncating variants in 15 probands, and other strong or moderate candidate variants in 69 additional patients. We found both coding and non-coding mutations, including structural variants. Candidate genes included known AIS genes (e.g., COL11A2, FBN1) and genes linked to other musculoskeletal disorders with scoliosis (e.g., RYR1). Association analysis confirmed four known AIS single-nucleotide polymorphisms in our cohort. Gene set enrichment analysis revealed four gene clusters related to skeletal muscle contraction, extracellular matrix, and gene expression regulation. This WGS-based approach identified clinically relevant genetic variations and biological pathways in AIS patients, offering valuable insights into its complex development.

Ryanodine receptor 1 (RYR1) is the sarcoplasmic reticulum (SR) Ca2+ release channel required for both skeletal muscle contraction and Ca2+ leak. Mutations in RYR1 cause malignant hyperthermia susceptibility (MHS) and enhanced sensitivity to heat stroke (ESHS), which can result in death due to excessive skeletal muscle thermogenesis upon exposure to volatile anesthetics or heat. Here, we investigated the molecular and physiological functions of phosphorylation of RYR1 at Ser2902 by the kinase striated muscle preferentially expressed protein (SPEG). Muscle from SPEG-deficient mice expressing RYR1 with a Ser2902→Asp2902 (S2902D) point mutation to mimic phosphorylation by SPEG showed decreased SR Ca2+ sparks. Muscle from mice homozygous for the S2902D point mutation had reduced SR Ca2+ transients and small changes in force generation but overall mild phenotypic changes. YS mice, which are heterozygous for a Tyr524→Ser524 point mutation in RYR1, show increased Ca2+ leak and are a model of MHS and ESHS. Crossing YS mice with S2902D mice led to decreased SR Ca2+ leak and desensitized the mice to both volatile anesthetics and heat. Thus, SPEG inhibits SR Ca2+ leak in skeletal muscle by phosphorylating Ser2902 on RYR1, and mutation of Ser2902 to Asp2902 to mimic this phosphorylation event rescues YS mice from heat-induced death.

Accurate and reproducible tools to evaluate skeletal muscle contractility help to advance the field of physiology by defining skeletal muscle function in the context of skeletal muscle development, disease, aging, and injury recovery. The biomechanical assessment of muscle strength can be accomplished by measuring in vivo muscle torque, specifically, the muscle force generated at a moment arm around a joint. This approach to assess skeletal muscle contractility in preclinical animal studies primarily measures muscle torque in anesthetized animals using noninvasive electrophysiological stimulation. This method is advantageous because skeletal muscle contractions are evoked in a controlled, quantifiable manner that is independent of subject motivation, allowing for maximal functional data and reproducible research outcomes. The purpose of this Cores of Reproducibility in Physiology (CORP) review is to discuss the underlying physiology of the in vivo method, to highlight common outcomes and their physiological importance, and to provide considerations for technical reproducibility and data interpretation. The hope is this CORP will provide skeletal muscle researchers with the foundational and practical knowledge to better incorporate the in vivo technique in their future studies.

Background/Objectives: Dülmen wild horses are kept in a fenced wooden and marsh area around Dülmen in Westphalia, Germany, since 1856. Previous analyses supported early genetic divergence from other domesticated horse populations and the Przewalski horse. Therefore, the objective of this study was to evaluate genetic diversity using high-density genomic data. Methods: We collected 337 one-year-old male Dülmen wild horses, captured at 12 annual auctions, for genotyping on the Illumina GGP Equine Plus Beadchip. All analyses were performed for 63,123 autosomal SNPs. Results: On average, each horse had 27.96 ROH with an average length of 8.237 Mb, resulting in an average genomic inbreeding coefficient FROH of 0.107. ROH with a length of 2–4 Mb were most frequent, and the next frequent ROH fall into the length categories of 4–8 and 8–16 Mb. The effective population size (Ne) steadily decreased in the last 100 generations by 4.57 individuals per generation from 498 to 41. We identified 10 ROH islands on equine chromosomes 1, 4, 5, 7, 9, and 10. Only one ROH island on ECA 1 was shared by 45% of the horses. Overrepresented genes of ROH islands were associated with glycerophospholipid catabolism through phospholipase A2 genes, skeletal muscle contraction (TNNI3, TNNT1), synapse activity and structure (CTTNBP2), regulation of inflammatory response (NLRP genes), and zinc finger protein genes, which are involved in many cellular processes and may also act as tumor suppressors and oncogenes. Conclusions: This study highlights the development of genomic inbreeding and shows the importance of the stallions selected for breeding on the genetic diversity of the Dülmen wild horses. The results of this study should be used to develop strategies to slow down increase in inbreeding and prevent transmitting unfavorable alleles from the stallions to the next generation.

In this paper, a model for the analysis of skeletal muscle contraction is presented, and its deformation properties are accounted for. It was also presented an analytical approach for analysis of the considered muscle contraction.

Background:Myofascial trigger points (MTrPs) for abnormal skeletal muscle contraction are the cause of myofascial pain. The G protein-coupled receptor family and tyrosine kinase receptor family regulate the contraction of vascular smooth muscle through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Phosphorylated myosin light chain (p-MLC) is associated with skeletal muscle contraction. The aim of the current study was to explore the effect and mechanism of the PI3K/AKT/MLC signalling pathway on myofascial pain in rats.Methods:A rat model of myofascial pain was established by a blunt strike to the gastrocnemius muscle combined with centrifugal exercise for 8 weeks, followed by recovery for 4 weeks. Different concentrations of the PI3K inhibitor LY294002 (0.01, 0.1, or 1 mg/ml) were subsequently injected into the MTrPs of rats with myofascial pain to observe the effects on the mechanical tenderness threshold at the MTrPs.Results:LY294002 (0.1 mg/ml) inhibited myofascial pain at 0.5, 1 and 2 h after injection, and LY294002 (1 mg/ml) inhibited myofascial pain at 0.5, 1, 2 and 4 h after injection. The expression of PI3K increased on the enlarged muscle fibre membrane at MTrPs. LY294002 (1 mg/ml) inhibited the expression of PI3K, p-AKT, and p-MLC and the abnormal contraction of muscle fibres at MTrPs and alleviated nerve fibre compression at MTrPs. Moreover, LY294002 inhibited the expression of Fos in the spinal dorsal horn of rats with myofascial pain.Conclusions:These findings suggested that the increased expression of PI3K/p-AKT/p-MLC was related to myofascial pain in rats and that the PI3K inhibitor LY294002 might alleviate myofascial pain in rats by inhibiting abnormal contraction at MTrPs.

Measuring the forces of individual muscles in a muscle group around a jointis non-trivial, and researchers have suggested using surrogates for individualmuscle forces instead. Traditionally, experimentalists have shown that theforce output of the skeletal muscle tissue can be correlated to theintra-muscular pressure (IMP) generated by the muscle belly. However, IMPproves difficult to measure in vivo, due to variations from sensor placementand invasiveness of the procedure. Numerical biomechanical simulations offer atool to analyze muscle contractions, enabling new insights into thecorrelations among non-invasive experimentally measurable quantities such asstrains, and the force output. In this work, we investigate the correlationsbetween the muscle force output, the principal, shear and volumetric strainsexperienced by the muscle, as well as the pressure developed within the musclebelly as the tissue undergoes isometric contractions with varying activationprofiles and magnitudes. It is observed that pressure does not correlate wellwith force output under higher sub-maximal and maximal activation levels,especially at locations away from the center of the muscle belly due topressure relaxation effects. This study reveals strong correlations betweenforce output and the strains at all locations of the belly, irrespective of thetype of activation considered. This observation offers evidence for further invivo studies using experimentally measurable principal and volumetric strainsin the muscle belly as proxies for the force generation by the individualmuscle and consequently enables the estimation on the contribution of variousmuscle groups to the total force.

Background: Congenital heart disease (CHD) exhibits significant genetic variability. Advancements in exome sequencing have identified several disease-associated genes, yet the precise roles of many genes are still not fully understood. We hypothesize that common molecular determinants underlie CHD, with different genes forming shared functional modules. Identifying these modules requires a comprehensive and integrative analysis approach, to get novel insights into disease mechanisms, and causal connections between biological processes and CHD pathogenesis. Methods: We used exome-sequencing data on 300 CHD patients to identify disease-associated genes linked to CHD phenotypes whose functional roles are not well characterized. Network based analysis was performed to investigate the functional relationships among these genes and to explore protein-protein interaction (PPI) networks. Functional enrichment analysis was conducted with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome. All analysis was done in R using ClusterProfiler and STRING packages to integrate and identify common pathways and strongly connected functional modu l es within the PPI networks. Results: GO enrichment analysis identified significant enrichment in biological processes such as cytoskeletal motor activity ((GO: 0003774; p = 6.67E-11), actin filament binding; (GO: 0051015; p = 9.53E-10), and DNA-binding transcription factor interactions (GO: 0140297; p = 1.22E-08), which highlight key events in cardiac structure and gene regulation. KEGG pathway analysis validated the process of cytoskeletal regulation in muscle cells (hsa04820, p = 3.74E-22) to CHD. Reactome pathways such as cilium assembly (R-HAS 561733, p = 9.05E-10) and striated muscle contraction (R-HSA 390522; p = 1.70E-08) were highlighted. PPI network analysis identified ten hub proteins—MYO15A, VCL, TGFB1, PLOD3, COL1A1, WFS1, FANCE, LAMA4, ISL1, and TNNC1—which form strongly connected modules involved in cytoskeletal remodeling, extracellular matrix integrity, and transcriptional regulation. Notably, MYO15A appears to be a key protein which may be an underexplored genetic contributor to CHD. Conclusion: Integrative n etwork-based approaches can uncover novel functional roles of underexplored disease genes in CHD. These findings offer new insights and provide a basis for future experimental validation and a potential to inform diagnostic and therapeutic strategies for this complex disease.