Sarcomere assembly and maintenance are essential physiological processes required for cardiac and skeletal muscle function and organism mobility. Over decades of research, components of the sarcomere and factors involved in the formation and maintenance of this contractile unit have been identified. Although we have a general understanding of sarcomere assembly and maintenance, much less is known about the development of the thin filaments and associated factors within the sarcomere. In the last decade, advancements in medical intervention and genome sequencing have uncovered patients with novel mutations in sarcomere thin filaments. Pairing this sequencing with reverse genetics and the ability to generate patient avatars in model organisms has begun to deepen our understanding of sarcomere thin filament development. In this review, we provide a summary of recent findings regarding sarcomere assembly, maintenance, and disease with respect to thin filaments, building on the previous knowledge in the field. We highlight debated and unknown areas within these processes to clearly define open research questions.

Heart failure is the leading cause of mortality worldwide, primarily associated with cardiovascular disease. Many heart muscle diseases are caused by mutations in genes that encode contractile proteins, including cardiac actin mutations. Zebrafish are an advantageous system for modelling cardiac diseases due to their ability to develop without a functional heart throughout embryonic development. However, genome duplication in the teleost lineage poses a unique challenge by increasing the number of genes involved in heart development. Four actin genes are expressed in the zebrafish heart: acta1b, actc2 , and duplicates of actc1a on chromosomes 19 and 20. In this study, we characterize the actin genes involved in early zebrafish heart development using in situ hybridization and CRISPR targeting to determine the most suitable gene for modelling actin changes observed in human patients with heart disease. The actc1a and acta1b genes are predominantly expressed during embryonic heart development, resulting in severe cardiac phenotypes when targeted with CRISPR. Considering the duplication of the actc1a gene, we recommend acta1b as the best gene for targeted cardiac actin research.

Heart failure is the number one cause of mortality in the world, contributed to by cardiovascular disease. Many diseases of the heart muscle are caused by mutations in genes encoding contractile proteins, including cardiac actin mutations. Zebrafish are an advantageous system for modeling cardiac disease since embryos can develop without a functional heart. However, genome duplication in the teleost lineage creates a unique obstacle by increasing the number of genes involved in heart development. Four actin genes are expressed in the zebrafish heart: acta1b; actc1c; and duplicates of actc1a on chromosome 19 and 20. Here, we characterize the actin genes involved in early zebrafish heart development using in situ hybridization and CRISPR targeting to determine which gene is best to model changes seen in human patients with heart disease. The actc1a and acta1b genes are predominant during embryonic heart development, resulting in severe cardiac phenotypes when targeted with CRISPRs. Targeting these two cardiac genes with CRISPRs simultaneously results in a more severe phenotype than their individual counterparts, with the results suggesting compensation for lost actin genes by other actin paralogues. Given the duplication of the actc1a gene, we recommend acta1b as the best gene for targeted cardiac actin research.

Background/Objectives: Abnormal development of the second heart field significantly contributes to congenital heart defects, often caused by disruptions in tightly regulated molecular pathways. Smyd1, a gene encoding a protein with SET and MYND domains, is essential for heart and skeletal muscle development. Mutations in SMYD1 result in severe cardiac malformations and misregulation of Hand2 expression in mammals. This study examines the role of Smyd1b in zebrafish cardiac morphogenesis to elucidate its function and the mechanisms underlying congenital heart defects. Methods: Smyd1b (still heart) mutant embryos were analyzed for cardiac defects, and changes in gene expression related to heart development using live imaging, in situ hybridization, quantitative PCR and immunofluorescent comparisons and analysis. Results: Smyd1b mutants displayed severe cardiac defects, including failure to loop, severe edema, and an expansion of cardiac jelly linked to increased has2 expression. Additionally, the expression of key cardiac transcription factors, such as gata4, gata5, and nkx2.5, was notably reduced, indicating disrupted transcriptional regulation. The migration of cardiac progenitors was impaired and the absence of Islet-1-positive cells in the mutant hearts suggests a failed contribution of SHF progenitor cells. Conclusions: These findings underscore the essential role of Smyd1b in regulating cardiac morphogenesis and the development of the second heart field. This study highlights the potential of Smyd1b as a key factor in understanding the genetic and molecular mechanisms underlying congenital heart defects and cardiac development.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure with notable sex differences in susceptibility and progression. Although mutations within the sarcomere, such as the change of threonine (T) to isoleucine (I) at position 126 of actin alpha cardiac muscle 1 (ACTC1) p.T126I, contribute to familial DCM, the in vivo effects and sex-specific consequences remain unclear. We generated a zebrafish model carrying the orthologous Acta1b p.T126I mutation and conducted longitudinal sex-stratified analyses of cardiac function, morphology and gene expression. Mutants showed variable onset of cardiac dysfunction, with progressive DCM, pericardial effusion, ventricular dilation and reduced survival in adults. Female mutants exhibited earlier and sustained diastolic dysfunction, greater cardiac remodeling and significantly lower survival compared to males, revealing pronounced sexual dimorphism. Molecular profiling at a pre-symptomatic stage identified upregulation of nppb, downregulation of hypertrophic transcription factors (gata4, mef2ca), and sex-specific alterations in Ca2+-handling genes (serca2, pln1, slc8a1a) and proteostasis regulators (hsf1, bag3). Assessment at later stages demonstrated a variable shift of gene expression to cardiac remodeling and decompensation in individual fish. These findings demonstrate that the Acta1b p.T126I mutation drives progressive sex-specific DCM in zebrafish, highlighting the biological sex as a critical modifier of sarcomeric cardiomyopathy progression and targeted therapy development.

/Summary: Dilated cardiomyopathy (DCM) is a leading cause of heart failure with notable sex differences in disease susceptibility and progression. Mutations in sarcomeric proteins such as cardiac actin (ACTC1) contribute to familial DCM, yet the in vivo consequences of the ACTC1 p.T126I variant and its sex-specific effects remain unclear. We generated a zebrafish model carrying the orthologous Acta1b p.T126I mutation and conducted longitudinal analyses of cardiac phenotype, function, morphology, and gene expression stratified by sex. Mutants showed variable onset of cardiac dysfunction; surviving adults developed progressive DCM characterized by pericardial effusion, ventricular dilation, and reduced survival. Female mutants exhibited earlier and sustained diastolic dysfunction, more severe cardiac remodeling and significantly lower survival compared to males, revealing pronounced sexual dimorphism. Molecular profiling identified upregulation of the cardiac stress marker nppb , downregulation of hypertrophic transcription factors ( gata4, mef2ca ), stable or reduced sarcomeric gene expression, and sex-specific alterations in calcium handling genes ( serca2, pln1, slc8a1a ) and proteostasis regulators ( hsf1, bag3 ). These findings demonstrate that the Acta1b p.T126I thin filament mutation drives progressive, sex-specific dilated cardiomyopathy in zebrafish, underscoring biological sex as a critical modifier of sarcomeric cardiomyopathy progression and providing a valuable platform for investigating sex-dependent mechanisms and developing targeted therapies relevant to women’s cardiovascular health.