Cardiac diseases continue to be one of the major causes of death worldwide. Ischemic heart disease such as myocardial infarction can result in an irreversible replacement of healthy, contractile heart tissue with a non-contractile, collagen-rich fibrotic scar. As the intrinsic proliferative capacity of adult cardiomyocytes is limited, future therapeutic approaches to mend ischemic heart diseases can hugely benefit from a better understanding of the regulatory mechanisms involved in cardiomyocyte proliferation.
Searching for novel regulators of cardiomyocyte proliferation, we isolated the mutant zebrafish line weiches herz (whz) in an ENU mutagenesis screen. Whz mutant embryos exhibit a cardiac phenotype, with a thin ventricular myocardial layer. Positional cloning identified a missense mutation in the tbx20 gene as cause for the whz phenotype.
Although, tbx20 mRNA was not found to be down-regulated in whz mutant embryos, we found a severe reduction in Tbx20 protein levels. This suggests that the whz mutation leads to destabilization and subsequent degradation of Tbx20 in whz zebrafish embryos. Furthermore, injection of a morpholino antisense oligonucleotide against tbx20 mimicked the whz phenotype, confirming that the whz mutation causes a loss of Tbx20 function.
Interestingly, counting of cardiomyocytes in whz mutant ventricles revealed significantly reduced numbers of cardiomyocytes compared to wild-type littermates at 72 hours post fertilization. To assess whether decreased cardiomyocyte numbers is due to less cardiomyocyte precursors in the Anterior Lateral Plate Mesoderm, we evaluated the cardiac precursor cell population by cmlc2-specific In situ hybridization at 14 and 20 somite stages. We found no reduction of cardiomyocyte precursors in whz mutant embryos, suggesting that impaired cardiomyocyte proliferation later during cardiogenesis might account for reduced cardiomyocyte numbers in whz mutants. To address this question, we performed EdU staining at 72 hours post fertilization to measure proliferating cardiomyocytes in whz embryos and found that indeed cardiomyocyte proliferation was severely reduced in the mutant embryos compared to the wild-type littermates. We also performed TUNEL staining to analyze the apoptosis in our whz mutants and found no difference in cell death compared to the wild-type littermates.
Taken together, our data indicate that the loss of tbx20 in zebrafish embryos result in a reduced number of cardiomyocytes caused by impaired proliferation of cardiomyocytes.