RESUMO
The Heart of Glass-Cerebral Cavernous Malformation (Heg-CCM) pathway is essential for normal cardiovascular development in zebrafish and mouse. In zebrafish, the Heg-CCM pathway mutants santa(ccm1/san), valentine (ccm2/vtn), and heart of glass (heg) exhibit severely dilated hearts and inflow tracts and a complete absence of blood circulation. We identified a novel gene based on its sequence identity with ccm2, which we have named ccm2-like (ccm2l), and characterized its role in cardiovascular development. Disruption of ccm2l by morpholino injection causes dilation of the atrium and inflow tract and compromised blood circulation. Morpholino co-injection experiments identify ccm2l as an enhancer of the characteristic Heg-CCM dilated heart phenotype, and we find that ccm2 overexpression can partially rescue ccm2l morphant defects. Finally, we show that Ccm2l binds Ccm1 and perform deletion and mutational analyses to define the regions of Ccm1 that mediate its binding to Ccm2l and its previously established interactors Ccm2 and Heg. These genetic and biochemical data argue that ccm2l is a necessary component of the Heg-CCM pathway.
Assuntos
Sistema Cardiovascular/embriologia , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Cardiovascular/metabolismo , Primers do DNA/genética , Immunoblotting , Hibridização In Situ , Proteína KRIT1 , Camundongos , Proteínas dos Microfilamentos/genética , Microscopia , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Morfolinos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transdução de Sinais/genéticaRESUMO
The zebrafish mutant silent partner is characterized by a dysmorphic, non-contractile ventricle resulting in an inability to generate normal blood flow. We have identified the genetic lesion in the zebrafish homolog of the slow twitch skeletal/cardiac troponin C gene. Although human troponin C1 (TNNC1) is expressed in both cardiac and skeletal muscle, duplication of this gene in zebrafish has resulted in tissue-specific partitioning of troponin C expression and function. Mutation of the zebrafish paralog tnnc1a, which is expressed predominantly in the heart, results in a loss of contractility and myofibrillar organization within ventricular cardiomyocytes, while skeletal muscle remains functional and intact. We further show that defective contractility in the developing heart results in abnormal atrial and ventricular chamber morphology. Together, our results suggest that tnnc1a is required both for the function and structural integrity of the contractile machinery in cardiomyocytes, helping to clarify potential mechanisms of troponin C-mediated cardiomyopathy.
