Abstract
Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts. Functional analysis of Tmem65 both in vitro using lentiviral shRNA-mediated knockdown in mouse cardiomyocytes and in vivo using morpholino-based knockdown in zebrafish show marked alterations in gap junction function and cardiac morphology. Molecular analyses suggest that Tmem65 interaction with connexin 43 (Cx43) is required for correct localization of Cx43 to the intercalated disc, since Tmem65 deletion results in marked internalization of Cx43, a shorter half-life through increased degradation, and loss of Cx43 function. Our data demonstrate that the membrane protein Tmem65 is an intercalated disc protein that interacts with and functionally regulates ventricular Cx43.
Highlights
Membrane proteins are crucial to heart function and development
We isolated a membranedepleted homogenate (H) and a membrane-enriched pellet (P) fraction, and both sets were subjected to MudPIT (Multidimensional Protein Identification Technology)[11] analysis, database searching and statistical filtering on the peptide and protein levels (o1% false discovery rate (FDR)), followed by relative quantification based on spectral counts (Fig. 1a)
Crucial to the understanding of human disease mechanisms is the understanding of normal physiology, which has proven to be difficult since healthy human cardiac tissue is not available
Summary
Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. Spontaneous heartbeats rely on proteins embedded in or associated with the cardiac sarcolemmal membrane This membrane system includes an extensive transverse-tubular structure enriched in proteins involved in excitation–contraction coupling and specialized regions abundant in lipid rafts and caveolae required for transport and signalling. As a result of these analyses, we uncover Tmem[65], a novel evolutionarily conserved intercalated disc protein (ICD) that interacts with connexin 43 (Cx43), the major gap junction protein in the heart. We show that both in vivo and in vitro knockdown cause dysregulation and internalization of Cx43, a mechanism common to cardiac arrhythmias
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