Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

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.

REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects

“…REEP5 is a conserved cardiac-enriched membrane protein. Our previous proteomic experiments of mouse and human cardiac myocytes, integrated with microarray tissue expression profiles and phenotype ontology information identified poorly characterized, evolutionary conserved, cardiac-enriched membrane proteins 27 . Rank-ordered evaluation of these protein candidates identified that REEP5 was one of these most highly ranked proteins.…”

“…Given its identification in both mouse and human myocyte proteomic membrane isolations 27 , we first performed a detailed multispecies amino-acid sequence analysis of REEP5 which showed 96% homology between human and mouse REEP5 and 73% between human and zebrafish ( Fig. 1a).…”

“…1b). To confirm these predictions for human REEP5, we employed a membrane yeast two-hybrid (MYTH) system, as previously described 27,28 (Fig. 1c); which takes advantage of the ability of ubiquitin fragments (NubG and NubI) reconstitution, N-terminal TF transcription factor tagged (TF-Cub) bait protein and ubiquitin fragments tagged (NubI) prey protein were tested for protein-protein interactions.…”

Mussel‐Inspired Conductive Cryogel as Cardiac Tissue Patch to Repair Myocardial Infarction by Migration of Conductive Nanoparticles

“…[40] The connexin-43 (CX-43) protein is mainly responsible for the electric-contraction coupling of the myocardium. [41,42] Accordingly, we studied the functional difference between the DOPA-based MA-G/PEGDA and DOPA-based MA-G/PEGDA/Ppy cryogels for functionalization of cardiomyocytes mainly by observing the expression of cardiac-specific proteins and by studying their ultra-microstructure. Figure 3 shows the expression of cardiacspecific proteins in cardiomyocytes in the DOPA-based MA-G/PEGDA cryogel, DOPAbased MA-G/PEGDA/Ppy cryogel, and on plain tissue culture plates (TCPs).…”

Inhibition of Mir-128-3p by Tongxinluo Protects Human Cardiomyocytes From Ischemia/Reperfusion Injury via Upregulation of P70s6k1/P-P70s6k1

“…Although our prior studies proved that TXL had infarct-sparing effect during I/R in animals, it remained unclear whether TXL could protect human cardiomyocytes from reperfusion injury. As a consequence, for our in vitro experiments, we used human cardiomyocytes, which are widely employed in cardiovascular research (Albrecht-Schgoer et al, 2012;Boon et al, 2013;Baker et al, 2015;Kuo et al, 2015;Nehra et al, 2015;Sharma et al, 2015), rather than cardiomyocytes isolated from animals.…”