Abstract Body: The cardiomyocyte transverse-axial tubular system (TATS) is key for excitation-contraction coupling. In chronic heart failure, the TATS is remodeled and its density decreases, which is detrimental to cardiac function. The underlying signals and processes, however, remain unclear. We investigated if and how protein kinase C (PKC) its downstream pathways are involved in t-tubule (TT) loss.
Isolated rat and human cardiomyocytes as well as rat and rabbit cardiac slices in organotypic culture were treated with phorbol 12-myristate 13-acetate (PMA) to activate PKC. Downstream pathways were blocked by specific inhibitors. T-system density was assessed by the mean intracellular TT distance (dTT) after membrane staining and confocal microscopy. Ca²⁺ imaging was performed with Fluo-4 for confocal line scans and FURA-2 for simultaneous contraction analysis by epifluorescence microscopy. TATS degradation was investigated with fluorophore-conjugated dextran in the presence of PMA +/- different inhibitors of endocytosis. RNA sequencing was performed in rat myocytes treated with PMA, PMA+NFkB inhibitor or vehicle as control.
PMA treatment resulted in a striking loss of the TATS after 1 d in culture. This was rescued by inhibition of PKC, protein kinase D (PKD) or NFκB. Inhibiting other pathways, e.g. NFAT or AKT, was not effective. This was reproduced in human isolated cardiomyocytes and in beating rat and rabbit cardiac slices cultured with PMA for 4-5d. PMA increased the amount of internalized dextran exhibiting t-tubule shapes. Macropinocytosis inhibitors significantly reduced the amount of internalized TTs, while Dynasore, an inhibitor of dynamin and clathrin- mediated endocytosis, was less effective. Linear regression demonstrated a positive relationship between TT loss and dextran internalization (p=<0.01, R²=0.54). Ca²⁺ release and sarcomere shortening in rat myocytes treated with PMA were substantially reduced when compared with control or cells treated with PMA+NFkB inhibitors. Transcriptomics confirmed NFκB as the major signal and suggested the involvement of genes related to endocytosis, cytoskeletal organization and extracellular anchoring.
We conclude that PKC activation causes TT loss via the PKD-NFκB axis through an internalization mechanism related to macropinocytosis. This deteriorates Ca²⁺ signaling and contraction. We suggest that PKC/NFkB signaling contributes to TATS remodeling in heart disease.