PKC Promotes T-Tubule Membrane Loss by Activating a PKD– NFκB Endocytic Pathway
Abstract
Background
In heart disease, the membrane of the cardiomyocyte transverse-axial tubular system (TATS) deteriorates. This impairs contractility, hinders recovery and predisposes to arrhythmia. However, the key signals and cellular processes driving TATS loss are not understood. We investigated protein kinase C (PKC) and its downstream signals in animal and human cardiomyocytes.
Methods
Ventricular cardiomyocytes were isolated from healthy adult rat, rabbit and failing human hearts and treated with the PKC activator phorbol 12-myristat 13-acetat (PMA) or receptor-mediated agonists, alongside inhibitors targeting PKC, PKD, NFκB, MKK1–ERK1/2, NFAT, or endocytic pathways. TATS density was analyzed by confocal microscopy using lipophilic membrane dyes. Signaling pathway activation was determined by Western blotting and RNA sequencing. Ca2+signals and contractility were assessed in rat cells. Mechanisms of TATS loss were studied using endocytosis assays involving fixable dextran.
Results
PMA induced severe TATS loss, which was prevented by inhibiting PKC, PKD, NFκB or MKK1, but not by blocking NFAT or p38 MAPK. Receptor-mediated PKC activation also decreased TATS density. All effective inhibitors suppressed IκBαexpression. RNA sequencing indicated PMA-mediated activation of the NFκB and MAPK/ERK pathways and genes related to endocytosis. NFκB inhibition did not suppress the MAPK pathway, but MKK inhibition suppressed NFκB. PMA decreased Ca2+transient amplitudes and contractility, whereas NFκB inhibitors preserved both. Dextran assays revealed that TATS membranes were internalized via a macropinocytic process that followed saturation kinetics was upregulated by PMA, downregulated by NFκB inhibition, and required PI3K, myosin I, and clathrin-independent endocytosis and correlated with the rate of TATS loss. Key findings were consistent in human cardiomyocytes and in ex-vivo rat and rabbit myocardial slice culture.
Conclusions
PKC activation drives TATS loss in human and animal myocytes via PKC–PKD–NFκB, T-tubules are degraded by endocytic internalization, offering a new perspective on how cardiomyocyte membranes may deteriorate in heart disease.
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