NFκB signaling induces t-tubule loss by an endocytosis-related process and disrupts excitation-contraction coupling in rat cardiomyocytes

Introduction:
The cardiomyocyte transverse-axial tubular system (TATS) is crucial for excitation-contraction coupling. In chronic heart failure (HF) the TATS is remodeled and its density decreases, which is detrimental to cardiac function. The underlying mechanisms and signals, however, remain unclear. Because protein kinase C (PKC) and inflammatory signaling pathways are activated in HF, we investigated if and how PKC and downstream pathways, such as NFAT and NFkB, are involved in t-tubule (TT) loss and subsequently impaired excitation-contraction coupling.

Materials and Methods:
Isolated rat and human cardiomyocytes as well as rat and rabbit cardiac slices 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 Di-8-ANEPPS or WGA staining and confocal microscopy. Ca2+ imaging was performed with Fluo-4 on a confocal microscope (line scans) and with FURA-2 and simultaneous contraction analysis by epifluorescence microscopy. The degradation of t-tubules was investigated with fluorophore-conjugated dextran in the extracellular medium and measured 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.

Results:
PMA treatment resulted in a marked loss of the TATS which led to an increase of dTT after 1 d in culture, and this effect was rescued by inhibition of PKC (dTT in μm 1.69±0.01 in PMA vs 0.85±0.03 in PMA+BISXI, p=<0.001), PKD (dTT in μm 1.43±0.18 in PMA vs 0.54±0.02 in PMA+CRT0066101, p=<0.01), and NFκB (dTT in μm 1.5±0.11 in PMA vs 0.9±0.09 in PMA+BMS-345541, p=<0.05). Inhibitors of other pathways, e.g. NFAT or AKT, showed no effects. We could reproduce t-system loss by PMA in human isolated myocytes (volume density control: 0.04±0.02%, n=49/6, PMA: 0.03±0.01%, n=39/6, p<0.05). T-tubule loss was also confirmed in rat and rabbit cardiac slices treated with PMA for 4-5 days. PMA increased the amount of internalized dextran, which exhibited TT shapes and a lack of membrane staining. Inhibition of PI3K, or the use of pitstop or amiloride significantly reduced the amount of internalized TTs, while Dynasore, an inhibitor of dynamin and clathrin- mediated endocytosis, was less effective. Linear regression across multiple inhibitors demonstrated a positive relationship between TT loss and dextran internalization (p=<0.01, R2=0.54). Ca2+ release and sarcomere shortening measured at 2 Hz in rat myocytes treated with PMA were strikingly reduced (0.09±0.01 AU and 0.01±0.02 µm, n=40/5) when compared with control (0.16±0.01 AU and 0.056±0.007µm, p<0.01, n=40/5) or cells treated with PMA+CRT0066101 (0.19±0.01 AU and 0.044±0.005, p<0.01, n=40/5). Bulk mRNA expression analysis confirmed NFκB activation and revealed that TT-related proteins like dysferlin, BIN1, Ehd4, cavin4, nexilin, CACNA1c and desmin were differentially regulated in PMA-treated cells.

Conclusion:
PKC activation causes TT loss via PKD/NFκB signaling through an internalization mechanism related to macropinocytosis. This deteriorates Ca2+ signaling and contraction. We therefore suggest that PKC/NFkB signaling contributes to TATS remodeling in heart disease.