Background

Chronic heart failure (HF) causes maladaptive remodelling and loss of the transverse tubular system (t-system), a dense network of tubular membrane structures in ventricular cardiomyocytes important for excitation-contraction (EC) coupling. This contributes to progression of heart failure and prevents recovery. The signalling pathways involved in t-system loss are, however, poorly understood. Here we investigated the role of protein kinase C (PKC), protein kinase D (PKD) and protein kinase B (PKB/AKT).

Methods

Rat and human left-ventricular (LV) cardiomyocytes were isolated enzymatically from adult animals and LV samples obtained from chronic HF patients undergoing heart transplantation or ventricular assist device implantation. All patients gave their written informed consent according to the declaration of Helsinki principles. Myocytes were treated with the PKC activator Phorbol-12-myristat-13-acetat (PMA, 50 nM) and different inhibitors of PKC, PKD and PKB and kept in culture for up to three days. Subsequently, the t-system was stained with lipophilic dyes and analyzed by confocal microscopy and automated image analysis. In addition, intracellular Ca²⁺ levels and contractility were determined in rat cells at 37˚ C with FURA-2 as a Ca²⁺ indicator and Fourier transform-based assessment of sarcomere shortening, respectively. 

Results

After two days in culture, the t-system in rat myocytes treated with 50nM PMA had almost completely disappeared (volume density 0.007±0.004% vs 0.16±0.05% in control, n=48/6 cells/hearts, p<0.001). We identified an EC₅₀ for PMA of 5 nM. Addition of the PKC inhibitors BMS345541 (10 µM) and GÖ6983 (1µM) prevented or significantly reduced PMA-induced t-system loss (0.17±0.02% PMA+BMS, 0.10±0.01% PMA+GÖ, p<0.001 each vs PMA, n=24/3 cells animals). In a second experimental series, cells were treated with PMA ± PKD inhibitors CRT0066101 (2.5 µM) or kb-NB142-70 (5µM) for 1d. PMA+CRT cells exhibited a dense t-system (0.24±0.01%, n=38/5, p<0.001 vs PMA) and no visible difference from control cells (0.25±0.01%, n=38/5, p=0.29). PMA+kb-NB142-70 (5µM), resulted in intermediate density (0.13±0.01, n=22/3, p<0.001 vs PMA, p<0.001 vs control). Inhibitors and activators of PKB/AKT did not reduce t-system loss. The specificity of activators and inhibitors was verified by phosphorylation-specific Western blotting of known target proteins. Autophosphorylation of PKD at Ser916, a necessary step for PKD activation, was inhibited by CRT0066101 and other substances that prevented or reduced PMA-induced t-system loss, but not by substances without effect. 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). 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, paired t-test).

Conclusions