1Friedrich-Alexander Universität Erlangen-Nürnberg Institut für Zelluläre und Molekulare Physiologie Erlangen, Deutschland
Background
The transverse tubular system (t-system) plays an important role in excitation-contraction coupling, and its loss in heart failure contributes to cardiac dysfunction. Recently, PKC signalling has been shown to cause acute t-system remodelling and t-tubule (TT) loss in cardiac myocytes, and may underly this process also in heart failure. Here, we explore the role and relevance of the different PKC isoforms and their downstream effectors to elaborate on how PKC/PKD signalling causes t-system deterioration.
Methods
Left ventricular rat cardiomyocytes were isolated and cultured for a maximum of 3 days, wherein different inhibitors were applied to block different PKC isoforms or specific PKC downstream pathways of interest. In most cases, these inhibitors were combined together with the broad PKC activator phorbol-12-myristat-13-acetate (PMA) at 50 nM. Treated cells were stained with the lipophilic dye Di-8-ANEPPS and imaged with confocal microscopy, after which the integrity of the t-system was evaluated quantitatively using customized software for image segmentation and analysis. Inhibition of the different signaling cascades was verified by phosphorylation-specific Western blotting.
Results
PMA reduced t-system density strikingly after 1d of treatment (mean TT distance, dTT in µm, 0.63±0.02 in CTRL vs 1.3±0.05 in PMA, p=<0.001). Addition of GÖ6893 (1µM), which inhibits the α, β, γ, δ and ζ PKC isoforms, yielded a marked preservation of the t-system compared to PMA alone (dTT 1.5±0.11 in PMA vs 0.68±0.03 in PMA+GO6893, p=0.04). The broad-spectrum inhibitors sotrastaurin and staurosporin, which target PKC α, β and δ, and PKC α, γ and δ respectively, had no significant effect. The PKC β inhibitor enzastaurin, at a concentration at which it also inhibits PKC α, γ and ϵ (500nM), showed only a small effect. Ruboxistaurin, another PKC β inhibitor, showed no effect. Inhibition of NFkB, a transcription factor downstream of nPKCs and PKD, resulted in a reliable conservation of the t-system. A combination of TPCA1 (10µM) and NIKSMI1 (500nM), which respectively block the canonical and non-canonical NFkB pathway, prevented PMA-induced t-system loss (dTT 1.35±0.14 in PMA vs 0.59±0.05 in PMA+TPCA1+NIKSMI1, p=0.006). Additionally, it also hindered spontaneous t-system loss after 3 days of culture (dTT 1.21±0.04 in CTRL vs 0.75±0.06 in TPCA1+NIKSMI1, p=0.003). Blocking the translocation of NFkB to the nucleus with SN50 (canonical) and SN52 (non-canonical) also preserved the TT in the presence of PMA (dTT 1.15±0.09 in PMA vs 0.76±0.04 in PMA+SN50, p=0.006; dTT 1.18±0.08 in PMA vs 0.81±0.06 in PMA+SN52, p=0.003). PI3K inhibitors prevented PMA-induced t-system loss as well (dTT 1.34±0.08 in PMA vs 0.61±0.03 in PMA+PI-103, p<0.001; dTT 1.33±0.14 in PMA vs 0.78±0.14 in PMA+Wortmannin, p=0.04). Blocking the ERK1/2 activating kinase MEK1/2 with PD184352 and Mirdametinib preserved TT in the presence of PMA (dTT 1.18±0.1 in PMA vs 0.67±0.03 in PMA+PD184352, p=0.01; dTT 1.18±0.1 in PMA vs 0.63±0.03 in PMA+Mirdametimib, p=0.009). Inhibition of NFAT with CsA, Tacrolimus, INCA-6 and VIVIT had little to no effect. Similarly, no effects were observed with AKT inhibition by AKTi-1/2.
Conclusions
We found evidence for the PKC-PKD-NFkB signaling cascade as the main pathway for PKC-related loss of the t-system. We also found an association with PI3K activation and ERK1/2 signaling, while the contributions of NFAT and AKT appear to be minimal.