Internalization of t-tubules as a cellular mechanism of remodelling and loss of the t-system

Ann-Katrin Pfeuffer (Erlangen)1, A. Martinez-Vilchez (Erlangen)1, J. Weßolowski (Erlangen)1, D. Fiegle (Erlangen)1, P. Andrä (Erlangen)1, T. Volk (Erlangen)1, T. Seidel (Erlangen)1

1Friedrich-Alexander Universität Erlangen-Nürnberg Institut für Zelluläre und Molekulare Physiologie Erlangen, Deutschland


Adverse remodelling in chronic heart failure (HF) includes the loss of the transverse tubular system (t-system), a dense network of membrane invaginations in cardiomyocytes (CM) important for excitation-contraction coupling. However, the cellular processes involved in t-system remodelling and t-tubule degradation are poorly understood. Here, we investigated endocytosis-related mechanisms causing t-system loss in rat and human CMs and the influence of protein kinase C (PKC) on those processes.
Left-ventricular cardiomyocytes were isolated enzymatically from adult rat hearts and myocardial 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-acetate (PMA, 50 nM), other PKC activators and different inhibitors of PKC, PKD and endocytosis, and kept in culture for up to three hours. Subsequently, cells were incubated in fluorescent dextran to track endocytosis. After washout of dextran, the t-system was stained with the lipophilic, fixable membrane dye AM4-66. Both dyes were visualized with confocal microscopy after fixation and analyzed by automated image analysis. 
Rat CMs internalized dextran-filled structures resembling t-tubules, which were not stained by AM4-66 (overlap 0.103±0.005, n=252/35 cells/hearts) suggesting an endocytosis-related mechanism of t-tubule internalization. After 3 h of incubation with PMA, CMs showed a higher internalization rate (IR) of dextran (volume ratio 0.009±0.0002 vs. 0.004±0.0001 in control, n=207/27, p<0.001). PMA also caused t-system loss. The simultaneous incubation with the PKC activators angiotensin (1 µM), endothelin (100 nM) and phenylephrine (10 µM) also increased the IR (0.006±0.0003 vs. 0.004±0.0002 in control, p<0.001, n=62/6). In a second experimental series cells were treated with PMA ± inhibitors of PKC GÖ6983 (1 µM), sotrastaurin (500 nM) and PKD CRT0066101 (2.5 µM) for 3 h. This resulted in a decreased IR of dextran compared to PMA (PMA+GÖ+Sotra 0.003±0.0003, PMA+CRT 0.006±0.0006, p<0.001). The addition of endocytosis inhibitors to PMA decreased the IR (Pitstop+PMA 0.001±0.0002, amilorid+PMA 0.006±0.0005, p<0.001, n=38/5). We conducted time-based experiments and live-cell imaging to examine the course of internalized tubules. We observed an accumulation of internalized dextran over time when CMs were incubated with dextran for different intervals (5-80 min) and a decrease after the dextran was washed out (10-40 min). Additionally, in a long-term live-cell experiment, we noted the disappearance of dextran and simultaneous reappearance of AM4-66, suggesting that dextran was released back into the extracellular medium due to tubular membrane reintegration. Furthermore, we confirmed the principle of t-tubule internalization in isolated human CMs, both in those with and without pathologically remodeled t-system, showing dextran internalizations with t-system-like shape and mutual exclusion of dextran and AM4-66 stainings (overlap 0.279±0.0272, n=64/6). 
We provide evidence that t-tubules in isolated CMs are degraded by an endocytosis-related process, which is accelerated by PKC activation and results in t-system loss. Furthermore, our results suggest that reintegration of t-tubules into the cell membrane is possible.
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