Functional and structural effects of PKC activation on rabbit and human myocardium in vitro

Linda Katharina Küpfer (Erlangen)1, Z. Iqbal (Erlangen)1, P. Potue (Erlangen)1, H. Milting (Bad Oeynhausen)2, T. Volk (Erlangen)1, T. Seidel (Erlangen)1

1Friedrich-Alexander Universität Erlangen-Nürnberg Institut für Zelluläre und Molekulare Physiologie Erlangen, Deutschland; 2Herz- und Diabeteszentrum NRW E.& H. Klessmann-Institut f. kardiovask. Forschung Bad Oeynhausen, Deutschland


PKC activity is upregulated in heart failure, but PKC-induced functional and structural effects on the myocardium are not clearly understood. Animal studies on PKC reported controversial results, e.g., regarding its effect on contractility. Here, we cultured beating healthy rabbit and failing human myocardial slices cultured with or without the DAG mimetic phorbol-12-myristat-13-acetat (PMA) to investigate the functional and microstructural effects of PKC.
We obtained 5 myocardial tissue samples from the left ventricle of explanted hearts after the patients’ written informed consent and approval by the local Ethics committee according to the declaration of Helsinki principles. Myocardial slices from the human samples and from 15 healthy rabbit hearts were cultured with permanent electrical stimulation and measurement of contraction force. After 1-2 d in culture, rabbit slices were treated with 50nM PMA or vehicle (CTRL) and were further cultured for 4 d. Human slices were treated with 10-50nM PMA after 8-15 d in culture. β-adrenergic response was investigated by adding 100nM isoprenaline. The amount of PKD phosphorylated at the PKC-specific site ser744-748 was quantified by western blotting (WB). To assess Ca2+ cycling and sensitivity, we examined intracellular Ca2+ via Calbryte in rabbit slices and increased extracellular Ca2+ by adding 4mM CaCl2, respectively. We tested passive stiffness and used confocal microscopy to analyze the structural effects of PMA.
Phosphorylation-specific WB confirmed PKC activation by PMA. PMA reduced contraction amplitudes of rabbit slices at 1Hz pacing from 2530±297µN to 1649±237µN after 24 h (p=0.0001, n=61/10 slices/samples), while CTRL showed no significant change (1775±182µN vs 2142±288µN, p=0.14, n=59/10). After 5d of PMA treatment, contraction force was further reduced (2683±425µN vs 366±66µN, p<0.0001, n=15/5), while CTRL slices remained stable (2279±372µN vs 2371±465µN, p=0.84, n=19/5). This was replicated in PMA-treated human slices after 1d, showing significantly reduced force (2560±731µN vs 525±207µN, p=0.0095, n=7/4), while the human CTRL slices remained unaltered (2585±461µN vs 2855±524µN, p= 0.087, n=8/5). PMA-induced reduction in contractility manifested only after 2 h in both the species. However, the relative response to isoprenaline was higher in PMA than in CTRL after 1 d (PMA 3.99±0.47, p=0.003, n=5/3 vs CTRL 1.75±0.14, p=0.003, n=6/3) and also after 5 d. Before treatment, the response of both groups was similar (PMA 2.19±0.31 and 2.30±0.27). Similarly, the relative response to 4 mM Ca2+ was comparable in both groups at baseline (PMA 2.21±0.17, CTRL 2.01±0.14), but more pronounced after 1d of PMA treatment (PMA 2.41±0.23, p=0.002, n=6/3 vs CTRL 1.84±0.15, p=0.006, n=5/2).  Ca2+ imaging did not indicate any effects of PMA on intracellular Ca2+ transients, either with or without 10nM isoprenaline (PMA 1.54±0.11, CTRL 1.50±0.05, n=11/10). No differences concerning fibrosis or cell size were found. Passive stiffness was higher in PMA-treated slices (10% stretch: PMA 9531±2371µN, n=11/7, CTRL 4661±1505µN, n=13/7, p=0.02).
PKC activation reduces contractile force in living rabbit and human myocardium and increases the relative response to extracellular Ca2+ and β-adrenergic stimulation. Maximum contraction force was unaltered after 24 h of PKC activation. As underlying cause, we suggest decreased Ca2+ sensitivity.
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