Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Platelets protect cultured cardiomyocytes from apoptosis in an experimental model of tachypacing

D. Gramlich1, D. Heinzmann1, M. Sigle1, V. Haug1, M. Gawaz1

1Innere Medizin III, Kardiologie und Kreislauferkrankungen, Universitätsklinikum Tübingen, Tübingen;

Introduction: Platelets interact with a variety of nucleated cells and modify their function. Upon activation platelets release several pro- and anti-apoptotic factors including chemokines. The effect of platelets on function of cardiomyocytes is poorly understood. Atrial fibrillation is associated with enhanced apoptosis and fibrosis of the atrial myocardium. We aimed to analyze the effect of platelets on the function of immortalized atrial cardiomyocytes (HL-1 cells) in an in vitro model of tachypacing (TP).   Methods: HL-1 cells were cultured until subconfluency was obtained. Cell cultures were treated with intermittent electrostimulation using graphite electrodes (IonOptix C-pace) with pre-specified pacing protocols (amplitude 10V, pulse width 10ms, frequency 3Hz).  Isolated human platelets (200 platelets per HL-1 cell) were incubated  with subconfluent HL-1 cells for the indicated time. After TP treatment cell supernatant was removed, washed and adherent cells were further analyzed by immunofluorescence, FACS, TUNEL assay and immunoblotting.   Results: Treatment of cultured HL-1 cells by TP for 2 or 4 hours induced substantial cell apoptosis as indicated by TUNEL staining, Annexin-V binding, and TMRE-fluorescence. Co-incubation of HL-1 cells with human platelets lead to a significant, almost 20% reduction of apoptosis markers after 4 h of TP (Figure A n=6 for TUNEL TP vs TP+Plt 23±8 % vs 6±3 %, p<0.01,  Figure B n=6 for Annexin V positive cells TP vs TP+Plt 42±12 % vs 3±2 %). Incubation of HL-1 cells with supernatant derived from activated platelets decreases H202-induced phosphatidylserine exposure on HL-1 cells (Annexin V signals). This indicates that platelets release a single or multiple survival factors that preserve HL-1 cells from apoptosis. Further, treatment of isolated platelets co-incubated with HL-1 cells during 2 to 6 h TP showed elevated markers of degranulation (P-selectin expression, n=5, Control medium vs TP medium 25±2 mean fluorescent intensity [MFI] vs 92±12 MFI, p<0.05). Conclusion: Our data show that tachypacing induces apoptosis in immortalized atrial cardiomyocytes. The antiapoptotic effect of platelets on TP-induced HL-1 apoptosis implies a protective role of platelets. Interaction of platelets with cardiomyocytes may play an important to our knowledge unrecognized role for cardiomyocyte function which needs further elaboration.

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