Abstract 160: Reperfusion-induced Hypercontracture Of Cardiomyocytes Is Secondary To A Sarcoplasmic-Mitochondrial Interaction Involving The Mitochondrial Permeability Transition Pore (mptp)
Objective: We showed previously that reperfusion causes hypercontracture and necrosis of cardiomyocytes due to oscillatory elevations of cytosolic Ca2+ originating from the sarcoplasmic reticulum (SR). It has also been shown that opening of mitochondrial permeability transition pore (MPTP) induces cell death in reperfused myocardium. We now investigated if SR-triggered Ca2+ oscillations and MPTP are causally connected in signalling pathways leading to reperfusion-induced cardiomyocyte hypercontracture.
Methods: To simulate ischemia and reperfusion, isolated cardiac myocytes from adult rat were superfused anoxically (pH 6.4) and then reperfused with a normoxic puffer (pH 7.4). Induction of MPT was detected in whole cells by the calcein loading procedure. Cytosolic [Ca2+] was measured by use of fura-2.
Results: During simulated ischemia, no significant change in calcein fluorescence was observed. Reperfusion induced a marked decline in calcein fluorescence, which reflects induction of MPT. Concomitantly, reperfused cells developed hypercontracture. Application of the MPT inhibitor cyclosporine (500 nM), inhibition of SR-Ca2+ release with ryanodine (3 μM), or inhibition of mitochondrial Ca2+ uptake with Ru 360 (1 μM) reduced the decline in calcein fluorescence and hypercontracture (calcein fluorescence as % after 5 min reperfusion: control: 42.2 ± 2.4; cyclosporine: 90.5 ± 1.8*; ryanodine: 86 ± 1.5*; Ru 360: 82 ± 3.0, n=20; *p<0.05 vs. control; cell length % after 5 min reperfusion; control: 46.8 ± 1.6; cyclosporine: 75.0 ± 1.8*; ryanodine: 94.8 ± 2.0*, Ru 360: 86 ± 4.0; n=20; *p<0.05). All three treatments also reduced the frequency of reperfusion-induced Ca2+ oscillation (control: 27 ± 3; cyclosporine: 13 ± 4*; ryanodine: 6 ± 3*; Ru 360: 9 ± 4; n=20; *p<0.05 vs. control).
Conclusions: Induction of MPT is involved in the development of reperfusion-induced hypercontracture of cardiac myocytes. It is triggered by oscillations of cytosolic Ca2+ originating from the SR during the early phase of reperfusion that favour the opening of MPTP.