mPTP Formation Research Articles

Vardenafil limits infarction at reperfusion in isolated rat hearts dependent on guanylyl cyclase and PKG

The type 5 phosphodiesterase inhibitor vardenafil (VAR) reduces myocardial infarct size following ischemia/ reperfusion injury when applied prior to ischemia or at reperfusion in models of in situ hearts. Little is known about the underlying protective signaling. Here, we test whether VAR is protective in a cell model of calcium stress as well as in isolated rat hearts. HL-1 cardiomyocytes were loaded with tetramethylrhodamine ethyl ester (TMRE, 100 nM) which causes cells to fluoresce proportional to their mitochondrial membrane potential (Ψm). A reduction of fluorescence serves as an indicator of collapse of Ψm, and, presumably to permeability transition pore (mPTP) formation. Cells were subjected to the calcium ionophore calcimycin (CAL) (100 μM) which causes mPTP formation due to calcium overload. Fluorescence intensity was measured after 80 minutes of calcium stress with FACS technique. Treatment with CAL reduced the mean cell fluorescence (903 ± 28 arbitrary units (a.u.), vs. 548 ± 29 a.u. in untreated cells, p < 0.001) while VAR (1 nM) could protect the cells (698 ± 40 a.u., p < 0.001). This protection could be blocked by either the guanylyl cyclase (GC) inhibitor ODQ (20 μM) (519 ± 41 a.u.) or the protein kinase G blocker KT5823 (1 μM) (513 ± 22 a.u.), while inhibition of NO-synthase with L-NAME had no effect. To further support the results, isolated rat hearts were subjected to 30 min regional ischemia followed by 120 min reperfusion. As expected, VAR (10 nM) at 5 min prior to reperfusion reduced infarct size as percentage of the ischemic zone from 46.6 ± 2.1% in control hearts to 26.2 ± 2.7% (p < 0.001). This protective effect could be blocked with co-administration of ODQ (43.9 ± 2.2%), while LNAME was not effective, confirming a role for GC but not for NOS in VAR's protection. Taken together, these results further support the hypothesis that PDE-5 inhibitors induce protective effects in the ischemic heart in addition to their well known clinical effects in the treatment of erectile dysfunction in men. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007 Published: 25 July 2007

NECA given at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase

The A1/A2 adenosine agonist 5′-(N-ethylcarboxamido) adenosine (NECA) limits myocardial infarction when administered at reperfusion. We tested whether p70S6 kinase is involved in this cardioprotective effect. Adult rat ventricular myocytes were loaded with TMRE (100 nM) which causes cells to fluoresce in proportion to their mitochondrial membrane potential (Ψm). A reduction of fluorescence serves as an indicator of collapse of Ψm, and, presumably to permeability transition pore (mPTP) formation. Cells were subjected to H2O2 (200 μM) which causes mPTP formation. Ψm was considered as irreversibly collapsed when fluorescence reached less than 60% of the starting value. Treatment with NECA (1 mM) delayed the collapse of Ψm (48.0 ± 3.2 min vs. 29.5 ± 2.2 min in untreated cells, P < 0.01), and that protection was blocked with the co-administration of the mTOR/p70S6 inhibitor rapamycin (5 nM). Inhibition of GSK-3β has been proposed to be the downstream suppressor of mPTP formation. The GSK-3β inhibitor SB216763 (3 μM) also prolonged the time to collapse of Ψm (49.2 ± 2.1 min, P < 0.01). This protection was unaffected by rapamycin suggesting that GSK-3β resides downstream of p70S6. NECA treatment (100 nM) of isolated rabbit hearts at reperfusion after 30 min of regional ischemia decreased infarct size from 33.0 ± 3.8% of the risk zone in control hearts to 11.8 ± 2.0% (P < 0.001), and rapamycin blocked this protection. A comparable effect was seen for SB216763 (1 μM) with infarct size reduction to 13.5 ± 2.3% (P < 0.001). In summary, these experiments reveal a critical role for p70S6 upstream of mPTP in the signaling pathway of NECA's cardioprotection at reperfusion.