Reactive oxygen species vs. NHE1 reactivation in myocardial reperfusion injury

PÉREZ G; FANTINELLI JC; GARCIARENA C; ENNIS I; MOSCA SM; CINGOLANI HE

Colby-Sawyer College, New London, USA

Conferencia; Gordon Research Conference on Cardiac Regulatory Mechanisms; 2009

Restoration of blood flow to ischemic myocardium is the most effective therapeutic strategy for reducing infarct size (IS) but it causes the so called reperfusion injury. NHE1 reactivation leading to intracellular Na+ and Ca2+ increase and/or reactive oxygen species (ROS) burst at the onset of reperfusion are the two most accepted mechanisms to explain this injury. This study was aimed to further explore the underlying mechanism of reperfusion injury. Regional infarction was induced in isolated perfused rat hearts by 40 minutes coronary occlusion followed by 2 hours reperfusion. IS and risk area (RA) were assessed by TTC and Evans blue staining. Maximal doses of three pharmacologic interventions, ROS scavenging by 2-mercaptopropionylglycine (MPG, 2 mmol/L), NHE1 inhibition by cariporide (10 mmol/L) and phosphodiesterase5A (PDE5A) inhibition by sildenafil (1 mmol/L), were applied at the onset of reperfusion. At the end of reperfusion, their effects on IS and myocardial TBARS and on phosphorylation of ERK1/2, p90rsk and NHE1 (assessed by a phosphospecific antibody which recognizes the Ser phosphorylated 14-3-3 protein binding motif of the NHE1) were analysed. Control IS was 28 ± 2 % of risk area (n=10). 10 mmol/L HOE642, 2 mmol/L MPG or 1 mmol/L sildenafil decreased IS similarly (in % of RA): 13 ± 2 (n=7), 13 ± 2 (n=6), 10 ± 3 (n=8) (P<0.05). No further protection was found by combining HOE642 + MPG (14 ± 2 %, n=10) or HOE642 + sildenafil (9 ± 3 %, n=5) or MPG + sildenafil (13 ± 3 %, n=6). TBARS were 10 ± 1 nmol/g of tissue in infarcted hearts (n=10) and were decreased similarly by MPG (4 ± 1 nmol/g, n=6) or HOE642 (5 ± 1 nmol/g, n=9) (P<0.05) but unaffected by sildenafil (9 ± 1 nmol/g, n=5) suggesting miscorrelation between ROS and IS. Since ROS activate NHE1 through MAP kinases phosphorylation and we recently suggested that sildenafil inhibits NHE1, we analyzed MAP kinases phosphorylation in our experimental conditions. Control infarcted hearts showed increased ERK1/2, p90rsk and NHE1 phosphorylation (235 ± 57, 232 ± 57 and 207 ± 30 % of non-infarcted control respectively, n=4, P<0.05) effects that were cancelled by MPG (89 ± 9, 97 ± 15 and 122 ± 15 % of non-infarcted control respectively, n=4) or HOE642 (118 ± 19, 109 ± 20 and 128 ± 15 % of non-infarcted control respectively, n=4). Interestingly, sildenafil did not reduce ERK1/2 or p90rsk phosphorylation (264 ± 26 and 223 ± 18 % of non-infarcted control respectively, n=4, P<0.05) but blunted NHE1 phosphorylation (123 ± 6 % non-infarcted control, n=4). If we consider recent reports showing that PDE5A inhibition decreases NHE1 activity and that HOE642 decreases ROS formation by a mitochondrial effect we may conclude that reperfusion injury results from NHE1 reactivation, being the burst of ROS at the onset of reperfusion the upstream necessary step for this reactivation. The PDE5A inhibition by sildenafil allowed us to show protection by decreasing NHE1 phosphorylation in spite of high levels of ROS.