Dual role of nNOS in ischemic injury and preconditioning

Abstract

Background: Nitric oxide (NO) is cardioprotective and a mediator of ischemic preconditioning (IP). Endothelial nitric oxide synthase (eNOS) is protective against myocardial ischemic injury and a component of IP but the role and location of neuronal nitric oxide synthase (nNOS) remains unclear. Therefore, the aims of these studies were to: (i) investigate the role of nNOS in ischemia/reoxygenation-induced injury and IP, (ii) determine whether its effect is species-dependent, and (iii) elucidate the relationship of nNOS with mitoKATP channels and p38MAPK, two key components of IP transduction pathway.

Results: Ventricular myocardial slices from rats and wild and nNOS knockout mice, and right atrial myocardial slices from human were subjected to 90 min ischemia and 120 min reoxygenation (37 degrees C). Specimens were randomized to receive various treatments (n = 6/group). Both the provision of exogenous NO and the inhibition of endogenous NO production significantly reduced tissue injury (creatine kinase release, cell necrosis and apoptosis), an effect that was species-independent. The cardioprotection seen with nNOS inhibition was as potent as that of IP, however, in nNOS knockout mice the cardioprotective effect of non-selective NOS (L-NAME) and selective nNOS inhibition and also that of IP was blocked while the benefit of exogenous NO remained intact. Additional studies revealed that the cardioprotection afforded by exogenous NO and by inhibition of nNOS were unaffected by the mitoKATP channel blocker 5-HD, although it was abrogated by p38MAPK blocker SB203580.

Conclusions: nNOS plays a dual role in ischemia/reoxygenation in that its presence is necessary to afford cardioprotection by IP and its inhibition reduces myocardial ischemic injury. The role of nNOS is species-independent and exerted downstream of the mitoKATP channels and upstream of p38MAPK.

Figure 1

CK release (a), and cell necrosis (b) and apoptosis (c) in rat ventricular myocardium subjected to 90 min ischemia followed by 120 min of reoxygenation (n = 6/group). Specimens were incubated with SNAP, L-NAME or TRIM for 20 min prior to ischemia. *P < 0.05 vs. ischemia/reoxygenation (I/R) alone; †P < 0.05 vs. SNAP and TRIM treated groups.

Figure 2

CK release (a), and cell necrosis (b) and apoptosis (c) in mouse ventricular myocardium and CK release (d), and cell necrosis (e) and apoptosis (f) in human right atrial myocardium subjected to 90 min ischemia followed by 120 min of reoxygenation (n = 6/group). Specimens were incubated with SNAP, L-NAME or TRIM for 20 min prior to ischemia. *P < 0.05 vs. ischemia/reoxygenation (I/R) alone; †P < 0.05 vs. SNAP and TRIM treated groups.

Figure 3

CK release (a), and cell necrosis (b) and apoptosis (c) in nNOS knockout mice ventricular myocardium subjected to 90 min ischemia followed by 120 min of reoxygenation (n = 6/group). Specimens were incubated with SNAP, L-NAME or TRIM for 20 min prior to ischemia. For comparison, some muscles were subjected to ischemic preconditioning (IP). *P < 0.05 vs. ischemia/reoxygenation (I/R) alone.

Figure 4

CK release (a), and cell necrosis (b) and apoptosis (c) in rat ventricular myocardium subjected to 90 min ischemia followed by 120 min of reoxygenation (n = 6/group). Specimens were incubated with SNAP or TRIM for 20 min prior to ischemia in the absences and presence of 5-HD. For comparisons, some muscles were subjected to ischemic preconditioning (IP) in the absences and presence of 5-HD. *P < 0.05 vs. ischemia/reoxygenation (I/R) alone.

Figure 5

CK release (a), and cell necrosis (b) and apoptosis (c) in rat ventricular myocardium subjected to 90 min ischemia followed by 120 min of reoxygenation (n = 6/group). Specimens were incubated with SNAP or TRIM for 20 min prior to ischemia in the absences and presence of SB203580 (SB). For comparisons, some muscles were subjected to ischemic preconditioning (IP) in the absences and presence of SB203580. *P < 0.05 vs. ischemia/reoxygenation (I/R) alone.