Ischemia/reperfusion-induced death of cardiac myocytes: possible involvement of nitric oxide in the coordination of ATP supply and demand during ischemia

Abstract

Nitric oxide (NO) has been known to play various functional and pathological roles as an intracellular or intercellular messenger in the heart. In this study, we investigated whether NO produced during ischemia was involved in the coordination of ATP supply and demand, and also in protection from cell death using cultured cardiac myocytes. Unexpectedly, the survival rate of myocytes for 3 h simulated ischemia (SI) was increased as compared with that for 2 h SI at 24 h after reperfusion. The cellular ATP level at 3 h after the start of SI was increased compared with that at 2 h, and was almost the same as that before the start of SI. The cellular ATP level at 3 h SI was significantly reduced by either the inhibition of nitric oxide synthase (NOS) or scavenging of NO. Either the inhibition of NOS or the scavenging of NO during SI for 3 h also resulted in a significant decrease in the survival rate of myocytes.Immunocytochemical and Western blot analyses revealed that the expression of nNOS was most evident in cardiac myocytes, but no significant change was observed in the expression of all three NOS isoforms at 2 h SI and at 3 h SI. The fluorescent intensity of DAF-FM was significantly increased at 3 h SI as compared with that at 2 h SI, and 2 the increase in DAF fluorescence during SI was almost completely suppressed by treatment with L-VNIO, a potent specific inhibitor of nNOS. In addition, treatment with L-VNIO decreased the cellular ATP level and survival rate. This study suggested that the enhanced production of NO was critical in balancing ATP supply and demand during ischemia, and also in protecting cells from ischemia/reperfusion injury.

Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia

“…In this study, the extracellular ATP-purinoceptor system was possibly involved in cardiomyocytes [11]. In addition, Feron et al [26] have revealed that eNOS is found in caveolin-3-containing lipid rafts in cardiomyocytes, and that elevations in intracellular Ca 2+ cause the activation of eNOS, further supporting our inference that NO was involved in the suppression of ATP release via Cx43 hemichannels.…”

“…We first investigated the underlining mechanisms for this transient ATP increase caused by the exposure to OGD. The concentration of intracellular ATP did not decrease, but rather increased probably due to a reduction in ATP utilization, since spontaneous and rhythmic contractions were almost completely abolished at 1 h and 2 h after the onset of OGD [11]. However, the intracellular ATP was markedly decreased at 3 h OGD due to continued suppression of the production of ATP.…”

“…The method of culturing cardiomyocytes is described in detail in our previous papers [10][11][12][13]. In brief, cardiomyocytes were prepared from the ventricles of Wistar rats aged 2-3 days, removed after decapitation.…”

“…OGD procedure is described elsewhere in detail [11,14]. In short, serum-containing incubation medium was exchanged with serum-free DMEM (Gibco BRL, Invitrogen Corp., Carlsbad, CA) 2 h before the start of experiments.…”

“…ATP concentrations of the cultures were analyzed with a CheckLite TM 250 (Kikkoman, Chiba, Japan) or ATP-assay kit (Toyo B-net, Tokyo, Japan) according to each manufacturer's instructions, based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin [3,11]. For the measurement of the concentration of intracellular ATP, the cells were placed in 400 l of Tris-HCl buffer, and lysis reagent was added 20 min later.…”