Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically, and have the properties of a non-linear oscillator. In this study, we have analyzed the relationship between the fluctuation of contraction rhythm of spontaneously beating cultured cardiac myocytes, and the coupling strength among them. The coefficient of variation of contraction intervals increased transiently in the early stages of incubation, and then decreased almost monotonically with time. The contraction rhythm of the myocytes became synchronized in the late stage of the culture. The day on which synchronization occurred almost coincided with the day when the coefficient of variation reached its lowest value. In addition, we have performed a mathematical analysis using interacting Bonhoeffer-van der Pol oscillators to clarify the mechanisms underlying the changes in the fluctuation of contraction rhythm with time. As the coupling strength among oscillators increased, the coefficient of variation of oscillation periods increased temporarily, but then decreased rapidly when the oscillators showed synchronization. These results suggest that the changes in the fluctuation of beating rhythm result from the increase in strength of electrical coupling among spontaneously beating cardiac myocytes.
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.
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