Methods that increase cardiomyocyte survival upon exposure to
ischemia,
hypoxia and reoxygenation
injuries are required to improve the efficacy of cardiac
cell therapy and enhance the viability and function of engineered tissues. We investigated the effect of combined
hypoxia/NaNO2 pretreatment on rat neonatal cardiomyocyte (CM), cardiac fibroblast, and human embryonic stem cell-derived CM (hESC-CM) survival upon exposure to
hypoxia/reoxygenation (H/R) injury in vitro. Cells were pretreated with and without
hypoxia and/or various concentrations of NaNO2 for 20 min, then incubated for 2 h under hypoxic conditions, followed by 2 h in normoxia. The control cells were maintained under normoxia for 4 h. Pretreatment with either
hypoxia or NaNO2 significantly increased CM viability but had no effect on cardiac fibroblast viability. Combined
hypoxia/NaNO2 pretreatment significantly increased CM viability but significantly decreased cardiac fibroblast viability. In rat neonatal CMs, cell death, as determined by
lactate dehydrogenase (LDH) activity, was significantly reduced with
hypoxia/NaNO2 pretreatment; and in hESC-CMs,
hypoxia/NaNO2 pretreatment increased the BCL-2/BAX gene expression ratio, suggesting that
hypoxia/NaNO2 pretreatment promotes cell viability by downregulating apoptosis. Additionally, we found a correlation between the prosurvival effect of
hypoxia/NaNO2 pretreatment and the
myoglobin content of the cells by comparing neonatal rat ventricular and atrial CMs, which express high and low
myoglobin respectively. Functionally,
hypoxia/NaNO2 pretreatment significantly improved the excitation threshold upon H/R injury to the level observed for uninjured cells, whereas pretreatment did not affect the maximum capture rate. Hence,
hypoxia/NaNO2 pretreatment may serve as a strategy to increase CM survival in cardiac regenerative
therapy applications and tissue engineering.