© 2015, Springer Science+Business Media New York.Oxidants increase intracellular free Zn2+ concentration ([Zn2+]i) in ventricular myocytes, which contributes to oxidant-induced alterations in excitation-contraction coupling (ECC). However, it is not clear whether increased [Zn2+]i in cardiomyocytes via increased reactive nitrogen species (RNS) has a role on heart function under pathological conditions, such as hyperglycemia. In this study, first we aimed to investigate the role of increased [Zn2+]i under in vitro condition in the development of both electrical and mechanical dysfunction of isolated papillary muscle strips from rat heart via exposed samples to a Zn2+-ionophore (Zn-pyrithione; 1 μM) for 20 min. Under simultaneous measurement of intracellular action potential and contractile activity in these preparations, Zn-pyrithione exposure caused marked prolongation in action potential repolarization phase and slowdown in both contraction and relaxation rates of twitch activity. Second, in order to demonstrate an association between increased [Zn2+]i and increased RNS, we monitored intracellular [Zn2+]i under an acute exposure of nitric oxide (NO) donor sodium nitroprusside, SNP, in freshly isolated quiescent cardiomyocytes loaded with FluoZin-3. Resting level of free Zn2+ is significantly higher in cardiomyocytes under hyperglycemic condition compared to those of the controls, which seems to be associated with increased level of RNS production in hyperglycemic cardiomyocytes. Western blot analysis showed that Zn-pyrithione exposure induced a marked decrease in the activity of protein phosphatase 1 and 2A, member of macromolecular protein complex of cardiac ryanodine receptors, RyR2, besides significant increase in the phosphorylation level of extracellular signal-regulated kinase1/2 as a concentration-dependent manner. Overall, the present data demonstrated that there is a cross-relationship between increased RNS production and increased [Zn2+]i level in cardiomyocytes under pathological conditions such as hyperglycemia.