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Insulin has shown to have cardioprotective effect in diabetic patient after digoxin intoxication. The latter, prompted us to study whether insulin interacts directly with Na⁺/K⁺-ATPase. The interaction of insulin with Na⁺/K⁺-ATPase was explored using enzyme activity, Biacore and Western blot. We also used, flow cytometry, immunohistochemistry and chronotropy on both neonatal and adult rats cardiomyocytes. Insulin at concentration 1.7e⁻⁷ M blunted the effect of digoxin on Na⁺/K⁺-ATPase activity. In Western blot, the same insulin concentration decreased enzyme α subunit immunoreactivity. Insulin and digoxin decreased both enzyme α subunit immunoreactivity but insulin/digoxin co-treatment did not. Biacore confirmed a direct interaction between insulin and Na⁺/K⁺-ATPase. In neonatal rat cardiomyocytes, insulin plus digoxin induced cell apoptosis but not alone. In adult rat cardiomyocytes, insulin at optimal dose did not induce apoptosis but prevented the one induced by digoxin. In immunocytochemsitry both insulin and digoxin altered Na⁺/K⁺-ATPase α subunit immunoreactivity while their association did not. Finally, insulin increased the beating rate of neonatal rat cardiomyocytes (45±7 beats/min); so did digoxin (36±13 beats/min). The effect of insulin was prevented after pre-treated with digoxin. These results demonstrate that insulin interacts directly with Na⁺/K⁺-ATPase pump and alters the effect of digoxin. This would have important clinical relevance in cardiac complications related to type I and II diabetes., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)