Contractility and protein phosphorylation in cardiomyocytes: effects of isoproterenol and AR-L57

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Abstract
The cardiotonic drugs AR-L57 [2-(2,4-dimethoxyphenyl)-1H-imidazo[4,5b]pyridine] and isoproterenol stimulated contractility in cultured heart cells in concentration-dependent manners; only the effects of isoproterenol were blocked by propranolol. Isoproterenol but not AR-L57, enhanced the phosphorylation state of 7 protein bands [relative MW 155,000, 96,000, 27,000, 24,000, 20,000, 16,000, 12,000] and resulted in the dephosphorylation of 1 protein band (MW 21,000). Also, only isoproterenol increased the activation states of adenosine 3'',5''-cAMP-dependent protein kinase and glycogen phosphorylase. The 8 protein bands resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and detected by autoradiography were altered by isoproterenol in time- and concentration-dependent manners. The 24,000-MW protein substrate phosphorylated in response to isoproterenol was converted to a 12,000-MW species by heating in the presence of SDS prior to electrophoresis, suggesting that the 2 substrates were in fact identical proteins. A comparison of the 2-min responses to varying concentrations of isoproterenol resulted in excellent correlations between the phosphorylation states of individual protein bands and contractility. This was true even for the 21,000-MW species that was dephosphorylated. Only the 27,000-, 24-12,000-, and 16,000-MW substrates were phosphorylated rapidly enough to be associated with the onset of the inotropic response. Cultured myocytes are an important feature of these studies as they are 84% pure ventricular cells that remain 100% viable throughout an experiment. Because this system is suitable for biochemical measurements and the effects of agents on heart cell contractility can be determined, it is possible to correlate changes in biochemical parameters with alterations in physiological state.