Influence of ATP-sensitive potassium channel modulators on ischemia-induced fibrillation in isolated rat hearts.

We have confirmed the findings of Kantor and colleagues that ischemia-induced fibrillation in isolated Langendorff-perfused rat hearts can be prevented by glyburide, a blocker of ATP-dependent K channels. These data suggest that block of ATP-dependent K current [IK(ATP)] is a novel antiarrhythmic mechanism. This hypothesis was further tested by evaluating the effects of another sulfonylurea IK(ATP) blocker, tolbutamide (1 mM) and two agents known to activate these channels in cardiac tissue, BRL 34915 (10 microM) and pinacidil (30 microM). Similar to glyburide, tolbutamide was also effective in preventing fibrillation in this isolated rat heart model. The IK(ATP) activators enhanced the rate of tachycardia and shortened the time required for the hearts to develop fibrillation. Coadministration of glyburide with either IK(ATP) activator prevented their effects. It is concluded that activation of IK(ATP) during global ischemia contributes to the development of fibrillation in the perfused rat heart model.

Use of adult rat cardiomyocytes to study cardiac glycogen metabolism.

The use of adult rat cardiomyocytes to model cardiac glycogen metabolism was investigated by monitoring the response of glycogen phosphorylase and glycogen synthase to epinephrine and insulin treatment. Cardiomyocytes derived from normal rats respond to epinephrine in the range of 1 X 10(-7) to 5.5 X 10(-6) M epinephrine with an increase in the percent of phosphorylase in the AMP-independent form from 11.5 to 24.8%. In the same cells, insulin in the range of 10(-9) to 10(-7) M increased the glucose 6-phosphate independent form of glycogen synthase from 30.5 to 40.5%. Cells derived from alloxan-diabetic hearts exhibit a hypersensitive phosphorylase activation and a refractile synthase inactivation in response to epinephrine treatment. This pattern is similar to that recorded using perfused heart preparations. The data presented suggests that adult rat cardiomyocytes represent a valid model of glycogen metabolism in both the normal and alloxan-diabetic rat.

Phosphorylation of rat heart glycogen synthase: studies in cardiomyocytes and in vitro phosphorylations with cAMP-dependent kinase and protein phosphatase-1.

The phosphorylation of glycogen synthase has been studied in freshly isolated adult rat cardiomyocytes. Six peaks of 32P-labeled tryptic peptides are recovered via C-18 high performance liquid chromatography (HPLC) when synthase is immunoprecipitated from 32P-labeled cardiomyocytes and digested with trypsin. When epinephrine treated cells are used as a source of enzyme, the same HPLC profile is obtained with a dramatic enhancement of 32P recovered in two of the HPLC peaks. In vitro phosphorylation of rat heart synthase by cAMP-dependent protein kinase stimulates the conversion of synthase from the I to the D form and results in the recovery of the same tryptic peptides from the C-18 as is the case for synthase derived from cardiomyocytes. Treatment of cAMP-dependent kinase phosphorylated synthase with protein phosphatase-1 leads to a reactivation of the enzyme and a dephosphorylation of the same tryptic peptides that are selectively phosphorylated in epinephrine treated cardiomyocytes. These results are discussed in relation to hormonal control of glycogen metabolism in cardiac tissue.

Immunologic identification of a glycogen synthase 93,000-dalton subunit from rat heart and liver.

A polyclonal sheep antibody to rat heart glycogen synthase has been used for immunoblot analysis and immunoprecipitation of both rat heart and liver synthase. The purified antibody completely inhibits glycogen synthase activity in rat heart preparations and specifically blots to a 93-kDa band in the 10,000 X g supernatants of both heart and liver homogenates. Immunoprecipitation of in vitro translation products from rat heart or liver poly(A+) RNA yields a unique band with a molecular mass of 93 kDa. Thus the subunit molecular mass of active glycogen synthase in rat heart is 93 kDa. In rat liver at least one form of glycogen synthase also appears to have a molecular mass of 93 kDa. Protocols used to purify rat liver synthase yield a subunit of 80-87 kDa, which retains activity, but which is no longer recognized by the antibody. This suggests that 1) a specific antigenic sequence has been proteolytically removed from the NH2 or COOH terminus of the protein, or 2) that limited proteolysis has led to a conformational change in the enzyme such that the antibody binding site is no longer recognized. Either or both of these possibilities represent a significant alteration in the enzyme due to proteolysis. In vitro studies using synthase preparations having molecular masses less than 93 kDa must be interpreted with caution due to possible structural changes which occur during purification which may alter the regulation or covalent modification of synthase.