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Hearts from female animals have been reported to be more resistant to ischaemia than those from males. Using rat isolated cardiac myocytes, I investigated gender differences in ischaemic resistance and whether it could be correlated with increased ATP-sensitive K+ (KATP) current density. Cells from female animals showed greater resistance to simulated ischaemia and reperfusion measured by an increase in the proportion of cells that recovered contractile function in response to stimulation and a reduction the number of hypercontracted cells. Female cells also had a greater ability to maintain calcium homeostasis in response to ischaemic challenge.;To measure KATP current density, whole-cell KATP currents were measured using patch clamp and normalised to cell capacitance. The KATP openers pinacidil or P-1075 elicited a higher KATP current density in female myocytes. However currents elicited by metabolic inhibition (NaCN + iodoacetate) were much larger than those produced by openers, and did not differ between genders. Similarly in cell-attached patch experiments there was no difference in the number of channels observed or their time to activation.;I also investigated the possibility, proposed recently, that protein kinase C may lead to internalisation of the KATP channel. In response to metabolic inhibition, whole-cell KATP currents are activated and then decline with time. Pre-incubation of myocytes with phorbol myristate acetate (PMA) did not affect the time course of the decline in contrast to the PKC inhibitor chelerythrine. Further, in cell attached patches, the single-channel open probability declined in the same way as did whole cell current, suggesting that the decline of whole-cell current reflects a fall in open probability rather than channel internalisation.

The aim of these studies was to investigate the changes in gene expression that occur in H9c2 myoblast cells subjected to the metabolic stresses associated with myocardial ischaemia/reperfusion. The expression of Vascular Endothelial Growth Factor-A (VEGF-A) and Myocyte Stress 1 protein (ms1) were examined. VEGF-A has a physiological role in stimulating angiogenesis following cardiac ischaemia/reperfusion in vivo, whereas ms1 expression is induced following left ventricular hypertrophy, but has not been examined previously during ischaemia/reperfusion. No increase in VEGF-A, or ms1 mRNA was detected during simulated ischaemia but both were upregulated following reperfusion. The induction in VEGF-A mRNA was caused by an increase in mRNA stability regulated by ERK and JNK pathways. A reporter system was used to demonstrate that simulated ischaemia/reperfusion exerts its effect on both the 5' and 3'UTR of VEGF-A mRNA. The induction in ms1 mRNA was caused by an increase in transcription that was dependent on activation of the JNK pathway. An increase in the amount of ms1 protein expression was also detected during reperfusion. Regulation of translation also influences the expression of proteins during ischaemia/reperfusion. The effect of simulated ischaemia on message translation was investigated using cDNA microarrays. Simulated ischaemia inhibited CAP dependent translation and induced eIF2a phosphorylation. Some messages were more highly represented in the polysome fraction from ischaemic cells, compared to that from resting cells, including FAD oxidoreductase and a sub complex of NADH dehydrogenase, proteins involved in electron transport.

This study investigated cytoplasmic inter-subunit interactions between the Kir6.2 and SUR2A subunits of the cardiac ATP-sensitive potassium channel. The channels are a heterooligomeric complex of pore-forming Kir6.2 subunits and sulphonylurea receptor (SUR2A) subunits. Interactions between the cytoplasmic loops, the nucleotide binding domains (NBF1 and NBF2) of SUR2A and the full length of Kir6.2 were determined. In co-immunoprecipitation experiments, fragments from the C-terminal of SUR2A containing residues 1294-1358 tagged with Maltose-binding protein (MBP) showed binding with the full length Kir6.2 subunit, while residues between 1358-1545 did not. This indicated involvement of a 65 amino acid domain in the proximal C-terminal of SUR2A in forming a direct interaction with Kir6.2. When HEK 293 cells stably expressing Kir6.2/SUR2A channels were transiently transfected with SUR2A fragments containing residues 1294-1359, KATP current was decreased. This current reduction was due to a decreased number of channel subunits in the cell membrane; this was demonstrated by using immunocytochemistry, which showed that anti-K ATP channel subunit-associated fluorescence was lower in the cell membrane and increased in the intracellular compartment in the presence of the binding region.;Use of SUR2A/MRP1 chimaeras of the putative binding domain showed that the last eleven amino acids of the binding region were important for binding activity but that they do not contain all the elements necessary for binding. Co-immunoprecipitation and assays of disruption of functional channels with the binding domain chimaeras suggested an important role for the residues between 1318 and 1337 in the Kir6.2 binding motif within the SUR2A C-terminal domain.

In freshly isolated adult rat cardiac myocytes, adrenaline decreased AMPK activity and Thrl72 phosphorylation in AMPK a-subunits. This was associated with a decrease in AMPK-driven phosphorylation of acetyl-CoA carboxylase. The effect of adrenaline on AMPK was rapid with a half-time of approximately 4 minutes. The inactivation of AMPK by adrenaline was not associated with detectable changes in the myocyte contents of ATP, ADP, AMP, creatine, and creatine phosphate. The effect of adrenaline on AMPK was preserved under conditions where AMPK was activated by palmitate or sorbitol, but it was markedly diminished when AMPK was activated by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), oligomycin, or phenformin. The effect of adrenaline was partially blocked by propranolol (0-adrenergic antagonist) or phentolamine (ai-adrenergic antagonist) while it was essentially abolished when both blockers were present, suggesting involvement of both p and ai adrenergic receptors. Isoproterenol (P-adrenergic agonist) and phenylephrine (ai-adrenergic agonist) could also decrease AMPK activity and Thrl72 phosphorylation. Adrenaline did not increase phosphorylation of Ser485/491 in the AMPK a-subunit, but incubation of a catalytically inactive AMPK complex (aipiyl) with a cell lysate from adrenaline-treated myocytes increased phosphorylation of the AMPK pi subunit. The effect of adrenaline was not mimicked by conditions that activated cAMP-pathways and was not blocked by an inhibitor of calcium/calmodulin-dependent kinase II. However, a phorbol ester could mimic the effect of adrenaline on AMPK, suggesting the possible involvement of PKC isoforms.