okadaic-acid and Myocardial-Infarction

In this study, we investigated the effects of a PKA stimulating cocktail on sodium currents from normal epicardial cells (NZs) and on those from cells dispersed from the epicardial zone of the 5-day infarcted canine heart (IZs). To do so, we used whole-cell voltage-clamp techniques.. During superfusion with the PKA activator cocktail, peak sodium current (I(Na)) density significantly increased by 32+/-5.3% (NZs) and 17+/-5.4% (IZs). However, despite this increase, IZ peak I(Na) still was not fully restored to NZ values. In both cell types, the density effect was accompanied by a shift in I/I(max) curves, as well as a slowing in recovery from inactivation. Inactivation from a closed state was accelerated. Furthermore, in the presence of chloroquine, which is known to interrupt intracellular vesicular traffic, PKA activator effects to augment I(Na) were only partially inhibited in NZs but abolished in IZs. To understand whether the phosphorylation status of basal Na(+) channels in the two cell groups differed, the effects of okadaic acid and PP2A1 were studied. Results suggest that in IZs, Na(+) channels in the basal state are already phosphorylated.. PKA stimulation of I(Na) of the remodeled IZ does augment current density possibly by augmenting the trafficking of channels to an active site on the membrane. However, the resulting I(Na), while partially rescued, is not similar to the potentiated I(Na) of NZs. Specific kinetic changes also occur with the PKA stimulation of IZs and results with okadaic acid and PP2A1 suggest that in their remodeled state, Na(+) channels in IZs are already phosphorylated.

Topics: Analysis of Variance; Animals; Calcium Channel Agonists; Cyclic AMP-Dependent Protein Kinases; Dogs; Enzyme Activation; Myocardial Infarction; Myocardium; Okadaic Acid; Patch-Clamp Techniques; Sodium Channels; Ventricular Remodeling

Downregulation of the L-type Ca(2+) current (I(Ca)) is an important determinant of the electrical remodeling of diseased atria. Using a rat model of heart failure (HF) due to ischemic cardiopathy, we studied I(Ca) in isolated left atrial myocytes with the whole-cell patch-clamp technique and biochemical assays. I(Ca) density was markedly reduced (1.7+/-0.1 pA/pF) compared with sham-operated rats (S) (4.1+/-0.2 pA/pF), but its gating properties were unchanged. Calcium channel alpha(1C)-subunit quantities were not significantly different between S and HF. The beta-adrenergic agonist isoproterenol (1 micromol/L) had far greater stimulatory effects on I(Ca) in HF than in S (2.5- versus 1-fold), thereby suppressing the difference in current density. Dialyzing cells with 100 micromol/L cAMP or pretreating them with the phosphatase inhibitor okadaic acid also increased I(Ca) and suppressed the difference in density between S and HF. Intracellular cAMP content was reduced more in HF than in S. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine had a greater effect on I(Ca) in HF than in S (76.0+/-11.2% versus 15.8+/-21.2%), whereas the inhibitory effect of atrial natriuretic peptide on I(Ca) was more important in S than in HF (54.1+/-4.8% versus 24.3+/-8.8%). Cyclic GMP extruded from HF myocytes was enhanced compared with S (55.8+/-8.0 versus 6.2+/-4.0 pmol. mL(-1)). Thus, I(Ca) downregulation in atrial myocytes from rats with heart failure is caused by changes in basal cAMP-dependent regulation of the current and is associated with increased response to catecholamines.

Topics: 1-Methyl-3-isobutylxanthine; Adrenergic beta-Agonists; Animals; Atrial Natriuretic Factor; Calcium; Calcium Channels, L-Type; Catecholamines; Cell Separation; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Down-Regulation; Enzyme Inhibitors; Heart Atria; Heart Failure; Male; Myocardial Infarction; Myocardium; Okadaic Acid; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Rats; Rats, Wistar; Signal Transduction

We report that okadaic acid (OA), a known inhibitor of Ser/Thr phosphatases, protects pig myocardium against ischemic injury in an in vivo model and stimulates the activities of stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs). When OA was directly infused into the subsequently ischemic myocardium for 60 min before a 60-min period of coronary occlusion followed by reperfusion, infarct size was reduced from a control value of 83.4 +/- 2.8% of the risk region to 40.7 +/- 9.1%. When OA was infused for 10 min before a 5-min occlusion and during 45 min thereafter, infarct size was reduced to 26.5%. In a separate set of similar experiments, we pretreated pig hearts in vivo with the protein-synthesis inhibitor and known activator of SAPK/JNK, anisomycin (AN), and found that this compound also significantly reduced infarct size from 83.4 +/- 2.8.1% to 48.1 +/- 5.1%. For in vitro assays, OA (600 nM), AN (500 microM), or solvent (KHB) were locally infused into the left ventricular myocardium, and biopsies from in situ beating hearts were obtained after 10, 30, and 60 min of infusion. The activities of Ser/Thr phosphatases (PPases), especially PP-2A, were significantly decreased after OA infusion. OA infusion increased the activity (in-gel phosphorylation of N-terminal c-Jun1-135) of both 46- and 55-kDa SAPK/JNKs (twofold to threefold, 30 and 60 min of infusion), and this increase correlated well with the observed decrease of PPase activities. Western blot analysis with a phosphospecific SAPK/JNK (Thr 183/Tyr 185) antibody showed an increased content of the phosphorylated forms after OA treatment. We observed significant stimulation of SAPK/JNK activity also after AN treatment (threefold to fourfold, after 30 min of infusion). In contrast to the SAPK/JNKs, the infusion of both OA and AN did not significantly change the activities and phosphorylation of extracellular signal-related kinases (ERKs) and p38-MAPK. The findings that the protective effect of both OA and AN correlates with increased activity of SAPK/JNKs suggest the involvement of these enzymes in the mechanism of cardioprotection.

Topics: Animals; Anisomycin; Calcium-Calmodulin-Dependent Protein Kinases; Hemodynamics; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Myocardial Infarction; Myocardial Ischemia; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Protein Kinases; Swine