Reperfusion-activated Akt kinase prevents apoptosis in transgenic mouse hearts overexpressing insulin-like growth factor-1.

Abstract -Hearts of wild-type and insulin-like growth factor-1 overexpressing (Igf-1(+/-)) transgenic mice were subjected to Langendorff perfusions and progressive periods of ischemia followed by reperfusion. Apoptosis was measured by DNA nucleosomal cleavage and a hairpin probe labeling assay to detect single-base overhang. Transgenic hearts subjected to 20 minutes of ischemia and 4 hours of reperfusion (I/R) sustained a rate of apoptosis of 1.8+/-0.3% compared with 4.6+/-1.1% for wild-type controls (n=4; P<0.03). Phosphorylation of the protein kinase Akt/protein kinase B was elevated 6.2-fold in transgenic hearts at baseline and increased another 4.4-fold within 10 minutes of reperfusion, remaining elevated for up to 2 hours. I/R activated Akt in wild-type hearts but to a lesser extent (1.6+/-0.3-fold). Pretreatment of transgenic hearts with wortmannin immediately before and during ischemia eliminated reperfusion-mediated activation of Akt and neutralized the resistance to apoptosis. The stress-activated kinase p38 was also activated during ischemia and reperfusion in both wild-type and transgenic hearts. Perfusion with the p38 inhibitor SB203580 (10 micromol/L) blocked both p38 activation and phosphorylation of Akt and differentially modulated apoptosis in wild-type and transgenic hearts. Pretreatment with SB203580 reduced apoptosis in wild-type hearts but increased apoptosis in transgenic hearts. These results demonstrate that Akt phosphorylation during I/R is modulated by IGF-1 and prevents apoptosis in hearts that overexpress the IGF-1 transgene.

Characteristics of I(K) and its response to quinidine in experimental healed myocardial infarction.

INTRODUCTION: Mechanisms and drug treatment of serious ventricular arrhythmias in patients with healed myocardial infarction (HMI) are incompletely understood, in part because the electrophysiology and pharmacology of myocytes from noninfarcted regions of HMI hearts are not well characterized. METHODS AND RESULTS: We studied the delayed rectifier potassium current (I(K)) and quinidine responsiveness of single left ventricular subendocardial myocytes isolated from the region remote to the border zone of healed infarct myocardium (4 to 6 mm from scar edge) in cat hearts 2 months after coronary artery occlusion. Subendocardial cells isolated from corresponding regions of normal cat hearts provided controls. I(K) activation and tail currents were recorded using whole cell, voltage clamp techniques. Membrane capacitance of cells remote to HMI (187 +/- 7 pF) was significantly greater than normal (155 +/- 6 pF; P < 0.001). Action potential durations (APDs) recorded from myocytes in remote regions were prolonged (APD90 = 247 +/- 10 msec) compared to normal (214 +/- 11 msec; P < 0.05). Quinidine (1 microM) significantly prolonged APD90 in normal cells but not in remote cells. Density of I(K) (tail current) was significantly decreased in remote cells (3.1 +/- 0.3 pA/pF) compared to normal (3.9 +/- 0.3 pA/pF; P < 0.05), and voltage-dependent activation of I(K) was shifted in the positive direction. Quinidine had significantly less incremental blocking effect on I(K) already blunted by regional hypertrophy compared to its effect on normal cells in remote cells. IC50 shifted to 0.95 microM in remote cells compared with 0.50 microM in normal cells. CONCLUSION: Cells in noninfarct region remote from the scar are hypertrophied and display altered electrophysiology. Their reduced I(K) responsiveness to quinidine may explain, in part, failure of quinidine to prolong APD in such cells. Moreover, dispersion of repolarization may be decreased by the effect of quinidine on normal cells.

Hypertrophy decreases cardiac KATP channel responsiveness to exogenous and locally generated (glycolytic) ATP.

This study tests the hypothesis that glycolytic regulation of KATP channel activity is altered in myocardial hypertrophy. Left ventricular (LV) subendocardial myocytes were isolated from cats with normal or left ventricular hypertrophied hearts (LVH). Saponin-permeabilized open cell-attached patch configurations of normal and LVH cells were exposed to an exogenous ATP consuming system containing hexokinase and 2-deoxyglucose. Phosphoenol pyruvate (PEP, substrate for the last ATP producing step in glycolysis) was applied extracellularly; ADP was present. In both cell types, KATP channels were activated in the absence of PEP, inhibited when PEP was added and activated again when PEP was removed, indicating the cells retained metabolic integrity and generated ATP in the proximity of their KATP channels. Single channel conductance in the absence of PEP was similar (70 pS, normal; 66 pS, LVH). However, LVH KATP channels showed enhanced activity (P0=0.50+/-0.03); normal (0.41+/-0.03) in PEP absence (P<0. 05). PEP responsiveness was reduced in LVH, with IC50, PEP increased to 23 microM; (11 microM normal). Lactate failed to activate KATP channels in both cell types. The concentration-P0 response curves obtained during exposure of open cells to exogenous ATP also revealed reduced responsiveness to ATP of LVH KATP channels (IC50, ATP=283 microM LVH; 93 microM normal). Our data indicate myocardial hypertrophy increases the maximal activity of KATP channels in the absence of ATP and reduces their responsiveness to ATP, including locally generated glycolytic ATP. These alterations in metabolic regulation of myocardial electrophysiology may contribute to diversity of action potential shortening in hypertrophied hearts during acute ischemia.

Regional gradation of L-type calcium currents in the feline heart with a healed myocardial infarct.

INTRODUCTION: Abnormal action potentials in myocytes adjacent to > 2-month-old feline LV myocardial infarcts (MI) may reflect alterations in Ca2+ currents (Ica). METHODS AND RESULTS: We compared ICa, at 36 degrees C, in subendocardial myocytes isolated from areas adjacent to MI and to ICa in cells from remote areas (> 4 mm away; REM) and control cells from similar regions in normal hearts. Control (CON) myocytes had membrane capacitance of 234 +/- 10 pF (n = 81 cells) compared to 305 +/- 14 pF in REM (71 cells; P < 0.05 from CON) and 237 +/- 11 pF (n = 55 cells) in MI (not different from CON). From Vh = -40 mV; peak ICa elicited by test potentials (-35 to +70 mV) were significantly larger in CON (-1746 +/- 123 pA) and REM (-1795 +/- 142 pA) compared to MI (-1352 +/- 129 pA) (P < 0.05). Peak ICa density was significantly reduced in REM (-6.0 +/- 0.4 pA/pF) or MI (-5.7 +/- 0.4 pA/pF, P < 0.05) compared to CON (-7.5 +/- 0.4 pA/pF). Double exponential ICa decay was similar among groups. Half-inactivation potential (V0.5) was significantly shifted (hyperpolarizing direction) for MI (-29.1 +/- 2.6 mV) and REM (-24.6 +/- 1.2 mV) myocytes compared to -20.3 +/- 1.0 mV in CON. MI slope factor (k; 9.0 +/- 0.5) was significantly different from CON (6.8 +/- 0.3) and REM (7.3 +/- 0.4). No differences in time course of recovery from inactivation were noted. Five millimolar Ba2+o produced significant increases in ICa in CON and REM but an attenuated response in MI. Bay K8644 (1 microM) produced similar ICa increase in all groups. ICa increase due to isoproterenol (1 microM) in MI and REM was half that in CON, but there were no differences in increased ICa responses among groups following phenylephrine (10 microM). CONCLUSION: Reduced ICa density in REM reflects cell hypertrophy, whereas altered ICa of MI may reflect altered channel structure and/or function.