Inhibition of p38 MAPK alpha/beta reduces ischemic injury and does not block protective effects of preconditioning

We examined the effect of inhibition of p38 mitogen-activated protein kinase (MAPK) alpha/beta during ischemia and preconditioning by using the inhibitor SB-202190. Isolated rat hearts were perfused with Krebs-Henseleit buffer, while left ventricular developed pressure (LVDP) and (31)P nuclear magnetic resonance spectra were acquired continuously. After 20 min of ischemia and 25 min of reperfusion, recovery of LVDP in untreated hearts was 32 +/- 4%, whereas hearts treated with SB-202190 5 min before ischemia recovered 59 +/- 7% of their pretreatment LVDP. Preconditioning improved functional recovery to 65 +/- 5%, which was unaffected by SB-202190 treatment, added either throughout the preconditioning protocol (56 +/- 5% recovery) or during the final reperfusion period of preconditioning (71 +/- 11% recovery). Necrosis was assessed after 40 min of ischemia and 2 h of reperfusion using 2,3,5-triphenyltetrazolium chloride (TTC) staining and creatine kinase release. The untreated group had 54 +/- 8% necrotic myocardium, whereas the SB-202190-treated group had 32 +/- 7% and the preconditioned group had 21 +/- 4% necrotic tissue by TTC staining.     

p38 mitogen-activated protein kinase mediates adenosine-induced alterations in myocardial glucose utilization via 5'-AMP-activated protein kinase

Adenosine-induced acceleration of glycolysis in hearts stressed by transient ischemia is accompanied by suppression of glycogen synthesis and by increases in activity of adenosine 5'-monophosphate-activated protein kinase (AMPK). Because p38 mitogen-activated protein kinase (MAPK) may regulate glucose metabolism and may be activated downstream of AMPK, this study determined the effects of the p38 MAPK inhibitors SB202190 and SB203580 on adenosine-induced alterations in glucose utilization and AMPK activity. Studies were performed in working rat hearts perfused aerobically following stressing by transient ischemia (2 x 10-min ischemia followed by 5-min reperfusion). Phosphorylation of AMPK and p38 MAPK each were increased fourfold by adenosine, and these effects were inhibited by either SB202190 or SB203580. Neither of these inhibitors directly affected AMPK activity. Attenuation of the adenosine-induced increase in AMPK and p38 MAPK phosphorylation by SB202190 and SB203580 occurred independently of any change in tissue ATP-to-AMP ratio and did not alter glucose uptake, but it was accompanied by an increase in glycogen synthesis and glycogen content and by inhibition of glycolysis and proton production. There was a significant inverse correlation between the rate of glycogen synthesis and AMPK activity and between AMPK activity and glycogen content. These data demonstrate that AMPK is likely downstream of p38 MAPK in mediating the effects of adenosine on glucose utilization in hearts stressed by transient ischemia. The ability of p38 MAPK inhibitors to relieve the inhibition of glycogen synthesis and to inhibit glycolysis and proton production suggests that these agents may restore adenosine-induced cardioprotection in stressed hearts.     

Cardioprotection induced by cardiac-specific overexpression of fibroblast growth factor-2 is mediated by the MAPK cascade

Our laboratory showed previously that cardiac-specific overexpression of FGF-2 [FGF-2 transgenic (Tg)] results in increased recovery of contractile function and decreased infarct size after ischemia-reperfusion injury. MAPK signaling is downstream of FGF-2 and has been implicated in other models of cardioprotection. Treatment of FGF-2 Tg and wild-type hearts with U-0126, a MEK-ERK pathway inhibitor, significantly reduced recovery of contractile function after global low-flow ischemia-reperfusion injury in FGF-2 Tg (86 +/- 2% vehicle vs. 66 +/- 4% U-0126; P < 0.05) but not wild-type (61 +/- 7% vehicle vs. 67 +/- 7% U-0126) hearts. Similarly, MEK-ERK inhibition significantly increased myocardial infarct size in FGF-2 Tg (12 +/- 3% vehicle vs. 31 +/- 2% U-0126; P < 0.05) but not wild-type (30 +/- 4% vehicle vs. 36 +/- 7% U-0126) hearts. In contrast, treatment of FGF-2 Tg and wild-type hearts with SB-203580, a p38 inhibitor, did not abrogate FGF-2-induced cardioprotection from postischemic contractile dysfunction. Instead, inhibition of p38 resulted in decreased infarct size in wild-type hearts (30 +/- 4% vehicle vs. 11 +/- 2% SB-203580; P < 0.05) but did not alter infarct size in FGF-2 Tg hearts (12 +/- 3% vehicle vs. 14 +/- 1% SB-203580). Western blot analysis of ERK and p38 activation revealed signaling alterations in FGF-2 Tg and wild-type hearts during early ischemia or reperfusion injury. In addition, MEK-independent ERK inhibition by p38 was observed during early ischemic injury. Together these data suggest that activation of ERK and inhibition of p38 by FGF-2 is cardioprotective during ischemia-reperfusion injury.     

Stress kinase phosphorylation is increased in pacing-induced heart failure in rabbits

In hearts with chronic left ventricular (LV) systolic dysfunction secondary to hypertension or myocardial infarction, MAPK phosphorylation and/or activity are increased. Whether other settings of LV dysfunction not associated with ischemia-reperfusion are also characterized by increased MAPK phosphorylation or activity is unknown. After 3 wk of rapid LV pacing (400 beats/min), eight rabbits displayed clinical signs of heart failure (HF), and echocardiography revealed an increase in LV end-diastolic diameter from 15.6 +/- 0.7 (means +/- SE) to 18.8 +/- 0.7 mm and a reduced shortening fraction from 31 +/- 1to10 +/- 2% (both P < 0.05). Morphological alterations in HF included increased numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cardiomyocytes, extent of fibrosis, and cross-sectional cardiomyocyte area. Total p38 MAPK did not differ between failing and normal hearts (n = 8). However, p38 MAPK phosphorylation [164,488 +/- 29,323 vs. 43,565 +/- 14,817 arbitrary units (AU), P < 0.05, densitometry] and the activities of p38 MAPK-alpha and -beta were increased in failing compared with normal hearts (149,441 +/- 38,381 and 170,430 +/- 32,952 vs. 68,815 +/- 28,984 and 81,788 +/- 22,774 AU, respectively, both P < 0.05). In failing compared with normal hearts, total and phosphorylated JNK46 and JNK54 MAPK were increased, whereas total and phosphorylated ERK MAPK remained unchanged. In pacing-induced HF, p38 and JNK MAPK phosphorylation as well as p38 MAPK activity was increased. Further studies will have to define whether or not chronic specific blockade of MAPK activity can interfere with apoptosis/fibrosis and thereby attenuate the progression of HF.     

Adenosine-induced late preconditioning in mouse hearts: role of p38 MAP kinase and mitochondrial K(ATP) channels

We investigated the role of p38 mitogen-activated protein kinase (MAPK) phosphorylation and opening of the mitochondrial ATP-sensitive K(+) [(K(ATP))(mito)] channel in the adenosine A(1) receptor (A(1)AR)-induced delayed cardioprotective effect in the mouse heart. Adult male mice were treated with vehicle (5% DMSO) or the A(1)AR agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 0.1 mg/kg ip). Twenty-four hours later, hearts were subjected to 30 min of global ischemia and 30 min of reperfusion in the Langendorff mode. Genistein or SB-203580 (1 mg/kg i.p.) given 30 min before CCPA treatment was used to block receptor tyrosine kinase or p38 MAPK phosphorylation, respectively. 5-Hydroxydecanoate (5-HD; 200 microM) was used to block (K(ATP))(mito) channels. CCPA produced marked improvement in left ventricular function, which was partially blocked by SB-203580 and 5-HD and completely abolished with genistein. CCPA caused a reduction in infarct size (12.0 +/- 2.0 vs. 30.3 +/- 3.0% in vehicle), which was blocked by genistein (29.4 +/- 2.3%), SB-203580 (28.3 +/- 2.6%), and 5-HD (33.9 +/- 2.4%). CCPA treatment also caused increased phosphorylation of p38 MAPK during ischemia, which was blocked by genistein, SB-203580, and 5-HD. The results suggest that A(1)AR-triggered delayed cardioprotection is mediated by p38 MAPK phosphorylation. Blockade of cardioprotection with 5-HD concomitant with decrease in p38 MAPK phosphorylation suggests a potential role of (K(ATP))(mito) channel opening in phosphorylation and ensuing the late preconditioning effect of A(1)AR.     

Activation of p38 MAPK and increased glucose transport in chronic hibernating swine myocardium

In preconditioned myocardium, activation of the mitogen-activated protein kinase (MAPK) p38 leads to increased glucose uptake via enhanced GLUT-4 translocation. Glucose uptake is also increased in chronic hibernating myocardium, but the role of p38 MAPK and GLUT-4 translocation has not been studied. Nine swine underwent instrumentation of the proximal left anterior descending coronary artery (LAD) with a small, external constrictor. At 3 mo after instrumentation, myocardial glucose uptake by PET imaging was higher in the LAD than in the remote region under basal, fasted conditions (0.08 +/- 0.02 vs. 0.04 +/- 0.01 micromol.min(-1).g(-1), P < 0.05). Compared with the remote region, the LAD region demonstrated increased membrane-bound GLUT-4 relative to total content (61 +/- 04 vs. 45 +/- 06%, P < 0.05), higher glycogen (28.37 +/- 4.41 vs. 19.26 +/- 1.87 mg/g wet wt, P < 0.05), and increased inducible nitric oxide synthase (NOS) activity (1.43 +/- 0.34 vs. 0.51 +/- 0.21 activity/mg protein, P < 0.05). p38 MAPK was 47 +/- 14% higher in the LAD than in the remote region (P < 0.05) and correlated well with the absolute degree of GLUT-4 membrane-bound translocation (r = 0.81, P < 0.01), relative increase in glycogen (r = 0.70, P < 0.05), and total NOS activity (r = 0.68, P < 0.05). In chronic hibernating myocardial tissue, p38 MAPK activation is increased under basal fasted conditions and correlates well with the increased degree of GLUT-4 translocation, glycogen accumulation, and NOS activity. As in preconditioned myocardium, activation of p38 MAPK may play an important role in the metabolic adaptations that characterize chronic hibernating myocardium.     

Inconsistent relation of MAPK activation to infarct size reduction by ischemic preconditioning in pigs

The importance of the activation of mitogen-activated protein kinases (MAPK) for the cardioprotection achieved by ischemic preconditioning (IP) is still controversial. We therefore measured infarct size and p38, extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) MAPK phosphorylation (by biopsies) in enflurane-anesthetized pigs. After 90 min low-flow ischemia and 120 min reperfusion, infarct size averaged 18.3 +/- 12.4 (SD)% (group 1, n = 14). At similar subendocardial blood flows, IP by 10 min ischemia and 15 min reperfusion (group 2, n = 14) reduced infarct size to 6.2 +/- 5.1% (P < 0.05). An inconsistent increase in p38, ERK, and p54 JNK phosphorylation (by Western blot) was found during IP; p46 JNK phosphorylation increased with the subsequent reperfusion. At 8 min of the sustained ischemia, p38, ERK, and p54 JNK phosphorylation were increased with no difference between groups (medians: p38: 207% of baseline in group 1 vs. 153% in group 2; ERK: 142 vs. 144%; p54 JNK: 171 vs. 155%, respectively). MAPK phosphorylation and reduction of infarct size by IP were not correlated, thus not supporting the concept of a causal role of MAPK in mediating cardioprotection by IP.     

Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.     

Preconditioning enhanced glucose uptake is mediated by p38 MAP kinase not by phosphatidylinositol 3-kinase

Ischemia is reported to stimulate glucose uptake, but the signaling pathways involved are poorly understood. Modulation of glucose transport could be important for the cardioprotective effects of brief intermittent periods of ischemia and reperfusion, termed ischemic preconditioning. Previous work indicates that preconditioning reduces production of acid and lactate during subsequent sustained ischemia, consistent with decreased glucose utilization. However, there are also data that preconditioning enhances glucose uptake. The present study examines whether preconditioning alters glucose transport and whether this is mediated by either phosphatidylinositol 3-kinase (PI3K) or p38 MAP kinase. Langendorff-perfused rat hearts were preconditioned with 4 cycles of 5 min of ischemia and 5 min of reperfusion, with glucose as substrate. During the last reflow, glucose was replaced with 5 mM acetate and 5 mM 2-deoxyglucose (2DG), and hexose transport was measured from the rate of production of 2-deoxyglucose 6-phosphate (2DG6P), using (31)P nuclear magnetic resonance. Preconditioning stimulated 2DG uptake; after 15 min of perfusion with 2DG, 2DG6P levels were 165% of initial ATP in preconditioned hearts compared with 96% in control hearts (p < 0.05). Wortmannin, an inhibitor of PI3K, did not block the preconditioning induced stimulation of 2DG6P production, but perfusion with SB202190, an inhibitor of p38 MAP kinase, did attenuate 2DG6P accumulation (111% of initial ATP, p < 0. 05 compared with preconditioned hearts). SB202190 had no effect on 2DG6P accumulation in nonpreconditioned hearts. Preconditioning stimulation of translocation of GLUT4 to the plasma membrane was not inhibited by wortmannin. The data demonstrate that ischemic preconditioning increases hexose transport and that this is mediated by p38 MAP kinase and is PI3K-independent.     

A1 adenosine receptor overexpression attenuates ischemia-reperfusion-induced apoptosis and caspase 3 activity

We tested the hypothesis that myocardial ischemia-reperfusion (I/R)-induced apoptosis is attenuated in transgenic mice overexpressing cardiac A(1) adenosine receptors. Isolated hearts from transgenic (TG, n = 19) and wild-type (WT, n = 22) mice underwent 30 min of ischemia and 2 h of reperfusion, with evaluation of apoptosis, caspase 3 activity, function, and necrosis. I/R-induced apoptosis was attenuated in TG hearts. TG hearts had less I/R-induced apoptotic nuclei (0.88 +/- 0.10% vs. 4.22 +/- 0.24% terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in WT, P < 0.05), less DNA fragmentation (3.30 +/- 0.38-fold vs. 4.90 +/- 0.39-fold over control in WT, P < 0.05), and less I/R-induced caspase 3 activity (145 +/- 25% over nonischemic control vs. 234 +/- 31% in WT, P < 0.05). TG hearts also had improved recovery of function and less necrosis than WT hearts. In TG hearts pretreated with LY-294002 (3 microM) to evaluate the role of phosphosinositol-3-kinase in acute signaling, there was no change in the functional protection or apoptotic response to I/R. These data suggest that cardioprotection with transgenic overexpression of A(1) adenosine receptors involves attenuation of I/R-induced apoptosis that does not involve acute signaling through phosphoinositol-3-kinase.     

Protection against ventricular arrhythmias and cardiac death using adenosine and lidocaine during regional ischemia in the in vivo rat

Despite decades of research, there are few effective ways to treat ventricular fibrillation (VF), ventricular tachycardia (VT), or cardiac ischemia that show a significant survival benefit. Our aim was to investigate the combined therapeutic effect of two common antiarrhythmic compounds, adenosine and lidocaine (AL), on mortality, arrhythmia frequency and duration, and infarct size in the rat model of regional ischemia. Sprague-Dawley rats (n = 49) were anesthetized with pentobarbital sodium (60 mg.ml(-1).kg(-1) i.p.) and instrumented for regional coronary occlusion (30 min) and reperfusion (120 min). Heart rate, blood pressure, and a lead II electrocardiogram were recorded. Intravenous pretreatment began 5 min before ischemia and extended throughout ischemia, terminating at the start of reperfusion. After 120 min, hearts were removed for infarct size measurement. Mortality occurred in 58% of saline controls (n = 12), 50% of adenosine only (305 microg.kg(-1).min(-1), n = 8), 0% in lidocaine only (608 microg.kg(-1).min(-1), n = 8), and 0% in AL at any dose (152, 305, or 407 microg.kg(-1).min(-1) adenosine plus 608 microg.kg(-1).min(-1) lidocaine, n = 7, 8, and 6). VT occurred in 100% of saline controls (18 +/- 9 episodes), 50% of adenosine-only (11 +/- 7 episodes), 83% of lidocaine-only (23 +/- 11 episodes), 60% of low-dose AL (2 +/- 1 episodes, P < 0.05), 57% of mid-dose AL (2 +/- 1 episodes, P < 0.05), and 67% of high-dose AL rats (6 +/- 3 episodes). VF occurred in 75% of saline controls (4 +/- 3 episodes), 100% of adenosine-only-treated rats (3 +/- 2 episodes), and 33% lidocaine-only-treated rats (2 +/- 1 episodes) of the rats tested. There was no deaths and no VF in the low- and mid-dose AL-treated rats during ischemia, and only one high-dose AL-treated rat experienced VF (25.5 sec). Infarct size was lower in all AL-treated rats but only reached significance with the mid-dose treatment (saline controls 61 +/- 5% vs. 38 +/- 6%, P < 0.05). We conclude that a constant infusion of a solution containing AL virtually abolished severe arrhythmias and prevented cardiac death in an in vivo rat model of acute myocardial ischemia and reperfusion. AL combinational therapy may provide a primary prevention therapeutic window in ischemic and nonischemic regions of the heart.     

Modulation of PGF2alpha- and hypoxia-induced contraction of rat intrapulmonary artery by p38 MAPK inhibition: a nitric oxide-dependent mechanism

The mechanisms through which p38 mitogen-activated protein kinase (p38 MAPK) is involved in smooth muscle contraction remain largely unresolved. We examined the role of p38 MAPK in prostaglandin F(2alpha) (PGF(2alpha))-induced vasoconstriction and in hypoxic pulmonary vasoconstriction (HPV) of rat small intrapulmonary arteries (IPA). The p38 MAPK inhibitors SB-203580 and SB-202190 strongly inhibited PGF(2alpha)-induced vasoconstriction, with IC(50)s of 1.6 and 1.2 microM, whereas the inactive analog SB-202474 was approximately 30-fold less potent. Both transient and sustained phases of HPV were suppressed by SB-203580, but not by SB-202474 (both 2 microM). Western blot analysis revealed that PGF(2alpha) (20 microM) increased phosphorylation of p38 MAPK and of heat shock protein 27 (HSP27), and this was abolished by SB-203580 but not by SB-202474 (both 2 microM). Endothelial denudation or blockade of endothelial nitric oxide (NO) synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly suppressed the relaxation of PGF(2alpha)-constricted IPA by SB-203580, but not by SB-202474. Similarly, the inhibition of HPV by SB-203580 was prevented by prior treatment with L-NAME. SB-203580 (2 microM), but not SB-202474, enhanced relaxation-induced by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in endothelium-denuded IPA constricted with PGF(2alpha). In alpha-toxin-permeabilized IPA, SB-203580-induced relaxation occurred in the presence but not the absence of the NO donor sodium nitroprusside (SNP); SB-202474 was without effect even in the presence of SNP. In intact IPA, neither PGF(2alpha)- nor SNAP-mediated changes in cytosolic free Ca(2+) were affected by SB-203580. We conclude that p38 MAPK contributes to PGF(2alpha)- and hypoxia-induced constriction of rat IPA primarily by antagonizing the underlying Ca(2+)-desensitizing actions of NO.     

Inhibition of p38 reduces myocardial infarction injury in the mouse but not pig after ischemia-reperfusion

The MAPK family member p38 is activated in the heart after ischemia-reperfusion (I/R) injury. However, the cardioprotective vs. proapoptotic effects associated with p38 activation in the heart after I/R injury remain unresolved. Another issue to consider is that the majority of past studies have employed the rodent as a model for assessing p38's role in cardiac injury vs. protection, while the potential regulatory role in a large animal model is even more uncertain. Here we performed a parallel study in the mouse and pig to directly compare the extent of cardiac injury after I/R at baseline or with the selective p38 inhibitor SB-239063. Infusion of SB-239063 5 min before ischemia in the mouse prevented ischemia-induced p38 activation, resulting in a 25% reduction of infarct size compared with vehicle-treated animals (27.9 +/- 2.9% vs. 37.5 +/- 2.7%). In the pig, SB-239063 similarly inhibited myocardial p38 activation, but there was no corresponding effect on the degree of infarction injury (43.6 +/- 4.0% vs. 41.4 +/- 4.3%). These data suggest a difference in myocardial responsiveness to I/R between the small animal mouse model and the large animal pig model, such that p38 activation in the mouse contributes to acute cellular injury and death, while the same activation in pig has no causative effect on these parameters.     

Effects of cortisol on cardiac myocytes and on expression of cardiac genes in fetal sheep

In 17 fetal sheep aged 129 days, the effects of large-dose infusions of cortisol (72.1 mg/day for 2-3 days) on proliferation, binucleation, and hypertrophy of cardiac myocytes, cardiac expression of angiotensinogen, angiotensin receptor subtypes 1 and 2, Glut-1, glucocorticoid and mineralocorticoid receptors, proteins of the MAPK pathways and calcineurin were studied. Cortisol levels were 8.7 +/- 2.3 nM (SE) in 8 control and 1,028 +/- 189 nM in 9 treated fetuses (P < 0.001). Cortisol had no effect on myocyte binucleation. Left ventricular free wall (LVFW) uni- and binucleated myocytes were larger in cortisol-treated fetuses (P < 0.001, P < 0.05). Cortisol-treated fetuses had higher right ventricular free wall (RVFW) and LVFW angiotensinogen (Aogen) mRNA levels (treated: 2.30 +/- 0.37, n = 8 and 2.05 +/- 0.45, n = 7 vs. control: 0.94 +/- 0.12, n = 8 and 0.67 +/- 0.09, n = 7, P < 0.02). Levels of the glucose transporter Glut-1 mRNA were lower in the LVFW of treated fetuses (0.83 +/- 0.23 vs. 1.47 +/- 0.30 in control, P < 0.05, n = 7, 8). The higher the cortisol level, the greater the Aogen mRNA level (RVFW, r = 0.61, P < 0.01, n = 16; LVFW, r = 0.83, P < 0.0003, n = 14). There were no other changes in mRNA levels nor in levels of extracellular kinase, JNK, p38, their phosphorylated forms, and calcineurin. Thus high levels of cortisol such as occur after birth do not affect fetal cardiac myocyte binucleation or number but are associated with higher levels of ventricular Aogen mRNA, lower levels of Glut-1 mRNA, and hypertrophy of LVFW myocytes. These effects could impact on postnatal cardiac development.     

Moderate alcohol consumption induces sustained cardiac protection by activating PKC-epsilon and Akt

C57BL/6 mice were fed 18% ethanol (vol/vol) in drinking water for 12 wk. Isovolumic hearts were subjected to 20 min of ischemia and 30 min of reperfusion on a Langendorff apparatus. There were no differences in baseline hemodynamic function between hearts from ethanol (EtOH)-fed mice and controls. However, prior alcohol consumption doubled recovery of left ventricular developed pressure (68 +/- 8 vs. 33 +/- 8 mmHg for controls; n = 10, P < 0.05) and reduced creatine kinase release by half (0.26 +/- 0.04 vs. 0.51 +/- 0.08 U x min(-1) x g wet wt(-1) for controls; n = 10, P < 0.05). EtOH feeding doubled expression of activated protein kinase C epsilon (PKC)epsilon (n = 6, P < 0.05); whereas PKC inhibition blocked protection during ischemia-reperfusion. EtOH feeding also increased expression of Akt three- to fivefold (n = 6, P < 0.05), whereas PKC inhibition prevented increases in Akt kinase activity. We conclude that signaling pathways involving PKC-epsilon are critical for sustained EtOH-mediated cardioprotection and that Akt may be a downstream effector of resistance to myocardial reperfusion injury.     

Altered sinus nodal and atrioventricular nodal function in freely moving mice overexpressing the A1 adenosine receptor

To investigate whether altered function of adenosine receptors could contribute to sinus node or atrioventricular (AV) nodal dysfunction in conscious mammals, we studied transgenic (TG) mice with cardiac-specific overexpression of the A1 adenosine receptor (A1AR). A Holter ECG was recorded in seven freely moving littermate pairs of mice during normal activity, exercise (5 min of swimming), and 1 h after exercise. TG mice had lower maximal heart rates (HR) than wild-type (WT) mice (normal activity: 437 +/- 18 vs. 522 +/- 24 beats/min, P < 0.05; exercise: 650 +/- 13 vs. 765 +/- 28 beats/min, P < 0.05; 1 h after exercise: 588 +/- 18 vs. 720 +/- 12 beats/min, P < 0.05; all values are means +/- SE). Mean HR was lower during exercise (589 +/- 16 vs. 698 +/- 34 beats/min, P < 0.05) and after exercise (495 +/- 16 vs. 592 +/- 27 beats/min, P < 0.05). Minimal HR was not different between genotypes. HR variability (SD of RR intervals) was reduced by 30% (P < 0.05) in TG compared with WT mice. Pertussis toxin (n = 4 pairs, 150 microg/kg ip) reversed bradycardia after 48 h. TG mice showed first-degree AV nodal block (PQ interval: 42 +/- 2 vs. 37 +/- 2 ms, P < 0.05), which was diminished but not abolished by pertussis toxin. Isolated Langendorff-perfused TG hearts developed spontaneous atrial arrhythmias (3 of 6 TG mice vs. 0 of 9 WT mice, P < 0.05). In conclusion, A1AR regulate sinus nodal and AV nodal function in the mammalian heart in vivo. Enhanced expression of A1AR causes sinus nodal and AV nodal dysfunction and supraventricular arrhythmias.     

Metabolic actions of metformin in the heart can occur by AMPK-independent mechanisms

The metabolic actions of the antidiabetic agent metformin reportedly occur via the activation of the AMP-activated protein kinase (AMPK) in the heart and other tissues in the presence or absence of changes in cellular energy status. In this study, we tested the hypothesis that metformin has AMPK-independent effects on metabolism in heart muscle. Fatty acid oxidation and glucose utilization (glycolysis and glucose uptake) were measured in isolated working hearts from halothane-anesthetized male Sprague-Dawley rats and in cultured heart-derived H9c2 cells in the absence or in the presence of metformin (2 mM). Fatty acid oxidation and glucose utilization were significantly altered by metformin in hearts and H9c2 cells. AMPK activity was not measurably altered by metformin in either model system, and no impairment of energetic state was observed in the intact hearts. Furthermore, the inhibition of AMPK by 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyyrazolo[1,5-a] pyrimidine (Compound C), a well-recognized pharmacological inhibitor of AMPK, or the overexpression of a dominant-negative form of AMPK failed to prevent the metabolic actions of metformin in H9c2 cells. The exposure of H9c2 cells to inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) or protein kinase C (PKC) partially or completely abrogated metformin-induced alterations in metabolism in these cells, respectively. Thus the metabolic actions of metformin in the heart muscle can occur independent of changes in AMPK activity and may be mediated by p38 MAPK- and PKC-dependent mechanisms.     

p38 MAPK-dependent small HSP27 and αB-crystallin phosphorylation in regulation of myocardial function following cardioplegic arrest

We previously demonstrated that myocardial p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27) are phosphorylated following cardioplegic arrest in patients undergoing cardiac surgery and correlate with reduced cardiac function. The following studies were performed to determine whether inhibition of p38 MAPK and/or overexpression of nonphosphorylatable HSP27 improves cardiac function following cardioplegic arrest. Langendorff-perfused isolated rat hearts were subjected to 2 h of intermittent cold cardioplegia followed by 30 min of reperfusion. Hearts were treated with (CP+SB) or without (CP) the p38 MAPK inhibitor SB-203580 (5 μM) supplied in the cardioplegia. Sham-treated hearts served as controls. In separate experiments, isolated rat ventricular myocytes infected with either green fluorescent protein (GFP) or a nonphosphorylatable HSP27 mutant (3A-HSP27) were subjected to 3 h of cold hypoxic cardioplegia and simulated reperfusion (CP) followed by video microscopy and length change measurements. Baseline parameters of cardiac function were similar between groups [left ventricular developed pressure (LVDP), 119 ± 4.9 mmHg; positive and negative first derivatives of LV pressure (± dP/dt), 3,139 ± 245 and 2, 314 ± 110 mmHg/s]. CP resulted in reduced cardiac function (LVDP, 72.2 ± 5.8 mmHg; ± dP/dt, 2,076 ± 231 and -1,317 ± 156 mmHg/s) compared with baseline. Treatment with 5 μM SB-203580 significantly improved CP-induced cardiac function (LVDP, 101.9 ± 0 mmHg; ± dP/dt, 2,836 ± 163 and -2,108 ± 120 mmHg/s; P = 0.03, 0.01, and 0.04, CP+SB vs. CP). Inhibition of p38 MAPK significantly lowered CP-induced p38 MAPK, HSP27, and αB-crystallin (cryAB) phosphorylation. In vitro CP decreased myocyte length changes from 10.3 ± 1.5% (GFP) to 5.7 ± 0.8% (GFP+CP). Infection with 3A-HSP27 completely rescued CP-induced decreased myocyte contraction (11.1 ± 1.0%). However, infection with 3A-HSP27 did not block the endogenous HSP27 response. We conclude that inhibition of p38 MAPK and subsequent HSP27 and cryAB phosphorylation and/or overexpression of nonphosphorylatable HSP27 significantly improves cardiac performance following cardioplegic arrest. Modulation of HSP27 phosphorylation may improve myocardial stunning following cardiac surgery.