Studies on the role of apoptosis after transient myocardial ischemia: genetic deletion of the executioner caspases-3 and -7 does not limit infarct size and ventricular remodeling.

Although it is widely accepted that apoptosis may contribute to cell death in myocardial infarction, experimental evidence suggests that adult cardiomyocytes repress the expression of the caspase-dependent apoptotic pathway. The aim of this study was to analyze the contribution of caspase-mediated apoptosis to myocardial ischemia-reperfusion injury. Cardiac-specific caspase-3 deficient/full caspase-7-deficient mice (Casp3/7DKO) and wild type control mice (WT) were subjected to in situ ischemia by left anterior coronary artery ligation for 45 min followed by 24 h or 28 days of reperfusion. Heart function was assessed using M-mode echocardiography. Deletion of caspases did not modify neither infarct size determined by triphenyltetrazolium staining after 24 h of reperfusion (40.0 ± 5.1 % in WT vs. 36.2 ± 3.6 % in Casp3/7DKO), nor the scar area measured by pricosirius red staining after 28 days of reperfusion (41.1 ± 5.4 % in WT vs. 44.6 ± 8.7 % in Casp3/7DKO). Morphometric and echocardiographic studies performed 28 days after the ischemic insult revealed left ventricular dilation and severe cardiac dysfunction without statistically significant differences between WT and Casp3/7DKO groups. These data demonstrate that the executioner caspases-3 and -7 do not significantly contribute to cardiomyocyte death induced by transient coronary occlusion and provide the first evidence obtained in an in vivo model that argues against a relevant role of apoptosis through the canonical caspase pathway in this context.

cGMP/PKG pathway mediates myocardial postconditioning protection in rat hearts by delaying normalization of intracellular acidosis during reperfusion.

Ischemic postconditioning has been demonstrated to limit infarct size in patients, but its molecular mechanisms remain incompletely understood. Low intracellular pH (pHi) inhibits mitochondrial permeability transition, calpain activation and hypercontracture. Recently, delayed normalization of pHi during reperfusion has been shown to play an important role in postconditioning protection, but its relation with intracellular protective signaling cascades is unknown. The present study investigates the relation between the rate of pHi normalization and the cGMP/PKG pathway in postconditioned myocardium. In isolated Sprague-Dawley rat hearts submitted to transient ischemia both, postconditioning and acidic reperfusion protocols resulted in a similar delay in pHi recovery measured by (31)P-NMR spectroscopy (3.6±0.2min and 3.5±0.2min respectively vs. 1.4±0.2min in control group, P<0.01) and caused equivalent cardioprotection (48% and 41% of infarct reduction respectively, P<0.01), but only postconditioning increased myocardial cGMP levels (P=0.02) and activated PKG. Blockade of cGMP/PKG pathway by the addition of the guanylyl cyclase inhibitor ODQ or the PKG inhibitor KT5823 during reperfusion accelerated pHi recovery and abolished cardioprotection in postconditioned hearts, but had no effect in hearts subjected to acidic reperfusion suggesting that PKG signaling was upstream of delayed pHi normalization in postconditioned hearts. In isolated cardiomyocytes the cGMP analog 8-pCPT-cGMP delayed Na(+)/H(+)-exchange mediated pHi normalization after acidification induced by a NH(4)Cl pulse. These results demonstrate that the cGMP/PKG pathway contributes to postconditioning protection at least in part by delaying normalization of pHi during reperfusion, probably via PKG-dependent inhibition of Na(+)/H(+)-exchanger.

Calpain translocation and activation as pharmacological targets during myocardial ischemia/reperfusion.

Calpains contribute to reperfusion-induced myocardial cell death. However, it remains controversial whether its activation occurs during ischemia or reperfusion. We investigated the regulation and time-course of calpain activation secondary to transient ischemia and the efficacy of its inhibition at reperfusion as a therapeutic strategy to limit infarct size. In isolated rat hearts (Sprague-Dawley), ischemia induced a time-dependent translocation of m-calpain to the membrane that was not associated with calpain activation as assessed by proteolysis of its substrate alpha-fodrin. Translocation of calpain was dependent on Ca(2+) entry through reverse mode Na(+)/Ca(2+)-exchange and was independent of acidosis. Calpain activation occurred during reperfusion, but only after intracellular pH (pHi) normalization, and was not prevented by inhibiting its translocation during ischemia with methyl-beta-cyclodextrin. The intravenous infusion of MDL-28170 in an in vivo rat model with transient coronary occlusion during the first minutes of reperfusion resulted in a reduction of infarct size (43.9+/-3.9% vs. 60.2+/-4.7, P=0.046, n=18) and alpha-fodrin degradation. These results suggest that (1) Ca(2+)-induced calpain translocation to the membrane during ischemia is independent of its activation, (2) intracellular acidosis inhibits calpain activation during ischemia and pHi normalization allows activation upon reperfusion, and (3) calpain inhibition at the time of reperfusion appears as a potentially useful strategy to limit infarct size.

Delayed phospholamban phosphorylation in post-conditioned heart favours Ca2+ normalization and contributes to protection.

AIMS: It has been shown that sarcoplasmic reticulum calcium ATPase (SERCA) plays a critical role in reperfusion injury. Moreover, ischaemic post-conditioning (PoCo) results in protein kinase G (PKG) activation which has been proposed to modulate phospholamban (PLB) and SERCA. We assessed whether PLB phosphorylation contributes to the cardioprotective effects of PoCo. METHODS AND RESULTS: Isolated Sprague-Dawley rat hearts were submitted to 40 min of ischaemia and reperfusion with and without a PoCo protocol that reduced infarct size by 48%. Reperfusion caused a rapid phosphorylation in PLB at Ser16 and Thr17 that was delayed by PoCo. NO-independent activation of soluble guanylate cyclase (sGC) (ataciguat) and cAMP-dependent protein kinase (PKA) inhibition (KT5720) mimicked the reduction in Ser16 phosphorylation in reperfused control hearts, while in PoCo hearts the inhibitors of PKG (KT5823) and phosphodiesterase 2 (BAY-60-7550) reverted it. CaMKII activity measured by Thr287 phosphorylation was reduced in PoCo. In reperfused control hearts, inhibition of PLB phosphorylation or SERCA (thapsigargin) simulated the cardioprotective effects of PoCo. Ataciguat reduced cytosolic Ca(2+) oscillations and improved Ca(2+) recovery in cardiomyocytes subjected to anoxia-reoxygenation and infarct size by 32% in rats with 30 min of the left anterior descending coronary artery occlusion and 2 h of reperfusion. Blockade of Na(+)/Ca(2+)-exchanger (NCX; KB-R7943) impaired Ca(2+) control in cardiomyocytes and abolished cardioprotection in PoCo hearts. CONCLUSIONS: PoCo reduces SERCA activity at the onset of reperfusion by delaying PLB phosphorylation through activation of PKG and inhibition of PKA and CaMKII. This effect contributes to PoCo protection by favouring cytosolic Ca(2+) extrusion through NCX, and it may be mimicked by pharmacological stimulation of sGC.

Activation of cGMP/protein kinase G pathway in postconditioned myocardium depends on reduced oxidative stress and preserved endothelial nitric oxide synthase coupling.

BACKGROUND: The cGMP/protein kinase G (PKG) pathway is involved in the cardioprotective effects of postconditioning (PoCo). Although PKG signaling in PoCo has been proposed to depend on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt cascade, recent data bring into question a causal role of reperfusion injury signaling kinase (RISK) in PoCo protection. We hypothesized that PoCo increases PKG activity by reducing oxidative stress-induced endothelial nitric oxide synthase (NOS) uncoupling at the onset of reperfusion. METHODS AND RESULTS: Isolated rat hearts were submitted to 40 minutes of ischemia and reperfusion with and without a PoCo protocol. PoCo reduced infarct size by 48% and cGMP depletion. Blockade of cGMP synthesis (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and inhibition of PKG (KT5823) or NOS (l-NAME) abolished protection, but inhibition of PI3K/Akt cascade (LY294002) did not (n=5 to 7 per group). Phosphorylation of the RISK pathway was higher in PoCo hearts. However, this difference is due to increased cell death in control hearts because in hearts reperfused with the contractile inhibitor blebbistatin, a drug effective in preventing cell death at the onset of reperfusion, RISK phosphorylation increased during reperfusion without differences between control and PoCo groups. In these hearts, PoCo reduced the production of superoxide (O2(-)) and protein nitrotyrosylation and increased nitrate/nitrite levels in parallel with a significant decrease in the oxidation of tetrahydrobiopterin (BH4) and in the monomeric form of endothelial NOS. CONCLUSIONS: These results demonstrate that PoCo activates the cGMP/PKG pathway via a mechanism independent of the PI3K/Akt cascade and dependent on the reduction of O2(-) production at the onset of reperfusion, resulting in attenuated oxidation of BH4 and reduced NOS uncoupling.