Ravingerová T

Mitogen-activated protein kinases: A new therapeutic target in cardiac pathology

Eukaryotic cells respond to different external stimuli by activation of mechanisms of cell signaling. One of the major systems participating in the transduction of signal from the cell membrane to nuclear and other intracellular targets is the highly conserved mitogen-activated protein kinase (MAPK) superfamily. The members of MAPK family are involved in the regulation of a large variety of cellular processes such as cell growth, differentiation, development, cell cycle, death and survival. Several MAPK subfamilies, each with apparently unique signaling pathway, have been identified in the mammalian myocardium. These cascades differ in their upstream activation sequence and in downstream substrate specifity. Each pathway follows the same conserved three-kinase module consisting of MAPK, MAPK kinase (MAPKK, MKK or MEK), and MAPK kinase kinase (MAPKKK, MEKK). The major groups of MAPKs found in cardiac tissue include the extracellular signal-regulated kinases (ERKs), the stress-activated/c-Jun NH2-terminal kinases (SAPK/JNKs), p38-MAPK, and ERK5/big MAPK 1 (BMK1). The ERKs are strongly activated by mitogenic and growth factors and by physical stress, whereas SAPK/JNKs and p38-MAPK can be activated by various cell stresses, such as hyperosmotic shock, metabolic stress or protein synthesis inhibitors, UV radiation, heat shock, cytokines, and ischemia. Activation of MAPKs family plays a key role in the pathogenesis of various processes in the heart, e.g. myocardial hypertrophy and its transition to heart failure, in ischemic and reperfusion injury, as well in the cardioprotection conferred by ischemia- or pharmacologically-induced preconditioning. The following approaches are currently utilized to elucidate the role of MAPKs in the myocardium: (i) studies of the effects of myocardial processes on the activity of these kinases; (ii) pharmacological modulations of MAPKs activity and evaluation of their impact on the (patho)physiological processes in the heart; (iii) gene targeting or expression of constitutively active and dominant-negative forms of enzymes (adenovirus-mediated gene transfer).This review is focused on the regulatory role of MAPKs in the myocardium, with particular regard to their involvement in pathophysiological processes, such as myocardial hypertrophy and heart failure, ischemia/reperfusion injury, as well as in the mechanisms of cardioprotection. In addition, it summarizes current information on pharmacological modulations of MAPKs activity and their impact on the cardiac response to pathophysiological processes.

Ischemic tolerance of rat hearts in acute and chronic phases of experimental diabetes

Different from clinical studies of diabetes mellitus (DM), experimental data reveal both, higher and lower vulnerability of the heart to ischemic injury. We have previously demonstrated an enhanced resistance to ischemia-induced arrhythmias in isolated rat hearts in the acute phase of DM. Our objectives were thus to extend our knowledge to the effects of DM of different duration on myocardial infarction, in conjunction with susceptibility to arrhythmias, in the in vivo model. DM was induced by streptozotocin (45 mg/kg, i.v.) and following 1 week (acute phase) and 8 weeks (chronic phase), anesthetized open-chest diabetic and age-matched control rats were subjected to 30-min regional ischemia (occlusion of LAD coronary artery) followed by 4-h reperfusion for the evaluation of the infarct size (tetrazolium staining). In the control rats, ventricular tachycardia (VT) represented 45.4% of total arrhythmias and occurred in 90% of the animals. In the acute phase of DM, arrhythmia profile was similar to that in the control animals, and the incidence and severity of arrhythmias were not enhanced. On the other hand, the size of infarct area normalized to the size of area at risk was significantly smaller in the diabetics than in the controls (47.2 ± 2.8 vs. 70.2 ± 2.1%, respectively; p < 0.05). In the chronic phase, only 17.7% of arrhythmias occurred as VT in 44% of the diabetics (p < 0.05 vs. controls). Severity of arrhythmias was also lower (arrhythmia score: 2.1 ± 0.3 vs. 2.9 ± 0.3 in the controls, respectively; p < 0.05). This effect was not due to a smaller infarct size, since the latter did not differ from that in the controls. In conclusion: diabetic rat hearts exhibit rather lower, than higher sensitivity to ischemia. In acute phase of DM, diabetic hearts are more resistant to irreversible cell damage, whereas in the chronic phase they exhibit reduced susceptibility to arrhythmias; these discrepancies might reflect different pathogenesis of arrhythmias and myocardial infarction. (Mol Cell Biochem 249: 167–174, 2003)

Free oxygen radicals contribute to high incidence of reperfusion-induced arrhythmias in isolated rat heart.

Early period of reperfusion of ischemic myocardium is associated with a high incidence of severe tachyarrhythmias including ventricular tachycardia and fibrillation (VT and VF). Free oxygen radicals (FOR) have been identified as one of the principal factors responsible for reperfusion-induced events. However, their role in arrhythmogenesis is not clear. In the present study, in isolated Langendorff-perfused rat hearts subjected to 30 min global ischemia, the onset of reperfusion induced 100% incidence of both VT and VF with their gradual cessation over 5 min of reperfusion. Generation of H2O2 in the myocardium in the first minutes of reperfusion was visualized by means of cerium cytochemistry and confirmed by X-ray microanalysis. The mechanism of the arrhythmogenic effect of FOR may involve inhibition of the sarcolemmal Na+/K+-ATPase, as demonstrated in the rat heart sarcolemmal fraction subjected to FOR-generating system (H2O2 + FeSO4).

Changes in PPAR gene expression and myocardial tolerance to ischaemia: relevance to pleiotropic effects of statins

Peroxisome proliferator-activated receptors (PPAR), which are key transcriptional regulators of lipid metabolism and energy production, have been suggested to play an important role in myocardial ischaemia-reperfusion (I/R) injury. Their role in cardioprotection, however, is not yet fully elucidated. Statins have shown beneficial effects on I/R damage beyond lipid lowering, and some of their cardioprotective cholesterol-independent effects may be related to the regulation of PPAR. To clarify this issue, we explored a potential link between a response to I/R and changes in cardiac PPARalpha protein and gene expression in simvastatin-treated normocholesterolaemic rats. After 5 days of treatment with simvastatin (10 mg/kg per day, p.o.), Langendorff-perfused hearts were subjected to 30 min regional ischaemia (occlusion of the left anterior descending coronary artery) or global ischaemia and 2 h reperfusion for the evaluation of the infarct size (triphenyltetrazolium chloride and planimetry; as percentage of risk area), ischaemic arrhythmias, and postischaemic contractile recovery. Baseline PPARalpha mRNA and protein levels were increased by 3-fold and 2-fold, respectively, in simvastatin-treated hearts compared with the untreated controls. Simvastatin-treated hearts exhibited smaller size of infarction (11.5% +/- 0.4% vs. 33.7% +/- 4% in controls; p < 0.01), improved postischaemic contractile recovery, and lower severity of arrhythmias during ischaemia and early reperfusion. Enhanced resistance to I/R injury was associated with preservation of mRNA and protein levels of PPARalpha in contrast to their marked downregulation in controls. In conclusion, statin-induced changes in the expression of PPARalpha may contribute to attenuation of myocardial I/R injury and thus suggest the involvement of cardioprotective mechanisms independent of inhibition of HMG-CoA reductase.

Mitochondrial KATP opening confers protection against lethal myocardial injury and ischaemia-induced arrhythmias in the rat heart via PI3K/Akt-dependent and -independent mechanisms.

Opening of mitochondrial KATP channels (mitoKATP) has been reported to underlie protection against ischaemia-reperfusion injury induced by ischaemic preconditioning (I-PC); however, the molecular mechanisms of its antiarrhythmic effect have not been fully elucidated. We explored the involvement of phosphatidylinositol 3-kinase (PI3K)/Akt in the PC-like effect of mitoKATP opener diazoxide with particular regard to its role in protection against ischaemia-induced arrhythmias. Langendorff-perfused rat hearts were subjected to 30 min LAD occlusion with or without a prior 15 min of perfusion with diazoxide (50 micromol/L) given either alone (D-PC) or in combination with the PI3K/Akt inhibitor wortmannin (100 nmol/L). In an additional protocol, ischaemia was followed by 2 h reperfusion for infarct size (IS) determination (tetrazolium staining). The total number of premature ventricular complexes over the whole period of ischaemia, episodes of ventricular tachycardia and its duration were significantly lower in the D-PC group than in the non-preconditioned controls (158 +/- 20, 2 +/- 0.6 and 4.6 +/- 1.8 s vs. 551 +/- 61, 11 +/- 2 and 42 +/- 8 s, respectively; p < 0.05), concomitant with a 62% reduction in the size of infarction. Wortmannin modified neither arrhythmogenesis nor IS in the non-preconditioned hearts. Bracketing of diazoxide with wortmannin did not reverse the antiarrhythmic protection, whereas the IS-limiting effect was blunted. The results indicate that in contrast with the positive role of PI3K/Akt in protection against lethal myocardial injury, its activity is not involved in suppression of ischaemia-induced arrhythmias conferred by mitoKATP opening in the rat heart.

Diabetic cardiomyopathy in rats: alleviation of myocardial dysfunction caused by Ca2+ overload

There is some evidence that diabetic hearts are more resistant to ischaemia/reperfusion injury due to alterations in Ca2+ handling. Our objective was to explore this hypothesis in the model of Ca2+ overloaded heart (calcium paradox, CaP). Diabetes was induced by streptozotocin (45 mg/kg, i.v.). Despite regular insulin treatment blood glucose was increased. After a diabetes duration of 9 weeks the heart/body weight ratio was higher than in age-matched controls, and the heart rate, the coronary flow (CF) and the rate of contraction and relaxation was reduced as assessed in Langendorff preparation. Depressed function was accompanied by a lower content of high energy phosphates and ultrastructural alterations, such as an increased number of glycogen granules, lipid droplets and changes in the walls of capillaries leading to the narrowing of their lumen. In controls, readmission of Ca2+ into Ca(2+)-depleted hearts resulted in extensive deterioration of heart function, development of contraction bands, ultrastructural damage and loss of ATP. Diabetic hearts, despite impaired performance before CaP, showed an improved recovery of heart function manifested by restoration of electrical and contractile activity, as well as CF after Ca2+ repletion. This corresponded to better maintenance of energy metabolism and preservation of ultrastructure. In conclusion, diabetic hearts exhibit greater resistance to Ca2+ overload. Depressed heart function may account for this protective effect: bradycardia facilitates saving ATP; lower CF results in a slower rate of Ca2+ washout from the heart during Ca2+ depletion thus causing less damage to the cell membrane and maintenance of its integrity.

Prolonged oxytocin treatment in rats affects intracellular signaling and induces myocardial protection against infarction.

Oxytocin is a hormone, which is released into the circulation in response to acute or chronic stress stimuli. One of the important targets of oxytocin is cardiovascular system. Present studies were aimed at testing the hypothesis that prolonged treatment with oxytocin (simulation of stress-induced rise in circulating oxytocin) activates intracellular signaling pathways playing a role in ischemia/reperfusion injury. Furthermore, we tested protective effects of oxytocin treatment in vivo against cardiac injury induced by ischemia/reperfusion of isolated hearts. Male Wistar rats were treated with oxytocin or vehicle continuously via osmotic minipumps for 2 weeks. The hearts were used for biochemical measurements or isolated for Langendorff perfusion. Treatment with oxytocin resulted in a significant increase in specific phosphorylation (activation) of p38-MAPK and Akt kinase, an increase in phosphorylated Hsp27 and an elevation in atrial natriuretic peptide (ANP) levels in left ventricular heart tissue. There were no significant changes in the activation of MMP-2 and ERK in the left heart ventricle of oxytocin-treated rats. Postischemic recovery of functional parameters LVDP, RPP, +dP/dtmax and -dP/dtmax was better in the hearts of oxytocin-treated rats compared to that in the controls. Oxytocin treatment significantly reduced infarct size to 15.1 + 3.2% as compared to 32.4 + 3.5% in vehicle-treated rats (p < 0.01). This is the first evidence for cardioprotective effects of oxytocin administered in vivo simulating chronic stress-induced elevation in plasma oxytocin. The present results show that positive effects of oxytocin that may ameliorate negative consequences of stress on the heart are, at least in part, mediated through p38-MAPK and Akt kinase pathways.

Energy transfer in acute diabetic rat hearts: adaptation to increased energy demands due to augmented calcium transients.

Abstract: Objectives—Hearts of rats with diabetes mellitus (DM) are characterized by energy demands exceeding their energy production, but they might also exhibit decreased vulnerability to ischemia and calcium overload. This indicates adaptation in cardiac energetics (CE), where energy transport is not rate‐limiting. Aim—This study was designed to elucidate the functional significance of the DM‐induced adaptation in CE by investigating the formation of mitochondrial contact sites (MiCS), facilitating the Ca‐dependent/high‐capacity energy transfer from mitochondria, in conjunction with testing the ischemic tolerance (IT) of hearts. Methods—After 1 week of streptozotocin‐induced DM (45 mg/kg iv), the hearts of male diabetic and age‐matched control rats (C) were isolated and Langendorff‐perfused with either 1.6 or 2.2 mmol/L of CaCl2. MiCS formation was assessed by cytochemical detection of mCPK octamers and was quantified stereologically as MiCS to mitochondrial surface ratio (SS). IT was evaluated in anesthetized open‐chest animals subjected to 30‐min occlusion of the LAD coronary artery followed by 4‐h reperfusion, by monitoring ischemic arrhythmias and by measuring the size of infarction (tetrazolium double staining). Results—In C hearts, increasing Ca2+ induced both positive inotropic response (dP/dt increase from 2270 ± 220 to 2955 ± 229, p < 0.01) and elevated MiCS formation (SS increase from 0.070 ± 0.011 to 0.123 ± 0.012, p < 0.01). In DM hearts, basic MiCS formation was already comparable with that induced by elevated Ca2+ in C hearts and could not be further stimulated by Ca2+. In C, ventricular tachycardia represented 55.4% of the total arrhythmias and occurred in 90% of the animals. In DM rats, the arrhythmia profile was similar to that in C, and the incidence of tachyarrhythmias and their severity were not enhanced (arrhythmia score: 3.18 ± 0.4 vs. 3.30 ± 0.3 in C). The infarct size normalized to the size of area at risk was smaller in the DM than in C hearts (52.3 ± 5.8% vs. 69.2 ± 2.2%, respectively; p < 0.05). Conclusions—Ca‐signaling represents the link between energy delivery from mitochondria (via MiCS) and energy requirements of the heart. In DM hearts, energy transport via MiCS is elevated to the maximum value. This contributes to increased resistance of DM hearts to irreversible cell damage.

Resistance of diabetic rat hearts to Ca overload-related injury. Histochemical and ultrastructural study

The enzymatic histochemical and ultrastructural alterations of the rat heart during development of streptozotocin (STZ) induced diabetic cardiomyopathy were studied. Moreover, the response of the isolated diabetic hearts to Ca overload-Ca paradox-was investigated. In the early stage of diabetes (1 week of diabetes), no apparent histochemical changes were observed but gentle alterations of the ultrastructure of the myocytes and particularly capillaries were found. Structural changes of the myocytes and microangiopathy accompanied by decreased activities of some enzymes (phosphorylase, various dehydrogenases, ATPase) progressed with time and were more pronounced late in diabetes (9 weeks). Ca paradox induced severe structural damage of the majority of cardiomyocytes and loss of the cellular integrity, and marked decrease in activities of all enzymes. However, in acute diabetic heart only partial Ca paradox was observed. It was manifested by transmural heterogeneity of structural and enzymatic histochemical changes. Evident preservation of the ultrastructure and enzyme activities of the myocardium was revealed in late stage (9 weeks) of diabetes. It can be concluded that diabetes results in prevention of the Ca overload in rat myocardium in vitro. Disturbances in coronary perfusion associated with microangiopathy as well as altered Ca handling and depressed heart function may account for delayed development of Ca paradox in diabetic heart.

Prevention of processes coupled with free radical formation prevents also the development of calcium-resistance in the diabetic heart.

Recently it was shown that besides their negative role in pathogenesis of diabetes, reactive oxygen species (ROS) and particularly the products of non-enzymatic glycation of proteins (NEGP) may also participate in some processes of adaptation of the myocardium to diabetes, such as in the mechanism of development of calcium resistance of the heart. Our study revealed that the hearts of rats with experimentally induced diabetes (single dose of streptozotocin, 45 mg/kg i.v., 6 U/kg insulin daily) develop considerable resistance against calcium overload (induced by means of Ca-paradox). On the day 63 after the beginning of experiment, when the diabetic cardiomyopathy became fully developed but the hearts were still not failing, their calcium resistance was increased to 83.33%. Our results provide evidence that, when applied in a special regimen, resorcylidene aminoguanidine (RAG, 4 mg/kg) prevented both, the formation of fructosamine (a source of ROS generation), and also that of the advanced Maillard products, in the heart sarcolemma of diabetic rats. The effect of RAG was accompanied by a decrease in calcium resistance in the group of rats with chronic diabetes (63 days) from 83.3 to 46.7%. It is concluded that NEGP and ROS formation are inevitably needed for development of calcium resistance in the diabetic hearts.