Jan Slezak

IL-10 attenuates TNF-α-induced NFκB pathway activation and cardiomyocyte apoptosis

AIMS We have recently reported that tumour necrosis factor-alpha (TNF-alpha) increases oxidative stress and apoptosis in cardiomyocytes by upregulating p38 mitogen-activated protein (MAP) kinase (MAPK) phosphorylation. Interleukin-10 (IL-10) blocked these effects of TNF-alpha by upregulating extracellular signal-regulated kinase 1/2 (ERK 1/2) MAPK phosphorylation. However, the precise site of this IL-10 action is still unknown, and this is investigated in the present study. METHODS AND RESULTS Cardiomyocytes isolated from adult Sprague-Dawley rats were exposed to TNF-alpha (10 ng/mL), IL-10 (10 ng/mL), and IL-10+TNF-alpha (ratio 1) for 4 h. Hydrogen peroxide and antioxidant trolox were used as positive controls. Exposure to TNF-alpha resulted in an increase in the production of reactive oxygen species, the number of apoptotic cells, caspase-3 activation, and poly-ADP ribose polymerase (PARP) cleavage. Increased oxidative stress by using hydrogen peroxide also caused apoptosis. The changes due to TNF-alpha were associated with an increase in the inhibitor of kappaB kinase (IKK) and nuclear factor kappa-B (NF kappaB) phosphorylation. IL-10 by itself had no effect, but it prevented the above mentioned TNF-alpha-induced changes. Trolox also mitigated TNF-alpha induced changes. Pre-exposure of cells to an IKK inhibitor (PS-1145) prevented TNF-alpha-induced caspase-3 and PARP cleavage. Inhibition of ERK 1/2 MAPK with PD98059 attenuated the protective role of IL-10 against TNF-alpha-induced activation of IKK and NF kappaB as well as cardiomyocyte apoptosis. CONCLUSION The present study shows that TNF-alpha-induced activation of the NF kappaB pathway plays a critical role in cardiomyocyte apoptosis. IL-10 prevents TNF-alpha-induced NF kappaB activation and pro-apoptotic changes in cardiomyocytes by inhibiting IKK phosphorylation through the activation of ERK 1/2 MAPK.

Biology of TNFα and IL-10, and their imbalance in heart failure

Our understanding of the multiple in vivo functions of the proinflammatory cytokine, tumor necrosis factor (TNFα), is advancing at a rapid pace. In addition to its antitumor effects, overproduction of TNFα provokes tissue injury and organ failure. TNFα has also been shown to be cardiodepressent and responsible for various cardiovascular complications. It appears that still much needs to be learned for a full comprehension of the role of TNFα in heart biology. Another cytokine, interleukin-10 (IL-10), has been shown to have anti-inflammatory properties. It is suggested to counterbalance many adverse effects of TNFα. IL-10 suppresses the production of TNFα and many other proinflammatory cytokines. TNFα-induced oxidative stress is also known to be mitigated by IL-10. Moreover, improvement in cardiac function after treatment with various drugs is also shown to be associated with an increase in IL-10 content. Based on the data reviewed in here, it is suggested that an optimal balance between IL-10 and TNFα may be a new therapeutic strategy for a healthier heart.

Hibernating myocardium: pathophysiology, diagnosis, and treatment

Comprehensive management of patients with chronic ischemic disease is a critically important component of clinical practice. Cardiac myocytes have the potential to adapt to limited flow conditions by adjusting contractile function, reducing metabolism, conserving resources, and preserving myocardial integrity to cope with an oxygen and (or) nutrition shortage. A prime metabolic feature of cardiac myocytes affected by chronic ischemia is the return to a fetal gene pattern with predominance of carbohydrates as the substrate for energy. Structural adaptation with multiple intracellular changes is part of the remodeling process in hibernating myocardium. Transmural heterogeneity, which defines the pattern of injury in ventricular cardiomyocytes and the response to chronic ischemia, is a multifactorial process originating from functional, metabolic, and flow differences in subendocardial and subepicardial regions. Autophagy is typically activated in hibernating myocardium and has been identified as a prosurvival mechanism. Chronic ischemia is associated with changes in the number, size, and distribution of gap junctions and may give rise to conduction disturbances and arrhythmogenesis. Differentiation between viable and nonviable myocardium by assessing sensitivity of inotropic reserve is a crucial diagnostic tool that is correlated with the prognosis and outcome for improved contractility after restoration of blood perfusion in afflicted myocardium.Reliable and accurate diagnosis of ischemic, scar, and viable tissues is critical for recover strategies. Although early surgical reinstitution of blood flow is most effective in restoring physiologic function of the hibernating myocardium, several new approaches offer promising alternatives. Among others, vascular endothelial growth factor and fibroblast growth factor-2 (FGF-2), especially its lo-FGF-2 isoform, have been shown to be effective in rapid neovascularization. Substances such as statins, resveratrol, some hormones, and omega-3 fatty acids can improve recovery effect in chronically underperfused hearts. For patients with drug-refractory ischemia, intramyocardial transplantation of stem cells into predefined areas of the heart can enhance vascularization and have beneficial effects on cardiac function. This review of ischemic injury, its heterogeneity, accurate diagnosis, and newer methods of treatment, shows there is much information and tremendous hope for better management of patients with coronary heart disease.

Gap junction remodelling is involved in the susceptibility of diabetic rats to hypokalemia-induced ventricular fibrillation.

The objective of the present study was to examine the susceptibility of diabetic rats with cardiomyopathy to hypokalemia-induced ventricular fibrillation and to localize gap junction protein connexin-43 as well as subcellular changes that may be involved in the development of severe arrhythmia. Our results showed a significantly higher incidence of sustained ventricular fibrillation in diabetic hearts as compared with control hearts, 80% vs 20%, respectively. Diabetic cardiomyopathy itself was accompanied by a distinct decrease in connexin-43-immunopositive gap junctions. Moreover, interstitial fibrosis and subcellular alterations to various degrees were observed in diabetic hearts, and a further deterioration of the ultrastructure and impairment of intercellular junctions, and a stronger local decrease in connexin-43 levels due to hypokalemia were found. These changes were heterogeneously distributed throughout the myocardium and occurred earlier and were more pronounced in diabetic hearts than control hearts. In conclusion, our results indicate that diabetic cardiomyopathy is associated with down-regulation of gap junction proteins and may account for the higher vulnerability of diabetic rats to ventricular fibrillation in combination with impairment of intercellular communication due to hypokalemia.

Dietary omega-3 fatty acids attenuate myocardial arrhythmogenic factors and propensity of the heart to lethal arrhythmias in a rodent model of human essential hypertension.

Objective: Hypertension-induced myocardial remodeling is known to be associated with increased risk for malignant arrhythmias and alterations in electrical coupling protein, connexin-43 (Cx43), may be involved. We investigated whether omega-3 fatty acids intake affects abnormalities of Cx43 as well as protein kinase C (PKC) signaling and myosin heavy chain (MyHC) profile at the early and late stage of hypertension in the context of the hearts susceptibility to ventricular fibrillation and ability to restore sinus rhythm. Methods: Untreated young and old male spontaneously hypertensive rats (SHRs) and age-matched normotensive rats were compared with animals supplemented by omega-3 (eicosapentaneoic acid + docosahexaneoic acid, 200 mg/kg body weight/day) for 2 months. Left ventricular tissues were taken for examination of subcellular integrity of gap junctions, Cx43 mRNA and protein expression, PKC&egr; and PKC&dgr; as well as MyHC determination. Electrically inducible ventricular fibrillation and sinus rhythm restoration (SRR) were examined on Langedorff-perfused heart preparation. Results: Omega-3 intake significantly reduced cardiovascular risk factors, suppressed inducible ventricular fibrillation, and facilitated SRR in hypertensive rats. Supplementation attenuated lateralization and internalization of Cx43, suppressed elevated Cx43 mRNA, enhanced total Cx43 protein expression and/or expression of its functional phosphorylated forms as well as the expression of cardioprotective PKC-&egr; and suppressed pro-apoptotic PKC-&dgr; isoform. Moreover, the omega-3 diet normalized MyHC profiles in SHR at early stage of disease and old nonhypertensive rats, but failed to do so in old SHR at late stage of disease. Conclusion: Findings suggest that amelioration of myocardial Cx43-related abnormalities, positive modulation of PKC pathways, and normalization of MyHC can significantly contribute to the antiarrhythmic effects of omega-3 in rat model mimicking human essential hypertension. Our results support the prophylactic use of omega-3 to minimize cardiovascular risk and sudden arrhythmic death.

Cytochemical distribution of cyclic AMP-dependent 3′,5′-nucleotide phosphodiesterase in the rat myocardium

SummaryThe cytochemical localization of cAMP-dependent phosphodiesterase was studied in the rat myocardium. Slices 40 μm thick from perfusion-fixed rat hearts were incubated in the medium with cAMP as a substrate and Pb ions as a capture metal of the reaction product. After the incubation in the basic medium the specific precipitate of cAMP phosphodiesterase was localized on the sarcolemma of cardiomyocytes. In addition, it was localized on the plasmalemma of endothelia cells of capillaries and small coronary arteries as well as on the membrane of smooth muscle cells. Using selective inhibitors SK&F 94120 for phosphodiesterase III and Rolipram for the IV isoenzyme, both isoforms were detected on the membrane of smooth muscle cell. In addition, phosphodiesterase III was localized on the sarcolemma only and phosphodiesterase IV on the sarcolemma of cardiomyocytes and the plasmalemma of endothelial cells.

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.

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.

Protective effect of 7-oxo-prostacyclin on myocardial function and metabolism during postischemic reperfusion and calcium paradox

SummaryThe effect of 7-oxo PGI2 on function and metabolism of postischemic reperfused (30-min ischemia and 30-min reperfusion) rat hearts was studied with special regard to calcium overload as one of the main factors of the postischemic reperfusion damage to the heart. The drug (50 μg/kg i.p.) was applied 48 h prior to starting the experiments on isolated rat hearts (Langendorff preparation at 37 °C and constant perfusion pressure of 65 mm Hg). A late protective effect of 7-oxo PGI2 was manifested by an improved recovery of heart function during reperfusion and calcium overload, better preservation of myocardial ATP contents during ischemia and also after calcium overload, as well as by a normalization of the lactate content, otherwise extremely increased during ischemia. Electron microscopic data also supported the above results. The beneficial effect of pretreatment with PGI2 may be explained not only by its vasodilating action, but more by its membrane stabilizing effect with a consequently decreased sodium accumulation, potassium loss, as well as intracellular calcium overload.

Nitric oxide synthase in muscular dystrophies: a re-evaluation

Duchenne and Becker muscular dystrophies (DMD and BMD) are associated with decreased total nitric oxide (NO). However, mechanisms leading to NO deficiency with consequent muscle-cell degeneration remain unknown. To address this issue, we examined skeletal muscles of DMD and BMD patients for co-expression of NO synthase (NOS) with nitrotyrosine and transcription factor CREB, as well as with enzymes engaged in NO signaling. Employing immunocytochemical labeling, Western blotting and RT-PCR, we found that, in contrast to the most commonly accepted view, neuronal NOS was not restricted to the sarcolemma and that muscles of DMD and BMD patients retained all three NOS isoforms with an up-regulation of the inducible NOS isoform, CREB and nitrotyrosine. We suggest that enhanced nitrotyrosine immunostaining in muscle fibers as well as in the vasculature of DMD and BMD specimens reflects massive oxidative stress, resulting in withdrawal of NO from its regular physiological course via the scavenging actions of superoxides.