Aging attenuates cardiac tolerance to ischemia/reperfusion (I/R) associated with defects in protective cell signaling, however, the onset of this phenotype has not been completely investigated. This study aimed to compare changes in response to I/R and the effects of remote ischemic preconditioning (RIPC) in the hearts of younger adult (3 months) and mature adult (6 months) male Wistar rats, with changes in selected proteins of protective signaling. Langendorff-perfused hearts were exposed to 30 min I/120 min R without or with prior three cycles of RIPC (pressure cuff inflation/deflation on the hind limb). Infarct size (IS), incidence of ventricular arrhythmias and recovery of contractile function (LVDP) served as the end points. In both age groups, left ventricular tissue samples were collected prior to ischemia (baseline) and after I/R, in non-RIPC controls and in RIPC groups to detect selected pro-survival proteins (Western blot). Maturation did not affect post-ischemic recovery of heart function (Left Ventricular Developed Pressure, LVDP), however, it increased IS and arrhythmogenesis accompanied by decreased levels and activity of several pro-survival proteins and by higher levels of pro-apoptotic proteins in the hearts of elder animals. RIPC reduced the occurrence of reperfusion-induced ventricular arrhythmias, IS and contractile dysfunction in younger animals, and this was preserved in the mature adults. RIPC did not increase phosphorylated protein kinase B (p-Akt)/total Akt ratio, endothelial nitric oxide synthase (eNOS) and protein kinase Cε (PKCε) prior to ischemia but only after I/R, while phosphorylated glycogen synthase kinase-3β (GSK3β) was increased (inactivated) before and after ischemia in both age groups coupled with decreased levels of pro-apoptotic markers. We assume that resistance of rat heart to I/R injury starts to already decline during maturation, and that RIPC may represent a clinically relevant cardioprotective intervention in the elder population.
- MeSH
- Phosphorylation MeSH
- Hemodynamics MeSH
- Ischemic Preconditioning, Myocardial * MeSH
- Glycogen Synthase Kinase 3 beta genetics metabolism MeSH
- Rats MeSH
- Myocardium metabolism MeSH
- Rats, Wistar MeSH
- Protein Kinase C-epsilon genetics metabolism MeSH
- Proto-Oncogene Proteins c-akt genetics metabolism MeSH
- Myocardial Reperfusion Injury metabolism pathology MeSH
- Aging MeSH
- Nitric Oxide Synthase Type III genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
We aimed to explore whether specific high-sucrose intake in older female rats affects myocardial electrical coupling protein, connexin-43 (Cx43), protein kinase C (PKC) signaling, miR-1 and miR-30a expression, and susceptibility of the heart to malignant arrhythmias. Possible benefit of the supplementation with melatonin (40 μg/ml/day) and omega-3 polyunsaturated fatty acids (Omacor, 25 g/kg of rat chow) was examined as well. Results have shown that 8 weeks lasting intake of 30% sucrose solution increased serum cholesterol, triglycerides, body weight, heart weight, and retroperitoneal adipose tissues. It was accompanied by downregulation of cardiac Cx43 and PKCε signaling along with an upregulation of myocardial PKCδ and miR-30a rendering the heart prone to ventricular arrhythmias. There was a clear benefit of melatonin or omega-3 PUFA supplementation due to their antiarrhythmic effects associated with the attenuation of myocardial Cx43, PKC, and miR-30a abnormalities as well as adiposity. The potential impact of these findings may be considerable, and suggests that high-sucrose intake impairs myocardial signaling mediated by Cx43 and PKC contributing to increased susceptibility of the older obese female rat hearts to malignant arrhythmias.
- MeSH
- Anti-Arrhythmia Agents metabolism pharmacology MeSH
- Connexin 43 metabolism MeSH
- Dietary Sucrose adverse effects MeSH
- Rats MeSH
- Melatonin metabolism pharmacology MeSH
- MicroRNAs metabolism MeSH
- Myocardium metabolism MeSH
- Obesity chemically induced complications drug therapy metabolism MeSH
- Fatty Acids, Omega-3 metabolism pharmacology MeSH
- Rats, Wistar MeSH
- Protein Kinase C-delta metabolism MeSH
- Protein Kinase C-epsilon metabolism MeSH
- Signal Transduction drug effects MeSH
- Heart drug effects MeSH
- Arrhythmias, Cardiac etiology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Reduced tolerance to ischemia/reperfusion (IR) injury has been shown in elder human and animal hearts, however, the onset of this unfavorable phenotype and cellular mechanisms behind remain unknown. Moreover, aging may interfere with the mechanisms of innate cardioprotection (preconditioning, PC) and cause defects in protective cell signaling. We studied the changes in myocardial function and response to ischemia, as well as selected proteins involved in "pro-survival" pathways in the hearts from juvenile (1.5 months), younger adult (3 months) and mature adult (6 months) male Wistar rats. In Langendorff-perfused hearts exposed to 30-min ischemia/2-h reperfusion with or without prior PC (one cycle of 5-min ischemia/5-min reperfusion), we measured occurrence of reperfusion-induced arrhythmias, recovery of contractile function (left ventricular developed pressure, LVDP, in % of pre-ischemic values), and size of infarction (IS, in % of area at risk size, TTC staining and computerized planimetry). In parallel groups, LV tissue was sampled for the detection of protein levels (WB) of Akt kinase (an effector of PI3-kinase), phosphorylated (activated) Akt (p-Akt), its target endothelial NO synthase (eNOS) and protein kinase Cepsilon (PKCepsilon) as components of "pro-survival" cascades. Maturation did not affect heart function, however, it impaired cardiac response to lethal IR injury (increased IS) and promoted arrhythmogenesis. PC reduced the occurrence of malignant arrhythmias, IS and improved LVDP recovery in the younger animals, while its efficacy was attenuated in the mature adults. Loss of PC protection was associated with age-dependent reduced Akt phosphorylation and levels of eNOS and PKCepsilon in the hearts of mature animals compared with the younger ones, as well as with a failure of PC to upregulate these proteins. Aging-related alterations in myocardial response to ischemia may be caused by dysfunction of proteins involved in protective cell signaling that may occur already during the process of maturation.
- MeSH
- Time Factors MeSH
- Phosphorylation MeSH
- Ventricular Function, Left MeSH
- Adaptation, Physiological MeSH
- Myocardial Infarction metabolism pathology physiopathology prevention & control MeSH
- Ischemic Preconditioning, Myocardial methods MeSH
- Ventricular Pressure MeSH
- Coronary Circulation MeSH
- Disease Models, Animal MeSH
- Myocardium metabolism pathology MeSH
- Recovery of Function MeSH
- Rats, Wistar MeSH
- Isolated Heart Preparation MeSH
- Protein Kinase C-epsilon metabolism MeSH
- Proto-Oncogene Proteins c-akt metabolism MeSH
- Myocardial Reperfusion Injury metabolism pathology physiopathology prevention & control MeSH
- Signal Transduction MeSH
- Arrhythmias, Cardiac etiology physiopathology prevention & control MeSH
- Heart Rate MeSH
- Aging metabolism pathology MeSH
- Nitric Oxide Synthase Type III metabolism MeSH
- Age Factors MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
Previous data suggest that type 1 diabetes mellitus leads to the deterioration of myocardial intercellular communication mediated by connexin-43 (Cx43) channels. We therefore aimed to explore Cx43, PKC signaling and ultrastructure in non-treated and omega-3 fatty acid (omega-3) treated spontaneously diabetic Goto-Kakizaki (GK) rats considered as type 2 diabetes model. Four-week-old GK and non-diabetic Wistar-Clea rats were fed omega-3 (200 mg/kg/day) for 2 months and compared with untreated rats. Real-time PCR and immunoblotting were performed to determine Cx43, PKC-epsilon and PKC-delta expression. In situ Cx43 was examined by immunohistochemistry and subcellular alterations by electron microscopy. Omega-3 intake reduced blood glucose, triglycerides, and cholesterol in diabetic rats and this was associated with improved integrity of cardiomyocytes and capillaries in the heart. Myocardial Cx43 mRNA and protein levels were higher in diabetic versus non-diabetic rats and were further enhanced by omega-3. The ratio of phosphorylated (functional) to non-phosphorylated Cx43 was lower in diabetic compared to non-diabetic rats but was increased by omega-3, in part due to up-regulation of PKC-epsilon. In addition, pro-apoptotic PKC-delta expression was decreased. In conclusion, spontaneously diabetic rats at an early stage of disease benefit from omega-3 intake due to its hypoglycemic effect, upregulation of myocardial Cx43, and preservation of cardiovascular ultrastructure. These findings indicates that supplementation of omega-3 may be beneficial also in the management of diabetes in humans.
- MeSH
- Diabetes Mellitus, Experimental metabolism pathology prevention & control MeSH
- Drug Combinations MeSH
- Connexin 43 metabolism MeSH
- Rats MeSH
- Eicosapentaenoic Acid pharmacology therapeutic use MeSH
- Docosahexaenoic Acids pharmacology therapeutic use MeSH
- Myocardium metabolism ultrastructure MeSH
- Dietary Supplements MeSH
- Drug Evaluation, Preclinical MeSH
- Protein Kinase C-delta metabolism MeSH
- Protein Kinase C-epsilon metabolism MeSH
- Heart drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Continuous normobaric hypoxia (CNH) renders the heart more tolerant to acute ischemia/reperfusion injury. Protein kinase C (PKC) is an important component of the protective signaling pathway, but the contribution of individual PKC isoforms under different hypoxic conditions is poorly understood. The aim of this study was to analyze the expression of PKCepsilon after the adaptation to CNH and to clarify its role in increased cardiac ischemic tolerance with the use of PKCepsilon inhibitory peptide KP-1633. Adult male Wistar rats were exposed to CNH (10 % O(2), 3 weeks) or kept under normoxic conditions. The protein level of PKCepsilon and its phosphorylated form was analyzed by Western blot in homogenate, cytosolic and particulate fractions; the expression of PKCepsilon mRNA was measured by RT-PCR. The effect of KP-1633 on cell viability and lactate dehydrogenase (LDH) release was analyzed after 25-min metabolic inhibition followed by 30-min re-energization in freshly isolated left ventricular myocytes. Adaptation to CNH increased myocardial PKCepsilon at protein and mRNA levels. The application of KP-1633 blunted the hypoxia-induced salutary effects on cell viability and LDH release, while control peptide KP-1723 had no effect. This study indicates that PKCepsilon is involved in the cardioprotective mechanism induced by CNH.
- MeSH
- Adaptation, Physiological genetics MeSH
- Hypoxia enzymology genetics physiopathology MeSH
- Protein Kinase Inhibitors pharmacology MeSH
- Myocytes, Cardiac drug effects metabolism MeSH
- Rats MeSH
- L-Lactate Dehydrogenase metabolism MeSH
- RNA, Messenger biosynthesis genetics MeSH
- Rats, Wistar MeSH
- Protein Kinase C-epsilon antagonists & inhibitors biosynthesis genetics MeSH
- Myocardial Reperfusion Injury enzymology genetics MeSH
- Cell Survival drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Ischemic postconditioning and remote conditioning are potentially useful tools for protecting ischemic myocardium. This study tested the hypothesis that 2,3-dehydrosilybin (DHS), a flavonolignan component of Silybum marianum, could attenuate cardiomyocyte damage following hypoxia/reoxygenation by decreasing the generation of reactive oxygen species (ROS). After 5-6 days of cell culture in normoxic conditions the rat neonatal cardiomyocytes were divided into four groups. Control group (9 h at normoxic conditions), hypoxia/reoxygenation group (3 h at 1 % O₂, 94 % N₂and 5 % CO₂followed by 10 min of 10 micromol·l⁻¹DHS and 6 h of reoxygenation in normoxia) and postconditioning group (3 h of hypoxia, three cycles of 5 min reoxygenation and 5 min hypoxia followed by 6 h of normoxia). Cell viability assessed by propidium iodide staining was decreased after DHS treatment consistent with increased levels of lactatedehydrogenase (LDH) after reoxygenation. LDH leakage was significantly reduced when cardiomyocytes in the H/Re group were exposed to DHS. DHS treatment reduced H₂O₂production and also decreased the generation of ROS in the H/Re group as evidenced by a fluorescence indicator. DHS treatment reduces reoxygenation-induced injury in cardiomyocytes by attenuation of ROS generation, H₂O₂and protein carbonyls levels. In addition, we found that both the postconditioning protocol and the DHS treatment are associated with restored ratio of phosphorylated/total protein kinase C epsilon, relative to the H/Re group. In conclusion, our data support the protective role of DHS in hypoxia/reperfusion injury and indicate that DHS may act as a postconditioning mimic.
- MeSH
- Antioxidants pharmacology MeSH
- Cytoprotection MeSH
- Phosphorylation MeSH
- Ischemic Preconditioning, Myocardial MeSH
- Protein Carbonylation drug effects MeSH
- Myocytes, Cardiac drug effects metabolism pathology MeSH
- Cells, Cultured MeSH
- L-Lactate Dehydrogenase metabolism MeSH
- Animals, Newborn MeSH
- Oxidative Stress drug effects MeSH
- Hydrogen Peroxide metabolism MeSH
- Rats, Wistar MeSH
- Protein Kinase C-epsilon metabolism MeSH
- Myocardial Reperfusion Injury metabolism pathology prevention & control MeSH
- Signal Transduction drug effects MeSH
- Silymarin pharmacology MeSH
- Cell Survival drug effects MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Disruption to the sensitive balance of long-chain fatty acids and glucose in the heart could cause cardiovascular diseases. Searching for a possible role of novel protein kinase C (nPKC) in heart with disrupted energy balance, we compared the insulin-resistant spontaneously hypertensive rats (SHR), which carry a nonfunctional variant of the fatty acid transporter FAT/CD36, with the less insulin-resistant congenic strain SHR-4 that is genetically identical except for a segment on chromosome 4 including a wild-type gene for a functional FAT/CD36. We analyzed expression of the nPKC-δ and -ε isoforms plus triacylglycerols (TAG) content in the myocardium of both FAT/CD36 strains and after a high sucrose diet (HSD). Two weeks before killing, males of both strains were randomly divided into two groups and fed either a standard laboratory chow or an HSD. PKC was determined by Western blotting in particulate and cytosolic fractions from left ventricles. The SHR-4 rats exhibited lower serum levels of insulin and free fatty acids than did SHR rats and higher amounts of PKC-ε in the heart particulate fraction. HSD caused accumulation of heart TAG in SHR but not in SHR-4. HSD increased PKC-δ and decreased PKC-ε expression in particulate fraction from left ventricles of SHR-4 while having no effects in SHR. These results demonstrate that reduced insulin resistance in SHR-4 rats with wild-type FAT/CD36 is associated with the insulin signaling pathway involving nPKCs.
- MeSH
- Enzyme Activation MeSH
- CD36 Antigens genetics metabolism MeSH
- Cytosol metabolism MeSH
- Insulin blood metabolism MeSH
- Insulin Resistance genetics physiology MeSH
- Blood Glucose analysis metabolism MeSH
- Rats MeSH
- Fatty Acids, Nonesterified blood metabolism MeSH
- Myocardium metabolism MeSH
- Rats, Inbred SHR MeSH
- Protein Kinase C-delta biosynthesis MeSH
- Protein Kinase C-epsilon biosynthesis MeSH
- Gene Expression Regulation MeSH
- Sucrose metabolism MeSH
- Signal Transduction MeSH
- Heart Ventricles metabolism MeSH
- Triglycerides blood metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Protein kinase C (PKC) appears to play a significant role in the signal transduction of cardiac growth and development. The aim of this study was to determine changes in the total PKC activity and the expression of PKC isoforms alpha, delta and epsilon in the rat heart that was affected by pressure overload imposed at postnatal day (d) 2. Three groups of Wistar rats were employed for the experiment: rats submitted to the abdominal aortic constriction (AC), sham-operated controls (SO) and intact controls. Animals were sacrificed at d2, d3, d5 and d10. The total PKC activity was measured by the incorporation of (32)P into histone IIIS and the expression of PKC was analyzed by immunoblotting in the homogenate of the left ventricular myocardium and in the cytosolic, membrane-enriched (10(5) g) and nuclear-cytoskeletal-myofilament-enriched (10(3) g) fractions. We observed the significant transient increase in both the total PKC activity and the expression of all isoforms at d5 (the third day after the operation) in the cardiac homogenate of AC rats as compared with SO animals. Aortic constriction did not significantly affect the distribution of activity and isoform abundance among individual cellular fractions except for PKCdelta, which increased significantly at d10 in the cytosolic fraction at the expense of the membrane-enriched fraction. It is concluded that PKCalpha, PKCdelta and PKCepsilon undergo transient upregulation associated with the accelerated cardiac growth induced by pressure overload imposed in the very early postnatal period.
- MeSH
- Time Factors MeSH
- Financing, Organized MeSH
- Phosphorylation MeSH
- Cell Fractionation MeSH
- Histones metabolism MeSH
- Hypertension MeSH
- Isoenzymes MeSH
- Blood Pressure MeSH
- Rats MeSH
- Disease Models, Animal MeSH
- Myocardium enzymology pathology MeSH
- Heart Diseases MeSH
- Animals, Newborn MeSH
- Rats, Wistar MeSH
- Protein Kinase C-alpha MeSH
- Protein Kinase C-delta metabolism MeSH
- Protein Kinase C-epsilon metabolism MeSH
- Protein Kinase C metabolism MeSH
- Up-Regulation MeSH
- Blotting, Western MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
We have examined the changes of intercellular electrical coupling protein connexin-43 (Cx43) and of PKC-epsilon in heart atria of diabetic rats and/or after the treatment with triiodothyronine (T(3)). Diabetes was induced in Wistar-Kyoto rats by streptozotocin (50 mg/kg, i.v.) and atria were examined after 5 (acute stage) and 10 (chronic stage) weeks. T(3) (10 microg/100 g/day) was applied via a gastric tube for the last 10 days prior to the end of the experiments to non-diabetic and to the half of diabetic rats. Expression and phosphorylated status of Cx43, as well as expression of PKC-epsilon, were analyzed by Western blots using mouse monoclonal anti-Cx43 and rabbit polyclonal anti-PKC-epsilon antibodies. We found that the Cx43 expression was significantly increased after the treatment with T(3) and in the acute diabetes. Both in diabetes and after T(3) treatment the phosphorylation of Cx43 isoforms was markedly suppressed compared to the non-diabetic and T(3)-untreated controls. Such a down-regulation was less pronounced in diabetic rats after the T(3)-treatment. The expression of atrial PKC-epsilon was increased in diabetic rats. This increase was suppressed after T(3) administration and the expression was decreased in T(3)-treated non-diabetic rats. We suggest that the reduced Cx43 phosphorylation in diabetic and hyperthyroid rats can deteriorate a cell-to-cell coupling and consequently facilitate a development of atrial tachyarrhythmia in diabetic or hyperthyroid animals.
- MeSH
- Diabetes Mellitus, Experimental complications metabolism MeSH
- Atrial Fibrillation complications metabolism MeSH
- Financing, Organized MeSH
- Phosphorylation MeSH
- Hyperthyroidism chemically induced complications metabolism MeSH
- Connexin 43 metabolism MeSH
- Blood Glucose metabolism MeSH
- Rats MeSH
- Myocardium metabolism MeSH
- Rats, Inbred WKY MeSH
- Protein Kinase C-epsilon metabolism MeSH
- Heart Atria metabolism MeSH
- Tachycardia, Supraventricular complications metabolism MeSH
- Triiodothyronine pharmacology MeSH
- Up-Regulation physiology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- MeSH
- CD36 Antigens * blood metabolism MeSH
- Diabetes Mellitus, Type 2 complications metabolism MeSH
- Financing, Organized MeSH
- Insulin metabolism MeSH
- Cardiovascular Diseases metabolism MeSH
- Metabolic Syndrome complications metabolism MeSH
- Rats, Inbred SHR MeSH
- Protein Kinase C-delta blood metabolism MeSH
- Protein Kinase C-epsilon blood metabolism MeSH
- Heart Ventricles * cytology chemistry metabolism MeSH
- Statistics as Topic MeSH
- Blotting, Western methods utilization MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
