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
- antiarytmika metabolismus farmakologie MeSH
- konexin 43 metabolismus MeSH
- konzumní sacharóza škodlivé účinky MeSH
- krysa rodu rattus MeSH
- kyseliny mastné omega-3 metabolismus farmakologie MeSH
- melatonin metabolismus farmakologie MeSH
- mikro RNA metabolismus MeSH
- myokard metabolismus MeSH
- obezita chemicky indukované komplikace farmakoterapie metabolismus MeSH
- potkani Wistar MeSH
- proteinkinasa C-delta metabolismus MeSH
- proteinkinasa C-epsilon metabolismus MeSH
- signální transdukce účinky léků MeSH
- srdce účinky léků MeSH
- srdeční arytmie etiologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The oxidized phospholipids (oxPl) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are cytotoxic components of oxidized LDL (oxLDL). Sustained exposure to oxLDL or isolated oxPl induces apoptotic signaling in vascular cells, which is a hallmark of the late phase of atherosclerosis. Activation of sphingomyelinase, the coordinate formation of ceramide and activation of caspase 3/7 as well as the activation of stress-associated kinases are causally involved in this process. Here, we provide evidence for a role of PKCδ in oxPl cytotoxicity. Silencing of the enzyme by siRNA significantly reduced caspase 3/7 activation in RAW 264.7 macrophages under the influence of oxPl. Concomitantly, PKCδ was phosphorylated as a consequence of cell exposure to PGPC or POVPC. Single molecule fluorescence microscopy provided direct evidence for oxPl-protein interaction. Both oxPl recruited an RFP-tagged PKCδ to the plasma membrane in a concentration-dependent manner. In addition, two color cross-correlation number and brightness (ccN&B) analysis of the molecular motions revealed that fluorescently labeled PGPC or POVPC analogs co-diffuse and are associated with the fluorescent protein kinase in live cells. The underlying lipid-protein interactions may be due to chemical bonding (imine formation between the phospholipid aldehyde POVPC with protein amino groups) and physical association (with POVPC or PGPC). In summary, our data supports the assumption that PKCδ acts as a proapototic kinase in oxPl-included apoptosis of RAW 264.7 macrophages. The direct association of the bioactive lipids with this enzyme seems to be an important step in the early phase of apoptotic signaling.
- MeSH
- aktivace enzymů MeSH
- apoptóza účinky léků MeSH
- časové faktory MeSH
- fosfolipidethery toxicita MeSH
- fosforylace MeSH
- kaspasa 3 metabolismus MeSH
- kaspasa 7 metabolismus MeSH
- makrofágy účinky léků enzymologie patologie MeSH
- myši MeSH
- oxidace-redukce MeSH
- proteinkinasa C-delta genetika metabolismus MeSH
- RAW 264.7 buňky MeSH
- regulace genové exprese enzymů MeSH
- reportérové geny MeSH
- RNA interference MeSH
- signální transdukce účinky léků MeSH
- transfekce MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- experimentální diabetes mellitus metabolismus patologie prevence a kontrola MeSH
- fixní kombinace léků MeSH
- konexin 43 metabolismus MeSH
- krysa rodu rattus MeSH
- kyselina eikosapentaenová farmakologie terapeutické užití MeSH
- kyseliny dokosahexaenové farmakologie terapeutické užití MeSH
- myokard metabolismus ultrastruktura MeSH
- potravní doplňky MeSH
- preklinické hodnocení léčiv MeSH
- proteinkinasa C-delta metabolismus MeSH
- proteinkinasa C-epsilon metabolismus MeSH
- srdce účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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
- aktivace enzymů MeSH
- antigeny CD36 genetika metabolismus MeSH
- cytosol metabolismus MeSH
- inzulin krev metabolismus MeSH
- inzulinová rezistence genetika fyziologie MeSH
- krevní glukóza analýza metabolismus MeSH
- krysa rodu rattus MeSH
- kyseliny mastné neesterifikované krev metabolismus MeSH
- myokard metabolismus MeSH
- potkani inbrední SHR MeSH
- proteinkinasa C-delta biosyntéza MeSH
- proteinkinasa C-epsilon biosyntéza MeSH
- regulace genové exprese MeSH
- sacharosa metabolismus MeSH
- signální transdukce MeSH
- srdeční komory metabolismus MeSH
- triglyceridy krev metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The adaptation to chronic hypoxia confers long-lasting cardiac protection against acute ischemia-reperfusion injury. Protein kinase C (PKC) appears to play a role in the cardioprotective mechanism but the involvement of individual PKC isoforms remains unclear. The aim of this study was to examine the effects of chronic intermittent hypoxia (CIH; 7,000 m, 8 h/day) and acute administration of PKC-δ inhibitor (rottlerin, 0.3 mg/kg) on the expression and subcellular distribution of PKC-δ and PKC-ε in the left ventricular myocardium of adult male Wistar rats by Western blot and quantitative immunofluorescence microscopy. CIH decreased the total level of PKC-ε in homogenate without affecting the level of phosphorylated PKC-ε (Ser729). In contrast, CIH up-regulated the total level of PKC-δ as well as the level of phosphorylated PKC-δ (Ser643) in homogenate. Rottlerin partially reversed the hypoxia-induced increase in PKC-δ in the mitochondrial fraction. Immunofluorescent staining of ventricular cryo-sections revealed increased co-localization of PKC-δ with mitochondrial and sarcolemmal membranes in CIH hearts that was suppressed by rottlerin. The formation of nitrotyrosine as a marker of oxidative stress was enhanced in CIH myocardium, particularly in mitochondria. The expression of total oxidative phosphorylation complexes was slightly decreased by CIH mainly due to complex II decline. In conclusion, up-regulated PKC-δ in CIH hearts is mainly localized to mitochondrial and sarcolemmal membranes. The inhibitory effects of rottlerin on PKC-δ subcellular redistribution and cardioprotection (as shown previously) support the view that this isoform plays a role in the mechanism of CIH-induced ischemic tolerance.
- MeSH
- chronická nemoc MeSH
- fosforylace MeSH
- hypoxie enzymologie MeSH
- inhibitory proteinkinas farmakologie MeSH
- krysa rodu rattus MeSH
- mitochondrie metabolismus MeSH
- myokard metabolismus patologie MeSH
- ochranné látky MeSH
- potkani Wistar MeSH
- proteinkinasa C-delta genetika metabolismus fyziologie MeSH
- sarkolema metabolismus MeSH
- tkáňová distribuce MeSH
- upregulace MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- časové faktory MeSH
- financování organizované MeSH
- fosforylace MeSH
- frakcionace buněk MeSH
- histony metabolismus MeSH
- hypertenze MeSH
- izoenzymy MeSH
- krevní tlak MeSH
- krysa rodu rattus MeSH
- modely nemocí na zvířatech MeSH
- myokard enzymologie patologie MeSH
- nemoci srdce MeSH
- novorozená zvířata MeSH
- potkani Wistar MeSH
- proteinkinasa C-alfa MeSH
- proteinkinasa C-delta metabolismus MeSH
- proteinkinasa C-epsilon metabolismus MeSH
- proteinkinasa C metabolismus MeSH
- upregulace MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- MeSH
- antigeny CD36 * krev metabolismus MeSH
- diabetes mellitus 2. typu komplikace metabolismus MeSH
- financování organizované MeSH
- inzulin metabolismus MeSH
- kardiovaskulární nemoci metabolismus MeSH
- metabolický syndrom komplikace metabolismus MeSH
- potkani inbrední SHR MeSH
- proteinkinasa C-delta krev metabolismus MeSH
- proteinkinasa C-epsilon krev metabolismus MeSH
- srdeční komory * cytologie chemie metabolismus MeSH
- statistika jako téma MeSH
- western blotting metody využití MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- MeSH
- acetofenony metabolismus terapeutické užití MeSH
- benzofenantridiny metabolismus terapeutické užití MeSH
- experimenty na zvířatech MeSH
- financování organizované MeSH
- infarkt myokardu * metabolismus MeSH
- ischemické přivykání * MeSH
- kardiomyocyty metabolismus účinky léků MeSH
- potkani Wistar MeSH
- proteinkinasa C-delta metabolismus účinky léků MeSH
- proteinkinasa C-epsilon metabolismus účinky léků MeSH
- proteinkinasa C antagonisté a inhibitory terapeutické užití MeSH
- reperfuzní poškození myokardu * metabolismus MeSH
- statistika jako téma MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
We examined the influence of dietary fatty acid (FA) classes on the expression of protein kinase C (PKC) delta and epsilon in relation to the cardioprotective effects of chronic intermittent hypoxia (CIH). Adult male Wistar rats were fed a nonfat diet enriched with 10% lard (saturated FA [SFA]), fish oil (n-3 polyunsaturated FA [n-3 PUFA]), or corn oil (n-6 PUFA) for 10 weeks. After 4 weeks on the diet, each group was divided into two subgroups that were either exposed to CIH in a barochamber (7000 m, 8 hrs/ day) or kept at normoxia for an additional 5-6 weeks. A FA phospholipid profile and Western blot analysis of PKC were performed in left ventricles. Infarct size was assessed in anesthetized animals subjected to 20-min coronary artery occlusion and 3-hr reperfusion. CIH decreased the n-6/n-3 PUFA ratio in all groups by 23% independently of the initial value set by various diets. The combination of n-3 diet and CIH had a stronger antiarrhythmic effect during reperfusion than the n-3 diet alone; this effect was less pronounced in rats fed the n-6 diet. The normoxic n-6 group exhibited smaller infarctions (by 22%) than the n-3 group. CIH decreased the infarct size in n-3 and SFA groups (by 20% and 23%, respectively) but not in the n-6 group. Unlike PKC epsilon, the abundance of PKC delta in the myocardial particulate fraction was increased by CIH except for the n-6 group. Myocardial infarct size was negatively correlated (r=- 0.79) with the abundance of PKC delta in the particulate fraction. We conclude that lipid diets modify the infarct size-limiting effect of CIH by a mechanism that involves the PKC delta-dependent pathway.
- MeSH
- dietní tuky farmakologie MeSH
- hypoxie enzymologie patofyziologie MeSH
- infarkt myokardu enzymologie patofyziologie MeSH
- kardiotonika farmakologie MeSH
- krevní tlak účinky záření MeSH
- krysa rodu rattus MeSH
- kyseliny mastné omega-3 farmakologie MeSH
- kyseliny mastné omega-6 farmakologie MeSH
- mastné kyseliny farmakologie MeSH
- myokard enzymologie MeSH
- potkani Wistar MeSH
- proteinkinasa C-delta metabolismus MeSH
- proteinkinasa C-epsilon metabolismus MeSH
- reperfuzní poškození myokardu enzymologie patofyziologie MeSH
- srdeční frekvence účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- MeSH
- acetylcystein analogy a deriváty terapeutické užití MeSH
- experimenty na zvířatech MeSH
- hypoxie * metabolismus MeSH
- infarkt myokardu * diagnóza etiologie metabolismus MeSH
- nadmořská výška MeSH
- náhlá srdeční smrt * prevence a kontrola MeSH
- oxidační stres fyziologie imunologie účinky léků MeSH
- potkani Wistar MeSH
- proteinkinasa C-delta farmakologie MeSH
- proteinkinasa C-epsilon farmakologie MeSH
- proteinkinasa C farmakologie klasifikace MeSH
- reaktivní formy kyslíku metabolismus škodlivé účinky MeSH
- statistika jako téma MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
