RESUMO
Inhibition of receptor-interacting protein kinase 1 (RIP1) has been recognized as a compelling tool for limiting necroptosis. Recent findings have indicated that RIP1 inhibitor, necrostatin-1 (Nec-1), is also able to modify heart function under non-cell death conditions. In this study, we investigated its underlying molecular mechanisms and compared with those of novel pharmacologically improved agents (Nec-1s and GSK'772) and its inactive analog (Nec-1i). Heart function was examined in Langendorff-perfused rat hearts. Certain proteins regulating myocardial contraction-relaxation cycle and oxidative stress (OS) were evaluated by immunoblotting and as the extent of lipid peroxidation, protein carbonylation and nitration, respectively. In spite of the increase of left ventricular developed pressure (LVDP) due to treatment by both Nec-1 and Nec-1i, only the former agent increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) at threonine 287 and cardiac myosin-binding protein-C (cMyBPc) at serine 282. In contrast, Nec-1s did not elicit such changes, while it also increased LVDP. GSK'772 activated CaMKIIδ-phospholamban (PLN) axis. Neither protein kinase A (PKA) nor its selected molecular targets, such as serine 16 phosphorylated PLN and sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), were affected by either RIP1 inhibitor. Nec-1, like other necrostatins (Nec-1i, Nec-1s), but not GSK'772, elevated protein tyrosine nitration without affecting other markers of OS. In conclusion, this study indicated for the first time that Nec-1 may affect basal heart function by the modulation of OS and activation of some proteins of contraction-relaxation cycle.
Assuntos
Coração/efeitos dos fármacos , Imidazóis/farmacologia , Indóis/farmacologia , Contração Miocárdica , Necrose , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Masculino , Ratos , Ratos Wistar , Proteína Serina-Treonina Quinases de Interação com ReceptoresRESUMO
Remote ischemic preconditioning (RIPC) represents one of the forms of innate cardioprotection. While being effective in animal models, its application in humans has not been always beneficial, which might be attributed to the presence of various comorbidities, such as hypertension, or being related to the confounding factors, such as patients' sex and age. RIPC has been shown to mediate its cardioprotective effects through the activation of Reperfusion Injury Salvage Kinase (RISK) pathway in healthy animals, however, scarce evidence supports this effect of RIPC in the hearts of spontaneously hypertensive (SHR) rats, in particular, in relationship with aging. The study aimed to investigate the effectiveness of RIPC in male SHR rats of different age and to evaluate the role of RISK pathway in the effect of RIPC on cardiac ischemic tolerance. RIPC was performed using three cycles of inflation/deflation of the pressure cuff placed on the hind limb of anesthetized rats aged three, five and eight months. Subsequently, hearts were excised, Langendorff-perfused and exposed to 30-min global ischemia and 2-h reperfusion. Infarct-sparing and antiarrhythmic effects of RIPC were observed only in three and five months-old animals but not in eight months-old rats. Beneficial effects of RIPC were associated with increased activity of RISK and decreased apoptotic signaling only in three and five months-old animals. In conclusion, RIPC showed cardioprotective effects in SHR rats that were partially age-dependent and might be attributed to the differences in the activation of RISK pathway and various aspects of ischemia/reperfusion injury in aging animals.
Assuntos
Hipertensão , Infarto do Miocárdio , Traumatismo por Reperfusão Miocárdica , Humanos , Masculino , Ratos , Animais , Lactente , Infarto do Miocárdio/metabolismo , Ratos Endogâmicos SHR , Traumatismo por Reperfusão Miocárdica/metabolismo , Isquemia , Hipertensão/prevenção & controleRESUMO
Iron is an essential mineral participating in numerous biological processes in the organism under physiological conditions. However, it may be also involved in the pathological mechanisms activated in various cardiovascular diseases including myocardial ischemia/reperfusion (I/R) injury, due to its involvement in reactive oxygen species (ROS) production. Furthermore, iron has been reported to participate in the mechanisms of iron-dependent cell death defined as "ferroptosis". On the other hand, iron may be also involved in the adaptive processes of ischemic preconditioning (IPC). This study aimed to elucidate whether small amounts of iron may modify the cardiac response to I/R in isolated perfused rat hearts and their protection by IPC. Pretreatment of the hearts with iron nanoparticles 15 min prior to sustained ischemia (iron preconditioning, Fe-PC) did not attenuate post-I/R contractile dysfunction. Recovery of left ventricular developed pressure (LVDP) was significantly improved only in the group with combined pretreatment with iron and IPC. Similarly, the rates of contraction and relaxation [+/-(dP/dt)max] were almost completely restored in the group preconditioned with a combination of iron and IPC but not with iron alone. In addition, the severity of reperfusion arrhythmias was reduced only in the iron+IPC group. No changes in protein levels of "survival" kinases of the RISK pathway (Reperfusion Injury Salvage Kinase) were found except for reduced caspase 3 levels in both preconditioned groups. The results indicate that a failure to precondition rat hearts with iron may be associated with the absent upregulation of RISK proteins and the pro-ferroptotic effect manifested by reduced glutathione peroxidase 4 (GPX4) levels. However, combination with IPC suppressed the negative effects of iron resulting in cardioprotection.
Assuntos
Precondicionamento Isquêmico Miocárdico , Precondicionamento Isquêmico , Traumatismo por Reperfusão Miocárdica , Ratos , Animais , Masculino , Ratos Wistar , Ferro/metabolismo , Coração , Traumatismo por Reperfusão Miocárdica/metabolismo , Precondicionamento Isquêmico/métodos , Miocárdio/metabolismoRESUMO
This study deals with changes, induced by hypertension and its treatment, in the function and properties of mitochondria in the heart and kidneys. Male, 16-week-old hypertensive rats were allocated to 3 groups: (i) animals treated daily for 4 weeks with captopril (CAP, 80 mg·(kg body mass)(-1), n = 45), (ii) animals treated with CAP + nifedipine (NIF, 10 mg·kg(-1), n = 45), or (iii) untreated hypertensive controls (n = 96). Wistar rats (n = 96) were used as normotensive controls. Systolic blood pressure (SBP), heart rate (HR), and heart mass / body mass (HW/BW) ratio were measured at the beginning and end of the experiments; measurements for mitochondrial Mg(2+)-ATPase activity, O(2)-consumption (QO(2)), respiratory control index (RCI), ADP/O, oxidative phosphorylation rate (OPR), conjugated diene content (CD), and membrane fluidity (MF) were also taken at different time intervals. In the heart, elevated SBP, HR, and HW/BW accompanied increased QO(2), OPR, and Mg(2+)-ATPase activity, indicating an adaptive response to hypertension-induced increase in the energy demands of the myocardium. Treatments with CAP or with CAP + NIF were very similar in their prevention of increase in SBP, HR, HW/BW, and the rise in OPR (all p < 0.05-0.01). In the kidneys, hypertension induced a drop in OPR; however, antihypertensive therapy aggravated the resulting energy deficiency, whereby treatment with CAP + NIF was more detrimental than treatment with CAP alone. Heart and kidney mitochondria exhibited negligible changes in CD and moderately increased MF, which was more potentiated by treatment with CAP alone than with CAP + NIF.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Rim/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Miocárdio/metabolismo , Animais , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Captopril/administração & dosagem , Captopril/farmacologia , Captopril/uso terapêutico , Quimioterapia Combinada , Frequência Cardíaca/efeitos dos fármacos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Rim/metabolismo , Masculino , Fluidez de Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Membranas Mitocondriais/metabolismo , Nifedipino/administração & dosagem , Nifedipino/farmacologia , Nifedipino/uso terapêutico , Ratos , Ratos Endogâmicos SHR , Ratos WistarRESUMO
The root extract of Hemidesmus indicus (Linn.) R. Br. (Asclepiadaceae) (HI) was studied for its cardioprotective effect in Langendorff-perfused rat hearts. HI was perfused for 15 min at a concentration of 0.09 g/L prior to 30 min global ischemia/120 min reperfusion (I/R). Recovery of functional parameters, reperfusion arrhythmias, and infarct size (TTC staining) served as the end-points. After 15 min of perfusion with HI, the left ventricular developed pressure (LVdevP) and HR (heart rate) were not altered significantly (p>0.05), as compared with the pre-drug values. During R, HI showed a significantly higher (p<0.05) recovery of LVdevP at nearly all time points. The recovery of maximal rate of pressure development (+dP/dtmax) and left ventricular end-diastolic pressure (LVEDP) at 40 min of R were significantly better than in non-treated controls. There was also a significant reduction in the total number of ventricular premature beats (VPB) and duration of ventricular tachycardia (VT). HI can protect ischemic myocardium against contractile dysfunction and reperfusion-induced arrhythmias and reduce the extent of irreversible tissue damage following I/R in rat hearts.
Assuntos
Cardiotônicos/farmacologia , Hemidesmus/química , Traumatismo por Reperfusão Miocárdica/complicações , Extratos Vegetais/farmacologia , Animais , Cardiotônicos/isolamento & purificação , Frequência Cardíaca/efeitos dos fármacos , Masculino , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/prevenção & controle , Raízes de Plantas , Ratos , Ratos Wistar , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/prevenção & controle , Fatores de Tempo , Complexos Ventriculares Prematuros/etiologia , Complexos Ventriculares Prematuros/prevenção & controleRESUMO
We examined the involvement of phosphatidylinositol 3-kinase (PI3K) and its effector protein kinase B (Akt) in cardioprotective effects of ischemic preconditioning (PC) with particular regards to its role in the protection against ischemia-induced arrhythmias in isolated perfused rat heart. PI3K/Akt inhibitor wortmannin (100 nM) was administered 15 min prior to 30-min regional (left anterior descending coronary artery occlusion) ischemia for the study of ischemic arrhythmias in the hearts perfused at constant coronary flow or prior to 30-min global ischemia followed by 2-h reperfusion for the infarct size (IS) determination (tetrazolium staining) in the hearts perfused at constant pressure. PC procedure (one cycle of ischemia/reperfusion, 5 min each) significantly reduced the total number of ventricular premature complexes (PVC) and severity of arrhythmias (arrhythmia score; AS) over the whole period of left anterior descending coronary artery occlusion in comparison with non-PC controls (PVC 166+/-40; AS 1.6+/-0.2 vs. 550+/-60 and 3.2+/-0.2; respectively; P<0.05). In a setting of global ischemia/reperfusion, PC decreased IS (in % of the left ventricle, LV) by 73 %. Pretreatment with wortmannin modified neither arrhythmogenesis nor IS in the non-PC hearts. Bracketing of PC with wortmannin did not abolish antiarrhythmic protection (PVC 92+/-25; AS 1.7+/-0.2; P<0.05 vs. non-PC hearts). On the other hand, wortmannin increased IS/LV in the PC hearts to 24+/-1.2 % as compared with 9 +/- 0.6 % in the untreated ones (P<0.05). In conclusion, PI3K/Akt inhibition did not affect reduced arrhythmogenesis during ischemia in the PC hearts indicating that in contrast to its positive role in the irreversible myocardial injury, PI3K/Akt activity is not required for protection induced by PC against ischemic arrhythmias in the rat heart.
Assuntos
Arritmias Cardíacas/prevenção & controle , Precondicionamento Isquêmico Miocárdico , Infarto do Miocárdio/prevenção & controle , Isquemia Miocárdica/terapia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Androstadienos/farmacologia , Animais , Arritmias Cardíacas/enzimologia , Arritmias Cardíacas/etiologia , Técnicas In Vitro , Masculino , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/etiologia , Isquemia Miocárdica/complicações , Isquemia Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/etiologia , Miocárdio/patologia , Perfusão , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ratos , Ratos Wistar , WortmaninaRESUMO
Ischemic preconditioning (I-PC) induced by brief episodes of ischemia and reperfusion (I/R) protects the heart against sustained I/R. Although activation of mitochondrial K(ATP) channels (mitoK(ATP)) interacting with reactive oxygen species (ROS) has been proposed as a key event in this process, their role in the antiarrhythmic effect is not clear. This study was designed: 1) to investigate the involvement of mito K(ATP) opening in the effect of I-PC (1 cycle of I/R, 5 min each) on ventricular arrhythmias during test ischemia (TI, 30-min LAD coronary artery occlusion) in Langendorff-perfused rat hearts and subsequent postischemic contractile dysfunction, and 2) to characterize potential mechanisms of protection conferred by I-PC and pharmacological PC induced by mito K(ATP) opener diazoxide (DZX), with particular regards to the modulation of ROS generation. Lipid peroxidation (an indicator of increased ROS production) was determined by measurement of myocardial concentration of conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) in non-ischemic controls, non-preconditioned and preconditioned hearts exposed to TI, I-PC alone, as well as after pretreatment with DZX, mito K(ATP) blocker 5-hydroxydecanoate (5-HD) and antioxidant N-acetylcysteine (NAC). Total number of ventricular premature beats (VPB) that occurred in the control hearts (518+/-71) was significantly (P<0.05) reduced by I-PC (195+/-40), NAC (290+/-56) and DZX (168+/-22). I-PC and NAC suppressed an increase in CD and TBARS caused by ischemia indicating lower production of ROS. On the other hand, I-PC and DZX themselves moderately enhanced ROS generation, prior to TI. Bracketing of I-PC with 5-HD suppressed both, ROS production during PC and its cardioprotective effect. In conclusion, potential mechanisms of protection conferred by mito K(ATP) opening in the rat heart might involve a temporal increase in ROS production in the preconditioning phase triggering changes in the pro/antioxidant balance in the myocardium and attenuating ROS production during subsequent prolonged ischemia.
Assuntos
Precondicionamento Isquêmico Miocárdico , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Canais de Potássio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Função Ventricular Esquerda , Complexos Ventriculares Prematuros/prevenção & controle , Acetilcisteína/farmacologia , Animais , Antiarrítmicos/farmacologia , Antioxidantes/farmacologia , Ácidos Decanoicos/farmacologia , Diazóxido/farmacologia , Hidroxiácidos/farmacologia , Técnicas In Vitro , Peroxidação de Lipídeos , Masculino , Contração Miocárdica/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Estresse Oxidativo , Perfusão , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Ratos , Ratos Wistar , Recuperação de Função Fisiológica , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Fatores de Tempo , Função Ventricular Esquerda/efeitos dos fármacos , Complexos Ventriculares Prematuros/metabolismo , Complexos Ventriculares Prematuros/fisiopatologiaRESUMO
Statins, the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are most frequently used drugs in the prevention of coronary artery disease due to their cholesterol-lowering activity. However, it is not exactly known whether these effects of statins or those independent of cholesterol decrease account for the protection against myocardial ischemia-reperfusion (I/R) injury. In this study, we investigated the effect of 5-day treatment with simvastatin (10 mg/kg) in Langendorff-perfused hearts of healthy control (C) and diabetic-hypercholesterolemic (D-H; streptozotocin + high fat-cholesterol diet, 5 days) rats subjected to 30-min global ischemia followed by 40-min reperfusion for the examination of postischemic contractile dysfunction and reperfusion-induced ventricular arrhythmias or to 30-min (left anterior descending) coronary artery occlusion and 2-h reperfusion for the infarct size determination (IS; tetrazolium staining). Postischemic recovery of left ventricular developed pressure (LVDP) in animals with D-H was improved by simvastatin therapy (62.7+/-18.2 % of preischemic values vs. 30.3+/-5.7 % in the untreated D-H; P<0.05), similar to the values in the simvastatin-treated C group, which were 2.5-fold higher than those in the untreated C group. No ventricular fibrillation occurred in the simvastatin-treated C and D-H animals during reperfusion. Likewise, simvastatin shortened the duration of ventricular tachycardia (10.2+/-8.1 s and 57.8+/-29.3 s in C and D-H vs. 143.6+/-28.6 s and 159.3+/-44.3 s in untreated C and D-H, respectively, both P<0.05). The decreased arrhythmogenesis in the simvastatin-treated groups correlated with the limitation of IS (in % of risk area) by 66 % and 62 % in C and D-H groups, respectively. However, simvastatin treatment decreased plasma cholesterol levels neither in the D-H animals nor in C. The results indicate that other effects of statins (independent of cholesterol lowering) are involved in the improvement of contractile recovery and attenuation of lethal I/R injury in both, healthy and diseased individuals.
Assuntos
Arritmias Cardíacas/prevenção & controle , Cardiotônicos/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Hipercolesterolemia/tratamento farmacológico , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/prevenção & controle , Isquemia Miocárdica/prevenção & controle , Miocárdio/patologia , Sinvastatina/farmacologia , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Colesterol/sangue , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipercolesterolemia/complicações , Hipercolesterolemia/patologia , Hipercolesterolemia/fisiopatologia , Masculino , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Isquemia Miocárdica/etiologia , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Perfusão , Ratos , Ratos Wistar , Recuperação de Função Fisiológica , Pressão Ventricular/efeitos dos fármacosRESUMO
Although physical exercise is known to reduce size of infarction, incidence of ventricular arrhythmias, and to improve heart function, molecular mechanisms of this protection are not fully elucidated. We explored the hypothesis that voluntary running, similar to adaptive interventions, such as ischemic or remote preconditioning, may activate components of pro-survival (RISK) pathway and potentially modify cell proliferation. Sprague-Dawley adult male rats freely exercised for 23 days in cages equipped with running wheels, while sedentary controls were housed in standard cages. After 23 days, left ventricular (LV) myocardial tissue samples were collected for the detection of expression and activation of RISK proteins (WB). The day before, a marker of cell proliferation 5-bromo-2'-deoxyuridine (BrdU) was given to all animals to detect its incorporation into DNA of the LV cells (ELISA). Running increased phosphorylation (activation) of Akt, as well as the levels of PKC? and phospho-ERK1/2, whereas BrdU incorporation into DNA was unchanged. In contrast, exercise promoted pro-apoptotic signaling - enhanced Bax/Bcl-2 ratio and activation of GSK-3ß kinase. Results suggest that in the rat myocardium adapted to physical load, natural cardioprotective processes associated with physiological hypertrophy are stimulated, while cell proliferation is not modified. Up-regulation of pro-apoptotic markers indicates potential induction of cell death mechanisms that might lead to maladaptation in the long-term.
Assuntos
Proliferação de Células/fisiologia , Mediadores da Inflamação/metabolismo , Miocárdio/metabolismo , Condicionamento Físico Animal/fisiologia , Animais , Masculino , Miocárdio/patologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fatores de Risco , Taxa de Sobrevida/tendênciasRESUMO
The study has been designed to characterize protein systems involved in the responses of rat hearts to chronic doxorubicin (DOX) treatment. We investigated the influence of DOX on cardiac function, mitogen-activated protein kinases (MAPKs) and heat stress proteins (HSPs). Doxorubicin was administered to rats by intraperitoneal injections over a period of 6 weeks. In control and DOX-treated hearts exposed to 20 min global ischemia and 40 min reperfusion the recovery of contractile function after ischemia/reperfusion (I/R) was determined. The levels and phosphorylation state of proteins in tissue samples were analyzed using specific antibodies. We found an activation of extracellular signal-regulated kinases (ERKs) in rat hearts exposed to DOX treatment and better recovery of contractile function after I/R. Analysis of HSPs showed that DOX induced up-regulation of the levels of HSP60 and down-regulation of HSP70 levels. The levels and/or specific phosphorylation of other studied proteins (p38-MAPK, HSP27, HSP90) were not influenced by DOX. The results point to the possible role of ERKs and some HSPs in mechanisms underlying the response of rat hearts to chronic DOX treatment.
Assuntos
Chaperonina 60/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Cardiopatias/enzimologia , Miocárdio/enzimologia , Animais , Antibióticos Antineoplásicos , Modelos Animais de Doenças , Doxorrubicina , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP90/metabolismo , Cardiopatias/induzido quimicamente , Cardiopatias/fisiopatologia , Proteínas de Choque Térmico/metabolismo , Masculino , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Proteínas de Neoplasias/metabolismo , Fosforilação , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Contrary to clinical trials, experimental studies revealed that diabetes mellitus (DM) may initiate, besides increased myocardial vulnerability to ischemia-reperfusion injury (I/R) and pro/antioxidant dysbalance, development of adaptation leading to an enhanced tolerance to I/R. The aims were to characterize 1) susceptibility to ischemia-induced ventricular arrhythmias in the diabetic rat heart 2) its response to antioxidant N-acetylcysteine (NAC) and a NOS inhibitor L-NAME, and 3) the effect of DM on endogenous antioxidant systems. Seven days after streptozotocin injection (65 mg/kg, i.p.), Langendorff-perfused control (C) and DM hearts were subjected to 30-min occlusion of the LAD coronary artery with or without prior 15-min treatment with L-NAME (100 microM) or NAC (4 mM). Total number of ventricular premature beats (VPB), as well the total duration of ventricular tachycardia (VT) were reduced in the DM group (from 533+/-58 and 37.9+/-10.2 s to 224.3+/-52.6 and 19+/-13.5 s; P<0.05). In contrast to the antiarrhythmic effects of L-NAME and NAC in controls group (VPB 290+/-56 and 74+/-36, respectively; P<0.01 vs. control hearts), application of both drugs in the diabetics did not modify arrhythmogenesis (L-NAME: VPB 345+/-136, VT 25+/-13 s; NAC: VPB 207+/-50, VT 12+/-3.9 s; P>0.05 vs non-treated diabetic hearts). Diabetic state was associated with significantly elevated levels of CoQ10 and CoQ9 (19.6+/-0.8 and 217.3+/-9.5 vs. 17.4+/- 0.5 and 185.0+/-5.0 nmol/g, respectively, in controls; P<0.05), as well as alpha-tocopherol (38.6+/-0.7 vs. 31.5+/-2.1 nmol/g in controls; P<0.01) in the myocardial tissue. It is concluded that early period of DM is associated with enhanced resistance to ischemia-induced arrhythmias. Diabetes mellitus might induce adaptive processes in the myocardium leading to lower susceptibility to antioxidant and L-NAME treatment.
Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Isquemia Miocárdica/tratamento farmacológico , Miocárdio/enzimologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Taquicardia Ventricular/prevenção & controle , Adaptação Fisiológica , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Masculino , Isquemia Miocárdica/complicações , Isquemia Miocárdica/enzimologia , Isquemia Miocárdica/fisiopatologia , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Wistar , Taquicardia Ventricular/enzimologia , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/fisiopatologia , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Regulação para Cima , Função Ventricular Esquerda , alfa-Tocoferol/metabolismoRESUMO
Intrinsic cardioprotective mechanisms become activated in the early diabetes mellitus (DM) and this may protect the heart from ischemia/reperfusion (I/R) similarly as in case of ischemic preconditioning. However, this protection may by blunted in the presence of cardiovascular risk factors. Assuming that hypercholesterolemia (HCH) frequently accompanies DM, this study extends findings from separate models of DM and HCH by investigation the impact of HCH on DM-induced changes, including those of compensatory nature, in rat heart and its mitochondria. We used a factorial design with all combinations of treatment factors DM and HCH: control rats (C) and streptozotocin-treated rats (DM), both on standard chow (C and DM) and fed fat-cholesterol diet (HCH and DM-HCH). Isolated, Langendorff perfused hearts were subjected to 30 min global ischemia followed by reperfusion. Significantly increased levels of cholesterol in DM-HCH after I/R injury abrogated compensatory fluidization characteristic of DM mitochondria membranes. Concomitantly, the mitochondrial Mg2+-ATPase activity in DM-HCH was depressed. In comparison with DM, which showed significantly reduced size of myocardial infarction with simultaneously improved recovery of contractile function due to conditioning, DM-HCH hearts exhibited attenuated resistance to I/R injury. Taken together, cholesterol-enriched diet was associated with inflicting damage and has been implicated in the mechanisms leading to suppression of cardiac protection presented in diabetic group. Apparently, DM and HCH are factors which are not additive in their effects, therefore, caution should be exercised, when interpreting findings from studies considering these factors in isolation. Our findings suggest that this complex condition could accelerate the development of late diabetic complications.
Assuntos
Colesterol na Dieta/administração & dosagem , Diabetes Mellitus Experimental/fisiopatologia , Coração/fisiologia , Hipercolesterolemia/fisiopatologia , Mitocôndrias Cardíacas/fisiologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Adaptação Fisiológica , Animais , Masculino , Ratos WistarRESUMO
Despite recent advances in pharmacotherapy of coronary artery disease and interventional cardiology, the management of myocardial ischemia still remains a major challenge for basic scientists and clinical cardiologists. An urgent need to combat ischemic heart disease, its forms, such as infarction, and complications including sudden cardiac death led to the development of an alternative strategy of myocardial protection based on the exploitation of the heart's own intrinsic protective mechanisms. A new concept relies on the evidence that the heart is able to protect itself by way of adaptation, either short-term or long-term, to transient episodes of stress (e.g., ischemia, hypoxia, free oxygen radicals, heat stress, etc.) preceding sustained ischemia. Preconditioning by brief episodes of ischemia (ischemic preconditioning, IP) represents the most powerful cardioprotective phenomenon. Apart from the short-lasting protection afforded by classical IP or its delayed ("second window") phase, adaptation to long-lasting physiological stimuli or pathological processes is also known to increase myocardial resistance to ischemic injury. Although molecular mechanisms of cardiac adaptation conferring a higher ischemic tolerance still remain not sufficiently elucidated, multiple cascades of intracellular signalization are suggested to be involved in this process. Experimental studies led to the observations that pharmacological modulations at different levels of signal transduction might mimic protective effects of the adaptive phenomena and thus provide a safer way of inducing cardioprotection in humans.
Assuntos
Precondicionamento Isquêmico Miocárdico/métodos , Isquemia Miocárdica/complicações , Isquemia Miocárdica/prevenção & controle , Estresse Fisiológico/prevenção & controle , Morte Súbita Cardíaca/patologia , Morte Súbita Cardíaca/prevenção & controle , Radicais Livres/metabolismo , Transtornos de Estresse por Calor/patologia , Transtornos de Estresse por Calor/prevenção & controle , Humanos , Hipóxia/patologia , Hipóxia/prevenção & controle , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Isquemia Miocárdica/patologia , Transdução de Sinais/fisiologia , Estresse Fisiológico/patologia , Fatores de TempoRESUMO
Pretreatment with diazoxide, mitochondrial K(ATP) channel opener, was found to protect the rat heart against ischemia/reperfusion injury. Our aim was also to characterize the effects of diazoxide on the alterations of regulatory myocardial proteins, on mitochondrial ultrastructure, integrity and induction of apoptotic responses. Isolated rat hearts were Langendorff perfused and subjected to index ischemia (II) induced by 25 min global ischemia and 35 min reperfusion. In diazoxide- treated hearts, diazoxide (50 micromol/l) was applied 15 min before II. The levels and activation of specific proteins were determined using specific antibodies, activities of matrix metalloproteinases by zymography using gelatin as a substrate. The ultrastructure of mitochondria was investigated by electron microscopy of ultrathin sections of mitochondrial fractions embedded in Epon812. In rat hearts pretreated with diazoxide we found better recovery of contractile function after II. Electron microscopy studies revealed that application of diazoxide was connected with better preservation of mitochondrial integrity at basal conditions and after II in comparison to control hearts. Ischemia induced activation of caspase-3 as well as decrease of mitochondria-associated Bcl-2 levels but diazoxide treatment did not significantly influence these changes. On the other hand, diazoxide pretreatment reduced the cytosolic levels of pro-apoptotic Bax protein. Western blot analysis revealed that application of diazoxide increased activation of both ERK-1 and ERK-2 as compared with control hearts. ERK-2 activities were also higher in diazoxide-treated hearts after II when compared to control hearts. Moreover, application of diazoxide inhibited the activities of tissue matrix metalloproteinases (MMP-2). The results suggest that the cardioprotection mediated by diazoxide in rats is associated with preservation of mitochondrial integrity and function. The effect of diazoxide on ERK pathway points to the involvement of this signaling cascade in diazoxide-mediated adaptive responses of myocardium to ischemia.
Assuntos
Diazóxido/administração & dosagem , Isquemia Miocárdica/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/genética , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas In Vitro , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Metaloproteinase 2 da Matriz/genética , Microscopia Eletrônica , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/patologia , Mitocôndrias Cardíacas/ultraestrutura , Contração Miocárdica/efeitos dos fármacos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Ratos Wistar , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Chronic intermittent high altitude (IHA) hypoxia results in long-term adaptation protecting the heart against acute ischemia/reperfusion injury; however, molecular mechanisms of this phenomenon are not completely elucidated so far. The present study was aimed at investigation of a modulating effect of IHA hypoxia on the expression and/or activation of selected regulatory proteins, with particular emphasis on differential responses in the right ventricle (RV) and left ventricle (LV). Adult male Wistar rats were exposed to IHA hypoxia of 7000 m simulated in a hypobaric chamber (8 h/day, 25 exposures), and protein contents and activities in myocardial fractions were determined by Western blot analysis. In markedly hypertrophic RV of hypoxic rats, gelatinolytic activity of MMP-2 and protein levels of carbonic anhydrase IX (a marker of hypoxia) were significantly enhanced. Study of mitogen-activated protein kinases (MAPKs) revealed no differences in the contents of total p38-MAPK in both ventricles between the IHA and normoxic control rats, whereas activation of p38-MAPK was decreased in the RV and moderately increased in the LV of IHA rats as compared to controls. Extracellular signal regulated kinase-2 (ERK-2) was partially up-regulated in the RV of IHA rats, and, in addition, expression of acidic fibroblast growth factor (aFGF), a potential activator of ERK cascade, was also significantly increased. In contrast, expression of ERKs in the LV as well as their activities in both ventricles, were not affected by IHA hypoxia. Differential effects of IHA hypoxia on c-Jun-N-terminal protein kinases (JNKs) in the RV and LV were also observed. As compared with the controls, total content of JNKs was increased in the RV of the IHA rats, while expression of JNKs in the LV was down-regulated. IHA hypoxia changed neither total levels of Akt kinase in both RV and LV, nor Akt kinase activity in the RV. However, increased levels of activated phospho-Akt kinase were found in the LV of IHA rats. The results demonstrate that adaptation of rat hearts to chronic IHA hypoxia is associated with disctinct changes in the levels and/or activation of several regulatory proteins in two ventricles. The latter could be attributed to both myocardial remodeling and cardioprotection induced by chronic hypoxia.
Assuntos
Anidrases Carbônicas/metabolismo , Ventrículos do Coração/enzimologia , Hipertrofia Ventricular Direita/enzimologia , Hipóxia/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Constituição Corporal , Doença Crônica , Hematócrito , Hipertrofia Ventricular Direita/etiologia , Hipóxia/complicações , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Rats with streptozotocin-diabetes develop mechanisms of endogenous protection (MEP) that participate actively in functional remodeling of cardiac sarcolemma. Remodeling of sarcolemma is a sign of damage but it also protects the cells of the diabetic heart (DH) against additional energy disbalance due to excessive Ca(2+) entry. Since yet, cardiac mitochondria (MIT) were investigated predominantly from the aspect of damage only. Aims of the present study were: i) to distinguish between acute diabetes-induced changes in function of rat heart MIT which clearly belong to damage from those that reflect the MEP and participate in functional remodeling of the MIT; ii) elucidate the significance of MEP-induced changes in heart MIT for cardiac energetics. Acute diabetes (8 days) was induced in adult male Wistar rats by streptozotocin (STZ, 65 mg.kg(-1) i.p., single dose). On the day 8 after STZ administration, the diabetic animals exhibited 300-330 % increase in blood glucose, triacylglycerols and cholesterol as well as 89.6 % increase in glycohemoglobin (all p < 0.01). The blood level of insulin dropped by 53 % (p < 0.02). State 3 and state 4 oxygen consumptions of DH MIT were decreased against the controls, leading to drop of the respiratory control index (17.9 and 7.3 %) and oxidative phosphorylation rate (OPR, 27.5 and 24.6 %; all p < 0.003-0.02). These effects of damage yielding in strained energy balance of the acute DH were partially alleviated by MEP. The latter involved temporary preservation of the ADP : O ratio, with participation of elevated MIT Mg(2+)-ATPase activity as well as increased formation of MIT substrate and energy transition pores (both p < 0.05). Hence, the energy disbalance of the acute DH was finally manifested in 13 % loss in its AMP content only (p < 0.05). Results indicate that MIT in STZ-DH are functionally remodeled. Defective O2 consumption by MIT renders molecular changes suggestive of a mild hypoxic state but an increase in Mg(2+)-ATPase activity and facilitated energy delivery from MIT to the cytoplasm indicate the presence of MEP acting in the MIT and alleviating the effect of decreased oxidative energy production in the acute DH.
Assuntos
Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Remodelação Ventricular/fisiologia , Doença Aguda , Adaptação Fisiológica , Animais , Células Cultivadas , Citoproteção , Diabetes Mellitus Experimental/induzido quimicamente , Masculino , Ratos , Ratos Wistar , EstreptozocinaRESUMO
The present state of knowledge unequivocally indicates that chronic diabetes is associated with impaired function of coronary vessels. Langendorff retrograde perfusion is one of the most frequently employed methods to study dysfunction of coronary vasculature in animal models of diabetes mellitus. However, because of methodological discrepancies in experimental protocols, the reliability of this technique is limited. In the current study, we propose the novel technique of vasoactive drug administration and aim to evaluate its usefulness in detecting coronary dysfunction in diabetes. Using Langendorff model, we compared the results of coronary endothelium-dependent (bradykinin) and -independent (diethylamine/nitric oxide, DEA/NO) vasodilatation obtained from experimental model utilizing automatically corrected-rate infusion with commonly used, constant-rate infusion of vasoactive drug. The infusion of bradykinin at constant rate failed to reveal coronary endothelium-dependent dysfunction typical for diabetes mellitus. Induction of endothelium-independent vasodilatation by constant infusion demonstrated augmented response in diabetic hearts. The administration of bradykinin or DEA/NO at the corrected rate was associated with significantly increased maximal responses in comparison with constant infusion experiments. This phenomenon was observed particularly in the control group. We conclude that only corrected-rate infusion of vasoactive agents to actual value of coronary flow enables the reliable detection of endothelial dysfunction in diabetes mellitus.
Assuntos
Vasos Coronários/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Endotélio Vascular/fisiopatologia , Animais , Glicemia/análise , Bradicinina/administração & dosagem , Bradicinina/farmacologia , Peptídeo C/análise , Colesterol/sangue , Circulação Coronária/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Endotélio Vascular/efeitos dos fármacos , Jejum/sangue , Coração/efeitos dos fármacos , Coração/fisiopatologia , Hidrazinas/administração & dosagem , Hidrazinas/farmacologia , Técnicas In Vitro , Infusões Intravenosas , Insulina/sangue , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Doadores de Óxido Nítrico/administração & dosagem , Doadores de Óxido Nítrico/farmacologia , Perfusão , Ratos , Ratos Wistar , Triglicerídeos/sangue , Vasodilatação/efeitos dos fármacos , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Quercetin, a polyphenolic compound present in various types of food, has been shown to exert beneficial effects in different cardiac as well as non-cardiac ischemia/reperfusion (I/R) models in adult animals. However, there is no evidence about the effects of quercetin on I/R injury in non-mature animals, despite the fact that efficiency of some interventions against I/R is age-dependent. This study was aimed to investigate the effects of chronic quercetin treatment on I/R injury in juvenile and adult rat hearts. Juvenile (4-week-old) as well as adult (12-week-old) rats were treated with quercetin (20 mg/kg/day) for 4 weeks, hearts were excised and exposed to 25-min global ischemia followed by 40-min reperfusion. Functional parameters of hearts and occurrence of reperfusion arrhythmias were registered to assess the cardiac function. Our results have shown that quercetin improved post-ischemic recovery of LVDP, as well as recovery of markers of contraction and relaxation, +(dP/dt)max and -(dP/dt)max, respectively, in juvenile hearts, but not in adult hearts. Quercetin had no impact on incidence as well as duration of reperfusion arrhythmias in animals of both ages. We conclude that the age of rats plays an important role in heart response to quercetin treatment in the particular dose and duration of the treatment. Therefore, the age of the treated subjects should be taken into consideration when choosing the dose of quercetin and duration of its application in prevention and/or treatment of cardiovascular diseases.
Assuntos
Antioxidantes/uso terapêutico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Quercetina/uso terapêutico , Fatores Etários , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/prevenção & controle , Técnicas In Vitro , Masculino , Traumatismo por Reperfusão Miocárdica/complicações , Ratos WistarRESUMO
Several studies have shown that diabetes mellitus modulates heart resistance to ischemia and abrogates effectivity of cardioprotective interventions, such as ischemic preconditioning (IP). The aim of this study was to evaluate whether the effect of hyperglycemic conditions on the severity of ischemia-reperfusion (I/R) injury in preconditioned and non-preconditioned hearts (controls, C) is related to changes in osmotic activity of glucose. Experiments were performed in isolated rat hearts perfused according to Langendorff exposed to 30-min coronary occlusion/120-min reperfusion. IP was induced by two cycles of 5-min coronary occlusion/5-min reperfusion, prior to the long-term I/R. Hyperosmotic (HO) state induced by an addition of mannitol (11 mmol/l) to a standard Krebs-Henseleit perfusion medium significantly decreased the size of infarction and also suppressed a release of heart fatty acid binding protein (h-FABP - biomarker of cell injury) from the non-IP hearts nearly to 50 %, in comparison with normoosmotic (NO) mannitol-free perfusion. However, IP in HO conditions significantly increased the size of infarction and tended to elevate the release of h-FABP to the effluent from the heart. The results indicate that HO environment plays a cardioprotective role in the ischemic myocardium. On the other hand, increased osmolarity, similar to that in the hyperglycemic conditions, may play a pivotal role in a failure of IP to induce cardioprotection in the diabetic myocardium.
Assuntos
Coração/fisiopatologia , Precondicionamento Isquêmico Miocárdico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Concentração Osmolar , Animais , Microambiente Celular , Circulação Coronária , Solução Hipertônica de Glucose/farmacologia , Hiperglicemia/fisiopatologia , Técnicas In Vitro , Masculino , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ratos , Ratos WistarRESUMO
Excessive production of oxygen free radicals has been regarded as a causative common denominator of many pathological processes in the animal kingdom. Hydroxyl and nitrosyl radicals represent the major cause of the destruction of biomolecules either by a direct reaction or by triggering a chain reaction of free radicals. Scavenging of free radicals may act preventively or therapeutically. A number of substances that preferentially react with free radicals can serve as scavengers, thus increasing the internal capacity/activity of endogenous antioxidants and protecting cells and tissues against oxidative damage. Molecular hydrogen (H(2)) reacts with strong oxidants, such as hydroxyl and nitrosyl radicals, in the cells, that enables utilization of its potential for preventive and therapeutic applications. H(2) rapidly diffuses into tissues and cells without affecting metabolic redox reactions and signaling reactive species. H(2) reduces oxidative stress also by regulating gene expression, and functions as an anti-inflammatory and anti-apoptotic agent. There is a growing body of evidence based on the results of animal experiments and clinical observations that H(2) may represent an effective antioxidant for the prevention of oxidative stress-related diseases. Application of molecular hydrogen in situations with excessive production of free radicals, in particular, hydroxyl and nitrosyl radicals is relatively simple and effective, therefore, it deserves special attention.