RESUMO
We have previously demonstrated that inhibition of extracellularly oriented carbonic anhydrase (CA) isoforms protects the myocardium against ischemia-reperfusion injury. In this study, our aim was to assess the possible further contribution of CA intracellular isoforms examining the actions of the highly diffusible cell membrane permeant inhibitor of CA, ethoxzolamide (ETZ). Isolated rat hearts, after 20 min of stabilization, were assigned to the following groups: (1) Nonischemic control: 90 min of perfusion; (2) Ischemic control: 30 min of global ischemia and 60 min of reperfusion (R); and (3) ETZ: ETZ at a concentration of 100 µM was administered for 10 min before the onset of ischemia and then during the first 10 min of reperfusion. In additional groups, ETZ was administered in the presence of SB202190 (SB, a p38MAPK inhibitor) or chelerythrine (Chel, a protein kinase C [PKC] inhibitor). Infarct size, myocardial function, and the expression of phosphorylated forms of p38MAPK, PKCε, HSP27, and Drp1, and calcineurin Aß content were assessed. In isolated mitochondria, the Ca2+ response, Ca2+ retention capacity, and membrane potential were measured. ETZ decreased infarct size by 60%, improved postischemic recovery of myocardial contractile and diastolic relaxation increased P-p38MAPK, P-PKCε, P-HSP27, and P-Drp1 expression, decreased calcineurin content, and normalized calcium and membrane potential parameters measured in isolated mitochondria. These effects were significantly attenuated when ETZ was administered in the presence of SB or Chel. These data show that ETZ protects the myocardium and mitochondria against ischemia-reperfusion injury through p38MAPK- and PKCε-dependent pathways and reinforces the role of CA as a possible target in the management of acute cardiac ischemic diseases.
Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Etoxzolamida/farmacologia , Coração/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Miocárdio/metabolismo , Animais , Benzofenantridinas/farmacologia , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Preparação de Coração Isolado , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Traumatismo por Reperfusão Miocárdica , Proteína Quinase C/antagonistas & inibidores , Piridinas/farmacologia , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Heart failure is the leading cause of death among diabetic people. Cellular and molecular entities leading to diabetic cardiomyopathy are, however, poorly understood. Coupling of cardiac carbonic anhydrase II (CAII) and Na+/H+ exchanger 1 (NHE1) to form a transport metabolon was analyzed in obese type 2 diabetic mice (ob-/-) and control heterozygous littermates (ob+/-). Echocardiography showed elevated systolic interventricular septum thickness and systolic posterior wall thickness in ob-/- mice at 9 and 16â¯weeks. ob-/- mice showed increased left ventricular (LV) weight/tibia length ratio and increased cardiomyocyte cross sectional area as compared to controls, indicating cardiac hypertrophy. Immunoblot analysis showed increased CAII expression in LV samples of ob-/-vs. ob+/- mice, and augmented Ser703 phosphorylation on NHE1 in ob-/- hearts. Reciprocal co-immunoprecipitation analysis showed strong association of CAII and NHE1 in LV samples of ob-/- mice. NHE1-dependent rate of intracellular pH (pHi) normalization after transient acid loading of isolated cardiomyocytes was higher in ob-/- mice vs. ob+/-. NHE transport activity was also augmented in cultured H9C2 rat cardiomyoblasts treated with high glucose/high palmitate, and it was normalized after CA inhibition. We conclude that the NHE1/CAII metabolon complex is exacerbated in diabetic cardiomyopathy of ob-/- mice, which may lead to perturbation of pHi and [Na+] and [Ca2+] handling in these diseased hearts.
Assuntos
Anidrase Carbônica II/metabolismo , Cardiomegalia/patologia , Diabetes Mellitus Tipo 2/complicações , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Anidrase Carbônica II/antagonistas & inibidores , Inibidores da Anidrase Carbônica/farmacologia , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Eletrocardiografia , Etoxzolamida/farmacologia , Feminino , Ventrículos do Coração/patologia , Concentração de Íons de Hidrogênio , Camundongos Mutantes , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Serina/metabolismoRESUMO
BACKGROUND: Recent studies from our laboratory show the cardioprotective action of benzolamide (BZ, carbonic anhydrase inhibitor) against ischemia-reperfusion injury. However, the mechanisms involved have not been fully elucidated. OBJECTIVE: To examine the participation of the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) in the effects of BZ in a model of regional ischemia. METHODS: Isolated rat hearts perfused by Langendorff technique were submitted to 40â¯min of coronary artery occlusion followed by 60â¯min of reperfusion (IC). Other hearts received BZ during the first 10â¯min of reperfusion in absence or presence of L-NAME, NOS inhibitor. The infarct size (IS) and the post-ischemic recovery of myocardial function were measured. Oxidative/nitrosative damage were assessed by reduced glutathione (GSH) content, thiobarbituric acid reactive substances (TBARS) and 3-nitrotyrosine levels. The expression of phosphorylated forms of Akt, p38MAPK and eNOS, and the concentration of inducible nitric oxide synthase (iNOS) were also determined. RESULTS: BZ significantly decreased IS (6.2⯱â¯0.5% vs. 34⯱â¯4%), improved post-ischemic contractility, preserved GSH levels and diminished TBARS and 3-nitrotyrosine. In IC hearts, P-Akt, P-p38MAPK and P-eNOS decreased and iNOS increased. After BZ addition the levels of P-kinases and P-eNOS increased and iNOS decreased. Except for P-Akt, P-p38MAPK and iNOS, the effects of BZ were abolished by L-NAME. CONCLUSIONS: Our data demonstrate that the treatment with BZ at the onset of reperfusion was effective to reduce cell death, contractile dysfunction and oxidative/nitrosative damage produced by coronary artery occlusion. These BZ-mediated beneficial actions appear mediated by eNOS/NO-dependent pathways.
Assuntos
Benzolamida/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Animais , Preparação de Coração Isolado , Masculino , Ratos , Ratos WistarRESUMO
The authors have retracted this article [1] because of modifications in the control lanes of Figs. 2a and 8a of the COX1 blot obtained for 18-week-old rats (rotation, horizontal flipping and re-use of the control lanes for the 35-week-old rats blot). In light of the concerns raised, the conclusions drawn in this article cannot be relied upon.
RESUMO
During ischemia, increased anaerobic glycolysis results in intracellular acidosis. Activation of alkalinizing transport mechanisms associated with carbonic anhydrases (CAs) leads to myocardial intracellular Ca2+ increase. We characterize the effects of inhibition of CA with benzolamide (BZ) during cardiac ischemia-reperfusion (I/R). Langendorff-perfused isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion. Other hearts were treated with BZ (5 µM) during the initial 10 min of reperfusion or perfused with acid solution (AR, pH 6.4) during the first 3 min of reperfusion. p38MAPK, a kinase linked to membrane transporters and involved in cardioprotection, was examined in hearts treated with BZ in presence of the p38MAPK inhibitor SB202190 (10 µM). Infarct size (IZ) and myocardial function were assessed, and phosphorylated forms of p38MAPK, Akt, and PKCε were evaluated by immunoblotting. We determined the rate of intracellular pH (pHi) normalization after transient acid loading in the absence and presence of BZ or BZ + SB202190 in heart papillary muscles (HPMs). Mitochondrial membrane potential (ΔΨm), Ca2+ retention capacity and Ca2+-mediated swelling after I/R were also measured. BZ, similarly to AR, reduced IZ, improved postischemic recovery of myocardial contractility, increased phosphorylation of Akt, PKCε, and p38MAPK, and normalized ΔΨm and Ca2+ homeostasis, effects abolished after p38MAPK inhibition. In HPMs, BZ slowed pHi recovery, an effect that was restored after p38MAPK inhibition. We conclude that prolongation of acidic conditions during reperfusion by BZ could be responsible for the cardioprotective benefits of reduced infarction and better myocontractile function, through p38MAPK-dependent pathways. NEW & NOTEWORTHY Carbonic anhydrase inhibition by benzolamide (BZ) maintains acidity, decreases infarct size, and improves postischemic myocardial dysfunction in ischemia-reperfusion (I/R) hearts. Protection afforded by BZ mimicked the beneficial effects elicited by an acidic solution (AR). Increased phosphorylation of p38MAPK occurs in I/R hearts reperfused with BZ or with AR. Mitochondria from I/R hearts possess abnormal Ca2+ handling and a more depolarized membrane potential compared with control hearts, and these changes were restored by treatment with BZ or AR.
Assuntos
Benzolamida/farmacologia , Infarto do Miocárdio/tratamento farmacológico , Isquemia Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Infarto do Miocárdio/metabolismo , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
NBC Na+/HCO3- cotransporter (NBCn1) and NHE1 Na+/H+ exchanger have been associated with cardiac disorders and recently located in coronary endothelial cells (CEC) and cardiomyocytes mitochondria, respectively. Mitochondrial NHE1 blockade delays permeability transition pore (MPTP) opening and reduces superoxide levels, two critical events exacerbated in cells of diseased hearts. Conversely, activation of NBCn1 prevented apoptosis in CEC subjected to ischemic stress. We characterized the role of the NHE1 and NBCn1 transporters in heart mitochondria from hypertrophic (SHR) and control (Wistar) rats. Expression of NHE1 was analyzed in left ventricular mitochondrial lysates (LVML), by immunoblots. NHE1 expression increased by ~40% in SHR compared to control (P < 0.05, n = 4). To examine NHE1-mediated Na+/H+ exchange activity in cardiac hypertrophy, mitochondria were loaded with BCECF-AM dye and the maximal rate of pHm change measured after the addition of 50 mM NaCl. SHR mitochondria had greater changes in pHm compared to Wistar, 0.10 ± 0.01 vs. 0.06 ± 0.01, respectively (P < 0.05, n = 5). In addition, mitochondrial suspensions from SHR and control myocardium were exposed to 200 µM CaCl2 to induce MPTP opening (light-scattering decrease, LSD) and swelling. Surprisingly, SHR rats showed smaller LSD and a reduction in mitochondrial swelling, 67 ± 10% (n = 15), compared to control, 100 ± 8% (n = 13). NBC inhibition with S0859 (1 µM) significantly increased swelling in both control 139 ± 10% (n = 8) and SHR 115 ± 10% (n = 4). Finally, NBCn1 Na+/HCO3- cotransporter increased by twofold its expression in SHR LVML, compared to normal (P < 0.05, n = 5). We conclude that increased NBCn1 activity may play a compensatory role in hypertrophic hearts, protecting mitochondria from Ca2+-induced MPTP opening and swelling.
Assuntos
Cardiomegalia/metabolismo , Mitocôndrias/patologia , Dilatação Mitocondrial , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Cardiomegalia/patologia , Modelos Animais de Doenças , Células HEK293 , Humanos , Immunoblotting , Imuno-Histoquímica , Microscopia Confocal , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Miócitos Cardíacos , Ratos , Ratos Endogâmicos SHR , Ratos WistarRESUMO
BACKGROUND: Two potent carbonic anhydrase (CA) inhibitors with widely differing membrane permeability, poorly diffusible benzolamide (BZ), and highly diffusible ethoxzolamide (ETZ) were assessed to determine whether they can reduce cardiac dysfunction in rats subjected to coronary artery ligation (CAL)-induced myocardial infarction. METHODS AND RESULTS: Rats with evidence of heart failure (HF) at 32 weeks following a permanent left anterior coronary artery occlusion were treated with placebo, BZ, or ETZ (4 mg kgday-1) for 4 weeks at which time left ventricular function and structure were evaluated. Lung weight/body weight (LW/BW) ratio increased in CAL rats by 17±1% vs. control, suggesting pulmonary edema. There was a trend for BZ and ETZ to ameliorate the increase in LW/BW by almost 50% (9±5% and 9±8%, respectively, versus CAL) (P=.16, NS). Echocardiographic assessment showed decreased left ventricular midwall shortening in HF rats, 21±1% vs. control 32±1%, which was improved by BZ to 29±1% and ETZ to 27±1%, and reduced endocardial shortening in HF rats 38±3% vs. control 62±1%, partially restored by BZ and ETZ to ~50%. Expression of the hypoxia-inducible membrane-associated CAIX isoform increased by ~60% in HF rat hearts, and this effect was blocked by ETZ. CONCLUSIONS: We conclude that CAL-induced myocardial interstitial fibrosis and associated decline in left ventricular function were diminished with BZ or ETZ treatment. The reductions in cardiac remodeling in HF with both ETZ and BZ CA inhibitors suggest that inhibition of a membrane-bound CA appears to be the critical site for this protection.
Assuntos
Benzolamida/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Etoxzolamida/farmacologia , Coração/efeitos dos fármacos , Infarto do Miocárdio/patologia , Animais , Vasos Coronários/cirurgia , Modelos Animais de Doenças , Immunoblotting , Ligadura , Masculino , Ratos , Ratos Wistar , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
The NBCn1 Na(+)/HCO3(-) cotransporter catalyzes the electroneutral movement of 1 Na(+):1 HCO3(-) into kidney cells. We characterized the intracellular pH (pHi) regulation in human embryonic kidney cells (HEK) subjected to NH4Cl prepulse acid loading, and we examined the NBCn1 expression and function in HEK cells subjected to 24-h elevated Pco2 (10-15%). After acid loading, in the presence of HCO3(-), â¼50% of the pHi recovery phase was blocked by the Na(+)/H(+) exchanger inhibitors EIPA (10-50 µM) and amiloride (1 mM) and was fully cancelled by 30 µM EIPA under nominally HCO3(-)-free conditions. In addition, in the presence of HCO3(-), pHi recovery after acid loading was completely blocked when Na(+) was omitted in the buffer. pHi recovery after acidification in HEK cells was repeated in the presence of the NBC inhibitor S0859, and the pHi recovery was inhibited by S0859 in a dose-dependent manner (Ki = 30 µM, full inhibition at 60 µM), which confirmed NBC Na(+)/HCO3(-) cotransporter activation. NBCn1 expression increased threefold after 24-h exposure of cultured HEK cells to 10% CO2 and sevenfold after exposure to 15% CO2, examined by immunoblots. Finally, exposure of HEK cells to high CO2 significantly increased the HCO3(-)-dependent recovery of pHi after acid loading. We conclude that HEK cells expressed the NBCn1 Na(+)/HCO3(-) cotransporter as the only HCO3(-)-dependent mechanism responsible for cellular alkaline loading. NBCn1, which expresses in different kidney cell types, was upregulated by 24-h high-Pco2 exposure of HEK cells, and this upregulation was accompanied by increased NBCn1-mediated HCO3(-) transport.
Assuntos
Dióxido de Carbono/farmacologia , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Regulação para Cima/efeitos dos fármacos , Amilorida/análogos & derivados , Amilorida/farmacologia , Bicarbonatos/metabolismo , Relação Dose-Resposta a Droga , Células HEK293/citologia , Humanos , Concentração de Íons de Hidrogênio , Fatores de TempoRESUMO
Mitochondrial damage has been associated with early steps of cardiac dysfunction in heart subjected to ischemic stress, oxidative stress and hypertrophy. A common feature for the mitochondrial deterioration is the loss of the mitochondrial membrane potential (ΔΨ m) with the concomitant irreversible opening of the mitochondrial permeability transition pore (MPTP) which follows the mitochondrial Ca(2+) overload, and the subsequent mitochondrial swelling. We have recently characterized the expression of the Na(+)/H(+) exchanger 1 (mNHE1) in mitochondrial membranes. This surprising observation provided a unique target for the prevention of the Ca(2+)-induced MPTP opening, based on the inhibition of the NHE1 m. In this line, inhibition of NHE1 m activity and/or reduction of NHE1 m expression decreased the Ca(2+)-induced mitochondrial swelling and the release of reactive oxygen species (ROS) in isolated cardiac mitochondria and preserved the ΔΨ m in isolated cardiomyocytes. Mitochondrial NHE1 thus represents a novel target to prevent cardiac disease, opening new avenues for future research.
RESUMO
Myocardial stretch is an established signal that leads to hypertrophy. Myocardial stretch induces a first immediate force increase followed by a slow force response (SFR), which is a consequence of an increased Ca(2+) transient that follows the NHE1 Na(+)/H(+) exchanger activation. Carbonic anhydrase II (CAII) binds to the extreme COOH terminus of NHE1 and regulates its transport activity. We aimed to test the role of CAII bound to NHE1 in the SFR. The SFR and changes in intracellular pH (pHi) were evaluated in rat papillary muscle bathed with CO2/HCO3(-) buffer and stretched from 92% to 98% of the muscle maximal force development length for 10 min in the presence of the CA inhibitor 6-ethoxzolamide (ETZ, 100 µM). SFR control was 120 ± 3% (n = 8) of the rapid initial phase and was fully blocked by ETZ (99 ± 4%, n = 6). The SFR corresponded to a maximal increase in pHi of 0.18 ± 0.02 pH units (n = 4), and pHi changes were blocked by ETZ (0.04 ± 0.04, n = 6), as monitored by epifluorescence. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation, using muscle lysates. CAII immunoprecipitated with an anti-NHE1 antibody and the CAII immunoprecipitated protein levels increased 58 ± 9% (n = 6) upon stretch of muscles, assessed by immunoblots. The p90(RSK) kinase inhibitor SL0101-1 (10 µM) blocked the SFR of heart muscles after stretch 102 ± 2% (n = 4) and reduced the binding of CAII to NHE1, suggesting that the stretch-induced phosphorylation of NHE1 increases its binding to CAII. CAII/NHE1 interaction constitutes a component of the SFR to heart muscle stretch, which potentiates NHE1-mediated H(+) transport in the myocardium.
Assuntos
Anidrase Carbônica II/antagonistas & inibidores , Inibidores da Anidrase Carbônica/farmacologia , Etoxzolamida/farmacologia , Fusos Musculares/metabolismo , Músculos Papilares/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Anidrase Carbônica II/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Imunoprecipitação , Medições Luminescentes , Masculino , Músculos Papilares/enzimologia , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Trocador 1 de Sódio-Hidrogênio , Fatores de TempoRESUMO
Na(+)/HCO(3)(-) cotransporter (NBC)e1 catalyze the electrogenic movement of 1 Na(+):2 HCO(3)(-) into cardiomyocytes cytosol. NBC proteins associate with carbonic anhydrases (CA), CAII, and CAIV, forming a HCO(3)(-) transport metabolon. Herein, we examined the physical/functional interaction of NBCe1 and transmembrane CAIX in cardiac muscle. NBCe1 and CAIX physical association was examined by coimmunoprecipitation, using rat ventricular lysates. NBCe1 coimmunoprecipitated with anti-CAIX antibody, indicating NBCe1 and CAIX interaction in the myocardium. Glutathione-S-transferase (GST) pull-down assays with predicted extracellular loops (EC) of NBCe1 revealed that NBCe1-EC4 mediated interaction with CAIX. Functional NBCe1/CAIX interaction was examined using fluorescence measurements of BCECF in rat cardiomyocytes to monitor cytosolic pH. NBCe1 transport activity was evaluated after membrane depolarization with high extracellular K(+) in the presence or absence of the CA inhibitors, benzolamide (BZ; 100 µM) or 6-ethoxyzolamide (ETZ; 100 µM) (*P < 0.05). This depolarization protocol produced an intracellular pH (pH(i)) increase of 0.17 ± 0.01 (n = 11), which was inhibited by BZ (0.11 ± 0.02; n = 7) or ETZ (0.06 ± 0.01; n = 6). NBCe1 activity was also measured by changes of pH(i) in NBCe1-transfected human embryonic kidney 293 cells subjected to acid loads. Cotransfection of CAIX with NBCe1 increased the rate of pH(i) recovery (in mM/min) by about fourfold (12.1 ± 0.8; n = 9) compared with cells expressing NBCe1 alone (3.1 ± 0.5; n = 7), which was inhibited by BZ (7.5 ± 0.3; n = 9). We demonstrated that CAIX forms a complex with EC4 of NBCe1, which activates NBCe1-mediated HCO(3)(-) influx in the myocardium. CAIX and NBCe1 have been linked to tumorigenesis and cardiac cell growth, respectively. Thus inhibition of CA activity might be useful to prevent activation of NBCe1 under these pathological conditions.
Assuntos
Anidrases Carbônicas/metabolismo , Miocárdio/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Bicarbonatos/metabolismo , Transporte Biológico Ativo , Anidrase Carbônica IX , Linhagem Celular , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Ratos , Simportadores de Sódio-Bicarbonato/químicaRESUMO
Two AE3 transcripts, full-length (AE3fl) and cardiac (AE3c) are expressed in the heart. AE3 catalyzes electroneutral Cl(-)/HCO(3)(-) exchange across cardiomyocyte sarcolemma. AE proteins associate with carbonic anhydrases (CA), including CAII and CAIV, forming a HCO(3)(-) transport metabolon (BTM), increasing HCO(3)(-) fluxes and regulating cardiomyocytes pH. CAXIV, which is also expressed in the heart's sarcolemma, is a transmembrane enzyme with an extracellular catalytic domain. Herein, AE3/CAXIV physical association was examined by coimmunoprecipitation using rodent heart lysates. CAXIV immunoprecipitated with anti-AE3 antibody and both AE3fl and AE3c were reciprocally immunoprecipitated using anti-CAXIV antibody, indicating AE3fl-AE3c/CAXIV interaction in the myocardium. Coimmunoprecipitation experiments on heart lysates from a mouse with targeted disruption of the ae3 gene, failed to pull down AE3 with the CAXIV antibody. Confocal images demonstrated colocalization of CAXIV and AE3 in mouse ventricular myocytes. Functional association of AE3fl and CAXIV was examined in isolated hypertrophic rat cardiomyocytes, using fluorescence measurements of BCECF to monitor cytosolic pH. Hypertrophic cardiomyocytes of spontaneously hypertensive rats (SHR) presented elevated myocardial AE-mediated Cl(-)/HCO(3)(-) exchange activity (J(HCO3-) mM.min(-1)) compared to normal (Wistar) rats (7.5±1.3, n=4 versus 2.9±0.1, n=6, respectively). AE3fl, AE3c, CAII, CAIV, and CAIX protein expressions were similar in SHR and Wistar rat hearts. However, immunoblots revealed a twofold increase of CAXIV protein expression in the SHR myocardium compared to normal hearts (n=11). Furthermore, the CA-inhibitor, benzolamide, neutralized the stimulatory effect of extracellular CA on AE3 transport activity (3.7±1.5, n=3), normalizing AE3-dependent HCO(3)(-) fluxes in SHR. CAXIV/AE3 interaction constitutes an extracellular component of a BTM which potentiates AE3-mediated HCO(3)(-) transport in the heart. Increased CAXIV expression and consequent AE3/CAXIV complex formation would render AE3 hyperactive in the SHR heart.
Assuntos
Anidrases Carbônicas/metabolismo , Cardiomegalia/enzimologia , Miocárdio/enzimologia , Animais , Antiporters/genética , Antiporters/metabolismo , Anidrases Carbônicas/genética , Cardiomegalia/genética , Linhagem Celular , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Ligação Proteica , Ratos , Ratos Endogâmicos SHR , Ratos WistarRESUMO
Myocardial stretch induces a biphasic force response: a first abrupt increase followed by a slow force response (SFR), believed to be the in vitro manifestation of the Anrep effect. The SFR is due to an increase in Ca²âº transient of unclear mechanism. We proposed that Naâº/H⺠exchanger (NHE-1) activation is a key factor in determining the contractile response, but recent reports challenged our findings. We aimed to specifically test the role of the NHE-1 in the SFR. To this purpose small hairpin interference RNA capable of mediating specific NHE-1 knockdown was incorporated into a lentiviral vector (l-shNHE1) and injected into the left ventricular wall of Wistar rats. Injection of a lentiviral vector expressing a nonsilencing sequence (scramble) served as control. Myocardial NHE-1 protein expression and function (the latter evaluated by the recovery of pH(i) after an acidic load and the SFR) were evaluated. Animals transduced with l-shNHE1 showed reduced NHE-1 expression (45 ± 8% of controls; P < 0.05), and the presence of the lentivirus in the left ventricular myocardium, far from the site of injection, was evidenced by confocal microscopy. These findings correlated with depressed basal pH(i) recovery after acidosis [(max)dpH(i)/dt 0.055 ± 0.008 (scramble) vs. 0.009 ± 0.004 (l-shNHE1) pH units/min, P < 0.05], leftward shift of the relationship between J(Hâº) (H⺠efflux corrected by the intrinsic buffer capacity), and abolishment of SFR (124 ± 2 vs. 101 ± 2% of rapid phase; P < 0.05) despite preserved ERK1/2 phosphorylation [247 ± 12 (stretch) and 263 ± 23 (stretch l-shNHE1) % of control; P < 0.05 vs. nonstretched control], well-known NHE-1 activators. Our results provide strong evidence to propose NHE-1 activation as key factor in determining the SFR to stretch.
Assuntos
Mecanorreceptores/metabolismo , Força Muscular , Contração Miocárdica , Músculos Papilares/metabolismo , Interferência de RNA , Trocadores de Sódio-Hidrogênio/metabolismo , Acidose/metabolismo , Acidose/fisiopatologia , Animais , Regulação para Baixo , Concentração de Íons de Hidrogênio , Injeções Intramusculares , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculos Papilares/fisiopatologia , Fosforilação , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Wistar , Transdução de Sinais , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: Na(+) /HCO(3) (-) co-transport (NBC) regulates intracellular pH (pH(i) ) in the heart. We have studied the electrogenic NBC isoform NBCe1 by examining the effect of functional antibodies to this protein. EXPERIMENTAL APPROACH: We generated two antibodies against putative extracellular loop domains 3 (a-L3) and 4 (a-L4) of NBCe1 which recognized NBCe1 on immunoblots and immunostaining experiments. pH(i) was monitored using epi-fluorescence measurements in cat ventricular myocytes. Transport activity of total NBC and of NBCe1 in isolation were evaluated after an ammonium ion-induced acidosis (expressed as H(+) flux, J(H) , in mmol·L(-1) min(-1) at pH(i) 6.8) and during membrane depolarization with high extracellular potassium (potassium pulse, expressed as ΔpH(i) ) respectively. KEY RESULTS: The potassium pulse produced a pH(i) increase of 0.18 ± 0.006 (n= 5), which was reduced by the a-L3 antibody (0.016 ± 0.019). The a-L-3 also decreased J(H) by 50%. Surprisingly, during the potassium pulse, a-L4 induced a higher pH(i) increase than control,(0.25 ± 0.018) whereas the recovery of pH(i) from acidosis was faster (J(H) was almost double the control value). In perforated-patch experiments, a-L3 prolonged and a-L4 shortened action potential duration, consistent with blockade and stimulation of NBCe1-carried anionic current respectively. CONCLUSIONS AND IMPLICATIONS: Both antibodies recognized NBCe1, but they had opposing effects on the function of this transporter, as the a-L3 was inhibitory and the a-L4 was excitatory. These antibodies could be valuable in studies on the pathophysiology of NBCe1 in cardiac tissue, opening a path for their potential clinical use.
Assuntos
Anticorpos/imunologia , Simportadores de Sódio-Bicarbonato/imunologia , Animais , Gatos , Fluorescência , Masculino , Microscopia Confocal , Miocárdio/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , CoelhosRESUMO
Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by â¼60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 µM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.
Assuntos
Mitocôndrias Cardíacas/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Antiarrítmicos/farmacologia , Cálcio/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/ultraestrutura , Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Guanidinas/farmacologia , Masculino , Mitocôndrias Cardíacas/ultraestrutura , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Wistar , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/genética , Sulfonas/farmacologiaRESUMO
Myocardial pH(i) recovery from intracellular alkalization results in part from the acid load (-J(H+)) carried by Cl(-)/HCO(3)(-) anion-exchangers (AE). Three AE isoforms, AE1, AE2 and AE3, have been identified in cardiac membranes, but the function of each isoform on pH(i) homeostasis is still under investigation. This work explored, by means of specific antibodies, the role of AE3 isoform in myocardial pH(i) regulation. We developed rabbit polyclonal antibodies against the extracellular "loops": one connecting the fifth to sixth and the other one the seventh to eighth transmembrane domains (loops 3 and 4, respectively) of AE3, and their effect on pH(i) regulation was studied in rat papillary muscles. The anti-AE3 loop 3 antibody decreased -J(H+) in response to myocardial alkalization (from a mean control value of 1.06+/-0.26 to 0.32+/-0.13 mmol/L/min, n=7, P<0.05) without affecting the baseline pH(i) (7.22+/-0.03 vs. 7.21+/-0.04). The anti-AE3 loop 4 antibody did not modify either pH(i) recovery or baseline pH(i). Under control conditions, endothelin-1 (ET-1) increased -J(H+) in response to myocardial alkalization from 1.30+/-0.18 to 2.01+/-0.33 mmol/L /min (n=5, P<0.05). This effect of ET-1 on -J(H+) was abolished by anti-AE3 loop 3 antibody. In addition, the MgATP-induced stimulation of AE activity was reduced by the anti-AE3 loop 3 antibody. These data support the key role of the AE3 isoform in myocardial pH(i) recovery from alkaline loads and also in the stimulatory effect of ET-1 on AE activity. To a lesser extent, it may also contribute to the effect of MgATP on pH(i).
Assuntos
Alcalose/metabolismo , Antiporters/antagonistas & inibidores , Antiporters/metabolismo , Miocárdio/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Anticorpos Bloqueadores/farmacologia , Especificidade de Anticorpos , Antiporters/agonistas , Soluções Tampão , Reações Cruzadas , Endotelina-1/farmacologia , Glutationa/metabolismo , Concentração de Íons de Hidrogênio , Masculino , Membranas/efeitos dos fármacos , Membranas/metabolismo , Coelhos , Ratos , Ratos Wistar , Estimulação QuímicaRESUMO
Previous work demonstrated that the slow force response (SFR) to stretch is due to the increase in calcium transients (Ca2+T) produced by an autocrine-paracrine mechanism of locally produced angiotensin II/endothelin activating Na+-H+ exchange. Although a rise in pHi is presumed to follow stretch, it was observed only in the absence of extracellular bicarbonate, suggesting pHi compensation through the Na+-independent Cl--HCO3- exchange (AE) mechanism. Because available AE inhibitors do not distinguish between different bicarbonate-dependent mechanisms or even between AE isoforms, we developed a functional inhibitory antibody against both the AE3c and AE3fl isoforms (anti-AE3Loop III) that was used to explore if pHi would rise in stretched cat papillary muscles superfused with bicarbonate after AE3 inhibition. In addition, the influence of this potential increase in pHi on the SFR was analyzed. In this study, we present evidence that cancellation of AE3 isoforms activity (either by superfusion with bicarbonate-free buffer or with anti-AE3Loop III) results in pHi increase after stretch and the magnitude of the SFR was larger than when AE was operative, despite of similar increases in [Na+]i and Ca2+T under both conditions. Inhibition of reverse mode Na+-Ca2+ exchange reduced the SFR to the half when the AE was inactive and totally suppressed it when AE3 was active. The difference in the SFR magnitude and response to inhibition of reverse mode Na+-Ca2+ exchange can be ascribed to a pHi-induced increase in myofilament Ca2+ responsiveness.
Assuntos
Antiporters/metabolismo , Bicarbonatos/metabolismo , Cloretos/metabolismo , Miocárdio/metabolismo , Músculos Papilares/fisiologia , Tioureia/análogos & derivados , Animais , Anticorpos/farmacologia , Antiporters/antagonistas & inibidores , Cálcio/metabolismo , Estimulação Cardíaca Artificial , Gatos , Líquido Extracelular/metabolismo , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Técnicas In Vitro , Líquido Intracelular/metabolismo , Contração Miocárdica/fisiologia , Músculos Papilares/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Sódio/metabolismo , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Mecânico , Tioureia/farmacologiaRESUMO
BACKGROUND: Sodium/hydrogen ion exchange is hyperactive in hypertension. Myocardial sodium/hydrogen ion exchange hyperactivity accompanies the regression of cardiac hypertrophy in spontaneously hypertensive rats (SHR) after long term control of blood pressure with enalapril. OBJECTIVES: To explore whether this effect is shared by other antihypertensive agents or is specific to angiotensin-converting enzyme inhibition. ANIMALS AND METHODS: SHR and normotensive Wistar Kyoto (WKY) rats were treated for five weeks with enalapril (20 mg/kg/day), nifedipine (10 mg/kg/day) or losartan (40 mg/kg/day). Sodium/hydrogen ion exchange activity was estimated in terms of both steady intracellular pH in HEPES buffer and the rate of intracellular pH recovery from intracellular acid loads in isolated superfused 2'-7'-bis(2-carboxyethyl)-5,-(and-6)-carboxyfluorescein, acetoxymethyl ester form-loaded papillary muscles. RESULTS: Enalapril, nifedipine and losartan decreased systolic blood pressure in SHR to about the same value (140 3, 140 2 and 146 3 mmHg, respectively, at the end the treatment). However, the index of cardiac hypertrophy (heart weight to body weight ratio) was decreased to a smaller value with losartan than with nifedipine or enalapril (2.66 0.09, 3.06 0.05 and 2.86 0.04 mg/g respectively; P<0.05 ANOVA). For the untreated SHR, the index of cardiac hypertrophy was 3.30 0.04 mg/g. Myocardial sodium/hydrogen ion exchange hyperactivity in SHR was normalized by all treatments. CONCLUSIONS: The three treatments regressed cardiac hypertrophy and normalized sodium/hydrogen ion exchange exchange activity in SHR, and losartan was the most effective treatment for reversing cardiac hypertrophy, despite producing effects on blood pressure and sodium/hydrogen exchange activity similar to that of other antihypertensive drugs.