RESUMO
Cardiac aging is characterized by alterations in contractility and intracellular calcium ([Ca2+]i) homeostasis. It has been suggested that oxidative stress may be involved in this process. We and others have reported that in cardiomyopathies the NADPH oxidase (NOX)-derived superoxide is increased, with a negative impact on [Ca2+]i and contractility. We tested the hypothesis that in the aged heart, [Ca2+]i handling and contractility are disturbed by NOX-derived superoxide. For this we used adults (≈5 month-old) and aged (20â»24 month-old) rats. Contractility was evaluated in isolated hearts, challenged with isoproterenol. To assess [Ca2+]i, isolated cardiac myocytes were field-stimulated and [Ca2+]i was monitored with fura-2. Cardiac concentration-response to isoproterenol was depressed in aged compared to adults hearts (p < 0.005), but was restored by NOX inhibitors apocynin and VAS2870. In isolated cardiomyocytes, apocynin increased the amplitude of [Ca2+]i in aged myocytes (p < 0.05). Time-50 [Ca2+]i decay was increased in aged myocytes (p < 0.05) and reduced towards normal by NOX inhibition. In addition, we found that myofilaments Ca2+ sensitivity was reduced in aged myocytes (p < 0.05), and was further reduced by apocynin. NOX2 expression along with NADPH oxidase activity was increased in aged hearts. Phospholamban phosphorylation (Ser16/Thr17) after isoproterenol treatment was reduced in aged hearts compared to adults and was restored by apocynin treatment (p < 0.05). In conclusion, ß-adrenergic-induced contractility was depressed in aged hearts, and NOX inhibition restored back to normal. Moreover, altered Ca2+ handling in aged myocytes was also improved by NOX inhibition. These results suggest a NOX-dependent effect in aged myocytes at the level of Ca2+ handling proteins and myofilaments.
Assuntos
Acetofenonas/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Benzoxazóis/farmacologia , Cálcio/metabolismo , Coração/efeitos dos fármacos , Isoproterenol/farmacologia , Contração Miocárdica/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , Triazóis/farmacologia , Envelhecimento/efeitos dos fármacos , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Feminino , Coração/fisiologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NADPH Oxidases/metabolismo , RatosRESUMO
The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit(+) cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit(+) cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex vivo expanded c-kit(+) cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together, these findings document a novel neonatal rat kidney c-kit(+) stem cell population that can be isolated, expanded, cloned, differentiated, and used for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Rim/citologia , Rim/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Feminino , Rim/embriologia , Rim/enzimologia , Córtex Renal/citologia , Córtex Renal/enzimologia , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
The enhanced activity of the cardiac Na+/H+ exchanger (NHE-1) after myocardial stretch is considered a key step of the intracellular signaling pathway leading to the slow force response to stretch as well as an early signal for the development of cardiac hypertrophy. We propose that the chain of events triggered by stretch begins with the release of small amounts of Angiotensin II (Ang II)/endothelin (ET) and ends with the increase in intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger in reverse mode (NCX(rev)), which triggers cardiac hypertrophy by activation of widely recognized Ca2+-dependent intracellular signaling pathways.
Assuntos
Cardiomegalia/fisiopatologia , Trocadores de Sódio-Hidrogênio/fisiologia , Adulto , Angiotensina II/fisiologia , Fenômenos Biomecânicos , Cardiomegalia/genética , Ventrículos do Coração/fisiopatologia , Humanos , Hipertrofia Ventricular Direita/fisiopatologia , Obesidade/genética , Pressorreceptores/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Função VentricularRESUMO
When the length of the myocardium is increased, a biphasic response to stretch occurs involving an initial rapid increase in force followed by a delayed slow increase called the slow force response (SFR). Confirming previous findings involving angiotensin II in the SFR, it was blunted by AT1 receptor blockade (losartan). The SFR was accompanied by an increase in reactive oxygen species (ROS) of approximately 30% and in intracellular Na(+) concentration ([Na(+)](i)) of approximately 2.5 mmol l(-1) over basal detected by H(2)DCFDA and SBFI fluorescence, respectively. Abolition of ROS by 2-mercapto-propionyl-glycine (MPG) and EUK8 suppressed the increase in [Na(+)](i) and the SFR, which were also blunted by Na(+)/H(+) exchanger (NHE-1) inhibition (HOE642). NADPH oxidase inhibition (apocynin or DPI) or blockade of the ATP-sensitive mitochondrial potassium channels (5HD or glybenclamide) suppressed both the SFR and the increase in [Na(+)](i) after stretch, suggesting that endogenous angiotensin II activated NADPH oxidase leading to ROS release by the ATP-sensitive mitochondrial potassium channels, which promoted NHE-1 activation. Supporting the notion of ROS-mediated NHE-1 activation, stretch increased the ERK1/2 and p90rsk kinases phosphorylation, effect that was cancelled by losartan. In agreement, the SFR was cancelled by inhibiting the ERK1/2 signalling pathway with PD98059. Angiotensin II at a dose that mimics the SFR (1 nmol l(-1)) induced an increase in .O(2)(-) production of approximately 30-40% detected by lucigenin in cardiac slices, an effect that was blunted by losartan, MPG, apocynin, 5HD and glybenclamide. Taken together the data suggest a pivotal role of mitochondrial ROS in the genesis of the SFR to stretch.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Músculos Papilares/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Gatos , Mecanotransdução Celular , Estresse MecânicoRESUMO
Na+/H+ exchanger-1 (NHE-1) inhibition induces cardiac hypertrophy regression and (or) prevention in several experimental models, although the intracellular events involved remain unclarified. We aimed to determine whether the calcineurin/NFAT pathway mediates this effect of NHE-1 inhibitors. Spontaneously hypertensive rats (SHR) with cardiac hypertrophy were treated with the NHE-1 inhibitors cariporide and BIIB723 for 30 days. Wistar rats served as normotensive controls. Their hearts were studied by echocardiography, immunoblotting, and real-time RT-PCR. Cytoplasmic Ca2+ and calcineurin Abeta expression were measured in cultured neonatal rat ventricular myocytes (NRVM) stimulated with endothelin-1 for 24 h. NHE-1 blockade induced cardiac hypertrophy regression (heart mass/body mass=3.63+/-0.07 vs. 3.06+/-0.05 and 3.02+/-0.13 for untreated vs. cariporide- and BIIB723-treated SHR, respectively; p<0.05) and decreased myocardial brain natriuretic peptide, calcineurin Abeta, and nuclear NFAT expressions. Echocardiographic evaluation demonstrated a reduction in left ventricular wall thickness without changes in cavity dimensions or a significant decrease in blood pressure. NHE-1-inhibitor treatment did not affect myocardial stiffness or endocardial shortening, but increased mid-wall shortening, suggesting that a positive inotropic effect develops after hypertrophy regression. Cariporide normalized the increased diastolic Ca2+ and calcineurin Abeta expression observed in ET-1-stimulated NRVM. In conclusion, our data suggest that inactivation of calcineurin/NFAT pathway may underlie the regression of cardiac hyper-trophy induced by NHE-1 inhibition.
Assuntos
Calcineurina/metabolismo , Cardiomegalia/metabolismo , Hipertensão/metabolismo , Fatores de Transcrição NFATC/metabolismo , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Antiarrítmicos/farmacologia , Cálcio/metabolismo , Células Cultivadas , Guanidinas/farmacologia , Masculino , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Peptídeo Natriurético Encefálico/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonas/farmacologiaRESUMO
Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na(+)/H(+) exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na(+) ([Na(+)](i)) mediated by NHE-1 activity may induce the reverse mode of Na(+)/Ca(2+) exchanger (NCX(rev)) increasing intracellular Ca(2+) ([Ca(2+)](i)) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX(rev) prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 mumol/L HOE 642 or 5 mumol/L KB-R7943. Cell surface area, (3)H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 +/- 3, 220 +/- 12 and 190 +/- 25% of control, respectively (P < 0.05) by ET-1. [Na(+)](i) and total [Ca(2+)](i) were higher (8.1 +/- 1.2 mmol/L and 636 +/- 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 +/- 1.3 and 346 +/- 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca(2+)](i) induced by extracellular Na(+) removal (NCX(rev)) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX(rev) inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and (3)H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX(rev) inhibition. Our results provide the first evidence that NCX(rev) is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs.
Assuntos
Animais Recém-Nascidos , Cardiomegalia/metabolismo , Endotelina-1/farmacologia , Miócitos Cardíacos/metabolismo , RNA Mensageiro/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Fator Natriurético Atrial/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Ratos , Ratos WistarRESUMO
BACKGROUND: Myocardial stretch promotes an increase in developed force (DF) in two phases: a rapid initial phase, and a slowly developing second phase called the slow force response (SFR) to myocardial stretch. The SFR results from an autocrine/paracrine mechanism of angiotensin II and endothelin (ET) release that is triggered by the stretch. OBJECTIVE: To explore whether exogenous ET-1 and/or ET-3 could mimic the SFR. METHODS: Experiments were performed in isometrically contracting (0.2 Hz) rat papillary muscles at 30 degrees C. DF was measured either after stretch or after the addition of ET-1 or ET-3 (in doses that increase contractility to a similar magnitude as does the SFR), with or without the selective ETA receptor antagonist BQ123 (300 nmol/L). RESULTS AND CONCLUSIONS: After 15 min, the SFR was 17.6+/-1.4% greater than the initial rapid phase (n=4; P<0.05) and was abolished by BQ123. ET-1 (5.0 nmol/L) increased DF by 25.9+/-1.7% (n=4; P<0.05) after 30 min, an effect that was not altered by BQ123 (22.6+/-3.9%; n=5). ET-3 (5.0 nmol/L) increased DF by 23.8+/-3.2% (n=5; P<0.05), an effect that was suppressed by BQ123 (-5.4+/-1.9%; n=5; P<0.05). Given that BQ123 eliminated the SFR and the inotropic response to ET-3 but not to ET-1, the results suggest that the SFR that follows myocardial stretch is due to the endogenous release of ET-3 acting in an autocrine/paracrine fashion.
Assuntos
Endotelina-1/farmacologia , Endotelina-3/farmacologia , Contração Miocárdica/efeitos dos fármacos , Músculos Papilares/efeitos dos fármacos , Vasoconstritores/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Antagonistas dos Receptores de Endotelina , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Masculino , Peptídeos Cíclicos/farmacologia , Ratos , Ratos WistarRESUMO
Myocardial stretch elicits a biphasic increase in developed force with a first rapid force response and a second slow force response (SFR). The rapid phase is due to an increase in myofilament Ca(2+) responsiveness; the SFR, analyzed here, is ascribed to a progressive increase in Ca(2+) transients. Experiments were performed in cat papillary muscles to further elucidate the signaling pathway underlying the SFR. Although the SFR was diminished by BQ-123, a similar endothelin (ET)-1-induced increase in force was not affected: 23 +/- 2 vs. 23 +/- 3% (not significant). Instead, BQ-123 suppressed the contractile effects of ET-2 or ET-3 (21 +/- 2 and 25 +/- 3% vs. -1 +/- 1 and -7 +/- 3% respectively, P < 0.05), suggesting that ET-2 or ET-3, but not ET-1, was involved in the SFR. Each isoform activated the Na(+)/H(+) exchanger (NHE-1), increasing intracellular Na(+) concentration by 2.0 +/- 0.1, 2.3 +/- 0.1, and 2.1 +/- 0.4 mmol/l for ET-1, ET-2, and ET-3, respectively (P < 0.05). The NHE-1 inhibitor HOE-642 prevented the increases in force and intracellular Na(+) concentration induced by all the ET isoforms, but only ET-2 and ET-3 effects were sensitive to BQ-123. Real-time RT-PCR measurements of prepro-ET-1, -ET-2, and -ET-3 were performed before and 5, 15, and 30 min after stretch. No changes in ET-1 or ET-2, but an increase of approximately 60% in ET-3, mRNA after 15 min of stretch were detected. Stretch-induced ET-3 mRNA upregulation and its mechanical counterpart were suppressed by AT(1) receptor blockade with losartan. These data suggest a role for AT(1)-mediated ET-3 released in the early activation of NHE-1 that follows myocardial stretch.
Assuntos
Endotelina-1/farmacologia , Endotelina-2/farmacologia , Endotelina-3/farmacologia , Endotelinas/farmacologia , Contração Miocárdica/fisiologia , Músculos Papilares/fisiologia , Animais , Gatos , Primers do DNA , Ventrículos do Coração/efeitos dos fármacos , Técnicas In Vitro , Contração Miocárdica/efeitos dos fármacos , Músculos Papilares/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Reação em Cadeia da Polimerase/métodos , Isoformas de Proteínas/farmacologia , RNA Mensageiro/genética , Estresse Mecânico , Função VentricularRESUMO
OBJECTIVE: This work was aimed to prove that release/formation of endogenous endothelin acting in an autocrine/paracrine fashion contributes to the increase in contractility promoted by a low dose of angiotensin II. METHODS: Isolated cat papillary muscles were used for force, pH(i), [Na(+)](i) and [Ca(2+)](i) measurements and isolated cat myocytes for patch-clamp experiments. RESULTS: In papillary muscles, 1.0 nmol/l angiotensin II increased force by 23+/-2% (n=4, P<0.05), [Na(+)](i) by 2.2+/-0.2 mmol/l (n=4, P<0.05), and peak (but not diastolic) Ca(2+) from 0.674+/-0.11 to 0.768+/-0.13 micromol/l (n=4, P<0.05), without affecting pH(i). Force and [Na(+)](i) increase were abolished by inhibition of the Na(+)/H(+) exchanger (NHE) with the inhibitor HOE642, blockade of endothelin receptors with the nonselective antagonist TAK044 and by inhibition of the endothelin-converting enzyme with phosphoramidon. Force but not [Na(+)](i) increase was abolished by inhibition of reverse Na(+)/Ca(2+) exchange (NCX) with the inhibitor KB-R7943. Similar increase in force (21+/-2%, n=4, P<0.05) and in [Na(+)](i) (2.4+/-0.4 mmol/l, n=4, P<0.05) that were also suppressed by TAK044 and HOE642 were induced by exogenous 5.0 nmol/l endothelin-1. KB-R7943 reverted the endothelin-1 effect on force but not on [Na(+)](i). In isolated myocytes, exogenous endothelin-1 dose-dependently increased the NCX current and shifted the NCX reversal potential (E(NCX)) to a more negative value (DeltaE(NCX): -10+/-3 and -17+/-5 mV, with 1 and 10 nmol/l endothelin-1, respectively, n=12). The latter effect was prevented by HOE642. CONCLUSION: Taken together, the results indicate that a low dose of angiotensin II induces release of endothelin, which, in autocrine/paracrine fashion activates the Na(+)/H(+) exchanger, increases [Na(+)](i) and changes E(NCX), promoting the influx of Ca(2+) that leads to a positive inotropic effect (PIE).
Assuntos
Angiotensina II/farmacologia , Endotelina-1/farmacologia , Músculos Papilares/efeitos dos fármacos , Trocador de Sódio e Cálcio/metabolismo , Tioureia/análogos & derivados , Bloqueadores do Receptor Tipo 1 de Angiotensina II , Animais , Gatos , Eletrofisiologia , Antagonistas dos Receptores de Endotelina , Inibidores Enzimáticos/farmacologia , Glicopeptídeos/farmacologia , Guanidinas/farmacologia , Técnicas In Vitro , Losartan/farmacologia , Contração Muscular/efeitos dos fármacos , Músculos Papilares/metabolismo , Técnicas de Patch-Clamp , Peptídeos Cíclicos/farmacologia , Sulfonas/farmacologia , Tioureia/farmacologiaRESUMO
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.