Myocardial stretch produces an increase in developed force (DF) that occurs in two phases: the first (rapidly occurring) is generally attributed to an increase in myofilament calcium responsiveness and the second (gradually developing) to an increase in [Ca(2+)](i). Rat ventricular trabeculae were stretched from approximately 88% to approximately 98% of L(max), and the second force phase was analyzed. Intracellular pH, [Na(+)](i), and Ca(2+) transients were measured by epifluorescence with BCECF-AM, SBFI-AM, and fura-2, respectively. After stretch, DF increased by 1.94+/-0.2 g/mm(2) (P<0.01, n = 4), with the second phase accounting for 28+/-2% of the total increase (P<0.001, n = 4). During this phase, SBFI(340/380) ratio increased from 0.73+/-0.01 to 0.76+/-0.01 (P<0.05, n = 5) with an estimated [Na(+)](i) rise of approximately 6 mmol/L. [Ca(2+)](i) transient, expressed as fura-2(340/380) ratio, increased by 9.2+/-3.6% (P<0.05, n = 5). The increase in [Na(+)](i) was blocked by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). The second phase in force and the increases in [Na(+)](i) and [Ca(2+)](i) transient were blunted by AT(1) or ET(A) blockade. Our data indicate that the second force phase and the increase in [Ca(2+)](i) transient after stretch result from activation of the Na(+)/H(+) exchanger (NHE) increasing [Na(+)](i) and leading to a secondary increase in [Ca(2+)](i) transient. This reflects an autocrine-paracrine mechanism whereby stretch triggers the release of angiotensin II, which in turn releases endothelin and activates the NHE through ET(A) receptors.
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 ∼30% and in intracellular Na + concentration ([Na + ] i ) of ∼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 ∼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.
Abstract-Myocardial stretch is a well-known stimulus that leads to hypertrophy. Little is known, however, about the intracellular pathways involved in the transmission of myocardial stretch to the cytoplasm and nucleus. Studies in neonatal cardiomyocytes demonstrated stretch-induced release of angiotensin II (Ang II). Because intracellular alkalinization is a signal to cell growth and Ang II stimulates the Na ϩ /H ϩ exchanger (NHE), we studied the relationship between myocardial stretch and intracellular pH (pH i ). Experiments were performed in cat papillary muscles fixed by the ventricular end to a force transducer. Muscles were paced at 0.2 Hz and superfused with HEPES-buffered solution. pH i was measured by epifluorescence with the acetoxymethyl ester form of the pH-sensitive dye 2Ј,7Ј-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM). Each muscle was progressively stretched to reach maximal developed force (L max ) and maintained in a length that was Ϸ92% L max (L i ). During the "stretch protocol," muscles were quickly stretched to L max for 10 minutes and then released to L i ; pH i significantly increased during stretch and came back to the previous value when the muscle was released to L i . The increase in pH i was eliminated by (1) Key Words: stretch, myocardial Ⅲ pH, intracellular Ⅲ Na ϩ /H ϩ exchange Ⅲ angiotensin Ⅲ endothelin A lthough it is well known that mechanical stimuli cause a variety of effects on the structure and function of the myocardial cells, little is known about how cells sense the mechanical stimuli, transmit the information to messenger systems, and finally regulate function and growth.1,2 Highly regarded experiments demonstrate that the release of angiotensin II (Ang II) contributes to stretch-induced hypertrophy in cultured neonatal cardiac myocytes, [2][3][4][5] and that the effect was suppressed by the AT 1 -receptor antagonist TCV-116. 4 The release of Ang II may involve an autocrine or paracrine mechanism because stretch-conditioned media mimicked the effect of stretch when transferred to nonstretched neonatal cardiomyocytes.2 An increase in PKC activity is an effect detected after stretching cultured neonatal cardiomyocytes 2 and the adult heart. 6 Because Ang II, by mechanisms still unresolved but probably linked to PKC, activates the Na ϩ /H ϩ exchanger (NHE), 7-8 the logical expectation is a rise in intracellular pH (pH i ) after myocardial stretch. Although we are unaware of measurements of myocardial pH i before and during stretch, pressure overload increased NHE-1 mRNA levels in hearts.9 Furthermore, the activity of mitogen-activated protein kinase (MAP kinase) was increased by stretch in cultured cardiomyocytes, and this increase was partially eliminated by NHE inhibition.9 Nevertheless, an unknown is whether stretch alters NHE activity in multicellular preparations from adult hearts. This point is critical, because Ang II released after stretch might increase NHE activity 10 -14 and promote the expression of endothelin (ET) as well as the upregulation of ET rec...
Myocardial stretch elicits a rapid increase in developed force, which is mainly caused by an increase in myofilament calcium sensitivity (Frank-Starling mechanism). Over the ensuing 10-15 min, a second gradual increase in force takes place. This slow force response to stretch is known to be the result of an increase in the calcium transient amplitude and constitutes the in vitro equivalent of the Anrep effect described 100 years ago in the intact heart. In the present review, we will update and discuss what is known about the Anrep effect as the mechanical counterpart of autocrine/paracrine mechanisms involved in its genesis. The chain of events triggered by myocardial stretch comprises 1) release of angiotensin II, 2) release of endothelin, 3) activation of the mineralocorticoid receptor, 4) transactivation of the epidermal growth factor receptor, 5) increased formation of mitochondria reactive oxygen species, 6) activation of redox-sensitive kinases upstream myocardial Na(+)/H(+) exchanger (NHE1), 7) NHE1 activation, 8) increase in intracellular Na(+) concentration, and 9) increase in Ca(2+) transient amplitude through the Na(+)/Ca(2+) exchanger. We will present the experimental evidence supporting each of the signaling steps leading to the Anrep effect and its blunting by silencing NHE1 expression with a specific small hairpin interference RNA injected into the ventricular wall.
-Conotoxins (-CTXs) specifically inhibit Na ؉ flux by occluding the pore of voltage-gated Na ؉ channels. Although the three-dimensional structures of -CTXs are well defined, the molecular configuration of the channel receptor is much less certain; even the fundamental question of whether the four homologous Na . These newly identified toxinchannel interactions within adjacent domains, interpreted in light of the known asymmetric toxin structure, fix the orientation of the toxin with respect to the channel and reveal that the four internal domains of Na ؉ channels are arranged in a clockwise configuration as viewed from the extracellular surface.
Myocardial stretch elicits a biphasic contractile response: the Frank-Starling mechanism followed by the slow force response (SFR) or Anrep effect. In this study we hypothesized that the SFR depends on epidermal growth factor receptor (EGFR) transactivation after the myocardial stretch-induced angiotensin II (Ang II)/endothelin (ET) release. Experiments were performed in isolated cat papillary muscles stretched from 92 to 98% of the length at which maximal twitch force was developed (L max ). The SFR was 123 ± 1% of the immediate rapid phase (n = 6, P < 0.05) and was blunted by preventing EGFR transactivation with the Src-kinase inhibitor PP1 (99 ± 2%, n = 4), matrix metalloproteinase inhibitor MMPI (108 ± 4%, n = 11), the EGFR blocker AG1478 (98 ± 2%, n = 6) or the mitochondrial transition pore blocker clyclosporine (99 ± 3%, n = 6). Stretch increased ERK1/2 phosphorylation by 196 ± 17% of control (n = 7, P < 0.05), an effect that was prevented by PP1 (124 ± 22%, n = 7) and AG1478 (131 ± 17%, n = 4). In myocardial slices, Ang II (which enhances ET mRNA) or endothelin-1 (ET-1)-induced increase in O 2 − production (146 ± 14%, n = 9, and 191 ± 17%, n = 13, of control, respectively, P < 0.05) was cancelled by AG1478 (94 ± 5%, n = 12, and 98 ± 15%, n = 8, respectively) or PP1 (100 ± 4%, n = 6, and 99 ± 8%, n = 3, respectively). EGF increased O 2 − production by 149 ± 4% of control (n = 9, P < 0.05), an effect cancelled by inhibiting NADPH oxidase with apocynin (110 ± 6% n = 7), mKATP channels with 5-hydroxydecanoic acid (5-HD; 105 ± 5%, n = 8), the respiratory chain with rotenone (110 ± 7%, n = 7) or the mitochondrial permeability transition pore with cyclosporine (111 ± 10%, n = 6). EGF increased ERK1/2 phosphorylation (136 ± 8% of control, n = 9, P < 0.05), which was blunted by 5-HD (97 ± 5%, n = 4), suggesting that ERK1/2 activation is downstream of mitochondrial oxidative stress. Finally, stretch increased Ser703 Na + /H + exchanger-1 (NHE-1) phosphorylation by 172 ± 24% of control (n = 4, P < 0.05), an effect that was cancelled by AG1478 (94 ± 17%, n = 4). In conclusion, our data show for the first time that EGFR transactivation is crucial in the chain of events leading to the Anrep effect.
The possibility of a direct mitochondrial action of Na(+)/H(+) exchanger-1 (NHE-1) inhibitors decreasing reactive oxygen species (ROS) production was assessed in cat myocardium. Angiotensin II and endothelin-1 induced an NADPH oxidase (NOX)-dependent increase in anion superoxide (O(2)(-)) production detected by chemiluminescence. Three different NHE-1 inhibitors [cariporide, BIIB-723, and EMD-87580] with no ROS scavenger activity prevented this increase. The mitochondria appeared to be the source of the NOX-dependent ROS released by the "ROS-induced ROS release mechanism" that was blunted by the mitochondrial ATP-sensitive potassium channel blockers 5-hydroxydecanoate and glibenclamide, inhibition of complex I of the electron transport chain with rotenone, and inhibition of the permeability transition pore (MPTP) by cyclosporin A. Cariporide also prevented O(2)(-) production induced by the opening of mK(ATP) with diazoxide. Ca(2+)-induced swelling was evaluated in isolated mitochondria as an indicator of MPTP formation. Cariporide decreased mitochondrial swelling to the same extent as cyclosporin A and bongkrekic acid, confirming its direct mitochondrial action. Increased O(2)(-) production, as expected, stimulated ERK1/2 and p90 ribosomal S6 kinase phosphorylation. This was also prevented by cariporide, giving additional support to the existence of a direct mitochondrial action of NHE-1 inhibitors in preventing ROS release. In conclusion, we report a mitochondrial action of NHE-1 inhibitors that should lead us to revisit or reinterpret previous landmark observations about their beneficial effect in several cardiac diseases, such as ischemia-reperfusion injury and cardiac hypertrophy and failure. Further studies are needed to clarify the precise mechanism and site of action of these drugs in blunting MPTP formation and ROS release.
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