María C. Camilión de Hurtado

Mechanisms Underlying the Increase in Force and Ca2+ Transient That Follow Stretch of Cardiac Muscle : A Possible Explanation of the Anrep Effect

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.

Reverse Mode of the Na+-Ca2+ Exchange After Myocardial Stretch : Underlying Mechanism of the Slow Force Response

Abstract— This study was designed to gain additional insight into the mechanism of the slow force response (SFR) to stretch of cardiac muscle. SFR and changes in intracellular Na+ concentration ([Na+]i) were assessed in cat papillary muscles stretched from 92% to ≈98% of Lmax. The SFR was 120±0.6% (n=5) of the rapid initial phase and coincided with an increase in [Na+]i. The SFR was markedly depressed by Na+-H+ exchanger inhibition, AT1 receptor blockade, nonselective endothelin-receptor blockade and selective ETA-receptor blockade, extracellular Na+ removal, and inhibition of the reverse mode of the Na+-Ca2+ exchange by KB-R7943. KB-R7943 prevented the SFR but not the increase in [Na+]i. Inhibition of endothelin-converting enzyme activity by phosphoramidon suppressed both the SFR and the increase in [Na+]i. The SFR and the increase in [Na+]i after stretch were both present in muscles with their endothelium (vascular and endocardial) made functionally inactive by Triton X-100. In these muscles, phosphoramidon also suppressed the SFR and the increase in [Na+]i. The data provide evidence that the last step of the autocrine-paracrine mechanism leading to the SFR to stretch is Ca2+ entry through the reverse mode of Na+-Ca2+ exchange.

pHi Regulation in Myocardium of the Spontaneously Hypertensive Rat : Compensated Enhanced Activity of the Na+-H+ Exchanger

To elucidate the mechanisms controlling pHi in myocardium of the spontaneously hypertensive rat (SHR), experiments were performed in papillary muscles (isometrically contracting at 0.2 Hz) from SHR and age-matched normotensive Wistar-Kyoto (WKY) rats loaded with the pH-sensitive fluorescent probe BCECF-AM. An enhanced activity of the Na(+)-H+ exchanger was detected in the hypertrophic myocardium of SHR. This conclusion was based on the following: (1) The myocardial pHi was more alkaline in SHR (7.23 +/- 0.03) than in WKY rats (7.10 +/- 0.03) (P < .05) in HEPES buffer. (2) SITS (0.1 mmol/L in HEPES buffer) did not alter pHi in the SHR (pHi 7.26 +/- 0.03 and 7.28 +/- 0.03 before and after SITS, respectively). (3) The fall in pHi observed after 20 minutes of Na(+)-H+ exchanger inhibition [5 mumol/L 5-(N-ethyl-N-isopropyl)amiloride (EIPA)] was greater in SHR (-0.16 +/- 0.01) than in WKY rats (-0.09 +/- 0.02, P < 0.05). (4) The velocity of pHi recovery from an intracellular acid load was faster in SHR than in WKY rats (0.068 +/- 0.02 versus 0.014 +/- 0.002 pH units/min at pHi 6.99, P < .05). (5) After EIPA inhibition, the rate of pHi recovery from the same acid load decreased to a similar value in both rat strains (0.0032 +/- 0.002 pH units/min in SHR and 0.0032 +/- 0.002 pH units/min in WKY rats). Under the more physiological HCO3(-)-CO2 buffer, no significant difference in steady state myocardial pHi was detected between rat strains (7.15 +/- 0.03 in SHR and 7.11 +/- 0.05 in WKY rats).(ABSTRACT TRUNCATED AT 250 WORDS)

Regression of Isoproterenol-Induced Cardiac Hypertrophy by Na+/H+ Exchanger Inhibition

Abstract— Cardiac hypertrophy is often associated with an increased sympathetic drive, and both in vitro and in vivo studies have demonstrated the development of cardiomyocyte hypertrophy in response to either [alpha]‐ or [beta]‐adrenergic stimulation. Because an association between the Na+/H+ exchanger and cellular growth has been proposed, this study aimed to analyze the possible role of the antiporter in isoproterenol‐induced cardiac hypertrophy. Isoproterenol alone (5 mg/kg IP once daily) or combined with a selective inhibitor of the Na+/H+ exchanger activity (3 mg · kg‐1 · d‐1 BIIB723) was given to male Wistar rats for 30 days. Sex‐ and age‐matched rats that received 0.9% saline IP daily served as controls. Echocardiographic follow‐up showed a 33% increase in left ventricular mass in the isoproterenol‐treated group, whereas it did not increase in the isoproterenol+BIIB723‐treated group. Heart weight–to–body weight ratio at necropsy was 2.44±0.11 in controls and increased to 3.35±0.10 (P <0.05) with isoproterenol, an effect that was markedly attenuated by BIIB723 (2.82±0.07). Intense cardiomyocyte enlargement and severe subendocardial fibrosis were found in isoproterenol‐treated rats, and both effects were attenuated by BIIB723. Myocardial Na+/H+ exchanger activity and protein expression significantly increased in isoproterenol‐treated rats compared with the control group (1.45±0.11 vs 0.91±0.05 arbitrary units, P <0.05). This effect was significantly reduced by BIIB723 (1.17±0.02, P <0.05). In conclusion, our results show that Na+/H+ exchanger inhibition prevented the development of isoproterenol‐induced hypertrophy and fibrosis, providing strong evidence in favor of a key role played by the antiporter in this model of cardiac hypertrophy.

Regression of cardiomyocyte hypertrophy in SHR following chronic inhibition of the Na+/H+ exchanger

OBJECTIVE Experiments were performed to examine the effect of chronic inhibition of the Na(+)/H(+) exchanger isoform-1 (NHE-1) on cardiac hypertrophy of spontaneously hypertensive rats (SHR). METHODS SHR were orally treated during 1 month with two different doses (0.3 and 3.0 mg/kg/day) of the NHE-1 inhibitor, cariporide, or nifedipine (10.0 mg/kg/day). RESULTS The two doses of cariporide did not differ in their effects after 1 month of treatment, since both induced a slight decrease in systolic blood pressure (SBP) of approximately 6 mmHg and regression of the heart weight to body weight ratio (mg/g) from 3.28+/-0.05 to 3.04+/-0.05 (0.3 mg) and 2.99+/-0.10 (3.0 mg, P<0.05). Nifedipine, given for the same period, produced similar reduction in the hypertrophy index (3.03+/-0.05), but with a much greater decrease in arterial pressure (35.6+/-7.4 mmHg). Chronic treatment with cariporide induced a complete regression of the augmented cross sectional area of left ventricular myocytes without significant changes in collagen content, serum procollagen 1 propeptide levels or myocardial distensibility. CONCLUSIONS NHE inhibition represents a novel approach to induce regression of pathological hypertrophy of the heart. The finding can be rationalized mechanistically by previous in vitro studies suggesting a role of the NHE in the development of myocardial hypertrophy.

The Positive Inotropic Effect of Angiotensin II: Role of Endothelin-1 and Reactive Oxygen Species

Many effects believed to be because of angiotensin II (Ang II) are attributable to the action of endothelin (ET)-1, which is released/produced by Ang II. We investigated whether Ang II elicits its positive inotropic effect (PIE) by the action of endogenous ET-1, in addition to the role played by reactive oxygen species (ROS) in this mechanism. Cat cardiomyocytes were used for: (1) sarcomere shortening measurements; (2) ROS measurements by epifluorescence; (3) immunohistochemical staining for preproET-1, BigET-1, and ET-1; and (4) measurement of preproET-1 mRNA by RT-PCR. Cells were exposed to 1 nmol/L Ang II for 15 minutes. This low concentration of Ang II increases sarcomere shortening by 29.2±3.7% (P<0.05). This PIE was abrogated by Na+/H+ exchanger or Na+/Ca2+ exchanger reverse mode inhibition. The production of ROS increased in response to Ang II treatment (&Dgr;ROS respect to control: 68±15 fluorescence units; P<0.05). The Ang II–induced PIE and ROS production were blocked by the Ang II type 1 receptor blocker losartan, the nonselective ET-1 receptor blocker TAK044, the selective ETA receptor blocker BQ-123, or the ROS scavenger N-(2-mercapto-propionyl)glycine. Exogenous ET-1 (0.4 nmol/L) induced a similar PIE and increase in ROS production to those caused by Ang II. Immunostaining for preproET-1, BigET-1, and ET-1 was positive in cardiomyocytes. The preproET-1 mRNA abundance increased from 100±4.6% in control to 241.9±39.9% in Ang II–treated cells (P<0.05). We conclude that the PIE after exposure to 1 nmol/L Ang II is due to endogenous ET-1 acting through the ETA receptor and triggering ROS production, Na+/H+ exchanger stimulation, and Na+/Ca2+ exchanger reverse mode activation.

Regression of Hypertensive Myocardial Fibrosis by Na+/H+ Exchange Inhibition

Abstract—We have recently reported that the inhibition of the Na+/H+ exchanger (NHE) during 1 month in spontaneously hypertensive rats (SHR) is followed by regression of cardiomyocyte hypertrophy but not of myocardial fibrosis. The aim of this study was to evaluate whether a treatment of longer duration could reduce myocardial fibrosis and stiffness. SHR received 3.0 mg/kg per day of the specific NHE-1 inhibitor cariporide; the effect on cardiomyocyte cross-sectional area, myocardial collagen volume fraction, collagen synthesis, and myocardial stiffness (length-tension relation in left papillary muscles) was evaluated at several time points (after 1, 2, or 3 months). A slight decrease of ≈5 mm Hg in systolic blood pressure was observed after 1 month of treatment with no further changes. After 2 and 3 months of treatment, the size of cardiomyocytes remained within normal values and myocardial fibrosis progressively decreased to normal level. Accordingly, myocardial stiffness and the serum levels of the carboxyterminal propeptide of procollagen type I, a marker of collagen type I synthesis, were normalized after 3 months. Left ventricular weight decreased from 910±43 (in untreated SHR) to 781±21 mg (treated SHR) after 3 months of treatment. No difference in body weight between treated and untreated SHR was observed after this period of treatment. The present data allow us to conclude that in the SHR the administration of an NHE-1 inhibitor for 2 or 3 months leads to the normalization of collagen type I synthesis, myocardial collagen volume fraction, and stiffness.

Angiotensin II Activates Na+-Independent Cl−-HCO3− Exchange in Ventricular Myocardium

The effect of angiotensin II (Ang II) on the activity of the cardiac Na+-independent Cl--HCO3- exchanger (anionic exchanger [AE]) was explored in cat papillary muscles. pHi was measured by epifluorescence with BCECF-AM. Ang II (500 nmol/L) induced a 5-(N-ethyl-N-isopropyl)amiloride-sensitive increase in pHi in the absence of external HCO3- (HEPES buffer), consistent with its stimulatory action on Na+-H+ exchange (NHE). This alkalinizing effect was not detected in the presence of a CO2-HCO3- buffer (pHi 7.07+/-0.02 and 7.08+/-0.02 before and after Ang II, respectively; n=17). Moreover, in Na+-free HCO3--buffered medium, in which neither NHE nor Na+-HCO3- cotransport are acting, Ang II decreased pHi, and this effect was canceled by previous treatment with SITS. These findings suggested that the Ang II-induced activation of NHE was masked, in the presence of the physiological buffer, by a HCO3--dependent acidifying mechanism, probably the AE. This hypothesis was confirmed on papillary muscles bathed with HCO3- buffer that were first exposed to 1 micromol/L S20787, a specific inhibitor of AE activity in cardiac tissue, and then to 500 nmol/L Ang II (n=4). Under this condition, Ang II increased pHi from 7.05+/-0.05 to 7.22+/-0.05 (P<.05). The effect of Ang II on AE activity was further explored by measuring the velocity of myocardial pHi recovery after the imposition of an intracellular alkali load in a HCO3--containing solution either with or without Ang II. The rate of myocardial pHi recovery was doubled in the presence of Ang II, suggesting a stimulatory effect on AE. The enhancement of the activity of this exchanger by Ang II was also detected when the AE activity was reversed by the removal of extracellular Cl- in a Na+-free solution. Under this condition, the rate of intracellular alkalinization increased from 0.053+/-0.016 to 0.108+/-0.026 pH unit/min (n=6, P<.05) in the presence of Ang II. This effect was canceled either by the presence of the AT1 receptor antagonist, losartan, or by the previous inhibition of protein kinase C with chelerythrine or calphostin C. The above results allow us to conclude that Ang II, in addition to its stimulatory effect on alkaline loading mechanisms, activates the AE in ventricular myocardium and that the latter effect is mediated by a protein kinase C-dependent regulatory pathway linked to the AT1 receptors.

Enalapril induces regression of cardiac hypertrophy and normalization of pHi regulatory mechanisms

Intracellular pH is under strict control in myocardium; H+ are extruded from the cells by sodium-dependent mechanisms, mainly Na+/H+ exchanger and Na+/HCO3- symport, whereas Na+-independent Cl-/HCO3- exchanger extrudes bases on intracellular alkalinization. Hypertrophic myocardium from spontaneously hypertensive rats (SHR) exhibits increased Na+/H+ exchange activity that is accompanied by enhanced extrusion of bases through Na+-independent Cl-/HCO3- exchange. The present experiments were designed to investigate the effect of enalapril-induced regression of cardiac hypertrophy on the activity of these exchangers. Male SHR and normotensive Wistar-Kyoto rats (WKY) received enalapril maleate (20 mg/kg per day) in the drinking water for 5 weeks. Gender- and age-matched SHR and WKY were used as untreated controls. Enalapril treatment significantly reduced systolic blood pressure in SHR and completely regressed cardiac hypertrophy. Na+/H+ activity was estimated in terms of both steady pHi value in HEPES buffer and the rate of pHi recovery from CO2-induced acid load. Na+-independent Cl-/HCO3- activity was assessed by measuring the rate of pHi recovery from intracellular alkalinization produced by trimethylamine exposure. Regression of cardiac hypertrophy was accompanied by normalization of Na+/H+ and Na+-independent Cl-/HCO3- exchange activities. Inhibition of protein kinase C (PKC) activity with chelerythrine (10 mmol/L) or calphostin C (50 nmol/L) returned both exchange activities to normal values. These results show that angiotensin-converting enzyme inhibition normalizes the enhanced activity of both exchangers while regressing cardiac hypertrophy. Because normalization of exchange activities could be also achieved by PKC inhibition, the data would suggest that PKC-dependent mechanisms play a significant role in the increased ion exchange activities of hypertrophic myocardium and in their normalization by angiotensin-converting enzyme inhibition.

Role of an Electrogenic Na+-HCO3− Cotransport in Determining Myocardial pHi After an Increase in Heart Rate

The contribution of electrogenic Na(+)-HCO3- cotransport to pHi regulation during changes in heart rate was explored in cat papillary muscles loaded with BCECF-AM in bicarbonate-free (HEPES) medium and in CO2/HCO3(-)-buffered medium. Stepwise increments in the frequency of contraction from 15 to 100 bpm induced a reversible increase in the pHi from 7.13 +/- 0.03 to 7.36 +/- 0.03 (P < .05, n = 5) in the presence of CO2/ HCO3- buffer. The same increase in the frequency of stimulation, however, decreased pHi from 7.10 +/- 0.02 to 6.91 +/- 0.06 (P < .05, n = 5), in the absence of bicarbonate. Moreover, in CO2/HCO3(-)-superfused muscles pretreated with SITS (0.1 mmol/L), this effect of increasing the contraction frequency was reversed, and a decrease of pHi from 7.03 +/- 0.04 to 6.88 +/- 0.06 (P < .05, n = 4) was observed when the pacing rate was increased stepwise from 15 to 100 bpm. High [K+]o-induced depolarization of cell membrane alkalinized myocardial cells in the presence of HCO3- ions, whereas acidification was observed as a consequence of hyperpolarization induced by low external [K+]o. Myocardial resting membrane potential became hyperpolarized upon exposure to HCO3(-)-buffered media. This HCO3(-)-induced hyperpolarization was not blocked by the inhibition of Na+,K(+)-ATPase activity by ouabain (0.5 mumol/L) but was prevented by SITS. The results suggested that membrane depolarization during cardiac action potential causes an increase in electrogenic Na(+)-HCO3- cotransport. Such depolarizations occurring as a consequence of increases in heart rate would thus, by means of elevated bicarbonate influxes, substantially increase the myocardial cells ability to recover from an enhanced proton production.