Abstract
Although inhibition of Na+/H+ exchanger isoform 1 (NHE-1) reduces cardiomyocyte hypertrophy, the mechanisms underlying this effect are not known. Recent evidence suggests that this may be associated with improved mitochondrial function. To understand the mechanistic bases for mitochondrial involvement in the antihypertrophic effect of NHE-1 inhibition, we examined the effect of the NHE-1-specific inhibitor N-[2-methyl-4,5-bis(methylsulphonyl)-benzoyl]-guanidine, hydrochloride (EMD, EMD87580; 5 μM) on the hypertrophic phenotype, mitogen-activated protein kinase (MAPK) activity, mitochondrial membrane potential (Δψm), permeability transition (MPT) pore opening, and superoxide generation in phenylephrine (PE)-treated neonatal rat cardiomyocytes. EMD significantly suppressed markers of cell hypertrophy, including cell surface area and gene expression of atrial natriuretic peptide and α-skeletal actin. EMD inhibited the PE-induced MPT pore opening, prevented the loss in Δψm, and attenuated superoxide generation induced by PE. Moreover, the activation of p38 MAPK (p38) and extracellular signal-regulated kinase (ERK) 1/2 MAPKs induced by PE was significantly attenuated in the presence of EMD as well as the antioxidant catalase. To examine the role of MPT and mitochondrial Ca2+ uniport in parallel with EMD, the effects of cyclosporin A (0.2 μM) and ruthenium red (10 μM) were evaluated. Both agents significantly attenuated PE-induced hypertrophy and inhibited both mitochondrial dysfunction and p38 and ERK1/2 MAPK activation. Our results suggest a novel mechanism for attenuation of the hypertrophic phenotype by NHE-1 inhibition that is mediated by a reduction in PE-induced MAPK activation and superoxide production secondary to improved mitochondrial integrity.
Sodium-hydrogen exchanger-1 (NHE-1), a critical intracellular pH regulator, plays an important role in mediating the hypertrophic response that follows myocardial injury, with the subsequent evolution to heart failure (Spitznagel et al., 2000; Yoshida and Karmazyn, 2000; Karmazyn et al., 2001). NHE-1 activity has been shown to be up-regulated during hypertrophic responses to various paracrine and autocrine factors such as α-adrenergic agonists, angiotensin II, and endothelin-1 (Karmazyn et al., 2001; Avkiran and Marber, 2002). Studies using different models of cardiac remodeling and heart failure demonstrated that NHE-1 inhibition improves heart performance, particularly ventricular dysfunction and hypertrophy (Yoshida and Karmazyn, 2000; Kusumoto et al., 2001; Cingolani et al., 2003; Marano et al., 2004). NHE-1 inhibitors have been shown to reduce the hypertrophic response to various extracellular stimuli in cultured cardiac cells (Hori et al., 1990; Yamazaki et al., 1998; Karmazyn et al., 2003).
The cellular mechanistic bases for the ability of NHE-1 inhibitors to suppress the hypertrophic response are not known with certainty but is likely multifaceted involving various processes including, as we recently reported, improved mitochondrial function (Javadov et al., 2005). Indeed, we have suggested in our study that defective mitochondrial function may represent a key basis for the transition between reversible and irreversible postinfarction myocardial remodeling (Javadov et al., 2005).
Mitochondrial oxidative phosphorylation represents a major ATP-synthetic pathway in cells. In addition to ATP synthesis, the electron transport chain of mitochondria is a significant source of ROS, mainly superoxide (), which is converted to H2O2 either by spontaneous dismutation or by the enzyme superoxide dismutase (SOD). The main sources of generation are two segments of the respiratory chain: the reduced flavin mononucleotide of NADH dehydrogenase in complex I and the ubisemiquinone radical intermediate (QH·), formed during the Q cycle at the Qo site of complex III (Brookes et al., 2004). Interestingly, the chronic release of ROS has been recently linked to the development of hypertrophy and heart failure (Ide et al., 1999; Amin et al., 2001; Sorescu and Griendling, 2002). High intracellular ROS level has been detected in cells stimulated with cytokines, growth factors, and angiotensin II. Furthermore, recent studies have demonstrated that mitochondrial-derived ROS may function as intracellular messengers to modulate cell signaling pathways (Cheng et al., 1999; Brookes et al., 2002). It was shown that angiotensin II causes hypertrophy in part via the generation of ROS in cardiomyocytes (Nakamura et al., 1998; Shih et al., 2001). Dysfunction in mitochondrial biogenesis may be responsible for excessive production, with subsequent development of hypertrophy, which, in turn, might progress to heart failure.
Although the precise mechanisms explaining the role of ROS in the development of hypertrophy are not well understood, MAPK has been shown to be activated by ROS generated intracellularly during hypoxia (Kulisz et al., 2002) and hypertrophy (Tanaka et al., 2001) as well as when administered exogenously (Tu et al., 2003; Purdom and Chen, 2005). Furthermore, ROS-mediated activation of MAPKs was observed in cardiomyocytes during α1-adrenergic-stimulated hypertrophy (Amin et al., 2001). Based on these observations, we hypothesized that the increase in generated by mitochondria might be responsible for MAPKs activation in phenylephrine (PE)-treated cardiomyocytes, and the purpose of our study was to investigate the role of mitochondrial-derived in the antihypertrophic effect of NHE-1 inhibition. Because mitochondrial production of is a result of respiratory chain dysfunction with subsequent alteration in inner membrane integrity, we have also evaluated the effect of NHE-1 inhibition on mitochondrial membrane potential (Δψm) and mitochondrial permeability transition (MPT) pore opening. Moreover, in parallel to NHE-1 inhibition with the selective and potent NHE-1 inhibitor EMD-87580 (EMD), we studied the contribution of mitochondrial Ca2+ and opening of MPT pores to cardiomyocyte hypertrophy by determining the effect of ruthenium red (RR), a blocker of the mitochondrial calcium uniporter, and cyclosporin A (CyA), an inhibitor of the MPT pore opening.
Materials and Methods
All procedures were performed in accordance with the University of Western Ontario animal care guidelines, which conform to the guidelines of the Canadian Council on Animal Care (Ottawa, ON, Canada).
Primary Culture of Neonatal Rat Cardiac Ventricular Myocytes. Cardiac ventricular myocytes were prepared from 4-day-old Sprague-Dawley rats (Charles River Canada, St. Constant, PQ, Canada) and cultured as described previously (Karmazyn et al., 2003). In brief, the hearts were isolated, and ventricles were minced and subjected to collagenase digestions four times at 37°C. All digestions were pooled and centrifuged for 5 min at 500g. The cell pellet was suspended in culture medium and plated on Primaria dishes. We have previously shown that approximately 95% of cells prepared by this method demonstrate sarcomeric myosin-heavy chain staining, indicating relatively low nonmyocyte contamination (Rajapurohitam et al., 2003). Cardiomyocytes prepared and cultured by this procedure are greater than 95% viable and stable for up to 6 days under the conditions tested (Regula et al., 2004).
Experimental Protocol. Cells were first serum-starved for 24 h and then were treated with 10 μM PE for a further 24 h. To assess the effect of different interventions, the cells were treated with either 5 μM EMD, 0.2 μM CyA (Sigma-Aldrich, Oakville, ON, Canada), and/or 10 μM RR (Sigma-Aldrich) 40 min before PE administration. EMD was a gift from Merck KGaA (Darmstadt, Germany).
Cell Area Measurement. The cells were plated at a density of 1 × 106 cells/6-cm dish to obtain individually plated cells. Cells were viewed using a Leica DM IL (Wetzlar, Germany) inverted microscope equipped with a Polaroid digital camera. Nine random photographs were taken from each dish, and at least 60 individual cell surface area measurements were made from each sample using Mocha software (SPSS Inc., Chicago, IL).
RNA Isolation and Reverse Transcription. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA (5 μg) was used to synthesize first strand of cDNA using SuperScript II RNase H-Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol and was used as a template in the following PCR reactions.
Real-Time PCR. The expression of ANP, α-skeletal actin, and 18S rRNA genes was performed in 20-μl reaction volume using SYBR Green Jumpstart Taq ReadyMix DNA polymerase (Sigma-Aldrich), and fluorescence was measured and quantified using DNA Engine Opticon 2 System (MJ Research, Waltham, MA). Amplification was performed using the following primers: 5′-CTGCTAGACCACCTGGAGGA-3′ (forward) and 5′-AAGCTGTTGCAGCCTAGTCC-3′ (reverse) for ANP, 5′-CACGGCATTATCACCAACTG-3′ (forward) and 5′-CCGGAGGCATAGAGAGACA-G-3′ (reverse) for α-skeletal actin, and 5′-GTAACCCGTTGAACCCCATT-3′ (forward) and 5′-CCATCCAATCGGTAGTAGCG-3 (reverse) for 18S rRNA. PCR conditions and cell cycle number was optimized for each set of primers. Melting-curve analysis showed a single PCR product for each gene amplification. PCR conditions to amplify all three genes were 30 s at 94°C followed by annealing at 54°C for 20 s for 18S rRNA and 60°C for 25 s for α-skeletal actin and ANP, with further elongation at 72°C for 30 s. ANP and α-skeletal actin were amplified for 40 cycles, whereas 18S rRNA was amplified for 35 cycles. 18S rRNA gene expression was used as a control.
Western Blotting. Cell lysates were transferred to a 1.5-ml Eppendorf tube, homogenized, and centrifuged at 10,000g for 5 min at 4°C. The supernatant was transferred to a fresh tube and the protein concentration determined by the Bradford protein assay kit (Bio-Rad, Hercules, CA). Thirty micrograms of protein were resolved on 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham Biosciences Inc., Piscataway, NJ). The membranes were blocked in 5% milk for 1 h and incubated with primary antibody for 1 h followed by secondary antibody for 1 h and then detected by enhanced chemiluminescence reagent (Amersham Biosciences Inc.). Antibodies were purchased from Cell Signaling Technology Inc., Beverly, MA (P-p38) or Santa Cruz Biotechnology, Santa Cruz, CA (p38, P-ERK1/2, ERK1/2) and all used at 1:1000 dilutions, except P-ERK1/2, which was used at 1:2000.
Mitochondrial Membrane Potential. To monitor Δψm, cells (5 × 105 cells/well) plated in a 24-well Primaria culture dish were incubated with the potential sensitive dye JC-1 (10 μg/ml, 5,5′,6,6′-tetraethyl-benzimidazolylcarbocyanine iodide; Molecular Probes, Eugene, OR) for 30 min. The intensity of fluorescence was immediately measured using a microplate monochromator reader (TECAN, Salzburg, Austria) at 527 and 590 nm emission (with excitation at 488 nm). For confocal microscopy, the cells plated on glass-bottom dishes were loaded with JC-1, and images were captured using a Zeiss LSM 510 (Carl Zeiss, Oberkochen, Germany) microscope.
Mitochondrial Permeability Transition Pore. To measure MPT pore opening, cardiomyocytes were loaded with 5 μM calcein-acetoxymethylester (calcein-AM; Molecular Probes) in the presence of 5 mM cobalt chloride to quench cytosolic and nuclear calcein loading (Petronilli et al., 1999). Cells were visualized with an Olympus AX-70 Research fluorescence microscope (Carsen Group, Markham, ON, Canada), and fluorescence was captured using a high-speed Sensys digital camera (Photometrics, Inc., Waterloo, ON, Canada). Data are expressed as mean ± S.E.M. integrated optical density. Confocal images of cells were collected using a Zeiss LSM 510 (Carl Zeiss) microscope at 488 nm (excitation) and 525 nm (emission).
Superoxide Production. To measure superoxide production, cells (0.5 × 106 cells/well) were plated in 24-well culture dish and incubated in culture medium containing 10 μM dihydroethidium (DHE; Molecular Probes). DHE fluorescence (excitation 518 nm/emission 605 nm) was measured at the indicated time points using a microplate monochromator reader (TECAN) as previously reported (Sayen et al., 2003).
Statistical Analysis. All values in the figures and text are presented as mean ± S.E.M. Multiple comparisons between groups were determined by one-way analysis of variance. An unpaired two-tailed Student's t test was used to compare mean differences between groups. Linear regression analysis was used to determine the relations between Δψm or superoxide production and hypertrophic gene expression or MAPK activation. Differences were considered to be statistically significant at a level of P < 0.05.
Results
Effect of EMD, CyA, and RR on the Hypertrophic Response. We first examined the effect of EMD, CyA, and RR on hypertrophy induced by treating myocytes with the α1-adrenergic receptor agonist PE for 24 h. As shown in Fig. 1A, myocytes displayed a significant increase (approximately 40%) in cell surface area (P < 0.01). The PE-induced hypertrophy was prevented by the NHE-1 inhibitor EMD as well as by CyA and RR (P < 0.01). Moreover, as shown in Fig. 1, B and C, the significant increase in expression of two hypertrophic gene markers, ANP and α-skeletal actin mRNA, was similarly abolished by all three agents. In control cells, treatment with EMD, CyA, and RR in the absence of PE did not exert a significant effect on cell size or on hypertrophic gene markers.
Effect of EMD, CyA, and RR on Δψm and MPT. Δψm represents an important marker of mitochondrial integrity, and its dissipation is a critical event in cell pathology. We have used JC-1 dye emission to measure quantitative changes in Δψm. Different sensitivities of JC-1 to Δψm changes as well as signal calibrations have been shown in isolated mitochondria (Di Lisa et al., 1995) as well as myocytes (Mathur et al., 2000). In the present study, we have demonstrated the specificity of responses using both JC-1 and tetramethylrhodamine methyl ester in the presence of the respiratory chain uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Results were normalized to control for each group and expressed as percent changes from the control condition.
To determine whether the antihypertrophic effect of EMD is associated with preservation of Δψm, we assessed the change in JC-1 fluorescence ratio in PE-treated cells in the presence or absence of EMD. As shown in Fig. 2, treatment of cells with PE induced a decrease of Δψm to 84.1% (P < 0.05) of control. The decrease in JC-1 fluorescence ratio was significantly suppressed by EMD, suggesting the preservation of Δψm (Fig. 2, A and B). Δψm was also preserved in PE-treated cells in the presence of CyA and seemed to be so for RR, although with respect to the latter, the effect failed to reach statistical significance (Fig. 2B). All three drugs demonstrated no effect on JC-1 fluorescence in control cells (without PE treatment).
A loss in Δψm represents one of the major contributors to MPT pore opening, and therefore, to determine whether MPT pore opening occurs in PE-stimulated cells, cardiomyocytes were loaded with calcein-AM in the presence of cobalt chloride to quench cytosolic and nuclear calcein. Because fluorescent calcein concentrates in the mitochondrial matrix, loss of calcein fluorescence has been used to detect the opening of the MPT pores. We have previously used dual staining of the cells with tetramethylrhodamine methyl ester and calcein to confirm that the mitochondria are, in fact, labeled. Confirmation of the specificity of responses was also demonstrated using bongkrekic acid, an inhibitor of the adenine nucleotide translocase (ANT) and MPT pore opening (Gurevich et al., 2001; Regula et al., 2004). As illustrated in Fig. 3, PE induced MPT pore opening, resulting in the release of fluorescent calcein from mitochondria, an effect which was significantly attenuated by both EMD and CyA (Fig. 3, A and B). Both agents failed to exert a significant influence on MPT pore opening in control cells in the absence of PE. Taken together, these data demonstrate that PE elicits Δψm loss, which is accompanied by the opening of MPT pores, although both effects were prevented by the NHE-1 inhibitor EMD.
Effect of EMD, CyA, and RR on Superoxide Generation. To study whether PE-induced cardiomyocyte hypertrophy is associated with generation of superoxide, cells were treated with 10 μM DHE, which is oxidized to ethidium-DNA (Et-DNA) by . Figure 4A shows the time-dependent effect of PE on relative Et-DNA fluorescence. Relative Et-DNA fluorescence was significantly stimulated 10 min after PE treatment, and values tended to remain higher through the 24-h treatment period.
We further examined the ability of interventions to affect superoxide generation and the possible relevance of the latter to early MAPK activation. Therefore, these studies were done in cells exposed to PE for 10 min, which represented the time for peak MAPK activation as well as significantly increased Et-DNA fluorescence. As shown in Fig. 4B, both EMD and RR significantly decreased PE-induced generation (105 ± 3 versus 119 ± 3% in PE group, P < 0.01), whereas CyA did not have significant effects, although a trend toward lower generation appeared evident. It should be noted that all three compounds as well as inhibitors of the reactions responsible for production did not induce any significant alteration of the superoxide level in control cardiomyocytes.
In parallel to mitochondria, is also produced intracellularly by activation of three main enzymatic processes in cytoplasm, including NADPH oxidases, nitric-oxide synthase, and xanthine oxidase. Therefore, the contribution of each of each of these processes to production was determined using the following specific metabolic inhibitors: apocynin (300 μM; NADPH oxidase inhibitor), Nω-nitro-l-arginine methyl ester (100 μM; l-NAME, nitric-oxide synthase inhibitor), and allopurinol (100 μM; xanthine oxidase inhibitor). However, as shown in Fig. 5A, none of these inhibitors given either individually or in combination attenuated PE-induced generation, suggesting that these cytoplasmic reactions do not play a significant role in the elevation of level in PE-treated cardiomyocytes.
To further study the role of the mitochondrial respiratory chain in the intracellular generation, PE-treated cells were incubated with either the complex I inhibitor rotenone (10 μM) or the complex III inhibitor myxothiazol (1 μM). As shown in Fig. 5B, rotenone failed to attenuate Et-DNA relative fluorescence, whereas a significant reduction in superoxide generation was seen with myxothiazol. When the results are taken together, they suggest that mitochondria represent the major source of generation in PE-treated cardiomyocytes, which occurs via complex III of the respiratory chain.
Our results are consistent with studies reported by others describing two potential sites of generation in the mitochondrial respiratory chain, including reduced flavin mononucleotide of NADH dehydrogenase in complex I and the ubisemiquinone site with the cytochrome b-c1 segment of complex III (Turrens et al., 1985; Becker et al., 1999; Ide et al., 1999) with no contribution of other superoxide-producing reactions in cytoplasm during ischemia (Becker et al., 1999).
Regulation of MAPK Activity in PE-Treated Myocytes. ROS have been shown to be implicated in the pathogenesis of hypertrophy and heart failure (Ide et al., 1999; Amin et al., 2001; Sorescu and Griendling, 2002). Studies using different models of hypertrophy have demonstrated that ROS at low concentrations serve as second messengers and are involved in the hypertrophic signaling in the early stage of cardiac hypertrophy. MAPKs have been found to be activated in cardiomyocytes treated with H2O2 (Tu et al., 2003; Purdom and Chen, 2005), growth factors (Amin et al., 2001), and intracellular ROS is produced during hypertrophy and hypoxia (Tanaka et al., 2001; Kulisz et al., 2002). To further study the importance of the mitochondria-derived for MAPK activation, we assessed p38 and ERK1/2 activities 10 min after PE addition. Western blot analysis demonstrated 2.0- (P < 0.01) and 3.1- (P < 0.01) fold increases in phosphorylated p38 and ERK1/2 activity in PE-treated cardiomyocytes (Fig. 6, A and B). As shown in Fig. 6, EMD as well as CyA and RR significantly attenuated activation of both kinases when the drugs were administered either alone or in combination. However, the combination protocol had no additive effect.
To directly examine the relationship between ROS production and MAPK activation, the PE-treated cells were incubated in the presence of 200 units/ml catalase. As shown in Fig. 6, catalase significantly attenuated the phosphorylation of both p38 and ERK1/2 induced by PE. The ability of catalase to attenuate MAPK activation akin to that shown for EMD, CyA, and RR suggests that up-regulation of MAPKs in response to hypertrophic stimuli is mediated, at least in part, by ROS generation in cardiomyocytes and that the antihypertrophic effect of NHE-1 inhibition could reflect down-regulation of MAPK activation.
Linear regression analyses were performed to determine the relationships between either Δψm or superoxide production and hypertrophic gene expression or MAPK activation PE-treated myocytes. An increase in levels was positively correlated with increased expression of hypertrophic genes (r = +0.87, P < 0.001) and phosphorylated MAPKs (r = +0.75, P < 0.001). Conversely, a negative relationship was observed with mitochondrial Δψm: its reduction was associated with elevated expression levels of hypertrophic genes (r = –0.83, P < 0.02) and MAPK activation (r = –0.74, P < 0.02).
Discussion
The primary focus of this study was to investigate the potential contribution of mitochondrial-derived in mediating the antihypertrophic effect of NHE-1 inhibition on PE-induced hypertrophy and to assess underlying mechanisms. Our study clearly demonstrates that the NHE-1-specific inhibitor EMD suppresses PE-induced hypertrophy as determined by cell size and molecular (ANP and α-skeletal actin) markers, an effect which was associated with inhibition of PE-induced loss in Δψm and mitochondrial-derived generation. EMD also decreased PE-induced phosphorylation of p38 and ERK1/2 MAPKs. The effects of EMD were generally mimicked by both CyA and RR. Coupled with the close correlation between mitochondrial function with either hypertrophic genes expression or MAPK activity, our results are suggestive for an essential role of Δψm and in hypertrophic response to PE.
The ability of both CyA and EMD to inhibit hypertrophy was associated with reversal of PE-induced MPT opening, thereby suggesting that MPT opening could participate in the hypertrophic response of cardiomyocytes to stimuli. Indeed, we have previously shown that in an in vivo model of postinfarction heart failure, the salutary effects of NHE-1 inhibition were associated with a mitigation in MPT opening and improved mitochondrial respiratory function, although a cause and effect relationship could not be established (Javadov et al., 2005). However, it should be noted that the antihypertrophic effects of CyA could be due to other factors; for example, the drug has also been shown to block the effect of calcineurin (Olson and Williams, 2000; Taigen et al., 2000). However, it has previously been shown that CyA directly inhibits MPT opening in cardiac mitochondria exposed to high calcium concentrations (Javadov et al., 2003).
One of the possible mechanisms underlying the antihypertrophic effect of EMD may involve inhibition of [Ca2+]m. Indeed, the mechanism for the acute involvement of NHE-1 in myocardial injury likely involves enhanced intracellular Ca2+ (reviewed in Karmazyn et al., 2001; Avkiran and Marber, 2002; Linz and Busch, 2003), and NHE-1 inhibition has been shown to prevent oxidative stress-induced [Ca2+]m overload (Teshima et al., 2003). Whether reduced [Ca2+]m overload contributes to the antihypertrophic effect of NHE-1 inhibition needs to be determined, however, we have recently demonstrated that EMD administration to chronically postinfarcted rats decreased mitochondrial vulnerability to exogenous Ca2+ ex vivo (Javadov et al., 2005). In the present study, treatment of the cardiomyocytes with the mitochondrial Ca2+ uniport blocker RR attenuated the hypertrophic phenotype and abolished mitochondrial dysfunction induced by PE, which is presumably a consequence of the prevention of [Ca2+]m overload induced by PE.
It has previously been demonstrated that NHE-1 inhibition preserves the mitochondrial proton gradient and delays ATP depletion during ischemia (Ruiz-Meana et al., 2003), protects the Δψm during H2O2-induced oxidative stress (Teshima et al., 2003), inhibits MPT pore opening, and improves respiratory function during postinfarcted remodeling (Javadov et al., 2005). Antioxidant effects of various NHE-1 inhibitors have been demonstrated in terms of their ability to quench superoxide and hydroxyl radicals production in guinea pig ischemia-reperfused heart (Hotta et al., 2004). The results of the present study show that Δψm dissipation in PE-stimulated cells is accompanied by superoxide production and MPT pore opening, which were nearly completely abrogated by EMD. A close relationship between Δψm loss and MPT pore opening has been demonstrated previously in neonatal cardiomyocytes during oxidative stress (Akao et al., 2003). It is currently unknown whether Δψm loss from increased ROS production results in inner mitochondrial membrane depolarization and, subsequently, MPT pore opening, or whether MPT alone disrupts the outer membrane and causes a loss in Δψm. Studies with isolated mitochondria titrated with uncoupling agents to manipulate the Δψm showed that MPT pore opening is increased with increased depolarization (Petronilli et al., 1993). It was also demonstrated that mitochondrial depolarization inhibits adenine nucleotide binding to ANT, leading to increased sensitization of the MPT to Ca2+ (Halestrap and Brennerb, 2003). Therefore, Δψm loss most likely plays an important role in MPT pore opening when it is accompanied by ROS production and Ca2+ overload.
ROS have been shown to be increased during cardiac hypertrophy and heart failure and probably contribute to these processes, especially at high concentrations (Ide et al., 1999; Siwik et al., 1999; Sorescu and Griendling, 2002). It has been demonstrated that MAPKs are activated by ROS generated intracellularly (Tanaka et al., 2001; Kulisz et al., 2002) as well as by exogenous H2O2 (Tu et al., 2003; Purdom and Chen, 2005). Moreover, stimulation of cardiac α1-adrenoreceptors has been shown to induce MAPK activation partly mediated by intracellular ROS generation (Amin et al., 2001) and represented primarily by mitochondrial respiratory chain-derived (Brookes et al., 2002; Kulisz et al., 2002; Ramachandran et al., 2002). Our study indeed demonstrates that mitochondria represent a primary source of production in PE-treated cardiomyocytes with virtually no contribution by other intracellular -generating processes. Furthermore, the reduced PE-induced phosphorylation of p38 and ERK1/2 by catalase indicates an important role of ROS in the MAPK activation and cell growth. In the present study, EMD significantly attenuated p38 and ERK1/2 phosphorylation. This was a surprising observation because it is generally considered that ERK is a major regulator of NHE-1 activity in cardiomyocytes at least under ischemic conditions (Moor et al., 2001). Thus, it seems that there may exist a reciprocal relationship between these two systems, and under certain conditions, such as PE-induced hypertrophy, NHE-1 could represent an important regulator of MAPK activity. Indeed, the ability of NHE-1 to regulate ERK activity in vascular smooth muscle in response to angiotensin II and 5-hydroxytryptamine has been recently reported with NHE-1 inhibition, preventing ERK activation induced by these two agents (Mukhin et al., 2004). Although the precise mechanism by which NHE-1 inhibition could decrease MAPK phosphorylation is not known, a logical basis involves both a reduced [Ca2+]m overload and mitochondrial-derived ROS, because the effects were mimicked by both RR and catalase.
Based on our studies, a hypothesis regarding the interrelationship between NHE-1 and mitochondrial function in the hypertrophied cardiomyocyte is presented in Fig. 7. PE induced activation of NHE-1 via α1-receptor stimulation, and the resultant increase in intracellular Na+ concentrations would increase intracellular Ca2+ levels via Na+-Ca2+ exchange. Increased cytosolic Ca2+ would be taken up by mitochondria through an RR-sensitive Ca2+ uniporter, resulting in an elevation in [Ca2+]m followed by Δψm loss, which in turn results in increased MPT opening, the latter being further enhanced by direct effects of high Ca2+. Moreover, respiratory chain rotenone-sensitive complex I and myxothiazol-sensitive complex III-derived generation contributes to the overall ROS burden produced within the mitochondria directly or via its dismutation to H2O2. Because catalase was found to markedly inhibit MAPK activation in the present study, coupled with previous reports demonstrating stimulation of MAPK by exogenous H2O2 in cultured cardiomyocytes (Tu et al., 2003; Purdom and Chen, 2005), it is likely that MAPK stimulation due to PE addition reflects dismutation of to H2O2, resulting in a hypertrophic response.
In conclusion, our results demonstrate a novel pathway that mediates the antihypertrophic effect of NHE-1 inhibition in cardiac myocytes. It further demonstrates the importance of mitochondria to the hypertrophic process in terms of its ability to regulate MAPK activity through ROS generation. This study, as well as our previous report demonstrating an antihypertrophic effect of the mitochondrial KATP channel opener diazoxide (Xia et al., 2004), further reinforces the concept of mitochondrial regulation of the cardiomyocyte hypertrophic response, at least to PE. However, whether the results obtained with diazoxide reflect identical mechanisms described in the present report requires further studies. Interestingly, diazoxide-induced attenuation of hypertrophy was associated with diminished NHE-1 expression (Xia et al., 2004). Aside from providing novel insights into the antihypertrophic effects of NHE-1 inhibition, the present results further suggest that mitochondrial preservation may represent a therapeutic target for the attenuation of hypertrophic responses.
Footnotes
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This study was supported by the Canadian Institutes of Health Research. M.K. holds a Canada Research Chair in Experimental Cardiology. L.A.K. holds a Canada Research Chair in Molecular Cardiology.
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doi:10.1124/jpet.105.100107.
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ABBREVIATIONS: NHE-1, sodium-hydrogen exchanger-1; ROS, reactive oxygen species; , superoxide anion; SOD, superoxide dismutase; MAPK, mitogen-activated protein kinase; PE, phenylephrine; Δψm, mitochondrial membrane potential; MPT, mitochondrial permeability transition; EMD, N-[2-methyl-4,5-bis(methylsulphonyl)-benzoyl]-guanidine, hydrochloride (EMD87580); RR, ruthenium red; CyA, cyclosporin A; PCR, polymerase chain reaction; ANP, atrial natriuretic peptide; ERK1/2, extracellular signal-regulated kinase 1 and 2; AM, acetoxymethyl ester; DHE, dihydroethidium; ANT, adenine nucleotide translocase; Et-DNA, ethidium-DNA; l-NAME, Nω-nitro-l-arginine methyl ester; p38, p38 MAPK.
- Received December 16, 2005.
- Accepted March 1, 2006.
- The American Society for Pharmacology and Experimental Therapeutics