Original Research Received: June 17, 2014 Accepted after revision: November 20, 2014 Published online: February 25, 2015
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
Nitric Oxide Synthase Inhibition Abolishes Exercise-Mediated Protection against Isoproterenol-Induced Cardiac Hypertrophy in Female Mice Jiling Ren a Lei Yang b Wencong Tian b Mengmeng Zhu c Jie Liu b Ping Lu a Jing Li b Liang Yang b Zhi Qi c
a c
Department of Parasitology, Basic Medical College, Tianjin Medical University, and Departments of b Pharmacology and Histology and Embryology, School of Medicine, Nankai University, Tianjin, China
For editorial comment see p. 172
Abstract Objective: Exercise training (ET) provides a cardioprotective effect against pathological cardiac hypertrophy. Nitric oxide (NO) plays an important role in modulating cardiac hypertrophy. However, few studies explore the relationship between NO signaling and the inhibitory effect of ET on pathological cardiac remodeling. Methods: In this study, we evaluated ET effects on isoproterenol (ISO)-induced cardiac hypertrophy in female mice. Moreover, L-NAME (Nω-nitro-L-arginine methyl ester), a nonselective NO synthase (NOS) inhibitor, was used to assess the involvement of NO signaling in cardiac hypertrophy. Morphological and echocardiographic variables were assessed. Cardiac hypertrophy-related gene expression was detected by real-time PCR and the protein levels of NOS signaling molecules were determined by Western blot. Results: L-NAME treatment prevented the beneficial effects of ET against the increase in heart weight (HW)/body weight (BW), HW/tibia length and lung weight/BW and echocardiographic variables following ISO injection. Also, L-NAME co-administration reversed ET-induced inhibition of myocardial fibrosis and fetal gene reactivation in ISO-treated mice. Furthermore, L-
© 2015 S. Karger AG, Basel 0008–6312/15/1303–0175$39.50/0 E-Mail [email protected] www.karger.com/crd
NAME treatment prevented ET-mediated up-regulation of phosphorylated endothelial NOS and plasma NO in ISO-treated mice. Conclusions: Our findings demonstrate that L-NAME treatment could abolish ET-induced cardioprotection against pathological cardiac hypertrophy and that NOS modulation may be involved in the antihypertrophic effects induced by ET. © 2015 S. Karger AG, Basel
Introduction
Accumulating evidence has shown that exercise training (ET) could protect against pathological cardiac hypertrophy in animal models [1, 2]. Further studies also showed that regular ET could attenuate left ventricular (LV) remodeling and improve cardiac function in patients with heart failure [3, 4]. However, the exact mechanism remains to be elucidated. Previous studies demonstrated that nitric oxide (NO) plays an important role in preventing hypertrophy and inhibiting cardiac fibrosis. An increase in endothelial NO synthase (eNOS) signaling induced by drugs, such as calcium antagonists or angiotensin-I-converting enzyme inhibitors, was associated with improvements in pathological myocardial remodeling [5, 6]. Administration of the NO precursor L-arginine could attenuate cardiac hypertrophy Liang Yang or Zhi Qi School of Medicine Nankai University Tianjin 300071 (China) E-Mail yangliang @ nankai.edu.cn or qizhi @ nankai.edu.cn
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Key Words Cardiac hypertrophy · Endothelial nitric oxide synthase · Exercise training · Nitric oxide · L-NAME
ET (2.5 h/day, 4 weeks)
Week 1
ISO (50 mg/kg, 1 week)
Week 2
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Fig. 1. Time course of experimental interventions and assessments. A nonlinear scale is shown to enable better identification of the various interventions.
24 h 12 h
Echocardiography
Animals Female C57BL/6 mice (6–8 weeks old) were purchased from the Military Academy of the Medical Science Laboratory Animal Center (Beijing, China). The study was approved by the Institute Research Ethics Committee (Nankai University, permit No. 10011), and all animal experiments were performed strictly according the Nankai University guidelines for laboratory animals. The animals were maintained in a temperature-controlled room (22–25 ° C) under a 12-hour light:12-hour dark cycle with free access to water and standard mouse chow. All animals were anesthetized with diethyl ether before each experiment and all efforts were made to minimize their suffering.
Groups and Treatments After 1 week of acclimatization, mice were randomly divided into eight experimental groups: controls (Con; n = 12), ISO (n = 8), L-NAME (n = 6), ET (n = 8), ET in combination with ISO (n = 10),
ET in combination with L-NAME (n = 10), ISO in combination with L-NAME (n = 10) and ISO in combination with ET and L-NAME (n = 10). Mice in ET, ISO + ET, ET + L-NAME and ISO + ET + L-NAME groups were subjected to a program of running on the motor-driven running wheels (China). Mice ran six times a week for 4 weeks; initially, sessions lasted 1.5 h but were increased by 15 min each day to reach 2.5 h on day 5. For each session, the running wheel speed was 18 m/min. The exercise volume is about 2,700 m for each day. The other groups remained caged with free movement during the mouse running wheel experiments. During the 4 weeks of exercise L mice in the L-NAME, ET + L-NAME, ISO + L-NAME and ISO + ET + L-NAME groups were administered L-NAME dissolved in drinking water at a concentration of 100 mg/l. Water intake was measured daily by dividing the total consumption by the number of mice in each cage. The dose of L-NAME chosen does not affect blood pressure, as described previously [13]. During the last week of exercise, mice in the ISO, ISO + ET, ISO + L-NAME and ISO + ET + L-NAME groups were injected ISO (50 mg/kg dissolved in normal saline) subcutaneously once daily for 7 days to induce cardiac hypertrophy. Mice in the other groups were injected with the same volume of normal saline. The time course of experimental interventions and assessments is shown in figure 1. Echocardiography As described previously [14], transthoracic echocardiography was performed using the Vevo 2100 (VisualSonics, Toronto, Ont., Canada) echocardiograph with a 30-MHz linear signal transducer 18–24 h after the last ISO injection. Briefly, mice were anesthetized with isoflurane/oxygen, and averaged M-mode measurements from parasternal long-axis images were recorded. Interventricular septum (IVS) and LV posterior wall (LVPW) dimensions were taken in diastole and systole, in addition to LV internal dimensions (LVIDd and LVIDs, respectively). Fractional shortening (FS) was calculated as (LVIDd – LVIDs/LVIDd) × 100 and ejection fraction (EF) as (LVIDd3 – LVIDs3)/LVIDd3 × 100. Hematoxylin-Eosin Staining Mice were anesthetized and blood samples were collected from the aorta 36–48 h after the last ISO injection. Then they were sacrificed, hearts were immediately removed and left ventricles were fixed in 4% paraformaldehyde for 24 h at room temperature. The tissues were dehydrated by sequential washes with 70, 80, 90 and 100% ethanol and embedded in Paraplast X-tra tissue embedding medium (McCormick Scientific). Transversal sections (5 mm)
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Materials and Methods
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
Week 4
Morphological variables Blood sample Heart sample
in spontaneously hypertensive rats by increasing myocardial NO production [7]. Further studies showed that eNOS overexpression in cardiomyocytes could improve cardiac function and attenuate hypertrophy in heart failure after myocardial infarction or chronic isoproterenol (ISO) infusion [8–10]. Intriguingly, ET was also reported to lead to an increase in NO generation and storage of NO metabolites in the mouse heart [11, 12]. However, few studies explored the relationship between eNOS signaling and the inhibitory effect of ET on pathological cardiac remodeling. Thus, we hypothesize that ET may inhibit pathological cardiac remodeling via the activation of the NOS signaling pathway. In order to test this hypothesis, we assessed the effect of a nonselective NOS inhibitor, L-NAME (Nω-nitro-L-arginine methyl ester), on ET-induced attenuation of cardiac hypertrophy in mice. Moreover, the expression of all NOS isoforms [eNOS, neuronal NOS (nNOS) and inducible NOS (iNOS)], the phosphorylation status of eNOS and plasma NO levels were also analyzed after L-NAME co-administration in ET mice.
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Fig. 2. The effect of L-NAME and ET on myocardial hypertrophy induced by ISO injection in the study groups. Animals were sacrificed 24 h after the last injection, and hearts were removed,
trimmed and washed. a HW/BW ratio. b HW/TL ratio. c LW/BW ratio. Means ± SD. * p < 0.05, ISO + ET vs. ISO, ** p < 0.05, ISO + ET + L-NAME vs. ISO + ET.
were cut starting from the base area of the left ventricle at 40-mm intervals and stained with hematoxylin and eosin (HE) for cell morphometry. Percent fibrosis was determined using ImageJ. The results are presented as percent change in fibrosis per image area (not the whole heart) in 30 sections from 5 mice per group.
ized to 18s rRNA expression. A standard curve was run with the dilution series of the amplified fragment allowing for mRNA copy number calculation.
NO Measurements Plasma NO was measured by plasma nitrite plus nitrate values using a modified Griess reaction. In brief, blood samples drawn from the aorta were centrifuged at 370 g for 20 min at 4 ° C. The supernatants were extracted three times. A total of 50 μl of the samples were incubated with 50 μl of the Griess reagent (part I: 1% sulfanilamide; part II: 0.1% naphthylethylenediamine dihydrochloride and 2% phosphoric acid) at room temperature. Ten minutes later, absorbance was measured at 540 nm using an automatic plate reader, and NOx concentrations were calculated (expressed as mmol/l) using a standard curve of NOx from commercially available kits (Beyotime Institution of Biotechnology, China).
RT-PCR and Quantitative Real-Time PCR Assays Total RNA in LV samples was extracted with TRIzol reagent (Invitrogen, Shanghai, China). First-strand cDNAs were generated from RNA samples by reverse transcription (Promega, Shanghai, China). RT-PCR was performed in a Genemate thermal cycler (Jinge Instr., Hangzhou, China). The following primers were used: ANF (atrial natriuretic factor), 5′-GGGGGTAGGATTGACAGG AT-3′ and 5′-CTCCAGGAGGGTATTCACCA-3′ and 18s rRNA, 5′-ACCGCAGCTAGGAATAATGGA-3′ and 5′-GCCTCAGTTC CGAAAACCA-3′. Procollagen IαI, 5′-CCGCCATCAAGGTCTA CTGC-3′ and 5′-GAATCCATCGGTCATGCTCT-3′; procollagen III, 5′-CCCACAGCCTTCTACACCT-3′ and 5′-CCACCCATT CCTCCCAC-3′, and fibronectin, 5′-CCCACTAACCTCCAGTTT GTC-3′ and 5′-CTCTGCTGGTTCCCTTTCAC-3′. The SYBR Green master mix kit (Promega) was used to perform amplifications with the two-step protocol as described in a Bio-Rad iQ5 detection system. All real-time PCR sample reactions were normal-
Effect of Exercise on Cardiac Hypertrophy in Mice
Western Blot Analysis Frozen LV samples were homogenized and lysed in ice-cold RIPA buffer and a protease/phosphatase inhibitor cocktail (5,872; Cell Signaling Technology, Boston, Mass., USA). The protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, Rockford, Ill., USA). Samples containing 30 μg of the homogenate were resolved in 10% SDS-PAGE and transferred to polyvinylidene fluoride membrane (Millipore). The membrane was blocked in TBST/milk (5% nonfat dry milk, 10 mM Tris-HCl, pH 7.6, 150 mM NaCl and 0.1% Tween 20) for 1.5 h at room temperature and then incubated overnight at 4 ° C with antibodies against eNOS, phosphorylated eNOS at Thr1179, nNOS, iNOS (Cell Signaling Technology) and β-actin (Santa Cruz Biotechnology, Dallas, Tex., USA), followed by TBST washes. The membranes were then probed with appropriate secondary antibodies at room temperature for 60 min and washed with TBST. Band densities were quantified using the ImageJ program.
Statistical Analysis Quantitative data were expressed as means ± SD. Statistical analyses were performed using one-way analysis of variance followed by Bonferroni’s post hoc comparisons. A value of p < 0.05 was accepted as statistically significant.
Results L-NAME Inhibits ET-Mediated Protection against
ISO-Induced Cardiac Hypertrophy As shown in figure 2, the administration of ISO over a 1-week period resulted in significant increases in heart Cardiology 2015;130:175–184 DOI: 10.1159/000370025
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Fig. 3. The effect of L-NAME and ET on echocardiographic parameters following ISO injection. Echocardiography was performed during isoflurane/oxygen anesthesia 24 h after the last drug injection. Structural heart changes were determined at the mid-papillary level. a Representative long-axis parasternal echocardiograph-
ic images. b IVSd and LVPWd represent end-diastolic IVS and LVPW measurements, respectively. c LVIDd/LVIDs measurements. d FS = [(LVIDd – LVIDs/LVIDd) × 100] and EF = [(LVIDd3 – LVIDs3)/LVIDd3 × 100]. Means ± SD. * p < 0.05, ISO + ET vs. ISO, ** p < 0.05, ISO + ET + L-NAME vs. ISO + ET.
weight (HW)/body weight (BW), HW/tibia length (TL) and lung weight (LW)/BW ratios between the ISO and control groups (p < 0.01, Con vs. ISO groups). ET or L-NAME alone did not note have a marked effect on myocardial mass. As expected, ET significantly prevented the ISO-induced increase in normalized HW and LW/BW (p < 0.05, ET + ISO vs. ISO groups). However, this protective effect was significantly abolished after co-administration of L-NAME, which resulted in increased HW/ BW, HW/TL and LW/BW ratios (p < 0.05, ET + ISO vs. ET + ISO + L-NAME groups). In contrast, L-NAME administration did not further increase cardiac hypertrophy
in ISO mice (p > 0.05, ISO vs. ISO + L-NAME groups). These data demonstrated that the cardioprotective effect of exercise against pathological cardiac hypertrophy was attenuated after L-NAME treatment.
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L-NAME Treatment Reverses ET-Induced
Cardioprotective Effects on Echocardiographic Parameters Transthoracic echocardiography disclosed that ISO treatment caused significant increases in IVS and LVPW thickness compared to the control group (p < 0.01; fig. 3b). This was associated with an increase in LVIDd and LVIDs Ren/Yang/Tian/Zhu/Liu/Lu/Li/Yang/Qi
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(fig. 3c) and impairment in cardiac function as reflected by decreasing FS and EF (fig. 3d). Conversely, compared to the ISO group, there was a marked reduction in IVS thickness in the ISO + ET group, consistent with the attenuation of LVIDd and LVIDs and the improved FS and EF (p < 0.05, ISO + ET vs. ISO groups). However, L-NAME co-administration resulted in re-increases in IVS and LVPW thickness. An enlarged LV chamber size, which is reflected by a significant increase in LVIDd and LVIDs, was also found in the ISO + ET + L-NAME group. Moreover, FS and EF deterioration were observed by co-administration of L-NAME (p < 0.05, ISO + ET vs. ISO + ET + L-NAME groups; fig. 3). L-NAME treatment did not sig-
nificantly affect echocardiographic parameters compared to the ISO group (p > 0.05, ISO vs. ISO + L-NAME groups). These results further confirm that ET-induced protection from pathological cardiac hypertrophy is attenuated after co-administration with L-NAME.
Effect of Exercise on Cardiac Hypertrophy in Mice
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
L-NAME Treatment Inhibits ET-Mediated Protection
against ISO-Induced Myocardial Fibrosis and Fetal Gene Reactivation Subsequently, myocardial fibrosis and gene reactivation were measured by HE staining and real-time PCR separately. As shown in figure 4, in response to ISO treatment, ANF expression was 5-fold increased compared to the Con 179
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hypertrophy and myocardial fibrosis with chronic ISO injection. Quantification of the mRNA transcript abundance for ANF (a), fibronectin (b), procollagen IαI (c) and procollagen IIIαI (d). n =
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Fig. 4. The effect of L-NAME and ET on the markers of cellular
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pared to the ISO + ET group. Large areas of interstitial fibrosis were found in the ISO + ET + L-NAME group by HE staining (fig. 5). However, there were no significant differences in interstitial fibrosis or ANF expression between the ISO and ISO + L-NAME groups (fig. 5), suggesting that LNAME prevents the ET-mediated protection against ISOinduced myocardial fibrosis and fetal gene reactivation. Regulation of eNOS Phosphorylation and NO Production by L-NAME and ET in ISO-Induced Cardiac Hypertrophy Finally, we examined the effects of ET and L-NAME on the expression and phosphorylation status of NOS in Ren/Yang/Tian/Zhu/Liu/Lu/Li/Yang/Qi
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group. Also, a significant increase in the area of myocardial fibrosis was found by histology and the up-regulation of genes encoding procollagens IαI and IIIαI, and fibronectin in the ISO-treated mice. There were no significant changes in these measurements in the ET or L-NAME groups. In contrast, a significant decrease in ANF expression and down-regulation of procollagen IαI and IIIαI, and fibronectin were observed in the ISO + ET group compared to the ISO group. Correspondingly, there was less interstitial fibrosis in the ISO + ET group compared to the ISO group (fig. 5). In contrast, mice in the ISO + ET + LNAME group displayed significant up-regulation of ANF, procollagen IαI and IIIαI, and fibronectin expression com-
0
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photomicrographs of HE-dyed heart slices (original magnification ×100): white arrows indicate fibrotic tissue. b Quantification of fibrotic area. Means ± SD from 30 sections (5 mice/group). * p < 0.05, ISO + ET vs. ISO group, ** p < 0.05, ISO + ET + L-NAME vs. ISO + ET group.
Effect of Exercise on Cardiac Hypertrophy in Mice
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
Discussion
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The results of the present study show that L-NAME treatment could reverse the exercise-mediated cardiac protection on cardiac hypertrophy and fetal gene reactivation induced by ISO injection. Furthermore, the increases in eNOS phosphorylation and NO production induced by ET were abolished by L-NAME co-administration. These observations suggest that NOS modulation is involved in the antihypertrophic effect induced by ET. There are three NOS types present in the heart (i.e. nNOS, iNOS and eNOS), and all of them have been implicated in pathological cardiac remodeling associated with cardiac hypertrophy [15]. We found increased expression of eNOS in ISO-treated mice, while the status of phosphorylated eNOS at Ser1177 did not change significantly. This could increase output of eNOS-derived NO and may be a compensatory mechanism against excessive β-adrenergic stimulation. However, there is no significant increase in NO production in the ISO group. In contrast, a significant increase in the phosphorylation status of eNOS and NO production were observed in the ISO + ET group. Previous studies reported that exercise could
increase the expression of phosphorylated eNOS at Ser1177 without altering the expression of total eNOS, which subsequently increased the production and bioavailability of NO throughout the body and protected the heart from myocardial ischemia-reperfusion injury [12, 14]. Our study confirmed this finding and demonstrated that the elevation in the phosphorylation status of eNOS and increased NO production were associated with a reduction in cardiac hypertrophy induced by chronic ISO injection. On the other hand, iNOS expression was increased in failing left ventricles. In a study by Krenek et al. [16], upregulation of iNOS was an early event in ISO-induced heart failure. Further studies showed that a 2-week ISO infusion in mice led to iNOS induction in the heart, which was partially prevented by a selective iNOS inhibitor and almost normalized in iNOS-deficient mice [17, 18]. Our data showed that the expression of iNOS was up-regulated in the ISO group compared to the Con group. However, iNOS expression was decreased in the ISO + ET group compared to the ISO group, which indicates that ET induces down-regulation of iNOS expression. However, Quindry et al. [19] found that myocardial iNOS expression was not influenced by 10-day ET in male rats. The varied conclusion may be related to differences in species, gender and ET regimens. In this study, to assess the involvement of NO signaling in cardiac hypertrophy, L-NAME was used to inhibit NO production in ET mice. Previous studies revealed that chronic oral administration of L-NAME alone (400 mg/l) can cause arterial hypertension and morphological abnormalities, such as moderate diffuse myocardial degeneration and necrosis with inflammatory infiltration in heart muscles [20–22]. Thus, L-NAME was added to the drinking water at dose of 100 mg/l in this study, which was chosen in order not to affect blood pressure or cardiac hypertrophy [13]. We found that ET could protect from myocardial fibrosis and fetal gene reactivation following ISO-induced hypertrophy. L-NAME co-administration abolished the protective effect of ET by decreasing the phosphorylation status of eNOS and NO production. It seems that L-NAME and exercise have opposite effects on heart function by regulating the phosphorylation status of eNOS and NO production. However, a previous study in a rat model showed that high-dose L-NAME induced hypertension, and ET had blood pressure-lowering effects by increasing eNOS expression and NO levels in female rats [23]. These results indicate that L-NAME alone could not totally reverse ET-induced blood pressure-lowering effects, whereas L-NAME could reverse ET-mediated protection against ISO-induced cardiac hypotrophy.
ISO-induced cardiac hypertrophy in mice. As shown in figure 6, total eNOS levels were significantly increased in ISO- and L-NAME-treated mice. However, no significant change in eNOS phosphorylation was observed in these mice compared to the Con group. In contrast, phosphorylation of eNOS at serine residue 1,177 is significantly increased (phosphorylation here increases enzyme activity) but not the expression of total eNOS in the ISO + ET group. However, eNOS phosphorylation did not significantly change in the ISO + ET + L-NAME group (fig. 6c). Additionally, there was a pronounced increase in iNOS expression in the ISO and ISO + L-NAME group compared to the Con group (fig. 6d). The expression of nNOS remained unchanged in response to ET or ISO injection (fig. 6e). Furthermore, we evaluated the effects of ET on the levels of total NO in the plasma. As shown in figure 6f, a significant increase in NO levels was observed in the plasma of the ISO + ET group compared to the Con and ISO groups. However, this elevation was significantly attenuated in the ET + L-NAME and ISO + ET + L-NAME groups (vs. the ISO + ET group, p < 0.01). It seems that the effects of ET on the expression of NO metabolites were abolished by L-NAME. Those results suggest that NO metabolites may be responsible for the cardioprotective effects of ET.
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phorylated eNOS at Ser1177 (c), iNOS (d) and nNOS (e) following 4 weeks of ET and/or L-NAME administration. f NOx production was quantified by measurements of nitrite and nitrate in mouse plasma. Means ± SD. * p < 0.05 vs. Con, ** p < 0.05 vs. ISO.
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Fig. 6. The effect of L-NAME and ET on eNOS, phosphorylated
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
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It is evident that chronic L-NAME administration could inhibit cardiac NOS activity and serum NOx levels [22, 24]. In our study, NOx levels were decreased but eNOS and nNOS levels were increased in L-NAME- and ISO-treated mice. Similar results have been reported before: rodents treated with different L-NAME doses (from 1 to 50 mg·kg–1·day–1) for different durations (2–60 days) may enhance compensatory eNOS [25], nNOS [26] and iNOS [26, 27] expression levels in the myocardium. Together, these results suggest that increased NOS expression by L-NAME treatment for days or weeks may be caused as a compensatory mechanism. In addition, a significant increase in eNOS phosphorylation was observed in the ISO + ET group. However, eNOS phosphorylation did not result in a marked change in the ISO + ET + L-NAME group. L-NAME is an L-arginine analog and should not, in theory, directly interfere with eNOS phosphorylation. Previous studies showed that 15 min after L-NAME treatment, eNOS phosphorylation did not significantly change in mice [28], which indicates that L-NAME has no direct effect on eNOS phosphorylation. It seems that the decrease in eNOS phosphorylation may
be related to both ISO and L-NAME. Further investigation is necessary to explain the interaction between the ET, L-NAME and ISO effects on cardiac eNOS phosphorylation. In conclusion, the current study demonstrates that 4 weeks of ET provides cardioprotection against cardiac hypertrophy induced by ISO injection, and this protective effect was lost after L-NAME co-injection. It seemed that the increase in eNOS signaling and NO production may contribute to the protective effect of exercise. However, the regulation of NOS-derived NO is complex and different NOS isoforms have independent, and even opposite, NO signaling effects. Further studies are needed to identify the role of NOS and NO on cardiac hypotrophy.
Acknowledgment This research was supported by the National Natural Science Foundation of China (81102436 to L.Y. and No. 81201206 to Z.Q; http://www.nsfc.gov.cn/nsfc/cen/bsdt/jggb.html) and the Natural Science Foundation of Tianjin (12JCQNJC08300 to L.Y.; http:// www.tstc.gov.cn/).
1 Serra AJ, Higuchi ML, Ihara SS, Antonio EL, Santos MH, Bombig MT, Tucci PJ: Exercise training prevents beta-adrenergic hyperactivity-induced myocardial hypertrophy and lesions. Eur J Heart Fail 2008; 10: 534–539. 2 Serra AJ, Santos MH, Bocalini DS, Antonio EL, Levy RF, Santos AA, Higuchi ML, Silva JA, Magalhaes FC, Barauna VG, et al: Exercise training inhibits inflammatory cytokines and more than prevents myocardial dysfunction in rats with sustained beta-adrenergic hyperactivity. J Physiol 2010;588:2431–2442. 3 Giannuzzi P, Temporelli PL, Corra U, Tavazzi L; ELVD-CHF Study Group: Antiremodeling effect of long-term exercise training in patients with stable chronic heart failure: results of the Exercise in Left Ventricular Dysfunction and Chronic Heart Failure (ELVD-CHF) Trial. Circulation 2003;108:554–559. 4 Andersen K, Jonsdottir S, Sigurethsson AF, Sigurethsson SB: The effect of physical training in chronic heart failure (in Icelandic). Laeknabladid 2006;92:759–764. 5 Sanada S, Node K, Minamino T, Takashima S, Ogai A, Asanuma H, Ogita H, Liao Y, Asakura M, Kim J, et al: Long-acting Ca2+ blockers prevent myocardial remodeling induced by chronic NO inhibition in rats. Hypertension 2003;41:963–967. 6 Linz W, Wohlfart P, Scholkens BA, Malinski T, Wiemer G: Interactions among ACE, ki-
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7
8
9
10
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nins and NO. Cardiovasc Res 1999; 43: 549– 561. Matsuoka H, Nakata M, Kohno K, Koga Y, Nomura G, Toshima H, Imaizumi T: Chronic L-arginine administration attenuates cardiac hypertrophy in spontaneously hypertensive rats. Hypertension 1996;27:14–18. Ozaki M, Kawashima S, Yamashita T, Hirase T, Ohashi Y, Inoue N, Hirata K, Yokoyama M: Overexpression of endothelial nitric oxide synthase attenuates cardiac hypertrophy induced by chronic isoproterenol infusion. Circ J 2002;66:851–856. Massion PB, Dessy C, Desjardins F, Pelat M, Havaux X, Belge C, Moulin P, Guiot Y, Feron O, Janssens S, Balligand JL: Cardiomyocyterestricted overexpression of endothelial nitric oxide synthase (NOS3) attenuates beta-adrenergic stimulation and reinforces vagal inhibition of cardiac contraction. Circulation 2004;110:2666–2672. Jones SP, Greer JJ, van Haperen R, Duncker DJ, de Crom R, Lefer DJ: Endothelial nitric oxide synthase overexpression attenuates congestive heart failure in mice. Proc Natl Acad Sci U S A 2003;100:4891–4896. Napoli C, Williams-Ignarro S, De Nigris F, Lerman LO, Rossi L, Guarino C, Mansueto G, Di Tuoro F, Pignalosa O, De Rosa G, et al: Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic
12
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15 16
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mice. Proc Natl Acad Sci U S A 2004; 101: 8797–8802. Calvert JW, Condit ME, Aragon JP, Nicholson CK, Moody BF, Hood RL, Sindler AL, Gundewar S, Seals DR, Barouch LA, Lefer DJ: Exercise protects against myocardial ischemia-reperfusion injury via stimulation of beta(3)-adrenergic receptors and increased nitric oxide signaling: role of nitrite and nitrosothiols. Circ Res 2011;108:1448–1458. Kurisu S, Ozono R, Oshima T, Kambe M, Ishida T, Sugino H, Matsuura H, Chayama K, Teranishi Y, Iba O, et al: Cardiac angiotensin II type 2 receptor activates the kinin/NO system and inhibits fibrosis. Hypertension 2003; 41:99–107. Yang L, Jia Z, Zhu M, Zhang J, Liu J, Wu P, Tian W, Li J, Qi Z, Tang X: Exercise protects against chronic beta-adrenergic remodeling of the heart by activation of endothelial nitric oxide synthase. PLoS One 2014; 9:e96892. Drexler H: Nitric oxide synthases in the failing human heart: a doubled-edged sword? Circulation 1999;99:2972–2975. Krenek P, Kmecova J, Kucerova D, Bajuszova Z, Musil P, Gazova A, Ochodnicky P, Klimas J, Kyselovic J: Isoproterenol-induced heart failure in the rat is associated with nitric oxide-dependent functional alterations of cardiac function. Eur J Heart Fail 2009; 11: 140– 146.
183
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References
184
21 Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R: Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension 1992;20:298–303. 22 Pfeiffer S, Leopold E, Schmidt K, Brunner F, Mayer B: Inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME): requirement for bioactivation to the free acid, NG-nitro-L-arginine. Br J Pharmacol 1996; 118: 1433–1440. 23 Kuru O, Senturk UK, Kocer G, Ozdem S, Baskurt OK, Cetin A, Yesilkaya A, Gunduz F: Effect of exercise training on resistance arteries in rats with chronic NOS inhibition. J Appl Physiol (1985) 2009;107:896–902. 24 Vergely C, Perrin-Sarrado C, Clermont G, Rochette L: Postischemic recovery and oxidative stress are independent of nitric-oxide synthases modulation in isolated rat heart. J Pharmacol Exp Ther 2002;303:149–157.
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
25 Sampaio RC, Tanus-Santos JE, Melo SE, Hyslop S, Franchini KG, Luca IM, Moreno H Jr: Hypertension plus diabetes mimics the cardiomyopathy induced by nitric oxide inhibition in rats. Chest 2002;122:1412–1420. 26 Suda O, Tsutsui M, Morishita T, Tanimoto A, Horiuchi M, Tasaki H, Huang PL, Sasaguri Y, Yanagihara N, Nakashima Y: Long-term treatment with N(omega)-nitro-L-arginine methyl ester causes arteriosclerotic coronary lesions in endothelial nitric oxide synthasedeficient mice. Circulation 2002; 106: 1729– 1735. 27 Darblade B, Batkai S, Causse E, Gourdy P, Fouque MJ, Rami J, Arnal JF: Failure of L-nitroarginine to inhibit the activity of aortic inducible nitric oxide synthase. J Vasc Res 2001; 38:266–275. 28 Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, Ma XL: Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation 2002;105: 1497–1502.
Ren/Yang/Tian/Zhu/Liu/Lu/Li/Yang/Qi
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17 Zhang P, Xu X, Hu X, van Deel ED, Zhu G, Chen Y: Inducible nitric oxide synthase deficiency protects the heart from systolic overload-induced ventricular hypertrophy and congestive heart failure. Circ Res 2007; 100: 1089–1098. 18 Hu A, Jiao X, Gao E, Koch WJ, Sharifi-Azad S, Grunwald Z, Ma XL, Sun JZ: Chronic betaadrenergic receptor stimulation induces cardiac apoptosis and aggravates myocardial ischemia/reperfusion injury by provoking inducible nitric-oxide synthase-mediated nitrative stress. J Pharmacol Exp Ther 2006; 318: 469–475. 19 Quindry JC, French J, Hamilton KL, Lee Y, Selsby J, Powers S: Exercise does not increase cyclooxygenase-2 myocardial levels in young or senescent hearts. J Physiol Sci 2010; 60: 181–186. 20 Bachhav SS, Bhutada MS, Patil SD, Baser B, Chaudhari KB: Effect of Viscum articulatum Burm. (Loranthaceae) in Nω-nitro-L-arginine methyl ester induced hypertension and renal dysfunction. J Ethnopharmacol 2012; 142: 467–473.
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
Nitric Oxide Synthase Inhibition Abolishes Exercise-Mediated Protection against Isoproterenol-Induced Cardiac Hypertrophy in Female Mice Jiling Ren a Lei Yang b Wencong Tian b Mengmeng Zhu c Jie Liu b Ping Lu a Jing Li b Liang Yang b Zhi Qi c
a c
Department of Parasitology, Basic Medical College, Tianjin Medical University, and Departments of b Pharmacology and Histology and Embryology, School of Medicine, Nankai University, Tianjin, China
For editorial comment see p. 172
Abstract Objective: Exercise training (ET) provides a cardioprotective effect against pathological cardiac hypertrophy. Nitric oxide (NO) plays an important role in modulating cardiac hypertrophy. However, few studies explore the relationship between NO signaling and the inhibitory effect of ET on pathological cardiac remodeling. Methods: In this study, we evaluated ET effects on isoproterenol (ISO)-induced cardiac hypertrophy in female mice. Moreover, L-NAME (Nω-nitro-L-arginine methyl ester), a nonselective NO synthase (NOS) inhibitor, was used to assess the involvement of NO signaling in cardiac hypertrophy. Morphological and echocardiographic variables were assessed. Cardiac hypertrophy-related gene expression was detected by real-time PCR and the protein levels of NOS signaling molecules were determined by Western blot. Results: L-NAME treatment prevented the beneficial effects of ET against the increase in heart weight (HW)/body weight (BW), HW/tibia length and lung weight/BW and echocardiographic variables following ISO injection. Also, L-NAME co-administration reversed ET-induced inhibition of myocardial fibrosis and fetal gene reactivation in ISO-treated mice. Furthermore, L-
© 2015 S. Karger AG, Basel 0008–6312/15/1303–0175$39.50/0 E-Mail [email protected] www.karger.com/crd
NAME treatment prevented ET-mediated up-regulation of phosphorylated endothelial NOS and plasma NO in ISO-treated mice. Conclusions: Our findings demonstrate that L-NAME treatment could abolish ET-induced cardioprotection against pathological cardiac hypertrophy and that NOS modulation may be involved in the antihypertrophic effects induced by ET. © 2015 S. Karger AG, Basel
Introduction
Accumulating evidence has shown that exercise training (ET) could protect against pathological cardiac hypertrophy in animal models [1, 2]. Further studies also showed that regular ET could attenuate left ventricular (LV) remodeling and improve cardiac function in patients with heart failure [3, 4]. However, the exact mechanism remains to be elucidated. Previous studies demonstrated that nitric oxide (NO) plays an important role in preventing hypertrophy and inhibiting cardiac fibrosis. An increase in endothelial NO synthase (eNOS) signaling induced by drugs, such as calcium antagonists or angiotensin-I-converting enzyme inhibitors, was associated with improvements in pathological myocardial remodeling [5, 6]. Administration of the NO precursor L-arginine could attenuate cardiac hypertrophy Liang Yang or Zhi Qi School of Medicine Nankai University Tianjin 300071 (China) E-Mail yangliang @ nankai.edu.cn or qizhi @ nankai.edu.cn
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Key Words Cardiac hypertrophy · Endothelial nitric oxide synthase · Exercise training · Nitric oxide · L-NAME
ET (2.5 h/day, 4 weeks)
Week 1
ISO (50 mg/kg, 1 week)
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L-NAME intake (100 mg/l in water, 4 weeks)
Fig. 1. Time course of experimental interventions and assessments. A nonlinear scale is shown to enable better identification of the various interventions.
24 h 12 h
Echocardiography
Animals Female C57BL/6 mice (6–8 weeks old) were purchased from the Military Academy of the Medical Science Laboratory Animal Center (Beijing, China). The study was approved by the Institute Research Ethics Committee (Nankai University, permit No. 10011), and all animal experiments were performed strictly according the Nankai University guidelines for laboratory animals. The animals were maintained in a temperature-controlled room (22–25 ° C) under a 12-hour light:12-hour dark cycle with free access to water and standard mouse chow. All animals were anesthetized with diethyl ether before each experiment and all efforts were made to minimize their suffering.
Groups and Treatments After 1 week of acclimatization, mice were randomly divided into eight experimental groups: controls (Con; n = 12), ISO (n = 8), L-NAME (n = 6), ET (n = 8), ET in combination with ISO (n = 10),
ET in combination with L-NAME (n = 10), ISO in combination with L-NAME (n = 10) and ISO in combination with ET and L-NAME (n = 10). Mice in ET, ISO + ET, ET + L-NAME and ISO + ET + L-NAME groups were subjected to a program of running on the motor-driven running wheels (China). Mice ran six times a week for 4 weeks; initially, sessions lasted 1.5 h but were increased by 15 min each day to reach 2.5 h on day 5. For each session, the running wheel speed was 18 m/min. The exercise volume is about 2,700 m for each day. The other groups remained caged with free movement during the mouse running wheel experiments. During the 4 weeks of exercise L mice in the L-NAME, ET + L-NAME, ISO + L-NAME and ISO + ET + L-NAME groups were administered L-NAME dissolved in drinking water at a concentration of 100 mg/l. Water intake was measured daily by dividing the total consumption by the number of mice in each cage. The dose of L-NAME chosen does not affect blood pressure, as described previously [13]. During the last week of exercise, mice in the ISO, ISO + ET, ISO + L-NAME and ISO + ET + L-NAME groups were injected ISO (50 mg/kg dissolved in normal saline) subcutaneously once daily for 7 days to induce cardiac hypertrophy. Mice in the other groups were injected with the same volume of normal saline. The time course of experimental interventions and assessments is shown in figure 1. Echocardiography As described previously [14], transthoracic echocardiography was performed using the Vevo 2100 (VisualSonics, Toronto, Ont., Canada) echocardiograph with a 30-MHz linear signal transducer 18–24 h after the last ISO injection. Briefly, mice were anesthetized with isoflurane/oxygen, and averaged M-mode measurements from parasternal long-axis images were recorded. Interventricular septum (IVS) and LV posterior wall (LVPW) dimensions were taken in diastole and systole, in addition to LV internal dimensions (LVIDd and LVIDs, respectively). Fractional shortening (FS) was calculated as (LVIDd – LVIDs/LVIDd) × 100 and ejection fraction (EF) as (LVIDd3 – LVIDs3)/LVIDd3 × 100. Hematoxylin-Eosin Staining Mice were anesthetized and blood samples were collected from the aorta 36–48 h after the last ISO injection. Then they were sacrificed, hearts were immediately removed and left ventricles were fixed in 4% paraformaldehyde for 24 h at room temperature. The tissues were dehydrated by sequential washes with 70, 80, 90 and 100% ethanol and embedded in Paraplast X-tra tissue embedding medium (McCormick Scientific). Transversal sections (5 mm)
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Materials and Methods
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
Week 4
Morphological variables Blood sample Heart sample
in spontaneously hypertensive rats by increasing myocardial NO production [7]. Further studies showed that eNOS overexpression in cardiomyocytes could improve cardiac function and attenuate hypertrophy in heart failure after myocardial infarction or chronic isoproterenol (ISO) infusion [8–10]. Intriguingly, ET was also reported to lead to an increase in NO generation and storage of NO metabolites in the mouse heart [11, 12]. However, few studies explored the relationship between eNOS signaling and the inhibitory effect of ET on pathological cardiac remodeling. Thus, we hypothesize that ET may inhibit pathological cardiac remodeling via the activation of the NOS signaling pathway. In order to test this hypothesis, we assessed the effect of a nonselective NOS inhibitor, L-NAME (Nω-nitro-L-arginine methyl ester), on ET-induced attenuation of cardiac hypertrophy in mice. Moreover, the expression of all NOS isoforms [eNOS, neuronal NOS (nNOS) and inducible NOS (iNOS)], the phosphorylation status of eNOS and plasma NO levels were also analyzed after L-NAME co-administration in ET mice.
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Fig. 2. The effect of L-NAME and ET on myocardial hypertrophy induced by ISO injection in the study groups. Animals were sacrificed 24 h after the last injection, and hearts were removed,
trimmed and washed. a HW/BW ratio. b HW/TL ratio. c LW/BW ratio. Means ± SD. * p < 0.05, ISO + ET vs. ISO, ** p < 0.05, ISO + ET + L-NAME vs. ISO + ET.
were cut starting from the base area of the left ventricle at 40-mm intervals and stained with hematoxylin and eosin (HE) for cell morphometry. Percent fibrosis was determined using ImageJ. The results are presented as percent change in fibrosis per image area (not the whole heart) in 30 sections from 5 mice per group.
ized to 18s rRNA expression. A standard curve was run with the dilution series of the amplified fragment allowing for mRNA copy number calculation.
NO Measurements Plasma NO was measured by plasma nitrite plus nitrate values using a modified Griess reaction. In brief, blood samples drawn from the aorta were centrifuged at 370 g for 20 min at 4 ° C. The supernatants were extracted three times. A total of 50 μl of the samples were incubated with 50 μl of the Griess reagent (part I: 1% sulfanilamide; part II: 0.1% naphthylethylenediamine dihydrochloride and 2% phosphoric acid) at room temperature. Ten minutes later, absorbance was measured at 540 nm using an automatic plate reader, and NOx concentrations were calculated (expressed as mmol/l) using a standard curve of NOx from commercially available kits (Beyotime Institution of Biotechnology, China).
RT-PCR and Quantitative Real-Time PCR Assays Total RNA in LV samples was extracted with TRIzol reagent (Invitrogen, Shanghai, China). First-strand cDNAs were generated from RNA samples by reverse transcription (Promega, Shanghai, China). RT-PCR was performed in a Genemate thermal cycler (Jinge Instr., Hangzhou, China). The following primers were used: ANF (atrial natriuretic factor), 5′-GGGGGTAGGATTGACAGG AT-3′ and 5′-CTCCAGGAGGGTATTCACCA-3′ and 18s rRNA, 5′-ACCGCAGCTAGGAATAATGGA-3′ and 5′-GCCTCAGTTC CGAAAACCA-3′. Procollagen IαI, 5′-CCGCCATCAAGGTCTA CTGC-3′ and 5′-GAATCCATCGGTCATGCTCT-3′; procollagen III, 5′-CCCACAGCCTTCTACACCT-3′ and 5′-CCACCCATT CCTCCCAC-3′, and fibronectin, 5′-CCCACTAACCTCCAGTTT GTC-3′ and 5′-CTCTGCTGGTTCCCTTTCAC-3′. The SYBR Green master mix kit (Promega) was used to perform amplifications with the two-step protocol as described in a Bio-Rad iQ5 detection system. All real-time PCR sample reactions were normal-
Effect of Exercise on Cardiac Hypertrophy in Mice
Western Blot Analysis Frozen LV samples were homogenized and lysed in ice-cold RIPA buffer and a protease/phosphatase inhibitor cocktail (5,872; Cell Signaling Technology, Boston, Mass., USA). The protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, Rockford, Ill., USA). Samples containing 30 μg of the homogenate were resolved in 10% SDS-PAGE and transferred to polyvinylidene fluoride membrane (Millipore). The membrane was blocked in TBST/milk (5% nonfat dry milk, 10 mM Tris-HCl, pH 7.6, 150 mM NaCl and 0.1% Tween 20) for 1.5 h at room temperature and then incubated overnight at 4 ° C with antibodies against eNOS, phosphorylated eNOS at Thr1179, nNOS, iNOS (Cell Signaling Technology) and β-actin (Santa Cruz Biotechnology, Dallas, Tex., USA), followed by TBST washes. The membranes were then probed with appropriate secondary antibodies at room temperature for 60 min and washed with TBST. Band densities were quantified using the ImageJ program.
Statistical Analysis Quantitative data were expressed as means ± SD. Statistical analyses were performed using one-way analysis of variance followed by Bonferroni’s post hoc comparisons. A value of p < 0.05 was accepted as statistically significant.
Results L-NAME Inhibits ET-Mediated Protection against
ISO-Induced Cardiac Hypertrophy As shown in figure 2, the administration of ISO over a 1-week period resulted in significant increases in heart Cardiology 2015;130:175–184 DOI: 10.1159/000370025
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ic images. b IVSd and LVPWd represent end-diastolic IVS and LVPW measurements, respectively. c LVIDd/LVIDs measurements. d FS = [(LVIDd – LVIDs/LVIDd) × 100] and EF = [(LVIDd3 – LVIDs3)/LVIDd3 × 100]. Means ± SD. * p < 0.05, ISO + ET vs. ISO, ** p < 0.05, ISO + ET + L-NAME vs. ISO + ET.
weight (HW)/body weight (BW), HW/tibia length (TL) and lung weight (LW)/BW ratios between the ISO and control groups (p < 0.01, Con vs. ISO groups). ET or L-NAME alone did not note have a marked effect on myocardial mass. As expected, ET significantly prevented the ISO-induced increase in normalized HW and LW/BW (p < 0.05, ET + ISO vs. ISO groups). However, this protective effect was significantly abolished after co-administration of L-NAME, which resulted in increased HW/ BW, HW/TL and LW/BW ratios (p < 0.05, ET + ISO vs. ET + ISO + L-NAME groups). In contrast, L-NAME administration did not further increase cardiac hypertrophy
in ISO mice (p > 0.05, ISO vs. ISO + L-NAME groups). These data demonstrated that the cardioprotective effect of exercise against pathological cardiac hypertrophy was attenuated after L-NAME treatment.
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L-NAME Treatment Reverses ET-Induced
Cardioprotective Effects on Echocardiographic Parameters Transthoracic echocardiography disclosed that ISO treatment caused significant increases in IVS and LVPW thickness compared to the control group (p < 0.01; fig. 3b). This was associated with an increase in LVIDd and LVIDs Ren/Yang/Tian/Zhu/Liu/Lu/Li/Yang/Qi
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(fig. 3c) and impairment in cardiac function as reflected by decreasing FS and EF (fig. 3d). Conversely, compared to the ISO group, there was a marked reduction in IVS thickness in the ISO + ET group, consistent with the attenuation of LVIDd and LVIDs and the improved FS and EF (p < 0.05, ISO + ET vs. ISO groups). However, L-NAME co-administration resulted in re-increases in IVS and LVPW thickness. An enlarged LV chamber size, which is reflected by a significant increase in LVIDd and LVIDs, was also found in the ISO + ET + L-NAME group. Moreover, FS and EF deterioration were observed by co-administration of L-NAME (p < 0.05, ISO + ET vs. ISO + ET + L-NAME groups; fig. 3). L-NAME treatment did not sig-
nificantly affect echocardiographic parameters compared to the ISO group (p > 0.05, ISO vs. ISO + L-NAME groups). These results further confirm that ET-induced protection from pathological cardiac hypertrophy is attenuated after co-administration with L-NAME.
Effect of Exercise on Cardiac Hypertrophy in Mice
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
L-NAME Treatment Inhibits ET-Mediated Protection
against ISO-Induced Myocardial Fibrosis and Fetal Gene Reactivation Subsequently, myocardial fibrosis and gene reactivation were measured by HE staining and real-time PCR separately. As shown in figure 4, in response to ISO treatment, ANF expression was 5-fold increased compared to the Con 179
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hypertrophy and myocardial fibrosis with chronic ISO injection. Quantification of the mRNA transcript abundance for ANF (a), fibronectin (b), procollagen IαI (c) and procollagen IIIαI (d). n =
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pared to the ISO + ET group. Large areas of interstitial fibrosis were found in the ISO + ET + L-NAME group by HE staining (fig. 5). However, there were no significant differences in interstitial fibrosis or ANF expression between the ISO and ISO + L-NAME groups (fig. 5), suggesting that LNAME prevents the ET-mediated protection against ISOinduced myocardial fibrosis and fetal gene reactivation. Regulation of eNOS Phosphorylation and NO Production by L-NAME and ET in ISO-Induced Cardiac Hypertrophy Finally, we examined the effects of ET and L-NAME on the expression and phosphorylation status of NOS in Ren/Yang/Tian/Zhu/Liu/Lu/Li/Yang/Qi
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group. Also, a significant increase in the area of myocardial fibrosis was found by histology and the up-regulation of genes encoding procollagens IαI and IIIαI, and fibronectin in the ISO-treated mice. There were no significant changes in these measurements in the ET or L-NAME groups. In contrast, a significant decrease in ANF expression and down-regulation of procollagen IαI and IIIαI, and fibronectin were observed in the ISO + ET group compared to the ISO group. Correspondingly, there was less interstitial fibrosis in the ISO + ET group compared to the ISO group (fig. 5). In contrast, mice in the ISO + ET + LNAME group displayed significant up-regulation of ANF, procollagen IαI and IIIαI, and fibronectin expression com-
0
Co
photomicrographs of HE-dyed heart slices (original magnification ×100): white arrows indicate fibrotic tissue. b Quantification of fibrotic area. Means ± SD from 30 sections (5 mice/group). * p < 0.05, ISO + ET vs. ISO group, ** p < 0.05, ISO + ET + L-NAME vs. ISO + ET group.
Effect of Exercise on Cardiac Hypertrophy in Mice
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Discussion
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The results of the present study show that L-NAME treatment could reverse the exercise-mediated cardiac protection on cardiac hypertrophy and fetal gene reactivation induced by ISO injection. Furthermore, the increases in eNOS phosphorylation and NO production induced by ET were abolished by L-NAME co-administration. These observations suggest that NOS modulation is involved in the antihypertrophic effect induced by ET. There are three NOS types present in the heart (i.e. nNOS, iNOS and eNOS), and all of them have been implicated in pathological cardiac remodeling associated with cardiac hypertrophy [15]. We found increased expression of eNOS in ISO-treated mice, while the status of phosphorylated eNOS at Ser1177 did not change significantly. This could increase output of eNOS-derived NO and may be a compensatory mechanism against excessive β-adrenergic stimulation. However, there is no significant increase in NO production in the ISO group. In contrast, a significant increase in the phosphorylation status of eNOS and NO production were observed in the ISO + ET group. Previous studies reported that exercise could
increase the expression of phosphorylated eNOS at Ser1177 without altering the expression of total eNOS, which subsequently increased the production and bioavailability of NO throughout the body and protected the heart from myocardial ischemia-reperfusion injury [12, 14]. Our study confirmed this finding and demonstrated that the elevation in the phosphorylation status of eNOS and increased NO production were associated with a reduction in cardiac hypertrophy induced by chronic ISO injection. On the other hand, iNOS expression was increased in failing left ventricles. In a study by Krenek et al. [16], upregulation of iNOS was an early event in ISO-induced heart failure. Further studies showed that a 2-week ISO infusion in mice led to iNOS induction in the heart, which was partially prevented by a selective iNOS inhibitor and almost normalized in iNOS-deficient mice [17, 18]. Our data showed that the expression of iNOS was up-regulated in the ISO group compared to the Con group. However, iNOS expression was decreased in the ISO + ET group compared to the ISO group, which indicates that ET induces down-regulation of iNOS expression. However, Quindry et al. [19] found that myocardial iNOS expression was not influenced by 10-day ET in male rats. The varied conclusion may be related to differences in species, gender and ET regimens. In this study, to assess the involvement of NO signaling in cardiac hypertrophy, L-NAME was used to inhibit NO production in ET mice. Previous studies revealed that chronic oral administration of L-NAME alone (400 mg/l) can cause arterial hypertension and morphological abnormalities, such as moderate diffuse myocardial degeneration and necrosis with inflammatory infiltration in heart muscles [20–22]. Thus, L-NAME was added to the drinking water at dose of 100 mg/l in this study, which was chosen in order not to affect blood pressure or cardiac hypertrophy [13]. We found that ET could protect from myocardial fibrosis and fetal gene reactivation following ISO-induced hypertrophy. L-NAME co-administration abolished the protective effect of ET by decreasing the phosphorylation status of eNOS and NO production. It seems that L-NAME and exercise have opposite effects on heart function by regulating the phosphorylation status of eNOS and NO production. However, a previous study in a rat model showed that high-dose L-NAME induced hypertension, and ET had blood pressure-lowering effects by increasing eNOS expression and NO levels in female rats [23]. These results indicate that L-NAME alone could not totally reverse ET-induced blood pressure-lowering effects, whereas L-NAME could reverse ET-mediated protection against ISO-induced cardiac hypotrophy.
ISO-induced cardiac hypertrophy in mice. As shown in figure 6, total eNOS levels were significantly increased in ISO- and L-NAME-treated mice. However, no significant change in eNOS phosphorylation was observed in these mice compared to the Con group. In contrast, phosphorylation of eNOS at serine residue 1,177 is significantly increased (phosphorylation here increases enzyme activity) but not the expression of total eNOS in the ISO + ET group. However, eNOS phosphorylation did not significantly change in the ISO + ET + L-NAME group (fig. 6c). Additionally, there was a pronounced increase in iNOS expression in the ISO and ISO + L-NAME group compared to the Con group (fig. 6d). The expression of nNOS remained unchanged in response to ET or ISO injection (fig. 6e). Furthermore, we evaluated the effects of ET on the levels of total NO in the plasma. As shown in figure 6f, a significant increase in NO levels was observed in the plasma of the ISO + ET group compared to the Con and ISO groups. However, this elevation was significantly attenuated in the ET + L-NAME and ISO + ET + L-NAME groups (vs. the ISO + ET group, p < 0.01). It seems that the effects of ET on the expression of NO metabolites were abolished by L-NAME. Those results suggest that NO metabolites may be responsible for the cardioprotective effects of ET.
1.5
*
eNOS (relative intensity)
eNOS eNOS-PS1177 iNOS nNOS
*
*
1.0
0.5
DŽ-Actin
5.0
iNOS (relative intensity)
3.0
*
2.0 1.0 0
*
1.5
1.0
0.5
E + E N T AM + E O
IS
L-
E
AM N
AM + IS
O
+ ET
L-
N
+
L-
O IS
L-
d
p = n.s.
ET
ET
E AM N
Co
IS O
n
E + E N T AM + E O
IS
IS
ET
O
L-
E
AM
N
AM
L-
+
L-
+
IS
L-
O
N
+
ET
ET
E AM N
Co
IS O
0
1.5
40
**
**
30
Plasma NOx (nm)
1.0
0.5
0
20
10
eNOS at Ser1177 (eNOS-PS1177), iNOS and nNOS expression and plasma NO in mice with chronic ISO injection. Representative immunoblots (a) and densitometric analysis of total eNOS (b), phos-
E + E N T AM + E
IS
L-
O
AM
N
E AM IS O
+ ET
+
L-
L-
N
+
ET
ET O IS
E
L-
N
AM
n Co
O
L-
IS
f
phorylated eNOS at Ser1177 (c), iNOS (d) and nNOS (e) following 4 weeks of ET and/or L-NAME administration. f NOx production was quantified by measurements of nitrite and nitrate in mouse plasma. Means ± SD. * p < 0.05 vs. Con, ** p < 0.05 vs. ISO.
Ren/Yang/Tian/Zhu/Liu/Lu/Li/Yang/Qi
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Fig. 6. The effect of L-NAME and ET on eNOS, phosphorylated
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
E + E N T AM + E
AM N
E AM IS O
+
L-
+
L-
N
+
ET
ET O
ET
e
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E AM
L-
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Co
n
0
IS O
nNOS (relative intensity)
IS
* *
*
c
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ET N AM O + E LN AM IS E O + LE N T AM + E
+
+
ET 2.0
*
4.0
L-
O IS
L-
b
n
iNOS Phosphorylated-eNOS/eNOS (relative intensity)
a
ET
E N
AM
IS O
Co n
0
It is evident that chronic L-NAME administration could inhibit cardiac NOS activity and serum NOx levels [22, 24]. In our study, NOx levels were decreased but eNOS and nNOS levels were increased in L-NAME- and ISO-treated mice. Similar results have been reported before: rodents treated with different L-NAME doses (from 1 to 50 mg·kg–1·day–1) for different durations (2–60 days) may enhance compensatory eNOS [25], nNOS [26] and iNOS [26, 27] expression levels in the myocardium. Together, these results suggest that increased NOS expression by L-NAME treatment for days or weeks may be caused as a compensatory mechanism. In addition, a significant increase in eNOS phosphorylation was observed in the ISO + ET group. However, eNOS phosphorylation did not result in a marked change in the ISO + ET + L-NAME group. L-NAME is an L-arginine analog and should not, in theory, directly interfere with eNOS phosphorylation. Previous studies showed that 15 min after L-NAME treatment, eNOS phosphorylation did not significantly change in mice [28], which indicates that L-NAME has no direct effect on eNOS phosphorylation. It seems that the decrease in eNOS phosphorylation may
be related to both ISO and L-NAME. Further investigation is necessary to explain the interaction between the ET, L-NAME and ISO effects on cardiac eNOS phosphorylation. In conclusion, the current study demonstrates that 4 weeks of ET provides cardioprotection against cardiac hypertrophy induced by ISO injection, and this protective effect was lost after L-NAME co-injection. It seemed that the increase in eNOS signaling and NO production may contribute to the protective effect of exercise. However, the regulation of NOS-derived NO is complex and different NOS isoforms have independent, and even opposite, NO signaling effects. Further studies are needed to identify the role of NOS and NO on cardiac hypotrophy.
Acknowledgment This research was supported by the National Natural Science Foundation of China (81102436 to L.Y. and No. 81201206 to Z.Q; http://www.nsfc.gov.cn/nsfc/cen/bsdt/jggb.html) and the Natural Science Foundation of Tianjin (12JCQNJC08300 to L.Y.; http:// www.tstc.gov.cn/).
1 Serra AJ, Higuchi ML, Ihara SS, Antonio EL, Santos MH, Bombig MT, Tucci PJ: Exercise training prevents beta-adrenergic hyperactivity-induced myocardial hypertrophy and lesions. Eur J Heart Fail 2008; 10: 534–539. 2 Serra AJ, Santos MH, Bocalini DS, Antonio EL, Levy RF, Santos AA, Higuchi ML, Silva JA, Magalhaes FC, Barauna VG, et al: Exercise training inhibits inflammatory cytokines and more than prevents myocardial dysfunction in rats with sustained beta-adrenergic hyperactivity. J Physiol 2010;588:2431–2442. 3 Giannuzzi P, Temporelli PL, Corra U, Tavazzi L; ELVD-CHF Study Group: Antiremodeling effect of long-term exercise training in patients with stable chronic heart failure: results of the Exercise in Left Ventricular Dysfunction and Chronic Heart Failure (ELVD-CHF) Trial. Circulation 2003;108:554–559. 4 Andersen K, Jonsdottir S, Sigurethsson AF, Sigurethsson SB: The effect of physical training in chronic heart failure (in Icelandic). Laeknabladid 2006;92:759–764. 5 Sanada S, Node K, Minamino T, Takashima S, Ogai A, Asanuma H, Ogita H, Liao Y, Asakura M, Kim J, et al: Long-acting Ca2+ blockers prevent myocardial remodeling induced by chronic NO inhibition in rats. Hypertension 2003;41:963–967. 6 Linz W, Wohlfart P, Scholkens BA, Malinski T, Wiemer G: Interactions among ACE, ki-
Effect of Exercise on Cardiac Hypertrophy in Mice
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9
10
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nins and NO. Cardiovasc Res 1999; 43: 549– 561. Matsuoka H, Nakata M, Kohno K, Koga Y, Nomura G, Toshima H, Imaizumi T: Chronic L-arginine administration attenuates cardiac hypertrophy in spontaneously hypertensive rats. Hypertension 1996;27:14–18. Ozaki M, Kawashima S, Yamashita T, Hirase T, Ohashi Y, Inoue N, Hirata K, Yokoyama M: Overexpression of endothelial nitric oxide synthase attenuates cardiac hypertrophy induced by chronic isoproterenol infusion. Circ J 2002;66:851–856. Massion PB, Dessy C, Desjardins F, Pelat M, Havaux X, Belge C, Moulin P, Guiot Y, Feron O, Janssens S, Balligand JL: Cardiomyocyterestricted overexpression of endothelial nitric oxide synthase (NOS3) attenuates beta-adrenergic stimulation and reinforces vagal inhibition of cardiac contraction. Circulation 2004;110:2666–2672. Jones SP, Greer JJ, van Haperen R, Duncker DJ, de Crom R, Lefer DJ: Endothelial nitric oxide synthase overexpression attenuates congestive heart failure in mice. Proc Natl Acad Sci U S A 2003;100:4891–4896. Napoli C, Williams-Ignarro S, De Nigris F, Lerman LO, Rossi L, Guarino C, Mansueto G, Di Tuoro F, Pignalosa O, De Rosa G, et al: Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic
12
13
14
15 16
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
mice. Proc Natl Acad Sci U S A 2004; 101: 8797–8802. Calvert JW, Condit ME, Aragon JP, Nicholson CK, Moody BF, Hood RL, Sindler AL, Gundewar S, Seals DR, Barouch LA, Lefer DJ: Exercise protects against myocardial ischemia-reperfusion injury via stimulation of beta(3)-adrenergic receptors and increased nitric oxide signaling: role of nitrite and nitrosothiols. Circ Res 2011;108:1448–1458. Kurisu S, Ozono R, Oshima T, Kambe M, Ishida T, Sugino H, Matsuura H, Chayama K, Teranishi Y, Iba O, et al: Cardiac angiotensin II type 2 receptor activates the kinin/NO system and inhibits fibrosis. Hypertension 2003; 41:99–107. Yang L, Jia Z, Zhu M, Zhang J, Liu J, Wu P, Tian W, Li J, Qi Z, Tang X: Exercise protects against chronic beta-adrenergic remodeling of the heart by activation of endothelial nitric oxide synthase. PLoS One 2014; 9:e96892. Drexler H: Nitric oxide synthases in the failing human heart: a doubled-edged sword? Circulation 1999;99:2972–2975. Krenek P, Kmecova J, Kucerova D, Bajuszova Z, Musil P, Gazova A, Ochodnicky P, Klimas J, Kyselovic J: Isoproterenol-induced heart failure in the rat is associated with nitric oxide-dependent functional alterations of cardiac function. Eur J Heart Fail 2009; 11: 140– 146.
183
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References
184
21 Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R: Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension 1992;20:298–303. 22 Pfeiffer S, Leopold E, Schmidt K, Brunner F, Mayer B: Inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME): requirement for bioactivation to the free acid, NG-nitro-L-arginine. Br J Pharmacol 1996; 118: 1433–1440. 23 Kuru O, Senturk UK, Kocer G, Ozdem S, Baskurt OK, Cetin A, Yesilkaya A, Gunduz F: Effect of exercise training on resistance arteries in rats with chronic NOS inhibition. J Appl Physiol (1985) 2009;107:896–902. 24 Vergely C, Perrin-Sarrado C, Clermont G, Rochette L: Postischemic recovery and oxidative stress are independent of nitric-oxide synthases modulation in isolated rat heart. J Pharmacol Exp Ther 2002;303:149–157.
Cardiology 2015;130:175–184 DOI: 10.1159/000370025
25 Sampaio RC, Tanus-Santos JE, Melo SE, Hyslop S, Franchini KG, Luca IM, Moreno H Jr: Hypertension plus diabetes mimics the cardiomyopathy induced by nitric oxide inhibition in rats. Chest 2002;122:1412–1420. 26 Suda O, Tsutsui M, Morishita T, Tanimoto A, Horiuchi M, Tasaki H, Huang PL, Sasaguri Y, Yanagihara N, Nakashima Y: Long-term treatment with N(omega)-nitro-L-arginine methyl ester causes arteriosclerotic coronary lesions in endothelial nitric oxide synthasedeficient mice. Circulation 2002; 106: 1729– 1735. 27 Darblade B, Batkai S, Causse E, Gourdy P, Fouque MJ, Rami J, Arnal JF: Failure of L-nitroarginine to inhibit the activity of aortic inducible nitric oxide synthase. J Vasc Res 2001; 38:266–275. 28 Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, Ma XL: Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation 2002;105: 1497–1502.
Ren/Yang/Tian/Zhu/Liu/Lu/Li/Yang/Qi
Downloaded by: University of Alabama, Lister Hill Library 138.26.31.3 - 3/2/2015 6:18:45 PM
17 Zhang P, Xu X, Hu X, van Deel ED, Zhu G, Chen Y: Inducible nitric oxide synthase deficiency protects the heart from systolic overload-induced ventricular hypertrophy and congestive heart failure. Circ Res 2007; 100: 1089–1098. 18 Hu A, Jiao X, Gao E, Koch WJ, Sharifi-Azad S, Grunwald Z, Ma XL, Sun JZ: Chronic betaadrenergic receptor stimulation induces cardiac apoptosis and aggravates myocardial ischemia/reperfusion injury by provoking inducible nitric-oxide synthase-mediated nitrative stress. J Pharmacol Exp Ther 2006; 318: 469–475. 19 Quindry JC, French J, Hamilton KL, Lee Y, Selsby J, Powers S: Exercise does not increase cyclooxygenase-2 myocardial levels in young or senescent hearts. J Physiol Sci 2010; 60: 181–186. 20 Bachhav SS, Bhutada MS, Patil SD, Baser B, Chaudhari KB: Effect of Viscum articulatum Burm. (Loranthaceae) in Nω-nitro-L-arginine methyl ester induced hypertension and renal dysfunction. J Ethnopharmacol 2012; 142: 467–473.
