Apoptosis (2014) 19:998–1005 DOI 10.1007/s10495-014-0978-z
ORIGINAL PAPER
Hydrogen sulfide improves left ventricular function in smoking rats via regulation of apoptosis and autophagy Xiang Zhou • Guoyin An • Jianchang Chen
Published online: 22 March 2014 Ó Springer Science+Business Media New York 2014
Abstract The present study was designed to investigate the protective effects of hydrogen sulfide (H2S) against cigarette smoking-induced left ventricular dysfunction in rats. Left ventricular structure and function were assessed using twodimensional echocardiography. Cardiomyocyte apoptosis was determined by Annexin V/PI and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Cardiac autophagy was evaluated by detection of autophagy-related protein expression and observation of autophagosomes. Our results indicated that administration of NaHS (a donor of H2S) could protect against smokinginduced left ventricular systolic dysfunction. H2S was found to exert anti-apoptotic effects in the myocardium of smoking rats by inhibiting JNK and P38 mitogen-activated protein kinases pathways and activating PI3K/Akt signaling. Moreover, H2S could also reduce smoking-induced autophagic cell death via regulation of AMPK/mTOR signaling pathway. In conclusion, our study demonstrates that H2S can improve left ventricular systolic function in smoking rats via regulation of apoptosis and autophagy.
mortality [1–3]. Cigarette smoke contains more than 4,000 chemical substances, including polycyclic aromatic hydrocarbons and oxidative gases, most of which exert cardiotoxic effects. In previous studies, we established a rat model of passive smoking and found that chronic exposure to cigarette smoke could eventually lead to left ventricular remodeling and dysfunction [4, 5]. Hydrogen sulfide (H2S), a new gaseous signal molecule identified after nitric oxide and carbon monoxide, is endogenously generated by cystathionine b-synthase, cystathionine c-lyase, and 3-mercaptopyruvate sulfurtransferase. Previous studies have reported that H2S could attenuate myocardial ischemia–reperfusion injury by preserving mitochondrial function and reducing cardiomyocyte apoptosis [6, 7]. In addition, H2S was found to be an inhibitor of L-type calcium channels and mechanical contraction in rat cardiomyocytes [8]. In the present study, we established a smoking rat model to investigate whether H2S has protective effects against smoking-induced left ventricular dysfunction.
Keywords Hydrogen sulfide Left ventricular function Smoking Apoptosis Autophagy
Materials and methods Groups and treatment
Introduction Smoking, which is a serious public health concern, contributes significantly to the cardiovascular morbidity and
X. Zhou (&) G. An J. Chen Department of Cardiology, The Second Affiliated Hospital of Soochow University, No. 1055 Sanxiang Road, Suzhou 215004, Jiangsu, China e-mail: [email protected]
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All experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health and were approved by the Animal Ethics Committee of Soochow University. Male Sprague–Dawley rats weighing 200–250 g were housed four per plastic cage in an animal room with an alternating 12 h light/dark cycle at 23 ± 2 °C and 50 ± 5 % relative humidity for 4 months. The rats were randomly divided into four groups: CS group
Apoptosis (2014) 19:998–1005
(n = 10; exposed to cigarette smoke at the rate of 40 cigarettes/day), NaHS group (n = 10; administered intragastrically with NaHS solution at a dose of 8 lmol/kg once daily in the morning), CS ? NaHS group (n = 10; exposed to cigarette smoke and administrated with NaHS), and control group (n = 10; neither exposed to cigarette smoke nor treated with NaHS). Rats were exposed to cigarette smoke in a chamber connected to a smoking device. The smoke was drawn out of commercial filtered cigarettes with a vacuum pump and then exhausted into the smoking chamber. During the first week, the number of cigarettes was gradually increased from five to ten cigarettes over a 30-min period, twice in the morning and twice in the afternoon. After that, ten cigarettes were used in each smoking trial, repeated 4 times/day for the rest of the 4-month experimental period. Measurement of H2S content After 4 months, the plasma and myocardial contents of H2S were measured by methylene blue and modified sulfide electrode methods described by Chunyu [9] and Su [10]. H2S content in the plasma was expressed as micromole per liter, while H2S content in the myocardial tissue was expressed in nanomole per milligram of protein.
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determine cardiomyocyte apoptosis according to the manufacturer’s instructions. Annexin V labeled with a fluorophore could identify cells in the early stage of apoptosis, and propidium iodine (PI), a fluorescent nucleic acid binding dye, was responsible for staining cells in the medium and late stages of apoptosis. After staining with Annexin V/PI, cardiomyocyte apoptosis was quantified by flow cytometric analysis. The apoptotic rate was calculated as the percentage of Annexin V-positive and PI-negative cells divided by the total number of cells in the gated region. TUNEL staining Left ventricular tissue was fixed in 10 % buffered formalin, embedded in paraffin and sectioned into 5-lm thick sections. Cardiomyocyte apoptosis was evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) In Situ Cell Death Detection Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. For each slide, the color images of ten separate fields were randomly captured and digitized. The cells with clear nuclear labeling were defined as TUNELpositive cells. The apoptotic index was calculated as the percentage of TUNEL-positive cells divided by the total number of cells. Transmission electron microscopy
Echocardiographic study Two-dimensional echocardiography was used to evaluate cardiac structure and function by the same experienced sonographer using the Philips Sonos 7500 ultrasound system (Philips Medical Systems, Andover, MA, USA). Rats were anesthetized by inhalation of isoflurane (2–5 %) in oxygen. The left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD) and left ventricular posterior wall thickness (LVPWT) were obtained from the parasternal long axis view. Left ventricular fractional shortening (LVFS) was calculated as (LVEDDLVESD)/LVEDD. Left ventricular ejection fraction (LVEF) was determined from the apical two- and four-chamber views using a modified Simpson’s method. The mitral peak flow velocities at early diastole (E) and atrial contraction (A) were recorded by pulsed Doppler technique and the E/A ratio was calculated. All measurements were averaged for three consecutive cardiac cycles in this study. Annexin V/PI staining Rat left ventricles were surgically removed, minced into small pieces, and then digested with trypsin solution at 37 °C for 10 min. The Annexin V-FITC apoptosis detection kit (BD Pharmingen, San Jose, CA, USA) was used to
Left ventricular tissue was cut into small pieces, and then fixed in 2.5 % glutaraldehyde, post-fixed in 1 % osmium tetroxide, dehydrated in an ascending series of alcohols, and embedded in epoxy resin. Ultrathin sections were cut and stained with uranyl acetate and lead citrate. Samples were observed using a Philips CM120 transmission electron microscope (FEI Company, Hillsboro, OR, USA). Phosphatidylinositol 3-kinase activity assay Lysates of left ventricular tissue were immunoprecipitated with antibody against phosphatidylinositol 3-kinase (PI3K; Millipore, Temecula, CA, USA). PI3K activity in the immunocomplexes was measured using PI3K ELISA kit (Echelon Biosciences, Salt Lake City, UT, USA) according to the manufacturer’s instructions. Western blot analysis Protein homogenates were prepared from left ventricular tissue, and protein concentrations were quantified with the BCA protein assay kit (Pierce, Thermo Scientific, Illinois, USA). Equal amounts of protein (50 lg) were loaded on 10 % SDS-PAGE gels, transferred onto nitrocellulose membranes and blocked with 5 % nonfat milk. The
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Fig. 1 H2S contents in plasma and myocardial tissue measured by methylene blue method (a, b) and modified sulfide electrode method (c, d). *P \ 0.05 versus control; **P \ 0.05 versus CS
membranes were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-cytochrome c, anti-caspase-3, anti-cleaved caspase-3 (Asp175), anti-caspase-9, anti-cleaved caspase-9 (Asp353), anti-Bax, anti-Bcl-2, anti-Beclin-1, anti-LC3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-phosphoJNK (Thr183/Tyr185), anti-JNK, anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-AMPK (Thr172), anti-AMPK, antiphospho-mTOR (Ser2448), anti-mTOR (Cell Signaling Technology, Beverly, MA, USA). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies at room temperature for 1 h. The immunocomplexes were visualized with an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). In this study, b-actin was used as loading control for the cytosolic fraction, while Porin-1 served as loading control for the mitochondrial fraction.
Results Hydrogen sulfide content was determined by methylene blue and modified sulfide electrode methods and the results are shown in Fig. 1. H2S contents in plasma and myocardial tissue were significantly lower in the CS group than in the control group, while in the CS ? NaHS group, H2S levels were markedly higher than those in the CS group. Echocardiography was used to assess cardiac structure and function and the results are presented in Table 1.
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LVEDD and LVESD were found to be significantly increased in the CS group and decreased in the CS ? NaHS group. Furthermore, LVFS and LVEF were remarkably lower in the CS group than in the control group, whereas these two indicators were markedly higher in the CS ? NaHS group than in the CS group. In addition, there were no significant differences in LVPWT and E/A ratio among these groups. Cardiomyocyte apoptosis was determined by Annexin V/PI and TUNEL staining and the results are shown in Fig. 2. The apoptotic cardiomyocytes were significantly increased in the CS group compared to the control group, whereas apoptotic cells were remarkably decreased in the CS ? NaHS group compared to the CS group. To evaluate whether H2S is involved in the regulation of mitochondrial apoptotic pathway, several apoptosis regulatory proteins were determined by Western blotting (Fig. 3). Cytosolic cytochrome c released from mitochondria was significantly increased in the CS group, while in the CS ? NaHS group, the translocation of cytochrome c into the cytosol was markedly decreased. Additionally, exposure to cigarette smoke induced a significant increase in the expression of cleaved caspase-9 and caspase-3, whereas administration of NaHS remarkably reduced the expression of these two proteins. Moreover, Bax/Bcl-2 ratio was significantly increased in the CS group and decreased in the CS ? NaHS group. To examine whether mitogen-activated protein kinases (MAPKs) were activated in the myocardial tissue of smoking rats, the phosphorylation of JNK and p38 MAPK was analysed by Western blotting (Fig. 4). Our results
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Table 1 Echocardiographic study Control
CS
CS ? NaHS
NaHS
LVEDD (mm)
7.45 ± 0.29
8.43 ± 0.50*
7.98 ± 0.44**
7.50 ± 0.31
LVESD (mm)
3.89 ± 0.23
4.99 ± 0.31*
4.46 ± 0.29**
4.00 ± 0.24
LVPWT (mm)
1.71 ± 0.09
1.79 ± 0.10
1.75 ± 0.08
1.69 ± 0.11
LVFS (%)
47.81 ± 1.67
40.79 ± 2.43*
44.03 ± 3.85**
46.72 ± 1.46
LVEF (%)
77.83 ± 2.02
69.53 ± 2.82*
73.30 ± 4.25**
76.97 ± 1.62
E/A ratio
1.49 ± 0.11
1.42 ± 0.15
1.44 ± 0.09
1.52 ± 0.14
Data are expressed as mean ± SD (n = 10) LVEDD left ventricular end-diastolic diameter, LVESD left ventricular end-systolic diameter, LVPWT left ventricular posterior wall thickness, LVFS left ventricular fractional shortening, LVEF left ventricular ejection fraction; E peak velocity of early ventricular filling, A peak velocity of transmitral flow during atrial contraction * P \ 0.05 versus control group; ** P \ 0.05 versus CS group Fig. 2 Representative images of cardiomyocyte apoptosis determined by Annexin V/PI (a) and TUNEL staining (b); apoptotic rate was calculated as the percentage of Annexin V-positive and PI-negative cells divided by the total number of cells (c); apoptotic index was calculated as the percentage of TUNEL-positive cells divided by the total number of cells (d). *P \ 0.05 versus control; **P \ 0.05 versus CS
showed that the protein levels of phospho-JNK and phospho-p38 were significantly elevated in the CS group. Conversely, administration of NaHS remarkably inhibited the activation of MAPKs in the CS ? NaHS group. The PI3K/Akt pathway plays a critical role in the regulation of apoptosis. In this study, PI3K activity was measured by ELISA and Akt phosphorylation was detected
by Western blotting (Fig. 5). Our findings indicated that PI3K activity was remarkably lower in the CS group than in the control group, while in the CS ? NaHS group, the PI3 K activity was markedly higher than that in the CS group. Furthermore, the protein level of phospho-Akt was reduced in the CS group and elevated in the CS ? NaHS group.
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Fig. 3 Western blot analysis of apoptosis regulatory proteins. NaHS administration significantly decreased the expression of Bax, cytosolic cytochrome c, cleaved caspase3 and caspase-9, and increased the expression of Bcl-2 and mitochondrial cytochrome c. *P \ 0.05 versus control; **P \ 0.05 versus CS
Fig. 4 Western blot analysis of JNK and p38 MAPK phosphorylation. The protein levels of phospho-JNK and phospho-p38 were significantly elevated in the CS group. In contrast, NaHS administration remarkably inhibited the activation of MAPKs in the CS ? NaHS group. *P \ 0.05, versus control; **P \ 0.05 versus CS
Cardiomyocyte autophagy was assessed by detection of autophagy-related protein expression and observation of autophagosomes under transmission electron microscope (Fig. 6). The protein expression of Beclin-1 was upregulated in the CS group and downregulated in the CS ? NaHS group. LC3-II/LC3-I ratio, which is correlated
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with the extent of autophagosome formation, was significantly increased in the smoking rats and decreased following the NaHS administration. In addition, cigarette smoking was associated with an increased number of autophagosomes in myocardium, whereas NaHS treatment was found to reduce the autophagosome numbers.
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Fig. 5 PI3 K activity measured by ELISA (a); representative immunoblots and densitometric analysis of phosphorylated Akt and total Akt (b). *P \ 0.05 versus control; **P \ 0.05 versus CS
Fig. 6 The protein expression of Beclin-1 and LC3-II was upregulated in the CS group and downregulated in the CS ? NaHS group (a, b); representative images of autophagosomes in myocardium (c). *P \ 0.05 versus control; **P \ 0.05 versus CS
The AMPK/mTOR pathway is known to be involved in the regulation of autophagy. As shown in Fig. 7, cigarette smoking was associated with an increase in AMPK phosphorylation and a decrease in mTOR phosphorylation, while NaHS administration could significantly inhibit AMPK activity and induce mTOR activation.
Discussion In the present study, we established a smoking rat model to investigate the protective effects of H2S against smokinginduced left ventricular dysfunction. Our findings indicated that endogenous levels of H2S were significantly decreased in the smoking rats, while exogenous administration of NaHS increased H2S contents in plasma and myocardial tissue of smoking rats. In this study, two-dimensional
echocardiography was used to evaluate cardiac structure and function in rats. The echocardiographic data showed that H2S could protect against left ventricular systolic dysfunction induced by cigarette smoking. In recent years, accumulating evidence has demonstrated that apoptosis and autophagy are both involved in the pathogenesis of chronic heart failure [11–15]. In this study, cardiomyocyte apoptosis was determined by Annexin V/PI and TUNEL staining, while cardiac autophagy was evaluated by detection of autophagy-related protein expression and observation of autophagosomes. Our findings suggested that H2S could significantly reduce cardiomyocyte apoptosis and autophagy in smoking rats, which might be important protective mechanisms against smoking-induced left ventricular systolic dysfunction. Apoptosis, also termed programmed cell death, is characterized by altered nuclear morphology including
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Fig. 7 Western blot analysis of AMPK and mTOR phosphorylation. Smoking was associated with an increase in AMPK phosphorylation and a decrease in mTOR phosphorylation, whereas NaHS administration significantly inhibited AMPK activity and induced mTOR activation.*P \ 0.05 versus control; **P \ 0.05 versus CS
chromatin condensation and DNA fragmentation, minor changes in cytoplasmic organelles, cell shrinkage, plasma membrane blebbing, and apoptotic body formation [16]. There are two major apoptotic signaling pathways: the death receptor pathway and the mitochondrial death pathway. During mitochondrial-mediated apoptosis, cytochrome c is released from mitochondrial into the cytosol and subsequently activates caspase-9. The activated caspase-9 cleaves pro-caspase-3 to produce active caspase-3 which leads to the cleavage of cellular target proteins [17, 18]. Bax and Bcl-2 are two important Bcl-2 family members which play critical roles in the regulation of mitochondrial apoptotic pathway [19, 20]. In the present study, our findings revealed that H2S could inhibit the activation of mitochondrial apoptotic pathway in smoking rats. The MAPK signaling cascades, which are evolutionarily conserved signal transduction pathways, respond to various extracellular factors and regulate diverse cellular processes such as differentiation, proliferation, apoptosis, survival and development [21, 22]. In this study, JNK and P38 MAPK signaling pathways were significantly activated in the myocardium of smoking rats, which might be an important molecular mechanism responsible for smokinginduced apoptosis. Furthermore, H2S was found to reduce cardiomyocyte apoptosis in smoking rats by inhibiting phosphorylation of JNK and p38 MAPK. The PI3K/Akt pathway plays a key role in the regulation of cell growth, proliferation, survival and metabolism [23, 24]. PI3K/Akt signaling generally acts to promote survival through inhibition of pro-apoptotic factors and activation of anti-apoptotic factors. In the present study, PI3K/Akt signaling was remarkably inhibited in the myocardium of smoking rats, which might be another molecular mechanism involved in smoking-induced apoptosis. Moreover, H2S was found to reduce cardiomyocyte apoptosis in smoking rats via activation of PI3K/Akt signaling.
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Autophagy, an evolutionarily conserved catabolic pathway of lysosome-dependent degradation of long-lived proteins and dysfunctional organelles, is known to be induced by AMPK signaling but inhibited by mTOR signaling [25, 26]. In this study, cigarette smoking was associated with an increase in AMPK phosphorylation and a decrease in mTOR phosphorylation, whereas NaHS administration was found to inhibit AMPK activity and induce mTOR activation in the myocardium of smoking rats, which suggested that H2S could protect against smoking-induced cardiac autophagy via regulation of AMPK/mTOR signaling pathway. In conclusion, our study demonstrates that H2S protects against cigarette smoking-induced left ventricular systolic dysfunction via regulation of apoptosis and autophagy. H2S may exert anti-apoptotic effects in the myocardium of smoking rats by inhibiting JNK and P38 MAPK pathways and activating PI3K/Akt signaling. Moreover, H2S can also reduce smoking-induced autophagic cell death via regulation of AMPK/mTOR signaling pathway.
Conflict of interest
The authors declare no conflict of interest.
References 1. Burns DM (2003) Epidemiology of smoking-induced cardiovascular disease. Prog Cardiovasc Dis 46(1):11–29 2. Tonstad S, Andrew Johnston J (2006) Cardiovascular risks associated with smoking: a review for clinicians. Eur J Cardiovasc Prev Rehabil 13(4):507–514 3. Erhardt L (2009) Cigarette smoking: an undertreated risk factor for cardiovascular disease. Atherosclerosis 205(1):23–32 4. Zhou X, Li C, Xu W, Chen J (2012) Trimetazidine protects against smoking-induced left ventricular remodeling via attenuating oxidative stress, apoptosis, and inflammation. PLoS ONE 7(7):e40424
Apoptosis (2014) 19:998–1005 5. Zhou X, Li C, Xu W, Chen J (2013) Protective effects of valsartan against cigarette smoke-induced left ventricular systolic dysfunction in rats. Int J Cardiol 167(3):677–680 6. Elrod JW, Calvert JW, Morrison J, Doeller JE, Kraus DW, Tao L, Jiao X, Scalia R, Kiss L, Szabo C, Kimura H, Chow CW, Lefer DJ (2007) Hydrogen sulfide attenuates myocardial ischemiareperfusion injury by preservation of mitochondrial function. Proc Natl Acad Sci USA 104(39):15560–15565 7. Yao LL, Huang XW, Wang YG, Cao YX, Zhang CC, Zhu YC (2010) Hydrogen sulfide protects cardiomyocytes from hypoxia/ reoxygenation-induced apoptosis by preventing GSK-3betadependent opening of mPTP. Am J Physiol Heart Circ Physiol 298(5):H1310–H1319 8. Sun YG, Cao YX, Wang WW, Ma SF, Yao T, Zhu YC (2008) Hydrogen sulphide is an inhibitor of L-type calcium channels and mechanical contraction in rat cardiomyocytes. Cardiovasc Res 79(4):632–641 9. Chunyu Z, Junbao D, Dingfang B, Hui Y, Xiuying T, Chaoshu T (2003) The regulatory effect of hydrogen sulfide on hypoxic pulmonary hypertension in rats. Biochem Biophys Res Commun 302(4):810–816 10. Su YW, Liang C, Jin HF, Tang XY, Han W, Chai LJ, Zhang CY, Geng B, Tang CS, Du JB (2009) Hydrogen sulfide regulates cardiac function and structure in adriamycin-induced cardiomyopathy. Circ J 73(4):741–749 11. Mughal W, Dhingra R, Kirshenbaum LA (2012) Striking a balance: autophagy, apoptosis, and necrosis in a normal and failing heart. Curr Hypertens Rep 14(6):540–547 12. Nishida K, Otsu K (2008) Cell death in heart failure. Circ J 72(Suppl A):A17–A21 13. Lee Y, Gustafsson AB (2009) Role of apoptosis in cardiovascular disease. Apoptosis 14(4):536–548
1005 14. Narula J, Haider N, Arbustini E, Chandrashekhar Y (2006) Mechanisms of disease: apoptosis in heart failure—seeing hope in death. Nat Clin Pract Cardiovasc Med 3(12):681–688 15. Nishida K, Kyoi S, Yamaguchi O, Sadoshima J, Otsu K (2009) The role of autophagy in the heart. Cell Death Differ 16(1):31–38 16. Edinger AL, Thompson CB (2004) Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol 16(6):663–669 17. Gupta S, Kass GE, Szegezdi E, Joseph B (2009) The mitochondrial death pathway: a promising therapeutic target in diseases. J Cell Mol Med 13(6):1004–1033 18. Riedl SJ, Shi Y (2004) Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol 5(11):897–907 19. Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9(1):47–59 20. Brunelle JK, Letai A (2009) Control of mitochondrial apoptosis by the Bcl-2 family. J Cell Sci 122(Pt 4):437–441 21. Chang L, Karin M (2001) Mammalian MAP kinase signalling cascades. Nature 410(6824):37–40 22. Raman M, Chen W, Cobb MH (2007) Differential regulation and properties of MAPKs. Oncogene 26(22):3100–3112 23. Franke TF (2008) PI3K/Akt: getting it right matters. Oncogene 27(50):6473–6488 24. Vasudevan KM, Garraway LA (2010) AKT signaling in physiology and disease. Curr Top Microbiol Immunol 347:105–133 25. Kim J, Kundu M, Viollet B, Guan KL (2011) AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 13(2):132–141 26. Jung CH, Ro SH, Cao J, Otto NM, Kim DH (2010) mTOR regulation of autophagy. FEBS Lett 584(7):1287–1295
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ORIGINAL PAPER
Hydrogen sulfide improves left ventricular function in smoking rats via regulation of apoptosis and autophagy Xiang Zhou • Guoyin An • Jianchang Chen
Published online: 22 March 2014 Ó Springer Science+Business Media New York 2014
Abstract The present study was designed to investigate the protective effects of hydrogen sulfide (H2S) against cigarette smoking-induced left ventricular dysfunction in rats. Left ventricular structure and function were assessed using twodimensional echocardiography. Cardiomyocyte apoptosis was determined by Annexin V/PI and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Cardiac autophagy was evaluated by detection of autophagy-related protein expression and observation of autophagosomes. Our results indicated that administration of NaHS (a donor of H2S) could protect against smokinginduced left ventricular systolic dysfunction. H2S was found to exert anti-apoptotic effects in the myocardium of smoking rats by inhibiting JNK and P38 mitogen-activated protein kinases pathways and activating PI3K/Akt signaling. Moreover, H2S could also reduce smoking-induced autophagic cell death via regulation of AMPK/mTOR signaling pathway. In conclusion, our study demonstrates that H2S can improve left ventricular systolic function in smoking rats via regulation of apoptosis and autophagy.
mortality [1–3]. Cigarette smoke contains more than 4,000 chemical substances, including polycyclic aromatic hydrocarbons and oxidative gases, most of which exert cardiotoxic effects. In previous studies, we established a rat model of passive smoking and found that chronic exposure to cigarette smoke could eventually lead to left ventricular remodeling and dysfunction [4, 5]. Hydrogen sulfide (H2S), a new gaseous signal molecule identified after nitric oxide and carbon monoxide, is endogenously generated by cystathionine b-synthase, cystathionine c-lyase, and 3-mercaptopyruvate sulfurtransferase. Previous studies have reported that H2S could attenuate myocardial ischemia–reperfusion injury by preserving mitochondrial function and reducing cardiomyocyte apoptosis [6, 7]. In addition, H2S was found to be an inhibitor of L-type calcium channels and mechanical contraction in rat cardiomyocytes [8]. In the present study, we established a smoking rat model to investigate whether H2S has protective effects against smoking-induced left ventricular dysfunction.
Keywords Hydrogen sulfide Left ventricular function Smoking Apoptosis Autophagy
Materials and methods Groups and treatment
Introduction Smoking, which is a serious public health concern, contributes significantly to the cardiovascular morbidity and
X. Zhou (&) G. An J. Chen Department of Cardiology, The Second Affiliated Hospital of Soochow University, No. 1055 Sanxiang Road, Suzhou 215004, Jiangsu, China e-mail: [email protected]
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All experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health and were approved by the Animal Ethics Committee of Soochow University. Male Sprague–Dawley rats weighing 200–250 g were housed four per plastic cage in an animal room with an alternating 12 h light/dark cycle at 23 ± 2 °C and 50 ± 5 % relative humidity for 4 months. The rats were randomly divided into four groups: CS group
Apoptosis (2014) 19:998–1005
(n = 10; exposed to cigarette smoke at the rate of 40 cigarettes/day), NaHS group (n = 10; administered intragastrically with NaHS solution at a dose of 8 lmol/kg once daily in the morning), CS ? NaHS group (n = 10; exposed to cigarette smoke and administrated with NaHS), and control group (n = 10; neither exposed to cigarette smoke nor treated with NaHS). Rats were exposed to cigarette smoke in a chamber connected to a smoking device. The smoke was drawn out of commercial filtered cigarettes with a vacuum pump and then exhausted into the smoking chamber. During the first week, the number of cigarettes was gradually increased from five to ten cigarettes over a 30-min period, twice in the morning and twice in the afternoon. After that, ten cigarettes were used in each smoking trial, repeated 4 times/day for the rest of the 4-month experimental period. Measurement of H2S content After 4 months, the plasma and myocardial contents of H2S were measured by methylene blue and modified sulfide electrode methods described by Chunyu [9] and Su [10]. H2S content in the plasma was expressed as micromole per liter, while H2S content in the myocardial tissue was expressed in nanomole per milligram of protein.
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determine cardiomyocyte apoptosis according to the manufacturer’s instructions. Annexin V labeled with a fluorophore could identify cells in the early stage of apoptosis, and propidium iodine (PI), a fluorescent nucleic acid binding dye, was responsible for staining cells in the medium and late stages of apoptosis. After staining with Annexin V/PI, cardiomyocyte apoptosis was quantified by flow cytometric analysis. The apoptotic rate was calculated as the percentage of Annexin V-positive and PI-negative cells divided by the total number of cells in the gated region. TUNEL staining Left ventricular tissue was fixed in 10 % buffered formalin, embedded in paraffin and sectioned into 5-lm thick sections. Cardiomyocyte apoptosis was evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) In Situ Cell Death Detection Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. For each slide, the color images of ten separate fields were randomly captured and digitized. The cells with clear nuclear labeling were defined as TUNELpositive cells. The apoptotic index was calculated as the percentage of TUNEL-positive cells divided by the total number of cells. Transmission electron microscopy
Echocardiographic study Two-dimensional echocardiography was used to evaluate cardiac structure and function by the same experienced sonographer using the Philips Sonos 7500 ultrasound system (Philips Medical Systems, Andover, MA, USA). Rats were anesthetized by inhalation of isoflurane (2–5 %) in oxygen. The left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD) and left ventricular posterior wall thickness (LVPWT) were obtained from the parasternal long axis view. Left ventricular fractional shortening (LVFS) was calculated as (LVEDDLVESD)/LVEDD. Left ventricular ejection fraction (LVEF) was determined from the apical two- and four-chamber views using a modified Simpson’s method. The mitral peak flow velocities at early diastole (E) and atrial contraction (A) were recorded by pulsed Doppler technique and the E/A ratio was calculated. All measurements were averaged for three consecutive cardiac cycles in this study. Annexin V/PI staining Rat left ventricles were surgically removed, minced into small pieces, and then digested with trypsin solution at 37 °C for 10 min. The Annexin V-FITC apoptosis detection kit (BD Pharmingen, San Jose, CA, USA) was used to
Left ventricular tissue was cut into small pieces, and then fixed in 2.5 % glutaraldehyde, post-fixed in 1 % osmium tetroxide, dehydrated in an ascending series of alcohols, and embedded in epoxy resin. Ultrathin sections were cut and stained with uranyl acetate and lead citrate. Samples were observed using a Philips CM120 transmission electron microscope (FEI Company, Hillsboro, OR, USA). Phosphatidylinositol 3-kinase activity assay Lysates of left ventricular tissue were immunoprecipitated with antibody against phosphatidylinositol 3-kinase (PI3K; Millipore, Temecula, CA, USA). PI3K activity in the immunocomplexes was measured using PI3K ELISA kit (Echelon Biosciences, Salt Lake City, UT, USA) according to the manufacturer’s instructions. Western blot analysis Protein homogenates were prepared from left ventricular tissue, and protein concentrations were quantified with the BCA protein assay kit (Pierce, Thermo Scientific, Illinois, USA). Equal amounts of protein (50 lg) were loaded on 10 % SDS-PAGE gels, transferred onto nitrocellulose membranes and blocked with 5 % nonfat milk. The
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Fig. 1 H2S contents in plasma and myocardial tissue measured by methylene blue method (a, b) and modified sulfide electrode method (c, d). *P \ 0.05 versus control; **P \ 0.05 versus CS
membranes were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-cytochrome c, anti-caspase-3, anti-cleaved caspase-3 (Asp175), anti-caspase-9, anti-cleaved caspase-9 (Asp353), anti-Bax, anti-Bcl-2, anti-Beclin-1, anti-LC3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-phosphoJNK (Thr183/Tyr185), anti-JNK, anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-AMPK (Thr172), anti-AMPK, antiphospho-mTOR (Ser2448), anti-mTOR (Cell Signaling Technology, Beverly, MA, USA). The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies at room temperature for 1 h. The immunocomplexes were visualized with an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). In this study, b-actin was used as loading control for the cytosolic fraction, while Porin-1 served as loading control for the mitochondrial fraction.
Results Hydrogen sulfide content was determined by methylene blue and modified sulfide electrode methods and the results are shown in Fig. 1. H2S contents in plasma and myocardial tissue were significantly lower in the CS group than in the control group, while in the CS ? NaHS group, H2S levels were markedly higher than those in the CS group. Echocardiography was used to assess cardiac structure and function and the results are presented in Table 1.
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LVEDD and LVESD were found to be significantly increased in the CS group and decreased in the CS ? NaHS group. Furthermore, LVFS and LVEF were remarkably lower in the CS group than in the control group, whereas these two indicators were markedly higher in the CS ? NaHS group than in the CS group. In addition, there were no significant differences in LVPWT and E/A ratio among these groups. Cardiomyocyte apoptosis was determined by Annexin V/PI and TUNEL staining and the results are shown in Fig. 2. The apoptotic cardiomyocytes were significantly increased in the CS group compared to the control group, whereas apoptotic cells were remarkably decreased in the CS ? NaHS group compared to the CS group. To evaluate whether H2S is involved in the regulation of mitochondrial apoptotic pathway, several apoptosis regulatory proteins were determined by Western blotting (Fig. 3). Cytosolic cytochrome c released from mitochondria was significantly increased in the CS group, while in the CS ? NaHS group, the translocation of cytochrome c into the cytosol was markedly decreased. Additionally, exposure to cigarette smoke induced a significant increase in the expression of cleaved caspase-9 and caspase-3, whereas administration of NaHS remarkably reduced the expression of these two proteins. Moreover, Bax/Bcl-2 ratio was significantly increased in the CS group and decreased in the CS ? NaHS group. To examine whether mitogen-activated protein kinases (MAPKs) were activated in the myocardial tissue of smoking rats, the phosphorylation of JNK and p38 MAPK was analysed by Western blotting (Fig. 4). Our results
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Table 1 Echocardiographic study Control
CS
CS ? NaHS
NaHS
LVEDD (mm)
7.45 ± 0.29
8.43 ± 0.50*
7.98 ± 0.44**
7.50 ± 0.31
LVESD (mm)
3.89 ± 0.23
4.99 ± 0.31*
4.46 ± 0.29**
4.00 ± 0.24
LVPWT (mm)
1.71 ± 0.09
1.79 ± 0.10
1.75 ± 0.08
1.69 ± 0.11
LVFS (%)
47.81 ± 1.67
40.79 ± 2.43*
44.03 ± 3.85**
46.72 ± 1.46
LVEF (%)
77.83 ± 2.02
69.53 ± 2.82*
73.30 ± 4.25**
76.97 ± 1.62
E/A ratio
1.49 ± 0.11
1.42 ± 0.15
1.44 ± 0.09
1.52 ± 0.14
Data are expressed as mean ± SD (n = 10) LVEDD left ventricular end-diastolic diameter, LVESD left ventricular end-systolic diameter, LVPWT left ventricular posterior wall thickness, LVFS left ventricular fractional shortening, LVEF left ventricular ejection fraction; E peak velocity of early ventricular filling, A peak velocity of transmitral flow during atrial contraction * P \ 0.05 versus control group; ** P \ 0.05 versus CS group Fig. 2 Representative images of cardiomyocyte apoptosis determined by Annexin V/PI (a) and TUNEL staining (b); apoptotic rate was calculated as the percentage of Annexin V-positive and PI-negative cells divided by the total number of cells (c); apoptotic index was calculated as the percentage of TUNEL-positive cells divided by the total number of cells (d). *P \ 0.05 versus control; **P \ 0.05 versus CS
showed that the protein levels of phospho-JNK and phospho-p38 were significantly elevated in the CS group. Conversely, administration of NaHS remarkably inhibited the activation of MAPKs in the CS ? NaHS group. The PI3K/Akt pathway plays a critical role in the regulation of apoptosis. In this study, PI3K activity was measured by ELISA and Akt phosphorylation was detected
by Western blotting (Fig. 5). Our findings indicated that PI3K activity was remarkably lower in the CS group than in the control group, while in the CS ? NaHS group, the PI3 K activity was markedly higher than that in the CS group. Furthermore, the protein level of phospho-Akt was reduced in the CS group and elevated in the CS ? NaHS group.
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Fig. 3 Western blot analysis of apoptosis regulatory proteins. NaHS administration significantly decreased the expression of Bax, cytosolic cytochrome c, cleaved caspase3 and caspase-9, and increased the expression of Bcl-2 and mitochondrial cytochrome c. *P \ 0.05 versus control; **P \ 0.05 versus CS
Fig. 4 Western blot analysis of JNK and p38 MAPK phosphorylation. The protein levels of phospho-JNK and phospho-p38 were significantly elevated in the CS group. In contrast, NaHS administration remarkably inhibited the activation of MAPKs in the CS ? NaHS group. *P \ 0.05, versus control; **P \ 0.05 versus CS
Cardiomyocyte autophagy was assessed by detection of autophagy-related protein expression and observation of autophagosomes under transmission electron microscope (Fig. 6). The protein expression of Beclin-1 was upregulated in the CS group and downregulated in the CS ? NaHS group. LC3-II/LC3-I ratio, which is correlated
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with the extent of autophagosome formation, was significantly increased in the smoking rats and decreased following the NaHS administration. In addition, cigarette smoking was associated with an increased number of autophagosomes in myocardium, whereas NaHS treatment was found to reduce the autophagosome numbers.
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Fig. 5 PI3 K activity measured by ELISA (a); representative immunoblots and densitometric analysis of phosphorylated Akt and total Akt (b). *P \ 0.05 versus control; **P \ 0.05 versus CS
Fig. 6 The protein expression of Beclin-1 and LC3-II was upregulated in the CS group and downregulated in the CS ? NaHS group (a, b); representative images of autophagosomes in myocardium (c). *P \ 0.05 versus control; **P \ 0.05 versus CS
The AMPK/mTOR pathway is known to be involved in the regulation of autophagy. As shown in Fig. 7, cigarette smoking was associated with an increase in AMPK phosphorylation and a decrease in mTOR phosphorylation, while NaHS administration could significantly inhibit AMPK activity and induce mTOR activation.
Discussion In the present study, we established a smoking rat model to investigate the protective effects of H2S against smokinginduced left ventricular dysfunction. Our findings indicated that endogenous levels of H2S were significantly decreased in the smoking rats, while exogenous administration of NaHS increased H2S contents in plasma and myocardial tissue of smoking rats. In this study, two-dimensional
echocardiography was used to evaluate cardiac structure and function in rats. The echocardiographic data showed that H2S could protect against left ventricular systolic dysfunction induced by cigarette smoking. In recent years, accumulating evidence has demonstrated that apoptosis and autophagy are both involved in the pathogenesis of chronic heart failure [11–15]. In this study, cardiomyocyte apoptosis was determined by Annexin V/PI and TUNEL staining, while cardiac autophagy was evaluated by detection of autophagy-related protein expression and observation of autophagosomes. Our findings suggested that H2S could significantly reduce cardiomyocyte apoptosis and autophagy in smoking rats, which might be important protective mechanisms against smoking-induced left ventricular systolic dysfunction. Apoptosis, also termed programmed cell death, is characterized by altered nuclear morphology including
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Fig. 7 Western blot analysis of AMPK and mTOR phosphorylation. Smoking was associated with an increase in AMPK phosphorylation and a decrease in mTOR phosphorylation, whereas NaHS administration significantly inhibited AMPK activity and induced mTOR activation.*P \ 0.05 versus control; **P \ 0.05 versus CS
chromatin condensation and DNA fragmentation, minor changes in cytoplasmic organelles, cell shrinkage, plasma membrane blebbing, and apoptotic body formation [16]. There are two major apoptotic signaling pathways: the death receptor pathway and the mitochondrial death pathway. During mitochondrial-mediated apoptosis, cytochrome c is released from mitochondrial into the cytosol and subsequently activates caspase-9. The activated caspase-9 cleaves pro-caspase-3 to produce active caspase-3 which leads to the cleavage of cellular target proteins [17, 18]. Bax and Bcl-2 are two important Bcl-2 family members which play critical roles in the regulation of mitochondrial apoptotic pathway [19, 20]. In the present study, our findings revealed that H2S could inhibit the activation of mitochondrial apoptotic pathway in smoking rats. The MAPK signaling cascades, which are evolutionarily conserved signal transduction pathways, respond to various extracellular factors and regulate diverse cellular processes such as differentiation, proliferation, apoptosis, survival and development [21, 22]. In this study, JNK and P38 MAPK signaling pathways were significantly activated in the myocardium of smoking rats, which might be an important molecular mechanism responsible for smokinginduced apoptosis. Furthermore, H2S was found to reduce cardiomyocyte apoptosis in smoking rats by inhibiting phosphorylation of JNK and p38 MAPK. The PI3K/Akt pathway plays a key role in the regulation of cell growth, proliferation, survival and metabolism [23, 24]. PI3K/Akt signaling generally acts to promote survival through inhibition of pro-apoptotic factors and activation of anti-apoptotic factors. In the present study, PI3K/Akt signaling was remarkably inhibited in the myocardium of smoking rats, which might be another molecular mechanism involved in smoking-induced apoptosis. Moreover, H2S was found to reduce cardiomyocyte apoptosis in smoking rats via activation of PI3K/Akt signaling.
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Autophagy, an evolutionarily conserved catabolic pathway of lysosome-dependent degradation of long-lived proteins and dysfunctional organelles, is known to be induced by AMPK signaling but inhibited by mTOR signaling [25, 26]. In this study, cigarette smoking was associated with an increase in AMPK phosphorylation and a decrease in mTOR phosphorylation, whereas NaHS administration was found to inhibit AMPK activity and induce mTOR activation in the myocardium of smoking rats, which suggested that H2S could protect against smoking-induced cardiac autophagy via regulation of AMPK/mTOR signaling pathway. In conclusion, our study demonstrates that H2S protects against cigarette smoking-induced left ventricular systolic dysfunction via regulation of apoptosis and autophagy. H2S may exert anti-apoptotic effects in the myocardium of smoking rats by inhibiting JNK and P38 MAPK pathways and activating PI3K/Akt signaling. Moreover, H2S can also reduce smoking-induced autophagic cell death via regulation of AMPK/mTOR signaling pathway.
Conflict of interest
The authors declare no conflict of interest.
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