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New mechanism of lipotoxicity in diabetic cardiomyopathy: Deficiency of Endogenous H2S Production and ER stress
Accepted Manuscript Title: New mechanism of lipotoxicity in diabetic cardiomyopathy: Deficiency of Endogenous H2 S Production and ER stress Author: Runmin Guo Zijun Wu Jiamei Jiang Chang Liu Bin Wu Xingyue Li Teng Li Hailiang Mo Songjian He Shanghai Li Hai Yan Ruina Huang Qiong You Keng Wu PII: DOI: Reference:
S0047-6374(16)30222-6 http://dx.doi.org/doi:10.1016/j.mad.2016.11.005 MAD 10898
To appear in:
Mechanisms of Ageing and Development
Received date: Revised date: Accepted date:
14-7-2016 1-11-2016 15-11-2016
Please cite this article as: Guo, Runmin, Wu, Zijun, Jiang, Jiamei, Liu, Chang, Wu, Bin, Li, Xingyue, Li, Teng, Mo, Hailiang, He, Songjian, Li, Shanghai, Yan, Hai, Huang, Ruina, You, Qiong, Wu, Keng, New mechanism of lipotoxicity in diabetic cardiomyopathy: Deficiency of Endogenous H2S Production and ER stress.Mechanisms of Ageing and Development http://dx.doi.org/10.1016/j.mad.2016.11.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
New mechanism of lipotoxicity in diabetic cardiomyopathy： Deficiency of Endogenous H2S Production and ER stress
Runmin Guo
1#
, Zijun Wu
1#
, Jiamei Jiang 1,Chang Liu 1, Bin Wu 1, Xingyue Li 1,
Teng Li 1, Hailiang Mo 1, Songjian He 1, Shanghai Li 1, Hai Yan 1, Ruina Huang 1, Qiong You 1, Keng Wu 1* 1. Department of Cardiology, The Affiliated Hospital, Guangdong Medical University, Zhanjiang, Guangdong Province,524001, P.R. China. #
Runmin Guo, Zijun Wu, contributed equally to this work.
*
Correspondence to: Keng Wu, Professor, Department of Cardiology, The Affiliated
Hospital, Guangdong Medical University, Zhanjiang, 524001, P.R. China., Tel:+86-759-2387412,
Fax:+86-759-2387412,
E-mail
address:
[email protected].
Highlights
Deficiency of endogenous H2S implicated in cardiac lipotoxicity of DCM. On the other hand, H2S is a cytoprotective agent. NaHS can also inhibit cardiac lipid droplet formation and cell apoptosis in STZ rats and AC16 human
cardiac
cells.
Exogenous
H2S
could
protect
against
cardiac lipotoxicity of DCM though regulation of ER stress.
In conclusion, we firstly have presented evidence showing that deficiency of endogenous H2S production and ER stress are new mechanisms of
cardiac lipotoxicity in diabetic cardiomyopathy, exogenous H2S could protection against cardiac lipotoxicity of DCM.
Abstract Objective: To investigate the roles and mechanisms of endogenous hydrogen sulfide (H2S) and endoplasmic reticulum (ER) stress in the development of diabetic cardiomyopathy (DCM). Methods: Blood of DCM patients included in the study were collected. The model of DCM rats was established using streptozotocin (STZ) injection. Cardiac lipotoxicity in vitro models were established using 500 μM palmitic acid (PA) treatment for 24 h in AC16 cardiomyocytes. Endogenous H2S production in plasma, culture supernatant and heart was measured by sulphur ion-selective electrode assay. Cell viability was tested by using the cell counting kit-8 (CCK-8) kit. Glucose regulated protein (GRP78), CCAAT/enhancer binding protein homologous transcription factor (C/EBP) homologous protein (CHOP), caspase-3 and caspase-12 expressions were measured using western blot analysis. Lipid droplet was evaluated by Oil Red O staining. Apoptosis in hearts of DCM rats was analyzed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results: H2S levels in serum of DCM patients and DCM rats were significant lower, H2S contents and cystathionine-γ-lyase (CSE) expression in heart tissues of DCM rats were also markedly lower. H2S levels in supernatants of PA-treated AC16 cardiac cells were decreased. Cardiac lipotoxicity demonstrated by increase in TUNEL positive cells and lipid deposit in vivo and in vitro accompanied by a decrease of H2S levels. Pretreatment AC16 cells with 100 µmol/L of NaHS (a donor of H2S) could suppress the PA-induced myocardial injury similar to the effects of 4-phenylbutyric
acid (4-PBA, an endoplasmic reticulum (ER) stress inhibitor), leading to an increase in cell viability and preventing lipid deposit. Meanwhile, administration diabetic rats with NaHS or 4-PBA alleviated cardiac lipotoxicity, as evidenced by decrease in TUNEL positive cells, cleaved caspase-3 expression and lipid accumulation. Conclusion: Deficiency of endogenous H2S was involved in lipotoxicity-induced myocardial injury. Exogenous H2S attenuates PA-induced myocardial injury though inhibition of ER stress. Keywords: lipotoxicity; diabetic cardiomyopathy; hydrogen sulfide; endoplasmic reticulum stress
Introduction Diabetes mellitus and its cardiovascular complications have resulted in an explosion over the last few decades [1]. There are approximately 340 million people with diabetes worldwide, of which 90% is Type 2 diabetes (T2D) [2], about 50% will die of cardiovascular diseases, primarily from either heart failure (HF) or myocardial infarction (MI). Thus, diabetic cardiomyopathy (DCM) is underlying pathogenic processes for diabetic cardiovascular complications, which is cardiac dysfunction independent of coronary artery disease or hypertension. DCM is characterized by the heart structural remodels and underlying diastolic rather than systolic dysfunction. Several mechanisms responsible for the diabetic cardiomyopathy have been proposed. Oxidative stress, cell apoptosis, cardiac insulin resistance, endoplasmic reticulum (ER) stress and mitochondrial dysfunction were regarded as major pathogenesis mechanisms [2]. Among them, ER stress is the accumulation of misfolded or unfolded
proteins in the ER lumen, leading to triggering the complex cellular response such as cell apoptosis and loss of function, and has been reported to be induced in the diabetic heart and participate in the pathogenesis of DCM [3]. More recently, accumulating evidences suggests that lower hydrogen sulfide (H2S) levels may play a role in the pathogenesis of diabetes mellitus and its related complications [4, 5], especially, some of the evidences shown that H2S might be involved in the development of DCM [6]. However, it is not known that the molecular mechanism underlying H2S action in the pathogenesis of DCM. Therefore, the purpose of this study was to investigate the roles and mechanisms of the endogenous H2S and ER stress.
Materials and Methods 2.1. Study Subjects and Blood Collection The study recruited 32 DCM patients (24 male and 8 femal), 62 diabetic patients with no LV dysfunction (38 male and 24 female) from the Department of Cardiology at the Affiliated Hospital of Guangdong Medical College in China from 2012 to 2015. Medical history recording and blood sample collections were performed at admission. Before any laboratory tests or imaging procedures all participant underwent a physical examination. Medical records in hospitals for all participant include a hypertension or myocardial infarction or coronary artery disease or congenital heart disease or valvular disease or diabetes mellitus history. All participant with either systolic or diastolic dysfunction, without a history of symptomatic or clinical Heart Failure were
included considering as preclinical ventricular dysfunction. Proposed diagnosed criteria for DCM based on previous definitions [1]: 1) a history of diabetes mellitus, 2) at least moderate diastolic or documented systolic dysfunction, 3) no history of clinical Heart Failure, 4) no history of coronary disease, hypertension, significant valvular disease, and congenital heart disease. Healthy controls without diabetes, LV dysfunction, hypertension, or coronary disease were selected from the age-matched Zhanjiang City population. Written informed consent was obtained from all patients according to the protocol approved by Ethics Committee of the Affiliated Hospital of Guangdong Medical University. 2.2. Experimental Animals and Treatments. Male Sprague-Dawley (SD) rats (180-220 g) were provided by Laboratory Animal Center of Guangdong Medical University. This study was approved by the Institutional Research Ethical Committee. Diabetes was induced by single intraperitoneal (ip) injections of 55 mg/kg streptozotocin (STZ, dissolved in 10 mmol/L sodium citrate buffer). Random blood glucose level ≥ 16.7 mmoL/ L in SD rats is defined as an experimental diabetic model after 48 h [4]. Rats were randomly divided into: normal control group (Control group), diabetic rats (4, 8 and 12 week DCM group), diabetic rats (DCM+NaHS treatment group, DCM+4-PBA treatment group). Rats in NaHS treatment groups were intraperitoneally injected daily with
100 μM of sodium hydrogen sulfide (NaHS, a donor of H2S),
Rats in 4-PBA (an ER stress inhibitor) treatment groups were was administered
intragastrically with 4-PBA solution once a day at a dose of 500 mg/kg / day for 20 days. NaHS, 4-PBA and STZ were purchased from Sigma-Aldrich (St. Louis, MO, USA). All rats were sacrificed at 12 weeks after the induction of diabetes for analysis of heart tissues. Blood samples were collected 4 weeks after STZ injections. All samples were kept at -80 °C refrigerator until needed. 2.3. Cardiac Lipotoxicity in vitro Model and Treatment Palmitic acid (PA, 50 mM) stock solution was freshly dissolved in 90 % ethanol. PA was bound to fatty acid free bovine serum albumin (BSA) in DMEM culture medium supplemented with 1 % fetal calf serum. The ratio between PA and BSA is 0.5 mM PA to 1 % BSA. PA was purchased from Sigma-Aldrich (St. Louis, MO). AC-16 cardiac cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12, Life Technologies) containing
10%
(v/v)
fetal
bovine
serum
(FBS,
Thermo
Fisher)
and
penicillin/streptomycin (100 units) and maintained in an 37℃ incubator with 5% CO2. Treatment human AC16 cardiac cells with PA (500 μM) for 24 h mimic cardiac lipotoxicity. Meanwhile, NaHS (100 μM) were added in the medium of the PA group. NaHS treatment was repeated every 6 h during the entire treatment period of 24 h. 2.4. Measurement of Endogenous H2S Production in Plasma and Culture Supernatant H2S contents were measured in plasma and culture supernatant as previously described (9, 10). Briefly, plasma was centrifuged after blood collection. Culture
medium was collected from human AC 16 cardiomyocytes flask. 25vml Erlenmeyer Pyrex flasks with a specially made glass chamber (diameter 1 cm and height 2 cm) were used for measurement. Cryovial test tubes (2 ml) containing 0.5 ml of 1 mol/l NaOH were used as the central wells. The Sulphur ion-selective electrode (PXS-270, Shanghai, China) was used to evaluate the endogenous H2S concentrations against a standard curve (3-250 μM) after calibrating with the protein concentration in the corresponding samples. All samples were tested in duplicate. 2.5. Cell Viability Assay. After AC 16 cells were received different treatments, which were cultured in 96-well plates, cell viability of AC 16 cells was measured by using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies) according to the manufacturer’s instructions. 10 µl of CCK-8 working solution was added to each well, followed by further 2 h incubation. The absorbance of CCK-8 was measured at 450 nm wavelenth with a microplate reader (Multiskan MK3 Microplate reader, Thermo Fisher Scientific Inc, USA). The mean optical density (OD) of five wells in the indicated groups was used to calculate the percentage of cell viability according to the formula below: Percentage of cell viability (of %) = OD treatment group/OD control group x 100%. Experiments were repeated for three times. 2.6. Western Blot Analysis. Frozen left ventricle tissues and AC 16 cells after the indicated treatments were lysed with ice-cold RIPA buffer, and the homogenate was centrifuged at 12,000 rpm for 10 min at 4 ˚C. The total protein in the supernatant was quantified using a BCA
protein assay kit (Thermo Fisher Scientific Inc, Rockford, IL, USA). Protein samples were separated on 10% SDS-PAGE gels, and then transferred to a polyvinyl Dene difluoride (PVDF) membrane (Millipore-Upstate). The membrane was blocked with 5% non-fat milk in TBS-T for 1 h at room temperature and then incubated with primary antibodies directed against CSE, GRP78, CHOP caspase-3 and caspase-12 (1:2,000) (Cell Signaling Technology, Beverly, MA, USA), or β-actine or GAPDH (loading control) with gentle agitation at 4 ˚C overnight. After washing with TBST, and
subsequently
the
membranes
were
incubated
with
horseradish
peroxidase-conjugated secondary antibodies for 1.5 h at room temperature. Following three washes with TBST, specific bands on membranes were detected with enhanced chemiluminescence (Thermo Scientific-Pierce) and exposed to X-ray films. To quantify protein expression, the X-ray films were scanned and semi-quantifiably analyzed with ImageJ 1.41o software (National Institutes of Health, USA). 2.7. Lipid Droplet Staining with Oil Red O The frozen heart was sectioned at 6μM thickness with a Leica cryomicrotome (Leica Microsystems) prior to fixation in 10% formalin for 10 min. AC-16 cells were cultured for 24 h and afterwards exposed to 500μM PA for another 24 h. Cells were fixed in paraformaldehyde for 15 min at room temperature. Following three washes cells or tissue sections with distilled water, the slides were placed in absolute propylene glycol for 5 min. Slides were then incubated in pre-warmed Oil Red O solution (Sigma-Aldrich, Munich, Germany) for 10 min. Slides were rinsed twice and coverslipped. Lipid accumulation was analyzed by Olympus BX-51 fluorescence
microscopy (Olympus America Inc., Melville, NY) and measured with ImageJ 1.41o software (National Institutes of Health, USA). 2.8. Terminal-deoxynucleotidyl Transferase-mediated Nick End Labeling (TUNEL) Cell apoptosis in rat heart tissue was detected by TUNEL staining using One Step TUNEL Apoptosis Assay Kit (Beyotime, Jiangsu, China) according to the manufacturer’s instructions. Briefly, heart tissues were paraffin embedded and sectioned and then dewaxed. The sections were washed 3 times with phosphate buffered saline (PBS), and then incubated with TUNEL-staining kit at 37 °C for 60 min. Nuclei were stained with DAPI (Beyotime, Jiangsu, China) after rinsed 3 times with PBS. Finally, the sections were visualized by Olympus BX-51 fluorescence microscopy (Olympus America Inc., Melville, NY) and TUNEL-positive cells were counted. The average number of TUNEL-positive cells in each section was defined as the apoptotic index. 2.9. Statistical Analyses. Data were expressed as mean ± SEM. The results for three or more groups were compared using one-way ANOVA analysis of variation (ANOVA) followed by a Tukey’s post hoc analysis. All statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.
3. Results
3.1. Decreased H2S Production in cardiac lipotoxicity in vivo and in vitro. Compared with control, H2S levels in serum of DCM patients and DCM rats were significantly lower (Fig 1A and 1C), H2S contents in heart tissues of DCM rats were also markedly lower (Fig 1B). Furthermore, because the H2S levels were low in the diabetic heart and cystathionine- γ -lyase (CSE) was regarded as a major H2S-producing enzymes in hearts, Western blot assay result shown that protein expression of CSE in heart of Streptozotocin (STZ)-induced DCM rats was gradually reduced with disease development (Fig 1D and 1E).
The levels of H2S in plasma of DCM patients (A) and DCM rats(C), and H2S contents in heart tissues of DCM rats (B) were measured by sulphur ion-selective electrode assay. The expression of H2S-producing enzymes (CSE) in heart of DCM rats (D and E) was detected by western blot assay. * P < 0.05, ** P < 0.01 versus control. 3.2. Deficiency of Endogenous H2S Involves in Lipotoxicity-Induced Myocardial Injury. The AC 16 cardiac cells were incubated in palmic acid (PA, 500 µmol/L) for 24 h to mimic the hyperlipidemia in DCM in vitro (cardiac lipotoxicity). H2S levels in supernatants of PA-treated AC 16 cardiac cells were decreased. As shown in Figure 2, AC 16 cells which were exposed to PA (500 µmol/L) resulted in a significant decrease in cell viability (Fig 2A) and increase in lipid deposit (Fig 2C). On the other side， similar results also were observed in hearts of DCM rats, as demonstrated by increase in TUNEL positive cells and lipid deposit (Fig 2D). Meanwhile, PA-induced
myocardial injury was accompanied by a decrease of H2S levels in AC 16 cells (Fig 2B). These results suggest that endogenous H2S is involved in the diabetic myocardial injury.
Cell viability of AC16 cardiomyocytes treated with different concentrations palmic acid (PA, 500 µmol/L) for 24 h (A) was detected by CCK-8 kit. H2S levels in supernatants of PA-induced AC16 cardiac cell injury model (B) was measured by sulphur ion-selective electrode assay. Lipid deposit in hearts of DCM rats (D) and AC 16 cells (C) were tested using oil red O staining. Cell apoptosis in hearts of DCM rats (D) was analysized by TUNEL staining. * P < 0.05, ** P < 0.01 versus control. 3.3. Exogenous H2S Attenuates PA-Induced Myocardial Injury. To determine whether exogenous H2S attenuated PA-induced myocardial injury, 100 µmol/L of NaHS (a donor of H2S) were added in the media of the PA (500 µmol/L) group. After adding NaHS in to the cell culture media, exogenous H2S could suppress the PA-induced myocardial injury similar to 4-PBA’s effects which is an ER stress inhibitor, leading to an increase in cell viability (Fig 3A) and preventing lipid deposit (Fig 2C). Meanwhile, administration diabetic rats with NaHS and 4-PBA could alleviated cardiac lipotoxicity, as evidenced by decrease in TUNEL positive cells (Fig 2D),cleaved caspase-3 expression (Fig 3B and 3C) and lipid accumulation (Fig 2D). 3.4. Exogenous H2S Inhibits Endoplasmic Reticulum (ER) Stress in Cardiac Cells. Next, in order to demonstrate the effect of NaHS on ER stress in presence or absence
of PA, we examined the expressions of ER stress marker protein in AC 16 cardiac cells. PA treatment for 24 h dramatically enhanced the expression ER stress marker proteins including GRP78, CHOP and caspase 12 (Fig. 4). However, pretreatment with NaHS (100 µmol/L) for 60 min following by PA (500 µmol/L) treatment significantly suppressed upregulation of ER stress marker proteins. At the same time, administration diabetic rats with NaHS could suppress cardiac ER stress as evidenced by decrease in the expressions of GRP78, CHOP and caspase-12 proteins (Fig 4D). These results indicated that ER Stress induced by PA can be mostly suppressed by NaHS treatment, exogenous H2S inhibits cardiac lipotoxicity via regulation of ER Stress.
Discussion In this study, we established a cardiac lipotoxicity in vitro model by treatment AC 16 cardiac cells with 500 μmol/L of palmic acid (PA) for 24 h to investigate the underlying mechanisms. Our results shown two important findings: (1) H2S production under cardiac lipotoxicity in vivo and in vitro was decreased, indicating deficiency of endogenous H2S implicated in PA-induced myocardial injury. (2) Exogenous H2S inhibited endoplasmic reticulum (ER) stress and myocardial injury induced by PA in AC 16 cardiac cells. There are multiple lines of evidence showing that oxidative stress, calcium homeostasis, apoptotic cell death, inflammation and reduced angiogenesis were regarded as major contributor to the development of diabetic cardiomyopathy (DCM)
[7, 8]. Hydrogen sulfide (H2S), which is a colorless water-soluble gas with the characteristic smell of rotten eggs, is widely known as the third gasotransmitter after nitric oxide and carbon monoxide. Endogenous H2S is endogenously synthesized by three enzymes: CSE (cystathionine-γ-lyase), CBS (cystathionine-β-synthase), and 3-MST (3-mercaptopyruvate sulfurtransferase) in mammalian tissues [9, 10, 11]. H2S is involved in numerous pathophysiological and physiological processes with its anti-apoptotic, anti-oxidative, anti-inflammatory and proangiogenic actions [12, 13]. Until recently, multiple lines of evidence implicate that endogenous H2S homeostasis contribute to the pathogenesis of diabetes mellitus and diabetic complications, dysregulation
of
endogenous
H 2S
production
under
hyperlipidemia
and
hyperglycemia circumstances might be involve in endothelial dysfunction which is a central pathogenesis process of diabetic complications [14, 15, 16, 17]. However, underlying role of endogenous H2S in DCM cardiac lipotoxicity is fairly limited. Our results indicated that circulating and heart H2S levels were lower than that in control, which were consistent with the results of previous studies of diabetic patients and animals models [4, 18,19]. Protein expression of CSE, a key H2S production enzyme in the hearts, was downregulated, which might explain the reduced H2S levels. The current article provides novel experimental evidence that endogenous H2S implicates in the pathogenesis of diabetic complications especially cardiac lipotoxicity in vitro and in vivo. Increasing evidence suggest that cardiac lipotoxicity and endoplasmic reticulum (ER) stress play a role in the pathogenesis of DCM [20, 21, 22]. Cardiac lipotoxicity
illustrate that excess lipid deposit may exert direct toxic effects on cardiomyocytes function, which may represent a significant component of the diabetic cardiomyopathy phenotype [22, 23]. ER stress was involved in the cardiac apoptosis of streptozotocin (STZ)-induced diabetic rat and high glucose treated cardiomyocytes [20, 21]. Furthermore, ER stress may play an important role in the development of lipotoxicity [24]. However, the underlying mechanisms whether ER stress involves in DCM cardiac lipotoxicity were not fully clear. In the hearts of STZ rats and AC cardiac cells treated with PA, a significant elevated of GRP78 and CHOP expression were observed, while the expressions of cleaved caspase-3 and caspase-12 were significantly elevated, indicating that ER stress and related apoptosis may also play an important role in diabetic cardiomyopathy. Meanwhile, lipid accumulation and correlated cardiac cell deaths in diabetic hearts tissues and cardiomyocytes treated with PA were observed. Thus, these results appear similar to previous reports [25, 26]. To clarify the relationship between ER stress and cardiac lipotoxicity in DCM, administration STZ rats and pretreatment AC16 human cardiac cells with 4-PBA can suppress cardiac lipid deposit and cell apoptosis. These results suggest that ER stress and related apoptosis mediates cardiac lipotoxicity in DCM. In addition, accumulating evidence confirm that ER stress-induced apoptosis, which involves in the pathological processes of cardiovascular diseases, is mainly mediated by CHOP, inositol-requiring enzyme 1 (IRE1, also called ERN1) and caspase-12 [27], suggesting the potential contribution of these pathway to cardiac lipotoxicity in DCM.
As is mentioned above,
deficiency
of
endogenous
H 2S
implicated
in
cardiac lipotoxicity of DCM. On the other hand, H2S is a cytoprotective agent. However, whether exogenous H2S could protect against cardiac lipotoxicity of DCM though regulation of ER stress are not yet known. We firstly reported that NaHS can also inhibit cardiac lipid droplet formation and cell apoptosis in STZ rats and AC16 human cardiac cells by inhibition of ER stress . In conclusion, we have presented evidence showing that deficiency of endogenous H2S production and ER stress are new mechanisms of cardiac lipotoxicity in diabetic cardiomyopathy, exogenous H2S could protection against cardiac lipotoxicity of DCM. Acknowledgements The present study was supported by grants from the Science and Technology Planning Project of Guangdong in China (no. 2012A080202020), the Guangdong Natural Science Foundation (no. 2015A030310359, S2011010002620) and Zhanjiang Municipal Financial Funds Special Competitive Project (2014A01033). Reference [1] Dandamudi S, Slusser J, Mahoney DW, Redfield MM, Rodeheffer RJ, Chen HH. The prevalence of diabetic cardiomyopathy: a population-based study in Olmsted County, Minnesota. J Card Fail. 2014;20(5):304-309. [2] Liu Q, Wang S, Cai L. Diabetic cardiomyopathy and its mechanisms: Role of oxidative stress and damage. J Diabetes Investig. 2014;5(6):623-634. [3] Yang L, Zhao D, Ren J, Yang J. Endoplasmic reticulum stress and protein quality
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Figure 1: Decreased H2S Production in cardiac lipotoxicity in vivo and in vitro.
Figure 2: Decreased Endogenous H2S Production Involves in cardiac lipotoxicity in vivo and in vitro.
Figure 3: Exogenous H2S Attenuates cardiac lipotoxicity in vivo and in vitro. Cell viability of AC16 cardiomyocytes treated with different treatments (A) was measured using CCK-8 kit. Cleaved caspase-3 protein expression in AC16 cells (B) and (C) were measured by western blot assay. ** P < 0.01 versus control; ## P < 0.01 versus PA group.
Figure 4: Exogenous H2S Inhibits Endoplasmic Reticulum (ER) Stress of Cardiac cells in hearts of DCM rats and AC16 cells treated with PA. GRP78, CHOP and caspase-12 protein expressions in cardiac cells (AC16 cells treated with PA (A, B, C) and DCM rats (D) were measured by western blot assay. P < 0.01 versus PA group.
**
P < 0.01 versus control; ##
S0047-6374(16)30222-6 http://dx.doi.org/doi:10.1016/j.mad.2016.11.005 MAD 10898
To appear in:
Mechanisms of Ageing and Development
Received date: Revised date: Accepted date:
14-7-2016 1-11-2016 15-11-2016
Please cite this article as: Guo, Runmin, Wu, Zijun, Jiang, Jiamei, Liu, Chang, Wu, Bin, Li, Xingyue, Li, Teng, Mo, Hailiang, He, Songjian, Li, Shanghai, Yan, Hai, Huang, Ruina, You, Qiong, Wu, Keng, New mechanism of lipotoxicity in diabetic cardiomyopathy: Deficiency of Endogenous H2S Production and ER stress.Mechanisms of Ageing and Development http://dx.doi.org/10.1016/j.mad.2016.11.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
New mechanism of lipotoxicity in diabetic cardiomyopathy： Deficiency of Endogenous H2S Production and ER stress
Runmin Guo
1#
, Zijun Wu
1#
, Jiamei Jiang 1,Chang Liu 1, Bin Wu 1, Xingyue Li 1,
Teng Li 1, Hailiang Mo 1, Songjian He 1, Shanghai Li 1, Hai Yan 1, Ruina Huang 1, Qiong You 1, Keng Wu 1* 1. Department of Cardiology, The Affiliated Hospital, Guangdong Medical University, Zhanjiang, Guangdong Province,524001, P.R. China. #
Runmin Guo, Zijun Wu, contributed equally to this work.
*
Correspondence to: Keng Wu, Professor, Department of Cardiology, The Affiliated
Hospital, Guangdong Medical University, Zhanjiang, 524001, P.R. China., Tel:+86-759-2387412,
Fax:+86-759-2387412,
address:
[email protected].
Highlights
Deficiency of endogenous H2S implicated in cardiac lipotoxicity of DCM. On the other hand, H2S is a cytoprotective agent. NaHS can also inhibit cardiac lipid droplet formation and cell apoptosis in STZ rats and AC16 human
cardiac
cells.
Exogenous
H2S
could
protect
against
cardiac lipotoxicity of DCM though regulation of ER stress.
In conclusion, we firstly have presented evidence showing that deficiency of endogenous H2S production and ER stress are new mechanisms of
cardiac lipotoxicity in diabetic cardiomyopathy, exogenous H2S could protection against cardiac lipotoxicity of DCM.
Abstract Objective: To investigate the roles and mechanisms of endogenous hydrogen sulfide (H2S) and endoplasmic reticulum (ER) stress in the development of diabetic cardiomyopathy (DCM). Methods: Blood of DCM patients included in the study were collected. The model of DCM rats was established using streptozotocin (STZ) injection. Cardiac lipotoxicity in vitro models were established using 500 μM palmitic acid (PA) treatment for 24 h in AC16 cardiomyocytes. Endogenous H2S production in plasma, culture supernatant and heart was measured by sulphur ion-selective electrode assay. Cell viability was tested by using the cell counting kit-8 (CCK-8) kit. Glucose regulated protein (GRP78), CCAAT/enhancer binding protein homologous transcription factor (C/EBP) homologous protein (CHOP), caspase-3 and caspase-12 expressions were measured using western blot analysis. Lipid droplet was evaluated by Oil Red O staining. Apoptosis in hearts of DCM rats was analyzed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results: H2S levels in serum of DCM patients and DCM rats were significant lower, H2S contents and cystathionine-γ-lyase (CSE) expression in heart tissues of DCM rats were also markedly lower. H2S levels in supernatants of PA-treated AC16 cardiac cells were decreased. Cardiac lipotoxicity demonstrated by increase in TUNEL positive cells and lipid deposit in vivo and in vitro accompanied by a decrease of H2S levels. Pretreatment AC16 cells with 100 µmol/L of NaHS (a donor of H2S) could suppress the PA-induced myocardial injury similar to the effects of 4-phenylbutyric
acid (4-PBA, an endoplasmic reticulum (ER) stress inhibitor), leading to an increase in cell viability and preventing lipid deposit. Meanwhile, administration diabetic rats with NaHS or 4-PBA alleviated cardiac lipotoxicity, as evidenced by decrease in TUNEL positive cells, cleaved caspase-3 expression and lipid accumulation. Conclusion: Deficiency of endogenous H2S was involved in lipotoxicity-induced myocardial injury. Exogenous H2S attenuates PA-induced myocardial injury though inhibition of ER stress. Keywords: lipotoxicity; diabetic cardiomyopathy; hydrogen sulfide; endoplasmic reticulum stress
Introduction Diabetes mellitus and its cardiovascular complications have resulted in an explosion over the last few decades [1]. There are approximately 340 million people with diabetes worldwide, of which 90% is Type 2 diabetes (T2D) [2], about 50% will die of cardiovascular diseases, primarily from either heart failure (HF) or myocardial infarction (MI). Thus, diabetic cardiomyopathy (DCM) is underlying pathogenic processes for diabetic cardiovascular complications, which is cardiac dysfunction independent of coronary artery disease or hypertension. DCM is characterized by the heart structural remodels and underlying diastolic rather than systolic dysfunction. Several mechanisms responsible for the diabetic cardiomyopathy have been proposed. Oxidative stress, cell apoptosis, cardiac insulin resistance, endoplasmic reticulum (ER) stress and mitochondrial dysfunction were regarded as major pathogenesis mechanisms [2]. Among them, ER stress is the accumulation of misfolded or unfolded
proteins in the ER lumen, leading to triggering the complex cellular response such as cell apoptosis and loss of function, and has been reported to be induced in the diabetic heart and participate in the pathogenesis of DCM [3]. More recently, accumulating evidences suggests that lower hydrogen sulfide (H2S) levels may play a role in the pathogenesis of diabetes mellitus and its related complications [4, 5], especially, some of the evidences shown that H2S might be involved in the development of DCM [6]. However, it is not known that the molecular mechanism underlying H2S action in the pathogenesis of DCM. Therefore, the purpose of this study was to investigate the roles and mechanisms of the endogenous H2S and ER stress.
Materials and Methods 2.1. Study Subjects and Blood Collection The study recruited 32 DCM patients (24 male and 8 femal), 62 diabetic patients with no LV dysfunction (38 male and 24 female) from the Department of Cardiology at the Affiliated Hospital of Guangdong Medical College in China from 2012 to 2015. Medical history recording and blood sample collections were performed at admission. Before any laboratory tests or imaging procedures all participant underwent a physical examination. Medical records in hospitals for all participant include a hypertension or myocardial infarction or coronary artery disease or congenital heart disease or valvular disease or diabetes mellitus history. All participant with either systolic or diastolic dysfunction, without a history of symptomatic or clinical Heart Failure were
included considering as preclinical ventricular dysfunction. Proposed diagnosed criteria for DCM based on previous definitions [1]: 1) a history of diabetes mellitus, 2) at least moderate diastolic or documented systolic dysfunction, 3) no history of clinical Heart Failure, 4) no history of coronary disease, hypertension, significant valvular disease, and congenital heart disease. Healthy controls without diabetes, LV dysfunction, hypertension, or coronary disease were selected from the age-matched Zhanjiang City population. Written informed consent was obtained from all patients according to the protocol approved by Ethics Committee of the Affiliated Hospital of Guangdong Medical University. 2.2. Experimental Animals and Treatments. Male Sprague-Dawley (SD) rats (180-220 g) were provided by Laboratory Animal Center of Guangdong Medical University. This study was approved by the Institutional Research Ethical Committee. Diabetes was induced by single intraperitoneal (ip) injections of 55 mg/kg streptozotocin (STZ, dissolved in 10 mmol/L sodium citrate buffer). Random blood glucose level ≥ 16.7 mmoL/ L in SD rats is defined as an experimental diabetic model after 48 h [4]. Rats were randomly divided into: normal control group (Control group), diabetic rats (4, 8 and 12 week DCM group), diabetic rats (DCM+NaHS treatment group, DCM+4-PBA treatment group). Rats in NaHS treatment groups were intraperitoneally injected daily with
100 μM of sodium hydrogen sulfide (NaHS, a donor of H2S),
Rats in 4-PBA (an ER stress inhibitor) treatment groups were was administered
intragastrically with 4-PBA solution once a day at a dose of 500 mg/kg / day for 20 days. NaHS, 4-PBA and STZ were purchased from Sigma-Aldrich (St. Louis, MO, USA). All rats were sacrificed at 12 weeks after the induction of diabetes for analysis of heart tissues. Blood samples were collected 4 weeks after STZ injections. All samples were kept at -80 °C refrigerator until needed. 2.3. Cardiac Lipotoxicity in vitro Model and Treatment Palmitic acid (PA, 50 mM) stock solution was freshly dissolved in 90 % ethanol. PA was bound to fatty acid free bovine serum albumin (BSA) in DMEM culture medium supplemented with 1 % fetal calf serum. The ratio between PA and BSA is 0.5 mM PA to 1 % BSA. PA was purchased from Sigma-Aldrich (St. Louis, MO). AC-16 cardiac cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12, Life Technologies) containing
10%
(v/v)
fetal
bovine
serum
(FBS,
Thermo
Fisher)
and
penicillin/streptomycin (100 units) and maintained in an 37℃ incubator with 5% CO2. Treatment human AC16 cardiac cells with PA (500 μM) for 24 h mimic cardiac lipotoxicity. Meanwhile, NaHS (100 μM) were added in the medium of the PA group. NaHS treatment was repeated every 6 h during the entire treatment period of 24 h. 2.4. Measurement of Endogenous H2S Production in Plasma and Culture Supernatant H2S contents were measured in plasma and culture supernatant as previously described (9, 10). Briefly, plasma was centrifuged after blood collection. Culture
medium was collected from human AC 16 cardiomyocytes flask. 25vml Erlenmeyer Pyrex flasks with a specially made glass chamber (diameter 1 cm and height 2 cm) were used for measurement. Cryovial test tubes (2 ml) containing 0.5 ml of 1 mol/l NaOH were used as the central wells. The Sulphur ion-selective electrode (PXS-270, Shanghai, China) was used to evaluate the endogenous H2S concentrations against a standard curve (3-250 μM) after calibrating with the protein concentration in the corresponding samples. All samples were tested in duplicate. 2.5. Cell Viability Assay. After AC 16 cells were received different treatments, which were cultured in 96-well plates, cell viability of AC 16 cells was measured by using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies) according to the manufacturer’s instructions. 10 µl of CCK-8 working solution was added to each well, followed by further 2 h incubation. The absorbance of CCK-8 was measured at 450 nm wavelenth with a microplate reader (Multiskan MK3 Microplate reader, Thermo Fisher Scientific Inc, USA). The mean optical density (OD) of five wells in the indicated groups was used to calculate the percentage of cell viability according to the formula below: Percentage of cell viability (of %) = OD treatment group/OD control group x 100%. Experiments were repeated for three times. 2.6. Western Blot Analysis. Frozen left ventricle tissues and AC 16 cells after the indicated treatments were lysed with ice-cold RIPA buffer, and the homogenate was centrifuged at 12,000 rpm for 10 min at 4 ˚C. The total protein in the supernatant was quantified using a BCA
protein assay kit (Thermo Fisher Scientific Inc, Rockford, IL, USA). Protein samples were separated on 10% SDS-PAGE gels, and then transferred to a polyvinyl Dene difluoride (PVDF) membrane (Millipore-Upstate). The membrane was blocked with 5% non-fat milk in TBS-T for 1 h at room temperature and then incubated with primary antibodies directed against CSE, GRP78, CHOP caspase-3 and caspase-12 (1:2,000) (Cell Signaling Technology, Beverly, MA, USA), or β-actine or GAPDH (loading control) with gentle agitation at 4 ˚C overnight. After washing with TBST, and
subsequently
the
membranes
were
incubated
with
horseradish
peroxidase-conjugated secondary antibodies for 1.5 h at room temperature. Following three washes with TBST, specific bands on membranes were detected with enhanced chemiluminescence (Thermo Scientific-Pierce) and exposed to X-ray films. To quantify protein expression, the X-ray films were scanned and semi-quantifiably analyzed with ImageJ 1.41o software (National Institutes of Health, USA). 2.7. Lipid Droplet Staining with Oil Red O The frozen heart was sectioned at 6μM thickness with a Leica cryomicrotome (Leica Microsystems) prior to fixation in 10% formalin for 10 min. AC-16 cells were cultured for 24 h and afterwards exposed to 500μM PA for another 24 h. Cells were fixed in paraformaldehyde for 15 min at room temperature. Following three washes cells or tissue sections with distilled water, the slides were placed in absolute propylene glycol for 5 min. Slides were then incubated in pre-warmed Oil Red O solution (Sigma-Aldrich, Munich, Germany) for 10 min. Slides were rinsed twice and coverslipped. Lipid accumulation was analyzed by Olympus BX-51 fluorescence
microscopy (Olympus America Inc., Melville, NY) and measured with ImageJ 1.41o software (National Institutes of Health, USA). 2.8. Terminal-deoxynucleotidyl Transferase-mediated Nick End Labeling (TUNEL) Cell apoptosis in rat heart tissue was detected by TUNEL staining using One Step TUNEL Apoptosis Assay Kit (Beyotime, Jiangsu, China) according to the manufacturer’s instructions. Briefly, heart tissues were paraffin embedded and sectioned and then dewaxed. The sections were washed 3 times with phosphate buffered saline (PBS), and then incubated with TUNEL-staining kit at 37 °C for 60 min. Nuclei were stained with DAPI (Beyotime, Jiangsu, China) after rinsed 3 times with PBS. Finally, the sections were visualized by Olympus BX-51 fluorescence microscopy (Olympus America Inc., Melville, NY) and TUNEL-positive cells were counted. The average number of TUNEL-positive cells in each section was defined as the apoptotic index. 2.9. Statistical Analyses. Data were expressed as mean ± SEM. The results for three or more groups were compared using one-way ANOVA analysis of variation (ANOVA) followed by a Tukey’s post hoc analysis. All statistical analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.
3. Results
3.1. Decreased H2S Production in cardiac lipotoxicity in vivo and in vitro. Compared with control, H2S levels in serum of DCM patients and DCM rats were significantly lower (Fig 1A and 1C), H2S contents in heart tissues of DCM rats were also markedly lower (Fig 1B). Furthermore, because the H2S levels were low in the diabetic heart and cystathionine- γ -lyase (CSE) was regarded as a major H2S-producing enzymes in hearts, Western blot assay result shown that protein expression of CSE in heart of Streptozotocin (STZ)-induced DCM rats was gradually reduced with disease development (Fig 1D and 1E).
The levels of H2S in plasma of DCM patients (A) and DCM rats(C), and H2S contents in heart tissues of DCM rats (B) were measured by sulphur ion-selective electrode assay. The expression of H2S-producing enzymes (CSE) in heart of DCM rats (D and E) was detected by western blot assay. * P < 0.05, ** P < 0.01 versus control. 3.2. Deficiency of Endogenous H2S Involves in Lipotoxicity-Induced Myocardial Injury. The AC 16 cardiac cells were incubated in palmic acid (PA, 500 µmol/L) for 24 h to mimic the hyperlipidemia in DCM in vitro (cardiac lipotoxicity). H2S levels in supernatants of PA-treated AC 16 cardiac cells were decreased. As shown in Figure 2, AC 16 cells which were exposed to PA (500 µmol/L) resulted in a significant decrease in cell viability (Fig 2A) and increase in lipid deposit (Fig 2C). On the other side， similar results also were observed in hearts of DCM rats, as demonstrated by increase in TUNEL positive cells and lipid deposit (Fig 2D). Meanwhile, PA-induced
myocardial injury was accompanied by a decrease of H2S levels in AC 16 cells (Fig 2B). These results suggest that endogenous H2S is involved in the diabetic myocardial injury.
Cell viability of AC16 cardiomyocytes treated with different concentrations palmic acid (PA, 500 µmol/L) for 24 h (A) was detected by CCK-8 kit. H2S levels in supernatants of PA-induced AC16 cardiac cell injury model (B) was measured by sulphur ion-selective electrode assay. Lipid deposit in hearts of DCM rats (D) and AC 16 cells (C) were tested using oil red O staining. Cell apoptosis in hearts of DCM rats (D) was analysized by TUNEL staining. * P < 0.05, ** P < 0.01 versus control. 3.3. Exogenous H2S Attenuates PA-Induced Myocardial Injury. To determine whether exogenous H2S attenuated PA-induced myocardial injury, 100 µmol/L of NaHS (a donor of H2S) were added in the media of the PA (500 µmol/L) group. After adding NaHS in to the cell culture media, exogenous H2S could suppress the PA-induced myocardial injury similar to 4-PBA’s effects which is an ER stress inhibitor, leading to an increase in cell viability (Fig 3A) and preventing lipid deposit (Fig 2C). Meanwhile, administration diabetic rats with NaHS and 4-PBA could alleviated cardiac lipotoxicity, as evidenced by decrease in TUNEL positive cells (Fig 2D),cleaved caspase-3 expression (Fig 3B and 3C) and lipid accumulation (Fig 2D). 3.4. Exogenous H2S Inhibits Endoplasmic Reticulum (ER) Stress in Cardiac Cells. Next, in order to demonstrate the effect of NaHS on ER stress in presence or absence
of PA, we examined the expressions of ER stress marker protein in AC 16 cardiac cells. PA treatment for 24 h dramatically enhanced the expression ER stress marker proteins including GRP78, CHOP and caspase 12 (Fig. 4). However, pretreatment with NaHS (100 µmol/L) for 60 min following by PA (500 µmol/L) treatment significantly suppressed upregulation of ER stress marker proteins. At the same time, administration diabetic rats with NaHS could suppress cardiac ER stress as evidenced by decrease in the expressions of GRP78, CHOP and caspase-12 proteins (Fig 4D). These results indicated that ER Stress induced by PA can be mostly suppressed by NaHS treatment, exogenous H2S inhibits cardiac lipotoxicity via regulation of ER Stress.
Discussion In this study, we established a cardiac lipotoxicity in vitro model by treatment AC 16 cardiac cells with 500 μmol/L of palmic acid (PA) for 24 h to investigate the underlying mechanisms. Our results shown two important findings: (1) H2S production under cardiac lipotoxicity in vivo and in vitro was decreased, indicating deficiency of endogenous H2S implicated in PA-induced myocardial injury. (2) Exogenous H2S inhibited endoplasmic reticulum (ER) stress and myocardial injury induced by PA in AC 16 cardiac cells. There are multiple lines of evidence showing that oxidative stress, calcium homeostasis, apoptotic cell death, inflammation and reduced angiogenesis were regarded as major contributor to the development of diabetic cardiomyopathy (DCM)
[7, 8]. Hydrogen sulfide (H2S), which is a colorless water-soluble gas with the characteristic smell of rotten eggs, is widely known as the third gasotransmitter after nitric oxide and carbon monoxide. Endogenous H2S is endogenously synthesized by three enzymes: CSE (cystathionine-γ-lyase), CBS (cystathionine-β-synthase), and 3-MST (3-mercaptopyruvate sulfurtransferase) in mammalian tissues [9, 10, 11]. H2S is involved in numerous pathophysiological and physiological processes with its anti-apoptotic, anti-oxidative, anti-inflammatory and proangiogenic actions [12, 13]. Until recently, multiple lines of evidence implicate that endogenous H2S homeostasis contribute to the pathogenesis of diabetes mellitus and diabetic complications, dysregulation
of
endogenous
H 2S
production
under
hyperlipidemia
and
hyperglycemia circumstances might be involve in endothelial dysfunction which is a central pathogenesis process of diabetic complications [14, 15, 16, 17]. However, underlying role of endogenous H2S in DCM cardiac lipotoxicity is fairly limited. Our results indicated that circulating and heart H2S levels were lower than that in control, which were consistent with the results of previous studies of diabetic patients and animals models [4, 18,19]. Protein expression of CSE, a key H2S production enzyme in the hearts, was downregulated, which might explain the reduced H2S levels. The current article provides novel experimental evidence that endogenous H2S implicates in the pathogenesis of diabetic complications especially cardiac lipotoxicity in vitro and in vivo. Increasing evidence suggest that cardiac lipotoxicity and endoplasmic reticulum (ER) stress play a role in the pathogenesis of DCM [20, 21, 22]. Cardiac lipotoxicity
illustrate that excess lipid deposit may exert direct toxic effects on cardiomyocytes function, which may represent a significant component of the diabetic cardiomyopathy phenotype [22, 23]. ER stress was involved in the cardiac apoptosis of streptozotocin (STZ)-induced diabetic rat and high glucose treated cardiomyocytes [20, 21]. Furthermore, ER stress may play an important role in the development of lipotoxicity [24]. However, the underlying mechanisms whether ER stress involves in DCM cardiac lipotoxicity were not fully clear. In the hearts of STZ rats and AC cardiac cells treated with PA, a significant elevated of GRP78 and CHOP expression were observed, while the expressions of cleaved caspase-3 and caspase-12 were significantly elevated, indicating that ER stress and related apoptosis may also play an important role in diabetic cardiomyopathy. Meanwhile, lipid accumulation and correlated cardiac cell deaths in diabetic hearts tissues and cardiomyocytes treated with PA were observed. Thus, these results appear similar to previous reports [25, 26]. To clarify the relationship between ER stress and cardiac lipotoxicity in DCM, administration STZ rats and pretreatment AC16 human cardiac cells with 4-PBA can suppress cardiac lipid deposit and cell apoptosis. These results suggest that ER stress and related apoptosis mediates cardiac lipotoxicity in DCM. In addition, accumulating evidence confirm that ER stress-induced apoptosis, which involves in the pathological processes of cardiovascular diseases, is mainly mediated by CHOP, inositol-requiring enzyme 1 (IRE1, also called ERN1) and caspase-12 [27], suggesting the potential contribution of these pathway to cardiac lipotoxicity in DCM.
As is mentioned above,
deficiency
of
endogenous
H 2S
implicated
in
cardiac lipotoxicity of DCM. On the other hand, H2S is a cytoprotective agent. However, whether exogenous H2S could protect against cardiac lipotoxicity of DCM though regulation of ER stress are not yet known. We firstly reported that NaHS can also inhibit cardiac lipid droplet formation and cell apoptosis in STZ rats and AC16 human cardiac cells by inhibition of ER stress . In conclusion, we have presented evidence showing that deficiency of endogenous H2S production and ER stress are new mechanisms of cardiac lipotoxicity in diabetic cardiomyopathy, exogenous H2S could protection against cardiac lipotoxicity of DCM. Acknowledgements The present study was supported by grants from the Science and Technology Planning Project of Guangdong in China (no. 2012A080202020), the Guangdong Natural Science Foundation (no. 2015A030310359, S2011010002620) and Zhanjiang Municipal Financial Funds Special Competitive Project (2014A01033). Reference [1] Dandamudi S, Slusser J, Mahoney DW, Redfield MM, Rodeheffer RJ, Chen HH. The prevalence of diabetic cardiomyopathy: a population-based study in Olmsted County, Minnesota. J Card Fail. 2014;20(5):304-309. [2] Liu Q, Wang S, Cai L. Diabetic cardiomyopathy and its mechanisms: Role of oxidative stress and damage. J Diabetes Investig. 2014;5(6):623-634. [3] Yang L, Zhao D, Ren J, Yang J. Endoplasmic reticulum stress and protein quality
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Figure 1: Decreased H2S Production in cardiac lipotoxicity in vivo and in vitro.
Figure 2: Decreased Endogenous H2S Production Involves in cardiac lipotoxicity in vivo and in vitro.
Figure 3: Exogenous H2S Attenuates cardiac lipotoxicity in vivo and in vitro. Cell viability of AC16 cardiomyocytes treated with different treatments (A) was measured using CCK-8 kit. Cleaved caspase-3 protein expression in AC16 cells (B) and (C) were measured by western blot assay. ** P < 0.01 versus control; ## P < 0.01 versus PA group.
Figure 4: Exogenous H2S Inhibits Endoplasmic Reticulum (ER) Stress of Cardiac cells in hearts of DCM rats and AC16 cells treated with PA. GRP78, CHOP and caspase-12 protein expressions in cardiac cells (AC16 cells treated with PA (A, B, C) and DCM rats (D) were measured by western blot assay. P < 0.01 versus PA group.
**
P < 0.01 versus control; ##