Biochemical Pharmacology 203 (2022) 115179 Contents lists available at ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm Alpha-lipoic acid treatment improves adverse cardiac remodelling in the diabetic heart – The role of cardiac hydrogen sulfide-synthesizing enzymes George J. Dugbartey *, Quinsker L. Wonje, Karl K. Alornyo, Ismaila Adams, Deborah E. Diaba Department of Pharmacology and Toxicology, School of Pharmacy, College of Health Sciences, University of Ghana, Legon, Accra, Ghana A R T I C L E I N F O A B S T R A C T Keywords: Introduction: Alpha-lipoic acid (ALA) is a licensed drug for the treatment of diabetic neuropathy. We recently Alpha-lipoic acid (ALA) reported that it also improves diabetic cardiomyopathy (DCM) in type 2 diabetes mellitus (T2DM). In this study, Type 2 diabetes mellitus (T2DM) we present evidence supporting our hypothesis that the cardioprotective effect of ALA is via upregulation of Diabetic cardiomyopathy (DCM) cardiac hydrogen sulfide (H2S)-synthesizing enzymes. Hydrogen sulfide (H2S) H S-synthesizing enzymes Methods: Following 12 h of overnight fasting, T2DM was induced in 23 out of 30 male Sprague-Dawley rats by 2 intraperitoneal administration of nicotinamide (110 mg/kg) followed by streptozotocin (55 mg/kg) while the rest served as healthy control (HC). T2DM rats then received either oral administration of ALA (60 mg/kg/day; n = 7) or 40 mg/kg/day DL-propargylglycine (PAG, an endogenous H2S inhibitor; n = 7) intraperitoneally for 6 weeks after which all rats were sacrificed and samples collected for analysis. Untreated T2DM rats served as diabetic control (DCM; n = 9). Results: T2DM resulted in weight loss, islet destruction, reduced pancreatic β-cell function and hyperglycemia. Histologically, DCM rats showed significant myocardial damage evidenced by myocardial degeneration, car- diomyocyte vacuolation and apoptosis, cardiac fibrosis and inflammation, which positively correlated with elevated levels of cardiac damage markers compared to HC rats (p < 0.001). These pathological alterations worsened significantly in PAG-treated rats (p < 0.05). However, ALA treatment restored normoinsulemia, nor- moglycemia, prevented DCM, and improved lipid and antioxidant status. Mechanistically, ALA significantly upregulated the expression of cardiac H2S-synthesizing enzymes and increased plasma H2S concentration compared to DCM rats (p < 0.001). Conclusion: ALA preserves myocardial integrity in T2DM likely by maintaining the expression of cardiac H2S- synthezing enzymes and increasing plasma H2S level. 1. Introduction heart failure in diabetic patients. It is the leading cause of morbidity and mortality among the diabetic population [4,5]. While the molecular Used for the first time in human post-mortem in 1972 by Rubler et al. mechanism underlying the pathogenesis and progression of DCM is [1] to distinguish it from other forms of cardiomyopathy, diabetic car- multifactorial, persistent hyperglycemia-induced overproduction of diomyopathy (DCM) is a specific cardiac manifestation in diabetic pa- reactive oxygen species (ROS; a destructive tissue mediator) principally tients characterized by pathological alterations mainly in the from the mitochondria of cardiomyocytes, has been shown to be a major myocardial interstitium in the early stages of the disease. As DCM pro- contributing factor [6–8]. Overproduction of ROS in DCM overwhelms gresses, there is ventricular cardiomyocyte hypertrophy, increased the body’s antioxidant defense system, leading to oxidative stress, which interstitial and perivascular fibrosis, thickening of capillary basement further potentiates cardiac tissue injury [6–8]. In addition to persistent membrane as well as systolic and diastolic dysfunction, with preserved hyperglycemia and ROS-induced oxidative stress, other mechanisms are ejection fraction and reduced cardiomyocyte contraction [1–3]. These being investigated with novel pharmacological agents and approaches pathological changes occur without traditional risk factors such as hy- with the aim of preventing or improving DCM [9,81,82,94]. pertension and coronary atherosclerosis. DCM occurs in both type 1 We recently observed improvement in glycemic control and DCM (T1DM) and type 2 diabetes mellitus (T2DM), with increased risk of following supplementation of conventional anti-diabetic therapy with * Corresponding author. E-mail address: gjdugbartey@ug.edu.gh (G.J. Dugbartey). https://doi.org/10.1016/j.bcp.2022.115179 Received 1 June 2022; Received in revised form 26 June 2022; Accepted 12 July 2022 Available online 16 July 2022 0006-2952/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). G.J. Dugbartey et al. B i o c h e m i c a l P h a r m a c o l o g y 203 (2022) 115179 alpha-lipoic acid (ALA) [9]. ALA is a disulfide compound and a multi- Ashanti Region, Ghana) and tap water, and housed in cages in Depart- functional antioxidant produced from cysteine (as a source of sulfur) by ment of Animal Experimentation of the same Institute under normal lipoic acid synthase in the mitochondria of cardiomyocytes and other light/dark conditions (L:D cycle 12:12 h) at an ambient temperature of tissues [10,11], with beneficial effect in other diabetic complications 21–25 ◦C. Rats were randomly assigned to one of four groups, being [12–14,95]. It functions as a cofactor for several mitochondrial enzymes healthy control (HC; n = 7), diabetic control (DCM; n = 9), diabetic rats in mitochondrial bioenergetics and amino acid metabolism [15]. Apart treated with DL-propargylglycine (DCM + PAG, CSE inhibitor; n = 7) from its production in the body, ALA can also be obtained from plants and diabetic rats treated with alpha-lipoic acid (DCM + ALA; n = 7). and animal sources, and also given as a dietary supplement [16]. Following its exogenous administration, ALA is absorbed and converted 2.3. Experimental procedures enzymatically to dihydrolipoic acid (DHLA) [17]. There are studies showing that DHLA releases hydrogen sulfide (H2S) from sulfane sulfur As illustrated in Fig. 1 and previously described [9], all 30 rats were in rat heart tissue homogenate after ALA administration [18,19]. subjected to 12-hours of overnight fasting one day before induction of Therefore, it can be inferred that an association exists between ALA and type 2 diabetes mellitus (T2DM), and fasting blood glucose (FBG) was H2S. H2S is a gas with a distinctive smell of rotten-egg, and notoriously measured the following day by tail puncture with a portable hand-held known for several centuries for its toxicity and death at high concen- glucometer (One Touch Select Plus®; LifeScan Inc., Zug, Switzerland). trations [20,21]. However, it has been recently found to be a signalling T2DM was induced in 23 of these rats by intraperitoneal (i.p.) admin- molecule with therapeutic potentials at low physiological concentra- istration of 110 mg/kg nicotinamide (Nanjing Yasnt Bio-Tech Co. Ltd - tions [22,23]. H2S biogenesis in mammalian cells including car- Nanjing, China). Fifteen minutes later, 55 mg/kg streptozotocin (Sigma diomyocytes is catalyzed by two cytosolic enzymes cystathionine Aldrich, Missouri, USA) was administered i.p. Hyperglycemia was β-synthase (CBS) and cystathionine γ-lyase (CSE), a mitochondrial defined as FBG > 13.9 mmol/L (250 mg/dL) on day 3 after T2DM in- enzyme 3-mecaptopyruvate sulfurtransferase (3-MST) and a peroxi- duction and rats were considered diabetic as we previously reported [9]. somal enzyme D-amino acid oxidase (DAO). While the distribution of Two groups of the diabetic rats (DCM + PAG and DCM + ALA) then these enzymes is subcellular and tissue-specific, CBS, CSE and 3-MST are received i.p. administration of 40 mg/kg/day DL-propargylglycine abundantly expressed in cardiomyocytes, and their pharmacological (Sigma Aldrich Co, St. Louis, MO, USA) and oral administration of 60 inhibition or genetic deletion leads to adverse heart conditions such as mg/kg/day alpha-lipoic acid (Nanjing Yasnt Bio-Tech Co. Ltd - Nanjing, hypertension and cardiac hypertrophy [24–27]. H2S can also be China) respectively for 6 weeks. Untreated diabetic rats received administered exogenously via H2S donor compounds as pharmacolog- distilled water and served as diabetic control (DCM) while non-diabetic ical supplementation to endogenous H2S level [28–35]. rats were used as healthy control (HC). The dose of ALA was determined Although several empirical evidence have shown therapeutic bene- from our previous study [15] and that of PAG was determined from Jia fits of H2S in various pathologies, which we have reviewed recently et al. [44]. Body weights (BW) of all rats were measured and blood [36–43], no study has so far established a link between H2S and ALA in samples were obtained by tail snipping with a pair of clean and sharp the context of DCM. Therefore, the present study aims to determine scissors for measurement of glycosylated hemoglobin A1c (HbA1c) whether there is an association between H2S and ALA from the levels every week using an automatic biochemical analyzer (Nycocard perspective of DCM. Reader, Axis Shield, Oslo, Norway) at Tema General Hospital, Tema, Ghana. Following 6 weeks of treatment and after blood sample collec- 2. Materials and methods tion by cardiac puncture, rats were sacrificed by rapid cardiectomy after they were anesthetized with a mixture of ketamine (60 mg/kg) and 2.1. Ethical statement xylazine (10 mg/kg) i.p. The heart was weighed and heart weight (HW)/ BW ratio (%) was computed as an index of diabetic cardiomyopathy. All the animal work in this study was conducted according to rele- Cardiac apices were snap-frozen and stored in − 80 ◦C freezer for west- vant national and international guidelines, and was approved by the ern blot analysis while the midventricular section and pancreas were Institutional Animal Care and Use Committee of the University of fixed in 10 % neutral buffered formalin for immunohistochemical Ghana, Legon, Accra, Ghana. analysis. 2.2. Experimental animals and grouping 2.4. Plasma preparation and biochemical analysis Prior to the experiment, 30 male Sprague-Dawley rats (Rattus Plasma samples were prepared by centrifugation of whole blood at novergicus, 180 ± 20 g) between 6 and 8 weeks old from Noguchi Me- 3000 rpm for 15 min at 4 ◦C. Plasma insulin levels were measured using morial Institute for Medical Research, University of Ghana, Legon, Accra a radioimmunoassay (RIA) kit from Atom High-Tech Co., Ltd., Beijing, were fed ad libitum with standard rat chow (Agricare Ltd, Kumasi, China, while plasma triglycerides, total cholesterol and high-density Fig. 1. The diabetic cardiomyopathy (DCM) model in type 2 diabetes mellitus (T2DM) rats. Rats underwent 12 h of overnight fasting one day before induction of T2DM, and fasting blood glucose (FBG) was measured the following day. T2DM was induced by injection of nicotinamide (NIC; 110 mg/kg) and streptozotocin (STZ; 55 mg/kg) intraperitoneally (i.p.). Upon confirmation of T2DM on day 3 following T2DM induction, T2DM rats received either oral administration of alpha- lipoic acid (ALA; 60 mg/kg/day) or i.p. injection of 40 mg/kg/day DL-propargylglycine (PAG, a CSE inhibitor) for 6 weeks. The rats were then euthanized and samples were collected for analysis. 2 G.J. Dugbartey et al. B i o c h e m i c a l P h a r m a c o l o g y 203 (2022) 115179 lipoproteins-cholesterol (HDL) were measured with Mindray BS-200 and the residue was dissolved in radioimmunoassay buffer and assayed Biochemistry Auto-analyzer, Shenzhen, China, following the manufac- in accordance with instructions from the manufacturer. The myocardial turer’s instructions and as we previously reported [9,12]. The levels of content of BNP was computed and expressed as pmol/g wet weight for plasma interleukin-1β (IL-1β), IL-6 and tumor necrosis factor (TNF-α) tissues [48]. For myocardial creatine kinase-MB, the tissue homogenate were also measured by ELISA as described by Bortolon et al. [45] using a was centrifuged at 5000 rpm for 20 min at 4 ◦C and the supernatant was DuoSet Kit and according to the manufacturer’s instructions (Quanti- analysed for CK-MB using ELISA kit (MyBiosource, Inc, San Diego. USA) kine, R&D Systems, Minneapolis, MN, USA). Plasma levels of cardiac [49,50]. damage markers (brain natriuretic peptide [BNP] and creatine kinase- myocardial band [CK-MB]) were measured with an RA 50 semi-auto 2.7. Western blotting analyser while cardiac troponin I (cTnI) level in plasma was measured using a commercially available sandwich ELISA kit (High Sensitivity Rat The expressions of cardiac H2S-synthesizing enzymes (CBS, CSE and Cardiac Troponin-I ELISA Kit, Life Diagnostics Inc., PA, USA) and as we 3-MST) and myocardial content of the anti-apoptotic proteins Bcl-xL and previously described [9]. Pancreatic β-cell function was assessed by Bcl-2 as well as house-keeping protein GAPDH were determined from computing HOMA-β using the formula: 20 × Insulin (μ IU/mL) ÷ about 50 mg of cardiac tissue in all groups of rats by western blot as we Glucose (mmol/L) − 3.5) [12]. previously described [51]. Briefly, the cardiac expression of these pro- teins on the nitrocellulose membranes were detected with CBS (1:1000; 2.5. Determination of cardiac antioxidant status Santa Cruz, The Netherlands) CSE (1:1000; Abnova, USA), 3-MST (1:1000; Santa Cruz, The Netherlands), Bcl-xL (1;1000; Cell Signaling, 2.5.1. Measurement of cardiac malondialdehyde level Danvers, USA), Bcl-2 (1:1000; Bio Vision, Milpitas, USA) and GAPDH Measurement of the levels of malondialdehyde (MDA; a by-product (1:5000; Abcam, Canada) primary antibodies after overnight incubation of lipid peroxidation and an indicator of ROS production) in cardiac at 4 ◦C. The membranes were washed three times with Tris buffered tissue of each group was done by the thiobarbituric acid reactive sub- saline (TBS) with 0.004 % Tween and incubated with respective HRP- stances (TBARS) method as we previously described [9]. In brief, about linked secondary antibodies (1:1000) in TBS + Tween-20 supple- 50 mg of heart tissue was homogenized in 100 mL phosphate buffered mented with 3 % bovine serum albumin (w/v) for 1 h at room tem- saline (PBS) containing butylated hydroxytoluene (Cell Biolabs, perature. Protein bands were visualized using a Gene Genome after the Netherlands) and centrifuged at 10,000g for 5 min at 4 ◦C. The super- blots were developed with SuperSignal West Dura Extended Duration natant (50 mL) was collected and a volume of 50 mL SDS-Lysis solution Substrate (Thermo Scientific, USA). The band intensities were quantified (Cell Biolabs) was then added to MDA standards, and incubated for 5 using Gene Tools software (Westburg B.V., Leusden, The Netherlands). min at room temperature. Next, a volume of 125 mL of TBA reagent (Cell Biolabs) was added and incubated for 60 min at 95 ◦C, and then cooled 2.8. Measurement of plasma H2S concentration to room temperature for 5 min followed by centrifugation at 1000g for 15 min at 4 ◦C. The supernatant was collected and 2-Butanol (150 μL, To measure plasma H2S concentration as we previously described Merck, Darmstadt, Germany) was added to it, mixed for 2 min and [51,52], a sulfide antioxidant buffer was prepared from 25 g of sodium centrifuged at 10,000g for 5 min at 4 ◦C. Lipid peroxidation was deter- salicylate, 6.5 g of ascorbic acid and 8.5 g of sodium hydroxide in 100 mined from 200 μL of the butanol fraction by measuring optical density mL of distilled water at pH ≥ 13. Next, 100 μL of the sufide antioxidant at 532 nm and expressed as nmol/mg of heart tissue. buffer was added to 100 μL of plasma and mixed thoroughly. A sulfide ion selective microelectrode was immersed into the mixture to measure 2.5.2. Measurement of cardiac glutathione content and superoxide the electrode potential. Finally, the plasma H2S concentration was dismutase activity calculated from a standard curve of Na2S.9H20 in the sulfide antioxidant Measurements of cardiac glutathione (GSH) content of each group buffer in accordance with the manufacturer’s guide (Lazar Research was done spectrophotometrically according to the test kit from Promega Laboratories, Inc., Los Angeles, CA, USA). (Madison, WI, USA). In brief, about 50 mg frozen-kept heart tissue from each experimental group was homogenized in 1 mL of ice-cold 0.5 % 2.9. Histopathology and immunohistochemical staining potassium chloride and sonicated for 1 min. The homogenate was centrifuged at 3000 rpm for 10 min at 4 ◦C after which the supernatant Midventricular heart sections and pancreas tissue samples that were was used together with the GSH standard provided in the test kits. fixed in 10 % neutral buffered formalin were processed for histological Cardiac GSH content was quantified by chemiluminescence in a Spec- examination as we previously described [9]. In brief, the tissue samples traMax 2 plate reader. In measuring SOD activity in heart tissue of each were dehydrated in an increasing order of alcohol concentration (70 %, experimental group, about 50 mg of frozen-kept heart tissue was used 80 %, 90 % and 100 %) followed by dehydration in xylene and finally according to a previously described method Xu et al. [46] and according embedded in molten paraffin wax. The paraffin-embedded heart sec- to instructions from the test kit (Nanjing Kaiji Bio, Nanjing, China). tions cut from similar site of the tissue and in similar plane at 4 μm, were dewaxed and stained with periodic acid-Schiff (PAS) reagent and 2.6. Measurement of myocardial content of injury markers counterstained briefly with Meyer’s hematoxylin. The sections were examined under light microscope and were scored independently in a To determine myocardial content of troponin I (cTnI) as a tissue double-blinded fashion by two experienced pathologists at 400x injury marker in diabetic hearts, a detection kit (Life Diagnostics, Koln, magnification based on the degree of myocardial degeneration and Germany) and ELISA method were used in accordance with the manu- cardiomyocyte vacuolation as previously described [53,54]. facturer’s instructions and as previously described by Bayrami et al. Heart sections were stained for Masson’s trichrome (diluted 1:100; [47]. To measure myocardial content of brain natriuretic peptide (BNP), Abcam, Canada), a marker for tissue fibrosis, caspase-3 (diluted 1:50; another cardiac damage marker, about 20 mg of heart tissue was boiled Abnova, USA), a marker for apoptosis, BAX (diluted 1:100; Santa Cruz in l M acetic acid for 10 min and homogenized with a Polytron set at 4 Biotechnology, USA), another apoptotic marker, interleukin‑6 (diluted ◦C. The homogenate was centrifuged at 24,000g for 30 min at 4 ◦C. The 1:500; Abcam, USA), a pro-inflammatory cytokine, and tumor necrosis supernatant from the centrifugation step was loaded onto a Sep-Pak C18 factor-α (diluted 1 µg/mL; Abcam, USA), another pro-inflammatory cartridge (Waters, MA, USA) and pre-equilibrated with 0.5 mM acetic cytokine. The stained sections were observed under a light microscope acid, and then eluted with 4 mL of 50 % CH3CN containing 0.1 % tri- and images were captured with a digital camera attached to it. Fibrotic fluoroacetic acid (TFA). Following this step, the sample was lyophilized, areas in the Masson’s trichrome stain were quantified with a color image 3 G.J. Dugbartey et al. B i o c h e m i c a l P h a r m a c o l o g y 203 (2022) 115179 analyzer (Leica Microsystems, Ltd., Milton Keynes, UK), and the ratio of apoptotic markers, and worse lipid profile and antioxidant status fibrotic area to total area was calculated to evaluate the degree of (Fig. 3A-Q Fig. 4A-F; Fig. 5A,C,D; p < 0.05). However, treatment with myocardial fibrosis. The stainings for BAX, caspase-3, interleukin-6 and ALA improved all these pathological changes to near HC levels (Fig. 3A- tumor necrosis factor-α were quantified using Image-Pro Plus v6 anal- Q; Fig. 4A-F; Fig. 5A, C, D). In summary, ALA administration of ALA in ysis software (Media Cybernetics, Inc., Rockville, MD, USA). T2DM rats preserved myocardial integrity. 2.10. Statistical analysis 3.3. ALA upregulated expression of cardiac H2S-synthesizing enzymes and increased plasma H2S concentration under diabetic condition Data are presented as mean ± standard error of the mean (SEM). Differences between groups were evaluated with one-way analysis of To investigate the mechanism by which ALA provided myocardial variance (ANOVA) followed by Tukey post-hoc test using Prism software protection in T2DM rats, we evaluated cardiac expression of H2S-syn- (Prism 8, GraphPad Software, Inc., San Diego, CA, USA). P-values < 0.05 thesizing enzymes (CBS, CSE and 3-MST) and also measured plasma H2S between groups were considered statistically significant. concentration. The expression levels of all three H2S-producing enzymes were strongly downregulated by over threefold in the cardiac tissues of 3. Results DCM rats, which corresponded with over threefold reduction plasma H2S concentration in comparison with HC rats (Fig. 5B, E-H; p < 0.001). 3.1. ALA maintained body weight and normoglycemia, and preserved While these H2S-generating enzymes were further downregulated along pancreas structure and β-cell function under diabetic condition with further reduction in plasma H2S concentration following treatment with PAG (Fig. 5B, E-H; p < 0.05), ALA administration upregulated the We measured body weight (BW), glycated hemoglobin (HbA1c; a expression of these enzymes in the diabetic heart to HC levels (Fig. 5B, E; marker to assess blood glucose level) before and after T2DM induction, p > 0.05) and markedly increased plasma H2S concentration by over pancreatic β-cell function, plasma insulin and performed histological twofold compared to that in HC rats (Fig. 5H; p < 0.01). Thus, ALA assessment of the pancreas to determine the effect of ALA on these pa- contributed to cardiac protection under diabetic condition by upregu- rameters during TD2DM. We also measured heart weight (HW) and lating cardiac H2S-synthesizing enzymes along with increased H2S computed relative heart weight (HW/BW ratio) as an index of DCM. production. Following induction of T2DM, the BW of rats reduced by threefold, which corresponded to markedly reduced HW and threefold decrease in 4. Discussion relative HW compared to healthy control (HC) rats (Fig. 2A-2C; p < 0.01). Also, the level of plasma insulin decreased by fivefold in untreated This study provides the first evidence that establishes an association T2DM (DCM) group, resulting in hyperglycemia, which correlated with between alpha-lipoic acid (ALA) and hydrogen sulfide (H2S) in the a fivefold decline in β-cell function relative to healthy control rats context of diabetic cardiomyopathy (DCM), in which ALA administra- (Fig. 2D-2F; p < 0.001). These observations suggest destruction of tion upregulated expression of cardiac H2S-producing enzymes cys- pancreatic islets of Langerhans as revealed by our histological assess- tathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3- ment, which affected insulin-producing β-cells in comparison with the mecaptopyruvate sulfurtransferase (3-MST), and increased H2S pro- intact pancreatic islets in HC group (Fig. 2G). Pharmacological blockage duction, leading to protection against adverse cardiac remodelling of endogenous H2S with PAG further worsened all these parameters under diabetic condition. In this study, induction of type 2 diabetes (Fig. 2A-2G). However, treatment of T2DM rats with ALA reversed the mellitus (T2DM) with intraperitoneal administration of nicotinamide levels of these parameters to HC levels (Fig. 2A-2G; p > 0.05), implying and streptozocin caused pancreatic islet destruction, which resulted in that ALA preserved pancreatic islet structure and β-cell function, leading reduced β-cell function, hypoinsulinemia and hyperglycemia. Histo- to maintenance of normoinsulinemia, normoglycemia and body weight. pathological examination of the diabetic heart in the present study revealed significant adverse cardiac remodelling characterized by 3.2. ALA preserved myocardial integrity under diabetic condition myocardial degeneration, fibrosis, inflammation, cardiomyocyte vacu- olation and apoptosis along with impaired cardiac antioxidant status, To assess myocardial injury induced by T2DM and the effect of ALA, lipid profile and weight loss. Interestingly, inhibition of the endogenous we performed histological staining of cardiac tissue and measured car- H2S production with DL-propargylglycine (PAG) caused further hypo- diac damage markers (BNP, CK-MB and cTnI), lipid profile (triglyceride, insulinemia, hyperglycemia and myocardial damage along with reduced total cholesterol and HDL), antioxidant status (MDA, GSH and SOD) and plasma H2S concentration, which were improved following treatment plasma levels of pro-inflammatory cytokines (IL-1β; IL-6 and TNF-α). with ALA. Induction of T2DM resulted in significant myocardial degeneration, Persistent hyperglycemia is a well-known factor that promotes about tenfold increase in vacuole formation in cardiomyocytes, hyper- adverse cardiac remodelling, and has been reported to inhibit the ac- trophy, deformed nuclei and interstitial edema in the heart tissues of tivity of adenosine monophosphate-activated protein kinase (AMPK), a DCM rats, which positively correlated with over threefold increase in the key enzyme that regulates cellular energy homeostasis, and thereby levels of cardiac damage markers with impaired lipid profile compared leading to DCM [55–57]. Also, impaired insulin metabolic signalling in to heart tissues from HC rats (Fig. 3A-K; p < 0.001). Consistent with T1DM and T2DM has been reported to decrease translocation of glucose these observations, DCM rats also showed over twofold increase in the transporter protein subtype 4 (GLUT 4) to cardiomyocyte plasma levels of plasma inflammatory markers and impaired cardiac antioxi- membrane, resulting in increased intracellular Ca2+ release from dant status (Fig. 3L-3Q; p < 0.001). To further determine the degree of sarcoplasmic reticulum and subsequent myocardial hypertrophy [3,58]. myocardial damage, we performed immunohistochemical staining for Interesting, the anti-hyperglycemic effect of ALA as observed in the fibrosis, apoptosis and inflammation. Compared to heart tissues of HC present study has been linked to its insulin-mimetic activity, which ac- rats, immunohistochemical examination of cardiac tissue of DCM rats tivates insulin receptors and enhances their activity, and thereby leading revealed myocardial fibrosis, inflammation and apoptosis of car- to cytoprotection against apoptosis [59]. Additionally, several preclin- diomyocytes, which were characterized by over fivefold increase in ical studies have implicated ALA in glucose-regulating mechanisms, as it deposition of collagen, expression of IL-6, TNF-α, caspases-3 and BAX functions as an insulin sensitizer in skeletal muscles, promotes trans- (Fig. 4A-F; p < 0.001). Interestingly, treatment with PAG caused further location of GLUT 4 to the plasma, activates AMPK and phosphoinositide- myocardial damage evidenced by further increase in the levels of car- 3-kinase (PI3K) signalling pathways, and also promotes tyrosine phos- diac damage makers, pro-inflammatory cytokines, collagen deposition, phorylation in the insulin receptor, which improves PI3K-dependent 4 G.J. Dugbartey et al. B i o c h e m i c a l P h a r m a c o l o g y 203 (2022) 115179 Fig. 2. Changes in body weight, plasma insulin and glucose levels and pancreas histology. (A) Percentage change in body weight, (B) heart weight, (C) relative heart weight, (D) HbA1c, (E) plasma insulin (F) β-cell function and (G) representative photomicrographs of PAS-stained images of pancreatic tissues at 400x magnification. Arrows point to pancreatic islets. HC = Healthy control; DCM = Diabetic control; DCM + PAG = Diabetic rats treated with propargylglycine; DCM + ALA = Diabetic rats treated with alpha-lipoic acid. *p < 0.05 vs. DCM, **p < 0.01 vs. DCM, ***p < 0.001 vs. DCM. 5 G.J. Dugbartey et al. B i o c h e m i c a l P h a r m a c o l o g y 203 (2022) 115179 Fig. 3. Cardiac histology, lipid profile, inflammation and antioxidant status. (A) Representative photomicrograph of mid-ventricular sections of all groups showing PAS stain at 400x magnification. Arrows indicate cardiomyocyte vacuolation. (B) Quantification of cardiac histology, (C) plasma BNP, (D) plasma CK-MB, (E) plasma cTnI, (F) cardiac BNP, (G) cardiac CK-MB, (H) cTnI in cardiac tissue, (I) plasma triglyceride, (J) total cholesterol, (K) plasma HDL, (L) plasma IL-1β, (M) plasma IL-6, (N) plasma TNF-α, (O) cardiac MDA level, (P) cardiac GSH content and (Q) cardiac SOD activity. HC = Healthy control; DCM = Diabetic control; DCM + PAG = Diabetic rats treated with propargylglycine; DCM + ALA = Diabetic rats treated with alpha-lipoic acid. *p < 0.05 vs. DCM, **p < 0.01 vs. DCM, ***p < 0.001 vs. DCM. 6 G.J. Dugbartey et al. B i o c h e m i c a l P h a r m a c o l o g y 203 (2022) 115179 Fig. 4. Immunohistochemical staining of cardiac tissue and quantification. (A) Immunohistochemistry of cardiac tissue at 400x magnification showing collagen deposition (myocardial fibrosis), BAX and caspases-3 (cardiomyocyte apoptosis), IL-6 and TNF-α (myocardial inflammation), and quantification of (B) collagen deposition, (C) BAX expression, (D) caspases-3 expression, (E) IL-6 expression and (F) TNF-α expression. Yellow arrows point to positively stained fibrotic areas. HC = Healthy control; DCM = Diabetic control; DCM + PAG = Diabetic rats treated with propargylglycine; DCM + ALA = Diabetic rats treated with alpha-lipoic acid. *p < 0.05 vs. DCM, **p < 0.01 vs. DCM, ***p < 0.001 vs. DCM. glucose uptake [59–63]. Although we did not investigate these glucose- attenuating high glucose- and hyperglycemia-induced cytotoxicity, regulating mechanisms of ALA in the present study, our observation of cardiomegaly, cardiac hypertrophy and fibrosis in in vitro and in vivo maintenance of normoinsulemia, normoglycemia and preservation of models of DCM [55,56,65]. These preclinical observations including pancreatic islet structure and β-cell function following 6 weeks of ours, suggest that the glucose-regulating mechanisms of ALA is, at least treatment with ALA in T2DM rats suggests that in addition to these in part, dependent on H2S system, and therefore H2S therapy may be glucose-regulating mechanisms, ALA may have also stimulated insulin important in the pharmacological treatment or management of diabetic release from functioning β-cells by reducing potassium ion permeability, complications including cardiomyopathy. a well-established mechanism which decreases insulin resistance and The main focus of attention in the present study is the observation of increases peripheral tissue sensitivity to insulin [64]. Induction of T2DM adverse cardiac remodelling after induction of T2DM, which was in the present study impaired cardiac H2S system, which was worsened worsened following pharmacological inhibition of endogenous H2S with following PAG administration, and contributed to the development and PAG. However, treatment of T2DM with ALA upregulated the expression progression of DCM. As ALA treatment activated and improved cardiac of cardiac H2S-generating enzymes and increased endogenous H2S H2S system, leading to cardioprotection in the present study, there are production, and thus resulted in cardioprotection against the patho- studies also showing that activation of the H2S system with H2S donor logical changes with improved antioxidant status in the diabetic heart. It compounds, in turn activates AMPK signalling pathway, and thereby is worth noting that ALA (enzymatically synthesized from cysteine) 7 G.J. Dugbartey et al. B i o c h e m i c a l P h a r m a c o l o g y 203 (2022) 115179 Fig. 5. Western blot images and cardiac H2S system. Images of Western blot showing cardiac expression of (A) anti-apoptotic proteins Bcl-xL and Bcl-2, (B) H2S- synthesizing enzymes CBS, CSE and 3-MST. Quantification of (C) cardiac Bcl-xL, (D) cardiac Bcl-2 expression (E) cardiac CBS expression (F) cardiac CSE expression and (G) cardiac 3-MST expression. (H) Plasma H2S concentration. HC = Healthy control; DCM = Diabetic control; DCM + PAG = Diabetic rats treated with propargylglycine; DCM + ALA = Diabetic rats treated with alpha-lipoic acid. *p < 0.05 vs. DCM, **p < 0.01 vs. DCM, ***p < 0.001 vs. DCM. contains sulfane sulfur, a sulfur reservoir which releases H2S in response multifunctional cytokine and a central mediator of myocardial fibrosis. to biosignals, and contributes to the biological actions and therapeutic It is important to note that ROS promotes pathogenesis and progression effects of ALA [18,19,66,67]. Hence, ALA administration was reported of myocardial fibrosis in DCM via activation of TGF-β1 and its down- to augment sulfane sulfur level and increased H2S concentration in the stream proteins Smad2 and 3 [72–74]. Hence, overexpression of TGF-β1 cardiac tissue of rats [18,66], which is in agreement with the finding in increased ROS-induced oxidative stress [75], and genetic inhibition of the present study. As already mentioned, persistent hyperglycemia is a Smad3 decreased myocardial oxidative stress, cardiomyocyte hyper- major pathophysiological factor in the development and progression of trophy and myocardial fibrosis, resulting in improved cardiac compli- DCM, as it induces oxidative stress, inflammation and cardiomyocyte ance in mice [76]. Moreover, the decrease in GLUT4 translocation as apoptosis, culminating in myocardial fibrosis [4]. As a potent antioxi- discussed above, causes excessive uptake of fatty acid by the cardiac dant, ALA treatment in T2DM rats significantly improved cardiac anti- mitochondria, which promotes ROS production to toxic levels, resulting oxidant status and decreased myocardial inflammation, fibrosis and in subsequent oxidative stress damage [77]. cardiomyocyte apoptosis, which were characterized by reduced pro- As we observed reduced endogenous H2S production in untreated duction of reactive oxygen species (ROS), increased glutathione (GSH) T2DM and PAG-treated rats and activation of cardiac H2S system by content and superoxide dismutase (SOD) activity, reduced IL-1β, IL-6, ALA, leading to cardioprotection in the present study, recent in vitro and TNF-α, collagen deposition, decreased expression of pro-apoptotic pro- in vivo models of DCM showed reduced ROS-induced oxidative stress and teins and increased expression of anti-apoptotic proteins in the diabetic attenuation of myocardial inflammation, fibrosis and cardiomyocyte heart. Our observation aligns with findings from previous preclinical apoptosis and necroptosis following activation of the H2S system with and clinical models of DCM in which treatment with ALA decreased exogenous H2S donor compounds via inhibition of various molecular mitochondrial ROS generation, increased SOD and GSH content in mechanisms such as JAK/STAT, STAT3/HIF-1α, Wnt/β-catenin and mitochondria of cardiomyocytes and reduced collagen deposition as TLR4/NF-ĸB signalling pathways [78–82]. Although we did not explore well as transforming growth factor-beta 1 (TGF-β1) and mitochondria- these signalling pathways, it can be inferred that the activation of the dependent cardiomyocyte apoptosis [68–71]. TGF-β1 is a cardiac H2S system by ALA might have inhibited these molecular 8 G.J. Dugbartey et al. B i o c h e m i c a l P h a r m a c o l o g y 203 (2022) 115179 mechanisms, and thus contributed to the observed cardioprotection in from other tissues such as kidney, liver, brain, lung and pancreas, which the present study. In addition to the antioxidant pathway by H2S, it is also express H2S-producing enzymes. Therefore, future investigations also known to interact with other gaseous signalling molecules such as should attempt to measure H2S content in cardiac tissue to distinguish its nitric oxide and carbon monoxide in the mitochondrial electron trans- contribution in H2S production and cardioprotection under diabetic port chain, leading to opening of adenosine triphosphate (ATP)-depen- condition from those of other tissues. Another limitation in the present dent potassium (KATP) channels, and subsequently activating their study is the lack of data on cardiac function due to technical challenges. individual antioxidant activities [83–85]. Such interaction produces The inclusion of such important data in future studies, would provide synergism, which increases the antioxidant potential and effect of H2S. association between DCM and end-systolic diameters, global systolic Moreover, it has been recently shown that ALA also activates KATP function and diastolic function. channels, resulting in cardioprotection in vitro and in vivo [66,86]. Taken together, the findings from the present study provide the first Therefore, it is plausible that the potent antioxidant activity of ALA is experimental evidence that establishes an association between ALA and partly due to its ability to activate KATP channels in the diabetic heart, H2S in the context of DCM, in which ALA activated cardiac H2S-syn- leading to activation of antioxidant activities of H2S. Dudek et al. [87] thesizing enzymes and increased endogenous H2S production, leading to also linked KATP channel activation by ALA to its anti-inflammatory ef- preservation of myocardial integrity under diabetic condition. There- fect. In their study, they observed abrogation of the anti-inflammatory fore, our result suggests that ALA therapy may be of clinical importance action of ALA in vivo following pharmacological blockage of KATP in the pharmacological treatment or management of DCM and other channel with glibenclamide and reduced H2S level [87], suggesting that cardiovascular complications of diabetes by activating cardiac H2S KATP channel opening by ALA is crucial in attenuating inflammation in system. the diabetic heart. Among the three H2S-synthesizing enzymes (CBS, CSE and 3-MST) Declaration of Competing Interest measured in the present study, 3-MST is known to be the most abun- dant in the heart and localized largely in the mitochondrion, and ac- The authors declare that they have no known competing financial counts principally for its H2S content [26,88]. Although CBS and CSE are interests or personal relationships that could have appeared to influence cytosolic enzymes, they can translocate to the mitochondria in response the work reported in this paper. to specific stressful stimuli, and together with 3-MST, increase mito- chondrial H2S generation and ATP production to improve mitochondrial Acknowledgement bioenergetics in multiple ways under stressful conditions such as DCM [89–91]. Thus, all three H2S-generating enzymes regulate mitochondrial We thank the laboratory technicians at Noguchi Memorial Institute function and bioenergetics. As the mitochondria are a key target in for Medical Research, University of Ghana, for providing technical cardiomyopathies due to their role in ATP production, which is needed support. for the heart’s contractile function and regulation of important cellular functions including Ca2+ signalling, ROS generation and cell death, References pharmacological inhibition or genetic deletion of 3-MST has been recently reported to increase mitochondrial ROS production and [1] S. Rubler, J. Dlugash, Y.Z. Yuceoglu, T. Kumral, A.W. Branwood, A. Grishman, New decreased cardiac antioxidant status, resulting in cardiac hypertrophy type of cardiomyopathy associated with diabetic glomerulosclerosis, Am. J. and hypertension in adult mice [26,88]. This finding supports our Cardiol. 30 (6) (1972) 595–602. [2] S. Penpargkul, F. Fein, E.H. Sonnenblick, J. Scheuer, Depressed cardiac western blot result, which showed downregulation of 3-MST (and the sarcoplasmic reticular function from diabetic rats, J. Mol. Cell. Cardiol. 13 (3) other two H2S-producing enzymes) and reduced endogenous H2S pro- (1981) 303–309. duction in untreated T2DM and PAG-treated rats, and thus contributed [3] S.U. Trost, D.D. Belke, W.F. Bluhm, M. Meyer, E. Swanson, W.H. Dillmann, Overexpression of the sarcoplasmic reticulum Ca(2+)-ATPase improves myocardial to reduced cardiac antioxidant status and adverse cardiac remodelling. contractility in diabetic cardiomyopathy, Diabetes 51 (4) (2002) 1166–1171. It is well-established that ROS-induced oxidative stress in the cardiac [4] S. Boudina, E.D. Abel, Diabetic cardiomyopathy revisited, Circulation 115 (25) mitochondria enhances the opening of mitochondrial permeability (2007) 3213–3223. [5] M.N. Tillquist, T.M. Maddox, Update on diabetic cardiomyopathy: inches forward, transition pore (MPTP), which causes ATP breakdown rather than syn- miles to go, Curr. Diab. Rep. 12 (3) (2012) 305–313. thesis, and thus results in compromised contractile function of the heart, [6] V.P. Singh, B. Le, R. Khode, K.M. Baker, R. Kumar, Intracellular angiotensin II impaired Ca2+ signalling and a series of pathological events and sub- production in diabetic rats is correlated with cardiomyocyte apoptosis, oxidative sequent cell death [92,93]. It is possible that the reduced H S level in stress, and cardiac fibrosis, Diabetes 57 (12) (2008) 3297–3306. 2 [7] J. Das, V. Vasan, P.C. Sil, Taurine exerts hypoglycemic effect in alloxan-induced untreated diabetic rats and the PAG-treated group might have promoted diabetic rats, improves insulin-mediated glucose transport signaling pathway in the opening of more MPTPs and its subsequent effects. Therefore, the heart and ameliorates cardiac oxidative stress and apoptosis, Toxicol. Appl. upregulation of cardiac 3-MST and the other two H S-producing en- Pharmacol. 258 (2) (2012) 296–308. 2 [8] C.G. Tocchetti, B.A. Stanley, V. Sivakumaran, D. Bedja, B. O’Rourke, N. Paolocci, zymes by ALA and the subsequent increased H2S in the present study, S. Cortassa, M.A. Aon, Impaired mitochondrial energy supply coupled to increased might have increased mitochondrial H2S and ATP production to prevent H2O2 emission under energy/redox stress leads to myocardial dysfunction during MPTP opening and occurrence of mitochondrial ROS-induced oxidative Type I diabetes, Clin. Sci. (Lond.). 129 (7) (2015) 561–574. [9] G.J. Dugbartey, Q.L. Wonje, K.K. Alornyo, L. Robertson, I. Adams, V. Boima, S. stress in the heart of T2DM rats. Moreover, ALA itself is an essential D. Mensah, Combination Therapy of Alpha-Lipoic Acid, Gliclazide and Ramipril cofactor for several mitochondrial multienzyme complexes in ATP pro- Protects Against Development of Diabetic Cardiomyopathy via Inhibition of TGF- duction [15]. Therefore, it is not surprising that its administration in the β/Smad Pathway, Front. Pharmacol. 13 (2022) 850542. [10] I. Padmalayam, S. Hasham, U. Saxena, S. Pillarisetti, Lipoic acid synthase (LASY): a present study increased the expression levels of the cardiac H2S-gener- novel role in inflammation, mitochondrial function and insulin resistance, Diabetes ating enzymes including mitochondrial 3-MST. 58 (2000) 600–608. While the present study holds a great clinical promise, a major lim- [11] M. Szeląg, D. Mikulski, M. Molski, Quantum-chemical investigation of the structure and the antioxidant properties of α-lipoic acid and its metabolites, J. Mol. Model. itation is our inability to directly measure H2S content in the cardiac 18 (7) (2012) 2907–2916. tissue due to lack of techniques with the sensitivity and selectivity to [12] G.J. Dugbartey, K.K. Alornyo, B.B. N’guessan, S. Atule, S.D. Mensah, S. Adjei, detect tissue content of H S in real-time. However, as ALA upregulated Supplementation of conventional anti-diabetic therapy with alpha-lipoic acid 2 prevents early development and progression of diabetic nephropathy, Biomed. the expression of cardiac H2S-synthesizing enzymes, we can infer that Pharmacother. 149 (2022) 112818, https://doi.org/10.1016/j. the high plasma H2S concentration in ALA-treated rats was due to its biopha.2022.112818. high production and release from the cardiac tissue, which was strongly [13] P.R. Verma, Effect of alpha-lipoic acid and its nano-formulation on streptozotocin inhibited in untreated T2DM and PAG-treated rats. This inference, induced diabetic neuropathy in rats, Pharma. Innovation. J. 7 (1) (2018) 482–485. [14] D. Ziegler, P.A. Low, R. Freeman, H. Tritschler, A.I. Vinik, Predictors of however, does not preclude the possibility of H2S production and release improvement and progression of diabetic polyneuropathy following treatment with 9 G.J. Dugbartey et al. B i o c h e m i c a l P h a r m a c o l o g y 203 (2022) 115179 alpha-lipoic acid for 4 years in the NATHAN 1 trial, J. Diabetes Complications 30 transplantation, Pharmacol. Res. 172 (2021) 105842, https://doi.org/10.1016/j. (2) (2016) 350–356. phrs.2021.105842. [15] J. Bustamante, J.K. Lodge, L. Marcocci, H.J. Tritschler, L. Packer, B.H. Rihn, Alpha- [42] G.J. Dugbartey, K.K. Alornyo, B.O. Ohene, V. Boima, S. Antwi, A. Sener, Renal lipoic acid in liver metabolism and disease, Free Radic. Biol. Med. 24 (6) (1998) consequences of the novel coronavirus disease 2019 (COVID-19) and hydrogen 1023–1039. sulfide as a potential therapy, Nitric Oxide 120 (2022) 16–25. [16] K.P. Shay, R.F. Moreau, E.J. Smith, A.R. Smith, T.M. Hagen, Alpha-lipoic acid as a [43] M.Y. Zhang, G.J. Dugbartey, S. Juriasingani, A. Sener, Hydrogen Sulfide dietary supplement: molecular mechanisms and therapeutic potential, BBA 1790 Metabolite, Sodium Thiosulfate: Clinical Applications and Underlying Molecular (10) (2009) 1149–1160. Mechanisms, Int. J. Mol. Sci. 22 (12) (2021) 6452. [17] A. Bast, G.R.M.M. Haenen, Lipoic acid: A multifunctional antioxidant, BioFactors [44] Q. Jia, S. Mehmood, X. Liu, S. Ma, R. Yang, Hydrogen sulfide mitigates myocardial 17 (1-4) (2003) 207–213. inflammation by inhibiting nucleotide-binding oligomerization domain-like [18] A. Bilska, M. Dudek, M. Iciek, I. Kwiecień, M. Sokołowska-Jezewicz, B. Filipek, receptor protein 3 inflammasome activation in diabetic rats, Exp. Biol. Med. L. Włodek, Biological actions of lipoic acid associated with sulfane sulfur (Maywood). 245 (3) (2020) 221–230. metabolism, Pharmacol. Rep. 60 (2) (2008) 225–232. [45] J.R. Bortolon, A.J.d.A. Silva Junior, G.M. Murata, P. Newsholme, R. Curi, T. [19] A. Bilska-Wilkosz, M. Iciek, D. Kowalczyk-Pachel, M. Górny, M. Sokołowska- C. Pithon-Curi, E. Hatanaka, Persistence of inflammatory response to intense Jeżewicz, L. Włodek, Lipoic Acid as a Possible Pharmacological Source of Hydroge exercise in diabetic rats, Exp. Diabetes Res. 2012 (2012) 1–8. Sulfide/Sulfane Sulfur, Molecules 22 (3) (2017) 388. [46] G. Xu, X. Zhao, J. Fu, X. Wang, Resveratrol increase myocardial Nrf2 expression in [20] R.O. Beauchamp Jr, J.S. Bus, J.A. Popp, C.J. Boreiko, D.A. Adjelkovich, A critical type 2 diabetic rats and alleviate myocardial ischemia/reperfusion injury (MIRI), review of the literature on hydrogen sulfide toxicity, Crit. Rev. Toxicol. 3 (1) Ann. Palliat. Med. 8 (5) (2019) 565–575. (1984) 25–97. [47] G. Bayrami, P. Karimi, F. Agha-Hosseini, S. Feyzizadeh, R. Badalzadeh, Effect of [21] D.S. Gabbay, F. De Roos, J. Perrone, Twenty-foot fall averts fatality from massive Ischemic Postconditioning on Myocardial Function and Infarct Size Following hydrogen sulfide exposure, J. Emerg. Med. 20 (2) (2001) 141–144. Reperfusion Injury in Diabetic Rats Pretreated With Vildagliptin, J. Cardiovasc. [22] K. Abe, H. Kimura, The possible role of hydrogen sulfide as an endogenous Pharmacol. Ther. 23 (2) (2018) 174–183. neuromodulator, J. Neurosci. 16 (3) (1996) 1066–1071. [48] W. Jiang, D.-Y. Cai, C.-S. Pan, Y.-F. Qi, H.-F. Jiang, B. Geng, C.-S. Tang, Changes in [23] E. Blackstone, M. Morrison, M.B. Roth, H2S induces a suspended animation-like production and metabolism of brain natriuretic peptide in rats with myocardial state in mice, Science 308 (5721) (2005) 518. necrosis, Eur. J. Pharmacol. 507 (1-3) (2005) 153–162. [24] M. Watanabe, J. Osada, Y. Aratani, K. Kluckman, R. Reddick, M.R. Malinow, [49] S. Goyal, S. Arora, R. Mittal, S. Joshi, T.C. Nag, R. Ray, S. Kumari, D.S. Arya, N. Maeda, Mice deficient in cystathionine beta-synthase: Animal models for mild Myocardial salvaging effect of telmisartan in experimental model of myocardial and severe homocysteinemia, PNAS 92 (1995) 1585–1589. infarction, Eur. J. Pharmacol. 619 (1-3) (2009) 75–84. [25] G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng, A.K. Mustafa, W. Mu, [50] T.M. Abdulsalam, A.H. Hasanin, R.H. Mohamed, A. El Sayed Badawy, Angiotensin S. Zhang, S.H. Snyder, R. Wang, H2S as a physiologic vasorelaxant: hypertension in receptor-neprilysin inhibitior (thiorphan/irbesartan) decreased ischemia- mice with deletion of cystathionine gammalyase, Science 322 (2008) 587–590. reperfusion induced ventricular arrhythmias in rat; in vivo study, Eur. J. [26] M. Peleli, S.-I. Bibli, Z. Li, A. Chatzianastasiou, A. Varela, A. Katsouda, S. Zukunft, Pharmacol. 882 (2020) 173295, https://doi.org/10.1016/j.ejphar.2020.173295. M. Bucci, V. Vellecco, C.H. Davos, N. Nagahara, G. Cirino, I. Fleming, D.J. Lefer, [51] G.J. Dugbartey, F. Talaei, M.C. Houwertjes, M. Goris, A.H. Epema, H.R. Bouma, R. A. Papapetropoulos, Cardiovascular phenotype of mice lacking 3-mercaptopyru- H. Henning, Dopamine treatment attenuates acute kidney injury in a rat model of vate sulfurtransferase, Biochem. Pharmacol. 176 (2020) 113833, https://doi.org/ deep hypothermia and rewarming - The role of renal H2S-producing enzymes, Eur. 10.1016/j.bcp.2020.113833. J. Pharmacol. 15 (769) (2015) 225–233. [27] B. Trautwein, T. Merz, N. Denoix, C. Szabo, E. Calzia, P. Radermacher, O. McCook, [52] G.J. Dugbartey, H.R. Bouma, A.M. Strijkstra, A.S. Boerema, R.H. Henning, J. ΔMST and the Regulation of Cardiac CSE and OTR Expression in Trauma and A. Joles, Induction of a torpor-like state by 5’-AMP does not depend on H2S Hemorrhage, Antioxidants (Basel). 10 (2) (2021) 233. production, PLoS ONE 10 (8) (2015) e0136113, https://doi.org/10.1371/journal. [28] L. Li, M. Whiteman, Y.Y. Guan, K.L. Neo, Y. Cheng, S.W. Lee, Y. Zhao, R. Baskar, C.- pone.013611310.1371/journal.pone.0136113.g00110.1371/journal. H. Tan, P.K. Moore, Characterization of a novel water-soluble hydrogen sulfide- pone.0136113.g002. releasing molecule (GYY4137): new insight into the biology of hydrogen sulfide, [53] T. Moriyama, M. Kemi, C. Okumura, K. Yoshihara, T. Horie, Involvement of Circulation 117 (18) (2008) 2351–2360. advanced glycation end-products, pentosidine and N(epsilon)-(carboxymethyl) [29] K. Kashfi, K.R. Olson, Biology and therapeutic potential of hydrogen sulfide and lysine, in doxorubicin-induced cardiomyopathy in rats, Toxicology 268 (1–2) hydrogen sulfide-releasing chimeras, Biochem. Pharmacol. 85 (5) (2013) 689–703. (2010) 89–97. [30] D.J. Polhemus, Z. Li, C.B. Pattillo, G. Gojon Sr., G. Gojon Jr., T. Giordano, et al., [54] M.S. Atta, A.H. El-Far, F.A. Farrag, M.M. Abdel-Daim, S.K. Al Jaouni, S.A. Mousa, A novel hydrogen sulfide prodrug, SG1002, promotes hydrogen sulfide and nitric Thymoquinone Attenuates Cardiomyopathy in Streptozotocin-Treated Diabetic oxide bioavailability in heart failure patients, Cardiovasc. Ther. 33 (4) (2015) Rats, Oxid. Med. Cell Longev. 2018 (2018) 1–10. 216–226. [55] W.-B. Wei, X. Hu, X.-D. Zhuang, L.-Z. Liao, W.-D. Li, GYY4137, a novel hydrogen [31] M.M. Safar, R.M. Abdelsalam, H2S donors attenuate diabetic nephropathy in rats: sulfide-releasing molecule, likely protects against high glucose-induced modulation of oxidant status and polyol pathway, Pharmacol. Rep. 67 (1) (2015) cytotoxicity by activation of the AMPK/mTOR signal pathway in H9c2 cells, Mol. 17–23. Cell. Biochem. 389 (1-2) (2014) 249–256. [32] D. Gerő, R. Torregrossa, A. Perry, A. Waters, S. Le-Trionnaire, J.L. Whatmore, [56] F. Yang, L. Zhang, Z. Gao, X. Sun, M. Yu, S. Dong, J. Wu, Y. Zhao, C. Xu, W. Zhang, M. Wood, M. Whiteman, The novel mitochondria-targeted hydrogen sulfide (H2S) F. Lu, Exogenous H2S Protects Against Diabetic Cardiomyopathy by Activating donors AP123 and AP39 protect against hyperglycemic injury in microvascular Autophagy via the AMPK/mTOR Pathway, Cell. Physiol. Biochem. 43 (3) (2017) endothelial cells in vitro, Pharmacol. Res. 113 (Pt A) (2016) 186–198. 1168–1187. [33] X. Qian, X. Li, F. Ma, S. Luo, R. Ge, Y. Zhu, Novel hydrogen sulfide-releasing [57] Y. Sun, S. Zhou, H. Guo, J. Zhang, T. Ma, Y. Zheng, Z. Zhang, L.u. Cai, Protective compound, S-propargyl-cysteine, prevents STZ-induced diabetic nephropathy, effects of sulforaphane on type 2 diabetes-induced cardiomyopathy via AMPK- Biochem. Biophys. Res. Commun. 473 (4) (2016) 931–938. mediated activation of lipid metabolic pathways and NRF2 function, Metabolism. [34] S. Juriasingani, A. Jackson, M.Y. Zhang, A. Ruthirakanthan, G.J. Dugbartey, 102 (2020) 154002, https://doi.org/10.1016/j.metabol.2019.154002. E. Sogutdelen, M. Levine, M. Mandurah, M. Whiteman, P. Luke, A. Sener, [58] G. Jia, V.G. DeMarco, J.R. Sowers, Insulin resistance and hyperinsulinaemia in Evaluating the effects of subnormothermic perfusion with AP39 in a novel blood- diabetic cardiomyopathy, Nat. Rev. Endocrinol. 12 (3) (2016) 144–153. free model of ex vivo kidney preservation and reperfusion, Int. J. Mol. Sci. 22 (13) [59] B. Diesel, S. Kulhanek-Heinze, M. Höltje, B. Brandt, H.D. Höltje, A.M. Vollmar, A. (2021) 7180, https://doi.org/10.3390/ijms22137180. K. Kiemer, Alpha-lipoic acid as a directly binding activator of the insulin receptor: [35] M.Y. Zhang, G.J. Dugbartey, S. Juriasingani, M. Akbari, W. Liu, A. Haig, protection from hepatocyte apoptosis, Biochemistry 46 (8) (2007) 2146–2155. P. McLeod, J. Arp, A. Sener, Sodium thiosulfate-supplemented UW solution [60] M. Khamaisi, R. Potashnik, A. Tirosh, E. Demshchak, A. Rudich, H. Trischler, protects renal grafts against prolonged cold ischemia-reperfusion injury in a K. Wessel, N. Bashan, Lipoic acid reduces glycemia and increases muscle GLUT4 murine model of syngeneic kidney transplantation, Biomed. Pharmacother. 145 content in streptozotocin-diabetic rats, Metabolism. 46 (7) (1997) 763–768. (2022) 112435, https://doi.org/10.1016/j.biopha.2021.112435. [61] W.J. Lee, K.H. Song, E.H. Koh, J.C. Won, H.S. Kim, H.S. Park, M.S. Kim, S.W. Kim, [36] G.J. Dugbartey, Diabetic nephropathy: A potential savior with ’rotten-egg’ smell, K.U. Lee, J.Y. Park, Alpha-lipoic acid increases insulin sensitivity by activating Pharmacol. Rep. 69 (2) (2017) 331–339. AMPK in skeletal muscle, Biochem. Biophys. Res. Commun. 332 (3) (2005) [37] G.J. Dugbartey, H.R. Bouma, I. Lobb, A. Sener, Hydrogen sulfide: A novel 885–891. nephroprotectant against cisplatin-induced renal toxicity, Nitric Oxide 57 (2016) [62] K. Yaworsky, R. Somwar, T. Ramlal, H.J. Tritschler, A. Klip, Engagement of the 15–20. insulin-sensitive pathway in the stimulation of glucose transport by alpha-lipoic [38] G.J. Dugbartey, H2S as a possible therapeutic alternative for the treatment of acid in 3T3-L1 adipocytes, Diabetologia 43 (3) (2000) 294–303. hypertensive kidney injury, Nitric Oxide 64 (2017) 52–60. [63] H. Moini, O. Tirosh, Y.C. Park, K.J. Cho, L. Packer, R-alpha-lipoic acid action on [39] G.J. Dugbartey, The smell of renal protection against chronic kidney disease: cell redox status, the insulin receptor, and glucose uptake in 3T3-L1 adipocytes, Hydrogen sulfide offers a potential stinky remedy, Pharmacol. Rep. 70 (2) (2018) Arch. Biochem. Biophys. 397 (2) (2002) 384–391. 196–205. [64] N.K. Agrawal, U. Gupta, Evaluation of ramipril on blood sugar level and interaction [40] G.J. Dugbartey, H.R. Bouma, M.N. Saha, I. Lobb, R.H. Henning, A. Sener, with the oral anti-diabetic drugs in alloxan-induced diabetic rats, Int. J. Pharm. Sci. A Hibernation-Like State for Transplantable Organs: Is Hydrogen Sulfide Therapy Res. 4 (8) (2013) 2933–2938. the Future of Organ Preservation? Antioxid. Redox Signal. 28 (16) (2018) [65] L.A. Barr, Y. Shimizu, J.P. Lambert, C.K. Nicholson, J.W. Calvert, Hydrogen sulfide 1503–1515. attenuates high fat diet-induced cardiac dysfunction via the suppression of [41] G.J. Dugbartey, S. Juriasingani, M.Y. Zhang, A. Sener, H2S donor molecules against endoplasmic reticulum stress, Nitric Oxide 46 (2015) 145–156. cold ischemia-reperfusion injury in preclinical models of solid organ [66] M. Dudek, J. Knutelska, M. Bednarski, L. Nowiński, M. Zygmunt, A. Bilska-Wilkosz, M. Iciek, M. Otto, I. Żytka, J. Sapa, L. Włodek, B. Filipek, Alpha lipoic acid protects 10 G.J. Dugbartey et al. B i o c h e m i c a l P h a r m a c o l o g y 203 (2022) 115179 the heart against myocardial post ischemia-reperfusion arrhythmias via KATP [81] M. Zhang, M. Ye, Hydrogen Sulfide Attenuates High Glucose-induced Myocardial channel activation in isolated rat hearts, Pharmacol. Rep. 66 (3) (2014) 499–504. Injury in Rat Cardiomyocytes by Suppressing Wnt/beta-catenin Pathway, Curr. [67] M. Iciek, A. Bilska-Wilkosz, M. Górny, Sulfane sulfur - new findings on an old topic, Med. Sci. 39 (6) (2019) 938–946. Acta Biochim. Pol. 66 (4) (2019) 533–544. [82] W. Gong, S. Zhang, Y. Chen, J. Shen, Y. Zheng, X. Liu, M. Zhu, G. Meng, Protective [68] A.E. Midaoui, A. Elimadi, L. Wu, P.S. Haddad, J. de Champlain, Lipoic acid role of hydrogen sulfide against diabetic cardiomyopathy via alleviating prevents hypertension, hyperglycemia, and the increase in heart mitochondrial necroptosis, Free Radic. Biol. Med. 181 (2022) 29–42. superoxide production, Am. J. Hypertens. 16 (3) (2003) 173–179. [83] M.E. Murphy, J.E. Brayden, Nitric oxide hyperpolarizes rabbit mesenteric arteries [69] C.J. Li, Q.M. Zhang, M.Z. Li, J.Y. Zhang, P. Yu, D.M. Yu, Attenuation of myocardial via ATP-sensitive potassium channels, J. Physiol. 486 (Pt 1) (1995) 47–58. apoptosis by alpha-lipoic acid through suppression of mitochondrial oxidative [84] W. Zhao, J. Zhang, Y. Lu, R. Wang, The vasorelaxant effect of H(2)S as a novel stress to reduce diabetic cardiomyopathy, Chin. Med. J. (Engl.). 122 (21) (2009) endogenous gaseous K(ATP) channel opener, EMBO J. 20 (21) (2001) 6008–6016. 2580–2586. [85] M.A. Pareira de Avila, A. Giusti-Paiva, G. de Oliveira, C. Nascimento, The [70] J.E. Lee, C.-o. Yi, B.T. Jeon, H.J. Shin, S.K. Kim, T.S. Jung, J.Y. Choi, G.S. Roh, peripheral antinociceptive effect induced by the heme oxygenase/carbon Alpha-Lipoic acid attenuates cardiac fibrosis in Otsuka Long-Evans Tokushima monoxide pathway is associated with ATP-sensitive K+ channels, Eur. J. Fatty rats, Cardiovasc Diabetol. 11 (1) (2012), https://doi.org/10.1186/1475- Pharmacol. 726 (2014) 41–48. 2840-11-111. [86] M. Dudek, K. Razny, A. Bilska-Wilkosz, M. Iciek, J. Sapa, L. Wlodek, B. Filipek, [71] S.K. Hegazy, O.A. Tolba, T.M. Mostafa, M.A. Eid, D.R. El-Afify, Alpha-lipoic acid Hypotensive effect of alpha-lipoic acid after a single administration in rats, Anatol. improves subclinical left ventricular dysfunction in asymptomatic patients with J. Cardiol. 16 (5) (2016) 306–309. type 1 diabetes, Rev. Diabet. Stud. 10 (1) (2013) 58–67. [87] M. Dudek, A. Bilska-Wilkosz, J. Knutelska, S. Mogilski, M. Bednarski, M. Zygmunt, [72] Y. Purnomo, Y. Piccart, T. Coenen, J.S. Prihadi, P.J. Lijnen, Oxidative stress and M. Iciek, J. Sapa, D. Bugajski, B. Filipek, L. Włodek, Are anti-inflammatory transforming growth factor-beta1-induced cardiac fibrosis, Cardiovasc. Hematol. properties of lipoic acid associated with the formation of hydrogen sulfide? Disord.: Drug Targets 13 (2) (2013) 165–172. Pharmacol. Rep. 65 (4) (2013) 1018–1024. [73] S.Q. Wang, D. Li, Y. Yuan, Long-term moderate intensity exercise alleviates [88] Z. Li, H. Xia, T. Sharp, H. Hidalgo, N. Noriyuki, J.W. Elrod, D.J. Lefer, Deficiency of myocardial fibrosis in type 2 diabetic rats via inhibitions of oxidative stress and 3-mecaptopyruvate sulfurtransferase results in impaired mitochondrial function TGF-beta1/Smad pathway, J. Physiol. Sci. 69 (6) (2019) 861–873. and increased heart failure severity, Circulation 140 (2019) A15987. [74] Y. Wang, K. Yu, C. Zhao, L. Zhou, J. Cheng, D.W. Wang, C. Zhao, Follistatin [89] J. Ohta, T. Ubuka, H. Kodama, K. Sugahara, K. Yao, N. Masuoka, M. Kinuta, Attenuates Myocardial Fibrosis in Diabetic Cardiomyopathy via the TGF-beta- Increase in cystathionine content in rat liver mitochondria after D, L- Smad3 Pathway, Front. Pharmacol. 12 (2021) 683335. propargylglycine administration, Amino Acids. 9 (2) (1995) 111–122. [75] C. Michaeloudes, M.B. Sukkar, N.M. Khorasani, P.K. Bhavsar, K.F. Chung, TGF-β [90] M. Fu, W. Zhang, L. Wu, G. Yang, H. Li, R. Wang, Hydrogen sulfide (H2S) regulates Nox4, MnSOD and catalase expression, and IL-6 release in airway smooth metabolism in mitochondria and its regulatory role in energy production, Proc. muscle cells, Am. J. Physiol. Lung Cell. Mol. Physiol. 300 (2) (2011) L295–L304. Natl. Acad. Sci. USA 109 (8) (2012) 2943–2948. [76] A. Biernacka, M. Cavalera, J. Wang, I. Russo, A. Shinde, P. Kong, C. Gonzalez- [91] C. Szabo, C. Coletta, C. Chao, K. Módis, B. Szczesny, A. Papapetropoulos, M. Quesada, V. Rai, et al., Smad3 Signaling Promotes Fibrosis While Preserving R. Hellmich, Tumor-derived hydrogen sulfide, produced by cystathionine- Cardiac and Aortic Geometry in Obese Diabetic Mice, Circ. Heart Fail. 8 (4) (2015) β-synthase, stimulates bioenergetics, cell proliferation, and angiogenesis in colon 788–798. cancer, Proc. Natl. Acad. Sci. USA 110 (30) (2013) 12474–12479. [77] S. Boudina, E.D. Abel, Diabetic cardiomyopathy, causes and effects, Rev. Endocr. [92] S. Shanmuganathan, D.J. Hausenloy, M.R. Duchen, D.M. Yellon, Mitochondrial Metab. Disord. 11 (1) (2010) 31–39. permeability transition pore as a target for cardioprotection in the human heart, [78] Z. Huang, X. Zhuang, C. Xie, X. Hu, X. Dong, Y. Guo, S. Li, X. Liao, Exogenous Am. J. Physiol. Heart Circ. Physiol. 289 (1) (2005) H237–H242. Hydrogen Sulfide Attenuates High Glucose-Induced Cardiotoxicity by Inhibiting [93] M. Federico, S. De la Fuente, J. Palomeque, S.-S. Sheu, The role of mitochondria in NLRP3 Inflammasome Activation by Suppressing TLR4/NF-κB Pathway in H9c2 metabolic disease: a special emphasis on heart dysfunction, J. Physiol. 599 (14) Cells, Cell. Physiol. Biochem. 40 (6) (2016) 1578–1590. (2021) 3477–3493. [79] M. Liu, Y. Li, B. Liang, Z. Li, Z. Jiang, C. Chu, J. Yang, Hydrogen sulfide attenuates [94] Q. Ge, L.i. Zhao, X.-M. Ren, P. Ye, Z.-Y. Hu, LCZ696, an angiotensin receptor- myocardial fibrosis in diabetic rats through the JAK/STAT signaling pathway, Int. neprilysin inhibitor, ameliorates diabetic cardiomyopathy by inhibiting J. Mol. Med. 41 (4) (2018) 1867–1876. inflammation, oxidative stress and apoptosis, Exp. Biol. Med. (Maywood). 244 (12) [80] J. Li, Y.Q. Yuan, L. Zhang, H. Zhang, S.W. Zhang, Y. Zhang, X.X. Xuan, M.J. Wang, (2019) 1028–1039. J.Y. Zhang, Exogenous hydrogen sulfide protects against high glucose-induced [95] G.J. Dugbartey, K.K. Alornyo, D.E. Diaba, I. Adams, Activation of renal CSE/H2S apoptosis and oxidative stress by inhibiting the STAT3/HIF-1α pathway in H9c2 pathway by alpha-lipoic acid protects against histological and functional changes cardiomyocytes, Exp. Ther. Med. 18 (5) (2019) 3948–3958. in the diabetic kidney, Biomed Pharmacother 153 (2022), 113386. 11