S-Propargyl-Cysteine (SPRC, purity > 99%) was synthesized as described previously (17) and provided by Professor Zhu Yi-Zhun (State Key Laboratory of Quality Research in Chinese Medicine and School of Pharmacy, Macau University of Science and Technology, Macau, China). Chemical formula of SPRC is shown in Supplementary Figure 1A.

All experimental procedures were performed in accordance with Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) of the United States and approved by the Ethics Committee of Experimental Research, Fudan University Shanghai Medical College. Male C57BLKS/J db/db mice and their wild type littermates (7-week-old) were purchased from Nanjing Biomedical Research Institute of Nanjing University and were housed in a climate-controlled environment (22 ± 2°C, 45–75% relative humidity) with a 12 h light-dark cycle and unrestricted access to food and water. After acclimatization for 1 weeks, wild-type (WT) mice were used as a normal control group and db/db mice were randomly divided into four different groups (n = 15–16 per group): diabetic model group, low-dose SPRC treatment group (20 mg/kg per day), medium-dose SPRC treatment group (40 mg/kg per day), and high-dose SPRC treatment group (80 mg/kg per day). Both WT control group and diabetic model group were orally administered an equal volume of vehicle (ddH₂O). After 12 weeks of administration, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital and blood samples were collected. Heart weights (HW) and tibia lengths (TL) were measured and heart tissue specimens were obtained.

At the first and last week of SPRC treatment, the hair was removed from the chest of mice using depilatory cream. The mice were then anesthetized with 1.5% isoflurane and placed in a supine position on the test bench, with ultrasound gel applied onto the chest. Mouse two-dimensional echocardiography was performed using a Vevo3100 ultrasound device (VisualSonics Inc., Canada), as previously described (18). B-mode, M-mode, and Power Doppler Mode ultrasound images of the left ventricle were recorded. All measurements were averaged for five consecutive cardiac cycles.

The FBG levels and BW of mice were monitored every 2 weeks after food was removed for 12 h. Glucose measurements were performed with venous blood collected from mice tails by glucose monitors (ONETOUCH, Johnson and Johnson, USA). After food was removed overnight for 16 h, FINS were detected by enzyme-linked immunosorbent assay (ELISA) kit (Mercodia, Sweden). The formula of homeostasis model assessment for insulin resistance index (HOMA-IR) is [(HOMA-IR) = (FBG × FINS)/22.5] (19).

Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed in the morning on nonfasted mice that had their food removed 1 h prior to intraperitoneal injection (IPGTT 1 g glucose per kg body weight and IPITT 1unit recombinant human insulin (Humulin 70/30, Lilly, USA) per kg body weight), Blood glucose levels were measured before the injection (time 0) and 15, 30, 60, and 120 min after the injection. Areas under the curve (AUC) were determined using the trapezoidal rule.

The total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), non-esterified fatty acid (NEFA) in plasma were determined using commercial kits (Nanjing Jiancheng, China) according to the manufacturer's instructions. Briefly, full-wavelength microplate reader (Infinite® 200 PRO, TECAN, Switzerland) was used to detect the absorbance of plasma samples at 510 nm for TC and TG or at 546 nm for HDL-C, LDL-C and NEFA.

Heart specimens were fixed in 4% paraformaldehyde solution. After being embedded in paraffin, the specimens were cut into 5-μm-thick sections and stained with wheat germ agglutinin (WGA) and Masson's trichrome. The other heart tissue specimens were frozen and stained with Oil Red O lipid stain. The size of myocardial cells, fiber area fraction, and myocardial lipid content were determined using Image J software (Bethesda, MA, USA).

To observe myocardial ultrastructure, heart tissues were cut into 1 mm transverse sections and immersed in 2% glutaraldehyde overnight. The sections were then immersed in 1% osmium tetroxide for 2 h, dehydrated in graded ethanol, and embedded in epoxy resin. Ultrathin sections (60–70 nm) were obtained, stained with uranyl acetate and lead citrate, and examined using a Tecnai G20 Twin transmission electron microscope (FEI, USA).

Primary cultures of cardiomyocytes were prepared from neonatal mice hearts. In brief, hearts were excised from 1- to 2-day-old mice pups. Ventricles were minced by small scissors and digested using 1 mg/ml collagenase type II (Worthington, USA). The digested solution was collected. The process was repeated 3–4 times until no chunks of tissue were visible. The final pooled solution was centrifuged and the cell pellet re-suspended in Dulbecco's Modified Eagle Medium (DMEM, Hyclone, USA), high Glucose containing 10% fetal bovine serum (FBS, Gibco, USA). The cells were pre-plated for 1 h to allow the attachment and removal of fibroblasts. The unattached cardiomyocytes remaining in suspension were then collected and plated in DMEM, high Glucose containing 10% FBS. After 48 h Cardiomyocytes cultures were used for subsequent experiments.

Cell viability was determined by cell counting kit-8 assay according to manufacturer's instructions (DOJINDO, Japan). Cardiomyocytes were cultured in a 96-well-culture plate and treated with different concentrations of SPRC (0–1,000 μM) for 24 h. Cells were subsequently incubated with 10 ml CCK-8 solution at 37°C for 4 h. The absorbance at 450 nm was measured.

Total RNA was extracted by Trizol reagent from heart tissue or cardiomyocytes. RNA was reverse-transcribed using a cDNA synthesis kit (Toyobo Life Science, Japan). Real-Time PCR was performed using a StepOnePlus Real-Time PCR Detection System (Applied Biosystems Inc., CA, USA). A total volume of 20 μL reaction mix containing 2 μL cDNA, 10 μL SYBR Green PCR Master Mix (Toyobo, Japan) and 1 μL each primer (10 μM). Gapdh was used for normalization and the relative expression of mRNA was calculated according to the ^ΔΔCt method. The specific primers were as follows: Myh7: 5′-ATCAATGCAACCCTGGAGAC-3′, 5′-CGAACATGTGGTGGTTGAAG-3′; InsR: 5′-GCTTCTGCCAAGACCTTCAC-3′, 5′-CACTCGGGGATGCACTTATT-3′; Glut4: 5′-ACCCTGGGCTCTGTATCCC-3′, 5′-CCCTGACCACTGAGTGCAAA-3′; Gapdh: 5′-TTCACCACCATGGAGAAGGC-3′, 5′-GGCATGGACTGTGGTCATGA-3′; β-actin: 5′- GACAGGATGCAGAAGGAGATTACT-3′, TGATCCACATCTGCTGGAAGGT-3′.

Left ventricular tissue and cell samples were lysed with Cell Lysis Buffer for Western (P0031; Beyotime Biotechnology, China) containing a protease inhibitor cocktail (049693132001; Roche, Basel, Switzerland). The crude cell lysate was centrifuged and the supernatant was harvested. The concentration of protein in the supernatant was quantified by standard bicinchoninic acid assay (BCA, P0012, Beyotime Biotechnology, China). For western blotting, equal amounts of proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000; A0208; Beyotime Biotechnology, China) for 2 h, and immunoreactive bands were visualized by chemiluminescence. And the gray value of the captured bands was determined by ImageJ software. The primary antibodies used included: anti-Insulin Receptor β (#3025), anti-Phospho-Insulin Receptor β Tyr1150/1151(#3024), anti-Akt (#9272), anti-phospho-Akt Ser473 (#4060), anti-GSK-3β (#9315), anti-Phospho-GSK-3β Ser9 (#5558) were purchased from Cell Signaling Technology (Beverly, MA, USA); anti-GAPDH (60004-1), anti-β-Actin (66009-1), and anti-α-Tubulin (11224-1) purchased from Proteintech Group (Chicago, USA), and anti-CSE(sc-374249) were purchased from Santa Cruz Biotechnology (Texas, USA).

Primary mice cardiomyocytes were incubated with 33.3 mM glucose (HG), 100 nM insulin (HI), or 33.3 mM glucose and 100 nM insulin (HG++HI) for 48 h. Among them, the HG+HI group treated with vehicle or different doses of SPRC (25–100 μM) for 24 h. then the uptake of 2-deoxyglucose by cardiomyocytes was measured as previously described (20). The cardiomyocytes were rinsed with pre-warmed KRP buffer (128 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl₂, 1.25 mM MgSO₄, 5 mM NaH₂PO₄, 5 mM Na₂HPO₄, and 10 mM HEPES, pH 7.4) for three times and then treated with 100 nM insulin in KRP buffer without glucose for 30 min in 37°C. Subsequently, the cardiomyocytes were incubated with 2-deoxy-D[³H]-glucose (1 μCi/ml) dissolved in KRP buffer with 100 nM insulin for 10 min in 37°C. After washing with pre-cold phosphate-buffered saline (PBS) containing 10 mM glucose for 3 times, the cells were lysed with 0.4 mM NaOH for 2 h. The radioactivity of ³H was measured in a liquid scintillation counter (Beckman LS6500) and the concentration of protein were detected by BCA method for standardization.

H₂S levels in heart tissues and plasma were measured as previously described (21). Briefly, heart tissues were homogenized in Tris-HCl (100 mmol/L, pH 8.5), then centrifuged at 12,000 g for 20 min at 4°C. The plasma sample were directly centrifuged at 3,500 rpm for 15 min at 4°C. And 30 μl of the supernatant from heart tissues and plasma were derivatized by MBB and detected by HPLC-MS. The concentration of protein from heart tissues were detected by BCA method for standardization.

All data were expressed as the means ± SEM from at least three independent experiments. SPSS 13.0 software (SPSS, Inc., Chicago, USA) was used for statistical analysis. Student's unpaired t-test was performed to statistically analyze individual group data. Multiple-group comparisons were evaluated by one-way analysis of variance (ANOVA) followed by Fisher's Least Significant Difference (LSD) and the non-parametric test was used when the variance was not equal. Values of P < 0.05 were considered to be statistically significant.

To explore the effect of SPRC on cardiac function of db/db diabetic mice, echocardiograph images were recorded before and after 12 weeks of SPRC treatment (Figure 1A). Compared with WT littermates, db/db mice preserved cardiac function at 8-week-old, but developed diastolic and systolic dysfunction at 20-week-old characterized by elevated deceleration time (DT, Figure 1B), reduced left ventricular ejection fraction (EF, Figure 1D) and decreased fractional shortening (FS, Figure 1E, P = 0.054) with a consistent heart rate (HR, Figure 1C). All of three parameters examined were improved by SPRC treatment (Supplementary Tables 1, 2). The beneficial effect of SPRC treatment on cardiac hypertrophy was confirmed by histopathological examinations and RT-PCR (Figure 2). Heart weight to tibia length (HW/TL) ratio was significantly increased in db/db mice compared with WT mice (Figure 2C). Consistently, cardiomyocyte cross-sectional area examined in db/db mice by wheat germ agglutinin (WGA) was markedly enlarged (Figure 2D). Administration of SPRC to db/db mice decreased HW/TL ratios and cardiomyocyte cross-sectional area, reduced mRNA level of beta-myosin heavy chain (Myh7) gene, marker for cardiac hypertrophy (Figure 2B). Masson staining revealed that obvious fibrosis in the interstitial of myocardium was observed in db/db mice, but significantly alleviated by SPRC treatment (Figure 2E). These data suggest that SPRC treatment improves cardiac function, and alleviates cardiac hypertrophy and fibrosis in the db/db diabetic model.

SPRC treatment protected db/db mice from abnormalities in the ultrastructure of cardiomyocytes. The mitochondria in the cardiomyocytes of WT mice were regularly arranged and exerted integral cristae, and the myofilaments were arranged tightly, while a portion of mitochondria in the myocardium of db/db mice were swollen, disorderly arranged with broken cristae, and the myofilaments were loose. After SPRC treatment, fewer abnormal ultrastructure of mitochondria and myofilaments were observed in diabetic mice (Figure 3).

Oil red O staining showed a predictable and significantly increase in lipid droplets of db/db hearts, which was markedly decreased in response to SPRC (Figures 4A,B). Meanwhile, systemic dyslipidemia was observed in db/db mice characterized by markedly increased TC, TG, LDL-C, and NEFA. SPRC treatment inhibited the increased plasma TC levels, indicating that SPRC has a certain lipid-lowering effect (Figures 4C–G).

Throughout the experiment, the body weight and fasting blood glucose of db/db mice were significantly increased compared to their WT littermates. However, SPRC treatment showed no significant impact on body weight and blood glucose (Figures 5A,B). To investigate the effect of SPRC on systemic insulin resistance, IPGTT and IPITT were preformed and the results demonstrated SPRC treatment at a dosing of 80 mg·kg⁻¹·day⁻¹ improved glucose tolerance to a certain extent, but has no significant impact on IPITT and its area under curve (Figures 5C–F). Furthermore, fasting plasma insulin levels and HOMA-IR index were remarkably elevated, with SPRC treatment failed to alleviate (Figures 5G,H).

Primary neonatal mice cardiomyocytes were cultured and identified to explore the mechanisms of SPRC on diabetic cardiomyopathy in vitro (Supplementary Figure 1B). Incubation of cardiomyocytes with SPRC (0.1–1,000 μM) for 24 h had no detectable cytotoxicity. Moreover, the cell viability was significantly increased at doses of 0.1 and 10 μM of SPRC treatment (Figure 6A). SPRC improved both mRNA and protein expression level of IR in cardiomyocytes, with the classical downstream pathway protein kinase B (Akt/PKB) and glycogen synthase kinase 3β (GSK3β) activated in the dose-dependently manner (Figures 6B–F).

The cardiac inulin receptor signaling in vivo were also determined. As shown in Figures 7A–D, SPRC treatment increased the mRNA of IR in myocardium, and it also improved protein expression of IR to a certain extent (P = 0.095). It is noteworthy that IR in the myocardium of diabetic mice was significantly phosphorylated, which is exacerbated by SPRC treatment. The phosphorylation levels of Akt and GSK3β in myocardium of db/db mice were significantly decreased, SPRC (80 mg/kg) significantly increased the phosphorylation levels of Akt and GSK3β in myocardium of DCM mice (Figures 7E,F).

We then exposed neonatal mice cardiomyocytes to 33.3 mM glucose and 100 nM insulin to mimic hyperglycemia and hyperinsulinemia in cardiomyocyte of diabetic heart. High concentrations of glucose and insulin stimulation significantly reduced the phosphorylation levels of Akt and GSK3β and the expression level of IR. However, after incubation with 50 μM SPRC for 24 h, the phosphorylation level of IR was preserved, and the expression level of IR was partly increased without statistical significance (P = 0.077). Consistently, the downstream of IR signaling including the phosphorylation levels of Akt and GSK3β were preserved (Figures 7G–K).

GLUT4 transporter is the most abundant glucose transporter in the heart, which is also the major glucose transporter translocating to the plasma membrane in response to insulin (22). As shown in Figures 8A,B, the protein expression level of GLUT4 were significantly decreased in diabetic heart, and SPRC treatment increased the mRNA and protein expression level of GLUT4. SPRC also enhanced the glucose uptake in vitro (Figure 8C).

CSE is the primary H₂S-generating enzyme in the cardiovascular system, catalyzing the synthesis of endogenous H₂S from L-cysteine, and SPRC is an activator of CSE. We confirmed that SPRC increases the protein expression of CSE in primary cardiomyocytes (Figure 9A). Moreover, SPRC enhanced CSE expression in the left ventricular tissues of db/db mice (Figure 9B). Furthermore, H₂S concentration in plasma and myocardium were detected. SPRC treatment at the doses of 40 and 80 mg·kg⁻¹·day⁻¹ improved H₂S levels in the myocardium of db/db mice (Figure 9C). And H₂S levels in plasma of db/db mice was significantly lower than that of WT mice, with SPRC treatment failed to rescue (Figure 9D).