In this study, we extracted purified morroniside from C. Officinalis sarcocarp provided by Tong-Ren-Tang (Beijing, China), in accordance with a previously described method (Wang et al., 2010). High-performance liquid chromatography (HPLC) was used to obtain a final purity of 98.5%.

Animal experiments were approved by the Animal Care and Welfare Committee of Xuanwu Hospital, Capital Medical University, China (approval number XW-20210501-01). All surgeries were performed under anesthesia, and animal suffering was minimized as much as possible. Male Sprague-Dawley rats, aged 6–8 weeks and weighing 260-280 g, were provided by the Beijing Vital River Experimental Animal Co. (Beijing, China) and raised at 22°C ± 2°C with a 12-h/12-h light/dark cycle. The rats were provided free access to food and water.

NRCMs were obtained from newborn rats (1–2 days after birth). Briefly, heart tissues were collected from newborn rats and washed with D-Hank’s solution. Subsequently, the heart tissues were minced prior to digestion with trypsin (0.075%; Gibco, Thermo Fisher Scientific Inc., Waltham, MA, United States) and type II collagenase (0.1%; Solarbio, Beijing, China). To suspend the cells, high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fisher Scientific) containing 100 U/mL of penicillin, 100 mg/mL streptomycin (Gibco, Thermo Fisher Scientific), and 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit-Haemek, Israel) was added. Then, cells were seeded on 10-cm plastic dishes in a humid incubator for a 1.5-h period under 5% CO₂ and 37°C conditions. DMEM-suspended cells were inoculated in glass-bottom cell culture dishes or 48-well plates at 20,000 cells/well in 5% CO₂ in a humidified atmosphere at 37°C.

To simulate ischemia in vitro, we exposed cells to hypoxia (95% N₂ together with 5% CO₂) prior to incubation for 6 h in glucose-free DMEM (Gibco, Thermo Fisher Scientific), and exposed cells in the control group to normoxia (95% air and 5% CO₂) for 6-h incubation. The cells were classified into six groups. We then cultured control cells normally and treated OGD cells with OGD for a 6-h period. Cells in the control + morroniside group were pretreated with 100 μM morroniside for 24 h. Cells in the three OGD + morroniside groups were subjected to 24-h pretreatment with 1, 10, or 100 μM morroniside prior to 6-h of OGD. After cell harvesting, radioimmunoprecipitation assay (RIPA) lysis buffer (Applygen, Beijing, China) containing phosphatase inhibitors (Roche, Indianapolis, IN, United States) was added to lyse the cells for Western blot analysis. For immunofluorescence staining analysis, the cells were fixed with 4% paraformaldehyde (PFA) for 20 min before immunofluorescence analysis.

As previously described (Liu et al., 2020), the rats underwent tracheal intubation, followed by ventilation with 3% isoflurane for induction and 2% isoflurane for anesthesia maintenance. Thoracotomy was performed in the fourth left intercostal space to expose the heart. We permanently ligated the left anterior descending (LAD) coronary artery 2 mm beneath the left atrium using a 6-0 prolene suture and closed the chest postoperatively. The rats were warmed for 15 min until they recovered. Rapid anterior surface discoloration and ST-segment elevation on electrocardiography (ECG) indicated a successful operation. Rats in the sham operation, AMI, and morroniside-treated AMI groups were administered morroniside at 60, 120, or 240 mg/kg. Specifically, morroniside was dissolved in normal saline before intragastric administration once daily from 3-h post-AMI for 7 and 28 days. Equivalent volumes of normal saline were administered to the sham-operation and AMI groups. The hearts were dissected 7 and 28 days after the operation for further analysis.

NRCMs were cultured in a medium containing 10 μM BrdU (Sigma-Aldrich, St. Louis, MO, United States) a day before OGD or normal culture, followed by another 6-h incubation with BrdU. In the adult heart section BrdU assay, BrdU at 500 μg/mL (Sigma-Aldrich) was administered to each rat via intraperitoneal injection twice a day after surgery for a 7-day period.

After 15-min of fixation using 4% PFA, cultured cells were rinsed with 0.1 M phosphate-buffered saline (PBS) three times for 5 min each. Later, 20-min permeabilization was performed with 0.1% Triton X-100 at ambient temperature. Subsequently, 5% donkey serum (Jackson ImmunoResearch Laboratories) was added to block the cells at ambient temperature for 1 h, followed by overnight incubation with primary antibodies at 4°C. After washing with PBS, the cells were incubated for 2 h with Alexa Fluor-488- (1:400, A21206, Life Technologies, Carlsbad, CA, United States) or Alexa Fluor-594-conjugated (1:400, A21203, Life Technologies) secondary antibodies at ambient temperature. After washing thrice with PBS, diamidino-2-phenylinidole (DAPI; Abcam, Cambridge, MA, United States) was added for nuclear staining. For BrdU staining, cells were permeabilized with 2 N HCl for 17 min at 37°C prior to blocking with donkey serum.

For heart slides, rats were killed at 7 and 28 days post-AMI, and the obtained myocardial samples were subjected to optimal cutting temperature (OCT) compound (Sigma-Aldrich) embedding and rapid freezing within liquid nitrogen prior to preservation at −80°C; 6-μm cryostat sections were cut. Ice-cold sections and neonatal rat primary cardiomyocytes were permeabilized with 0.1% Triton X-100 in PBS for 30 min, followed by 2 h of blocking using 5% donkey serum (Jackson ImmunoResearch Laboratories) at ambient temperature, as well as overnight primary antibody incubation at 4°C followed by incubation with Alexa Fluor-488 (1:400; Life Technologies) or Alexa Fluor-594-conjugated (1:400; Life Technologies) secondary antibodies. In addition, the nuclei were stained with mounting medium containing DAPI (Abcam). A Nikon 80i fluorescence microscope and Leika MICA confocal microscopy was used to visualize fluorescence signals. New positively stained cardiomyocytes per unit area were quantified using the Image-Pro Plus software package (version 6.0; IPP, Media Cybernetics, Maryland, United States).

The primary antibodies used in this assay were mouse anti-cTnT (1:200, ab8295, Abcam), rabbit anti-cTnT (1:200, ab209813, Abcam), rabbit anti-Ki67 (1:200, ab15580, Abcam), mouse anti-BrdU (1:200, 11170376001, Roche, Indianapolis, IN, United States), rabbit anti-histone H3 (1:200, ab5176, Abcam), rabbit anti-Aurora B (1:200, ab2254, Abcam) and anti-α-actinin (1:200, ab9465, Abcam).

NRCMs and hearts were homogenized and lysed in pre-chilled RIPA buffer (Applygen) containing phosphatase inhibitors (Roche) to extract proteins. The protein content was analyzed using a bicinchoninic acid protein assay kit (Applygen). Subsequently, equivalent amounts of total proteins (50 μg cardiac proteins and 70 μg cellular proteins) were blended prior to dissolution in 5 × sodium dodecyl-sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and then heated to 95°C for 5 min. Following SDS-PAGE for protein separation, the separated proteins were transferred onto nitrocellulose membranes. Primary antibodies against cyclin D1 (1:1,000, ab134175; Abcam), cyclin-dependent kinase 4 (CDK4) (1:1,000, 09173-4F11; Sigma-Aldrich), cyclin A2 (1:1,000, ab181591; Abcam), and cyclin B1 (1:1,000, 4135S; Cell Signaling Technology, Beverly, MA, United States) were used for immunodetection. Subsequently, horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies were added. Finally, an ECL kit (Millipore, Temecula, CA, United States) was used to visualize immunoreactive proteins on the membrane.

After ventilation with 3% and 2% isoflurane for anesthesia induction and maintenance, respectively, the rats were injected with needle electrodes in the lead II position. ECGs were recorded for each experimental mouse (MP150; BIOPAC Co., United States).

After dissection, heart tissue was immersed in liquid nitrogen for snap-freezing using an OCT compound, cut into 6-μm sections, and fixed with acetone. After washing with PBS, the sections were blocked with 5% donkey serum before incubation with Alexa-488-conjugated WGA (1:500, W11261, Thermo Fisher Scientific). ImageJ software was used to measure cell size.

After collection, the heart tissue was subjected to 4% PFA fixation, embedded in OCT, and sliced into 6-μm sections. Subsequently, four sections were prepared on one slide. Masson’s trichrome staining was carried out in line with specific instructions. Serial sections from the apex to the ligation site were examined. The average percent fibrotic area of the total area was calculated using ImageJ software based on Masson’s trichrome staining to quantify scar size.

The results are presented as the mean ± standard error of the mean (SEM). Statistical analyses were conducted using SPSS 23.0 and Prism 5.0 (GraphPad Software). One-way ANOVA and Tukey’s test were used to compare multiple groups. The significance levels are indicated at p < 0.05, p < 0.01, and p < 0.001.

To investigate whether morroniside induces cardiomyocyte cell cycle activity, we first labeled cell cycle activated NRCMs with Ki67 and cTnT using immunofluorescence staining. The number of cell cycle activated cardiomyocytes was notably higher in the OGD group than that in the control group. In addition, the number of Ki67⁺/cTnT⁺ cells was higher in the 10 and 100 μM morroniside-treated groups than in the OGD group (Figures 1A, C). We labeled the S phase with BrdU to further demonstrate that morroniside induced cardiomyocyte cell cycle activity (Yu et al., 2022). The 10 and 100 μM morroniside-treated groups exhibited a pronounced increase in the ratio of BrdU⁺/cTnT⁺ cells relative to the OGD group (Figures 1B, D). To determine whether increased DNA replication in cardiomyocytes ultimately leads to increased karyokinesis and cytokinesis, we stained the cells with phospho-histone H3 (pH3), a marker of late G2-phase/mitosis (Bhaskar et al., 2018), and Aurora B, a kinase localized in the midbody during cytokinesis (Gang et al., 2017). As indicated in Figures 1E–H, the percentages of pH3⁺/cTnT⁺ and Aurora B⁺/cTnT⁺ cells in the 10 μM and 100 μM morroniside-treated groups were higher than those in the OGD group. These results suggested that morroniside induced NRCM cell cycle activity after OGD in vitro.

We further determined whether morroniside induces cardiomyocyte cell cycle activity after AMI in vivo. We evaluated Ki67, BrdU, pH3, and Aurora B expression in cTnT⁺ cardiomyocytes in vivo (Gao et al., 2019; Magadum et al., 2020; Liu et al., 2021; Wu et al., 2021; Li et al., 2023). As shown in Figures 2, 3, the number of cell cycle activated cardiomyocytes increased at the infarct border in the AMI group. Compared to the AMI group, morroniside treatment at doses of 120 and 240 mg/kg notably elevated Ki67⁺ (Figure 2B) and Aurora B⁺ (Figure 3B) cardiomyocytes, whereas morroniside treatment at 240 mg/kg notably elevated the number of BrdU⁺ (Figure 2C) and pH3⁺ cardiomyocytes (Figure 2D). These results suggest that morroniside induces cardiomyocyte cell cycle activity in adult rat hearts 7 days after AMI in vivo.

Several cell cycle proteins are vital for generating cardiomyocytes (Ponnusamy et al., 2017). Cyclins and cyclin-dependent kinases (CDKs) are positive modulators of cell cycle progression. Their increased levels serve as an index of mitotic events in cardiomyocytes. To elucidate the mechanism underlying the effect of morroniside on induction of cardiomyocyte cell cycle activity, we evaluated the expression of cyclin D1, CDK4, cyclin A2, and cyclin B1 proteins using Western blotting. As shown in Figures 4A–D, OGD increased cyclin D1, CDK4, cyclin A2, and cyclin B1, and the differences between cyclin A2 and cyclin B1 were significant. Cyclin D1 expression was notably elevated in the 10 and 100 μM morroniside-treated groups relative to that in NRCMs in the OGD group (Figure 4A). There was also a tendency of increased CDK4 expression in the 100 μM morroniside-treated group relative to the OGD group (Figure 4B). Similar to the levels of cyclin D1 and CDK4, the expression of cyclin A2 was elevated in the 10 μM and 100 μM morroniside-treated groups relative to that in the AMI group (Figure 4C), and the level of cyclin B1 was increased in the 100 μM morroniside-treated group (Figure 4D). To validate the effects of morroniside on the expression of these cell cycle proteins, we assessed the expression of cyclin D1, CDK4, cyclin A2, and cyclin B1 in rats with AMI. As shown in Figures 4E–H, cyclin D1, CDK4, cyclin A2, and cyclin B1 levels were elevated in the AMI group, but not significantly. In comparison with that in the AMI group, the level of cyclin D1 in the 240 mg/kg morroniside-treated group increased obviously, and the 120 and 240 mg/kg morroniside-treated groups showed significant increases in CDK4, cyclin A2, and cyclin B1 levels relative to those in the AMI group. According to these results, proteins associated with the cell cycle mediate the effects of morroniside on cardiomyocytes following AMI.

Finally, we evaluated the function of morroniside in cardiac repair using a rat model of AMI. As shown in Figure 5A, the sham group showed no abnormal ECG patterns. Compared with the sham group, a pathological Q wave was observed in the ECG of the AMI group 7 days post-surgery. Our data showed that the 120 and 240 mg/kg morroniside-treated groups presented an obvious reduction in Q wave amplitude relative to the AMI group (Figure 5B). Figure 5C illustrates the incidence rate of pathological Q waves 7 days post-surgery; the incidence in the 240 mg/kg morroniside-treated group was notably lower than that in the AMI group. Analysis of Masson’s trichrome-stained heart cross-sections and quantification of scar size at 28 days post-surgery demonstrated that the infarcts were notably smaller in the 120 and 240 mg/kg morroniside-treated groups than in the AMI group (Figures 5D, E). In addition, WGA staining revealed the size of the cardiomyocytes in the border zone. The findings demonstrated that the cardiomyocyte size was larger in the AMI group than in the sham group, and cardiomyocytes in the 120 mg/kg and 240 mg/kg morroniside-treated groups were smaller than those in the AMI group (Figures 5F, G), suggesting a reduction in cardiomyocyte hypertrophy. We also examined the ratio of heart to body weight. The results showed that morroniside treatment (120 or 240 mg/kg) resulted in a dramatic reduction in the heart-to-body weight ratio in rats with AMI compared to the model group (Figures 5H, I). These data demonstrate that morroniside improves myocardial fibrosis and hypertrophy, and promotes heart repair in rats with AMI.