The alkaloid components of Yanhusuo were obtained from The Traditional Chinese Medicine Systems Pharmacology Database (TCMSP, https://old.tcmsp-e.com/index.php), along with literature databases. The collection of targets has been completed. The TCMSP database was searched with “yanhusuo” as the keyword, and all the alkaloid components were recorded in the results by selecting the classification of “ingredients.” Subsequently, the detailed information of each alkaloid was checked, its related targets were recorded, and the duplicate values were removed, and the results were used as CYTA-related targets. The gene names corresponding to all target proteins were standardized using the UniProtKB database (https://www.uniprot.org/). The GeneCards database (https://www.genecards.org/) was searched for relevant targets with the keyword “myocardial ischemia,” and all the target results were exported as MI-related genes.

The targets of CYTA and MI were inputted into the online Venn analysis tool (https://bioinfogp.cnb.csic.es/tools/venny/) to identify overlapping targets to be used as anti-MI targets for CYTA. The collected target information was then imported into the STRING database (https://string-db.org/), with the attribute set as Homo sapiens and the threshold set to 0.4, resulting in the retrieval of a protein interaction network (PPI) in TSV format. Subsequently, the files were imported into Cytoscape 3.6.1 to construct the PPI network diagram. The Network Analyzer tool within the software was utilized to analyze the topological parameters of the network interaction graph, such as degree and betweenness centrality (BC), which indicate the degree of correlation between nodes in the network graph. By applying parameter values, key nodes in the network graph were identified, leading to the investigation of CYTA’s mechanism on MI. Finally, the intersecting target genes underwent protein molecular function (MF), cell population (CC), biological process (BP), and KEGG analysis, which were performed using Metascape (https://metascape.org/gp/index.html). Throughout the analysis, a confidence level of p < 0.01 was employed.

CYTA (purity ≥90%), purchased from Chengdu Dexter Biotechnology Co., LTD., was synthesized and dissolved in dimethyl sulfoxide (DMSO) as 100 mM stock solutions. It was diluted to a specific concentration when used. Cell Counting Kit-8 (CCK-8) was provided by Beijing Zhuo Man Biotechnology Co. in Beijing, China. Type II collagenase was obtained from Worthington Biochemical in Lakewood, United States. Isoprenaline (ISO) and verapamil (VER) were supplied by Hefeng Pharmaceutical Co. in Shanghai, China. Unlabeled reagents of analytical purity were obtained from Sigma Chemical Co. in Missouri, United States. All experiment kits, including CK (catalog: A0321-1), LDH (catalog: A020-1-2), SOD (catalog: A001-3-2), CAT (catalog: A007-1), MDA (catalog: A020-1-2), GSH-Px (catalog: A005-1-2), ATP (catalog: A095-11), Ca²⁺-ATPase (catalog: A016-1-2), and BCA (catalog: A045-3), were purchased from Jiancheng BioEngineering Institute in Nanjing, China.

H9c2 cells (Bluefbio, Shanghai, China; catalog: BFN60804388) were cultured in High-sugar Dulbecco’s Modified Eagle Medium (DMEM) (Gibco; Thermo Fisher Scientific, Waltham, United States) supplemented with 10% inactivated fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Waltham, United States) and 100 U/mL penicillin/streptomycin (Leagene Biotechnology; Beijing, China). The incubator maintained a stable temperature (37°C), CO₂ level (5%), pH (7.2–7.4), and high relative humidity (95%). Cells reached 80% confluency or higher after 2–3 days of growth in culture flasks. After washing with Phosphate-buffered saline (PBS) (Solarbio Life Sciences; Beijing, China), the cells were detached using 0.25 g/L trypsin (Leagene Biotechnology; Beijing, China). Cells with appropriate density were uniformly seeded in 96-well plates, 24-well plates, or 6-well plates for subsequent experiments.

H9c2 cells were exposed to various concentrations of CoCl₂ (200, 400, 600, 800, and 1,000 μM) for 22 h and different concentrations of CYTA (1, 3, 10, 30, 100, and 300 μg/mL) to assess their impact on cell viability and determine the optimal concentrations of CoCl₂ and CYTA. At the end of the treatment, 10 μL of CCK-8 was added to each well to measure the absorbance value. Based on the aforementioned procedure, the optimal pretreatment concentration and time for CYTA were determined as 10 and 30 μg/mL for 4 h, respectively. The optimal concentration of CoCl₂ was found to be 800 μM. The experimental groups were categorized as follows: 1) Control (Con); 2) CoCl₂; 3) CoCl₂ + L-CYTA; 4) CoCl₂ + H-CYTA.

After the treatment, the supernatant was collected to measure the release of CK and LDH, following the instructions provided with the kit. H9c2 cells were scraped and centrifuged to obtain the cell pellet. The pellet was resuspended in an appropriate amount of PBS and sonicated for fragmentation. The protein concentrations of each cell group were determined using the BCA kit and used for subsequent assays of SOD, MDA, CAT, GSH-Px, ATP, and Ca²⁺-ATPase, following the instructions provided with the respective kits.

Following the treatment, the medium was aspirated, and H9c2 cells cultured in 24-well plates were washed with PBS. Next, 500 μL of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Cayman Chemical Company, Michigan, United States) was added to each well and incubated for 20 min in a dark environment. Subsequently, the remaining dye was washed off with PBS, and fluorescence images were captured using a fluorescence microscope. The acquired images were analyzed quantitatively using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

Following the treatment of H9c2 cells in 24-well plates, the plates were washed with PBS. Subsequently, 500 μL of 10 μmol/L Fluo-3 AM dye (Life Technologies, Carlsbad, United States) was added and incubated for 20 min in a light-free environment. After washing off the excess dye with PBS, fluorescent images were captured using a fluorescence microscope. The acquired images were subjected to quantitative analysis using Image-Pro Plus 6.0 software.

After treating the cells in 24-well plates, 500 μL of Rhodamine 123 (Rh123, Yuanye Biotechnology, Shanghai, China) dye was added to each well. The cells were then incubated at 37°C in the dark for 20 min. Subsequently, the remaining dye in each well was washed off with PBS, and fluorescence images were captured using a fluorescence microscope. The acquired images were subjected to quantitative analysis using Image-Pro Plus 6.0 software.

Following the treatment of cells cultured in 24-well plates, 500 μL of Hoechst-33258 staining solution (Beijing Solarbio Science & Technology, Beijing, China; Catalog: IH0060) at a concentration of 10 μg/mL was added to each well. The cells were then incubated in the dark at 37°C for 20 min. Subsequently, the residual dye in the cells was washed off with PBS. The cells were placed under a fluorescence microscope to capture fluorescence images. Finally, the acquired images were analyzed quantitatively using Image-Pro Plus 6.0 software.

Forty male Sprague-Dawley (SD) rats aged 6–8 weeks and weighing 200 ± 20 g were obtained from the Experimental Animal Centre of Hebei Medical University. The rats were housed in a controlled environment with an ambient temperature of 21°C ± 1°C and a humidity level of 40%–60%. Rats were housed under 12/12 h light/dark cycle and up to five animals per cage. They had free access to food and water and were acclimated for 1 week before the experiments. All procedures followed the ethical guidelines for experimental animals set by the Hebei University of Traditional Chinese Medicine (approval number: 2103062). The rats were randomly divided into 2 groups: CON and ISO. To establish the myocardial infarction (MI) model, each rat received subcutaneous injections of ISO for two consecutive days (85 mg/kg/day) (Hosseini et al., 2022; Wahid et al., 2022). Subsequently, cardiomyocytes were acutely isolated from a single rat at a specific time point. This study was carried out following the recommendations of the Declaration of Helsinki. Animal experiments and methods were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

The rats were anesthetized by intraperitoneal injection of sodium heparin (500 IU/KG) for 15 min, followed by an intraperitoneal injection of ethyl carbamate (1.0 g/KG). After the anesthesia was completed, the rat’s thorax was opened to separate the aorta. The heart was quickly removed and placed in a Ca²⁺-free Tyrode ice solution, and the cardiac aorta was inserted into the Langendorff device. The Ca²⁺-free Tyrode solution was injected into the heart for 5 min in a thermostatic device at 37°C. The prepared enzyme solution was then perfused recurrently for 15–20 min to digest the myocardium, followed by rinsing with Ca²⁺-free Tyrode solution to terminate digestion. Subsequently, the heart was placed in the Kreb’s buffer, and the ventricular muscle was quickly torn to yield a final KB suspension of individual ventricular myocytes. This suspension was left to stand for 1 h at 4°C before the remaining procedures were conducted. The optimal time for the cells to be available is 8 h. All solutions used were continuously oxygenated at a perfusion rate of 4 mL/min to ensure a 100% oxygen content. The configuration details of the solutions used are presented in Table 1.

A suspension of ventricular myocytes was transferred to the cell recording chamber of the membrane clamp. Once the cells became stable and attached to the bottom of the recording chamber, an external solution of cells was added to suppress any interference from other currents. Cells with intact and clean edges, free from surface particles, clear striations, and at rest, were selected for the experiment. The whole-cell membrane clamp recording method was employed to hold the cells in place. After zeroing the internal and external potential difference, a borosilicate glass electrode (filled with internal solution, resistance 3–5 MΩ) was applied to the cell membrane and then sealed. The recorded streams with a 2 kHz filter were amplified using an Axopatch 200B amplifier (Axon Instruments, Union City, CA, United States) and analyzed using pCLAMP 10.2 software (protocol: pulse waveform of 10 mV, 1 ms; frequency duration of 10 Hz).

To assess cardiomyocyte contractility, the cell suspension was gently introduced into the cell bath of the microscope. While the cells settled at the bottom of the bath, a calcium-free solution was perfused through the cells at a rate of 1 mL/min. Subsequently, the cardiomyocytes were stimulated to contract regularly at a frequency of 0.5 Hz (duration 2 ms) using a cell stimulator (MyoPacer, IonOptix), and the cell contractions were continuously recorded without any treatment using the IonOptix Myocardial Assay System (IonOptix, Milton, MA, United States). Following the recordings, the cells were returned to regular solution conditions.

Cardiomyocyte Ca²⁺ transients were recorded by observing changes in the fluorescent indicator. The cardiomyocytes were incubated with the fluorescent Ca²⁺ indicator, fluo2-AM (2 mM), for a duration of 10 min. The cell suspensions were then gently added to the bath, and cell excitation was induced by applying electric field stimulation through a sheet platinum electrode at a frequency of 0.5 Hz and a duration of 2 ms. The light from a 75 W UV xenon lamp was modulated by the electric field stimulation through a 340 or 380 nm filter to reach the ventricular myocytes, causing them to emit 510 nm light. The resulting fluorescence signal was examined using the IonOptix system.

The resulting experimental data were subjected to a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using Origin 9.1 software. The results were expressed as mean ± standard error (SEM). A p-value of less than 0.05 was considered statistically significant.

A comprehensive search was conducted on a total of 46 CYTA components using the TCMSP database and relevant literature (Table 2). Figure 1 illustrates the 549 targets associated with each component of CYTA retrieved from the TCMSP database. The MI-related genes were identified from the GeneCards database. Duplicate targets were removed, resulting in a total of 2649 MI-related targets. The Venn diagram (Figure 2A) demonstrates the intersection of potential targets between CYTA and MI, revealing 262 identical genes. To further examine the relationship among these 262 genes, the PPI network (Figure 2B) was generated using Cytoscape 3.6.1. The network represents proteins as nodes and their relationships as connecting lines, where nodes and connectors withhigh degree and BC values tend to be brighter in color and vice versa.

The GO enrichment analysis yielded 185 BP items, 130 CC items, and 108 MF items. The BP analysis highlighted topics such as positive regulation of cell death, response to oxygen levels, regulation of neuron death, and apoptotic signaling pathways. Regarding CC, the targets were primarily associated with the cell membrane, nucleus, mitochondria, and endoplasmic reticulum. The MF analysis revealed that the intersecting targets were linked to kinase binding, protein domain-specific binding, and transcription factor binding, as depicted in Figure 3A.

In relation to the KEGG enrichment analysis of CYTA for MI, it identified 174 pathways. Further analysis of these pathways showed a strong association with apoptosis and the calcium signaling pathway. The top 20 results, based on the p-value, were selected and presented in a bubble diagram (Figure 3B). The results indicate the involvement of apoptosis, mitochondrial energy metabolism, and oxidative stress in the preventive effects of CYTA against MI. Additionally, based on the KEGG enrichment analysis, numerous targets were found to be closely associated with the calcium signaling pathway. Therefore, we extracted targets associated with the calcium signaling pathway (Figure 2C), where nodes and connecting lines with high degree values and BC tended to be larger and brighter in color, and vice versa.

According to Figure 4B, concentrations of 1–100 μg/mL of CYTA did not exhibit significant inhibition of normal H9c2 cell proliferation (p > 0.05). However, at a 300 μg/mL concentration, an inhibition of normal H9c2 cell proliferation was observed (p < 0.01). Furthermore, at a concentration of 30 μg/mL, no inhibition of H9c2 cells was observed from 0 to 36 h (p > 0.05, Figure 4C). Based on these findings, dosing concentrations of 10 μg/mL and 30 μg/mL were selected.

The concentration of CoCl₂ that induced hypoxia was determined to be 800 μmol/L, as depicted in Figure 4A. Figure 4D shows that the survival of H9c2 cells was significantly reduced in the CoCl₂ group compared to the CON group (p < 0.01). However, pretreatment with either 10 or 30 μg/mL CYTA demonstrated a mitigating effect on hypoxic injury (p < 0.05 or 0.01).

Figures 4E, F demonstrate that the release of CK and LDH into the extracellular medium was significantly elevated in the CoCl₂ group compared to the CON group (p < 0.01). In contrast, CYTA exhibited the ability to significantly decrease the levels of CK and LDH in the supernatant of hypoxic H9c2 cells (p < 0.05 or 0.01), indicating a mitigating effect of CYTA on cell injury.

The results obtained from the measurement of ROS can be interpreted such that a higher average fluorescence intensity value corresponds to increased ROS production in the cells. Compared to the normal group, the fluorescence intensity of ROS in H9c2 cells was significantly elevated in the CoCl₂ group (p < 0.01). However, in both the high and low-dose groups of CYTA, the fluorescence intensity of ROS in H9c2 cells was significantly reduced compared to the CoCl₂ group (p < 0.05 or 0.01). The results are depicted in Figures 5A, B.

As depicted in Figures 5C–F, the activities of SOD, GSH-Px, and CAT were decreased, while the activity of MDA was increased in the CoCl₂ group compared to the CON group (p < 0.01). Conversely, in the CYTA group, the activities of SOD, GSH-Px, and CAT were increased, and the level of MDA was decreased in the supernatant compared to the CoCl₂ group (p < 0.05 or 0.01).

As shown in Figures 6A, B, damage to the mitochondrial membrane resulted in a decrease in membrane potential. Rh123 fluorescence staining allowed the detection of changes in intracellular mitochondrial membrane potential. Compared to the CON group, the mitochondrial membrane potential of H9c2 cells was significantly reduced after hypoxic injury, and the difference was statistically significant (p < 0.01). However, after pretreatment with CYTA, the reduction in mitochondrial membrane potential was significantly improved in H9c2 cells compared to the hypoxic injury group (p < 0.05 or 0.01).

Both ATP content and Ca²⁺-ATPase activity are direct and indirect indicators of mitochondrial function. Treatment with CoCl₂ significantly reduced the ATP content and Ca²⁺-ATPase activity in H9c2 cells (p < 0.01). However, pretreatment with CYTA resulted in varying degrees of increased intracellular ATP content and Ca²⁺-ATPase activity in H9c2 cells (p < 0.05 or 0.01) (Figures 6C, D).

Under fluorescence microscopy, the CON group showed no obvious apoptotic cells, with nuclei appearing light blue and having low fluorescence. In the CoCl₂ group, the nuclei exhibited uneven coloring, and apoptotic cells appeared bright blue with high fluorescence (p < 0.01). After CYTA treatment, the number of apoptotic cells gradually decreased (Figure 7A, p < 0.05 or 0.01). The apoptotic status of each cell group is depicted in Figure 7B.

The results are depicted in Figure 8. It is evident that CoCl₂ significantly increased the cytoplasmic Ca²⁺ content in H9c2 cells (p < 0.01), while pretreatment with CYTA significantly attenuated the increase in Ca²⁺ content (p < 0.01 or 0.05). These findings suggest that CYTA can regulate and maintain normal Ca²⁺ content in the cytoplasm of cardiomyocytes.

The high sensitivity of T-type Ca²⁺ channels to Ni²⁺ makes NiCl₂ a commonly employed specific blocker for T-type Ca²⁺ channels. Furthermore, it exhibits near-complete inhibition of the T-type calcium current while exerting minimal impact on the L-type calcium current (Lee et al., 1999). NiCl₂ was used to investigate its effect on the current at a concentration of 100 μM. Interestingly, the current was not inhibited by 100 μM NiCl₂, indicating that the recorded current was not a T-type Ca²⁺ current (Figures 9A, B) (Guan et al., 2015). In contrast, 1 μM VER almost completely blocked the current (p < 0.01), suggesting that the recorded current was I_Ca-L (Figures 9C, D) (Zhu et al., 2016).

The results demonstrate that CYTA at a concentration of 100 μg/mL inhibited I_Ca-L in both normal and ischemic rat ventricular myocytes, resulting in inhibition rates of 61.60% ± 1.49% and 50.79% ± 2.26%, respectively (p < 0.01). Furthermore, following the administration of CYTA, extracellular fluid was used for washout, and the currents were partially restored to their pre-administration levels. This suggests that the inhibitory effect of CYTA on I_Ca-L is reversible (Figures 10A–F).

The results presented in these figures show a sequential increase in the inhibitory effect of CYTA on I_Ca-L at concentrations of 1, 3, 10, 30, 100, and 300 μg/mL, resulting in inhibition rates of 2.12% ± 0.30%, 6.50% ± 0.56%, 14.45% ± 0.89%, 33.01% ± 1.95%, 62.17% ± 1.23%, and 75.65% ± 1.13%, respectively. This indicates a concentration-dependent effect of CYTA on I_Ca-L, with a half inhibition rate (IC50) of 43.60 ± 8.61 μg/mL (Figures 10G–I).

Figure 11A shows the effect of CYTA (10, 30 and 100 μg/ml) and VER on the relationship between the I-V curves. As shown in Figure 11B, CYTA shifts the I-V curve in a concentration-dependent manner. At −20 mV, the amplitude of I_Ca-L began to increase. In addition, the reversal potential of I_Ca-L and the I-V curve did not change significantly.

The results presented in Figures 11C, D indicate that CYTA at concentrations of 30 and 100 μg/mL did not significantly affect the steady-state activation-deactivation curve of I_Ca-L. The calculated values for V_1/2/slope (k) of the activation curves were −7.29 ± 1.14 mV/5.87 ± 0.99 mV, −9.64 ± 1.22 mV/5.70 ± 1.11 mV, and −10.84 ± 1.14 mV/5.56 ± 1.04 mV for 0, 30, and 100 μg/mL CYTA, respectively. Similarly, the V_1/2/slope (k) of the inactivation curves were −23.61 ± 0.73 mV/5.40 ± 0.23 mV, −29.25 ± 0.72 mV/3.97 ± 0.24, and −29.89 ± 0.11 mV/5.81 ± 0.69 mV, respectively.

Cell contractility was assessed at CYTA concentrations of 30 and 100 μg/mL, as depicted in Figures 12A, B. Figure 12C demonstrates that these concentrations of CYTA resulted in the inhibition of cell contraction by 25.27% ± 1.55% and 39.83% ± 1.68%, respectively (p < 0.01). Notably, the administration of extracellular fluid for washout partially restored cell contraction, indicating a reversible inhibitory effect of CYTA on ventricular myocyte contractility.

The time to 50% peak (Tp) is an important measure of cell contraction or Ca²⁺ elevation, while the time to 50% of baseline (Tr) is crucial for assessing cellular relaxation or Ca²⁺ reabsorption. Figures 12D, E show that both Tp and Tr were significantly lower in the CYTA group compared to the CON group (p < 0.05).

Figure 13A illustrates the impact of CYTA (100 μg/mL) on the peak change in Ca²⁺ transients. In Figure 13B, it is evident that a concentration of 100 μg/mL CYTA significantly inhibits the peak Ca²⁺ transient. Furthermore, Figure 13C demonstrates that CYTA at a concentration of 100 μg/mL resulted in a notable inhibition of the peak amplitude of cellular Ca²⁺ transient, with an inhibition rate of 38.48% ± 4.19% (p < 0.01).