Experiments were performed on 8-week-old male Sprague Dawley (SD) rats (weight, 180-220 g; Rong Heng Biological Co., Ltd). A total of 30 SD rats were randomly assigned to five rat cages (n=6/cage) after one week of acclimation, feeding in an indoor animal room with a 12 h light/dark cycle, a temperature of 22±2˚C and 40-60% relative humidity with free access to food and water. All procedures were performed with the approval of the Animal Care and Use Committee of Hebei University of Chinese Medicine (approval no. DWLL202206006).

Animal experiments were performed based on scientific rationale according to the 3R principles of animal experimentation. To reduce pain in rats, pentobarbital sodium was used as an anesthetic during surgery. The rats were randomized into five groups (n=6/group) as follows: Control group, CLP group, CLP + CVB-D group, CVB-D group and CLP + Ferr-1 (Ferrostatin-1, ferroptosis inhibitor) group. CLP surgery was performed to induce septic cardiac dysfunction. CVB-D was dissolved in methanol (final concentration, 10 mM) and stored at 4˚C as a stock solution. CVB-D (cumulative dose of 4 mg/kg; Meilunbio Co., Ltd.) (11) or saline was administered by gavage in rats for four consecutive days before CLP surgery. The rats received an intraperitoneal injection of Ferr-1 (2 µmol/kg; Xcess Biosciences) or saline once daily for four days before surgery.

The CLP surgery was performed as described previously (18). Briefly, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg) then fixed on an operating table and incised 1.5 cm along the midline of the abdomen under aseptic conditions. After the colon of the rat was exposed and ligated at 1/2 to the root, the distal cecum was then punctured twice with a 9-gauge needle. A small volume of fecal contents from the punctured cecum was spilled into the abdominal cavity. The control group underwent the same surgical procedure, but without cecal puncture. All animals were placed on the operating table and immobilized with ketamine (40 mg/kg; intramuscular injection in the front of the thigh) and xylazine (10 mg/kg; intraperitoneally administered) anesthesia, after which 5 ml blood was obtained from the abdominal aorta. After rats were sacrificed by exsanguination from the carotid artery, blood, heart and liver tissues were obtained, rats were sacrificed by exsanguination from the carotid artery. Blood (centrifuged at 3,000 x g for 15 min at 4˚C) was collected to obtain serum for measuring cardiac function biomarker levels. After the hearts were isolated, tissue samples from each heart were used for hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), reverse transcription-quantitative PCR (RT-qPCR) and biochemistry testing. Liver samples were also collected for RT-qPCR.

Heart tissues were soaked in 4% paraformaldehyde at 4˚C for two days, then the tissue samples were gradually dehydrated in an ascending gradient of ethanol 30 min each at room temperature, embedded in paraffin and cut into 4 µm sections. Slides were stained with H&E 5 min each at room temperature and observed under a light microscope (Leica Microsystems GmbH).

Biochemical indicators of myocardial injury and lipid peroxidation were detected by creatine kinase-muscle and brain (CK-MB; cat. no. H197-1-1; Nanjing Jiancheng Institute of Bioengineering), lactate dehydrogenase (LDH; cat. no. A020-2-2; Nanjing Jiancheng Institute of Bioengineering) and cardiac troponin I (cTnI; cat. no. H149-2; Nanjing Jiancheng Institute of Bioengineering) content in serum, as well as malondialdehyde (MDA; cat. no. A003-1-1; Nanjing Jiancheng Institute of Bioengineering), superoxide dismutase (SOD; cat. no. A001-3-2; Nanjing Jiancheng Institute of Bioengineering) and reduced glutathione (GSH; cat. no. A006-2-1; Nanjing Jiancheng Institute of Bioengineering) content in tissue. The content and activity of each sample indicator was measured spectrophotometrically using biochemical detection kits (Nanjing Jiancheng Bioengineering Institute).

Cardiac function was examined in rats 24 h after CLP. Rats were anesthetized by inhalation 2.5% isoflurane in 5% carbon dioxide and 95% oxygen and their chest fur removed with depilation cream (cat. no. VT-200; Veet; Reckitt Benckiser). A coupling agent (Kofu Medical Technology Development Co., Ltd) was applied evenly to the skin then M-mode echocardiograms were recorded and analyzed using high-frequency echocardiography (Vevo 2100, VisualSonics, Inc.). Left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic volume (LVESV) and left ventricular end-diastolic volume (LVEDV) were measured.

Cardiac tissue samples were cut into 1 mm cubes and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for at least 24 h at room temperature. All samples were postfixed in 1% osmium tetroxide for 2 h at room temperature, dehydrated in an ascending acetone gradient, embedded in Epon 812 for 72 h at 60˚C, cut into 70 nm-thick sections, mounted and double-stained with uranyl acetate and lead citrate for 20 min at room temperature. The samples were then imaged using a transmission electron microscope (H-7600; Hitachi, Ltd.).

H9C2 cells were collected and pooled together to achieve sufficient RNA. TRIzol (cat. no. RR047A; Takara Bio, Inc.) total RNA extraction reagent was used for RNA extraction and a PrimeScript™ RT reagent kit (cat. no. RR047A; Takara Bio, Inc.) was used to synthesize cDNA according to the manufacture's protocol. Detection of the mRNA expression levels of the hepcidin gene (hamp) was performed against GAPDH (19). The following thermocycling conditions were used for PCR: R67 Initial denaturation stage, 95˚C for 5 min. Cycling stages were as follows: Denaturation, 95˚C for 30 sec; annealing, 60˚C for 30 sec; and extension, 72˚C for 30 sec, for a total of 40 cycles. Final extension stage: 72˚C for 5 min. Relative mRNA expression levels were determined using the 2^-ΔΔCq method (20). The primers sequences used were as follows: Hamp forward (F), 5'-CCTGAGCAGCGGTGCCTATC-3' and reverse (R), 5'-TGGTGTCTCGCTTCCTTCGC-3'; Prostaglandin-endoperoxide synthase 2 (Ptgs2) F, 5'-ATGTTCGCATTCTTTGCCCAG-3' and R, 5'-TACACCTCTCCACCGATGAC-3'; and GAPDH F, 5'-GAGTCAACGGATTTGGTCGT-3' and R, 5'-GACAAGCTTCCCGTTCTCAG-3'.

Non-heme levels of iron in the heart were measured. Samples of heart tissue weighing ~50 mg were obtained then incubated with 10 times the volume of tissue digestive liquid (3M hydrochloric acid + 0.61M trichloroacetic acid) for 60 h at 65˚C. After centrifugation (3,000 x g for 15 min at 4˚C), the supernatant was collected for spectrophotometric quantification of the formation of the Fe(II)-bathophenanthroline disulfonate complex. Supernatant (0.5 ml) was added to 1.5 ml of iron chromogen and boiled in a water bath for 5 min. Iron developing color working solution (cat. no. A039-1-1; Nanjing Jiancheng Bioengineering Institute) was freshly prepared and the absorbance was measured at 535 nm. A standard curve was generated and the non-heme iron content of samples was calculated according to a formula provided by the manufacturer.

H9C2 cardiomyoblasts purchased from Procell Life Science & Technology Co., Ltd. were cultured in DMEM (cat. no. C11995500BT; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (cat. no. 10270106; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin in an incubator with a 5% CO₂ atmosphere at 37˚C. Cell culture medium was changed every other day. A stock solution of CVB-D (final concentration, 10 mM) (cat. no. 860-79-7; Meilunbio Co., Ltd.) was prepared in methanol. Cells were pretreated with CVB-D at doses of 0.1, 1, 10 or 100 µmol/l at 37˚C for 24 h, while the control groups were treated with an equivalent volume of PBS. To establish a model of septic myocardial injury in vitro, H9C2 cells were incubated at 37˚C for 12 h with LPS at doses of 0.1, 1, 10 or 100 µg/ml (Shanghai Aladdin Biochemical Technology Co., Ltd.) (21). Cells were then harvested for analysis.

The proliferation capacity of the cells was assessed in a 96-well plate (2x10⁵ cells/well) using the CCK-8 assay (22). The cells were incubated in 96-well plates overnight before the addition of CCK-8. After stimulation with CVB-D or LPS (cat. no. 93572-42-0; MilliporeSigma) for 24 h, 10 µl of CCK-8 reagent was added and incubated for 2 h at 37˚C under dark conditions. The absorbance value at 450 nm of each well was recorded with a microplate reader.

The level of fluorescence produced by the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA; cat. no. S0033S; Beyotime Institute of Biotechnology) was used to quantify ROS generation. Groups of H9C2 cells were loaded with 1:1,000 DCFH-DA probes diluted with serum-free medium and incubated for 20 min in a 37˚C cell incubator after 24 h of administration. DCFH-DA is hydrolyzed into dichlorodihydrofluorescein (DCFH) after entering the membrane of the H9C2 cell. DCFH, which is not fluorescent, can be oxidized by intracellular ROS to fluorescent DCF. The level of ROS was detected according to the fluorescence intensity. The fluorescence intensity was imaged using a fluorescence microscope (IX73; Olympus Corporation) and quantified using ImageJ (version 1.5.1) software (National Institutes of Health).

Intracellular labile iron was quantified using calcein-acetoxymethyl ester (Calcein-AM) (23). Cellular cytoplasmic esterase converts a nonfluorescent acetoxy ester fraction, Calcein-AM, into fluorescent calcein, which is impenetrable to other cells. When iron binds to calcein, its fluorescence is quenched and then restored when the iron loses its stronger chelating agent. Cells were incubated in 24-well plates at 37˚C for 24 h. Then CVB-D or LPS was given to incubate for 24 h. Calcein-AM (cat. no. 40719ES50; Shanghai Yeasen Biotechnology Co., Ltd.) was added to cells in a 24-well plate at a final concentration of 0.25 µM and incubated for 30 min at 37˚C. The fluorescence level was then measured using an inverted fluorescence microscope at the excitation and emission wavelengths of 488 and 525 nm, respectively (IX73; Olympus Corporation).

As previously described (24), total protein in myocardial tissue and H9C2 cells were precipitated in RIPA lysis buffer solution (cat. no. SL1020; Coolaber, Ltd.) containing protease inhibitors. Tissue and cells were centrifuged at 4˚C for 20 min at 12,000 x g to remove the insoluble residue. The supernatant was removed and the concentration of total protein was determined using a BCA kit (cat. no. CW0014; Beijing Kangwei Century Biotechnology Co., Ltd.). Equal amounts of total protein were separated by electrophoresis using 10 or 12% gel. Separated proteins were subsequently transferred to polyvinylidene fluoride (EMD Millipore) membranes and blocked overnight at 37˚C with TBS-0.1% Tween 20 in 5% skimmed milk. Membranes were incubated with GPX4 (1:2,000; cat. no. ET1706-45; HUABIO), SLC7A11 (1:1,000; cat. no. DF12509; Affinity Biosciences), transferrin receptor 1 (TfR1; 1:2,000; cat. no. abs131442; Absin), ferroportin 1 (FPN1; 1:3,000; cat. no. GTX100573; Absin), divalent metal transporter 1 (DMT1; 1:2,000; cat. no. abs112967; Absin), ferritin heavy chain (FtH; 1:1,000; cat. no. AB183781; Abcam), IL-6 (1:1,000; cat. no. GB11117; Wuhan Servicebio Technology Co., Ltd.), IL-1β (1:1,000; cat. no. AF5103; Affinity Biosciences), tumor necrosis factor-α (TNF-α; 1:1,000; cat. no. AF7014; Affinity Biosciences), phosphorylated-STAT3 (p-STAT3; 1:1,000; cat. no. AF3294; Affinity Biosciences), STAT3 (1:1,000; cat. no. AF6293; Affinity Biosciences), Nrf2 (1:1,000; cat. no. M200-3; MBL International Co.), Lamin B1 (1:1,500; cat. no. SI17-06; HUABIO) and β-tubulin (1:2,000; cat. no. A01857-1; Wuhan Boster Biological Technology, Ltd.) antibodies primary antibodies at 4˚C overnight. Goat anti-rabbit IgG (H+L)-HRP (1:10,000; cat. no. S0001) or goat anti-mouse IgG(H+L)-HRP (1:1,000; cat. no. S0002; both from Affinity Biosciences) were then placed at room temperature for 2 h. The cell membranes were then washed again and Visioncapt (V16.12; Vilber Lourmat) was used for quantification.

The concentration of Ca²⁺ was assessed using a Fluo-4 AM kit (cat. no. F8501; Beijing Solarbio Science & Technology Co., Ltd) according to manufacturer's instructions. 1x10⁵ H9C2 cells were seeded into a 24-well plate and cultured at 37˚C for 24 h. Fluo-4 AM (1 ml, 4 µM) was added to each well and incubated in darkness at 37˚C for 20 min. Hank's Balanced Salt Solution (HBSS; cat. no. F8501; Beijing Solarbio Science & Technology Co.) containing 1% fetal bovine serum was added and incubated at 37˚C for 40 min. After being washed three times with HBSS and FBS (1 ml per well) was used each time, cells were imaged to detect fluorescent calcium ions using excitation and emission wavelengths of 494 and 516 nm, respectively, with an inverted fluorescence microscope (IX73; Olympus Corporation).

To further analyze whether CVB-D inhibits calcium inwards flow through LTCCs, a diaphragm clamp technique was utilized to detect L-type calcium current changes. With the use of L-type calcium channel inhibitors to block calcium channels, the inwards flow of calcium ions is reduced, resulting in protection against myocardial injury. Verapamil (VER) is a typical calcium channel blocker. The expression of the L-type calcium channel (LTCC) in ventricular myocytes was analyzed utilizing the whole-cell patch-clamp method. The intracellular solution consisted of tetraethylammonium chloride (cat. no. 56-34-8; TCL), MgCl₂ (cat. no. 10012818; Sinopharm Group Chemical Reagent Co., Ltd), CaCl₂ (cat. no. 10005861; Sinopharm Group Chemical Reagent Co., Ltd), Glucose (cat. no. 56-86-0; Sigma-Aldrich; Merck KGaA) and N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid (cat. no. 7365-45-9, Beijing Bailingwei Technology Co., Ltd), and the extracellular solution consisted of CsCl (cat. no. 7647-17-8; Shanghai Yi En Chemical Technology Co., Ltd), tetraethylammonium chloride, ATP-Mg (cat. no. 7480-12-9; Sigma-Aldrich; Merck KGaA), N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid and ethylene glycol tetraacetic acid (cat. no. 67-42-5; Sigma-Aldrich; Merck KGaA). Currents under filtered at 2 kHz were recorded with Axon patch 200 B amplifier after the cells were connected with glass electrodes (Sutter Instrument). The Axon Patch 200B amplifier analyzed the current and pCLAMP™ software (version 10.0) was used for data analysis (Molecular Devices, LLC). The contraction of myocardial cells was measured. Cells were placed on an inverted microscope (Ion Optix) platform and perfused with the external solution to induce cardiac myocyte shortening 2 msec each time of 0.5 Hz. Cardiomyocytes with clear transverse lines and in good condition were selected to measure contraction.

Experimental data are presented as the mean ± standard deviation. Data were processed using GraphPad Prism software (version 8.0; Dotmatics) using one-way ANOVA followed by Tukey's test for comparisons among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

LPS is commonly used to generate pathophysiological conditions of septic cardiomyopathy in investigation studies. CCK-8 assay results demonstrated that H9C2 cell viability was significantly reduced by LPS in a dose-dependent manner (Fig. 1A). A dose of 10 µg/ml LPS at 37˚C for 12 h was selected to induce a cellular sepsis model for further use in the present study, as previously described (21,25,26). Additionally, a CCK-8 assay was performed to assess the effects of CVB-D on H9C2 cell viability. The results demonstrated that CVB-D had no cytotoxic effect on H9C2 cells at 0.1 or 1 µM, but significantly reduced H9C2 survival at 10 and 100 µM (Fig. 1A). Therefore, a dose of 1 µM CVB-D was selected for use in the present study. Moreover, pretreatment of H9C2 cells with 1 µM CVB-D significantly enhanced LPS-damaged cell viability (Fig. 1B). As expected, LPS distinctly weakened CK-MB, LDH activity and cTnI levels (P<0.01), whereas the inhibitory effect was blocked by CVB-D pretreatment (Fig. 1C).

The effect of CVB-D on H9C2 lipid peroxidation levels and production of first line defense antioxidant enzymes GSH and SOD were also assessed. LPS treatment of H9C2 cells significantly increased production of MDA, but significantly reduced GSH levels and significantly increased SOD activity; however, CVB-D significantly reversed these effects (Fig. 1D). The enhancement of ptgs2 mRNA is used as a putative marker of ferroptosis (9). CVB-D pretreatment significantly reduced the stimulatory effects of LPS on ptgs2 mRNA (P<0.01) (Fig. 1E). ROS production was analyzed using the ROS sensitive DCFH-DA probe. The images from this experiment demonstrated that LPS-treated H9C2 cells generated increased ROS compared with controls. CVB-D significantly decreased the ROS generation induced by LPS (Fig. 1F). Since the SLC7A11-GSH-GPX4 antioxidant axis is important in ferroptosis, the protein expression levels of SLC7A11 and GPX4 were analyzed in LPS-induced H9C2 cells pretreated with CVB-D. The protein expression levels of SLC7A11 and GPX4 were significantly reduced after LPS treatment but were elevated by treatment with CVB-D (Fig. 1G).

Excess cellular iron has been previously reported to promote the generation of both soluble and lipid ROS via the Fenton reaction, which favors the ferroptotic effect (27). Thus, it was hypothesized that CVB-D inhibited oxidative stress, lipid peroxidation and ferroptosis by modulating the abnormal iron metabolism induced by LPS. In the LPS-treated group of H9C2 cells, the significant decrease in calcein fluorescence intensity indicated a high level of calcein-bound iron, while CVB-D pretreatment significantly reversed this change (Fig. 2A). In addition, the presence of iron transporter proteins, such as DMT1, TfR1, FPN1 and FtH, was detected by western blotting (Fig. 2B). Compared with the LPS-treated group, CVB-D treatment significantly reduced DMT1, TfR1 and FtH protein expression levels but significantly increased FPN1 protein expression levels, which suggested that CVB-D may suppress iron uptake and storage while increasing iron export. As FPN1 is the only known iron exporter in mammalian cells, the expression levels of hamp mRNA. The stimulation of LPS in cells has been reported to induce the release of many inflammatory cytokines. Indeed, compared with the LPS group, CVB-D pretreatment significantly decreased the levels of hamp mRNA in H9C2 cells (Fig. 2C). Compared with LPS-treated cells, CVB-D pretreatment significantly decreased the protein expression levels of pro-IL-1β, TNF-α and IL-6 and the p-STAT3/STAT3 ratio in H9C2 cells (Fig. 2D). These findings demonstrated that CVB-D treatment could significantly inhibit iron-overload by modulating iron metabolism. In particular, CVB-D could eliminate LPS-induced hepcidin by blocking of the IL-6/STAT3 axis.

Nrf2 has previously been reported to promote intracellular iron accumulation by downregulating FPN1(28). To confirm whether Nrf2 serves an important role in protecting cells from septic harm, H9C2 cells were treated with the Nrf2 inhibitor ML385 at a dose of 1 µM/l for 12 h. Nrf2, SLC7A11 and GPX4 protein expression levels in H9C2 cells were analyzed (Fig. 3A). LPS significantly downregulated Nrf2 expression, and Nrf2 expression was significantly increased when LPS was combined with CVB-D treatment (Fig. 3B). Therefore, the hypothesis that an enhancement of Nrf2 would be harmful to cells by increasing iron by downregulating FPN1 was not supported. SLC7A11 and GPX4 protein expression levels were significantly upregulated in septic cells pretreated with CVB-D compared with controls, while ML385 significantly reversed this change (Fig. 3C and D). This indicated that Nrf2 serves a role in the protective effects of CVB-D against ferroptosis, but not via FPN1.

To evaluate whether CVB-D was beneficial for septic heart injury, cardiac structure and function in rats were assessed. H&E staining demonstrated that CVB-D treatment reduced the myocardial tissue damage caused by CLP (Fig. 4A). The rats in the CLP surgery group exhibited marked cardiomyocyte disorders, myocardial abnormalities, inflammatory cell infiltration, visible fibrosis and fiber breakage compared with the control surgery group. In contrast, the rats in the CLP surgery group pretreated with CVB-D demonstrated no obvious inflammatory cell infiltration or fibrosis and the arrangement of myocardial fibers was neat. Serum levels of CK-MB, LDH and cTnI, consistent with the degree of myocardial cell injury, were significantly increased in the myocardium of CLP rats compared with controls (Fig. 4B). Furthermore, CVB-D pretreatment significantly inhibited CLP-induced increases in CK-MB, LDH and cTnI (Fig. 4B). Type M-mode sonograms were performed to measure left ventricular function (Fig. 1C). The left ventricular ejection fraction shortening fraction was significantly decreased in the CLP-treated group compared with the control group (Fig. 4D). LVESV and LVDEV were both significantly increased in the CLP-treated group compared with the control. This change was significantly reversed in the CLP + CVB-D treatment group, but no significant differences were observed between the CVB-D group and the control. This suggested that CVB-D demonstrated a significant ameliorative effect on left ventricular dysfunction caused by CLP surgery.

To evaluate whether ferroptosis was involved in the cardioprotective effect of CVB-D on septic heart injury, levels of ferroptosis related markers were examined in myocardial tissues. TEM was used to image the ultrastructure of myocardial mitochondria (Fig. 5A). In the control group, the mitochondrial membrane was complete and the cristae were clear. Cardiomyocytes in the CLP-treated rats demonstrated some shrunken mitochondria with solidified mitochondrial membranes and decreased mitochondrial cristae. Pretreatment with CVB-D prevented these changes to mitochondrial ultrastructure, while CVB-D did not change the mitochondrial ultrastructure in control rats (Fig. 5A). Prostaglandin-endoperoxide synthase 2 (PTGS2) is considered a biomarker of ferroptosis (29). The significant CLP-induced increase in ptgs2 mRNA expression levels compared with controls, was significantly reversed by CVB-D pretreatment (Fig. 5B). Moreover, CVB-D attenuated sepsis-induced lipid peroxidation, as demonstrated by the significant decrease in MDA levels and the significant increase in GSH levels (Fig. 5C and D). Analysis of non-heme iron levels demonstrated that CVB-D prevented ferroptosis by inhibiting septic iron-overload (Fig. 5E). CVB-D intervention downregulated the liver hamp mRNA expression levels in CLP-treated rats (Fig. 5F). These changes indicated that CVB-D pretreatment effectively inhibited cardiac ferroptosis in septic rats. To determine whether ferroptosis was closely related to the pathophysiological processes of septic heart injury, pretreatment with Ferr-1 was applied before CLP surgery. It was subsequently demonstrated that inhibition of ferroptosis could significantly elevate both ejection fraction and fractional shortening compared with the CLP-treated group (Fig. 5G).

LTCCs and T-type calcium channels also serve a role in iron uptake into the heart (30). Therefore, the present study further analyzed the effect of CVB-D on calcium channels in septic cells. The influx of calcium ions was analyzed by detecting the fluorescence intensity of Ca²⁺ using Fluo-4 AM staining. The intracellular Ca²⁺ concentration was markedly increased after LPS stimulation compared with the control (Fig. 6A). Intracellular calcium levels were markedly reduced by CVB-D treatment.

Calcium channels are ion channels which selectively allow for the passage of calcium ions. Voltage-gated calcium channels and ligand-gated calcium channels are two categories of calcium channels. Voltage-gated channels include L-type, T-type and N-type calcium channels. LTCCs are the main source of inward calcium flow in cardiac myocytes (31).

Measurement of I_Ca-L. VER (1 µM), a specific L-type calcium current blocker, almost completely blocked LTCC, which indicated that the flowing current was Ca²⁺ current. Partial recovery of LTCC was observed after external fluid administration (Fig. 6B and C).

CVB-D inhibits I_Ca-L currents in normal and ischemic cardiomyocytes. During CVB-D (10 µM) exposure, I_Ca-L decreased significantly, by 69.43±0.42%. The partial recovery of I_Ca-L after topical solution washing indicated that the effect of CVB-D on I_Ca-L was reversible (Fig. 6D-F). The peak amplitude of I_Ca-L in CLP group ventricular myocytes was reduced by 70.22±1.98% at 10 µM CVB-D. The L-type Ca²⁺ current was partially recovered after the current was stabilized using an external solution, which indicated that CVB-D inhibited I_Ca-L in normal and post-infected rat ventricular myocytes with a partially reversible effect (Fig. 6G-I).

CVB-D concentration-dependent inhibition of I_Ca-L. The current trajectories from the test of potential depolarization from -80 mV to 0 mV under the action of various concentrations of CVB-D were analyzed (Fig. 6J). With increasing CVB-D concentrations (0.1-10 µmol/l), I_Ca-L was gradually inhibited. CVB-D (10 µM) significantly decreased I_Ca-L in normal cells and LPS-treated ventricular myocytes by increasing the concentration of CVB-D, and the time course of I_Ca-L was gradually decreased (Fig. 6K). The time dependence of CVB-D on I_Ca-L was measured (Fig. 6L). The inhibition rates of CVB-D at 0.1, 0.3, 1, 3 and 10 µM were 1.48±0.12, 14.41±0.76, 30.51±1.15, 51.09±0.96 and 72.04±1.31%, respectively.

Effect of CVB-D on the I-V relationship of I_Ca-L. The current-voltage curves of the control and 0.1, 0.3, 1, 3 and 10 µM CVB-D and VER (1 µM) treatment groups recorded during the steady-state activation procedure were measured (Fig. 7A). Potentials from -60-60 mV were measured at different treatment concentrations and a representative trace was generated. In addition, CVB-D demonstrated a marked dose-dependent effect on the current-voltage (I-V) curve (Fig. 7B).

Effects of CVB-D on the homeostatic activation and inactivation of I_Ca-L. The effect of CVB-D concentrations (1 and 10 µmol/l) on the steady-state activation caused a significant left shift and inactivation curve did not change significantly. In the I_Ca-L activation and inactivation curves, CVB-D caused a marked leftward movement. The V2/1 value/slope factor (k) of activated CON and CVB-D (1 and 10 µmol/l) were -9.685±1.774/8.880±1.631, -15.609±1.565/8.541±1.486 and -20.646±0.7345/6.1189±0.668, respectively (Fig. 7C). The V2/1/slope factor (k) values of the inactivation curves in CON and CVB-D (from 1 and 10 µmol/l) were -35.037±0.066/4.831±0.054, -36.649±0.091/5.333±0.080 and -38.624±0.262/5.712±0.241, respectively (Fig. 7D).

Recordings of the process of cardiomyocyte shortening were performed (Fig. 7E) and the effect of CVB-D treatment (10 µmol/l) on myocyte shortening was analyzed (Fig. 7F). CVB-D significantly inhibited myocyte shortening by 48.87±5.44%. After washing out, the contractility partially returned (Fig. 7G).