Hirsutine ameliorates myocardial ischemia-reperfusion injury through improving mitochondrial function via CaMKII pathway

ABSTRACT

Acute myocardial infarction (AMI) is the leading cause of death worldwide. Ischemia-reperfusion (I/R) injury is considered the most common contributor to AMI. Hirsutine has been shown to protect cardiomyocytes against hypoxic injury. The present study investigated whether hirsutine improved AMI induced by I/R injury and the underlying mechanisms. In our study, we used a rat model of myocardial I/R injury. The rats were given hirsutine daily (5, 10, 20 mg/kg) by gavage for 15 days before the myocardial I/R injury. Detectable changes were observed in myocardial infarct size, mitochondrial function, histological damage, and cardiac cell apoptosis. According to our findings, hirsutine pre-treatment reduced the myocardial infarct size, enhanced cardiac function, inhibited cell apoptosis, reduced the tissue lactate dehydrogenase (LDH) and reactive oxygen species (ROS) content, as well as enhanced myocardial ATP content and mitochondrial complex activity. In addition, hirsutine balanced mitochondrial dynamics by increasing Mitofusin2 (Mfn2) expression while decreasing dynamin-related protein 1 phosphorylation (p-Drp1), which was partially regulated by ROS and calmodulin-dependent protein kinase II phosphorylation (p-CaMKII). Mechanistically, hirsutine inhibited mitochondrial-mediated apoptosis during I/R injury by blocking the AKT/ASK-1/p38 MAPK pathway. This present study provides a promising therapeutic intervention for myocardial I/R injury.

KEYWORDS:

• Hirsutine
• myocardial ischemia-reperfusion injury
• mitochondria
• apoptosis

Introduction

Acute myocardial infarction (AMI) is one of the leading causes of disability and mortality in the world (1)). It results from endothelial thrombosis, which blocks the coronary arteries. Percutaneous coronary intervention (PCI) or thrombolysis is the major clinical treatment (2). However, coronary blood restoration aggravates tissue damage, which is called ischemia-reperfusion (I/R) injury (2). It has been reported that I/R injury accounts for up to 50% of the myocardial infarct size (3). Currently, improving myocardial resistance to reperfusion injury is a key field of research. Such approaches have the potential to improve the outcomes of patients suffering from AMI (4–6).

Mitochondria are abundant in cardiomyocytes and supply crucial energy for continuous cardiomyocyte contraction and relaxation (7). It is known that myocardial I/R injury could cause mitochondrial dysfunction, which is characterized by abnormal ATP synthesis disorder, aberrant mitochondrial dynamics, excessive permeability transition pore opening and so on (8). The disruption of mitochondrial dynamics leads to mitochondrial fragmentation (9). Fragmented mitochondria are potential sources of reactive oxygen species (ROS), cytochrome C, and mitochondrial DNA, which lead to cell death (10). Previous research has demonstrated that blocking mitochondrial fission could significantly lessen the infarct size of myocardial I/R injury (11,12). Thereafter, research into mitochondrial protection may become a new therapeutic focus for heart reperfusion injury.

Uncaria rhynchophylla, a member of the Uncaria genus of the family Rubiaceae, is one type of traditional Chinese herbal medicine, and it has been used extensively to treat cardiovascular diseases in the long history of China (13,14). Previous studies have confirmed that the total effective rate of the extracts from Uncaria rhynchophylla in patients with mild to moderate hypertension is 83% (13). In addition, the extracts have been reported to prevent myocardial I/R injury by reducing infarct size and alleviating oxidative stress (15). Hirsutine, one of the principal Uncaria extracts, has been demonstrated to protect neonatal rat cardiomyocytes against hypoxic injury by reducing oxidative stress and cell apoptosis (15). However, whether hirsutine is involved in the protective effects of myocardial I/R injury is still unknown. Consequently, it is necessary to further explore the capacity of hirsutine to regulate myocardial I/R injury.

Thereafter, the present study investigated whether hirsutine protected the myocardial I/R model and explored the underlying mechanisms. We provided evidence that hirsutine protected the heart against I/R injury by improving mitochondrial dysfunction, which was regulated by reactive oxygen species (ROS) and calmodulin-dependent protein kinase II phosphorylation (p-CaMKII). The underlying mechanism was related to the inhibition of the AKT/ASK-1/p38 MAPK-mediated apoptosis pathway.

Materials and methods

Reagents

Hirsutine was purchased from Must Biotechnology Company, Chengdu, China. The TUNEL Cell Apoptosis Detection Kit-POD was obtained from Boster, Wuhan, China. Hematoxylin and eosin (H&E) staining kit, Western and IP cell lysates, ROS detection kit and ATP content assay kit were obtained from Beyotime, Jiangsu, China. Kits for detecting the activity of mitochondrial complex I ~ IV were purchased from Solarbio, Beijing, China. The MitoSOX red for measurement of mitochondrial ROS was from Invitrogen, Waltham, MA, USA. TTC dyeing and kits for measurement of LDH, SOD, and MDA were obtained from the Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Evans blue, succinate dehydrogenase activity kit and succinate kit were purchased from Sigma Aldrich, St Louis, MO, USA. Recombinant anti-DRP1 antibody (ab184247), recombinant anti-DRP1 (phosphor S637) antibody (ab193216), recombinant anti-Mitofusin 2 antibody (ab124773), recombinant anti-AKT (phosphor T308) antibody (ab38449), recombinant anti-CaMKII alpha + CaMKII beta (phosphor T286) antibody (ab124880), recombinant anti-p38 antibody (ab170099), recombinant anti-p38 (phosphor T180 + Y182) antibody (ab4822), Goat Anti-Rabbit IgG H&L (HRP) (ab205718) and goat anti-mouse IgG H&L (HRP) (ab205719) were purchased from Abcam, Cambridge, United Kingdom. Rabbit Anti-CAMK2A + CAMK2B + CAMK2D antibody (bs-0541 R), Rabbit Anti-AKT1 + 2 + 3 antibody (bs-6951 R), Mouse Anti-Bax antibody (bs-0127 M), Rabbit Anti-Bcl-2 antibody (bs-0032 R), Rabbit Anti-ASK1 antibody (bs-1425 R), Rabbit Anti-phospho-ASK1 (Thr845) antibody (bs-3031 R) and Mouse Anti-Active Caspase-3 antibody (bsm -33 199 M) were obtained from Bioss, Beijing, China. Beta-actin antibody was purchased from Sino Biological, Beijing, China.

Animals

SD male rats aged 8–10 weeks (280–320 g) were purchased from Animal Experimental Center of Xinjiang Medical University and kept in a SPF room where is 23 ± 3°C and of 40–70% humidity under a 12-h light/dark cycle. All rats were supplied with a standard rodent diet and were free to access water. All procedures were approved by the Committee on Animal Care of the Xinjiang Medical University.

The candidate dosage of hirsutine was as previously described (16). Thirty rats were randomly divided into five groups: 1. the sham operation group (sham); 2. the I/R group (I/R); 3. the I/R group treated with a low dose of hirsutine (5 mg/kg, I/R + hirsutine); 4. the I/R group treated with a medium dose of hirsutine (10 mg/kg, I/R + hirsutine); 5. the I/R group treated with a high dose of hirsutine (20 mg/kg, I/R + hirsutine). I/R + hirsutine groups were treated with hirsutine intragastrically once a day for 15 consecutive days, while the sham and I/R groups were treated with normal saline. After 10 minutes from the last intragastric administration, the sham operation or myocardial I/R injury was induced.

I/R surgery

The rat model of myocardial I/R injury was carried out in accordance with the published protocol (4,17). Briefly, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg). All the rats underwent tracheotomy and tracheal intubation. A polyvine-50 (PE-50) tube was inserted into the trachea and connected to a rodent ventilator with a tidal volume of 1.2 L/kg and a respiration rate of 70/min for ventilation. The body temperature was maintained at 37°C, and the heart rate and cardiac electrophysiological activity were observed by electrocardiogram. After thoracotomy, the left coronary artery was ligated and encircled with a 6/0 silk suture slipknot for 45 min, followed by 120 min of reperfusion. The sham group underwent the same surgical procedures without LAD ligation. We confirmed successful occlusion by the color change of the vessel (from light red to dark violet). In addition, the color of the myocardium supplied by the LCA changed from bright red to pale and the connected ECG showed ST-segment elevation.

Evans blue/TTC staining

The extension of myocardial infarction was evaluated by Evans blue perfusion and TTC staining, as previously reported (18,19). Blood samples were collected from rats in each group after 4 h of reperfusion. 2 ml of 2% Evans blue dye were injected into the aorta. After 1 min of reperfusion, all rats were sacrificed and the hearts were then isolated, washed, and frozen at −20°C for 1 hour, wrapped in plastic. The hearts were chopped into 2 mm-thick slices in a direction parallel to the atrioventricular sulcus. The area of myocardial infarction was detected by TTC staining. Images were acquired with the Image-Pro Plus 6.0 software, discriminating between healthy areas (blue) from the area at risk (AAR, dark red) and the infarcted size (IS, while). The ratio of AAR/LV (left ventricle) reflects the degree of myocardial ischemia, while infarct size (IS)/AAR reflects the level of dead myocardium.

Measurement of cardiac function

The cardiac structure and function of rats in each group were evaluated by an ultrasound imaging system before the animals were sacrificed. The left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), left ventricular end systolic diameter (LVEsD) and left ventricular end-diastolic diameter (LVEDD) were calculated. Image acquisition and analysis were performed by a technician who was blinded to the grouping scheme.

Myocardial ATP content and mitochondrial complex activity detection

Following the manufacturer’s instructions, an ATP assay kit based on firefly luciferase was used to determine the ATP content of the myocardium. Meanwhile, the Micro Mitochondrial Respiratory Chain Complex I, II, III or IV Activity Assay Kits were used to measure the activity of complexes I~IV in the mitochondrial electron respiratory chain.

Cellular and mitochondrial ROS detection

For detection of the superoxide anions in cellular and mitochondria, myocardium was isolated into single cells and tagged with 10 M DCFH-DA and 5 M MitoSOX Red at 37°C for 1 h. according to the manufacturer’s instructions. DCFH-DA/MitoSOX-labeled cardiomyocytes were collected after being centrifuged for 10 min at 1000 g. The absorbance was measured with a microplate reader (Bio-Rad xMarkTM Microplate Absorbance Spectrophotometer, Hercules, USA).

TUNEL staining

TUNEL staining was performed with the TUNEL Cell Apoptosis Detection Kit-POD according to the manufacturer’s instructions. Slides were finally incubated with DAPI to visualize all nuclei. The Nikon E200 fluorescent photomicroscope (Nikon E200, Tokyo, Japan) was used to capture fluorescence images and the percent of TUNEL-positive cells was calculated.

Histological analysis

The hearts were fixed in 4% paraformaldehyde overnight and then dehydrated in ethanol. Thereafter, the samples were clarified in xylene and embedded in paraffin. Subsequently, samples were cut 5 μm apart transversely and stained with an H&E staining kit. The images were captured by the Nikon E200 fluorescent photomicroscope (Nikon E200, Tokyo, Japan).

Western blotting

Tissues were lysed with Western and IP cell lysates containing protease inhibitor cocktails. After centrifugation at 12 000 rpm for 15 min 4°C. Tissue homogenates (40 mg of protein) were separated by 10–12% SDS-PAGE and transferred onto nitro-cellulose membranes. The blots were then washed with tris-buffered saline with Tween 20 (TBST), blocked with 5% milk powder in TBST buffer for 1 hour at room temperature, and incubated with the recombinant anti-DRP1 (phosphor S637) antibody (1: 1,000), anti-DRP1 antibody (1: 1,000), recombinant anti-Mitofusin 2 (1: 1,000), recombinant anti-DDIT3 antibody (1: 1,000), recombinant anti-pan-AKT (phosphor T308) antibody (1: 1,000), recombinant anti-CaMKII alpha + CaMKII beta (phosphor T286) antibody (1: 1,000), recombinant anti-p38 antibody (1: 1,000), recombinant anti-p38 (phosphor T180 + Y182) antibody (1: 1,000), Rabbit Anti-CAMK2A + CAMK2B + CAMK2D antibody (1: 1,000), Rabbit Anti-AKT1 + 2 + 3 antibody (1: 1,000), Mouse Anti-Bax antibody (1: 1,000), Rabbit Anti-Bcl-2 antibody (1: 1,000), Rabbit Anti-ASK1 antibody (1: 1,000) and Rabbit Anti-phospho-ASK1 (Thr845) antibody (1: 1,000) at 4°C overnight. The next day, the membranes were washed and primary antibodies were detected with goat anti-Rabbit IgG H&L (HRP) and goat anti-mouse IgG H&L (HRP). The bands were visualized by the Chemiscope 3,000 (CliNX Science Instruments, Shanghai, China) and the images were quantified by ImageJ software.

Biochemistry indicators measurement

After reperfusion, the arterial blood samples collect. The levels of serum MDA, SOD, and LDH were measured by corresponding assay kits. The levels of succinate in heart tissues were detected by assay kits. The levels of SDH in mitochondria were measured by the Succinate Dehydrogenase Activity Colorimetric Assay Kit. All biochemical indicators were tested in accordance with the manufacturer’s instructions. The absorbance was measured with a microplate reader.

Statistical analysis

The data were presented as mean ± standard deviation (SD) values. The data were analyzed using GraphPad PRISM software version 5.0 (GraphPad Software, Inc., San Diego, CA, United States) and SPSS 19.0 software (SPSS Inc., Chicago, IL, United States). Statistical significance was determined by the ANOVA test, followed by Sidak’s multiple comparisons test for a multi-group (>2) comparison. Differences were considered statistically significant when P < .05 as specified in the figure legends.

Result

Effect of hirsutine on the ratio of infarct size area after I/R injury

To evaluate the effect of hirsutine on myocardial I/R injury, C57BL/6J mice were given hirsutine via gavage for 15 consecutive days at doses of 5, 10, or 20 mg/kg B.W/day before the myocardial I/R injury (Figure 1a). Evans blue/TTC staining was used to evaluate myocardium infarct size after I/R injury in rats (Figure 1b). The ratio of AAR/LV was used to represent the degree of myocardial ischemia, and it was maintained at a relatively constant level between I/R groups (Figure 1c). Then the ratio of IS/AAR was calculated, which reflected the level of dead myocardium. We found that hirsutine reduced the infarct size compared with the I/R group in a dose-dependent manner (Figure 1d). These results above demonstrate that hirsutine reduces the myocardial infarct size after I/R injury.

Figure 1. Effect of hirsutine on the ratio of infarct size area after I/R injury. (a) Schematic diagram of IR-induced cardiac injury (I/R: ischemia-reperfusion). (b) Evans blue/TTC staining was used to detect the infarct size of myocardial I/R rats (n = 3). (c) AAR/LV reflects the degree of ischemia, (d) IS/AAR reflects the degree of infarction (AAR: area at risk, LV: left ventricle, IS: infarcted size, n = 6). Data are presented as mean ± SD. *P < .05 vs. Sham group and #P < .05 vs. I/R group.

Effect of hirsutine irsutine on the cardiac function and pathological damage after I/R injury

To further identify the protective effect of hirsutine on myocardial I/R injury, H&E staining was first performed. The results showed 20 mg/kg hirsutine pre-treatment reduced the degree of myocardial cell edema, fracture, necrosis and interstitial inflammatory cell infiltration (Figure 2a). Besides, echocardiography results demonstrated that the LVEF and LVFS were remarkably higher in the 20 mg/kg group compared with the I/R group (Figure 2b-d). Meanwhile, hirsutine pre-treated with 10 mg/kg and 20 mg/kg essentially alleviated the increased LVEsD and LVEDD during I/R injury (Figure 2e,f). Overall, these results indicate the protective effect of hirsutine on cardiac function and pathological damage after I/R injury.

Figure 2. Effect of hirsutine on cardiac function and pathological damage after I/R injury. (a) Histopathological changes in the I/R-injured heart of rats pre-treated with or without hirsutine (I/R: ischemia-reperfusion, scale bars, upper: 200 μm, lower: 50 μm). (b) Representative echocardiographic images. (c-f) the effect of hirsutine on the left ventricular injection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic dimension (LVEsD) and left ventricular end-diastolic dimension (LVEDD) after myocardial I/R injury (n = 6). Data are presented as mean ± SD. *P < .05 vs. Sham group and #P < .05 vs. I/R group.

Effect of hirsutine on the oxidative stress and apoptosis after I/R injury

Advances in our knowledge of the complicated mechanisms involved in the etiology of myocardial I/R injury point to the central role of oxidative stress (20). Thus, we first assessed the effect of hirsutine on the myocardial oxidative stress indicators. The results showed that the levels of MDA, SDH, succinate and tissue ROS content were upregulated during I/R injury, which were all downregulated in a way that was dependent on dosage (Figure 3a-d). SOD, by contrast, was decreased in I/R injury while increasing by hirsutine in 10 mg/kg and 20 mg/kg groups (Figure 3e). Besides, our results demonstrated that hirsutine inhibited the upregulated LDH level in a dose-dependent manner (Figure 3f). Moreover, TUNEL staining results revealed that 20 mg/kg hirsutine pre-treatment decreased the apoptotic cells upon I/R injury (Figure 3g,h). These results suggest that hirsutine protects myocardial I/R injury through anti-oxidation and anti-apoptosis.

Figure 3. Effect of hirsutine on the apoptosis and oxidative stress after I/R injury. (a) the concentration of ROS in heart tissues (I/R: ischemia-reperfusion, n = 6). (b) the concentration of MDA in serum (n = 6). (c, d) the concentrations of succinate and SDH in heart tissues (n = 6). (e, f) the concentrations of SOD and LDH in serum (n = 6). (g, h) Representative TUNEL staining images and the statistical analysis automatically counted by the ImageJ software (scale bars = 50 μm, n = 6). Data are presented as mean ± SD. *P < .05 vs. Sham group and #P < .05 vs. I/R group).

Effect of hirsutine on the mitochondria dysfunction in I/R Injury

It has been shown that mitochondrial dynamics play a crucial role in regulating mitochondrial functions during I/R injury (21). In our study, ATP content and mitochondrial respiratory complex (I~IV) activity were reduced following I/R damage, whereas mitochondrial ROS production was increased, which was all reversed in the 10 mg/kg and 20 mg/kg hirsutine groups (Figure 4a-f). Furthermore, Drp1 phosphorylation for fission was upregulated and MFN2 for fusion was downregulated in I/R injury and the two indicators were reversed by hirsutine in the 10 mg/kg and 20 mg/kg groups. Recent evidence reveals that CaMKII phosphorylation regulates mitochondrial dynamics (16,22,23). Thus, we detected CaMKII phosphorylation and found that it was upregulated in I/R injury while downregulated significantly in 10 mg/kg and 20 mg/kg groups (Figure 4f-j). These data imply the protective role of hirsutine in mitochondria dysfunction in myocardial I/R injury.

Figure 4. Effect of hirsutine on the mitochondria dysfunction in I/R injury. (a) the ATP level in heart tissue (I/R: ischemia-reperfusion, n = 6). (b-e) the activities of mitochondrial complex I~IV (n = 6). (f) Mitochondrial ROS levels were detected by staining with MitoSOX Red (n = 6). (g) Representative images of Western blots results. Hirsutine increased the p-DRP1/DRP1 (h) and p-CaMKII/CaMKII (i) protein expressions, while decreased the MFN2 (j) protein expression after I/R injury in myocardial tissue. The immunoblots were calculated by densitometric analysis using β-actin as the internal reference (n = 3). Data are presented as mean ± SD. *P < .05 vs. Sham group and #P < .05 vs. I/R group.

Effect of hirsutine on apoptosis induced by the AKT/ASK-1/p38 MAPK pathway in I/R injury

To further understand the protective effect mechanisms of hirsutine, the Akt/ASK-1/p38 MAPK pathway was assessed since it has been shown to be involved in mitochondrial apoptosis (24,25). We observed the increased phosphorylation of AKT, ASK-1 and p38 in I/R injury, while these molecules were all decreased in the 10 mg/kg and 20 mg/kg groups. In addition, the anti-apoptotic protein B-cell lymphoma-2 (BCL-2) was downregulated while pro-apoptotic BCL2-associated X (BAX) and active caspase-3 were upregulated during I/R injury, which were reversed in the 10 mg/kg and 20 mg/kg groups (Figure 5a-g). Therefore, hirsutine depends on activating the AKT/ASK-1/p38 MAPK pathway to inhibit apoptosis in myocardial I/R injury.

Figure 5. Effect of hirsutine on apoptosis induced by the AKT/ASK-1/p38 MAPK pathway in I/R injury. (a) Representative images of Western blots results (I/R: ischemia-reperfusion). Hirsutine increased the p-AKT/AKT (b), p-ASK/ASK (c), p-P38/P38 (d), BAX (e) and active caspase-3 (g) protein expressions, while decreased the BCL-2 (f) protein expression after I/R injury in myocardial tissue. The immunoblots were calculated by densitometric analysis using β-actin as the internal reference (n = 3). Data are presented as mean ± SD. *P < .05 vs. Sham group and #P < .05 vs. I/R group.

Discussion

Coronary heart disease (CHD) is the leading cause of death and disability worldwide (1). According to the World Health Organization, 8.9 million deaths worldwide (16% of global deaths) resulted from CHD in 2019 (26). Myocardial I/R injury typically arises in patients presenting with an acute ST-segment elevation myocardial infarction (STEMI), and in these patients, the most effective therapeutic intervention is timely and effective myocardial reperfusion therapy (27). However, treatment for STEMI patients undergoing reperfusion therapy to prevent fatal myocardial reperfusion injury is limited and narrowed to symptomatic and supportive care (28). Hirsutine, extracted from Uncaria rhynchophylla, has been shown to have a protective effect on hypertension and cardiomyocyte hypoxic injury in previous studies (15,29). However, the role of hirsutine in myocardial I/R injury has not been reported. In our study, hirsutine was treated for 15 days by gavage before inducing the I/R model. Our results showed that hirsutine dramatically reduced myocardial infarct size in a dose-dependent manner. Besides, 20 mg/kg hirsutine pre-treatment significantly improved the destruction of tissue and the infiltration of inflammatory cells caused by I/R injury. Furthermore, 20 mg/kg hirsutine could improve the reduction of LVEF, LVFS and the upregulation of LVEDD and LVEsD during I/R injury, which were consistent with the echocardiography results in myocardial I/R injury of previous studies (30–32). Therefore, we conclude that hirsutine could improve myocardial tissue structure and reduce infarct size after I/R injury, thereby improving cardiac function.

Hirsutine had protective effects on cardiomyocytes against hypoxic injury in previous study, which is related to its anti-oxidant and anti-apoptotic characteristics (15). Our study also indicated that hirsutine played a protective role in inhibiting apoptosis in I/R injury. Hirsutine decreased the apoptotic cells in cardiac tissue and also drastically reduced the classical apoptotic pathway (BAX, BCL-2 and active caspase-3) in I/R injury. Mitochondria are an intracellular energy source as well as a key regulator of cell death (33). A balance between mitochondrial biogenesis, fission, fusion, and mitophagy is necessary for mitochondria to maintain homeostasis, which is a network of mechanisms that control ATP production (34,35). It is widely accepted that a precursor to myocardial I/R injury is mitochondrial dysfunction (36,37). In our research, we found hirsutine restored the ATP content and the mitochondrial respiratory complex (I~IV) activity, while reduced the mitochondrial ROS levels during I/R injury to rebalance the mitochondrial function. In addition, mitochondrial dysfunction is caused by an imbalance of the fission and fusion ratios in myocardial I/R injury (12). As expected, we found hirsutine decreased DRP1 phosphorylation while increasing MFN2 expression in myocardial I/R injury. Furthermore, AKT is always activated in response to mitochondrial dysfunction (38), which regulates a number of downstream apoptotic pathways, including ASK-1/p38 MAPK (39–41). Similarly, our results showed hirsutine dramatically prevented apoptosis mediated by the AKT/ASK-1/p38 MAPK signal pathway during I/R injury. Summarily, hirsutine reduces cell apoptosis by stabilizing mitochondrial function and therefore inhibiting the AKT/ASK-1/p38 MAPK pathway.

Oxidative stress is caused by the overproduction of ROS and the relative deficiency of antioxidants in myocardial I/R injury (42). In the early stages of I/R injury, ROS generation and release magnify the signal via cytokines, leading to an increase in intracellular Ca²⁺ (43). Subsequently, CaMKII is phosphorylated at the autophosphorylation site of the regulatory domain when Ca²⁺ is elevated (44). The activated CaMKII then promotes calcium leakage and results in mitochondrial dysfunction, while the intracellular ROS content is further increased by the mitochondrial ROS produced by succinate oxidation (45,46). In our study, we found that hirsutine reduced the CaMKII phosphorylation in I/R injury. And we discovered that I/R damaged tissues had lower ROS levels after receiving hirsutine pre-treatment. Meanwhile, succinate and SDH levels were all decreased in the presence of hirsutine. These data suggest that hirsutine improves mitochondrial dysfunction by inhibiting CaMKII activation, reducing tissue oxidative stress levels to protect against myocardial I/R injury.

Conclusion

Our results indicate that hirsutine improved I/R-induced cardiac dysfunction by regulating the balance of mitochondrial dynamics, partially through inhibiting ROS generation and CaMKII activation. The underlying mechanism of hirsutine may be related to the inhibition of the AKT/ASK-1/p38 MAPK-mediated apoptosis pathway. These findings provide a new sight into the treatment of myocardial I/R injury.

Author contributions

WJ, YZ and JC designed the study. WJ, YZ, WZ and JC wrote the manuscript. WJ and JC acquired funding for the study. WJ, YZ, WZ, XP, JL and QC acquired and analyzed the data.

Availability of data and materials

The data sets used and/or analyzed during this study are available from the corresponding author on reasonable request.

Acknowledgments

The authors acknowledge that the Central Laboratory of Xinjiang Medical University has provided the professional assistance in ultrasound imaging system.

Disclosure statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Additional information

Funding

The National Natural Science Foundation: Mechanisms of the Protective Effects of Tilianin Against Myocardium Ischemia/Reperfusion Injury Through Ox-CaMKII-mediated Mitochondrial Regulating Pathway (No. 81760045).