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Rhodiola pre-conditioning reduces exhaustive exercise-induced myocardial injury of insulin resistant mice

Myocardial injury reduction and recovery under acute cardiac stress are adversely impacted by insulin resistance (IR). We previously demonstrated that Rhodiola improved cardiac anti-stress capacity in mice. Thus, this study focuses on the preventive efficacy of Rhodiola on exhaustive exercise (EE)-induced myocardial injury of IR mice. An 8-week high-fat diet (HFD) model of IR mice was established. Rhodiola was administrated by garaging. After the 8-week intervention, half of the mice performed EE to simulate acute cardiac stress, and determine myocardial injury; The remaining mice were sacrificed following fasting to assess metabolic disorder. We found myocardial injury induced by EE in IR mice was worse and was alleviated by Rhodiola pre-conditioning. Further, the nuclear factor erythroid 2-related factor 2 (Nrf2)-related antioxidant system was impaired by HFD, while mitochondrial dynamic fusion and fission were activated by HFD as a physiological protective compensation. The Rhodiola administration rescued Nrf2 impairment and further facilitated mitochondrial fusion and fission. All these results indicate that Rhodiola is a potential treatment for the prevention of cardiac events in type 2 diabetes mellitus and metabolic syndrome patients, and the Nrf2-related antioxidant activity and mitochondrial dynamics are the proposed mechanisms.

www.nature.com/scientificreports/ diabetic nephropathy 14 . All these indicate the correlation between mitochondrial dynamic and ROS is critical in maintaining mitochondrial hemostasis.
In the myocardium of diabetic mice, mitochondrial dynamic disorder, as well as increased oxidative stress has been observed 15 , which could be one of the reasons behind poor cardiac anti-stress capacity in IR patients. However, further research is needed to explore its mechanisms and to develop efficient therapies.
Rhodiola (RS), a natural herb historically used to improve body hypoxia resistance at high altitudes 16 , has been shown to be a mitochondrial protectant 17,18 . Our previous study found Rhodiola enhances mitochondrial quality including mitochondrial dynamic, to increase cardiac anti-stress capacity in healthy mice 5 . However, whether this improvement can be extended under IR conditions and its underlying mechanism remains unclear. Recently, Rhodiola was found to be a Nuclear factor erythroid 2-related factor 2 (Nrf2) activator, which enhances the antioxidant system to exert neuroprotective effects 19 or relieve hypoxia-induced liver injury 20 . Thus, we hypothesize that Rhodiola activates Nrf2 and the downstream antioxidant system to regulate mitochondrial dynamic homeostasis, finally strengthening cardiac anti-stress capacity under IR conditions.
In this study, a high-fat diet (HFD) mice model was established to simulate the vulnerability to cardiac stress in patients with IR, and exhaustive swimming exercise was used to simulate acute cardiac stress to induce myocardial injury. Then, we investigate Rhodiola pre-conditioning's effect on the cardiac anti-stress capacity of HFD mice and explore the role of the Nrf2 signaling pathway.

Results
Rhodiola reduces exhaustive exercise-induced myocardial mitochondrial injury of HFD Mice. After adaptive feeding, the mice were fed a normal diet (ND) or HFD with/without Rhodiola administration, then the mice underwent exhaustive swimming or not. The animal protocol is further described in Fig. 1A. We found the forced swimming time had decreased in the HFD group compared to ND. However, when accompanied by Rhodiola treatment, the mice following HFD demonstrated a comparable swimming time to that of the ND group (Fig. 1B). Following exhaustive swimming, myocardium damage was subsequently evaluated by serum creatine kinase (CK), blood urea nitrogen (BUN), and electron microscopy. We found that serum CK and BUN were increased in HFD mice (Fig. 1C,D). In parallel, damaged mitochondrial with edema and ruptured cristae (yellow arrows) in the myocardium were observed with greater abundance in HFD mice (Fig. 1E,F). However, neither the morphologic alteration in myocardium nor CK and BUN release occurred in HFD + RS groups (Fig. 1C-F), suggesting that Rhodiola prevents EE-induced cardiac mitochondrial injury in HFD mice.
Rhodiola relieves the insulin resistance of HFD mice. The HFD mouse model was characterized by dyslipidemia, which indicates lipotoxicity and energy oversupply. An 8-week HFD led to increased weight gain , and serum creatine kinase (CK) of mice were measured after EE; (E) The morphology of myocardium after EE was observed by transmission electron microscope (TEM), intact mitochondria (the letter m), as well as damaged mitochondrion (yellow arrows), were indicated in images, scale bar = 2 μm; (F) The ratio of damaged mitochondria number, which have ruptured cristae and edema, to total mitochondrial number per field-of-view was analyzed. Data are expressed as Mean ± SD, n = 6, *, ** represent P < 0.05, P < 0.01 in comparison with ND; #, ## represent P < 0.05, P < 0.01 in comparison with HFD. www.nature.com/scientificreports/ compared to ND mice, while Rhodiola administration restrained this tendency in HFD mice even comparable to that in ND ( Fig. 2A). In-keeping with the bodyweight result, visceral fat weight in HFD mice also increased, while Rhodiola prevented this change (Fig. 2B). As for glucolipid metabolism, we found serum cholesterol, triglyceride, low-density lipoprotein (LDL), blood glucose, and insulin increased in HFD mice, in which the HOMA-IR index also increased. However, Rhodiola administration prevented these changes in HFD mice ( Fig. 2C-H).
Rhodiola activates Nrf2-related antioxidants in HFD mice. Considering the close correlation between oxidative stress and acute exercise stress in the myocardium, we evaluated oxidative stress and Nrf2related antioxidant signaling. As shown in Fig. 3A,B, the activity of SOD2 decreased in HFD mice, while the content of malondialdehyde (MDA) increased, indicating oxidative stress. Rhodiola administration alleviated HFD-induced oxidative stress. To investigate the role of Nrf2 signaling, we assessed the protein expression of Nrf2 and its downstream HO-1. Our results showed that Rhodiola improved inhibited Nrf2/HO-1 pathway in HFD mice (Fig. 3C,D). Protein expression of GPX4 and SOD2 exhibited a similar tendency (Fig. 3E).
Rhodiola prevents mitochondrial dynamics disorder in high-fat diet mice. Mitochondrial dynamic plays a vital role in maintaining the mitochondrial function, which supplies energy for cardiac contractile function. To evaluate the degree of fusion and fission in the myocardium, we assessed fusion factors MFN1, MFN2, and OPA1, as well as fission factors DRP1 and FIS1 expression. As shown in Fig. 4A-C, mitochondrial fusion and fission were both activated in HFD mice. This might be a stress cellular condition due to HFD. However, Rhodiola administration changed this: mitochondrial fusion was further enhanced in HFD + RS mice, while fission was prevented. Moreover, the ratio of mitochondrial DNA and ATP content was assessed to evaluate mitochondrial content and function. We found that mitochondrial DNA and ATP content decreased in the myocardium of HFD mice when compared with ND mice, Rhodiola treatment reversed this (Fig. 4D).

Discussion
This study aimed to illustrate the mechanism through which Rhodiola improves cardiac anti-stress capacity impaired by HFD in an IR mice model. Our results showed that an HFD increases EE-induced cardiac injury in mice, inhibits Nrf2 signaling regulated antioxidant enzyme, and triggers mitochondrial dynamic disorder. However, all these were alleviated by Rhodiola pre-conditioning. These findings suggest that Rhodiola is a potential clinical therapy for IR patients to improve cardiac anti-stress capacity. www.nature.com/scientificreports/ There are several methods to stimulate cardiac stress, including myocardial infarction, ischemia/reperfusion, chemical drugs, and EE. In comparison with other models, EE would better represent a complicated and changeable condition in clinical settings. Thus, we investigated anti-stress capacity in an EE model. Research by Behringer and colleagues has demonstrated that mitochondrial dysfunction due to EE leads to muscle injury characterized by increased oxidative stress, cytoskeletal damage, and various markers 21 . In addition, it has been demonstrated that EE regulates the Bcl-2 family via PI3K/AKT signaling pathway to decrease the openness level of mPTP, consequently resulting in myocardial injury 22 . Similar results were observed in our previous study, where we found Rhodiola increased weight-load swimming time and reduce cardiac injury after EE in normal mice, indicating that Rhodiola enhanced cardiac anti-stress capacity 5 . We previously demonstrated that oxidative stress remission and mitochondrial quality control adaptation, including fusion and fission, activated by Rhodiola, were the target mechanisms.
Clinical research has demonstrated that the exercise capacity of patients with IR and type 2 diabetes mellitus (T2DM) is limited 23 , while further evidence has established poor exercise capacity as an independent and strong predictor for all-cause mortality 24 . In this study, HFD mice presented decreased exercise capacity and worsened cardiac injury post-EE, indicating worse cardiac anti-stress capacity, which was alleviated following Rhodiola treatment. In skeletal muscle, another important target organ of IR, HFD mice have also been found to be worse affected than normal mice 25 . Noticeably, the inhibitor of NADPH oxidase, which is an important resource of ROS, relieved HFD-impaired mitochondrion function and exercise capacity. However, the role of disorder in the physiological antioxidant system is not fully known.
Rhodiola and its active constituent salidroside were found to be a natural antioxidant that protects mitochondria against injury in multiple diseases 18,26 . Nrf2, one of the targets of Rhodiola, is a key regulator of the cellular antioxidant response 19 . Besides oxidative stress, the activity of Nrf2 is also regulated by energy-based stimuli, thus, HFD inhibits Nrf2 and impairs the antioxidant system 27 . In this study, we found the expression of Nrf2 and downstream HO-1 were inhibited in HFD mice, while the antioxidant enzyme SOD2 and GPX4 were also inhibited, and the lipid peroxidation product MDA had increased. This implies the presence of a disorder of the antioxidant system in the myocardium of HFD mice which may contribute to the impaired cardiac anti-stress www.nature.com/scientificreports/ capacity. As expected, we found Rhodiola treatment redeemed the Nrf2-regulated antioxidant system and reduced oxidative stress. Future studies may focus their attention on structure optimization of salidroside to improve the targeting ability or pharmaceutical effect 28 .
In consideration of the coadjustment of oxidative and mitochondrial dynamics, we assessed the mitochondrial fusion and fission levels to further illustrate the mechanism behind Rhodiola's protective effect. Fusion is a vital process preserving ATP production and ROS dissipation. Muscle from obese or T2DM patients show a reduced degree of fusion 29 . However, our results show that mitochondrial fusion marker MFN1, MFN2, and OPA1 increased in HFD mice after EE. Concerning the fission caused by damaged mitochondria, it was enhanced in response to HFD and EE stress in this study. We presume this discrepancy is a result of the difference in tissue type or pathological staging, as the fusion and fission level in this study was activated as physiological compensation for mitochondrial quality control and cardioprotection 30 . Futhermore, we found Rhodiola treatment, which improved ATP content in HFD mice myocardium, further facilitated mitochondrial fusion while inhibiting fission. This indicates that 8-week Rhodiola pre-intervened mice did not exhibit such severe injury in mitochondrial after EE or/and this injury was relieved by the self-protective quality control process including fusion. However, further studies with specific inhibitor administration or gene conditional knockout mice are needed to explore this issue.
In summary, this study used an EE mice model to simulate cardiac stress and explore Rhodiola's effect on the HFD-impaired cardiac anti-stress capacity in mice: Rhodiola activates Nrf2 signal and downstream antioxidant system and improves cardiac anti-stress capacity via regulating mitochondrial dynamic disorder, including fusion and fission (Fig. 5). The results conclude that Nrf2-mediated antioxidant enzyme and mitochondrial dynamic are the targets of Rhodiola, which may be a potential treatment to prevent cardiac event for T2DM and MS patients. After 1-week adaptive feeding, mice were randomly divided into three groups (n = 16 for each): normal diet (ND), high-fat diet (HFD), and HFD + Rhodiola (HFD + RS). In HFD feeding 45% of total calories came from fat, while in ND feeding this figure is 18%. Mice were administrated with the same quantity of Rhodiola solution (5 mg/0.1 mL/10 g body weight) or normal saline (0.1 mL/10 g body weight) as a placebo by gavage every day for 8 consecutive weeks.
After 8 weeks, mice in each group were further divided into two subgroups: with or without an exhaustive exercise (EE) test (n = 8). Mice in the non-EE group were anesthetized via 1% pentobarbital sodium (150 mg/kg, i.p.) and then sacrificed via exsanguination after fasting, and collect blood samples from the inferior vena cava for lipid profile assays, visceral fat including mesenteric, epididymal, and perirenal fat tissue were dissected and weighted; Mice in EE subgroups underwent an exhaustive exercise as previously described 5 , while the researchers are blind to the groups. 12 h after the experiments, mice were anesthetized with 150 mg/kg pentobarbitone by intraperitoneal injection and then sacrificed, blood and myocardium samples were then collected. This study was conducted under the ARRIVE guidelines and any unnecessary animal suffering was avoided. The protocol has been described in Fig. 1A. Rhodiola. The extract from the root of Rhodiola chrysanthemifolia subsp. sacra (Raym. -Hamet) H. Ohba (Rhodiola, RS) was provided by the Tibet Rhodiola Pharmaceutical Holding Company. The extract complied with The Chinese Pharmacopeia 2015 (inspection report number C1051612067). The extraction method and the HPLC-MS analysis were described previously 31 . Briefly, the root of R.sacra was decocted twice for 1.5 h and 1 h respectively. The decoction was collected and dehydrated to powder after filtration. HPLC-MS analysis showed the proportion of main effective components, salidroside (C14H20O7, 2.62%) and flavone (C27H30O16, 3.27%). This powder was dissolved in distilled water at 50 mg/mL. Lipid profile assays. Serum was isolated from fasting blood by centrifugation at 4000 rpm for 10 min. The supernatants were collected and stored at -80 °C. Serum levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol were measured by the enzymatic method on an automatic biochemical analyzer (Beckman, IN, USA). Exhaustive exercise test. After the 8-week intervention, forced weight-loaded swimming was performed as an exhaustive exercise (EE) test to assess cardiac anti-stress capacity 5,31 . Briefly, mice were forced to swim with a lead sheath of 5% of body weight till exhaustion, defined as the failure to rise above the surface of the water for 7 s.

Creatine kinase (CK), and blood urea nitrogen (BUN
Mitochondrial DNA ratio. The protocol was described previously 31 . Total DNA was isolated from myocardium tissue using a DNeasy Kit (Qiagen, GER). The succinate dehydrogenase complex subunit A (SDHA) and MtCO3 oligos were analyzed to evaluate the quantification of nuclear and mitochondrial genomes. The primers' sequences are listed in Table 1.
Adenosine triphosphate (ATP) content. The ATP content in the myocardium was determined via the phosphomolybdic acid colorimetric method (Jiancheng). Procedures were performed according to the manufacturer's instructions.
SOD2 and Malondialdehyde (MDA) assay. The xanthine oxidase method was used to determine the activity of SOD2 in myocardium tissues, and the thiobarbituric acid method was used to detect the content of MDA according to the manufacturer's instructions respectively (Jiancheng).
Statistical analysis. Statistical analyses were performed by Prism 6 software (GraphPad, Inc.).The results are expressed as Mean ± SD. One-way ANOVA plus the Student-Newman-Keuls test was used for statistical analysis. P < 0.05 represents statistical significance.

Data availability
The original contributions presented in the study are included in the article/ "Supplementary Material", further inquiries can be directed to the corresponding author and S.L..  GCA GGA TTC TTC TGA GCG TTCT  GTC AGC AGC CTC CTA GAT CATGT  67   M-DNA-Sdha  TAC TAC AGC CCC AAG TCT  TGG ACC CAT CTT CTA TGC  194