Effects of different types of exercise on skeletal muscle atrophy, antioxidant capacity and growth factors expression following myocardial infarction

Accepted Manuscript Effects of different types of exercise on skeletal muscle atrophy, antioxidant capacity and growth factors expression following myocardial infarction

Mengxin Cai, Qing'an Wang, Zhiwei Liu, Dandan Jia, Rui Feng, Zhenjun Tian PII: DOI: Reference:

S0024-3205(18)30632-5 doi:10.1016/j.lfs.2018.10.015 LFS 15995

To appear in:

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Received date: Revised date: Accepted date:

28 August 2018 20 September 2018 6 October 2018

Please cite this article as: Mengxin Cai, Qing'an Wang, Zhiwei Liu, Dandan Jia, Rui Feng, Zhenjun Tian , Effects of different types of exercise on skeletal muscle atrophy, antioxidant capacity and growth factors expression following myocardial infarction. Lfs (2018), doi:10.1016/j.lfs.2018.10.015

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ACCEPTED MANUSCRIPT Effects of different types of exercise on skeletal muscle atrophy, antioxidant capacity and growth factors expression following myocardial infarction

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Mengxin Cai1, Qing’an Wang1,2, Zhiwei Liu1, Dandan Jia1, Rui Feng1,3, Zhenjun Tian1*

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1. Institute of Sports and Exercise Biology, Shaanxi Normal University, Xi' an

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710119, P. R. China

Medicine, Jinan 250355, P. R. China

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2. School of Rehabilitation Medicine, Shandong University of Traditional Chinese

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3. College of Life Sciences, Shaanxi Normal University, Xi’an 710119, P. R. China

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*Corresponding Authors:

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Zhenjun Tian

Institute of Sports and Exercise Biology Institute of Physical Education

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Shaanxi Normal University

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620 West Chang'an Avenue Xi'an 710119 P. R. China Tel: 86-85310156

Fax: 86-85310156 Email: [email protected] A running title: Exercise inhibit myocardial infarction-induced skeletal muscle atrophy 1

ACCEPTED MANUSCRIPT Abstract Aims: Myocardial infarction (MI) is accompanied with skeletal muscle abnormalities. The aims are to explore an optimal exercise mode to improve cardiac function and prevent skeletal muscle atrophy, and detected the possible mechanisms of exercise-induced inhibition of muscle atrophy.

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Main methods: Rats were subjected to four weeks of different types of exercise after

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MI surgery (resistance training, RT; moderated-intensity continuous aerobic exercise,

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MCE and high-intensity intermittent aerobic exercise, HIA). Cardiac function, histological changes of heart and skeletal muscle, oxidative stress, antioxidant

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capacity and the expression of muscle atrophy-related factors were detected in skeletal muscle.

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Key findings: The three types of exercise improved heart function, reduced cardiac fibrosis and increased muscle weight and cross-section area (CSA) of muscle fibers in

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different degrees. The survival rates of MI rats intervened by RT and MCE were

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higher than HIA. Exercise down-regulated the mRNA levels of murf1 and atrogin-1, decreased reactive oxygen species level, increased antioxidant capacity, regulated the

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expression of insulin-like growth factor I (IGF1), mechano growth factor1 (MGF), neuregulin 1 (NRG1) and myostatin (MSTN), and activated Akt and Erk1/2 signalings

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in soleus muscle. Furthermore, CSA of muscle fibers and the expression of IGF1, MGF, NRG1 in skeletal muscle had correlations with cardiac function. Significance: RT and MCE are the first two choices for the early exercise rehabilitation following MI. All types of exercise can effectively inhibit skeletal muscle atrophy through increasing the antioxidant capacity, reducing oxidative stress and protein degradation, and regulating the growth factors expression in skeletal muscle. 2

ACCEPTED MANUSCRIPT Keywords: exercise training, myocardial infarction, skeletal muscle atrophy, growth factor, oxidative stress Introduction Myocardial infarction (MI) is one of the leading causes of death worldwide.

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After MI, ischemia and hypoxia induces oxidative stress [1,2], which leads to a

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number of cardiomyocytes loss, cardiac fibrosis and heart dysfunction, and finally,

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results in heart failure (HF). Several studies indicated that MI, especially HF, is accompanied with skeletal muscle abnormalities, such as skeletal muscle atrophy

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[3-7], and impaired performance of skeletal muscle could reduce exercise capacity in

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patients [8]. It has been identified that skeletal muscle atrophy would be an independent predictor of mortality in HF patients [9]. Therefore, therapies for

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improving cardiac function and inhibiting muscle atrophy after MI need to be studied

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detailedly.

After heart injury, increased inflammation and redox unbalance in skeletal

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muscle are direct factors of muscle fiber injury and catabolism [10, 11]. HF could increase oxidative stress in skeletal muscle [12-15] and the production of reactive

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oxygen species (ROS) induced irreversible modification of contractile proteins and formation of lipid peroxidation products [16-19], finally resulted in muscle fiber injury and death. Many studies, focused on the mechanisms of skeletal muscle atrophy, indicated the ubiquitin-proteasome and the autophagy-lysosome systems are the two conserved proteolytic mechanisms for skeletal muscle atrophy [20-23]. The E3 ubiquitin ligases, Atrogin-1/MAFbx and Muscle RING-Finger-1 (MuRF1) play 3

ACCEPTED MANUSCRIPT important roles in protein degradation [24, 25]. Besides, growth factors are also involved in the skeletal muscle growth and atrophy, such as Myostatin (MSYN) [26, 27], insulin-like growth factor 1 (IGF1) [28-30], mechano growth factor (MGF) [31, 32] and Neuregulin1 (NRG1) [33-35]. These factors activate Akt or Erk1/2 signaling

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to improve or prevent skeletal muscle growth [29, 36-38]. Accordingly, after MI,

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inhibiting the oxidative stress and protein degradation, improving the antioxidant

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ability and regulating the expression of growth factors might be the key factors to inhibit skeletal muscle atrophy.

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Increasing evidences confirmed exercise training could give a beneficial effect

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on cardiac function in cardiovascular disease [39-49]. It has been a consensus that exercise should be involved in the cardiac rehabilitation program. However, the

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feasible exercise prescription needs more attentions. It has been shown that aerobic

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exercise improved cardiac function and prevented skeletal muscle atrophy in patients with HF [50-52]. Compared with moderate-intensity aerobic exercise, high-intensity

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interval exercise is more effective on oxygen uptake and aerobic exercise ability [53].

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However, Moreira et al. [54] compared the high and moderate-intensity aerobic exercise effects on skeletal muscle of infarcted rats, and found there were no significant changes between the two protocols on skeletal muscle adaptations [54]. In contrast to aerobic exercise, resistance training (RT) has been well known to enhance muscle strength and endurance, and improve metabolic status [55, 56]. In this study, we compared the effects of RT and aerobic exercise (high-intensity intermittent aerobic exercise, HIA, and moderate-intensity continuous aerobic exercise, MCE) on 4

ACCEPTED MANUSCRIPT cardiac function and skeletal muscle atrophy, oxidative stress and the expression of factors related to muscle atrophy in rats with MI. The aims of this study are to find an optimized, individualized, and safety practiced exercise mode after MI, and reveal the possible mechanisms of the three types of exercise on skeletal muscle atrophy after

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MI. 2. Materials and methods

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2.1 Animal

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The animal protocol of this study strictly conformed to the National Institutes of

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Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). All animal care and experimental procedures were approved by

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the ethical committee of Shaanxi Normal University and performed following Chinese animal protection laws and institutional guidelines.

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75 sprague Dawley rats (purchased from the Laboratory Animal Centre of Xi'an

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Jiaotong University, Xi’an, China), three months old, weighting 200±10g, were used in this study. Rats were housed in a temperature-controlled animal room (22-24 °C),

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with free access to chow diet and water. After feeding one week, rats were randomly subjected to MI or Sham surgery.

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2.2 Myocardial infarction model The MI model was established by ligating the left anterior descending coronary artery (LADCA) of the heart as previously described [57]. Rats were anaesthetized (sodium pentobarbital, 80 mg/kg, i.p.) and fixed on the operating table. After opening chest and exposing heart, The LADCA was ligated approximately 2.0 mm from its origin. The whole operation was monitored by an electrocardiogram. Totally 13 rats died during or after the surgery, and 12 rats without ST segment elevation were not 5

ACCEPTED MANUSCRIPT used in this study. Rats in Sham group underwent the same procedure, except for LADCA ligation, viewed as control (Sham; n=10). Survival rats after MI surgery were randomly divided into four groups: sedentary MI group (MI; n=10), MI with RT group (MR; n=10), MI with high-intensity intermittent aerobic exercise group (MA; n=10) and MI with moderate-intensity

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continuous aerobic exercise group (ME; n=10). Rats in MR, MA and ME groups were

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subjected to RT, HIA or MCE from the second week after surgery.

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2.3 Exercise training protocols

Rats in MR group were subjected to RT by using a ladder (52 vertical steps, 2 cm

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apart from each other) [58]. Rats were gradually adapted to climbing at the first week with the load from 0% to 75% body weight (BW) on the tail, 20 climbs per session for

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three sessions, with 2 min rest between each session. From the second week, the initial load was 75% BW, and then an additional 15% BW was added to rats until they

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failed to climb the ladder completely. The training protocol was performed 20 climbs

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per session and three sessions per day with 2 min rest between each session, five days a week for four weeks.

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HIA and MCE protocols were performed on a motor-driven treadmill as described previously [45, 47]. Initially, rats in both MA and ME groups were exposed

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to adaptive training at 10 m/min for 30 min at the first week. Then, in the MA group, the speed was up to 15 m/min, 7 min and alternately 25 m/min, 3 min, totally 60 min per session and five days a week for four weeks. In the ME group, the speed was gradually increased to 16 m/min and 50 min per session (including a 5 min warm-up at 10 m/min) and maintained constant throughout the four weeks experiment. 2.4 Hemodynamic measurement At the next day after the last training or sedentary behavior, the resting cardiac 6

ACCEPTED MANUSCRIPT function of rats was assessed by a hemodynamic test method as pervious described [57]. Rats were anaesthetized as mentioned above and placed in the supine position. A pressure transducer was inserted into the left ventricular (LV) cavity from the right carotid artery. An intraventricular catheter recording (Powerlab 8/30™, ML 870; AD Instruments, Castle Hill, Australia) was used to evaluate cardiac function by detecting

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the LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), maximal

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positive and minimal negative first derivative of LV pressure (±dp/dtmax). Then the

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hearts, soleus and gastrocnemius muscles were picked out and fixed in liquid nitrogen or 4% formaldehyde solution for 48 hours at least.

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2.5 Histological analysis

Hearts, soleus and gastrocnemius muscles, fixed in paraformaldehyde, were

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utilized in histological analysis. After embedded in paraffin, all samples were sectioned 5-8 µm thick using a rotary microtome. Heart tissue sections stained with

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Masson's trichrome for quantitatively analyzing the collagen content in the middle of

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the infarcted area of LV or the same area of the sham-operated heart. Skeletal muscle tissue sections stained with Hematoxylin-eosin (HE) to calculate the cross-section

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area (CSA) of muscle fibers. After scanned, 20 fields per section were viewed and images were computerized by Image Pro-Plus (IPP) 6.0. The collagen volume fraction

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(CVF) of heart was defined as the sum fall the connective tissue areas of the entire section. CSA of skeletal muscle fiber was assessed by calculating 20 fibers per field. 2.6 DHE staining The soleus muscle tissues, fixed in liquid nitrogen, were used to detect the O2‾ level by Dihydroethidium (DHE) fluorescence examination to reflect the ROS level. Samples were frozen sectioned 4 mm thick with a freezing microtome (Leica CM1950, Germany) and incubated with DHE (Invitrogen, Carlsbad, CA, USA), 2 7

ACCEPTED MANUSCRIPT mM, in a light protected oven at 37°C for 30 min. The sections were washed with phosphate buffer saline (PBS, pH=7.2) and fluorescence was assessed using a fluorescence microscopy (Nikon Eclipse 55i, Tokyo, Japan). The intensity of ethidium fluorescence detection of O2‾ was analyzed by IPP 6.0. Quantification of the O2‾ level

2.7 Activities of antioxidant enzyme and citrate synthase

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on section was expressed as a percentage of the total area of the muscle cross-section.

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Skeletal muscle samples, fixed in liquid nitrogen, were used in biochemical

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index test, western blotting and RT-qPCR examination. Frozen muscle pieces (100 mg) were placed in ice-cold PBS (pH=7.2). Samples were homogenized in PBS and

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centrifuged for 10 min at 10,000 × g at 4°C. The supernatant was used for the assays. The activities of superoxide dismutase (SOD), glutathione (GSH), glutathione (GSSG)

and

citrate

synthase

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disulfide

(CS)

was

measured

by

using

spectrophotometric assay kits (Jiancheng Biotech Co., Ltd., Nanjing, China).

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Following the manufacturer's instruction, the sample absorbance was detected.

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2.8 Real-time quantitative PCR (RT-qPCR) Total RNA was extracted from frozen muscle pieces (100 mg) with Trizol

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reagent following the manufacturer’s protocol. RNA concentration was determined spectrophotometrically at 260/280 nm ratio by using Biotek (BioTek Epoch, BioTek,

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VT, USA). First strand cDNA was generated from mRNA using the Revertaid First Strand cDNA synthesis kit (Fermentas, Glen Burnie, MD, USA). In brief, for reverse transcription termination, total RNA (2 μg), RiboLock RNase inhibitor (20 units), 1 mM dNTP mix, oligo (dT, 0.5 μg), and Revert Aid Reverse Transcriptase (200 units) were mixed and incubated at 42°C for 1 h plus 10 min at 70°C. PCR reaction was performed with quantitative PCR instrument (CFX connectTM Real-Time system, Bio-Rad, CA, USA) by using the Maxima SYBR Green/ROX qPCR Master Mix 8

ACCEPTED MANUSCRIPT (Fermentas). Results were expressed using the comparative cycle threshold (Ct) method. The ΔCt values were calculated as Ct

gene of target

minus Ct

reporter gene;

gapdh

was the control gene. Relative changes in mRNA levels (ΔΔCt) was calculated by subtracting ΔCt value of Sham group from ΔCt values of other groups. The level of Sham group was set to 1. Primer sequences are shown as followed: murf1 (F:

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5′-GGCGGGAGGGT-TGGGTCTCACTC-3′, 5′-CCCCAATACCCAGCCCCTTCTGC-3′);

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atrogin-1

5′-GTTGAATCTTCTGGAATCCAG-GAT-3′);

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5′-TACTAAGGAGGCCCATGGATACT-3′,

gapdh

R: (F: R: (F:

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5′-CAGTGCCAGCCTCGTCTCAT-3′, R: 5′-AGGCCATCCACA-GTCTTC-3′). 2.9 Western blotting

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Total protein was extracted from the soleus muscle and samples were separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE, 120 V constant, 1.5 h,

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room temperature), electrotransferred to nitrocellulose membranes (Millipore, MA,

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USA, 300mA, 1.5 h, 4°C). The membrane was blocked by incubating with 5% bovine serum albumin (BSA, 1.5 h, room temperature) to prevent unspecific binding of

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antibodies. Then followed by incubation with the primary antibodies (overnight, 4°C): IGF1 (1:1000 dilution), MGF (1:400 dilution), MSTN (1:2000 dilution, Abcam, MA,

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USA), NRG1 (1:1000 dilution, Bioworld, MN, USA), phosphorylated Akt (pAkt) and Akt, phosphorylated Erk1/2 (pErk1/2) and Erk1/2 (1:1000 dilution, Cell Signaling Technology, MA, USA). Membranes were washed with tris-buffered saline-Tween 20 (TBST) three times (5 min each) and incubated with horseradish-peroxidase (HRP)-conjugated secondary antibodies (1:5000 dilution, Jackson ImmunoResearch, PA, USA, 2 h, room temperature). After washed with TBST three times, antibody detection was performed in a digitalizing unit and visualized with an enhanced 9

ACCEPTED MANUSCRIPT chemiluminescence detection kit (Millipore). GAPDH was used as an internal control. Quantitative assessment of band gray value was performed by densitometry software (Quantity One, BioRad). 2.10 Statistical analysis Statistical analysis and correlation analysis were performed using SPSS17.0

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statistical package. Values presented as mean ± SEM. Differences between mean

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values in the groups were determined by one-way ANOVA with Tukey HSD post hoc.

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P<0.05 or P<0.01 was considered significant. 3. Results

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3.1 Effects of aerobic exercise and resistance training on survival rate, heart function and cardiac fibrosis following myocardial infarction

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Initially, 75 rats were used in this study. After MI surgery, only 40 rats with similar electrocardiogram changes were selected to subject exercise training or

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sedentary, and 10 rats did the sham surgery as control. The results showed that no rat

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died in Sham, MR and ME groups (survival rates were 100%), the survival rates of MA and MI groups were 70% and 90% (Tab.1). This result implies that under the

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same living condition, the safety of RT and MCE intervention is better than HIA. In order to compare the effective of aerobic exercise and RT on cardiac function

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in rats with MI, the hemodynamic variables of LV were assessed. Compared with the Sham group, the LVEDP was increased and LVSP and ±dp/dtmax were reduced significantly in rats with MI (P＜0.01). Compared with the MI group, three types of exercise improved cardiac function in different degrees by increasing the LVSP (P＜ 0.05 versus with ME, P＜0.01 versus with MR and MA), +dp/dtmax (all P＜0.01) and -dp/dtmax (P＜0.05 versus with MR, P＜0.01 versus with MA and ME) and reducing

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ACCEPTED MANUSCRIPT the LVEDP (P＜0.05 versus with MR, P＜0.01 versus with ME and MA, Tab. 1). According to these results, both aerobic exercise and RT have beneficial effects on cardiac function in MI rats, and the HIA would be the most effective mode, followed by MCE and RT. The Masson's trichrome staining was used to analyze the cardiac fibrosis by

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calculating the myocardial CVF. The collagen fibers (blue), nucleus (brown) and

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cytoplasm of cardiomyocytes (red) were clearly stained. In the Sham group, the

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cardiomyocytes were arranged in order, and the proportion of CVF was about 10% in the LV. Compared to the Sham group, infarcted rats had an appreciable increase in

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fibrotic deposition, the collagen fibers from the infarcted area extended to the peri-infarcted and non-infarcted areas, which displayed robust elevation in CVF (P＜

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0.01). HIA and MCE reduced the CVF levels in the MI rats when compared with the MI group and MR group (P＜0.01, P＜0.05). However, RT-treated MI rats had no

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appreciable change in CVF (Fig. 1). These data supported the contention that aerobic exercise has a better effect on heart structure and cardiac function than RT. However, considering the safety, the MCE or RT were suggested as the first choice for the

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exercise rehabilitation following MI.

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3.2 Effects of aerobic exercise and resistance training on skeletal muscle atrophy following myocardial infarction It has been reported that MI was accompanied with skeletal muscle atrophy [6, 9, 11]. In the present study, the effects of aerobic exercise and RT on skeletal muscle atrophy were detected by calculated the ratio of skeletal muscle weight and BW, and the CSA of muscle fibers. Results showed that compared with the Sham group, soleus muscle atrophy was observed by reduced muscle weight/BW (P＜0.05, Fig. 2A) and

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ACCEPTED MANUSCRIPT the CSA of muscle fiber (P＜0.05, Fig. 2C and E), however, muscle atrophy was not observed in the gastrocnemius muscle (Fig. 2B, D and F). After four weeks of exercise intervention, increased muscle weight and CSA of soleus (Fig. 2A, C and E) and gastrocnemius muscle fibers (Fig. 2B, D and F) were observed in MR (P＜0.05,

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P＜0.01), MA (all P＜0.01) and ME groups (all P＜0.05), and no significant differences between the three exercise protocols. To explore the possible relationship

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between skeletal muscle growth and cardiac function, a correlation analysis was done

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between CSA of skeletal muscle fiber and LVEDP in the trained rats by pearson analysis. Results showed that LVEDP had obviously negative correlation with CSA of

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soleus (r=-0.569, P<0.01, Fig. 3A) and gastrocnemius muscle fibers (r=-0.697,

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P<0.01, Fig. 3B). These results indicated that five weeks after MI, soleus muscle atrophy occurred, exercise training could improve skeletal muscle growth, and increased CSA of muscle fibers by exercise may be further involved in the

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improvement of cardiac function. In the following experiments, we detected the oxidative stress, antioxidant capacity and the expression of factors-related to muscle atrophy in soleus muscle.

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3.3 Effects of different types of exercise on oxidative stress and antioxidant

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capacity of skeletal muscle following myocardial infarction To detect the possible mechanism of exercise-prevented skeletal muscle atrophy, the oxidative stress level was assessed in soleus muscle firstly. DHE fluorescence examination was used to detect the O2- level. Results showed that compared with the Sham group, the DHE fluorescence area (O2-) increased in the MI group (P＜0.01), compared with the MI groups, RT, MCE and HIA decreased the DHE fluorescence area (P＜0.05, P＜0.01), and there was no significant changes among the three 12

ACCEPTED MANUSCRIPT exercise protocols (Fig. 4A). These results indicated that exercise could improve the MI-induced oxidative stress in skeletal muscle. Then we detected the activity levels of SOD, GSH and GSSG and CS in soleus muscle to assess the redox balance. Results showed that HIA prevented MI-induced impairment of SOD significantly (P＜0.05), RT and MCE tend to increase the

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concentration of SOD, whereas, without significant changes (Fig. 4B). No differences

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of the concentration of GSH was shown among the five groups, however, different

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types of exercise increased the concentration of GSSG in soleus muscle (P＜0.05, Fig. 4C and D) when compared with the MI group. CS is an enzyme of the Krebs cycle

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and an indicator of cellular aerobic metabolism. Compared with the Sham group, a

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reduction of CS activity was detected in soleus muscle homogenates of MI rats (P＜ 0.05), whereas RT, HIA and MCE increased CS activity in soleus muscle (P＜0.05,

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Fig. 4E). These results indicated that exercise could improve the antioxidant capacity

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of soleus muscle following MI, and the effect of HIA on SOD level was better than RT and MCE.

3.4 Effects of different types of exercise on the expression of murf1 and atrogin-1

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of skeletal muscle following myocardial infarction

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Ubiquitin-proteasome system plays an important role in the protein degradation[36]. In this study, mRNA levels of ubiquitin ligases atrogin-1 and murf1 were assessed in soleus muscles by qRT-PCR. Compared with the Sham group, mRNA levels of murf1 (Fig. 5A) and atrogin-1 (Fig. 5B) increased in the MI group (all P＜0.01), and compared with the MI group, mRNA levels reduced in MR (P＜ 0.05, P＜0.01), MA (all P＜0.01) and ME (all P＜0.05) groups.

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ACCEPTED MANUSCRIPT 3.5 Exercise regulated the expression of growth factors and activated Akt and Erk1/2 in the skeletal muscle of MI rats. Growth factors, such as IGF1, MGF and MSTN have been well reported to increase (IGF1, NRG1, MGF) or inhibit (MSTN) muscle fiber growth or regeneration. In the present study, results showed that compared with the Sham group, MSTN

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expression increased significant in soleus muscle of MI rats (P＜0.05). RT, HIA and

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MCE down-regulated MSTN expression (all P＜0.05, Fig. 6A and C), up-regulated

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the expression of IGF1 (P＜0.05, P＜0.01, Fig. 6A and D), MGF (all P＜0.05, Fig. 6A and E) and NRG1 (all P＜0.05, Fig. 6A and F), and increased the phosphorylation

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of Akt (P＜0.05, P＜0.01, Fig. 6B and G) and Erk1/2 (all P＜0.01, Fig. 6B and H).

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There was no significant changes among the three types of exercise on the expression of growth factors. However, the effect of HIA on Akt activity was better than RT (P＜

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0.05). These results indicated that exercise could regulate the expression of growth

muscle growth.

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factors and activated Akt and Erk1/2 signalings, which play important roles in skeletal

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Recent studies indicated that the skeletal muscle could be viewed as an endocrine organ, exercise-induced skeletal muscle growth was accompanied with myokines

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secretion and release [59]. Based on these, the regulated expression of growth factors, especially IGF1, MGF and NRG1 would function like myokines to participated in the exercise-induced improvement of cardiac function. Therefore, correlation analysis between growth factors and LVEDP was did by pearson analysis. Results showed that LVEDP has obviously negative correlations with the expression of IGF1(r=-0.537, P=0.039, Fig. 7A), MGF (r=-0.627, P=0.012, Fig. 7B) and NRG1 (r=-0.701, P=0.004, Fig. 7C) in the soleus muscle. These results indicated that expression of 14

ACCEPTED MANUSCRIPT growth factors would be closely related to the exercise-induced improvement of cardiac function. 4. Discussion In the present study, MI rats were intervened with aerobic exercise and RT, and the effects of these types of exercise on cardiac function and skeletal muscle atrophy

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were detected. The major findings of this study are as follows: (1) the effects of

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aerobic exercise (MCE and HIA) on heart function and cardiac fibrosis are better than

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that of RT. RT, HIA and MCE had similar effects on skeletal muscle atrophy in infarcted rats. However, considering the mortality, MCE and RT will be the first two

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choices in the early stage of exercise rehabilitation after MI; (2) RT, HIA and MCE reduce oxidative stress, improve antioxidant capacity, down-regulate the expression of

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atrogin-1, murf1 and MSTN, up-regulate the expression of IGF1, MGF and NRG1, and activate Akt and Erk1/2 in soleus muscle following MI. In addition, there are

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correlations between the cardiac function and the CSA of skeletal muscle fibers or the

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expression of growth factors in skeletal muscle. Exercise training can effectively improve cardiac function in MI patients,

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however, the optimized exercise mode needs further exploration. The current concerning type of exercise in rehabilitation are RT and aerobic exercise training [45,

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47, 54, 60]. It has been widely reported that MCE could effectively improve cardiac function after MI by improving cardiomyocyte proliferation, angiogenesis, and sympathetic and parasympathetic nervous regulation, inhibiting cardiomyocyte apoptosis and cardiac fibrosis [41, 44, 46, 47, 57]. Research pointed out that HIA was more effective than MCE on reversing LV remodeling, improving the VO2max and the life quality of MI patients [61]. However, some studies indicated there was no difference between MCE and HIA on improvement of cardiac function and skeletal 15

ACCEPTED MANUSCRIPT muscle abnormality [54, 62]. In our pervious study, we confirmed eight weeks of MCE and HIA could improve cardiac function [52, 57]. The effects of four weeks exercise still needs to be determined. Considering the molecular changes of heart and skeletal muscle in the acute and developmental stages of MI are more significant than long-term stage, we intervened rats with four weeks of exercise from the second week

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after MI surgery, and detected the effects on skeletal muscle atrophy after MI and

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explored its possible mechanisms.

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In the present study, we confirmed four weeks of MCE and HIA improved heart function and cardiac fibrosis, however, there was no significant changes between

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them. We speculated four weeks was not enough to make a difference between the two exercise protocols. In addition, the survival rate of HIA-intervened rats was only

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70%, and MCE was 100%. This indicates that under the similar living and training conditions, the tolerance of MI rats to HIA is lower than that of MCE, and there is a

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certain risk of HIA for MI rats in the recovery period after exercise. Unfortunately, we

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haven’t detected the specific effects of HIA and MCE on the heart structure and the possible cause of death needs further verification. In addition, it is believed that early

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RT after MI resulted in a lower risk of cardiac complications than aerobic exercise, and RT is suitable for the elderly [60, 63]. Results of this study confirmed that RT was

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able to improve cardiac function, although the effective was less than MCE and HIA on reducing CVF. Therefore, considering the safety of the three types of exercise, it is proposed that MCE and RT may be more suitable for early exercise rehabilitation after MI. However, the possible mechanisms of different types of exercise on cardiac load and structural are still not clear. Moreover, the effects of combined exercise on cardiac function following MI will be studied and the possible molecular mechanisms may be clarified in future work. 16

ACCEPTED MANUSCRIPT MI-induced HF is a degenerative disease, which accompanies with other organs abnormalities, such as skeletal muscle. Skeletal muscle atrophy is the main cause of decrease of exercise capacity in HF patients [9]. Multiple atrophic stimulates induce different responses on slow muscle and fast muscle. Unloading stimulation could easily cause slow muscles damage, while fast muscles are more sensitive to skeletal

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muscle diseases [54, 64]. In the present study, five weeks after MI, compared with the

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Sham group, the relative weight and the CSA of muscle fibers decreased significantly

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in soleus muscle, but not the gastrocnemius muscle. It has been reported that heart injury induced the decrease of peripheral blood and increased the oxidative stress

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increased in skeletal muscle [65, 66]. Considering the proportion of slow-muscle fibers is higher than fast-muscle fibers in soleus, the occurrence of soleus muscle

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atrophy is easy under the ischemic condition. Meanwhile, our results confirmed that MI increased the ROS level and reduced the activities of SOD and CS, which

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indicated MI could induce oxidative stress and impaired antioxidant capacity of

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soleus muscle. RT, HIA and MCE increased the relative weight and the CSA of muscle fibers in both soleus and gastrocnemius muscles, and increase the activities of

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GSSG and CS. However, HIA increased SOD activity significantly, and RT and MCE showed an increased trend without significant changes. Therefore, the effect of HIA

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would be better than that of MCE on skeletal muscle growth, indicated that muscle growth was dependent on the exercise intensity. RT, as a kind of strength training, has a significant effect on skeletal muscle growth, which is closely related to promotion of protein synthesis [67]. These results indicated that all the three types of exercise could reduce oxidative stress, improve the antioxidant capacity of skeletal muscle following MI and prevent skeletal muscle atrophy. Interestingly, we also found there were correlations between the CSA of soleus or gastrocnemius muscle fibers and cardiac 17

ACCEPTED MANUSCRIPT function in the trained rats, which indicated that promoting skeletal muscle growth would play some roles in the improvement of cardiac function. During muscle atrophy, due to the changes in protein metabolism which favor proteolysis over protein synthesis, myofiber protein was lost and myofiber size reduced ultimately [68]. The ubiquitin proteasome system is one of the major

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proteolytic systems [21]. Atrogin-1 and MuRF-1 are two E3 ubiquitin ligases that are

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important regulators of ubiquitin-mediated protein degradation in skeletal muscle [24,

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69]. Suppression of atrogin-1 and MuRF1 could prevent skeletal muscle atrophy [70]. In our study, we found MI increased the mRNA expression of atrogin-1 and murf1 in

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soleus muscle, RT, HIA and MCE down-regulated their expression. These results

functioned in the muscle atrophy.

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indicated exercise could inhibit the progress of muscle protein degradation, which

Increasing protein synthesis is also one of methods to inhibit the pathogenesis of

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muscle atrophy. Hormones and growth factors regulated by the endocrine system play

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important roles in the balance between skeletal muscle anabolism and catabolism. Anabolic hormones and growth factors, such as IGF1 and MGF, could induce muscle

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growth by increasing protein synthesis [71]. In contrast, MSTN lead to skeletal muscle atrophy by promoting catabolism [71]. Previous studies demonstrated that

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MSTN expression modulated oxidative stress in skeletal muscle [72], and up-regulation of MSTN expression in skeletal muscle after MI may be one of the main factors of skeletal muscle atrophy [73]. This study confirmed that MSTN expression increased in soleus muscle after MI, and different types of exercise reduced its expression. The possible mechanism would be related to the improvement of the microenvironment and oxidative stress of skeletal muscle by exercise [74]. IGF1, MGF and NRG1 are positive factors on regulation of muscle growth [29, 32, 18

ACCEPTED MANUSCRIPT 34]. In the present study, compared with the Sham group, the protein expression of IGF1 and MGF decreased in skeletal muscle of MI group, but there was no significant differences. It has been reported that exercise could up-regulate the expression of IGF1 and MGF in skeletal muscle [75, 76] and contractile stimulation induced NRG1 expression in skeletal muscle cells in vitro [77]. In this study, results confirmed RT,

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HIA and MCE up-regulated the expression of IGF1, MGF and NRG1 in skeletal

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muscle significantly, meanwhile, activated the AKT and Erk1/2 signalings. These

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results indicated that exercise could regulate the expression of growth-related factors to promoting skeletal muscle growth and inhibit muscle atrophy, there is no

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significant differences between the three exercise programs.

After MI, promoting cardiac function, inhibiting skeletal muscle atrophy and

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improving the life quality of patients are the final purposes of clinical treatment and rehabilitation. The beneficial effects of appropriate exercise on cardiovascular disease

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patients has been confirmed, but the mechanisms and the exercise mode still need to

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be further explored. Cytokines, including growth factors in the microenvironment of the heart after MI worked on the injured myocardium and peripheral organs through

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autocrine, paracrine or endocrine effects. It has been reported that exercise could improve the microenvironment of injured heart, reduce the oxidative stress and

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inflammation and regulate the cytokines expression. Muscle contraction during exercise could regulate the expression of myokines.

Based on the cross talking

between heart and skeletal muscle and the endocrine function of skeletal muscle, we speculated that the up-regulation of the expression of IGF-1, MGF and NRG1 (cardioprotective factors and participates in myocardial repair after MI) in skeletal muscle would have some relationship with the cardiac function. Interestingly, results confirmed our speculation. We found the expression levels of IGF-1, MGF and NRG1 19

ACCEPTED MANUSCRIPT in skeletal muscle were negative correlated with the LVEDP. Accordingly, we will screen the effective myokines and explore the possible mechanism of the protective effects of these growth factors expressed in skeletal muscle on cardiac function after MI in our future work. 5. Conclusion

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RT, HIA and MCE could improve cardiac function and inhibit skeletal muscle

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atrophy in different degrees after MI. HIA leads to a higher mortality under the same

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pathological conditions when compared with RT and MCE. In view of the process of clinical rehabilitation, it should be appropriate to choose the RT and MCE at the early

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stage of rehabilitation. Meanwhile, RT, HIA and MCE could reduce oxidative stress, improve antioxidant capacity, down-regulate the expression of atrogin-1, murf1 and

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MSTN, up-regulate the expression of IGF1, MGF and NRG1, and activate the AKT and Erk1/2 in skeletal muscle. There are correlations between the cardiac function and

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the CSA of skeletal muscle fibers or the expression of growth factors in skeletal

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muscle, which further suggests that skeletal muscle growth might be involved in the

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improvement of cardiac function.

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ACCEPTED MANUSCRIPT Conflict of interest The authors declare that there is no conflict of interest.

Acknowledgments

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This research was supported by National Natural Science Foundation of China

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(Grant No. 31701039, 31371199).

Author Contributions

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M. Cai and Z. Tian designed research; M. Cai, Q. Wang and Z. Liu performed

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research; M. Cai and Z. Tian wrote the paper; M. Cai, R. Feng and D. Jia analyzed.

21

ACCEPTED MANUSCRIPT Referances [1] P. Korantzopoulos, D. Galaris, D. Papaioannides, K. Siogas, The possible role of oxidative stress in heart failure and the potential of antioxidant intervention, Med Sci Monit. 9 (2003) RA120-125. [2] Y. Sun, Oxidative stress and cardiac repair/remodeling following infarction, Am J Med Sci.

PT

334 (2007) 197-205. [3] H. Drexler, U. Riede, T. Munzel, H. Konig, E. Funke, H. Just, Alterations of skeletal muscle in

RI

chronic heart failure, Circulation. 85 (1992) 1751-1759.

SC

[4] P. Georgiadou, S. Adamopoulos, Skeletal muscle abnormalities in chronic heart failure, Curr Heart Fail Rep. 9 (2012) 128-132.

NU

[5] D.M. Mancini, G. Walter, N. Reichek, R. Lenkinski, K.K. McCully, J.L. Mullen, J.R. Wilson, Contribution of skeletal muscle atrophy to exercise intolerance and altered muscle metabolism

MA

in heart failure, Circulation. 85 (1992) 1364-1373.

[6] A. Simonini, C.S. Long, G.A. Dudley, P. Yue, J. McElhinny, B.M. Massie, Heart failure in rats causes changes in skeletal muscle morphology and gene expression that are not explained by

D

reduced activity, Circ Res. 79 (1996) 128-136.

PT E

[7] M.J. Sullivan, H.J. Green, F.R. Cobb, Skeletal muscle biochemistry and histology in ambulatory patients with long-term heart failure, Circulation. 81 (1990) 518-527.

CE

[8] K. Okita, K. Yonezawa, H. Nishijima, A. Hanada, M. Ohtsubo, T. Kohya, T. Murakami, A. Kitabatake, Skeletal muscle metabolism limits exercise capacity in patients with chronic heart

AC

failure, Circulation. 98 (1998) 1886-1891. [9] S.D. Anker, P. Ponikowski, S. Varney, T.P. Chua, A.L. Clark, K.M. Webb-Peploe, D. Harrington, W.J. Kox, P.A. Poole-Wilson, A.J. Coats, Wasting as independent risk factor for mortality in chronic heart failure, Lancet. 349 (1997) 1050-1053. [10] A. Musaro, S. Fulle, G. Fano, Oxidative stress and muscle homeostasis, Curr Opin Clin Nutr Metab Care. 13 (2010) 236-242. [11] K.J. Lavine, O.L. Sierra, Skeletal muscle inflammation and atrophy in heart failure, Heart Fail Rev. 22 (2017) 179-189. 22

ACCEPTED MANUSCRIPT [12] E. De Sousa, V. Veksler, X. Bigard, P. Mateo, R. Ventura-Clapier, Heart failure affects mitochondrial but not myofibrillar intrinsic properties of skeletal muscle, Circulation. 102 (2000) 1847-1853. [13] R. Hambrecht, V. Adams, S. Gielen, A. Linke, S. Mobius-Winkler, J. Yu, J. Niebauer, H. Jiang, E. Fiehn, G. Schuler, Exercise intolerance in patients with chronic heart failure and increased expression of inducible nitric oxide synthase in the skeletal muscle, J Am Coll Cardiol. 33

PT

(1999) 174-179.

RI

[14] H. Tsutsui, Oxidative stress in heart failure: the role of mitochondria, Intern Med. 40 (2001) 1177-1182.

SC

[15] R. Ventura-Clapier, E. De Sousa, V. Veksler, Metabolic myopathy in heart failure, News

NU

Physiol Sci. 17 (2002) 191-196.

[16] E. Barreiro, B. de la Puente, J. Minguella, J.M. Corominas, S. Serrano, S.N. Hussain, J. Gea,

MA

Oxidative stress and respiratory muscle dysfunction in severe chronic obstructive pulmonary disease, Am J Respir Crit Care Med. 171 (2005) 1116-1124. [17] I. Dalle-Donne, D. Giustarini, R. Colombo, R. Rossi, A. Milzani, Protein carbonylation in

D

human diseases, Trends Mol Med. 9 (2003) 169-176.

PT E

[18] M.J. Mihm, F. Yu, P.J. Reiser, J.A. Bauer, Effects of peroxynitrite on isolated cardiac trabeculae: selective impact on myofibrillar energetic controllers, Biochimie. 85 (2003)

CE

587-596.

[19] M. Canton, I. Neverova, R. Menabo, J. Van Eyk, F. Di Lisa, Evidence of myofibrillar protein

AC

oxidation induced by postischemic reperfusion in isolated rat hearts, Am J Physiol Heart Circ Physiol. 286 (2004) H870-877. [20] P.R. Jannig, J.B. Moreira, L.R. Bechara, L.H. Bozi, A.V. Bacurau, A.W. Monteiro, P.M. Dourado, U. Wisloff, P.C. Brum, Autophagy signaling in skeletal muscle of infarcted rats, PLoS One. 9 (2014) e85820. [21] D. Attaix, L. Combaret, D. Bechet, D. Taillandier, Role of the ubiquitin-proteasome pathway in muscle atrophy in cachexia, Curr Opin Support Palliat Care. 2 (2008) 262-266. [22] T.F. Cunha, A.V. Bacurau, J.B. Moreira, N.A. Paixao, J.C. Campos, J.C. Ferreira, M.L. Leal, C.E. Negrao, A.S. Moriscot, U. Wisloff, P.C. Brum, Exercise training prevents oxidative stress 23

ACCEPTED MANUSCRIPT and ubiquitin-proteasome system overactivity and reverse skeletal muscle atrophy in heart failure, PLoS One. 7 (2012) e41701. [23] S.H. Lecker, R.T. Jagoe, A. Gilbert, M. Gomes, V. Baracos, J. Bailey, S.R. Price, W.E. Mitch, A.L. Goldberg, Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression, FASEB J. 18 (2004) 39-51. [24] S.C. Bodine, E. Latres, S. Baumhueter, V.K. Lai, L. Nunez, B.A. Clarke, W.T. Poueymirou,

PT

F.J. Panaro, E. Na, K. Dharmarajan, Z.Q. Pan, D.M. Valenzuela, T.M. DeChiara, T.N. Stitt,

RI

G.D. Yancopoulos, D.J. Glass, Identification of ubiquitin ligases required for skeletal muscle atrophy, Science. 294 (2001) 1704-1708, http://www.ncbi.nlm.nih.gov/pubmed/11679633.

SC

[25] E.J. Stevenson, P.G. Giresi, A. Koncarevic, S.C. Kandarian, Global analysis of gene

NU

expression patterns during disuse atrophy in rat skeletal muscle, J Physiol. 551 (2003) 33-48. [26] S.J. Lee, A.C. McPherron, Regulation of myostatin activity and muscle growth, Proc Natl

MA

Acad Sci U S A. 98 (2001) 9306-9311.

[27] S. Lokireddy, C. McFarlane, X. Ge, H. Zhang, S.K. Sze, M. Sharma, R. Kambadur, Myostatin induces degradation of sarcomeric proteins through a Smad3 signaling mechanism

D

during skeletal muscle wasting, Mol Endocrinol. 25 (2011) 1936-1949.

PT E

[28] A. Musaro, K. McCullagh, A. Paul, L. Houghton, G. Dobrowolny, M. Molinaro, E.R. Barton, H.L. Sweeney, N. Rosenthal, Localized Igf-1 transgene expression sustains hypertrophy and

CE

regeneration in senescent skeletal muscle, Nat Genet. 27 (2001) 195-200. [29] S. Schiaffino, C. Mammucari, Regulation of skeletal muscle growth by the IGF1-Akt/PKB

AC

pathway: insights from genetic models, Skelet Muscle. 1 (2011) 4. [30] P.C. Schulze, J. Fang, K.A. Kassik, J. Gannon, M. Cupesi, C. MacGillivray, R.T. Lee, N. Rosenthal, Transgenic overexpression of locally acting insulin-like growth factor-1 inhibits ubiquitin-mediated muscle atrophy in chronic left-ventricular dysfunction, Circ Res. 97 (2005) 418-426. [31] S.Y. Yang, G. Goldspink, Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation, FEBS Lett. 522 (2002) 156-160. [32] P.K. Kandalla, G. Goldspink, G. Butler-Browne, V. Mouly, Mechano Growth Factor E peptide (MGF-E), derived from an isoform of IGF-1, activates human muscle progenitor cells 24

ACCEPTED MANUSCRIPT and induces an increase in their fusion potential at different ages, Mech Ageing Dev. 132 (2011) 154-162. [33] A. Guma, V. Martinez-Redondo, I. Lopez-Soldado, C. Canto, A. Zorzano, Emerging role of neuregulin as a modulator of muscle metabolism, Am J Physiol Endocrinol Metab. 298 (2010) E742-750. [34] N.J. Hellyer, C.B. Mantilla, E.W. Park, W.Z. Zhan, G.C. Sieck, Neuregulin-dependent protein

PT

synthesis in C2C12 myotubes and rat diaphragm muscle, Am J Physiol Cell Physiol. 291

RI

(2006) C1056-1061.

[35] M. Hirata, K. Sakuma, S. Okajima, H. Fujiwara, S. Inashima, M. Yasuhara, T. Kubo,

SC

Increased expression of neuregulin-1 in differentiating muscle satellite cells and in

NU

motoneurons during muscle regeneration, Acta Neuropathol. 113 (2007) 451-459. [36] P. Bonaldo, M. Sandri, Cellular and molecular mechanisms of muscle atrophy, Dis Model

MA

Mech. 6 (2013) 25-39.

[37] E.N. Fuentes, B.T. Bjornsson, J.A. Valdes, I.E. Einarsdottir, B. Lorca, M. Alvarez, A. Molina, IGF-I/PI3K/Akt and IGF-I/MAPK/ERK pathways in vivo in skeletal muscle are regulated by

D

nutrition and contribute to somatic growth in the fine flounder, Am J Physiol Regul Integr

PT E

Comp Physiol. 300 (2011) R1532-1542. [38] Y. Xu, X. Li, X. Liu, M. Zhou, Neuregulin-1/ErbB signaling and chronic heart failure, Adv

CE

Pharmacol. 59 (2010) 31-51.

[39] M. Haykowsky, J. Scott, B. Esch, D. Schopflocher, J. Myers, I. Paterson, D. Warburton, L.

AC

Jones, A.M. Clark, A meta-analysis of the effects of exercise training on left ventricular remodeling following myocardial infarction: start early and go longer for greatest exercise benefits on remodeling, Trials. 12 (2011) 92. [40] I. Korzeniowska-Kubacka, M. Bilinska, E. Michalak, B. Kusmierczyk-Droszcz, B. Dobraszkiewicz-Wasilewska, R. Piotrowicz, Influence of exercise training on left ventricular diastolic function and its relationship to exercise capacity in patients after myocardial infarction, Cardiol J. 17 (2010) 136-142. [41] D.G. Martinez, J.C. Nicolau, R.L. Lage, E. Toschi-Dias, L.D. de Matos, M.J. Alves, I.C. Trombetta, V.J. Dias da Silva, H.R. Middlekauff, C.E. Negrao, M.U. Rondon, Effects of 25

ACCEPTED MANUSCRIPT long-term exercise training on autonomic control in myocardial infarction patients, Hypertension. 58 (2011) 1049-1056. [42] D.J. Duncker, E.D. van Deel, M.C. de Waard, M. de Boer, D. Merkus, J. van der Velden, Exercise training in adverse cardiac remodeling, Pflugers Arch. 466 (2014) 1079-1091. [43] J.P. French, K.L. Hamilton, J.C. Quindry, Y. Lee, P.A. Upchurch, S.K. Powers, Exercise-induced

protection

against

myocardial

apoptosis

and

necrosis:

MnSOD,

PT

calcium-handling proteins, and calpain, FASEB J. 22 (2008) 2862-2871.

RI

[44] F. Giallauria, W. Acampa, F. Ricci, A. Vitelli, G. Torella, R. Lucci, G. Del Prete, E. Zampella, R. Assante, G. Rengo, D. Leosco, A. Cuocolo, C. Vigorito, Exercise training early after acute

SC

myocardial infarction reduces stress-induced hypoperfusion and improves left ventricular

NU

function, Eur J Nucl Med Mol Imaging. 40 (2013) 315-324.

[45] J. Kraljevic, J. Marinovic, D. Pravdic, P. Zubin, Z. Dujic, U. Wisloff, M. Ljubkovic, Aerobic

MA

interval training attenuates remodelling and mitochondrial dysfunction in the post-infarction failing rat heart, Cardiovasc Res. 99 (2013) 55-64. [46] D. Leosco, G. Rengo, G. Iaccarino, L. Golino, M. Marchese, F. Fortunato, C. Zincarelli, E.

D

Sanzari, M. Ciccarelli, G. Galasso, G.G. Altobelli, V. Conti, G. Matrone, V. Cimini, N. Ferrara,

PT E

A. Filippelli, W.J. Koch, F. Rengo, Exercise promotes angiogenesis and improves beta-adrenergic receptor signalling in the post-ischaemic failing rat heart, Cardiovasc Res. 78

CE

(2008) 385-394.

[47] X. Xu, W. Wan, A.S. Powers, J. Li, L.L. Ji, S. Lao, B. Wilson, J.M. Erikson, J.Q. Zhang,

AC

Effects of exercise training on cardiac function and myocardial remodeling in post myocardial infarction rats, J Mol Cell Cardiol. 44 (2008) 114-122. [48] M.T. La Rovere, C. Bersano, M. Gnemmi, G. Specchia, P.J. Schwartz, Exercise-induced increase in baroreflex sensitivity predicts improved prognosis after myocardial infarction, Circulation. 106 (2002) 945-949. [49] L. Anderson, N. Oldridge, D.R. Thompson, A.D. Zwisler, K. Rees, N. Martin, R.S. Taylor, Exercise-Based Cardiac Rehabilitation for Coronary Heart Disease: Cochrane Systematic Review and Meta-Analysis, J Am Coll Cardiol. 67 (2016) 1-12. [50] A.V. Bacurau, M.A. Jardim, J.C. Ferreira, L.R. Bechara, C.R. Bueno, Jr., T.C. Alba-Loureiro, 26

ACCEPTED MANUSCRIPT C.E. Negrao, D.E. Casarini, R. Curi, P.R. Ramires, A.S. Moriscot, P.C. Brum, Sympathetic hyperactivity differentially affects skeletal muscle mass in developing heart failure: role of exercise training, J Appl Physiol (1985). 106 (2009) 1631-1640. [51] P.C. Brum, A.V. Bacurau, A. Medeiros, J.C. Ferreira, A.S. Vanzelli, C.E. Negrao, Aerobic exercise training in heart failure: impact on sympathetic hyperactivity and cardiac and skeletal muscle function, Braz J Med Biol Res. 44 (2011) 827-835.

PT

[52] D.D. Jia, M.X. Cai, Y.Xi, S.J. Du, Z.J. Tian, Interval exercise training increases LIF

RI

expression and prevents myocardial infarction-induced skeletal muscle atrophy in rats, Life Sciences, 193 (2018) 77-86.

SC

[53] T. Moholdt, I.L. Aamot, I. Granoien, L. Gjerde, G. Myklebust, L. Walderhaug, L. Brattbakk,

NU

T. Hole, T. Graven, T.O. Stolen, B.H. Amundsen, H.E. Molmen-Hansen, A. Stoylen, U. Wisloff, S.A. Slordahl, Aerobic interval training increases peak oxygen uptake more than usual

MA

care exercise training in myocardial infarction patients: a randomized controlled study, Clin Rehabil. 26 (2012) 33-44.

[54] J.B. Moreira, L.R. Bechara, L.H. Bozi, P.R. Jannig, A.W. Monteiro, P.M. Dourado, U. Wisloff,

D

P.C. Brum, High- versus moderate-intensity aerobic exercise training effects on skeletal

PT E

muscle of infarcted rats, J Appl Physiol (1985). 114 (2013) 1029-1041. [55] M.J. Toth, M.S. Miller, P. VanBuren, N.G. Bedrin, M.M. LeWinter, P.A. Ades, B.M. Palmer,

CE

Resistance training alters skeletal muscle structure and function in human heart failure: effects at the tissue, cellular and molecular levels, J Physiol. 590 (2012) 1243-1259.

AC

[56] N.K. LeBrasseur, K. Walsh, Z. Arany, Metabolic benefits of resistance training and fast glycolytic skeletal muscle, Am J Physiol Endocrinol Metab. 300 (2011) E3-10. [57] M.X. Cai, X.C. Shi, T. Chen, Z.N. Tan, Q.Q. Lin, S.J. Du, Z.J. Tian, Exercise training activates neuregulin 1/ErbB signaling and promotes cardiac repair in a rat myocardial infarction model, Life Sci. 149 (2016) 1-9. [58] R.D. Leite, C. Durigan Rde, A.D. de Souza Lino, M.V. de Souza Campos, M. Souza, H.S. Selistre-de-Araujo, E. Bouskela, L.G. Kraemer-Aguiar, Resistance training may concomitantly benefit body composition, blood pressure and muscle MMP-2 activity on the left ventricle of high-fat fed diet rats, Metabolism. 62 (2013) 1477-1484. 27

ACCEPTED MANUSCRIPT [59] S. Schnyder, C. Handschin, Skeletal muscle as an endocrine organ: PGC-1alpha, myokines and exercise, Bone. 80 (2015) 115-125. [60] V. Chaloupka, L. Elbl, S. Nehyba, [Strength training in patients after myocardial infarct], Vnitr Lek. 46 (2000) 829-834. [61] U. Wisloff, A. Stoylen, J.P. Loennechen, M. Bruvold, O. Rognmo, P.M. Haram, A.E. Tjonna, J. Helgerud, S.A. Slordahl, S.J. Lee, V. Videm, A. Bye, G.L. Smith, S.M. Najjar, O. Ellingsen,

PT

T. Skjaerpe, Superior cardiovascular effect of aerobic interval training versus moderate

RI

continuous training in heart failure patients: a randomized study, Circulation. 115 (2007) 3086-3094.

SC

[62] F. Pereira, R. de Moraes, E. Tibirica, A.C. Nobrega, Interval and continuous exercise training

Biomed Res Int. 2013 (2013) 752817.

NU

produce similar increases in skeletal muscle and left ventricle microvascular density in rats,

MA

[63] J.L. Oliveira, C.M. Galvao, S.M. Rocha, Resistance exercises for health promotion in coronary patients: evidence of benefits and risks, Int J Evid Based Healthc. 6 (2008) 431-439. [64] D.A. Riley, J.L. Bain, J.L. Thompson, R.H. Fitts, J.J. Widrick, S.W. Trappe, T.A. Trappe, D.L.

D

Costill, Disproportionate loss of thin filaments in human soleus muscle after 17-day bed rest,

PT E

Muscle Nerve. 21 (1998) 1280-1289.

[65] P. Li, R.E. Waters, S.I. Redfern, M. Zhang, L. Mao, B.H. Annex, Z. Yan, Oxidative phenotype

CE

protects myofibers from pathological insults induced by chronic heart failure in mice, Am J Pathol. 170 (2007) 599-608.

AC

[66] Z. Yu, P. Li, M. Zhang, M. Hannink, J.S. Stamler, Z. Yan, Fiber type-specific nitric oxide protects oxidative myofibers against cachectic stimuli, PLoS One. 3 (2008) e2086. [67] J.L. Ruas, J.P. White, R.R. Rao, S. Kleiner, K.T. Brannan, B.C. Harrison, N.P. Greene, J. Wu, J.L. Estall, B.A. Irving, I.R. Lanza, K.A. Rasbach, M. Okutsu, K.S. Nair, Z. Yan, L.A. Leinwand, B.M. Spiegelman, A PGC-1alpha isoform induced by resistance training regulates skeletal muscle hypertrophy, Cell. 151 (2012) 1319-1331. [68] S.C. Kandarian, R.W. Jackman, Intracellular signaling during skeletal muscle atrophy, Muscle Nerve. 33 (2006) 155-165. [69] V.C. Foletta, L.J. White, A.E. Larsen, B. Leger, A.P. Russell, The role and regulation of 28

ACCEPTED MANUSCRIPT MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy, Pflugers Arch. 461 (2011) 325-335. [70] E. Castillero, N. Alamdari, S.H. Lecker, P.O. Hasselgren, Suppression of atrogin-1 and MuRF1 prevents dexamethasone-induced atrophy of cultured myotubes, Metabolism. 62 (2013) 1495-1502. [71] M. Sheffield-Moore, R.J. Urban, An overview of the endocrinology of skeletal muscle, Trends Endocrinol Metab. 15 (2004) 110-115.

PT

[72] S. Sriram, S. Subramanian, D. Sathiakumar, R. Venkatesh, M.S. Salerno, C.D. McFarlane, R.

RI

Kambadur, M. Sharma, Modulation of reactive oxygen species in skeletal muscle by myostatin is mediated through NF-kappaB, Aging Cell. 10 (2011) 931-948.

SC

[73] J. Heineke, M. Auger-Messier, J. Xu, M. Sargent, A. York, S. Welle, J.D. Molkentin, Genetic

NU

deletion of myostatin from the heart prevents skeletal muscle atrophy in heart failure, Circulation. 121 (2010) 419-425.

MA

[74] K. Lenk, R. Schur, A. Linke, S. Erbs, Y. Matsumoto, V. Adams, G. Schuler, Impact of exercise training on myostatin expression in the myocardium and skeletal muscle in a chronic heart failure model, Eur J Heart Fail. 11 (2009) 342-348.

D

[75] R. Hambrecht, P.C. Schulze, S. Gielen, A. Linke, S. Mobius-Winkler, S. Erbs, J. Kratzsch, A.

PT E

Schubert, V. Adams, G. Schuler, Effects of exercise training on insulin-like growth factor-I expression in the skeletal muscle of non-cachectic patients with chronic heart failure, Eur J

CE

Cardiovasc Prev Rehabil. 12 (2005) 401-406. [76] K.M. Heinemeier, J.L. Olesen, P. Schjerling, F. Haddad, H. Langberg, K.M. Baldwin, M.

AC

Kjaer, Short-term strength training and the expression of myostatin and IGF-I isoforms in rat muscle and tendon: differential effects of specific contraction types, J Appl Physiol (1985). 102 (2007) 573-581. [77] N. Juretic, G. Jorquera, P. Caviedes, E. Jaimovich, N. Riveros, Electrical stimulation induces calcium-dependent up-regulation of neuregulin-1beta in dystrophic skeletal muscle cell lines, Cell Physiol Biochem. 29 (2012) 919-930.

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ACCEPTED MANUSCRIPT Figure legends Fig. 1 Masson's trichrome staining and cardiac fibrosis analysis of myocardium in infarcted rat. Cardiomyocytes (red), collagen fibers (blue) and nuclei (brown) were shown. The level of cardiac fibrosis in the infarcted heart was evaluated by the collagen volume

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fraction (CVF). Scale bar = 100 µm. Values are expressed as mean ± SEM, n = 6.

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Fig. 2 Muscle weight and cross section area of fibers in soleus and gastrocnemius muscles in infarcted rat.

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A and B, Result of soleus weight/body weight (A) and gastrocnemius weight/body weight in each group (B); C and D, HE staining of soleus (C) and gastrocnemius (D)

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muscles, Scale bar=100 µm; E and F, Analysis of cross section area of soleus (E) and

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gastrocnemius (F) muscle fibers. Values are expressed as mean±SEM, n=10.

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Fig. 3 Pearson correlation analysis between LVEDP and CSA of muscle fibers. Pearson correlation analysis between LVEDP and CSA of soleus muscle fibers (A)

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and gastrocnemius muscle fibers (B). Data are shown as correlation coefficient

AC

(significance). LVEDP, left ventricular end diastolic pressure; CSA, cross section area.

Fig. 4 Effects of exercise training on the ROS level and activities of antioxidant enzyme in soleus muscle of infarcted rat. A, DHE staining and analysis of fluorescence area in each group. the O 2‾ level is detected by DHE fluorescence examination (red) to reflect the ROS level, Scale bar=100 µm; B-E, Result of activity of SOD (B), GSSG (C), GSH (D) and CS (E) in 30

ACCEPTED MANUSCRIPT each group. Values are expressed as mean±SEM, n=8. ROS, reactive oxygen species; DHE, Dihydroethidium; SOD, superoxide dismutase; GSSG, glutathione disulfide; GSH, glutathione; CS, citrate synthase.

Fig. 5 mRNA expression of atrogin-1 and murf1 in soleus muscle of infarcted rat.

PT

mRNA expression of atrogin-1 (A) and murf1 (B) in each group. Values are expressed

SC

RI

as mean±SEM, n=8.

Fig. 6 Protein expression of growth factors and activities of Akt and Erk1/2 in

NU

soleus muscle of infarcted rat.

A, Protein expression of MSTN, IGF1, MGF and NRG1 in soleus muscle was

MA

examined with western blotting; B, The protein expression of pAkt, total Akt, pErk1/2 and total Erk1/2 in soleus muscle was examined with western blotting. C-F, Analysis

D

of protein expression of MSTN (C), IGF1 (D), MGF(E) and NRG1(F); G and H,

PT E

Analysis of the phosphorylation of Akt (G) and Erk1/2 (H). Values are expressed as mean±SEM, n=6. MSYN, Myostatin; IGF1, insulin-like growth factor; MGF,

CE

mechano growth factor; NRG1, Neuregulin1.

AC

Fig. 7 Pearson correlation analysis between LVEDP and growth factors. A-C, Pearson correlation analysis between LVEDP and IGF1 (A), MGF (B) and NRG1 (C) in soleus muscle. Data are shown as correlation coefficient (significance). LVEDP, left ventricular end diastolic pressure; IGF1, insulin-like growth factor; MGF, mechano growth factor; NRG1, Neuregulin1.

31

ACCEPTED MANUSCRIPT Tab. 1 Survival rate and Hemodynamic parameters in rats (n=10) MI

MR

MA

ME

Survival rate

100%

90%

100%

70%

100%

BW (g)

308.50±20.75

313.67±19.81

315.14±27.07

3322.43±24.47

330.40±16.68

HW (g)

0.95±0.09

1.04±0.10

1.02±0.13

1.04±0.09

1.06±0.04

HW/BW(kg/g)

3.10±0.10

3.30±0.22

3.25±0.25

LVSP(mmHg)

134.81±4.31

96.08±3.47

LVEDP(mmHg)

2.43±0.80

10.43±0.85

PT

Sham

△△

△△

6.56±1.39*

)

NU

△△

**

MA

4731.69±69.6 3415.79±165.79 4015.30±235.55 dp/dtmax(mmHg/s)

△△

1 △

*

113.60±3.85*

4.00±3.58**

3.70±0.91**

4684.48±157.94*

4521.62±177.87

*

4567.87±101.38*

**

4249.97±159.61

▲

*

P＜0.01 vs Sham; *P＜0.05, **P＜0.01 vs MI;

** ▲

P＜0.05 vs

D

Data represented as mean ± SEM;

121.91±4.57**

SC

118.32±7.05**

+dp/dtmax(mmHg/s 5367.68±179. 3440.04±247.81 4274.45±162.06 16

3.22±0.15

RI

3.23±0.16

PT E

MR. MI, sedentary MI group; MR, MI with resistance training group; MA, MI with high-intensity intermittent aerobic exercise group; ME, MI with moderate-intensity continuous aerobic exercise group; BW, Body weight; HW, Heart weight; LVSP, left ventricle systolic pressure; LVEDP, left

CE

ventricle end-diastolic pressure; ±dp/dtmax, maximal positive and minimal negative first

AC

derivative of left ventricle pressure.

32

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7