All mice used in the experiments were bred and maintained at the SPF Animal Research Building of China Astronaut Research and Training Center (12-h light, 12-h dark cycles, temperature controlled for 23°C and free access to food and water). The mice used on this study were 3 month old males and in a C57BL/6N background. The experimental procedures were approved by the Animal Care and Use Committee of China Astronaut Research and Training Center, and all animal studies were performed according to approved guidelines for the use and care of live animals.

The hindlimb-unloading procedure was achieved by tail suspension, as described by Morey-Holton and Globus (2002). Briefly, the 3-month-old mice were individually caged and suspended by the tail using a strip of adhesive surgical tape attached to a chain hanging from a pulley. The mice were suspended at a 30° angle to the floor with only the forelimbs touching the floor, which allowed the mice to move and access to food and water freely. The mice were subjected to hindlimb unloading through tail suspension for 28 days, which we will identify as the “unloaded” state, for a total of 28 days, after which they were returned to the normal four-extremity weight bearing “reloaded” position (hindlimb reloading, HR). Similar numbers of control mice of the same strain background were instrumented and monitored in similar fashion under identical cage conditions but without tail suspension.

Sections were generated from paraffin embedded hearts, and were stained with H&E for gross morphology, Masson's trichrome for detection of fibrosis, as described before Ling et al. (2012).

Total RNA was extracted from heart tissues by using RNAiso Plus reagent (Takara) according to the manufacturer's protocol. The RNA (500 ng/sample) was reverse transcribed into cDNA and qPCR was performed using a SYBR Green PCR kit (Takara) in a Light Cycler (Eppendorf, Germany). PCR for each sample was carried out in duplicate for all cDNAs. The mRNA level of each gene was normalized to that of Gapdh, which served as an internal control. Primers (synthesized by Sunbiotech Co, China) for Col1a1 (Product size, 119 bp, Melting temperature, 61°C), Col3a1 (Product size, 98 bp, Melting temperature, 60°C), BNP (Product size, 185 bp, Melting temperature, 56°C), and Gapdh (Product size, 122 bp, Melting temperature, 59°C), were as follows:

Col1a1 sense primer: 5′-CTG ACTGGAAGAGCGGAGAGT-3′,

Animals were lightly anesthetized with 2,2,2-tribromoethanol (0.2 ml/10 g body weight of a 1.2% solution) and set in a supine position. Two dimensional (2D) guided M-mode echocardiography was performed using a high resolution imaging system (Vevo 770, Visual-Sonics Inc., Toronto, ON, Canada). Two-dimensional images are recorded in parasternal long- and short-axis projections with guided M-mode recordings at the midventricular level in both views. Left ventricular (LV) cavity size and wall thickness are measured in at least three beats from each projection. Averaged LV wall thickness [interventricular septum (IVS) and posterior wall (PW) thickness] and internal dimensions at diastole and systole (LVIDd and LVIDs, respectively) are measured. LV fractional shortening ((LVIDd – LVIDs)/LVIDd), relative wall thickness [(IVS thickness + PW thickness)/LVIDd], and LV mass [LV Mass = 1.053 × [(LVIDd + LVPWd + IVSd)3 – LVIDd3]] are calculated from the M-mode measurements. LV ejection fraction (EF) was calculated from the LV cross-sectional area (2-D short-axis view) using the equation LV %EF = (LV Vold – LV Vols)/LV Vold × 100%. For RV, Two-dimensional images are recorded in right parasternal long- and short-axis projections with guided M-mode recordings at the maximum diameter level in both views. Right ventricular (RV) cavity size and wall thickness are measured in at least three beats from each projection. Averaged Right Ventricle Anterior Wall and internal dimensions at diastole and systole (RVIDd and RVIDs, respectively) are measured. Right Ventricle Percent Fractional Shortening (RVIDd – RVIDs)/RVIDd × 100%). Right Ventricle Percent Ejection Fraction (EF) was calculated from the RV cross-sectional area (2-D short-axis view) using the equation RV %EF = (RV Vold – RV Vols)/RV Vold × 100%.

For the RV mass weight calculation, firstly, we measured the RV endocardial borders in diastole (five measures) and systole (five measures) in five consecutive cardiac cycles in each flat-image, generating 10 RV endocardial areas (RVendo). Then, we got the epicardial borders and corresponding RV epicardial areas (RVepi) on the same frames. Ten RV free wall areas were then calculated by subtracting RVendo from RVepi (RVepi-RVendo). Finally, the total RV free wall volume of each plane was calculated by Simpson's method using the mean of the RV free wall area. RV free wall mass was obtained by multiplying this volume by the specific density of the myocardium (1.05 g/cc).

Mouse hearts were crushed by homogenizer (Power Gen125, Fisher Scientific) and then lysed in lysis buffer (50 mM Tris, pH7.5, 250 mM NaCl, 0.1% SDS, 2 mM dithiothreitol, 0.5% NP-40, 1 mM PMSF, and protease inhibitor cocktail) on ice for 15 min. Protein fractions were collected by centrifugation at 12,000 g at 4°C for 15 min and then applied to 8–12% SDS-PAGE gels, electrophoresed at 80 V fof 30 min and 120 V for 90 min. After electrophoresis, protein was transfected to a polyvinylidene fluoride membrane using a Criterion blotter apparatus (Bio Rad). The membrane was then blocked in 5% non-fat dry milk (Becton, Dickinson, and Company) in TBST (10 mM Tris–Cl, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h. After that, the membrane was incubated with primary antibody overnight at 4°C followed by incubation with a secondary antibody conjugated to horseradish peroxidase (HRP), and visualized using an chemiluminescence kit (Thermo Pierce, No.32 109). Specific antibodies to p-HDAC4 (Cell Signaling Technology, #3443S), HDAC4 (Cell Signaling Technology, #5392S), p-AMPKα (Cell Signaling Technology, #2531S), AMPKα (Cell Signaling Technology, #2532), p-ERK1/2 (Cell Signaling Technology, #4370S), ERK1/2 (Cell Signaling Technology, #4696S), LC3-II/I (MBL, PM036-PN), mTOR (Cell Signaling Technology, #2983), p-mTOR (Cell Signaling Technology, #5536) Atrogin1 (ECM biosciences, # AP2041), MuRF1 (ECM biosciences, # MP3401), Gapdh (Santa Cruz Biotechnology, sc-25778) were used to detect protein levels. Gapdh was used as a loading control.

Data are presented as mean ± SEM per experimental condition. For the statistical differences among groups, considering the presence of unequal variance for the data, we firstly test the equality of variances across groups. If it shows the variances are unequal, we then use Welch's t-test for one-way analysis. Otherwise we use Student's t-test. Bonferroni adjustment was used for multiple comparisons. P < 0.05 is considered statistically significant. P < 0.01 is considered very significant. All the statistical tests are analyzed by Prism software (Graphpad prism for windows, version 5.01).

Three groups of mice were subjected to HU by tail suspension for 28 days following which two groups were allowed to recover for 7 or 14 days (HU-28d, n = 10; HR-7d, n = 10; HR-14d, n = 10). The control group (n = 10) was treated equally, with the exception of tail suspension (Figure 1A). Body weight and heart weight were recorded before sacrifice (Figures 1B,C), and the mass of the LV and RV were calculated by echocardiography (Figures 1E,F). Compared with the control group, body weight showed an overall decrease, while heart weight increased following HU and HR. Thus, the ratio of heart weight to body weight increased following 28 days of HU and 7 days of HR (Figure 1D). LV mass calculated by echocardiography remained unchanged following HU or HR (Figure 1E), but RV mass exhibited an overall decrease following HU, and recovered following HR (Figure 1F). All echocardiographic measurements were made while mice maintained heart rates of 450 ± 50 beats per minute (Figure 1G).

To validate the effects of HU and HR on the LV and RV, transthoracic echocardiography was performed to determine ventricular function following HU and HR. After 28 days of HU, left ventricular fractional shortening (LV-FS), and left ventricular ejection fraction (LV-EF) decreased significantly in HU mice compared with the control, and did not recover during the first few days of HR. Full recovery was only apparent after reloading for 14 days (Figure 2A). However, after 28 days of HU, RV-FS and RV-EF decreased significantly in HU mice compared with the control, and did not recover even when reloaded for 14 days (Figure 2B). The results indicate that simulated microgravity can induce a decline in left and right ventricular function, and that recovery is slower in the RV after reloading.

To validate the influence of HU and HR in the LV, we performed transthoracic echocardiography to determine the structure of the LV following HU and HR (Figure 3A). Compared with control, the end-systolic LV internal diameter (LVIDs), and the end-diastolic LV internal diameter (LVIDd) did not change in the HU-28d mice, but increased following HR for 7 days, and recovered after 14 days of HR (Figures 3B,C). Furthermore, the change in end-systolic LV volume (LV-Vols) and the end-diastolic LV volume (LV-Vold) was the same as for LVIDs and LVIDd (Figures 3D,E). The LV posterior wall thickness (LVPW) and the LV anterior wall thickness (LVAW) didn't change following HU or HR (Figures 3F–I). Together, these data show that reloading after HU can induce enlargement of the left ventricular internal diameter and volume.

To validate the influence of HU and HR in the RV, we performed transthoracic echocardiography to assess the structure and function of the RV following HU and HR (Figure 4A). After 28 days of HU, the end-diastolic RV internal diameter (RVIDd) and the end-diastolic RV volume (RV-Vold) decreased in the HU mice compared with control, but recovered to its normal state in the HR-7d and HR-14d groups (Figures 4B,D). Furthermore, the end-systolic RV internal diameter (RVIDs) and the end-systolic RV volume (RV-Vols) did not change following HU, but increased in the HR-7d and HR-14d groups (Figures 4C,E). The RV anterior wall thickness (RVAWd and RVAWs) and the interventricular septal thickness (IVSd and IVSs) did not change following HU or HR (Figures 4F–I). Together, these data show that HU and HR induce different structural changes in the LV and RV (Figure 4J) and, during the recovery process, cardiac remodeling may be intensified because of reloading.

To address the influence of HU and HR in the LV and RV, hearts from mice following HU and HR were assessed for changes in morphology and gene expression. Histological analysis showed heart remodeling occurred following HU and HR. In hematoxylin and eosin-stained (HE) sections, gross evidence of edema was easily observed by separation of the myofibers in the LV, the interventricular septum (IVS), and the RV following HU. Recovery to a normal state occurred following 14 days of HR (Figure 5A). Masson trichrome staining (MTT) showed a deeper staining of collagen in the LV, the interventricular septum, and the RV following HU. These changes also recovered after 14 days of reloading. In the HU-28d and HR-7d groups, relative Col1a1, Col3a1, and BNP mRNA levels increased in the LV, and recovered, although not significantly (Figure 5B). In the RV, the relative mRNA levels of Col1a1 and Col3a1 increased in the HU-28d group, and only the level of Col1a1 recovered to its normal state in the HR-14d group. The relative mRNA level of BNP increased following HU, and continued to increase after 7 days of HR (Figure 5C). These results demonstrate that HU leads to slight fibrosis and remodeling in both the left and right ventricle, and recovery occurs slowly after 14 days of reloading.

To gain more insight into the signaling pathways involved in the declining function of both the left and right ventricles, we examined several important signaling molecules involved in cardiac remodeling induced by external or intrinsic stimuli. As shown in Figure 6A, quantification of phosphorylation levels normalized to total protein in the LV revealed that HDAC4 phosphorylation at Ser246 did not change following HU but increased following 7 days of reloading, and was fully restored after 14 days. Compared with the control, Erk1/2 phosphorylation at Thr202/Tyr204 increased following HU, remained the higher level during the first 7 days of reloading, and was fully restored after 14 days. The phosphorylation level of AMPK at Thr172 decreased following HU, continued to decrease after 7 days of reloading, and was restored after 14 days. According to the research of Liu et al. (2015), autophagy is involved in HU-induced decline in heart function, so we assessed the change of LC3-II in our model. Quantification of LC3-II levels normalized to LC3-I revealed an increased ratio of LC3-II: LC3-I following HU, and this ratio did not return to its normal state until after 14 days of reloading. The changes of these signaling pathways in RV following HU and HR were also analyzed. As shown in Figure 6B, the level of HDAC4 phosphorylation at Ser246 in the RV increased following HU, however it was not restored to the normal level until 14 days of reloading, which is in contrast to the changes in LV. Phosphorylation of Erk1/2 at Thr202/Tyr204 was up-regulated increased following HU, and recovered after 7 days of reloading. The phosphorylation of AMPK at Thr172 was reduced following HU, and continued to decrease after 7 days of reloading, however, it was not restored to its normal state until 14 days after reloading, which is different from the changes in the LV. The changes of LC3-II: LC3-I was the same as that for the LV.

We also analyzed the changes of mTOR phosphorylation and MuRF1/Atrogin1 levels, which are involved in protein synthesis and degradation pathways, respectively. In LV, As is shown in Figure S1A, mTOR phosphorylation at S2448 was decreased following HU, and was restored following 7 days of reloading. In comparison with the control, the level of Atrogin1, an E3 ubiquitin ligase that mediates proteolysis events during muscle atrophy, obviously increased following HU, and remained much higher level during the first 7 days of reloading, then restored to the normal level after 14 days of reloading. The level of MuRF1, another ubiquitin ligases, was increased in HU group, however, it did not recover to the normal condition even after 14 days of reloading (Figure S1A). In RV, as shown in Figure S1B, the changes of phosphorylation level of mTOR was the same as that for the LV. The levels of Atrogin1 and MuRF1 were both substantially increased following HU, and restored till 14 days of reloading (Figure S1B). The changes of these signaling pathways were closely related to disorders of cardiac function observed both in the LV and RV and the differences between them.

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. The reviewer [JA] and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review