The experimental procedures in mice and protocol complied with the National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Animal Care and Use Committee of China Astronaut Research and Training Center (ACC-IACUC-2020-002). Myocardial-specific CKIP-1 transgenic mice and CKIP-1-deficient mice have been obtained in previous reports (Ling et al., 2012). Male mice aged 8–10 weeks (weight 21–25 g) of WT, CKIP-1 transgenic (TG) mice, and CKIP-1 knockout (KO) mice were used in the study.

We followed the previously described 3-week swimming protocol to induce physiological cardiac hypertrophy (Wilkins et al., 2004; Taniike et al., 2008; Heineke et al., 2010). It began with 8 days of training time before the formal swimming exercise that was increased gradually 10 min every day until two 90-min phases were finished on the ninth day. Subsequently, mice went on swimming for as long as 12 additional days (21 days total). Of note, swimming training on the first day included two 10-min sessions separated by at least 4 h. Rest mice served as controls, which did not undergo any swimming training.

Mice were anesthetized with isoflurane (EZVET, No.04574). Two-dimensional guided M-mode echocardiography was performed using a high-resolution imaging system (Vevo 1100, 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 fractional shortening (FS) and ejection fraction (EF), and other parameters, including end-diastolic left ventricular posterior wall thickness (LVPWd), end-systolic left ventricular posterior wall thickness (LVPWs), end-diastolic left ventricular anterior wall thickness (LVAWd), end-systolic left ventricular anterior wall thickness (LVAWs), end-diastolic left ventricular internal diameters (LVIDd), end-systolic left ventricular internal diameters (LVIDs), and left ventricular mass were determined. Structure and function of left ventricular were measured as previously described (Ling et al., 2012).

Myocardial tissues from the left ventricular of mice were harvested and fixed with 4% paraformaldehyde solution for 24 h. Then, tissues were paraffin embedded and sectioned serially according to routine procedures. The sections (4–5 μm) underwent hematoxylin-eosin (H&E) staining in preparation for histological staining and Masson’s trichrome staining to detect collagen deposition.

Heart tissues were 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 20 min. Equivalent protein lysates were used for 10% SDS-PAGE according to the molecular weight of target proteins, electro-transferred onto nitrocellulose membranes (Millipore, Billerica, MA, United States), and then blocked in 5% non-fat dry milk in TBST (10 mM Tris-Cl, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 2 h. Subsequently, the membrane was incubated overnight at 4°C with primary antibodies against HDAC4 (1:2,000, Cell Signaling Technology, United States, 5392S), p-HDAC4 (S632) (1:2,000, Cell Signaling Technology, United States, 3424S), p-HDAC4 (S246) (1:2,000, Cell Signaling Technology, United States, 3443S), CKIP-1 (1:2,000, Proteintech, United States, 24883-1), and GAPDH (1:5,000, Santa Cruz Biotechnology, United States, sc-25778). After incubating with the corresponding secondary antibodies conjugated to horseradish peroxidase (HRP), these were visualized using a chemiluminescence kit (Thermo Pierce, United States, No.32 109). The intensities of bands were analyzed with ImageJ software (NIH).

Total mRNA was isolated from left ventricular tissues with TRIzol reagent (Takara, Japan) and reverse transcribed into cDNA with a Transcriptor First Strand cDNA Synthesis kit (Takara, Japan). The relative expression of specific genes was detected using quantitative real-time PCR with a SYBR Green PCR kit (Takara, Japan) in a Light Cycler (Eppendorf, Germany). The mRNA level of each gene was normalized to Gapdh, which served as an internal reference. The sequences of the primers are:

Data are presented as mean ± SEM per experimental condition. Statistical differences between two groups were determined by the unpaired two-tailed Student’s t-test. Statistical differences among groups were analyzed by two-way analysis of variance (ANOVA) followed by the Bonferroni procedure. All the statistical tests are analyzed by Prism software (Graphpad prism for Windows, version 6.01) and SPSS (Version 14.0). Differences were considered statistically significant when P value was <0.05.

To investigate whether CKIP-1 expression was altered during physiological cardiac hypertrophy, we subjected WT mice to a high-intensity swimming exercise regime (Figure 1A). Swimming exercise training remarkably promotes physiological cardiac growth. In comparison with the rest mice, trained mice had higher heart weight-to-body weight ratios (Supplementary Figure 1A) and cardiac contractile function (ejection fractions and fraction shortening; Supplementary Figures 1B,C). Furthermore, exercise training does not affect pathological hypertrophy marker genes atrial natriuretic peptide ANP, brain natriuretic peptide BNP, and β-MHC expression (Supplementary Figures 1D–F).

We measured the mRNA and protein levels of CKIP-1 in the hearts of mice after the swimming exercise via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses, respectively. As shown in Figure 1B, Ckip-1 mRNA levels were significantly higher in the hearts of WT mice after 21 days of swimming exercise compared with resting control mice. Similarly, CKIP-1 protein levels were significantly higher in the hearts of mice subjected to swimming exercise compared with resting mice (Figures 1C,D). These results suggest a potential role for CKIP-1 in the progression of swim-induced physiological cardiac hypertrophy.

Next, we investigated the role of CKIP-1 in the regulation of heart growth after swimming exercise. Eight-week-old CKIP-1 KO mice and littermate controls were subjected to a swimming protocol to induce physiological cardiac hypertrophy. CKIP-1 KO mice exhibited cardiac fibrosis prior to exercise, which was significantly exacerbated after the 21-day swimming exercise program, as revealed by Masson trichrome staining of cardiac tissues and quantification of interstitial fibrosis (Figure 2A and Supplementary Figure 2A). The WT mice exhibited a remarkable hypertrophic response to the exercise stimuli; however, the heart weight to body weight ratio and left ventricular (LV) weight to body weight ratio were significantly lower in CKIP-1 KO mice after the swimming exercise (Figures 2B,C).

To further evaluate the effects of CKIP-1 ablation on cardiac function and structure following swimming exercise, we performed echocardiography on CKIP-1-TG and WT mice. Notably, CKIP-1 KO mice exhibited signs of cardiac dysfunction after swimming, such as decreased ejection fractions and fraction shortening, while WT mice displayed significant increases in cardiac function (Figures 2D–F). Swim-trained WT mice exhibited attenuated LV end-systolic internal diameters (LVIDs). Furthermore, the diastolic and systolic LV internal diameters (LVIDd and LVIDs, respectively) were significantly larger in the hearts of CKIP-1 KO mice after swimming compared with WT mice (Figures 2G,H). The diastolic and systolic left ventricular posterior walls (LVPWd and LVPWs, respectively) were significantly thicker in the hearts of WT mice after the swimming exercise compared with before; however, this hypertrophy phenotype was not present in CKIP-1 KO mice, which exhibited no significant difference in LVPWd or LVPWs thickness after the swimming exercise (Figures 2I,J). No significant difference in the LV end-diastolic anterior wall thickness (LVAWd) (Figure 2K) or LV end-systolic anterior wall thickness (LVAWs) (Figure 2L) was observed between the WT and CKIP-1 KO mice after swimming, whereas cardiac stroke volume (SV) was drastically reduced in CKIP-1 KO mice after swimming (Supplementary Figure 2B). Meanwhile, heart rate (HR) was also depressed in the hearts of CKIP-1 KO mice with swimming exercise (Supplementary Figure 2C). Our findings suggest that CKIP-1 KO mice underwent maladaptive cardiac remodeling in response to swimming exercise, leading to attenuated cardiac function and fibrosis.

To further explore the physiological function of cardiac CKIP-1 in vivo after swimming, we subjected cardiac-specific CKIP-1-TG transgenic mice and littermate controls to a swimming exercise program. We evaluated baseline cardiac morphology and hypertrophy prior to exercise. CKIP-1 TG mice showed no differences in individual cardiomyocyte size, cardiac fibrosis (Supplementary Figure 3A), and heart weight-to-body weight ratio or LV weight/body weight compared with the controls (Figures 3A–C). Moreover, the left ventricular ejection fractions and fraction shortening were not significantly different between CKIP-1 TG mice and controls. These results indicate a lack of adverse cardiac remodeling at baseline in CKIP-1 TG mice (Figures 3D–F). After the swimming program, an increase in hypertrophic growth and cardiac function was observed in CKIP-1 TG mice, similar to the WT controls.

Echocardiographic measurements revealed a reduction in the LVIDd and LVIDs after swimming in both CKIP-1 TG and WT mice (Figures 3G,H). The LVPWd was thicker in the hearts of swim-trained CKIP-1 TG mice compared with swim-trained WT hearts and CKIP-1 TG resting controls (Figure 3I). Compared with the resting controls, the LVPWs was significantly thicker in the hearts of CKIP-1 TG and WT mice following swimming exercise (Figure 3J). The diastolic and systolic LV anterior wall thickness (LVAWd and LVAWs) increased after the swimming program in the hearts of CKIP-1 TG mice, but were lower in CKIP-1 TG mice compared with the controls (Figures 3K,L). Stroke volume (SV) and heart rate (HR) were unchanged in CKIP-1 TG compared with WT groups after swimming (Supplementary Figures 3B,C). Taken together, these findings indicated that cardiac-specific overexpression of CKIP-1 in adult mice induced physiological cardiac hypertrophy, with increased heart weight and cardiac function without interstitial fibrosis after swimming.

Our previous studies demonstrated that CKIP-1 plays a key role in the suppression of cardiac hypertrophy by promoting HDAC4 dephosphorylation (Ling et al., 2012, 2018). CKIP-1 deficiency triggers translocation of HDAC4 from the nucleus into the cytoplasm in cardiomyocytes (Supplementary Figure 4). To better understand the cellular signaling pathways contributing to the CKIP-1 KO and CKIP-1 TG phenotypes after the swimming exercise, we examined the phosphorylation levels of HDAC4 in heart tissues. HDAC4 phosphorylation at Ser246 and Ser632 was significantly higher in CKIP-1 KO mice compared with WT mice, both at rest and during swimming exercise. HDAC4 activation was further increased in CKIP-1 KO mice after the 21-day swimming exercise (Figures 4A,B); HDAC5 phosphorylation levels were also higher after exercise (Supplementary Figure 5). Canonical heart failure-associated marker genes, including ANP, BNP, and myosin heavy chain β (β-MHC), were upregulated in the hearts of CKIP-1 KO mice compared with controls under both baseline and swim-stimulated conditions (Figures 4C–E). In contrast to the CKIP-1 KO mice, the Ser246 and Ser632 HDAC4 phosphorylation levels in the hearts of CKIP-1 TG mice were similar before and after swim training, and lower than in the WT controls (Figures 5A,B). qRT-PCR analysis revealed that the pathological hypertrophic markers (ANP, BNP, β-MHC) were not significantly different (Figures 5C–E). Together, these results indicated that CKIP-1 KO mice promoted HDAC4 activation and reactivation of the fetal gene program in adult hearts in response to swimming exercise, thereby stimulating pathological cardiac remodeling. On the other hand, myocardial-specific CKIP-1 overexpression did not affect HDAC4 phosphorylation levels following swimming exercise. Indeed, cardiac HDAC4 phosphorylation levels were lower in CKIP-1 TG mice compared with WT controls. No significant differences in the expression of pathological hypertrophic marker genes were observed after swimming exercise in CKIP-1 TG mice (Figure 6).