C1q-TNF-related protein-3 attenuates pressure overload-induced cardiac hypertrophy by suppressing the p38/CREB pathway and p38-induced ER stress

Fig. 1. CTRP3 deficiency aggravates cardiac hypertrophy induced by TAC. a Representative western blot of CTRP3 levels in different tissues of WT mice and Ctrp3-KO mice (n = 3 mice per group). b The HW/BW ratio in animals after 4 weeks of TAC (n = 5–8 mice per group). c–e The left ventricular ejection fraction (LVEF), IVSd, and LVPWd, accordingly, determined by analyzing the echocardiographic images (n = 12–14 mice per group). f Representative images of the gross murine heart and sections stained with hematoxylin and eosin (HE), and wheat germ agglutinin (WGA) (n = 5–8 mice per group). g The mean cross-sectional area of cardiomyocytes from the indicated groups (n ≥ 100 cells per group). h Representative images of the murine heart sections (after 4 weeks of TAC) stained with Masson stain, arranged with the perivascular area at the top and the interstitial area at the bottom (n = 5–8 mice per group). i The LV collagen volume in different groups (n ≥ 40 fields per group). j Real-time polymerase chain reaction (real-time PCR) analysis of the expression of genes encoding hypertrophic markers α-MHC, β-MHC, ANP, BNP, Acta-1, IL-6, and Rcan1.4, and the fibrotic markers TGF-β1, collagen-I, and…

Fig. 2. CTRP3 overexpression represses pressure overload-induced cardiac hypertrophy. a Representative western blot and quantification of CTRP3 levels in the heart tissue of WT mice and LV-CTRP3 mice (n = 3 mice per group). b CTRP3 serum levels in mice infected with LV-NULL or LV-CTRP3 (n = 3 mice per group). c The HW/BW ratio in animals after 4 weeks of TAC surgery (n = 5–7 mice per group). d–f LVEF, IVSd, and LVPWd, accordingly, determined by analyzing the echocardiographic images (n = 12–14 mice per group). g Representative images of the gross murine heart and sections stained with HE and WGA (n = 5–7 mice per group). h The mean cross-sectional area of cardiomyocytes from the indicated groups (n ≥ 100 cells per group). i Representative images of the murine heart sections (after 4 weeks of TAC) stained with Masson stain, arranged with the perivascular area at the top and the interstitial area at the bottom (n = 5–7 mice per group). j The LV collagen volume in different groups (n ≥ 40 fields per group). k Real-time PCR analysis for the expression of genes encoding the hypertrophic markers α-MHC, β-MHC, ANP, BNP, Acta-1, IL-6, and Rcan1.4, and the fibrotic markers TGF-β1, collagen-I, and…

Fig. 3. CTRP3 alters PE-induced cardiomyocyte hypertrophy in NRCMS. a Left: Representative immunofluorescence images of the NRCMs stained by α-actinin (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue) observed under a confocal microscope. Right: The mean cell surface area of NRCMs from the indicated groups (n ≥ 30 cells per group). b Real-time PCR analysis of the expression of genes encoding the hypertrophic markers β-MHC, ANP, and BNP in the indicated groups (n = 5 samples per group). c Representative western blot (left), and quantification (right) of β-MHC, ANP and CaN levels in the indicated groups (n = 5 samples per group). d Left: Representative immunofluorescence images of the NRCMs stained by α-actinin (red) and DAPI (blue), observed under a confocal microscope. Right: The mean cell surface area of NRCMs from the indicated groups (n ≥ 30 cells per group). e Real-time RT-PCR analysis of the expression of genes encoding the hypertrophic markers β-MHC, ANP, and BNP in the indicated groups (n = 5 samples per group). f Representative western blot (left), and quantification (right) of β-MHC, ANP and CaN levels in the indicated groups (n = 5 samples per group). The data were analyzed…

Fig. 4. CTRP3 regulates activation of the p38 MAPK/CREB pathway in TAC mouse. a, b Representative western blot (top) and quantification (bottom) of the p38-CREB signaling pathway activity in the hearts of mice with different genotypes (WT, Ctrp3-KO, and LV-CTRP3) 4 weeks after sham treatment or TAC surgery (n = 5–6 mice per group). c, d Representative immunofluorescence images of murine heart sections stained with p-p38 (red) and DAPI (blue) (left), and the percentage of p-p38–positive nuclei (right) in the hearts of mice with different genotypes (WT, Ctrp3-KO, and LV-CTRP3) 4 weeks after sham treatment or TAC surgery (n = 5–6 mice per group). The data were analyzed by one-way ANOVA. *p < 0.05, **p < 0.01 vs. SHAM, #p < 0.05 vs. TAC. In the bar graphs, the data are presented as the mean ± SEM

Fig. 5. P38 MAPK activation mediates CTRP3 deficiency-aggravated cardiac hypertrophy and fibrosis. a Ejection fraction (EF)% after 4 weeks of TAC in the indicated groups (n = 5–6 mice per group). b The HW/BW ratio in animals from indicated groups after 4 weeks of TAC (n = 5–6 mice per group). c Representative images of the heart sections stained with HE, WGA, and Masson stain (n = 5 mice per group). d The mean cross-sectional area of cardiomyocytes from the indicated groups (n ≥ 100 cells per group). e The LV collagen volume in different groups (n ≥ 40 fields per group). f Real-time PCR analysis of the expression of genes encoding the hypertrophic markers β-MHC, ANP, and BNP, and the fibrotic markers TGF-β1, collagen-I, and collagen-III in each group (n = 5 mice per group). g Representative western blot (top) and quantification (bottom) of the CREB activity in the hearts of mice from indicated groups (n = 5 mice per group). The data were analyzed by one-way ANOVA. *p < 0.05 between the two indicated groups; ns, not significant. In the bar graphs, the data are presented as the mean ± SEM

Fig. 6. CTRP3 alleviates PE-induced cardiomyocyte hypertrophy in vitro by inhibiting the p38 MAPK pathway. a Representative western blot (left) and quantification (right) of the p38-CREB signaling pathway activity in NRCMs from the indicated groups (n = 5 samples per group). b Representative western blot (left) and quantification (right) of the p38-CREB signaling pathway activity in NRCMs from the indicated groups (n = 5 samples per group). c Left: Representative immunofluorescence images of NRCMs transfected with siRNA or si-CTRP3, and stained with α-actinin (red) and DAPI (blue). The NRCMs were treated with dimethyl sulfoxide (DMSO) or a p38 inhibitor SB203580 (1 μM). Right: The mean cell surface area of NRCMs in the indicated groups (n ≥ 30 cells per group). d Real-time PCR analysis of the expression of genes encoding the hypertrophic markers β-MHC, ANP, and BNP in the indicated groups (n = 5 samples per group). e Representative western blot (left), and quantification (right) of p-CREB and CREB levels in NRCMs from the indicated groups (n = 5 samples per group). f Left: Representative immunofluorescence images of PBS- and CTRP3-treated NRCMs stained with α-actinin (red) and DAPI…

Fig. 7. Protein p38-induced activation of the ER stress pathway is involved in pathological cardiac hypertrophy. a Representative western blot of the activity of GRP78, eIF2α, IRE1α, CHOP, and ATF6 in mice hearts from indicated groups. b Statistical diagrams of GRP78, eIF2α, IRE1α, CHOP, and ATF6 protein expression in mice hearts from indicated groups (n = 6 mice per group). c Representative western blot of the activity of GRP78, eIF2α, IRE1α, CHOP, and ATF6 in mice hearts from indicated groups. d Statistical diagrams of GRP78, eIF2α, IRE1α, CHOP, and ATF6 protein expression in mice hearts from indicated groups (n = 6 mice per group). e, f Representative immunofluorescence images of murine heart sections stained with GRP78 (red) and DAPI (blue) in the hearts of mice with different genotypes (WT, Ctrp3-KO, and LV-CTRP3) 4 weeks after sham treatment or TAC surgery (n = 5–6 mice per group). g Representative western blot (left), and quantification (right) of GRP78, eIF2α, IRE1α, CHOP, and ATF6 in the hearts of mice from the indicated groups (n = 5 mice per group). The data were analyzed by one-way ANOVA. b, d *p < 0.05, **p < 0.01 vs. SHAM, #p < 0.05 vs. TAC. g *p < 0.05 and #p < 0.05…

Fig. 8. CTRP3 exerted its protective role on pathological cardiac hypertrophy through inhibiting p38/CREB/ER stress pathway. a Representative western blot (left) and quantification (right) of the ER stress signaling pathway activity in NRCMs from the indicated groups (n = 4 samples per group). b Representative western blot (left) and quantification (right) of the p38-CREB and ER stress signaling pathway activity in NRCMs from the indicated groups (n = 4 samples per group). c Representative western blot (left) and quantification (right) of ER stress and p38-CREB signaling pathway activity, protein expression levels of ANP and β-MHC in NRCMs from the indicated groups (n = 4 samples per group). *p < 0.05, **p < 0.01 between indicated groups. In the bar graphs, the data are presented as the mean ± SEM. d Proposed protective mechanism of the role of CTRP3 in pathological cardiac hypertrophy. CTRP3 inhibits the activation of the p38/CREB signaling pathway and ER stress pathway in cardiomyocytes under pressure overload, thereby alleviating pathological cardiac hypertrophy