Fig 2: Gsα deletion downregulated BMP-mediated signaling and cAMP/CREB1 signaling.A Heat map representing differentially expressed genes between GsαCMKO mice and control mice B Gene ontology analysis of differentially expressed genes C KEGG analysis of top ten enriched pathways D Quantitative analysis of the mRNA expression of differentially expressed genes involving Acta1, Nppa, Nppb, Myh7, Creb1, and Bmp10, n = 3 per group E Western blot analysis of protein level of total CREB1, p-CREB1 (at ser-133), and PKA between GsαCMKO mice and control mice. F Quantitative analysis of the expression of CREB1/GAPDH, p-CREB1/GAPDH, and PKA/GAPDH, n = 3 per group G Western blot analysis of key signaling molecules of Bmp10-mediated Smad-dependent signaling at between GsαCMKO mice and control mice. H Quantitative analysis of the expression of Bmp10/GAPDH, p-Smad1/5/8 (at Ser463/Ser465/Ser467)/GAPDH, Type I collagen/GAPDH, and Type III collagen/GAPDH, n = 3 per group I Western blot analysis of key signaling molecules of Bmp10-mediated Smad-independent signaling between GsαCMKO mice and control mice. Including Stat3, p-Stat3 (at Tyr705), p-AKT (at Ser473), AKT, p-P38 (at Thr180/Tyr182), and P38. J Quantitative analysis of the expression of p-AKT/total AKT, p-P38/total P38, n = 3 per group. Mean ± SEM, *p < 0.05, **p < 0.01, ****p < 0.0001 vs control group. Statistical analysis was carried out by a two-tailed Student’s t-test.

Fig 3: CREB1 binds with BMP10 promotor and regulates its expression.A Correlation of Gnas and CREB1, as well as the CREB1 and Bmp10 in the GTEx database. B Western blot analysis of CREB1 and Bmp10 on mice treated with forskolin for 2 days. n = 4 per group C Quantitative analysis of the expression of CREB1/GAPDH and Bmp10/GAPDH. n = 4 per group. D CREB1 binding motif in HOMER database and ChIP-Seq analysis of CREB1 binding site in Bmp10 promotor region. E ChIP-PCR analysis to verify the binding site of CREB1 in the Bmp10 promotor. F Luciferase reporter assay in 293 T cells. n = 3 per group G Schematic diagram of Gsα-regulated cardiac function and cardiac structure via CREB1/Bmp10-mediated signaling. Mean ± SEM, *p < 0.05, ****p < 0.0001. Statistical analysis was carried out by a two-tailed Student’s t-test.

Fig 4: Generation of recombinant human pBMP10. A, schematic diagram of BMP10 production and processing. B, FPLC chromatography gel filtration trace of purified pBMP10. C, purified pBMP10 shown as the peak fraction from the gel filtration in B on a non-reducing SDS-PAGE. A single asterisk denotes a nonspecific protein. D, both D- and M-forms of BMP10 GFD could be detected by monoclonal anti-BMP10 antibody. E, prodomain can be detected by anti-BMP10 prodomain antibody. BMP10 GFD from R&D Systems was used as a positive control in D and negative control in E.

Fig 5: Right-atrium-secreted active BMP10 is in the prodomain-bound form. Proteins from RA-conditioned medium were separated by gel filtration chromatography, and the BMP activities in the fractions were measured using the BRE-luciferase assay as described in “Experimental Procedures.” The relative BRE-luciferase activities in the fractions were plotted against their elution volumes (black line with circle symbols). The same process was repeated with diluted, purified recombinant human pBMP10, and the relative BMP activities in the fractions were also measured and plotted (gray line with square symbols). In addition, a pBMP10-specific ELISA was carried out to measure the proteins in the fractions from RA-conditioned medium gel filtration. Data were presented as optical densities (absorbance at 405 nm, right axis, dotted line with triangular symbols). Proteins from Gel Filtration Calibration Kit were run under identical conditions, and arrows below showed the elution volumes of the protein standards.