Figure 3. Abolished CNP/cGMP signaling in CMs from mice with CM-restricted deletion (KO) of the GC-B receptor (GC-B^fl/fl αMHC-Cre^Tg).

(A) qRT-PCR analyses. GC-B and GC-A mRNA expression levels in CMs isolated from GC-B^fl/fl mice without (controls) or with the αMHC-Cre^Tg (KO). Values are the ratio of target mRNA level relative to β₂ microglobulin. n = 10 samples from 5 mice/genotype, ^†P < 0.05 vs. GC-B^fl/fl mice (Mann-Whitney U test). (B and C) CNP (10 nM to 1 μM) and ANP (100 nM) enhanced cGMP levels of control myocytes and fibroblasts. CNP effects were abolished in myocytes but preserved in fibroblasts from KO mice; ANP effects did not significantly differ between genotypes. n = 12 samples from 6 mice/genotype (2-way ANOVA). (D and E). Immunoblotting. In control myocytes, CNP increased the phosphorylation of phospholamban at Ser₁₆ (D) and of titin at Ser₄₀₈₀ (E). In KO myocytes, these effects were abolished. (Top) Representative Western blots. (Bottom) Levels of phosphorylated phospholamban (P-phospholamban) pentamers were normalized to GAPDH and of P-titin to total titin. Ratios were calculated as x-fold respective vehicle samples. n = 3 samples/genotype. *P < 0.05 versus PBS (represented as 0), ^†P < 0.05 versus control cells (2-way ANOVA). All data shown as means ± SEM.