C-type natriuretic peptide moderates titin-based cardiomyocyte stiffness

Figures

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  Figure 1. CNP infusion to mice did not change LV titin isoform composition but enhanced titin phosphorylation. (A) Titin gels (1.8% SDS-PAGE). (B) Western blot analyses using antibodies against total Ser/Thr-phosphorylated titin (left panel) and phospho-site–specific antibodies recognizing titin phosphorylated at Ser4080 (the PKGI-specific site; right panel). Data are shown as mean ± SEM. n = 6–8/group, *P < 0.05 versus sham (Student’s t test). T2, titin degradation; P, phospho.

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  Figure 2. LV CNP expression increases in response to pressure overload provoked by TAC. (A and B) Real-time qRT-PCR and immunoblot: CNP mRNA levels were normalized to β2-microglobulin (A) and protein levels to GAPDH (B); ratios were calculated as x-fold versus sham. (C) Immunohistochemistry: representative pictures illustrate increased number of CNP-containing cells surrounding cardiomyocytes after TAC. (D and E) qRT-PCR: enrichment of cardiac CD31+ endothelial cells and PDGFR-β–positive pericytes with separation of PDGFR-α–positive fibroblasts (D) revealed TAC-induced CNP increases in endothelial cells (E). Target genes were normalized to β2-microglobulin. Data are shown as means ± SEM. n = 6–12 samples from 3–6 mice/group (in A and B), 26 samples from 13 mice (in D), and 10 sham (5 mice) versus 16 TAC (8 mice) (in E); *P < 0.05 vs. sham (A, B, and E) or other cell types (in D) (A: Kruskal-Wallis test; B: unpaired Student’s t test; D: 1-way ANOVA; and E: Mann-Whitney U test).

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  Figure 3. Abolished CNP/cGMP signaling in CMs from mice with CM-restricted deletion (KO) of the GC-B receptor (GC-Bfl/fl αMHC-CreTg). (A) qRT-PCR analyses. GC-B and GC-A mRNA expression levels in CMs isolated from GC-Bfl/fl mice without (controls) or with the αMHC-CreTg (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-Bfl/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…

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  Figure 4. Pressure overload–induced LV remodeling was not different in control and CM GC-B–KO littermates. (A) Systolic blood pressure in the ascending aorta, proximal to the TAC, measured by retrograde catheterization in anesthetized mice. (B) LVW/tibia length ratios. (C) LV CM cross-sectional areas (90–120 cells per heart were measured). (D) LV interstitial collagen fractions (3 sections per mouse). Data are shown as mean ± SEM. n = 11–19/group, *P < 0.05 versus sham (A, C, and D, tested by 2-way ANOVA with Bonferroni’s multiple comparison t test; B by Kruskal-Wallis analysis).

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  Figure 5. CM GC-B–KO mice with TAC during 3 or 14 days developed mild systolic and diastolic dysfunction and LV stiffening. (A and B) Invasive catheterizations showed that the maximal rates of LV contraction (A) and relaxation (B) were almost fully preserved in control mice subjected to 3 and 14 days of TAC but mildly impaired in CM GC-B–KO mice (in comparison with respective sham mice). (C–E) End-diastolic volumes (C) and pressures (D) as well as EDPVRs (E) were unaltered in control but increased in CM GC-B–KO mice with TAC. Data are shown as mean ± SEM. n = 11–19/group (A–D) or 4–7/group (E); *P < 0.05 versus sham, †P < 0.05 versus control mice. (A and D: Kruskal-Wallis analysis; B, C, and E: 2-way ANOVA with Bonferroni’s multiple comparisons).

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  Figure 6. TAC increased the LV levels of Ser4080-phosphorylated titin in control but not in CM GC-B–KO mice. (A) Titin gels (1.8% SDS-PAGE) revealed that titin isoform composition was not altered by genotype or condition. (B) Western blot analyses using antibodies against total Ser/Thr-phosphorylated titin. (C) Phospho-site–specific antibodies recognizing titin phosphorylated at Ser4080 (the PKGI-specific site). The levels of phosphorylated titin were normalized to total titin (PVDF) and calculated as percentage (%) from sham control mice. Data are shown as mean ± SEM. n = 8 mice/group tested in duplicates. *P < 0.05 versus sham-operated mice; †P < 0.05 versus control (GC-Bfl/fl) mice (2-way ANOVA).

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  Figure 7. Isolated LV skinned CMs from CM GC-B–KO mice with TAC showed enhanced stiffness, which was reversed by addition of recombinant PKGI. (A) Stretch protocol used in these experiments (sarcomere lengths 1.8–2.4 μm). (B–D) Single skinned CMs were prepared from sham control (CTR) and CM GC-B–KO (KO) mice (B) and from both genotypes after 3 days (C) or 14 days (D) of TAC for recordings of the force response to stepwise cell stretching in relaxing buffer. Top panels in C and D: examples of original recordings. (Bottom) In addition passive force (Fpassive) in relation to sarcomere length was recorded for control and GC-B–KO CMs from mice with TAC without or with pretreatment with recombinant PKGI. Data are shown as means ± SEM. n = 16–24 CMs from 4–5 hearts per group. *P < 0.05 KO versus CTR (1-tailed Student’s t test); #P < 0.05 KO without and with PKGI (2-tailed Student’s t test); †P < 0.05 CTR without and with PKGI (2-tailed Student’s t test).

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  Figure 8. Addition of recombinant PKGI enhanced total Ser/Thr- and Ser4080-titin phosphorylation in skinned CMs prepared from CM GC-B–KO mice after TAC. LV CMs were prepared from CM GC-B–KO mice after 3 days (A) or 14 days (B) of TAC. Western blot analyses were performed using an antibody against total Ser/Thr-phosphorylated titin (left panel) and a phospho-site–specific antibody recognizing titin phosphorylated at Ser4080 (the PKGI-specific site) (right panel). The levels of phosphorylated titin were normalized to total titin (PVDF) and calculated as percentage (%) from the mean value of the untreated (-PKGI) sample group. Data are shown as means ± SEM. n = 8/group, *P < 0.05 versus untreated samples (-PKGI) (2-tailed Student’s t test).