Protein kinase g iα inhibits pressure overload-induced cardiac remodeling and is required for the cardioprotective effect of sildenafil in vivo

Abstract

Background: Cyclic GMP (cGMP) signaling attenuates cardiac remodeling, but it is unclear which cGMP effectors mediate these effects and thus might serve as novel therapeutic targets. Therefore, we tested whether the cGMP downstream effector, cGMP-dependent protein kinase G Iα (PKGIα), attenuates pressure overload-induced remodeling in vivo.

Methods and results: The effect of transaortic constriction (TAC)-induced left ventricular (LV) pressure overload was examined in mice with selective mutations in the PKGIα leucine zipper interaction domain. Compared with wild-type littermate controls, in response to TAC, these Leucine Zipper Mutant (LZM) mice developed significant LV systolic and diastolic dysfunction by 48 hours (n=6 WT sham, 6 WT TAC, 5 LZM sham, 9 LZM TAC). In response to 7-day TAC, the LZM mice developed increased pathologic hypertrophy compared with controls (n=5 WT sham, 4 LZM sham, 8 WT TAC, 11 LZM TAC). In WT mice, but not in LZM mice, phosphodiesterase 5 (PDE5) inhibition with sildenafil (Sil) significantly inhibited TAC-induced cardiac hypertrophy and LV systolic dysfunction in WT mice, but this was abolished in the LZM mice (n=3 WT sham, 4 LZM sham, 3 WT TAC vehicle, 6 LZM TAC vehicle, 4 WT TAC Sil, 6 LZM TAC Sil). And in response to prolonged, 21-day TAC (n=8 WT sham, 7 LZM sham, 21 WT TAC, 15 LZM TAC), the LZM mice developed markedly accelerated mortality and congestive heart failure. TAC induced activation of JNK, which inhibits cardiac remodeling in vivo, in WT, but not in LZM, hearts, identifying a novel signaling pathway activated by PKGIα in the heart in response to LV pressure overload.

Conclusions: These findings reveal direct roles for PKGIα in attenuating pressure overload-induced remodeling in vivo and as a required effector for the cardioprotective effects of sildenafil.

Keywords: heart failure; nitric oxide; protein kinase G; remodeling heart failure; signal transduction.

Figure 1.

LZM hearts and lungs express LZM-PKGIα mutant protein. Representative immunoblots of heart and lung lysates from WT and LZM mice with antibodies recognizing an epitope common to both isoforms of PKGI (PKG I_COM), PKGI LZM protein only (PKG Iα_LZM), or WT PKGIβ only (PKG Iβ). Total PKGI and PKGIβ expression levels were unchanged by the presence of the targeted mutation. Relative protein expression detected by PKG I_COM or PKG Iα_LZM antibodies is also shown. *P<0.05; n=3 hearts per genotype.

Figure 2.

Impaired systolic and diastolic compensation after 48 hours of pressure overload in LZM left ventricles. Summary data of (A) LV Contractility Index (LV dP/dt_max normalized to instantaneous pressure, IP), and (B) Tau, obtained from invasive hemodynamic measurements from anesthetized adult male mice (n=6 WT sham, 6 WT TAC, 5 LZM sham, 9 LZM TAC). C, Summary data of percent change in fractional shortening in unanesthetized adult male mice from baseline to 48 hours post-TAC (n=10 WT, 10 LZM). D, Representative echocardiograms at baseline and 48 hours post-TAC. E, Direct measurement of gradient across TAC. *P<0.05; ^†P<0.05 vs WT TAC, LZM sham; ^‡P<0.01 vs WT TAC.

Figure 3.

Increased 7-day TAC-induced LV hypertrophy and contractile dysfunction in LZM mice. A, Summary data of LV mass/tibia length in 10- to 14-week-old male mice subjected to 7-day TAC (n=6 WT sham, 5 LZM sham, 11 WT TAC, 14 LZM TAC). *P<0.01 vs WT sham; ^†P<0.05 vs WT TAC, LZM sham. B, Direct measurement of gradient across TAC. C, Summary data of percent fractional shortening (FS) from 10- to 14-week-old male mice subjected to 7-day TAC. *P<0.01 vs WT TAC, WT sham, LZM sham (n=5 WT sham, 4 LZM sham, 8 WT TAC, 11 LZM TAC). D, Representative echocardiographic images. E, Quantification of cardiac myocyte cross-sectional area (CSA), n≥200 CMs per group. Cells were analyzed from 3 WT sham, 3 LZM sham, 4 WT TAC, and 5 LZM TAC hearts, >30 myocytes per heart; statistical comparison was with 1-way ANOVA. *P<0.001 vs WT sham; ^†P<0.05 vs LZM sham; ^‡P<0.001 vs LZM sham, WT TAC, WT sham. F, Representative H&E images of LV cross-sections obtained at the midpapillary level.

Figure 4.

Loss of sildenafil antiremodeling effect in PKGIα–LZM mice. A, Fractional shortening in WT and LZM mice 7 days post-TAC. B, Heart weight/tibia length quantified in WT and LZM mice 7 days post-TAC (n=3 WT sham, 4 LZM sham, 3 WT TAC vehicle, 6 LZM TAC vehicle, 4 WT TAC Sil, 6 LZM TAC Sil). Sh indicates sham; T, TAC. Statistical analysis was performed with 1-way ANOVA, followed by correction for multiple comparisons with the Holm–Sidak method.

Figure 5.

Accelerated LV pressure overload–induced mortality and congestive heart failure in PKGIα LZM mice. A, Kaplan–Meier curve showing markedly accelerated and increased mortality in 10- to 14-week-old male LZM mice subjected to 21-day TAC compared with WT littermate controls. Summary data of (B) LV mass/tibia length and (C) wet lung mass/tibia length in 10- to 14-week-old male mice subjected to 21-day TAC. *P<0.05 vs LZM sham, WT TAC, LZM TAC; ^†P<0.05 vs WT sham, LZM sham; ^‡P<0.05 vs LZM sham; ^§P<0.01 vs WT sham, LZ sham, WT TAC, LZM TAC (n=8 WT sham, 7 LZM sham, 17 WT TAC 21-day survivors [from 21 subjected to TAC], 6 LZM 21-day survivors [from 15 subjected to TAC], 3 WT early death, 6 LZM early death). TAC indicates 21-day survivors; TAC ED, early death induced by TAC. Statistical analysis was performed with 1-way ANOVA followed by correction for multiple comparisons with the Holm–Sidak method. D, Pressure gradient across the TAC determined through noninvasive echocardiographic measurement of aortic flow velocity in a cohort of surviving mice at 7 days (D) and at 21 days (E) post-TAC (n=15 WT TAC survivors at 7 and 21 days, 6 LZM survivors at 7 days, 4 LZM survivors at 21 days).

Figure 6.

TAC-induced phosphorylation of JNK is reduced in LZM hearts. Representative Western blot and quantitation by densitometry of (A) phosphorylated (P-JNK) and total JNK (T-JNK) from cardiac protein lysates 48 hours after TAC or sham surgery (n=6 hearts per surgical group). B, Phosphorylated (P-MKK4) and total (T-MKK4) from cardiac protein lysates 48 hours after surgery (n=7 WT sham, 6 LZM sham, 4 WT TAC, and 4 LZM TAC hearts per group). *P<0.001; ^†P<0.01.