Sacubitril/Valsartan Improves Left Ventricular Function in Chronic Pressure Overload Independent of Intact Cyclic Guanosine Monophosphate-dependent Protein Kinase I Alpha Signaling

Abstract

Background: Combined angiotensin receptor/neprilysin inhibition with sacubitril/valsartan (Sac/Val) has emerged as a therapy for heart failure. The presumed mechanism of benefit is through prevention of natriuretic peptide degradation, leading to increased cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) signaling. However, the specific requirement of PKG for Sac/Val effects remains untested.

Methods and results: We examined Sac/Val treatment in mice with mutation of the cGMP-dependent protein kinase I (PKGI)α leucine zipper domain, which is required for cGMP-PKGIα antiremodeling actions in vivo. Wild-type (WT) or PKG leucine zipper mutant (LZM) mice were exposed to 56-day left ventricular (LV) pressure overload by moderate (26G) transaortic constriction (TAC). At day 14 after TAC, mice were randomized to vehicle or Sac/Val by oral gavage. TAC induced the same degree of LV pressure overload in WT and LZM mice, which was not affected by Sac/Val. Although LZM mice, but not WT, developed LV dilation after TAC, Sac/Val improved cardiac hypertrophy and LV fractional shortening to the same degree in both the WT and LZM TAC mice.

Conclusion: These findings indicate the beneficial effects of Sac/Val on LV structure and function in moderate pressure overload. The unexpected finding that PKGIα mutation does not abolish the Sac/Val effects on cardiac hypertrophy and on LV function suggests that signaling other than natriuretic peptide- cGMP-PKG mediates the therapeutic benefits of neprilysin inhibition in heart failure.

Fig. 1.

Cyclic guanosine monophosphate-dependent protein kinase I (PKGI)α leucine zipper domain required for hemodynamic effects of brain natriuretic peptide (BNP). (A) Mean arterial pressure (MAP) response to intraperitoneal injection of 0.1 mg/kg BNP in male wild-type (WT) and PKGIα leucine zipper mutant (LZM) littermates, implanted with aortic telemetry. Mice injected at 0 minutes. (B) Summary data of MAP at 60 minutes after injection. 0 minutes, before injection; 60 minutes, 60 minutes after injection. n = 4 WT, 3 leucine zipper mutant. *P < .05.

Figure 2.. PKGIα leucine zipper domain mutation does not affect degree of TAC-induced pressure overload but induces LV dilation after chronic pressure overload.

A. LV systolic pressure at day 56 of pressure overload by 26-gauge transaortic constriction (TAC) in WT (left) and LZM (right). n=5 WT sham, 6 WT TAC Vehicle, 8 WT TAC sacubitril/valsartan; 3 LZM sham, 6 LZM TAC vehicle, 8 LZM TAC sacubitril/valsartan. B. Western blot for phosphorylated cardiac myosin binding protein C (cMyBP-C) on serine 273. Summary densitometry of p273 normalized to GAPDH is shown. n=7 WT TAC vehicle, 6 WT TAC sacubitril/valsartan; 6 LZM TAC vehicle, 7 LZM TAC sacubitril/valsartan. C. LV end diastolic diameter obtained by echocardiography at day 56 after pressure overload in WT (left) and LZM (right) mice. n=5 WT and 5 LZM sham; 8 WT TAC vehicle; 8 WT TAC sacubitril/valsartan; 11 LZM vehicle; 10 LZM TAC sacubitril/valsartan. *, p<0.05. Veh, vehicle; Sac/Val, sacubitril/valsartan. ADU, arbitrary densitometric units. EDD, end diastolic diameter.

Figure 3:. PKGI substrate phosphorylation in LVs of vehicle and sacubitril/valsartan treated mice subjected to pressure overload.

Western blot for vasodilator-stimulated phosphoprotein (VASP) phosphorylation on the PKGI-specific serine 239, and for loading control with GAPDH. Also shown is fold-increase of VASP phosphorylation on serine 239 normalized to GAPDH, in sacubitril/valsartan-treated TAC left ventricles, normalized to vehicle treated TAC of the same genotype. n=7 WT TAC vehicle, 6 WT TAC sacubitril/valsartan; 6 LZM TAC vehicle, 7 LZM TAC sacubitril/valsartan. *, p<0.05. ADU, arbitrary densitometric units.

Figure 4.. Sacubitril/valsartan (Sac/Val) prevents cardiac hypertrophy and improves left ventricular systolic function after pressure overload to the same degree in the presence of wild type or mutant PKGIα.

A. Summary data of heart mass normalized to tibia length in wild type and PKG Leucine Zipper (LZ) mutant mice on day 56 after transaortic constriction. B. LV Contractility Index (dP/dt normalized to instantaneous pressure) obtained by invasive hemodynamics at Day 56. C. LV Fractional shortening percentage (FS%) on Day 14 and Day 56 in WT and PKG LZ mutant mice treated with vehicle (Veh), or sacubitril/valsartan (Sac/Val). *, p<0.05; †, p=0.05; ‡, p<0.01 WT Day 14 vs WT Day 56; §, p<0.01 LZM Day 14 vs LZM Day 56. n=5 per sham group, 8 WT TAC Veh, 8 WT TAC Sac/Val; 11 LZM TAC Veh, 10 LZM TAC Sac/Val. A-B, data analyzed by One Way ANOVA with Tukey’s multiple comparisons test. C, data analyzed by Two Way Repeated Measures ANOVA with Sidak’s multiple comparisons test.