sGC(alpha)1(beta)1 attenuates cardiac dysfunction and mortality in murine inflammatory shock models

Abstract

Altered cGMP signaling has been implicated in myocardial depression, morbidity, and mortality associated with sepsis. Previous studies, using inhibitors of soluble guanylate cyclase (sGC), suggested that cGMP generated by sGC contributed to the cardiac dysfunction and mortality associated with sepsis. We used sGC(alpha)(1)-deficient (sGC(alpha)(1)(-/-)) mice to unequivocally determine the role of sGC(alpha)(1)beta(1) in the development of cardiac dysfunction and death associated with two models of inflammatory shock: endotoxin- and TNF-induced shock. At baseline, echocardiographic assessment and invasive hemodynamic measurements of left ventricular (LV) dimensions and function did not differ between wild-type (WT) mice and sGC(alpha)(1)(-/-) mice on the C57BL/6 background (sGC(alpha)(1)(-/-B6) mice). At 14 h after endotoxin challenge, cardiac dysfunction was more pronounced in sGC(alpha)(1)(-/-B6) than WT mice, as assessed using echocardiographic and hemodynamic indexes of LV function. Similarly, Ca(2+) handling and cell shortening were impaired to a greater extent in cardiomyocytes isolated from sGC(alpha)(1)(-/-B6) than WT mice after endotoxin challenge. Importantly, morbidity and mortality associated with inflammatory shock induced by endotoxin or TNF were increased in sGC(alpha)(1)(-/-B6) compared with WT mice. Together, these findings suggest that cGMP generated by sGC(alpha)(1)beta(1) protects against cardiac dysfunction and mortality in murine inflammatory shock models.

Fig. 1.

Soluble guanylate cyclase (sGC) enzyme activity. Unstimulated (baseline) and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO)-stimulated sGC enzyme activity in lung extracts of 10- to 12-wk-old wild-type (WT) and sGC-deficient (sGCα₁^−/−B6) mice. Values are means ± SE (n = 6 for all groups). *P < 0.001 vs. WT DEA-NO. #P < 0.001 vs. baseline.

Fig. 2.

Acute effects on left ventricular (LV) end-systolic pressure (LVESP) of compounds acting on the nitric oxide (NO)-cGMP pathway. A: effect of sodium nitroprusside (SNP, 2.5, 5, or 10 μg/kg iv) on LVESP in WT (n = 9) and sGCα₁^−/−B6 (n = 8) mice. Values are means ± SE. *P < 0.05. #P < 0.01 vs. WT. B: sGC stimulator BAY 41-2272 decreased LVESP in WT, but not sGCα₁^−/−B6, mice. Values are means ± SE. *P < 0.001 vs. WT BAY 41-2272. #P < 0.001 vs. WT vehicle.

Fig. 3.

Levels of sGCα₂ expression, as measured by quantitative RT-PCR, in extracts from aorta (A) and lung (B) of WT and sGCα₁^−/− mice on the 129S6 (S6) and the C57BL/6 (B6) backgrounds. Values are means ± SE of number of mice indicated below each bar.

Fig. 4.

Echocardiographic analysis of LV function. LV fractional shortening (FS, A), ejection fraction (EF, B), LV end-systolic internal diameter (LVID_s, C), and heart rate (HR, D) were measured in WT and sGCα₁^−/−B6 mice 14 h after challenge with saline or 25 mg/kg endotoxin (LPS). Values are means ± SE. *P < 0.001; #P < 0.01 vs. saline. $P < 0.01; &P < 0.05 vs. WT LPS.

Fig. 5.

Expression analysis of inflammatory markers. mRNAs encoding type 2 nitric oxide synthase (NOS2, A) and IL-6 (B) in the LV of WT and sGCα₁^−/−B6 mice were measured by quantitative RT-PCR 14 h after challenge with saline (n = 13 and 11, respectively) or 25 mg/kg endotoxin (LPS, n = 10 and 11, respectively). Values are means ± SE. *P < 0.01 vs. saline.

Fig. 6.

Quantification of immunoblot analyses of sGCα₁ (A), sGCα₂ (B), sGCβ₁ (C), and GAPDH (D) in LV protein extracts of WT and sGCα₁^−/−B6 mice 14 h after challenge with saline or 25 mg/kg endotoxin (LPS). Blots represent results from 3 mice in each group. Values are means ± SE (n = 5 per group). *P < 0.001 and #P < 0.05 vs. WT in the same treatment group.

Fig. 7.

Percent cell shortening (CS, A) and amplitude of Ca²⁺ transient (ΔCa_i, B) in cardiomyocytes isolated from WT and sGCα₁^−/−B6 mice 14 h after challenge with saline or 25 mg/kg endotoxin (LPS) and paced at 6 Hz. Values are means ± SE of number of cells and mice (cells/mice) indicated below each bar. *P < 0.05; #P < 0.01 vs. saline-challenged mice of the respective genotype. $P < 0.05 vs. endotoxin-challenged WT mice.

Fig. 8.

Survival analysis. Kaplan-Meier curve shows survival after challenge of WT and sGCα₁^−/−B6 mice with endotoxin (LPS, A) or TNF (B). #P < 0.05; *P < 0.01 vs. WT (by log-rank test).