The alpha1 isoform of soluble guanylate cyclase regulates cardiac contractility but is not required for ischemic preconditioning

Abstract

Nitric oxide (NO)-dependent soluble guanylate cyclase (sGC) activation is an important component of cardiac signal transduction pathways, including the cardioprotective signaling cascade induced by ischemic preconditioning (IPC). The sGCα subunit, which binds to the common sGCβ1 subunit, exists in two different isoforms, sGCα1 and sGCα2, but their relative physiological roles remain unknown. In the present study, we studied Langendorff-perfused isolated hearts of genetically engineered mice lacking functional sGCα1 (sGCα1KO mice), which is the predominant isoform in the heart. Our results show that the loss of sGCα1 has a positive inotropic and lusitropic effect on basal cardiac function, indicating an important role for sGCα1 in regulating basal myocardial contractility. Surprisingly, IPC led to a similar 35-40% reduction in infarct size and concomitant protein kinase Cε (PKCε) phosphorylation in both wild-type (WT) and sGCα1KO hearts subjected to 40 min of global ischemia and reperfusion. Inhibition of the activation of all sGC isoforms by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 μmol/L) completely abolished the protection by IPC in WT and sGCα1KO hearts. NO-stimulated cGMP production was severely attenuated in sGCα1KO hearts compared to WT hearts, indicating that the sGCα2 isoform only produces minute amounts of cGMP after NO stimulation. Taken together, our results indicate that although sGCα1 importantly regulates cardiac contractility, it is not required for cardioprotection by IPC. Instead, our results suggest that possibly only minimal sGC activity, which in sGCα1KO hearts is provided by the sGCα2 isoform, is sufficient to transduce the cardioprotective signal induced by IPC via phosphorylation of PKCε.

Fig. 1

Immunoblot analysis of the sGCα1 and sGCα2 isoforms, as well as tubulin in whole hearts and cardiomyocytes of WT and sGCα1KO mice. a Representative immunoblots of sGC isoforms in homogenates of isolated cardiomyocytes (CM) and perfused whole hearts (heart). The sGCα2-immunoreactive band was blocked by the corresponding immunization peptide, confirming the specificity of the immunostaining. b Quantitative analysis of sGCα2 expression levels in WT (N = 4) and sGCα1KO (N = 5) samples, normalized to the respective WT samples. No significant differences were found between groups

Fig. 2

Basal functional parameters in isolated hearts from male WT (N = 49), sGCα1KO (N = 32), and WT mice treated with 10 μmol/L ODQ (N = 40). a LV developed pressure (LVDevP); b maximum and c minimum rate of LV pressure change (dP/dt_max and dP/dt_min, respectively); d coronary flow rate. **P < 0.01 and ***P < 0.001 versus WT by Bonferroni's post hoc test after one-way ANOVA

Fig. 3

Infarct size measured after 40 min of global ischemia and 1 h of reperfusion with (IPC) or without (Control I/R) ischemic preconditioning. N = 6–8 per group; **P < 0.01 versus control I/R without IPC and ##P < 0.01 versus respective group without ODQ by Bonferroni's post hoc test after two-way ANOVA

Fig. 4

Western blots showing the phosphorylation of the Ser729 residue on PKCε after IPC in WT and sGCα1KO hearts. a Representative blots are shown for Ser729 phosphorylated PKCε (p-PKCε), total PKCε, and tubulin. Control samples subjected to 40 min ischemia and 20 min of reperfusion without IPC; IPC samples subjected to ischemia and reperfusion with IPC. b Graph showing the average ratio of the relative intensity of Ser729 phosphorylated PKCε to total PKCε (N = 4–6 samples per group). *P < 0.05 versus respective control by unpaired t test

Fig. 5

cGMP concentration in homogenates of WT and sGCα1KO isolated hearts perfused with buffer containing vehicle or DEA/NO (100 μmol/L). ***P < 0.001 versus vehicle and ###P < 0.001 versus WT, both by Bonferroni's post hoc test after two-way ANOVA