sGC{alpha}1 mediates the negative inotropic effects of NO in cardiac myocytes independent of changes in calcium handling

Abstract

In the heart, nitric oxide (NO) modulates contractile function; however, the mechanisms responsible for this effect are incompletely understood. NO can elicit effects via a variety of mechanisms including S-nitrosylation and stimulation of cGMP synthesis by soluble guanylate cyclase (sGC). sGC is a heterodimer comprised of a β(1)- and an α(1)- or α(2)-subunit. sGCα(1)β(1) is the predominant isoform in the heart. To characterize the role of sGC in the regulation of cardiac contractile function by NO, we compared left ventricular cardiac myocytes (CM) isolated from adult mice deficient in the sGC α(1)-subunit (sGCα(1)(-/-)) and from wild-type (WT) mice. Sarcomere shortening under basal conditions was less in sGCα(1)(-/-) CM than in WT CM. To activate endogenous NO synthesis from NO synthase 3, CM were incubated with the β(3)-adrenergic receptor (β(3)-AR) agonist BRL 37344. BRL 37344 decreased cardiac contractility in WT CM but not in sGCα(1)(-/-) myocytes. Administration of spermine NONOate, an NO donor compound, did not affect sarcomeric shortening in CM of either genotype; however, in the presence of isoproterenol, addition of spermine NONOate reduced sarcomere shortening in WT but not in sGCα(1)(-/-) CM. Neither BRL 37344 nor spermine NONOate altered calcium handling in CM of either genotype. These findings suggest that sGCα(1) exerts a positive inotropic effect under basal conditions, as well as mediates the negative inotropic effect of β(3)-AR signaling. Additionally, our work demonstrates that sGCα(1)β(1) is required for NO to depress β(1)/β(2)-AR-stimulated cardiac contractility and that this modulation is independent of changes in calcium handling.

Fig. 1.

Localization of soluble guanylate cyclase-α₁ (sGCα₁) in cardiac myocytes (CM). CM isolated from wild-type (WT; A–C and G–I) and sGCα₁^−/− (D–F and J–L) mice were reacted with primary antibodies recognizing sGCα₁ (B and E), sGCβ₁ (H and K), and α-actinin (A, D, G, and J), and bound antibody was visualized by confocal microscopy using fluorescently labeled secondary antibodies. α-Actinin was detected with FITC-labeled anti-mouse IgG (green), and sGCα₁ and sGCβ₁ were detected using Dylight 594-labeled anti-rabbit IgG (red). Merged view (C, F, I, and L) demonstrates that sGCα₁ colocalizes with α-actinin on Z-lines. Scale bars (white) = 10 μm.

Fig. 2.

Effect of BRL 37344 on WT and sGCα₁^−/− CM contractility and calcium handling. %Sarcomere shortening (top) and calcium transient amplitude (ΔCa_i; bottom) are shown for WT and sGCα₁^−/− CM incubated in the absence and presence of BRL 37344 (1 nM). CM were incubated for 10 min and perfused with Tyrode buffer containing 1 nM of the β₃-AR agonist BRL 37344. Cells were paced at 2 Hz. *P < 0.001 for WT BRL vs. WT basal. **P < 0.01 for sGCα₁^−/− basal vs. WT basal.

Fig. 3.

Effect of nadolol, nitro-l-arginine methyl ester (l-NAME), and 1H-(1,2,4)oxadiazolo(4,3-a)-quinoxalin-1-one (ODQ) on contractile function and calcium handling after β₃-adrenergic receptor stimulation in WT CM. %Sarcomere shortening (top) and calcium transient amplitude (bottom) from WT CM incubated with BRL 37344 and 10 μM nadolol, 10 mM l-NAME, or 10 μM ODQ and paced at 2 Hz. *P value of < 0.01 for indicated groups vs. WT basal in Bonferroni posttests.

Fig. 4.

Effect of nitric oxide on β-adrenergic receptor-stimulated sarcomere shortening in WT and sGCα₁^−/− CM. Representative tracing demonstrating time-dependent change in sarcomere length of a single isoproterenol (ISO)-stimulated, paced WT (top) and sGCα₁^−/− (bottom) CM during perfusion with 100 μM spermine NONOate.

Fig. 5.

Summary data of %sarcomere shortening and corresponding Ca²⁺ transient peak height amplitudes of WT and sGCα₁^−/− CM. All CM were preincubated with ISO (10 nM) for 10 min before imaging. Graphs represent percentage of sarcomere shortening (top) and Ca²⁺ transient amplitude (bottom) measured at 150 s after start of perfusion buffer with or without spermine NONOate (100 μM). Additional CM were preincubated and continuously perfused with 125 nM DT-2 where indicated before administration of spermine NONOate. *P < 0.05 WT vs. WT with spermine NONOate.