sGCα₁ 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 β₁- and an α₁- or α₂-subunit. sGCα₁β₁ 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 α₁-subunit (sGCα₁^−/−) and from wild-type (WT) mice. Sarcomere shortening under basal conditions was less in sGCα₁^−/− CM than in WT CM. To activate endogenous NO synthesis from NO synthase 3, CM were incubated with the β₃-adrenergic receptor (β₃-AR) agonist BRL 37344. BRL 37344 decreased cardiac contractility in WT CM but not in sGCα₁^−/− 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 sGCα₁^−/− CM. Neither BRL 37344 nor spermine NONOate altered calcium handling in CM of either genotype. These findings suggest that sGCα₁ exerts a positive inotropic effect under basal conditions, as well as mediates the negative inotropic effect of β₃-AR signaling. Additionally, our work demonstrates that sGCα₁β₁ is required for NO to depress β₁/β₂-AR-stimulated cardiac contractility and that this modulation is independent of changes in calcium handling.

nitric oxide (no) modulates cardiac contractile function. However, the precise mechanisms by which NO impacts cardiac contractility remain to be determined. In cardiac myocytes (CM), NO is generated by neuronal, inducible, and endothelial NO synthases (NOS1, NOS2, and NOS3, respectively). NO binds to and activates soluble guanylate cyclase (sGC) to synthesize the second messenger molecule cGMP. cGMP signals by activating cGMP-dependent protein kinase (PKG), as well as by modulating the activity of cGMP-regulated phosphodiesterases (PDEs; Refs. 9, 17). Cyclic nucleotides are degraded by PDEs, some of which are cGMP specific, such as PDE5 (9, 17, 25). The effects of NO may be sGC dependent, but NO has also been suggested to regulate cardiac contractile function by cGMP-independent mechanisms, including by nitrosylation of protein thiol groups (13).

Previous studies (19, 22, 33, 41, 50, 51) have demonstrated that NO, endogenously produced by NOS or released from NO-donor compounds, can attenuate the ability of β₁/β₂-adrenergic receptor (β₁/β₂-AR) agonists to augment cardiac contractility. In the heart, β₃-adrenergic receptor (β₃-AR) signaling activates NOS3 and inhibition of NOS3 blocks β₃-AR signaling (11). BRL 37344, a β₃-AR agonist, exerts a negative inotropic effect on ventricular myocardium through the activation of endogenous NOS3 and has been suggested to signal via sGC (11, 12, 23). In isolated myocytes, high NO levels appear to elicit negative inotropic effects under basal conditions, while low levels of NO have been associated with positive inotropic effects (20, 21, 27).

Whether alterations in calcium levels or myofilament sensitivity to calcium are responsible for the NO/cGMP-mediated impact on contractile function is controversial. The amplitude of the calcium transient has been reported to either decrease or remain unchanged after challenge with NO in the presence of β₁/β₂-AR stimulation in isolated CM (8, 49, 51). Alternatively, phosphorylation of the myofilament protein troponin I, possibly by PKG, has been suggested to be responsible for the ability of NO and cGMP to decrease calcium sensitivity (23, 37).

The specific intracellular compartment where cGMP is generated and metabolized appears to be critical for its impact on cardiac contractility (28, 42). However, the intracellular location of sGC in CM has not been demonstrated. Of the two heterodimeric sGC isoforms, sGCα₁β₁ appears to be the predominant form expressed in CM (4, 26). We (5) have previously generated mice deficient in sGCα₁ on an SV129 genetic background (sGCα₁^−/−), and we reported that both basal and NO-stimulated sGC enzyme activity of the left ventricle were greatly diminished in sGCα₁^−/− mice. Male sGCα₁^−/− mice are hypertensive and exhibit increased left ventricular (LV) systolic function, as well as delayed LV relaxation (5).

The aim of the current study was to further elucidate the role of sGC in cardiac contractile function. We isolated CM from wild-type (WT) and sGCα₁^−/− mice and compared the contractile properties of WT and sGCα₁^−/− CM under baseline conditions, as well as after NO stimulation. Because sGCα₁^−/− mice exhibited enhanced cardiac inotropy in vivo, we hypothesized that CM from mice with reduced sGC activity would exhibit increased contractile function (5). Surprisingly, sGCα₁ deficiency was associated with impaired sarcomere shortening under basal conditions. Our data furthermore suggest that activation of the β₃-AR depresses cardiac contractility via the NOS3/sGC pathway. Finally, we report that the ability of NO to depress β₁/β₂-AR cardiac contractility requires sGCα₁ without alterations in calcium handling.

MATERIALS AND METHODS

Reagents.

Spermine NONOate (Enzo Life Sciences, Plymouth Meeting, PA) was dissolved in ice-cold 10 mM NaOH and kept on ice. Before use, spermine NONOate was preincubated at 37°C for 45 min at a final concentration of 100 μM. BRL 37344, nadolol, Bay 41–2272, and dl-isoproterenol (ISO) HCl were purchased from Sigma-Aldrich (St. Louis, MO). 1H-(1,2,4)oxadiazolo(4,3-a)-quinoxalin-1-one (ODQ) was obtained from Enzo Life Sciences, and nitro-l-arginine methyl ester (l-NAME) was purchased from Acros Organics (Morris Plains, NJ). DT-2 was purchased from Axxora (San Diego, CA).

Isolation of adult mouse CM.

The animal experimental protocol was approved by the Subcommittee on Research Animal Care at Massachusetts General Hospital, which conforms to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). CM were isolated from 8- to 12-wk-old male WT SV129 mice (Taconic Farms, Germantown, NY) and from male and female sGCα₁^−/− mice generated on an SV129 background (5). The animals were heparinized and anesthetized with 100 mg/kg of pentobarbital sodium. The heart was rapidly excised and placed in ice-cold calcium-free Tyrode buffer consisting of 137 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl₂, and 10 mM HEPES (pH 7.4) and supplemented with 10 mM d-glucose, 10 mM 2,3-butanedione monoxime, and 5 mM taurine. LV myocytes were isolated by Langendorff perfusion, as previously described (24). CM were transferred through a series of increasing calcium solutions to a final concentration of 1.2 mM CaCl₂.

Immunocytochemistry of adult murine CM.

Freshly isolated CM were fixed and permeabilized with a solution of 2% paraformaldehyde/0.75% Triton X-100. Antibodies directed against sGCβ₁ (1:1,000, ab5033, rabbit) or α-actinin (1:1,000, ab18061, mouse) were purchased from AbCam (Cambridge, MA). sGCα₁ antibody was a gift from S. Janssens and was used at 1:100 (36). Bound primary antibodies were detected with DyLight 594-conjugated AffiniPure donkey anti-rabbit IgG (1:100) or FITC-conjugated AffiniPure donkey anti-rabbit IgG. (1:50; both from Jackson Immunochemicals). Cells were visualized with a Nikon E800 microscope equipped with a Bio-Rad Radiance 2000 confocal head.

Imaging of sarcomere length and calcium transients in CM.

Freshly isolated CM were loaded with 1 μM fura-2 AM (Molecular Probes) in Tyrode buffer for 20 min at 25°C. CM were perfused with Tyrode buffer supplemented with 1.2 mM CaCl₂, 5.5 mM d-glucose, and 0.5 mM probenecid and maintained at 35–37°C. Cells were field stimulated at 1, 2, 4, and 6 Hz by a MyoPacer Field Stimulator (IonOptix, Milton, MA). Sarcomere length data were acquired by SarcLen software (IonOptix), and intracellular calcium ratio data were acquired using a dual-excitation fluorescence photomultiplier system (Hyperswitch Light Source; IonOptix). Sarcomere length and calcium transient data were analyzed with IonWizard software.

RESULTS

sGCα₁ localizes to the sarcomeric Z-line in CM.

The sarcomeres of CM are organized into repeating myofibrillar structures: the Z-line, and the I-, M-, and A-bands. To determine where sGC resides within CM, we performed immunocytochemistry using antibodies directed against the α₁- and β₁-subunits of sGC and visualized the cells using confocal microscopy. Coimmunostaining with antibodies directed against sGCα₁ and α-actinin revealed that sGCα₁ localizes to the sarcomeric Z-line in WT CM (Fig. 1, A–C). The sGCα₁^−/− mice were generated by deleting the sixth exon of the sGCα₁ gene (5). The expressed, nonfunctional mutant enzyme remains localized to the Z-line in CM (Fig. 1, D–F). These results suggest that sequences other than those encoded by the sixth exon are responsible for sGCα₁ localization. Similarly, sGCβ₁, the obligate binding partner of sGCα₁, also colocalized to Z-lines in CM isolated from WT or sGCα₁^−/− mice (Fig. 1, G-L). Taken together, these results suggest that sGCα₁β₁ localizes to the compartment previously identified to contain NOS3 and PDE5 in CM (35, 43).

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.

sGCα₁ deficiency decreases contractile function in a calcium-independent manner.

To determine if basal sGCα₁β₁ activity is important for contractile function, isolated CM from WT and sGCα₁^−/− mice were electrically stimulated at various pacing frequencies, and sarcomere lengths and calcium transients were recorded and analyzed. CM from sGCα₁^−/− mice shortened 32% less than CM from WT mice at a pacing frequency of 2 Hz (P < 0.05; Fig. 2, top). Despite the defect in sarcomeric shortening, the amplitude of the calcium transients was similar in CM from both genotypes (Fig. 2, bottom).

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.

We considered the possibility that the impaired sarcomere shortening observed in CM from male sGCα₁^−/− was attributable to the hypertension seen in these mice. To examine this hypothesis, we studied CM from female sGCα₁^−/− mice that do not have hypertension. CM from normotensive female sGCα₁^−/− also exhibited a decreased ability to shorten compared with WT CM, indicating that the contractile defect in the sGCα₁^−/− mice is due to deficiency of sGCα₁ and not due to secondary effects of hypertension on the heart (Supplemental Fig. S1; Supplemental Material for this article is available online at the Am J Physiol Heart Circ Physiol website). These results suggest that under basal conditions sGC activity exerts a positive inotropic effect in isolated CM.

Activation of sGC by NOS3-derived NO in CM decreases contractility.

To test whether sGCα₁β₁ mediates the effect of NO on cardiac contractility, we activated endogenous NOS3 in WT and sGCα₁^−/− CM by stimulating the β₃-AR with BRL 37344 (1 nM). BRL 37344 diminished sarcomere shortening by 47% in WT cells (P < 0.01; Fig. 2, top). In contrast, BRL 37344 did not decrease sarcomere shortening in sGCα₁^−/− CM. The BRL 37344-induced decrease in sarcomere shortening in WT cells occurred without changes in the magnitude of calcium transients, indicating that BRL 37344 influences cardiac contractility without altering calcium flux (Fig. 2, bottom).

To confirm that the effect of BRL 37344 on sarcomere shortening was independent of β₁/β₂-AR signaling, we tested the effect of the β₁/β₂-AR antagonist nadolol on sarcomere shortening, both in the absence and presence of BRL 37344. Nadolol (10 μM) alone had no effect on sarcomere shortening in WT CM. In CM treated with both BRL 37344 (1 nM) and nadolol, the sarcomere shortening was indistinguishable from cells treated with BRL 37344 alone (Fig. 3, top), indicating β1/β2-AR activation did not modulate the observed sarcomere shortening response to BRL 37344.

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.

To confirm that BRL 37344 depressed contractility through activation of NOS and sGC, we examined the effects on CM contractility of l-NAME, a nonselective NOS inhibitor, as well as ODQ, an inhibitor of sGC. Incubation of WT CM with l-NAME (10 μM) prevented BRL 37344 from decreasing sarcomere shortening, confirming that BRL 37344 depressed contractility by activating NOS (Fig. 3, top). Similar to sGCα₁ deficiency, ODQ prevented the decrease in sarcomere shortening in response to BRL 37344 (Fig. 3, top). Taken together, these results confirm that β₃-AR signaling requires NOS3 and sGC to attenuate cardiac contractility. Of note, incubation of CM with BRL 37344, nadolol, l-NAME, or ODQ did not alter Ca²⁺ transients (Fig. 3, bottom). These results indicate that NO/cGMP signaling impacts cardiac contractility independently of calcium handling.

sGC activity inhibits β₁/β₂-AR-stimulated contractile function in CM.

To evaluate the role of sGCα₁ in the ability of NO to modulate β₁/β₂-AR-stimulated contractile function, we studied the impact of NO on WT and sGCα₁^−/− CM in the absence and presence of ISO. In the absence of ISO, perfusion of WT CM with 100 μM spermine NONOate did not alter sarcomere shortening (data not shown). Sarcomeric shortening and Ca²⁺ transients were similar in WT and sGCα₁^−/− CM in the presence of 10 nM ISO. This dose of ISO produced a submaximal increase in sarcomere shortening (data not shown) and was previously used to reveal the impact of NO/cGMP on CM function (43). In WT cells, perfusion with the NO donor compound elicited a dramatic reduction of ISO-stimulated sarcomere shortening. The maximal decrease (39%) in sarcomere shortening occurred at 150 ± 30 s after addition of the NO donor compound. As confirmation that the change in contractile function was due to perfusion of spermine NONOate, sarcomere contraction increased rapidly upon removal of the NO donor compound (Fig. 4, top). However, in sGCα₁^−/− CM, perfusion with spermine NONOate did not alter sarcomere shortening in cells stimulated with ISO (Fig. 4, bottom). The absence of a response to NO in sGCα₁^−/− CM suggests that sGCα₁ is required for NO to decrease β₁/β₂-AR-stimulated myocardial contractility.

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.

To confirm that sGC activity is able to decrease β₁/β₂-AR-stimulated cardiac contractility, CM were treated with the sGC activator Bay 41–2272 (39, 40). In ISO-stimulated WT CM, perfusion with either 5 or 10 μM Bay 41–2272 reduced sarcomere shortening without altering Ca²⁺ transients. Bay 41–2272 did not decrease sarcomere shortening in ISO-stimulated myocytes from sGCα₁^−/− mice (Supplemental Fig. S2), confirming that sGCα₁β₁ is able to attenuate β₁/β₂-AR-stimulated myocardial contractility.

The muscarinic-cholinergic receptor 2 (M2-Chr), the predominant muscarinic receptor in the heart, exerts negative inotropic effects via pertussis toxin-sensitive G_i/o proteins. Activation of G_i/o inhibits adenylate cyclase and lowers cAMP levels leading to decreased contractility (10, 15). To demonstrate that ISO-stimulated sarcomere shortening could be decreased in both genotypes by a cGMP-independent mechanism, we administered the muscarinic-cholinergic agonist carbachol (10 μM) to WT and sGCα₁^−/− CM incubated with ISO. In contrast to our experiments using an NO donor compound, carbachol decreased sarcomere shortening in both WT and sGCα₁^−/− myocytes (Supplemental Fig. S3). These results suggest that sGCα₁^−/− myocytes retain the ability to decrease contractility in response to negative inotropic signals from a cGMP-independent pathway.

PKG is required for the NO-induced decrease in cardiac contractility.

NO donor compounds were previously shown to decrease contractile force in myocardial tissues from WT mice but not in those from mice deficient in PKG I, a major downstream target of cGMP, indicating that PKG is critical to NO-mediated control of contractile force (48). We further investigated whether PKG is responsible for attenuating CM contractility in response to NO. Pretreatment of WT CM with 125 nM DT-2, a cell-permeable inhibitor of PKG, completely prevented the decrease in ISO-induced sarcomere shortening induced by perfusion with NO (7). These results confirm that PKG is a critical mediator of the negative inotropic effects of NO (Fig. 5, top). DT-2 alone had no effect on sarcomere shortening (data not shown).

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.

The addition of NO had no effect on Ca²⁺ transients in ISO-stimulated WT or sGCα₁^−/− CM (Fig. 5, bottom). Similarly, inhibition of PKG by DT-2 did not alter the height of the Ca²⁺ transient in WT CM incubated with 10 nM ISO alone (Fig. 5, bottom). Therefore, the negative inotropic effects of NO/cGMP signaling appear to be calcium independent.

DISCUSSION

Data from both animal and human studies have supported the idea that elevating cardiac cGMP levels may elicit beneficial effects in a variety of cardiovascular diseases, such as heart failure and cardiac hypertrophy (14, 44). Elucidating the precise functions of cGMP in the heart is important because of the development of cGMP-elevating drugs, including those that activate sGC or inhibit PDE5 (32, 34, 40). In vivo experiments comparing the cardiac function of WT and sGCα₁^−/− mice yield important physiologic insights, but interpretation of studies using intact animals is complicated by the impact of neurohumoral responses and non-CM cells in the heart. Studies using isolated CM allow the dissection of cGMP signaling in cardiac contractile function in the absence of compensatory responses and the influence of other cell types. The contractile performance of CM from sGCα₁^−/− mice yields insight into the role of NO and cGMP in cardiac function. We report the unexpected finding that contractile function is impaired in CM from sGCα₁^−/− mice at baseline. Additionally, sGCα₁β₁ is required for NO to regulate the ability of β₁/β₂-AR to stimulate contractile function in the heart. With the use of the β₃-AR agonist BRL 37344, we confirm that the β₃-AR utilizes the NOS/cGMP pathway to limit contractile amplitude. Additionally, we provide evidence that the sGC/cGMP pathway regulates cardiac contractile function independently of calcium modulation.

CM exhibit precise localization of proteins that modulate cyclic nucleotide levels and their signaling partners. Both NOS3 and PDE5 are expressed at the Z-line of CM, while NOS1 appears to be targeted to the sarcoplasmic reticulum (1, 29, 35, 43). The β₃-AR also appears to colocalize with NOS3 and is compartmentalized to the Z-line in CM (2, 18, 28). We demonstrate that sGCα₁β₁, similar to NOS3, PDE5, and the β₃-AR, is spatially restricted to the Z-line of CM. Mutation of the α₁-subunit does not disrupt Z-line localization of sGCβ₁, indicating that the deleted portion of sGCα₁ in our mutant mouse is not responsible for cellular localization of the enzyme.

While high levels of NO and cGMP depress β₁/β₂-AR-stimulated cardiac contractility (6, 43), considerable evidence suggests that a low level increase in cGMP can have a positive inotropic effect on the heart (20, 21, 27). In vivo both endogenous and exogenous NO have been shown to increase cardiac function (16, 30). In isolated heart studies, NO and cGMP also induced positive inotropy (21, 31). In isolated CM, the addition of low concentrations of cGMP increased contractility (20, 27). Similarly, our work demonstrates that deletion of sGCα₁ decreases basal contractility. Taken together, these results support the hypothesis that low levels of cGMP generated by sGCα₁β₁ augment cardiac contractile function.

We (5) previously reported that LV end systolic pressure and systemic arterial pressure were greater in male sGCα₁^−/− mice on an SV129 background than in female sGCα₁^−/− mice or WT mice of either gender. Since elevated blood pressure can induce LV hypertrophy and may decrease contractile function, we studied myocytes derived from nonhypertensive sGCα₁^−/− females to exclude the possibility that systemic hypertension was responsible for the impaired contractile function observed in CM isolated from male sGCα₁^−/− mice. Similar to their male littermates, CM from sGCα₁^−/− female mice contracted less than WT CM. These results confirm that the decreased contractile function observed in the sGCα₁^−/− CM is not a result of secondary effects of hypertension in these mice.

A possible explanation for the reduced basal contractile phenotype of the sGCα₁^−/− myocytes is reduced modulation of PDEs by cGMP (45) The cAMP-hydrolyzing enzyme PDE3 is competitively inhibited by cGMP (28). In a recent study (38), NO-induced increases in cGMP concentrations resulted in elevated cAMP levels in a specific compartment of myocytes. Therefore, a reduction in cGMP synthesis due to sGCα₁ deficiency may increase cAMP degradation leading to decreased basal contractile function.

The β₃-AR agonist BRL 37344 decreased sarcomere shortening in WT CM but not in sGCα₁^−/− cells. Furthermore, inhibiting NOS activity with l-NAME or sGC activity with ODQ prevented the BRL 37344-induced reduction in sacromere shortening in WT cells, confirming that the β₃-adrenergic receptor functions to depress cardiac contractility through NOS and sGC. These results further support the idea that the effects of NO on cardiac contractility are mediated by cGMP and not by S-nitrosylation or other cGMP-independent mechanisms.

In the absence of ISO, exposure to spermine NONOate did not alter basal contractility in either WT or sGCα₁^−/− CM. This finding is in agreement with a previous report (33) that NO donor compounds do not impact contractility of isolated CM. This insensitivity to exogenous NO is not surprising considering that CM are known to express high levels of the NO-scavenger myoglobin. In fact, CM from myoglobin-deficient mice have a greater sensitivity to NO donors (47). In contrast, BRL 37344 was able to exert negative inotropic effects in the absence of β₁/β₂-AR stimulation. We hypothesize that colocalization of β₃-ARs, NOS3, and sGCα₁β₁ in CM allows direct activation of sGC in response to β₃-AR stimulation. In contrast, when NO is not generated by NOS3 in direct proximity to β₃-AR and sGCα₁β₁, NO modulates cardiac contractility only in the context of β₁/β₂-AR stimulation.

Exposure to NO after β₁/β₂-AR stimulation with ISO decreased sarcomere shortening in WT but not sGCα₁^−/− CM. The finding that the ability of NO to blunt β-adrenergic signaling is impaired in sGCα₁^−/− CM may explain why load-independent indexes of cardiac contractile function in vivo are greater in sGCα₁^−/− mice than in WT mice (5), while contractility was decreased in CM isolated from sGCα₁^−/− mice than from WT mice. We speculate that in vivo adrenergic stimulation produces a phenotype of increased cardiac inotropy in sGCα₁^−/− mice, which have decreased cGMP signaling and, thus, are less equipped to oppose β₁/β₂-AR stimulation.

Inhibiting PKG with DT-2 prevented the ability of NO to reduce sarcomere shortening in WT CM. This result, as well as our data showing that either NO or Bay 41–2272 can depress β₁/β₂-AR stimulated CM contractile function in WT CM but not sGCα₁^−/− CM, suggests that NO and Bay 41–2272-mediated generation of cGMP by sGCα₁β₁ and activation of PKG are responsible for the NO-mediated inhibition of β₁/β₂-AR stimulated cardiac contractile function.

We observed no alterations in calcium handling after perfusion with an NO donor compound. Similar to our findings, PDE5 inhibition was shown to increase basal contractility in CM in the absence of concomitant changes in the Ca²⁺ transient (43, 46). It is conceivable that cGMP, via PKG, exerts a direct effect on the myofilament machinery to modulate contractility. Since the phosphorylation site serine 23/24 of the myofilament protein troponin I was suggested to regulate calcium sensitivity, we examined the phosphorylation status of this protein (23, 37). However, we did not detect differences in troponin I phosphorylation between WT and sGCα₁^−/− CM under the conditions that produced an NO-dependent decrease in β₁/β₂-AR-stimulated sarcomeric shortening in WT CM but not in sGCα₁^−/− CM (i.e., ∼150 s after perfusion with spermine NONOate in ISO-stimulated CM).

In summary, we utilized mice deficient in sGCα₁ to focus on long-standing questions regarding the roles of NO and cGMP in cardiac physiology. sGCα₁β₁-derived cGMP has a positive inotropic role in basal CM contractility. Furthermore, we report that sGCα₁β₁ mediates the negative inotropic effect of β₃-AR stimulation on cardiac contractile function. Additionally, our work demonstrates that sGCα₁β₁ is required for NO to depress β₁/β₂-AR-stimulated cardiac contractility and that this modulation is independent of changes in calcium handling.

GRANTS

S. M. Cawley was supported by a Ruth L. Kirschtein National Research Service Award Grant HL-07208-29, E. S. Buys was supported by a Scientist Development Grant 10SDG2610313 from the American Heart Association, F. Ichinose was supported by National Institute of General Medical Sciences Grant RO1-GM-079360, and K. D. Bloch was supported by National Heart, Lung, and Blood Institute R01 HL74352.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).