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(Circulation. 2001;104:2961.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Expression of Inducible Nitric Oxide Synthase Depresses ß-Adrenergic–Stimulated Calcium Release From the Sarcoplasmic Reticulum in Intact Ventricular Myocytes

Mark T. Ziolo, PhD; Hideki Katoh, MD; Donald M. Bers, PhD

From the Department of Physiology, Loyola University Medical Center, Maywood, Ill. Dr Katoh is now at Internal Medicine III, Hamamatsu University School of Medicine, Hamamatsu, Japan.

Correspondence to Donald M. Bers, Department of Physiology, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153. E-mail dbers{at}lumc.edu

	Abstract

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Background— ß-Adrenergic hyporesponsiveness in many cardiomyopathies is linked to expression of inducible nitric oxide synthase (iNOS) and increased production of NO. The purpose of this study was to examine whether iNOS expression alters the function of the sarcoplasmic reticulum (SR) Ca²⁺ release channel (ryanodine receptor, RyR) during ß-adrenergic stimulation.

Methods and Results— Expression of iNOS was induced by lipopolysaccharide (LPS) injection (10 mg/kg) 6 hours before rat myocyte isolation. Confocal microscopy (fluo-3) was used to measure Ca²⁺ spark frequency (CaSpF, reflecting resting RyR openings) and Ca²⁺ transients. CaSpF was greatly increased by the adenylate cyclase activator forskolin (100 nmol/L) in normal myocytes (iNOS not expressed), but this effect was suppressed (by 77%) in LPS myocytes (iNOS expressed). When NO production by iNOS was inhibited by aminoguanidine (1 mmol/L), there was a further increase in the forskolin-induced CaSpF in LPS myocytes (to levels similar to the forskolin-stimulated CaSpF in normal myocytes). This effect was also seen in myocytes isolated from a failing human heart. There was no effect of aminoguanidine on forskolin-stimulated CaSpF in normal myocytes. ODQ (10 µmol/L), an inhibitor of NO stimulation of guanylate cyclase, did not restore the forskolin-induced rise in CaSpF in LPS myocytes. Aminoguanidine also increased twitch Ca²⁺ transient amplitude in LPS myocytes after forskolin application (independent of changes in SR Ca²⁺ load).

Conclusions— iNOS/NO depresses ß-adrenergic–stimulated RyR function through a cGMP-independent pathway (eg, NO- and/or peroxynitrite-dependent redox modification). This mechanism limits ß-adrenergic responsiveness and may be an important signaling pathway in cardiomyopathies, including human heart failure.

Key Words: receptors, adrenergic, beta • nitric oxide • sarcoplasmic reticulum • calcium • heart failure

	Introduction

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Calcium regulates cardiac contraction via excitation-contraction (EC) coupling.¹ Diseased human hearts exhibit deterioration of contractile function, frequently seen as decreased ß-adrenergic responsiveness.^2,3 ß-Adrenergic activation stimulates adenylate cyclase, increases cAMP, and activates cAMP-dependent protein kinase (PKA). Many key EC coupling proteins are phosphorylated by PKA; for example, the sarcoplasmic reticulum (SR) Ca²⁺ release channels (ryanodine receptors, RyR). The RyR is responsible for Ca²⁺ sparks. Ca²⁺ sparks can occur stochastically during diastole (independent of Ca²⁺ influx),⁴ but the cellular Ca²⁺ transient is due to temporal and spatial summation of Ca²⁺ sparks synchronized by the L-type calcium channel.

Although there is downregulation of ß-adrenergic receptors in end-stage heart failure,⁵ many cardiomyopathies also increase cytokine production, which leads to the expression of inducible nitric oxide synthase (iNOS) in cardiac myocytes (eg, heart failure).⁶ NO is a signaling molecule that is important in regulating myocardial contractility.⁷ Ventricular myocytes possess 2 NOS isoforms, constitutive (cNOS, type III) and inducible (iNOS, type II). iNOS expression leads to continuous production of large amounts of NO, compared with the small amount of NO produced by cNOS. Once NO is produced, it has 2 signaling pathways, cGMP-dependent and cGMP-independent, and can contribute to cardiac dysfunction.^2,8 In fact, many studies have found that when cardiac myocytes express iNOS, contractile function is decreased.^9,10 The mechanism of iNOS-dependent contractile dysfunction in human and animal cardiomyopathies, however, is not yet clear. It has been shown that at least part of the contractile dysfunction associated with these various diseases is due to overproduction of NO caused by expression of iNOS.¹⁰

NO has been shown to have "antiadrenergic" effects on cardiac myocyte function. Depending on experimental conditions and/or species, NO typically does not affect basal function but causes a hyporesponsiveness to ß-adrenergic stimulation.^11,12,15

NO also affects RyR activity in lipid bilayers¹³ and thus could contribute to the antiadrenergic effect of NO. Little work has been done, however, on the effects of NO on SR function, especially during ß-adrenergic stimulation, in intact cardiac myocytes that express iNOS.

The objective of this study is to examine the effect of iNOS expression on Ca²⁺ sparks, Ca²⁺ transients, and RyR activity during ß-adrenergic stimulation. The results may have direct bearing on the ß-adrenergic hyporesponsiveness commonly seen in cardiomyopathies. We hypothesize that iNOS expression and consequent NO production decrease Ca²⁺ transients because of inhibition of RyR activity during ß-adrenergic stimulation.

	Methods

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Expression of iNOS and Rat Cardiac Myocyte Isolation
Expression of iNOS was induced by injection of lipopolysaccharide (LPS, 10 mg/kg IP) into adult Sprague-Dawley rats (250 to 300 g) as described by Luss et al.¹⁴ Ventricular myocytes were isolated 6 hours after LPS injection as previously described.¹⁵ All procedures and animal care were in accordance with American Association for Accreditation of Laboratory Animal Care guidelines.

Human Myocyte Isolation
Myocytes were isolated from a failing human heart (ischemic, 63-year-old man) obtained at the time of transplantation. A section of left ventricle was excised. A vessel was cannulated and perfused with cold Tyrode’s solution containing (in mmol/L) NaCl 120, KCl 20, MgCl₂ 1, glucose 10, HEPES 10, NaH₂PO₄ 0.33, taurine 20, and pyruvate 5, and 3 mmol/L 2,3-butanedione monoxime (BDM, inhibitor of contractile proteins) to arrest the heart. Tissue was then perfused with the above solution (37°C) (except 4 mmol/L KCl) for 10 minutes, then collagenase (0.8 mg/mL, Worthington type II) and protease (0.08 mg/mL, Sigma) were added for 40 minutes, followed by washout with Tyrode’s solution for 10 minutes. Then the tissue was taken down, minced, and triturated. Cells were allowed to gravity-settle twice for 10 minutes, and the supernatant was discarded. The cell pellet was resuspended in Tyrode’s solution with 200 µmol/L Ca²⁺ without BDM. Cells were used within 6 hours of isolation. The procedure was done according to Loyola University Medical Center Institutional Review Board–approved protocol.

Dye Loading and Fluorescence Imaging
Myocytes were loaded with fluo 3-AM, and fluorescence imaging was performed as previously described.⁴ Ca²⁺ transients were derived from changes in fluorescence intensities (F) and normalized to basal fluorescence (F₀) and expressed as F/F₀.

Experimental Protocols
Myocytes were field-stimulated via platinum electrodes (0.2 Hz) and superfused with normal Tyrode’s solution (room temperature). When Ca²⁺ transients reached steady state, stimulation was stopped, and resting Ca²⁺ sparks were measured for 13.4 seconds. Field stimulation was started again and the solution switched to normal Tyrode’s solution with forskolin (100 nmol/L). When the cell response reached steady state (4 minutes), resting Ca²⁺ sparks were measured. Field stimulation was started again and the solution switched to normal Tyrode’s solution with forskolin and aminoguanidine (1 mmol/L) until steady state (4 minutes), and resting Ca²⁺ sparks were measured. SR Ca²⁺ content was estimated by rapid application of 10 mmol/L caffeine dissolved in normal Tyrode’s solution.

Solutions
The Tyrode’s solution consisted of (in mmol/L) NaCl 140, KCl 4, MgCl₂ 1, CaCl₂ 1, glucose 10, L-arginine 1, and HEPES 5 (pH 7.4). Forskolin was dissolved in DMSO to make a 100-µmol/L stock. An equivalent concentration of DMSO was added to control solution ([DMSO] <0.1%). Isoproterenol stock (1 mmol/L) was prepared fresh each day. Caffeine (10 mmol/L) and aminoguanidine (1 mmol/L) were dissolved directly in Tyrode’s solution. All chemicals were purchased from Sigma.

Statistical Analysis
Results were expressed as mean±SEM. Statistical significance (P<0.05) was determined by ANOVA (followed by Newman-Keuls test).

	Results

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iNOS Expression and Ca²⁺ Spark Frequency After ß-Adrenergic Stimulation
We examined the effect of iNOS expression in cardiac myocytes and its effect on resting Ca²⁺ spark frequency (CaSpF) after ß-adrenergic stimulation. Figure 1 shows representative experiments performed on a single myocyte isolated from normal (control) and LPS rats. Shown are line-scan images obtained with confocal microscopy. During control superfusion, there were few Ca²⁺ sparks (Figure 1, top) in either normal or LPS myocytes. When the cell was superfused with forskolin (100 nmol/L), a direct activator of adenylate cyclase, there was a substantial increase in the CaSpF in normal myocytes but only a small increase in the LPS myocytes (Figure 1, middle). When the cell was superfused with forskolin plus aminoguanidine (1 mmol/L), a specific iNOS inhibitor, there was no further increase in CaSpF in the normal myocyte but a dramatic increase in CaSpF in the LPS myocyte (Figure 1, bottom). Pooled data (Figure 2) show that there was no statistical difference between normal and LPS myocyte basal CaSpF [15±7 versus 8±2 sparks/(pL · s), P=NS]. There was a significant attenuation, however, in the response to forskolin in LPS myocytes compared with the normal myocytes [29±9 versus 128±14 sparks/(pL · s), P<0.05]. Blocking iNOS with aminoguanidine had no further effect on forskolin-stimulated CaSpF in normal myocytes [131±21 sparks/(pL · s)] but significantly increased the LPS myocyte response to forskolin stimulation [to 115±30 sparks/(pL · s), P<0.05 compared with forskolin alone]. In fact, there was no statistical difference between normal and LPS myocyte CaSpF in the presence of forskolin + aminoguanidine, suggesting complete reversal of the dysfunction. Moreover, these results may indicate that NO (produced via iNOS) can limit the ß-adrenergic stimulation of CaSpF.

View larger version (91K): [in this window] [in a new window]   Figure 1. Longitudinal line-scan images of Ca2+ sparks in rat ventricular myocytes. [Ca]i line plots from selected sites (white bars) are shown under images (width of 5 pixels). Left, Myocyte from normal rat; right, myocyte from LPS-injected rat. CONT indicates resting Ca2+ sparks in control Tyrode’s solution. Normal and LPS myocytes have 1 spark/image; FORSK, Ca2+ sparks in 100 nmol/L forskolin (normal myocyte has 12 sparks, LPS myocyte 2 sparks); FORSK+AG, forskolin+1 mmol/L aminoguanidine, a specific iNOS inhibitor (normal 9 sparks, LPS 12 sparks).

View larger version (18K): [in this window] [in a new window]   Figure 2. Forskolin (FORSK, 100 nmol/L) and aminoguanidine (AG, 1 mmol/L) effects on rat CaSpF. Pooled data are mean±SEM (*P<0.05, ANOVA, n=6 to 14 cells).

Guanylate Cyclase Involvement in the iNOS-Induced Depression of Ca²⁺ Sparks
If the CaSpF limitation by iNOS expression is due to NO activation of guanylate cyclase and increased cGMP production, then inhibiting guanylate cyclase should ameliorate the reduced CaSpF (forskolin-stimulated). Figure 3 shows that ODQ (10 µmol/L), a specific inhibitor of NO stimulation of guanylate cyclase, had no effect on the forskolin-induced increase in CaSpF in myocytes isolated from LPS rats. This contrasts to when iNOS was inhibited with aminoguanidine. These data suggest that the effect of NO on CaSpF is independent of guanylate cyclase activation and cGMP but could be mediated by other actions (eg, nitrosylation or other NO- or peroxynitrite-dependent redox modification of RyR).

View larger version (19K): [in this window] [in a new window]   Figure 3. Forskolin (FORSK, 100 nmol/L) and ODQ (10 µmol/L) effects on rat CaSpF in LPS myocytes. Data are mean±SEM (ANOVA, *P<0.05 vs control [CONT], n=5 cells).

Effects of iNOS on Twitch Ca²⁺ Transients and SR Ca²⁺ Load After ß-Adrenergic Stimulation
We examined the effects of iNOS expression on twitch [Ca]_i and SR Ca²⁺ load in cardiac myocytes field-stimulated at 0.2 Hz. SR Ca²⁺ load was measured by rapid application of caffeine (10 mmol/L). Figure 4A shows typical Ca²⁺ transient recordings and SR Ca²⁺ load of a myocyte isolated from an LPS rat. Figure 4B shows the mean data of twitch [Ca]_i. Forskolin (100 nmol/L) doubled twitch [Ca]_i compared with control (5.4±0.4 versus 2.6±0.7 F/F₀, P<0.05). When the cell was superfused with aminoguanidine, however, there was an additional increase in twitch [Ca]_i (7.0±0.7 F/F₀, P<0.05 versus forskolin alone). A main determinant of SR Ca²⁺ release is SR Ca²⁺ load. We examined this by caffeine-induced [Ca]_i (Figure 4A and 4C). Figure 4C shows mean data of SR Ca²⁺ load. As expected, forskolin increased SR Ca²⁺ load compared with control in LPS myocytes (6.0±0.8 versus 8.5±0.7 F/F₀, P<0.05). Superfusion with aminoguanidine had no further effect on SR Ca²⁺ load (8.6±0.6 F/F₀, P=NS versus forskolin alone). We conclude that the aminoguanidine-induced increase in twitch [Ca]_i (and CaSpF in Figures 1 and 2) in the presence of forskolin is independent of changes in SR Ca²⁺ load.

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of forskolin (FORSK) and aminoguanidine (AG) on twitch and caffeine-induced Ca2+ transients. A, Typical recordings of twitch [Ca]i transients (expressed as F/F0) and transients induced by rapid caffeine application (to measure SR Ca2+ load). B, Pooled data for twitch [Ca]i transients (mean±SEM, ANOVA, *P<0.05 vs control [CONT], **P<0.05 vs CONT and FORSK, n=8 cells). C, Pooled data for SR Ca2+ load (mean±SEM, ANOVA, *P<0.05 vs control, n=8 cells).

iNOS Inhibition and CaSpF in Human Failing Myocytes
We investigated whether iNOS inhibition would enhance the ß-adrenergic response of myocytes isolated from a failing human heart. The same experimental protocol was followed as with the LPS rat. Figure 5A shows line-scan images obtained with confocal microscopy. During control superfusion, there were few Ca²⁺ sparks (top). Addition of isoproterenol (10 nmol/L), a ß-adrenergic agonist, caused a small increase in CaSpF (middle). When 1 mmol/L aminoguanidine was added, there was a further increase in the CaSpF (bottom). Figure 5B shows the mean±SEM of all sweeps.

View larger version (24K): [in this window] [in a new window]   Figure 5. Ca2+ sparks in failing human ventricular myocytes. Recordings of line-scan images of Ca2+ sparks (A). Line plots of [Ca]i taken from selected sites (white bars) are shown under images. Top, Resting Ca2+ sparks in presence of control Tyrode’s solution (CONT), 1 spark shown in image. Middle, In presence of 10 nmol/L isoproterenol (ISO), 1 spark shown in image. Bottom, With ISO and 1 mmol/L aminoguanidine (ISO+AG), 5 sparks shown in image. B, Pooled data of CaSpF (mean±SEM, n=48 sweeps from 2 cells).

	Discussion

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Many studies have found that iNOS expression and increased NO production decrease contractile function.¹⁰ This may be a major pathway in the diminished ß-adrenergic responsiveness seen in many cardiomyopathies. Little work has been done, however, to assess the cellular mechanism of iNOS/NO-induced dysfunction.

ß-Adrenergic stimulation leads to a positive inotropic effect through activation of PKA and phosphorylation of several proteins involved in EC coupling. SR Ca²⁺ release events mediated by RyR have been identified as Ca²⁺ sparks, and these are the elementary events underlying EC coupling.¹⁶

iNOS, NO Production, Myocardial Dysfunction, and LPS
The LPS model has been well characterized in cardiac myocytes.¹⁴ For example, it is known that exposure to LPS leads to iNOS expression in cardiac myocytes,^14,17–20 increased NO synthase activity,^14,17–19 and increased cardiac myocyte cGMP levels.²⁰

Exposure to LPS leads to depressed myocardial contractility (usually seen as a decreased response to ß-adrenergic stimulation) and has been shown to be mainly through induction of iNOS and the resultant increase in NO production. For example, a study using iNOS-knockout mice found that nearly all of the myocardial dysfunction due to LPS injection was attributable to iNOS expression.²¹ Other studies have also found that inhibition of NO production reversed the contractile dysfunction due to LPS injection.^17,20 Thus, it is well established that LPS injection leads to cardiac myocyte iNOS functional expression and increased NO production and that this causes myocardial dysfunction.

NO, ß-Adrenergic Stimulation, and Ca²⁺ Sparks
ß-Adrenergic stimulation increases resting CaSpF.¹⁵ This effect is most likely via increased SR Ca²⁺ load (secondary to phospholamban phosphorylation) and/or phosphorylation of RyR by PKA to increase P_o. To the best of our knowledge, this is the first study to show that iNOS expression limits the increase in CaSpF induced by ß-adrenergic stimulation. In intact ventricular myocytes isolated from the LPS rat model, which expresses iNOS,¹⁴ there was a 77% decrease of the ß-adrenergic–induced stimulation of CaSpF compared with control myocytes (not expressing iNOS). Specific inhibition of iNOS with aminoguanidine had no effect on normal myocytes (CaSpF was 102±8% of forskolin control) but fully restored (compared with normal myocytes) the increase in CaSpF induced by forskolin in the myocytes from LPS rats (Figures 1 and 2). These data indicate that we can completely reverse this dysfunction simply by short-term (<5 minutes) inhibition of iNOS. We have previously shown that aminoguanidine is a specific inhibitor of iNOS in cardiac myocytes.^10,22 Thus, decreasing RyR activity may be a key mechanism in NO-induced reduction of ß-adrenergic responsiveness.

cGMP-Independent or -Dependent Signaling?
NO has 2 signaling pathways: cGMP-dependent and cGMP-independent. Our results with ODQ, a specific inhibitor of NO stimulation of guanylate cyclase,²³ imply that the effect of NO on CaSpF is independent of guanylate cyclase and cGMP (Figure 3). Previous work has shown that ODQ is a specific inhibitor of guanylate cyclase in cardiac myocytes (ODQ selectively decreased cGMP levels in response to NO donors).²⁴ If NO inhibits CaSpF through a cGMP-dependent pathway, then ODQ should have further increased the forskolin response (as for aminoguanidine). Because ODQ did not have any effect, these data suggest that NO alteration of RyR activity is cGMP-independent. Other studies have shown that NO can alter RyR gating in lipid bilayers via nitrosylation.^13,25,26 It is also known that the iNOS expression and increased NO production leads to peroxynitrite formation, which also causes myocardial dysfunction.²⁷ We have shown that part of the antiadrenergic effect of NO is via NO_x (eg, peroxynitrite).²⁴ The NO_x pathway could also play a role in the depressed RyR activity seen in this LPS model. This present study does not delineate the cGMP-independent pathway of iNOS-induced reduction in RyR activity, but it could be via RyR nitrosylation, nitration, or some other NO-dependent pathway.

iNOS, Ca²⁺ Transients, and SR Ca²⁺ Load
In the present study, we found that iNOS expression limited the increase in twitch [Ca]_i induced by forskolin (Figure 4A and 4B), although this effect was not as profound as was seen for CaSpF (Figure 2). A main determinant of SR Ca²⁺ release is the Ca²⁺ load of the SR.²⁸ Figure 4C shows that forskolin caused an increase in SR Ca²⁺ load. Aminoguanidine, however, did not cause a further increase in SR Ca²⁺ load (Figure 4C). This implies that the decrease in [Ca]_i (Figure 4B, FORSK versus FORSK+AG) and CaSpF (Figure 2) with iNOS is not due to decreased SR Ca²⁺ load but may be a direct effect of NO (or peroxynitrite) on RyR gating during EC coupling. In addition, we showed that although NO donors do not alter basal CaSpF in normal myocytes, they do have prominent effects after ß-adrenergic stimulation (mediated by altered SR Ca²⁺ load).¹⁵ This differs from results in the present study. We speculate that iNOS might be localized near RyR, thereby causing more direct local effects on RyR. With exogenous NO donors, local [NO] may not be high enough to directly affect RyR (the short half-life of NO may also limit local [NO] in this context). Neuronal NOS (NOS I) might also be localized in the SR,²⁹ but this is unlikely to be relevant here, because it would not be aminoguanidine-sensitive.

NO and Human Heart Failure
It is known that NO (produced via NO donors and endotoxin-induced iNOS expression) depresses the ß-adrenergic response in human myocardium.^30,31 iNOS expression has been reported in cardiac myocytes from various human heart failure phenotypes.^6,32,33 In fact, there is a significant correlation between heart failure severity and NO production.³⁴ Myocytes isolated from failing human hearts also have a decreased contractile response to ß-adrenergic stimulation and also to forskolin, which would bypass downregulation of ß-adrenergic receptors and increased G_i-protein expression seen in heart failure.³⁵ The ß-adrenergic hyporesponsiveness seen in human heart failure can also be reversed by inhibition of NO.^12,33 Our data in failing human myocytes (Figure 5) show increased ß-adrenergic–stimulated CaSpF with specific iNOS inhibition. These human data must be regarded as preliminary at this stage, and a more detailed study is warranted. Thus, the iNOS/NO-induced depression of RyR activity may be a key mechanism of the ß-adrenergic hyporesponsiveness in human heart failure. Thus, although ß-adrenergic receptors are downregulated in some heart failure pathogeneses, iNOS and NO may be more ubiquitous in mediating reduced ß-adrenergic responsiveness in cardiomyopathies.

In summary, the results of the present study show that expression of iNOS leads to a decrease in ß-adrenergic stimulation of SR Ca²⁺ release channel (RyR) activity, seen as a reduction in diastolic CaSpF and an attenuation of the whole-cell Ca²⁺ transient. NO decreases RyR function through a cGMP-independent mechanism, most likely through NO- and/or peroxynitrite-dependent redox modification of RyR. The NO-induced decrease in CaSpF is also seen in cardiac myocytes isolated from a failing human heart. This mechanism may be responsible in part for the ß-adrenergic hyporesponsiveness seen in many cardiomyopathies, including heart failure. Thus, specific targeting of cardiac myocyte iNOS may be therapeutically beneficial.

	Acknowledgments

 
This study was supported by NIH grants HL-10122 (Dr Ziolo) and HL-64098 and HL-64724 (Dr Bers). The authors thank the Loyola Department of Thoracic/Cardiovascular Surgery for providing the human failing heart and Klaus Schlotthauer, MD, for help with the human myocyte isolation.

Received July 5, 2001; revision received September 28, 2001; accepted October 3, 2001.

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