This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kaye, D. M. Articles by Kelly, R. A. Search for Related Content PubMed PubMed Citation Articles by Kaye, D. M. Articles by Kelly, R. A.

(Circulation Research. 1996;78:217-224.)
© 1996 American Heart Association, Inc.

Articles

Frequency-Dependent Activation of a Constitutive Nitric Oxide Synthase and Regulation of Contractile Function in Adult Rat Ventricular Myocytes

David M. Kaye, Stephen D. Wiviott, Jean-Luc Balligand, William W. Simmons, Thomas W. Smith, Ralph A. Kelly

From the Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Dr Ralph A. Kelly, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115. E-mail rakelly@bics.bwh.harvard.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Cardiac myocytes have recently been shown to express a constitutive Ca²⁺-sensitive isoform of NO synthase (NOS3), although the mechanism(s) responsible for activation of NOS3 and its physiological function remain to be determined. Since the activity of NOS3 is known to be regulated in part by the intracellular Ca²⁺ activity ([Ca²⁺]_i) in endothelial cells, we determined whether increasing myocyte [Ca²⁺]_i by uniform electric field pacing was accompanied by an increase in NOS3 activity, detected as nitrite accumulation in the medium. A higher [Ca²⁺]_i with increasing pacing frequencies was shown to be accompanied by a time-dependent accumulation of nitrite in medium that bathed adult rat ventricular myocytes stimulated at 3 Hz. Nitrite release by paced cells was significantly attenuated by treatment with either the NO synthase inhibitor nitro-L-arginine (L-NA, 1 mmol/L) or the intracellular Ca²⁺ chelator BAPTA-AM (20 µmol/L). Paced myocytes also exhibited a frequency- and time-dependent increase in intracellular cGMP content that could be inhibited significantly by either L-NA or the soluble guanylate cyclase inhibitor LY83583 (5 µmol/L). To determine whether the increase in NOS3 activity with pacing affected contractile function, myocytes were sequentially paced at frequencies from 0.5 to 3 Hz. Methylene blue, L-NA, and LY83583 all increased the amplitude of shortening of myocytes paced at 3 Hz. Furthermore, a significantly greater positive inotropic response to high extracellular Ca²⁺ (3 mmol/L) was demonstrated by myocytes pretreated with L-NA compared with control cells. These data indicate that myocyte NOS3 activity is regulated in part by [Ca²⁺]_i, whether induced by changes in pacing frequency or [Ca²⁺]_o, and depresses myocyte contractile responsiveness to higher stimulation frequencies.

Key Words: nitric oxide synthase • nitric oxide • contractility • cardiac myocytes

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Since the original description of endothelium-derived relaxing factor by Furchgott and Zawadski¹ and its subsequent biochemical identification as NO_x, it is now known that NO_x participates in an unexpectedly large number of biological events in diverse organs and tissues. These include its role as an obligate intermediate in specific intracellular and intercellular signaling pathways ranging from the regulation of smooth muscle tone in the vasculature to its participation at higher concentrations in specific host defense mechanisms.^{2 3 4 5 6} Within the heart, NOS activity has been identified in a number of cellular constituents of cardiac muscle, including endocardial endothelium, endothelium and smooth muscle of the epicardial vessels and the microvasculature, specialized cardiac conduction and pacemaker tissue, and activated infiltrating inflammatory cells and in cardiac myocytes themselves.^{7 8 9 10 11 12}

Two types of NOS activity have been described in cardiac muscle cells: a constitutive Ca²⁺-sensitive activity and a Ca²⁺-insensitive cytokine-inducible activity. It is now recognized that there are at least three isoforms of NOS, each the product of separate genes. Two NOS isoforms are constitutively present in many cell types and are activated in part by Ca²⁺ within the physiological range for Ca²⁺ activity in the cytosol of most cells. These are NOS1, originally identified in brain tissue, and NOS3, originally identified in large-vessel endothelial cells. A third isoform, termed iNOS or NOS2, the activity of which is not regulated by intracellular Ca²⁺ activity, was originally identified in activated murine macrophages but is now known to be inducible by inflammatory mediators in a number of cell types. The three NOS isoforms share 50% to 60% amino acid sequence homology.⁴

Induction of NOS2 with a high capacity for NO_x production in cellular constituents of cardiac muscle, including cardiac myocytes,¹³ microvascular endothelium,¹⁴ and inflammatory cells, may play a part in the pathogenesis of heart failure accompanying cardiac allograft rejection¹⁵ and the systemic inflammatory response syndrome.^{16 17} In contrast, the physiological role of the constitutively present NOS3 in cardiac myocytes is less clear. Work from this laboratory^{9 11} and by other researchers¹⁸ has suggested that a constitutively present NO signaling pathway mediates muscarinic cholinergic signaling in cardiac myocytes and specialized pacemaker tissue, such as sinoatrial node cells, and modifies myocyte contractile responsiveness to ß-adrenergic stimulation.

In the present report, we examine the hypothesis that increased contractile activity, with its attendant rise in intracellular Ca²⁺ activity ([Ca²⁺]_i), is associated with the activation of NOS3 and the generation of cGMP in cardiac myocytes. We also examine the relevance of these pacing-induced increases in NOS3 activity to the relationship between pacing frequency and the amplitude of contraction in these cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of Adult Rat Ventricular Myocytes
Ca²⁺-tolerant ventricular myocytes were isolated from adult rat ventricular muscle, as described previously.¹⁹ In brief, hearts were rapidly excised from male Sprague-Dawley rats (175 to 250 g) under ether anesthesia and then retrogradely perfused with KHB buffer (mmol/L: NaCl 110, KCl 4.0, CaCl₂ 1.0, MgSO₄ 0.9, K₂HPO₄ 0.9, NaHCO₃ 19.2, and glucose 11) that had been equilibrated to pH 7.4 with 5% CO₂/95% O₂. After a 5-minute perfusion with KHB buffer, a nominally Ca²⁺-free KHB buffer was substituted as the perfusate until beating ceased. At this point, enzymatic digestion was begun by perfusing the hearts with nominally Ca²⁺-free KHB containing 0.05% collagenase (Worthington) and 0.03% hyaluronidase (Sigma Chemical Co) until the hearts became flaccid. The atria, great vessels, and valvular apparatus were then excised, and the remaining ventricular tissue was minced and further digested in nominally Ca²⁺-free collagenase/hyaluronidase–containing KHB buffer with additional trypsin (0.02 mg/mL, Sigma) and deoxyribonuclease (0.02 mg/mL, Sigma). After digestion of the ventricular tissue, ventricular myocytes were segregated from the nonmyocyte cellular fraction by a series of washes, followed by density gradient sedimentation through albumin.²⁰ The final cell suspension, which typically consisted of at least 93% myocytes, was then resuspended in DMEM (GIBCO) supplemented with albumin (2 mg/mL), L-carnitine (2 mmol/L), creatine (5 mmol/L), taurine (5 mmol/L), L-glutamine (1.3 mmol/L), insulin (0.1 µmol/L), triiodothyronine (0.1 nmol/L), pyruvate (2.5 mmol/L), and 0.1% penicillin/streptomycin. This medium, termed "ACCITT,"¹⁹ is referred to as "defined medium" throughout this report. Primary myocyte cultures were maintained at 37°C in 5% CO₂. The medium was changed 1 hour after plating to remove loosely attached cells. All experiments described in the present study were initiated within 4 to 6 hours of cell isolation.

Continual Electric Field Stimulation and Assessment of Contractility
To assess the influence of beating on NO synthesis and release by adult ventricular myocytes, two separate electrical stimulation formats were used. First, in experiments designed to assess the rate of NO release, as gauged by nitrite accumulation in the surrounding culture media, continual electrical field stimulation was performed according to methods previously established in this laboratory.¹⁹ In brief, 4x10⁶ cells were plated on laminin (20 µg/mL)–coated 175-cm² tissue culture flasks (Ezin, Nunc) to which a custom-designed electrostimulation device consisting of parallel graphite electrodes was attached. Electrical stimulation was initiated 4 to 6 hours after plating, and cells were maintained at 37°C. In all experiments, unless otherwise stated, the stimulation frequency was 3 Hz. The stimulus voltage was adjusted to maximize pacing capture, which was typically >70% at 150 to 180 V. Electrical impulses were triggered by computer, and the polarity of each successive stimulus was alternated to minimize electrolysis at each electrode. In these experiments, electrical stimulation was performed in phenol red–free DMEM (GIBCO) in order to allow estimation of nitrite concentration by the Griess reaction, with supplements as previously described for the defined medium described above.

A second series of pacing studies was devised for assessing the effect of uniform electric field stimulation on myocyte cGMP content. In these experiments, 3x10⁵ myocytes were plated onto laminin-coated six-well cluster plates (Costar) in defined medium as described above. Electrical stimulation was delivered to either three or six wells at a time by means of platinum electrodes to each of the distal two wells, the remainder of which were connected by 1% agarose bridges, as previously described by McDermott and Morgan.²¹

The contractile behavior of isolated adult ventricular myocytes was assessed using techniques previously described.^{22 23} Freshly isolated cells were plated onto laminin-coated glass coverslips and placed in a perfusion chamber on the temperature-controlled (37°C) stage of a microscope connected to a video motion analyzer. Electrical stimulation was begun at 0.5 Hz (3-millisecond pulse duration) for at least 60 seconds to ensure stable baseline contractile activity. Subsequently, the stimulation frequency was increased sequentially to 1, 2, and 3 Hz. Measurements of contractile amplitude were acquired after at least 30 seconds at each level to allow a stable change in contractility. Only one cell was studied per coverslip. The criteria for selection were as follows: (1) a rod-shaped appearance without sarcolemmal blebs, (2) the absence of spontaneous beating, (3) a symmetrical pattern of cell shortening, (4) a stable contractile response during each stimulation level, and (5) the ability of a cell to complete the entire stimulation protocol. For a given cell, the contractile response at each frequency was expressed as a percentage of the baseline contractile amplitude at 0.5 Hz. The superfusion buffer for these short-term contractility experiments was composed of (mmol/L) NaCl 140, KCl 4.0, MgCl₂ 0.5, CaCl₂ 1.0, HEPES 10, and glucose 10, pH 7.4 at 37°C.

Measurement of Medium Nitrite and Myocyte cGMP Concentrations
Nitrite content of the defined medium conditioned by continually paced adult myocytes was determined by established methods.¹⁴ Aliquots of control and paced myocyte-conditioned medium (ie, defined medium containing phenol red–free DMEM) were centrifuged at 3000 rpm for 10 minutes to remove cellular debris. The nitrite content in the supernatant was measured by combining 150 µL of medium with 900 µL of the Griess reagent (0.75% sulfanilamide in 0.5N HCl/0.075% naphthylethylenediamine), and the concentration of the resulting chromophore was determined spectrophotometrically at 543 nm. Nitrite concentration was calculated from a standard curve constructed over the linear range of the assay (0.1 to 50 µmol/L). Note that only nitrite (NO₂^-) and not nitrate (NO₃^-) was detected in myocyte-conditioned medium using the protocol described above.

Myocyte cGMP content was determined by collecting cells in a lysis buffer containing 0.1N HCl and 1 mmol/L IBMX. cGMP content was determined by radioimmunoassay using a commercially available kit (Biomedical Technologies) and is expressed as picomoles per milligram protein. In some experiments, myocytes were pretreated with 0.1 mmol/L IBMX to optimize the detection of changes in cGMP content during pacing. Total cellular protein was determined by the Bradford method (BioRad).

Fura 2 Fluorescence Spectroscopy
To assess the effect of increasing driving frequencies on [Ca²⁺]_i, we performed fura 2 fluorescence spectroscopy. Fluorescence measurements were performed according to methods previously described by our laboratory.²² In brief, freshly isolated adult ventricular myocytes were plated onto laminin-coated glass coverslips and incubated in superfusion buffer (mmol/L: NaCl 140, KCl 4.0, MgCl₂ 0.5, CaCl₂ 1.0, HEPES 10, and glucose 10, pH 7.4) containing 2 µmol/L fura 2-AM for 20 minutes at 37°C, followed by 20 minutes of washing. Experiments were conducted on the heated stage (37°C) of a light shielded microscope. Fluorescence measurements were performed with a SPEX CM2 dual-excitation spectrofluorimeter (SPEX Industries) by monitoring the 505-nm emission with dual 340- and 380-nm excitation. The protocols for cell stimulation and perfusion were essentially identical to those described above. Ca²⁺ transients were acquired for 10 seconds at rest, followed by stimulation at 1, 2, and 3 Hz. An index of the time-averaged intracellular Ca²⁺ was obtained by determining the area under the Ca²⁺ transient (340/380 ratio)–time relationship using SPEX DM3000 software.

Characterization of NOS Isoforms in Freshly Isolated Myocytes
Total cellular RNA was extracted from freshly isolated adult rat ventricular myocytes according to the method of Chomczynski and Sacchi.²⁴ Total RNA (15 µg) was electrophoresed through a 1% agarose/2.2% formaldehyde gel, followed by vacuum transfer to nylon membrane. Northern hybridizations were then performed using a 217-bp cDNA for NOS2¹³ and a 324-bp cDNA for NOS3¹¹ that were radiolabeled with [³²P]dCTP by random primer labeling (Boehringer Mannheim). Blots were washed in 0.25x SSC/0.1% SDS for 30 minutes at 65°C and then autoradiographed for 12 to 24 hours.

Materials
All chemicals were purchased from Sigma unless otherwise specified. BAPTA-AM was obtained from Molecular Probes, and LY83583 was from Calbiochem.

Statistical Methods
Data are presented as mean±SEM. Between-group comparisons were performed by Student's t test where data were normally distributed or by the Mann-Whitney test for nonparametric data. Comparison among multiple groups was performed by ANOVA where appropriate. Regression coefficients were obtained by least squares regression. The null hypothesis was rejected at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
NOS3 Activity in Cardiac Myocytes: Increased [Ca²⁺]_i, Nitrite Release, and cGMP Induced by Pacing
During continual electrical field stimulation of freshly isolated adult rat ventricular myocytes at a frequency of 3 Hz, a time-dependent rise in the accumulation of nitrite in the culture media was consistently observed (Fig 1A). To confirm that the production of nitrite indeed reflected release of NO_x through a NOS-dependent process, the rate of nitrite accumulation was also determined for myocytes that were preincubated for 4 hours and then electrically stimulated at 3 Hz in the presence of the NOS inhibitor L-NA (1 mmol/L). Under these conditions, the rate of nitrite accumulation was significantly attenuated (P<.05), returning toward that observed in defined medium exposed to repetitive electrical impulses in the absence of cells (Fig 1B). Similarly, treatment with 20 µmol/L BAPTA-AM, an agent that chelates intracellular Ca²⁺, reduced the rate of nitrite accumulation to a level that was significantly lower than that observed in medium conditioned by paced myocytes. A slow time-dependent generation of nitrite was detected in the absence of myocytes in defined medium when exposed to the same pacing protocol. This was substantially below the level of nitrite generated by myocytes under otherwise identical conditions.

View larger version (42K): [in this window] [in a new window]   Figure 1. Pacing increases NOS3 activity in adult rat ventricular myocytes (ARVM). A, Time-dependent accumulation of nitrite in medium conditioned by electrically stimulated cardiac myocytes (3 Hz, ) and in medium conditioned by quiescent control myocytes (). Nitrite accumulation in medium conditioned by 4x106 cells per chamber in each experimental group was significantly higher in paced cells (P<.001 by ANOVA). B, The rate of increase in nitrite accumulation (dN/dt) illustrated for medium conditioned by quiescent control myocytes (ARVM), for medium exposed to the pacing protocol in the absence of cells, and for medium conditioned by myocytes stimulated at 3 Hz in the absence and presence of the NOS inhibitor L-NA (1 mmol/L) or the intracellular Ca2+ chelator BAPTA (20 µmol/L). Values are mean±SEM (n=4 to 7 experiments for each condition). *P<.05 vs quiescent control cells and vs medium exposed to repetitive electrical stimuli in the absence of cells; +P<.05 vs myocytes paced at 3 Hz in the absence of L-NA or BAPTA. C, Northern blots of total RNA from freshly isolated myocytes or from myocytes pretreated for 24 hours with inflammatory cytokines as described previously13 that was hybridized with radiolabeled cDNA probes for rat NOS3 (lane 1) and NOS2 (lanes 2 and 3), as well as for 18S rRNA to normalize for RNA loading.

To identify the NOS isoform responsible for the nitrite production in these experiments, total RNA was obtained from freshly isolated adult rat ventricular myocytes and hybridized with cDNA probes for rat NOS2 or NOS3 as previously described.^{11 13} As shown in Fig 1C, NOS3 mRNA was readily detectable in fresh myocyte primary isolates. NOS2 mRNA could only be detected in these preparations after myocyte pretreatment with inflammatory cytokines interleukin-1ß and interferon gamma.

To demonstrate the effect of increasing driving frequencies on myocyte [Ca²⁺]_i, we performed fluorescence spectroscopy in myocytes loaded with fura 2-AM. Increasing beating rates were associated with progressive increases in both systolic and diastolic [Ca²⁺]_i, as assessed by the fura 2 emission spectra ratios (Fig 2A). There was a linear rise in the integrated time-averaged [Ca²⁺]_i with increasing pacing frequencies from 0 to 3 Hz (Fig 2B, r=.99, P<.01).

View larger version (27K): [in this window] [in a new window]   Figure 2. Pacing increases [Ca2+]i and intracellular cGMP content. A, The ratio of the fura 2 emission spectra at 340- and 380-nm excitation wavelengths is illustrated from a single adult rat ventricular myocyte at rest and after pacing at 1, 2, and 3 Hz. B, The time-averaged integral (AUC) of the Ca2+ transient at successively increasing pacing frequencies is illustrated (mean±SEM from five cells). C, The intracellular cGMP content (in picomoles per milligram protein [prot] for 5 minutes) is shown in myocytes paced at 1, 2, or 3 Hz for 5 minutes and in quiescent control cells (C) and in myocytes paced at 3 Hz in the presence of L-NA (1 mmol/L). All cells were treated with 0.1 mmol/L IBMX. Values are mean±SEM from three sets of experiments. *P<.05 vs control cells; #P<.05 vs paced cells at 1 or 2 Hz; and P<.05 vs paced cells at 3 Hz in the absence of L-NA.

To determine whether increased NOS3 activity with pacing was accompanied by an increase in myocyte cGMP content, short-term pacing experiments were conducted in the presence of the nonselective phosphodiesterase inhibitor IBMX (0.1 mmol/L). After 5 minutes of pacing, a significant increase in the intracellular cGMP content was noted for cells at all pacing frequencies compared with control quiescent cells. At 3 Hz, the cGMP content was significantly greater than that at 1 and 2 Hz and was substantially attenuated in cells pretreated with 1 mmol/L L-NA (Fig 2C). In the absence of IBMX, a significant increase in cGMP content could be detected in myocytes paced at 3 Hz for 30 minutes and was maintained at 120 minutes (Fig 3A). Accumulation of myocyte cGMP with pacing could be inhibited both by the NOS antagonist L-NA (1 mmol/L) and by the soluble guanylate cyclase inhibitor LY83583 (5 µmol/L), as shown in Fig 3B.

View larger version (29K): [in this window] [in a new window]   Figure 3. Time-dependent increases in cGMP content with pacing. A, In the absence of IBMX, a significant increase in cGMP content compared with quiescent control adult rat ventricular myocytes was not apparent in myocytes paced at 3 Hz until 30 minutes after initiation of pacing (see Fig 2C). B, Both L-NA (1 nmol/L) and the guanylate cyclase inhibitor LY83583 (5 µmol/L) prevented the rise in cGMP at 30 minutes in adult rat ventricular myocytes continually paced at 3 Hz. Values are mean±SEM from three sets of experiments. *P<.05 vs quiescent control myocytes; P<.05 vs myocytes paced at 3 Hz in the absence of either L-NA or LY83583.

NOS Inhibition Augments the Amplitude of Shortening of Paced Adult Myocytes
Since activation of either the constitutive NOS3 or the inducible NOS2 in cardiac myocytes has been demonstrated to decrease the contractile responsiveness of isolated paced myocytes to ß-adrenergic agonists,^{9 10 13} we determined whether activation of NOS3 by pacing exerted a tonic negatively inotropic influence on isolated adult rat ventricular myocytes in vitro. To investigate this, two strategies were used. First, the amplitude of shortening of isolated myocytes to electrical stimulation at frequencies ranging from 0.5 to 3 Hz was determined. In control cells, a positive staircase response in contractile amplitude was observed from 0.5 to 3 Hz. Addition of 10 µmol/L methylene blue, a nonselective antagonist of the NO signaling pathway, significantly augmented the amplitude of shortening of myocytes paced at 3 Hz compared with control myocytes (Fig 4). Similarly, incubation with L-NA (1 mmol/L) or with the guanylate cyclase inhibitor LY83583 (5 µmol/L) accentuated contractile performance at 3 Hz. Of note, there was no difference in the amplitude of shortening at 0.5 Hz between control cells and those of any of the three treatment groups (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 4. Activation of NOS3 depresses myocyte amplitude of shortening during pacing. Graphs show the contractile response of isolated adult rat ventricular myocytes stimulated at 1, 2, and 3 Hz for control cells () and cells treated with 10 µmol/L methylene blue (MeB, , top panel), 1 mmol/L L-NA (, middle panel), and 5 µmol/L LY83583 (, bottom panel). Data are presented as mean±SEM (n=6 to 12 cells per treatment group) and are expressed as percentage increments over baseline amplitude of shortening at 0.5 Hz. *P<.05 vs control.

To address further the influence of endogenously generated NO_x on myocyte contractile function, we compared the positively inotropic response elicited by exposure to high external Ca²⁺ (3 mmol/L) in control myocytes paced at 2 Hz with the inotropic response to high Ca²⁺ of myocytes pretreated with L-NA (1 mmol/L) and also paced at 2 Hz. In control cells, high [Ca²⁺]_o increased the amplitude of shortening by 31±6% (n=11). This was significantly less than the 57±9% increase in contractile amplitude following high Ca²⁺ observed in L-NA–treated cells (n=8) (P<.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrate that repetitive depolarization accompanied by contraction in isolated adult rat ventricular myocytes is associated with enhanced generation of NO_x, as detected by nitrite accumulation in myocyte-conditioned medium and by increased accumulation of cGMP. Unlike previous studies,^{9 10 18 25} the increase in NOS activity was observed in the absence of autonomic nervous system agonists or inflammatory cytokines, both of which have been found to be associated with activation of NOS in cardiac muscle.

Several laboratories, including ours, have shown that NOS2 can be induced in cardiac myocytes in vivo after parenteral injection of endotoxin in animals or during rejection of transplanted hearts in an experimental cardiac transplant model.^{7 13 15 16} NOS2 activity can also be induced in cardiac myocytes in vitro after exposure to inflammatory cytokines. This results in a decrease in the spontaneous beating rate of neonatal rat myocytes²⁶ and blunts contractile performance of isolated electrically paced adult rat ventricular myocytes.^{10 13 16 27 28} During regular screening of freshly isolated preparations of adult rat ventricular myocytes obtained from presumptively normal animals, we have infrequently detected expression of NOS2 by Northern analysis, presumably arising from an unrecognized infection in the animal. As shown in Fig 1C, only NOS3 could be routinely detected in fresh adult myocyte primary isolates; therefore, NOS2 activity is unlikely to have contributed to the accumulation of nitrate in myocyte-conditioned medium or to the activation of guanylate cyclase in the experiments reported here.

It is likely, therefore, that the frequency-dependent accumulation of nitrite we observed was due to activation of a constitutive isoform of NOS in these cells. A relatively Ca²⁺-dependent NOS activity was originally reported by Schulz et al⁷ in isolated ventricular muscle cells. Our laboratory has recently identified this activity as being due to expression of the NOS isoform originally described in endothelial cells (ie, NOS3) using several complementary techniques. These included exclusion of other NOS isoforms by reverse-transcription polymerase chain reaction, by identification of NOS3 on Western blots from isolated myocyte primary cultures, and by immunohistochemical detection of this isoform in adult rat ventricular myocytes in situ and in vitro using an isoform-selective antibody.¹¹

The physiological role(s) of this endogenous constitutively expressed NO signaling system remains unclear. Finkel et al²⁹ demonstrated that exposure of isolated paced hamster papillary muscles to some inflammatory cytokines was accompanied by a rapid decline in force generation, a decline that could be reversed by a NOS antagonist. Since this response to cytokines occurred too rapidly for NOS2 induction to be a factor, these data implied that activation of a constitutive NOS in one or more cell types in cardiac muscle was responsible. We have demonstrated that a NOS inhibitor would augment the inotropic response of isolated paced adult rat ventricular myocytes to isoproterenol, implying that increased NO generation in response to the ß-adrenergic agonist played a role as a countervailing negatively inotropic signal. We also linked an endogenous constitutively expressed NO signaling pathway to muscarinic cholinergic agonist–induced depression both of the spontaneous beating rate of neonatal rat ventricular myocytes⁹ and ß-adrenergic agonist–induced increases in L-type Ca²⁺ channel current and contractile amplitude of paced adult ventricular myocytes.¹¹ In agreement with these observations, Hare et al²⁵ have recently reported that in an anesthetized closed-chest dog preparation, intracoronary infusion of a NOS inhibitor blunts the vagally mediated depression in the inotropic response of the left ventricle to intracoronary dobutamine, an effect that could be reversed by L-arginine.

The electrical pacing experiments in the present report were carried out in the absence of autonomic nervous system agonists, indicating a different mechanism of activation of the constitutive NOS. Both NOS1 and NOS3 are activated by Ca²⁺ and calmodulin after an increase in intracellular Ca²⁺ activity. ß-Adrenergic agonists, among other actions, increase [Ca²⁺]_i, which could activate NOS3 in cardiac myocytes. The frequency dependence of nitrite accumulation in paced adult ventricular myocytes also suggests that NOS activation was due to the increase in time-averaged intracellular Ca²⁺ with higher pacing rates, an interpretation that is supported by the ability of the intracellular Ca²⁺ chelator BAPTA-AM to suppress pacing-induced nitrite accumulation (Fig 1). Interestingly, Finkel et al³⁰ have recently demonstrated that the NOS inhibitor N^G-monomethyl-L-arginine shifts upward the force-frequency relationship of isolated electrically stimulated hamster papillary muscle, an effect that could be mimicked by methylene blue. In contrast, 8-bromo-cGMP shifts the force-frequency relationship downward, implicating NO-dependent generation of cGMP in the regulation of the excitation-contraction coupling in the heart. Although these authors could not define the cellular source of the endogenous NO responsible for this effect, the data in the present report suggest that it is likely to be activation of NOS3 in cardiac myocytes.

Several recent studies that have been designed to examine the extent to which basal NO production influences myocardial contractility, in the absence of adrenergic agonists or other cardiotonic agents, have yielded conflicting results.^{30 31 32} In general, either these studies have used NO antagonists and/or NO synthase inhibitors to examine the role of endogenously synthesized NO, or they have used one or more pharmacological NO donors in an effort to mimic the effect of endogenous NO_x. The lack of specificity of some NO antagonists and of some NOS inhibitors probably contributes to those apparently conflicting results.

Although the use of pharmacological NO donors has provided useful insights into the potential role of NO_x and its derivatives in many biological contexts, they have several important limitations. The actual dose of bioactive NO delivered to a specific cellular or intracellular target depends on concentration, cellular redox state, and pH_i, among other variables, and is difficult to estimate. Even if this were known, the relevance of these concentrations and the time course of exposure compared with those values achieved after the activation of endogenous sources of NO_x remains unclear. In addition, several NO donors (such as SIN-1, 3-morpholinosydnonimine) may have additional pharmacological effects that are unrelated to NO release.³³ Also, these agents cannot model spatially restricted actions of endogenously derived NO within a cell that may be the result of intracellular compartmentation.^{11 34}

The specific experimental preparation and biophysical techniques used also will constrain the conclusions that can be drawn. Brady et al,³¹ for example, reported no effect of methylene blue alone on the amplitude of shortening of isolated electrically paced adult guinea pig ventricular myocytes, and an initial report by Balligand et al⁹ from this laboratory reported that L-NA had no effect on the basal amplitude of shortening of isolated electrically paced adult rat ventricular myocytes (ie, in the absence of a ß-adrenergic agonist). However, these conclusions were based on the examination of myocyte shortening at 0.5 Hz (for methylene blue) and 2 Hz (for L-NA), respectively. Both driving frequencies are below those reported in the present study to demonstrate consistently an effect of either reagent on the amplitude of shortening (Fig 4). In addition, Weyrich et al³² have recently reported that neither NOS inhibitors nor a variety of pharmacological NO donors delivered at "physiological" concentrations had any effect on the contractile force developed by isolated rat papillary muscles or isolated adult rat cardiac myocytes. However, these authors used a stimulation frequency of 0.25 Hz, a frequency of stimulation that, as noted above, is well below that at which we have observed an effect of NO antagonists or NOS and certainly well below physiological beating rates for the rat heart.

Several groups of investigators have studied the effects of either pharmacological NO donors or lipid-soluble analogues of cGMP (a downstream chemical messenger for some components of NO-dependent signaling) in isolated hamster papillary muscles and intact guinea pig and ferret hearts, in isolated adult rat ventricular myocytes, and in normal human subjects undergoing intracoronary infusions of the NO donor nitroprusside.^{30 35 36 37} These studies noted only minimal effects on peak amplitude of shortening (in isolated cells examined at 25°C at a driving frequency of 0.5 Hz) or on peak left ventricular pressure (in isolated ejecting guinea pig hearts), although a modest decline in peak systolic pressure was observed with intracoronary infusion of nitroprusside. Interestingly, however, these reports conclude that the most notable pharmacological effect of nitroprusside or of 8-bromo-cGMP was to facilitate diastolic relengthening and ventricular relaxation (ie, a positive lusitropic effect). A qualitatively similar effect was observed by Shah et al,³⁶ who used 8-bromo-cGMP at a faster driving frequency (2 Hz at 35°C) in isolated guinea pig myocytes. These data are consistent with our observations at pacing frequencies at or below 2 Hz, where only minimal effects of an NO antagonist or NOS inhibitor were observed. We cannot make any statement about the effects of these reagents on either the velocity of shortening or the time course and velocity of relengthening because of the limited frequency response of our video-based edge-detection system.

In addition to the specific mechanism(s) contributing to the activation of NOS3 in paced adult myocytes, there remain at least two additional important aspects of the role of increased NO production in regulating myocyte contractile responsiveness to increased frequency of contraction: (1) the downstream signaling pathways that mediate the observed changes in cell function and (2) the physiological relevance of these changes for normal cardiac function. NO-dependent effects can be broadly classified into cGMP-mediated effects and non–cGMP-mediated actions. The prompt twofold to threefold rise in adult myocyte cGMP content within 5 minutes of the onset of pacing (Fig 2C) suggests that cGMP may play a role in mediating a portion of the physiological response we observed, either by activating protein kinase G or by regulating intracellular cAMP levels by cGMP-regulated cAMP phosphodiesterases. Shah et al³⁶ have documented in isolated adult rat ventricular myocytes that 8-bromo-cGMP, a relatively selective activator of protein kinase G, mimicked some of the biophysical effects of an exogenous NO donor. Others have suggested that cGMP activation of protein kinase G may reduce L-type Ca²⁺ current^{38 39} or affect Ca²⁺ release by the sarcoplasmic reticulum Ca²⁺ release channel.³⁰

A number of non–cGMP-dependent effects of NO have now been described. Recently, Kobzik and colleagues^{40 41} reported the presence of NOS1 and NOS3 in skeletal muscle, with the majority of NOS expression in fast-twitch (type II) fibers. NOS inhibitors shift the force-frequency relationship of mixed fiber skeletal muscle (such as diaphragm) to the left, an effect that can be reversed by pharmacological NO donors. In these skeletal muscles, cGMP-dependent signaling was found to have a relatively modest impact in mediating the actions of endogenous NOS activation, and other non–cGMP-mediated effects of NO were investigated. They demonstrated that increased NOS activation is associated with increased generation of reactive oxygen intermediates, which may direct NO away from metal complexes (eg, guanylate cyclase, cytochrome complexes) toward proteins with regulatory sulfhydryl targets, such as those on the sarcoplasmic reticulum Ca²⁺ release channel. However, no data were reported in support of specific nonheme targets. Finally, reports from our own⁴² and other laboratories,^{43 44 45} which used both NOS inhibitors and pharmacological NO donors, implicate a potential role for NO in the regulation of cellular energetics.

On the basis of the evidence reported here, we conclude that activation of NOS3 in electrically stimulated adult rat ventricular myocytes accompanies the rise in [Ca²⁺]_i that initiates excitation-contraction coupling. The resulting generation of NO_x, by increasing intracellular cGMP and perhaps by other mechanisms, results in a decrease in the amplitude of shortening both at higher pacing frequencies and in the presence of high extracellular Ca²⁺. These data support the view that an endogenous NO signaling pathway participates in the regulation of contractile function of cardiac muscle.

	Selected Abbreviations and Acronyms

 

IBMX = 3-isobutyl-1-methylxanthine KHB = Krebs-Henseleit bicarbonate L-NA = nitro-L-arginine NOS = NO synthase NOS1 = "neuronal" NOS NOS2 (iNOS) = inducible NOS NOS3 = "endothelial" NOS NOx = nitrogen oxide

	Acknowledgments

 
This study was supported by grant IP50-HL-52320 (to Dr Smith) from the National Heart, Lung, and Blood Institute. Dr Kaye is the recipient of an Overseas Research Scholarship from the National Heart Foundation of Australia. The valuable contributions of T. Achtem to these studies are acknowledged.

	Footnotes

 
This manuscript was sent to Harold Strauss, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received May 12, 1995; accepted October 10, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Furchgott RF, Zawadski JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373-376. [Medline] [Order article via Infotrieve]

2. Stamler JS. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell. 1994;78:931-936. [Medline] [Order article via Infotrieve]

3. Bredt D, Snyder S. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem. 1994;63:175-195. [Medline] [Order article via Infotrieve]

4. Nathan C, Xie Q-W. Nitric oxide synthases: roles, tolls, and controls. Cell. 1994;78:915-918. [Medline] [Order article via Infotrieve]

5. Schmidt HHHW, Walter U. NO at work. Cell. 1994;78:919-925. [Medline] [Order article via Infotrieve]

6. Marletta MA. Nitric oxide synthase: aspects concerning structure and catalysis. Cell. 1994;78:927-930. [Medline] [Order article via Infotrieve]

7. Schulz R, Nava E, Moncada S. Induction and potential biological relevance of a Ca²⁺-independent nitric oxide synthase in the myocardium. Br J Pharmacol. 1992;105:575-580. [Medline] [Order article via Infotrieve]

8. Ursell PC, Mayes M. The majority of nitric oxide synthase in pig heart is vascular not neural. Cardiovasc Res. 1993;27:1920-1924. [Abstract/Free Full Text]

9. Balligand J-L, Kelly RA, Marsden PA, Smith TW, Michel T. Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proc Natl Acad Sci U S A. 1993;90:347-351. [Abstract/Free Full Text]

10. Balligand J-L, Ungureanu D, Kelly RA, Kobzik L, Pimental D, Michel T, Smith TW. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest. 1993;91:2314-2319.

11. Balligand J-L, Kobzik L, Han X, Kaye DM, Belhassen L, O'Hara DS, Kelly RA, Smith TW, Michel T. Nitric oxide-dependent parasympathetic signaling is due to activation of constitutive endothelial (type III) nitric oxide synthase in cardiac myocytes. J Biol Chem. 1995;270:14582-14586. [Abstract/Free Full Text]

12. Klimaschewski L, Kummer W, Mayer B, Couraud JY, Preissler U, Philippin B, Heym C. Nitric oxide synthase in cardiac nerve fibers and neurons of rat and guinea pig heart. Circ Res. 1992;71:1533-1537. [Abstract/Free Full Text]

13. Balligand J-L, Ungureanu-Longrois D, Simmons WW, Pimental D, Malinski TA, Kapturczak M, Taha Z, Lowenstein CJ, Davidoff AJ, Kelly RA, Smith TW, Michel T. Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. J Biol Chem. 1994;269:27580-27588. [Abstract/Free Full Text]

14. Balligand J-L, Ungureanu-Longrois D, Simmons WW, Kobzik L, Lowenstein CJ, Lamas S, Kelly RA, Smith TW, Michel T. Induction of NO synthase in rat cardiac microvascular endothelial cells by IL-1ß and IFN-. Am J Physiol. 1995;268:H1293-H1303. [Abstract/Free Full Text]

15. Yang X, Chowdhury N, Cai B, Brett J, Marboe C, Sciacca R, Michler R, Cannon P. Induction of myocardial nitric oxide synthase by cardiac allograft rejection. J Clin Invest. 1994;94:714-721.

16. Brady A, Poole-Wilson P, Harding S, Warren J. Nitric oxide production within cardiac myocytes reduces their contractility in endotoxemia. Am J Physiol. 1992;263:H1963-H1966. [Abstract/Free Full Text]

17. Ungureanu-Longrois D, Balligand J-L, Kelly RA, Smith TW. Myocardial contractile dysfunction in the systemic inflammatory response syndrome: role of a cytokine-inducible nitric oxide synthase in cardiac myocytes. J Moll Cell Cardiol. 1995;27:155-167. [Medline] [Order article via Infotrieve]

18. Han X, Shimoni Y, Giles WR. An obligatory role for nitric oxide in autonomic control of mammalian heart rate. J Physiol (Lond). 1994;476:309-314. [Abstract/Free Full Text]

19. Berger H-J, Prasad SK, Davidoff AJ, Pimental D, Ellingsen O, Marsh JD, Smith TW, Kelly RA. Continual electric field stimulation preserves contractile function of adult ventricular myocytes in primary culture. Am J Physiol. 1994;266:H341-H349. [Abstract/Free Full Text]

20. Eid H, Larson DM, Springhorn JP, Attawia MA, Nayak RC, Smith TW, Kelly RA. Role of epicardial mesothelial cells in the modification of phenotype and function of adult rat ventricular myocytes in primary culture. Circ Res. 1992;71:40-50. [Abstract/Free Full Text]

21. McDermott PJ, Morgan HE. Contraction modulates the capacity for protein synthesis during growth of neonatal heart cells in culture. Circ Res. 1989;64:542-553. [Abstract/Free Full Text]

22. Kelly RA, Eid H, Kramer BK, O'Neill M, Liang BT, Reers M, Smith TW. Endothelin enhances the contractile responsiveness of adult rat ventricular myocytes to calcium by a pertussis toxin-sensitive pathway. J Clin Invest. 1990;86:1164-1171.

23. Borzak S, Murphy S, Marsh JD. Mechanisms of rate staircase in rat ventricular cells. Am J Physiol. 1991;260:884-892.

24. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]

25. Hare JM, Keaney JF Jr, Balligand J-L, Loscalzo J, Smith TW, Colucci WS. Role of nitric oxide in parasympathetic modulation of beta-adrenergic myocardial contractility in normal dogs. J Clin Invest. 1995;95:360-366.

26. Roberts AB, Vodovotz Y, Roche NS, Sporn MB, Nathan CF. Role of nitric oxide in antagonistic effects of transforming growth factor-beta and interleukin-1 beta on the beating rate of cultured cardiac myocytes. Mol Endocrinol. 1992;6:1921-1930. [Abstract/Free Full Text]

27. Hung J, Lew YW. Cellular mechanisms of endotoxin-induced myocardial depression in rabbits. Circ Res. 1993;73:125-134. [Abstract]

28. Tao S, McKenna T. In vitro endotoxin exposure induces contractile dysfunction in adult rat cardiac myocytes. Am J Physiol. 1994;267:H1745-H1752. [Abstract/Free Full Text]

29. Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, Simmons RL. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science. 1992;257:387-389. [Abstract/Free Full Text]

30. Finkel MS, Oddis CV, Mayer OH, Hattler BG, Simmons RL. Nitric oxide synthase inhibitor alters papillary muscle force-frequency relationship. J Pharmacol Exp Ther. 1995;272:945-952. [Abstract/Free Full Text]

31. Brady AJB, Warren JB, Poole-Wilson PA, Williams TJ, Harding SE. Nitric oxide attenuates cardiac myocyte contraction. Am J Physiol. 1993;265:H176-H182. [Abstract/Free Full Text]

32. Weyrich A, Ma X-L, Buerke M, Murohara, T, Armstead VE, Lefer AM, Nicolas JM, Thomas AP, Lefer DJ, Vinten-Johansen J. Physiological concentrations of nitric oxide do not elicit an acute negative inotropic effect in unstimulated cardiac muscle. Circ Res. 1994;75:692-700. [Abstract/Free Full Text]

33. Feelisch M. The biochemical pathways of nitric oxide formation from nitrovasodilators: appropriate choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions. J Cardiovasc Pharmacol. 1991;17(suppl 3):S25-S33.

34. Busconi L, Michel T. Endothelial nitric oxide synthase membrane targeting: evidence against involvement of a specific myristate receptor. J Biol Chem. 1994;269:25016-25020. [Abstract/Free Full Text]

35. Grocott-Mason R, Anning P, Evans H, Lewis MJ, Shah AM. Modulation of left ventricular relaxation in isolated ejecting heart by endogenous nitric oxide. Am J Physiol. 1994;267:H1804-H1813. [Abstract/Free Full Text]

36. Shah AM, Spurgeon HA, Sollott SJ, Talo A, Lakatta EG. 8-Bromo-cGMP reduces the myofilament response to Ca²⁺ in intact cardiac myocytes. Circ Res. 1994;74:970-978. [Abstract/Free Full Text]

37. Paulus WJ, Vantrimpont PJ, Shah AM. Acute effects of nitric oxide on left ventricular relaxation and diastolic distensibility in humans: assessment by bicoronary sodium nitroprusside infusion. Circulation. 1994;89:2070-2078. [Abstract/Free Full Text]

38. Levi R, Alloatti G, Fischmeister R. Cyclic GMP regulates the Ca-channel current in guinea pig ventricular myocytes. Pflugers Arch. 1989;413:685-687. [Medline] [Order article via Infotrieve]

39. Mery P, Lohmann S, Walter U, Fischmeister R. Ca²⁺ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes. Proc Natl Acad Sci U S A. 1991;88:1197-1201. [Abstract/Free Full Text]

40. Kobzik L, Reid MB, Bredt DS, Stamler JS. Nitric oxide in skeletal muscle. Nature. 1994;372:546-548. [Medline] [Order article via Infotrieve]

41. Kobzik L, Stringer B, Balligand J-L, Reid MB, Stamler JS. Endothelial type nitric oxide synthase (ec-NOS) in skeletal muscle fibers: mitochondrial relationships. Biol Biochem Res Commun. 1995;211:375-381.

42. Gross WL, Balligand J-L, Kelly RA, Smith TW, Ingwall JS. Nitric oxide and the heart: infusion of an exogenous nitric oxide donor alters cardiac high-energy phosphate metabolism and contractile reserve. Circulation. 1994;90(suppl I):I-193. Abstract.

43. Shen W, Xu X, Ochoa M, Zhao G, Wolin MS, Hintze TH. Role of nitric oxide in the regulation of oxygen consumption in conscious dogs. Circ Res. 1994;75:1086-1095. [Abstract/Free Full Text]

44. Schweizer M, Richter C. Nitric oxide potently and reversibly deenergizes mitochondria at low oxygen tension. Biochem Biophys Res Commun. 1994;204:169-175. [Medline] [Order article via Infotrieve]

45. Mateo RB, Reichner JS, Mastrofrancesco B, Kraft-Stolar D, Albina JE. Impact of nitric oxide on macrophage glucose metabolism and glyceraldehyde-3-phosphate dehydrogenase activity. Am J Physiol. 1995;37:C669-C675.

This article has been cited by other articles:

				J.-L. Balligand, O. Feron, and C. Dessy eNOS Activation by Physical Forces: From Short-Term Regulation of Contraction to Chronic Remodeling of Cardiovascular Tissues Physiol Rev, April 1, 2009; 89(2): 481 - 534. [Abstract] [Full Text] [PDF]

				E. N. Dedkova and L. A. Blatter Characteristics and function of cardiac mitochondrial nitric oxide synthase J. Physiol., February 15, 2009; 587(4): 851 - 872. [Abstract] [Full Text] [PDF]

				M. Seddon, A. M. Shah, and B. Casadei Cardiomyocytes as effectors of nitric oxide signalling Cardiovasc Res, July 15, 2007; 75(2): 315 - 326. [Abstract] [Full Text] [PDF]

				E. N. Dedkova, Y. G. Wang, X. Ji, L. A. Blatter, A. M. Samarel, and S. L. Lipsius Signalling mechanisms in contraction-mediated stimulation of intracellular NO production in cat ventricular myocytes J. Physiol., April 1, 2007; 580(1): 327 - 345. [Abstract] [Full Text] [PDF]

				S. R. Martin, K. Emanuel, C. E. Sears, Y.-H. Zhang, and B. Casadei Are myocardial eNOS and nNOS involved in the {beta}-adrenergic and muscarinic regulation of inotropy? A systematic investigation Cardiovasc Res, April 1, 2006; 70(1): 97 - 106. [Abstract] [Full Text] [PDF]

				Y. G Wang, E. N Dedkova, X Ji, L. A Blatter, and S. L Lipsius Phenylephrine acts via IP3-dependent intracellular NO release to stimulate L-type Ca2+ current in cat atrial myocytes J. Physiol., August 15, 2005; 567(1): 143 - 157. [Abstract] [Full Text] [PDF]

				H. C. Champion, D. Georgakopoulos, E. Takimoto, T. Isoda, Y. Wang, and D. A. Kass Modulation of In Vivo Cardiac Function by Myocyte-Specific Nitric Oxide Synthase-3 Circ. Res., March 19, 2004; 94(5): 657 - 663. [Abstract] [Full Text] [PDF]

				E. N. Dedkova, X. Ji, Y. G. Wang, L. A. Blatter, and S. L. Lipsius Signaling Mechanisms That Mediate Nitric Oxide Production Induced by Acetylcholine Exposure and Withdrawal in Cat Atrial Myocytes Circ. Res., December 12, 2003; 93(12): 1233 - 1240. [Abstract] [Full Text] [PDF]

				P.B. Massion, O. Feron, C. Dessy, and J.-L. Balligand Nitric Oxide and Cardiac Function: Ten Years After, and Continuing Circ. Res., September 5, 2003; 93(5): 388 - 398. [Abstract] [Full Text] [PDF]

				S. A. Khan, M. W. Skaf, R. W. Harrison, K. Lee, K. M. Minhas, A. Kumar, M. Fradley, A. A. Shoukas, D. E. Berkowitz, and J. M. Hare Nitric Oxide Regulation of Myocardial Contractility and Calcium Cycling: Independent Impact of Neuronal and Endothelial Nitric Oxide Synthases Circ. Res., June 27, 2003; 92(12): 1322 - 1329. [Abstract] [Full Text] [PDF]

				U. Hofmann, E. Domeier, S. Frantz, M. Laser, B. Weckler, P. Kuhlencordt, S. Heuer, B. Keweloh, G. Ertl, and A. W. Bonz Increased myocardial oxygen consumption by TNF-alpha is mediated by a sphingosine signaling pathway Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2100 - H2105. [Abstract] [Full Text] [PDF]

				C. E. Sears, S. M. Bryant, E. A. Ashley, C. A. Lygate, S. Rakovic, H. L. Wallis, S. Neubauer, D. A. Terrar, and B. Casadei Cardiac Neuronal Nitric Oxide Synthase Isoform Regulates Myocardial Contraction and Calcium Handling Circ. Res., March 21, 2003; 92 (5): e52 - e59. [Abstract] [Full Text] [PDF]

				H. Degenhardt, J. Jansen, R. Schulz, D. Sedding, R. Braun-Dullaeus, and K.-D. Schluter Mechanosensitive release of parathyroid hormone-related peptide from coronary endothelial cells Am J Physiol Heart Circ Physiol, October 1, 2002; 283(4): H1489 - H1496. [Abstract] [Full Text] [PDF]

				E. A. Ashley, C. E. Sears, S. M. Bryant, H. C. Watkins, and B. Casadei Cardiac Nitric Oxide Synthase 1 Regulates Basal and {beta}-Adrenergic Contractility in Murine Ventricular Myocytes Circulation, June 25, 2002; 105(25): 3011 - 3016. [Abstract] [Full Text] [PDF]

				N. Shiono, V. Rao, R. D. Weisel, M. Kawasaki, R.-K. Li, D. A. G. Mickle, P. W. M. Fedak, L. C. Tumiati, L. Ko, and S. Verma L-Arginine protects human heart cells from low-volume anoxia and reoxygenation Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H805 - H815. [Abstract] [Full Text] [PDF]

				W. J. Paulus, S. Frantz, and R. A. Kelly Nitric Oxide and Cardiac Contractility in Human Heart Failure: Time for Reappraisal Circulation, November 6, 2001; 104(19): 2260 - 2262. [Full Text] [PDF]

				J. M. Cotton, M. T. Kearney, P. A. MacCarthy, R. M. Grocott-Mason, D. R. McClean, C. Heymes, P. J. Richardson, and A. M. Shah Effects of Nitric Oxide Synthase Inhibition on Basal Function and the Force-Frequency Relationship in the Normal and Failing Human Heart In Vivo Circulation, November 6, 2001; 104(19): 2318 - 2323. [Abstract] [Full Text] [PDF]

				T. Stumpe, U. K. M. Decking, and J. Schrader Nitric oxide reduces energy supply by direct action on the respiratory chain in isolated cardiomyocytes Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H2350 - H2356. [Abstract] [Full Text] [PDF]

				Y. G. Wang, W. J. Benedict, J. Huser, A. M. Samarel, L. A. Blatter, and S. L. Lipsius Brief rapid pacing depresses contractile function via Ca2+/PKC-dependent signaling in cat ventricular myocytes Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H90 - H98. [Abstract] [Full Text] [PDF]

				J. M. Hare, R. A. Lofthouse, G. J. Juang, L. Colman, K. M. Ricker, B. Kim, H. Senzaki, S. Cao, R. S. Tunin, and D. A. Kass Contribution of Caveolin Protein Abundance to Augmented Nitric Oxide Signaling in Conscious Dogs With Pacing-Induced Heart Failure Circ. Res., May 26, 2000; 86(10): 1085 - 1092. [Abstract] [Full Text] [PDF]

				Z Kassiri, R Myers, R Kaprielian, H S Banijamali, and P H Backx Rate-dependent changes of twitch force duration in rat cardiac trabeculae: a property of the contractile system J. Physiol., April 1, 2000; 524(1): 221 - 231. [Abstract] [Full Text] [PDF]

				G. Muller-Strahl, K. Kottenberg, H.-G. Zimmer, E. Noack, and G. Kojda Inhibition of nitric oxide synthase augments the positive inotropic effect of nitric oxide donors in the rat heart J. Physiol., January 15, 2000; 522(2): 311 - 320. [Abstract] [Full Text] [PDF]

				J. Ren, L. Jefferson, J. R. Sowers, and R. A. Brown Influence of Age on Contractile Response to Insulin-Like Growth Factor 1 in Ventricular Myocytes From Spontaneously Hypertensive Rats Hypertension, December 1, 1999; 34(6): 1215 - 1222. [Abstract] [Full Text] [PDF]

				M. V. Brahmajothi and D. L. Campbell Heterogeneous Basal Expression of Nitric Oxide Synthase and Superoxide Dismutase Isoforms in Mammalian Heart : Implications for Mechanisms Governing Indirect and Direct Nitric Oxide-Related Effects Circ. Res., October 1, 1999; 85(7): 575 - 587. [Abstract] [Full Text] [PDF]

				H. Drexler Nitric Oxide Synthases in the Failing Human Heart : A Doubled-Edged Sword? Circulation, June 15, 1999; 99(23): 2972 - 2975. [Full Text] [PDF]

				S. Kanno, N. Oda, M. Abe, S. Saito, K. Hori, Y. Handa, K. Tabayashi, and Y. Sato Establishment of a Simple and Practical Procedure Applicable to Therapeutic Angiogenesis Circulation, May 25, 1999; 99(20): 2682 - 2687. [Abstract] [Full Text] [PDF]

				A. P. Garner, M. J. I. Paine, I. Rodriguez-Crespo, E. C. Chinje, P. O. De Montellano, I. J. Stratford, D. G. Tew, and C. R. Wolf Nitric Oxide Synthases Catalyze the Activation of Redox Cycling and Bioreductive Anticancer Agents Cancer Res., April 1, 1999; 59(8): 1929 - 1934. [Abstract] [Full Text] [PDF]

				G. Kojda and K. Kottenberg Regulation of basal myocardial function by NO Cardiovasc Res, March 1, 1999; 41(3): 514 - 523. [Full Text] [PDF]

				H. D. Battarbee, J. H. Zavecz, M. B. Grisham, R. E. Maloney, L. J. Chandler, J. W. Mercer Jr., and F. M. Cady Cardiac impairment and nitric oxide synthase activity in the chronic portal vein-stenosed rat Am J Physiol Gastrointest Liver Physiol, February 1, 1999; 276(2): G363 - G372. [Abstract] [Full Text] [PDF]

				A. Kumar, R. Brar, P. Wang, L. Dee, G. Skorupa, F. Khadour, R. Schulz, and J. E. Parrillo Role of nitric oxide and cGMP in human septic serum-induced depression of cardiac myocyte contractility Am J Physiol Regulatory Integrative Comp Physiol, January 1, 1999; 276(1): R265 - R276. [Abstract] [Full Text] [PDF]

				Y. Fujii, Y. Guo, and S. N. A. Hussain Regulation of nitric oxide production in response to skeletal muscle activation J Appl Physiol, December 1, 1998; 85(6): 2330 - 2336. [Abstract] [Full Text] [PDF]

				F. H. Khadour, D. W. O'Brien, Y. Fu, P. W. Armstrong, and R. Schulz Endothelial nitric oxide synthase increases in left atria of dogs with pacing-induced heart failure Am J Physiol Heart Circ Physiol, December 1, 1998; 275(6): H1971 - H1978. [Abstract] [Full Text] [PDF]

				S. E. Harding, C. H. Davies, A. M. Money-Kyrle, and P. A. Poole-Wilson An inhibitor of nitric oxide synthase does not increase contraction or {beta}-adrenoceptor sensitivity of ventricular myocytes from failing human heart Cardiovasc Res, December 1, 1998; 40(3): 523 - 529. [Abstract] [Full Text] [PDF]

				B. Stein, T. Eschenhagen, J. Rudiger, H. Scholz, U. Forstermann, and I. Gath Increased expression of constitutive nitric oxide synthase III, but not inducible nitric oxide synthase II, in human heart failure J. Am. Coll. Cardiol., November 1, 1998; 32(5): 1179 - 1186. [Abstract] [Full Text] [PDF]

				J. Ren, M. F. Walsh, M. Hamaty, J. R. Sowers, and R. A. Brown Altered inotropic response to IGF-I in diabetic rat heart: influence of intracellular Ca2+ and NO Am J Physiol Heart Circ Physiol, September 1, 1998; 275(3): H823 - H830. [Abstract] [Full Text] [PDF]

				X Han, L Kobzik, D Severson, and Y Shimoni Characteristics of nitric oxide-mediated cholinergic modulation of calcium current in rabbit sino-atrial node J. Physiol., June 15, 1998; 509(3): 741 - 754. [Abstract] [Full Text] [PDF]

				U. Bayraktutan, Z.-K. Yang, and A. M Shah Selective dysregulation of nitric oxide synthase type 3 in cardiac myocytes but not coronary microvascular endothelial cells of spontaneously hypertensive rat Cardiovasc Res, June 1, 1998; 38(3): 719 - 726. [Abstract] [Full Text] [PDF]

				R. E Klabunde, J. Tse, and H. R Weiss Guanylyl cyclase inhibition reduces contractility and decreases cGMP and cAMP in isolated rat hearts Cardiovasc Res, March 1, 1998; 37(3): 676 - 683. [Abstract] [Full Text] [PDF]

				J.-L. Balligand and P. J. Cannon Nitric Oxide Synthases and Cardiac Muscle : Autocrine and Paracrine Influences Arterioscler. Thromb. Vasc. Biol., October 1, 1997; 17(10): 1846 - 1858. [Abstract] [Full Text]

				B. D. Prendergast, B M. Sci, V. F. Sagach, and A. M. Shah Basal Release of Nitric Oxide Augments the Frank-Starling Response in the Isolated Heart Circulation, August 19, 1997; 96(4): 1320 - 1329. [Abstract] [Full Text]

				N. Ito, J. Bartunek, K. W. Spitzer, and B. H. Lorell Effects of the Nitric Oxide Donor Sodium Nitroprusside on Intracellular pH and Contraction in Hypertrophied Myocytes Circulation, May 6, 1997; 95(9): 2303 - 2311. [Abstract] [Full Text]

				L. Belhassen, O. Feron, D. M. Kaye, T. Michel, and R. A. Kelly Regulation by cAMP of Post-translational Processing and Subcellular Targeting of Endothelial Nitric-oxide Synthase (Type 3) in Cardiac Myocytes J. Biol. Chem., April 25, 1997; 272(17): 11198 - 11204. [Abstract] [Full Text] [PDF]

				A. J. Sherman, C. A. Davis III, F. J. Klocke, K. R. Harris, G. Srinivasan, A. S. Yaacoub, D. A. Quinn, K. A. Ahlin, and J. J. Jang Blockade of Nitric Oxide Synthesis Reduces Myocardial Oxygen Consumption In Vivo Circulation, March 4, 1997; 95(5): 1328 - 1334. [Abstract] [Full Text]

				J. Bartunek, A. M. Shah, M. Vanderheyden, and W. J. Paulus Dobutamine Enhances Cardiodepressant Effects of Receptor-Mediated Coronary Endothelial Stimulation Circulation, January 7, 1997; 95(1): 90 - 96. [Abstract] [Full Text]

				R. D. Bernstein, F. Y. Ochoa, X. Xu, P. Forfia, W. Shen, C. I. Thompson, and T. H. Hintze Function and Production of Nitric Oxide in the Coronary Circulation of the Conscious Dog During Exercise Circ. Res., October 1, 1996; 79(4): 840 - 848. [Abstract] [Full Text]

				R. A. Kelly, J.-L. Balligand, and T. W. Smith Nitric Oxide and Cardiac Function Circ. Res., September 1, 1996; 79(3): 363 - 380. [Full Text]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kaye, D. M. Articles by Kelly, R. A. Search for Related Content PubMed PubMed Citation Articles by Kaye, D. M. Articles by Kelly, R. A.