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 Prendergast, B. D. Articles by Shah, A. M. Search for Related Content PubMed PubMed Citation Articles by Prendergast, B. D. Articles by Shah, A. M.

(Circulation. 1997;96:1320-1329.)
© 1997 American Heart Association, Inc.

Articles

Basal Release of Nitric Oxide Augments the Frank-Starling Response in the Isolated Heart

Bernard D. Prendergast, B Med Sci MRCP; Vadim F. Sagach, MD, PhD; ; Ajay M. Shah, MD, MRCP

From the Department of Cardiology, Cardiovascular Sciences Group, University of Wales College of Medicine, Cardiff, UK (B.D.P., A.M.S.), and A.A. Bogomolets Institute of Physiology, Kiev, Ukraine (V.F.S.).

Correspondence to Dr A.M. Shah, Department of Cardiology, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XN, UK. E-mail shaham2{at}cf.ac.uk

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background The Frank-Starling response contributes to the regulation of cardiac output. The major underlying subcellular mechanism is a length-dependent change in myofilament responsiveness to Ca²⁺. Recent studies indicate that nitric oxide decreases myofilament responsiveness to Ca²⁺ and modulates myocardial relaxation and left ventricular (LV) diastolic function. We therefore investigated the interaction between nitric oxide and the Frank-Starling response.

Methods and Results Isolated ejecting guinea pig hearts (constant afterload and heart rate) were studied before and after interventions. Elevation of filling pressure from 10 to 20 cm H₂O increased cardiac output, LV end-diastolic pressure (LVEDP), and peak LV pressure (LVP_max). In the presence of N^G-monomethyl-L-arginine (L-NMMA, 10 µmol/L; n=10) or free hemoglobin (1 µmol/L; n=8), preload-induced increases in cardiac output were significantly attenuated but baseline cardiac output was unaffected. The effects of L-NMMA were inhibited in the presence of excess L-arginine (100 µmol/L; n=6). These changes were not attributable to alterations in coronary flow. Prostaglandin F₂ (0.01 µmol/L; n=6), which reduced coronary flow, failed to alter the cardiac output response to preload elevation. The exogenous nitric oxide donor sodium nitroprusside (1 µmol/L; n=6) reduced cardiac output at the lowest preload but not at higher preloads. LVEDP was elevated after L-NMMA and hemoglobin but reduced after sodium nitroprusside.

Conclusions Basal intracardiac production of nitric oxide significantly augments preload-induced rises in cardiac output in the isolated ejecting guinea pig heart. The mechanism appears to be unrelated to changes in coronary flow and may involve direct effects of nitric oxide on myocardial diastolic and/or systolic function.

Key Words: endothelium-derived factors • myocardial contraction • cardiac output • ventricles

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The Frank-Starling response, ie, an increase in ventricular performance in response to increase in EDV, contributes to the regulation of cardiac output in vivo, interacting with mechanisms such as heart rate, neurohumoral systems, coronary flow, and cardiac load.^{1 2} In normal human subjects, it contributes significantly to increases in cardiac output during changes in posture³ and submaximal exercise,^{4 5} and it is particularly important in older subjects in whom the chronotropic response to exercise is impaired.¹ In disease states such as heart failure and dilated cardiomyopathy, the Frank-Starling mechanism has been shown to be impaired or "exhausted" such that acute increases in ventricular volume fail to augment stroke volume.^{6 7}

The underlying mechanisms of the Frank-Starling response have been intensively investigated.^{1 2} In isolated cardiac muscle preparations, it has been demonstrated that changes in initial muscle length influence the level of myofilament activation: so-called length-dependent activation.^{8 9 10 11} Suggested cellular mechanisms include changes in contractile protein responsiveness to Ca²⁺, altered intracellular Ca²⁺ release, changes in inositol trisphosphate and protein kinase C activity, and the opening of stretch-activated ion channels.^{1 2} In both isolated muscle^{8 10 11} and intact hearts,^{12 13} two phases of length-dependent changes in contractile performance are described: an initial change that occurs almost instantaneously within a few beats and a slower phase with a time course of a few minutes. The latter phase is less prominent at physiological temperatures (37°C), when it accounts for <10% to 25% of the overall change in contractile performance.^{8 12} Convincing evidence implicates an increase in myofilament responsiveness to Ca²⁺ as the major mechanism responsible for the initial stretch-induced augmentation of muscle contraction.^{11 14 15} At the organ level, ventricular diastolic properties, ie, the instantaneous relation between filling pressure and volume, will influence the manifestation of the Frank-Starling response. For instance, if diastolic ventricular stiffness is increased, a given increase in filling pressure will result in a smaller rise in end-diastolic volume.

Recent studies indicate that cardiac contractile performance may be influenced by substances (eg, nitric oxide, endothelin-1) released by endothelial cells, analogous to vascular endothelial regulation of vessel tone and blood flow.^{16 17 18} In addition to its release by coronary microvascular and endocardial endothelial cells,¹⁸ nitric oxide may be produced within cardiac myocytes themselves by NOS-3.^{19 20} Several actions of nitric oxide on myocardial contractile function have been reported, including changes in myocardial relaxation and diastolic properties,^{21 22 23 24 25 26} modulation of ß-adrenergic inotropic responses,^{19 20 27} mediation of cholinergic responses,^{19 20} positive inotropic effects,^{28 29} and modulation of the force-frequency relationship.^{30 31} Of particular relevance to the present study are the nitric oxide–induced changes in relaxation and diastolic function. In myocardium previously unstimulated by extrinsic agonists, endothelium-derived nitric oxide and exogenous nitric oxide donors or cGMP analogues characteristically enhance relaxation without major effects on peak systolic function. This relatively selective relaxant action has been observed in isolated ferret and cat papillary muscle preparations,^{21 29 32} ejecting guinea pig hearts,^{22 23} and rat cardiac myocytes.^{24 33} Similar effects have also been noted in normal human subjects after low-dose bicoronary infusions of substance P (a specific agonist for the release of nitric oxide from endothelial cells) or of sodium nitroprusside.^{25 26} These agents induced an earlier onset of LV relaxation and a reduction in peak LV pressure without significant change in LV dP/dt_max, as well as a downward shift in the LV end-diastolic pressure-volume relation consistent with a reduction in LV end-diastolic chamber stiffness. The latter changes were not accounted for by ventricular interaction or right ventricular unloading, and systemic infusions of identical doses of drugs also failed to alter contractile performance. These effects were thus attributed to a direct action of nitric oxide on the heart. The subcellular mechanism was suggested to be a reduction in myofilament responsiveness to Ca²⁺ secondary to elevation of intracellular cGMP and activation of cGMP-dependent protein kinase. Evidence for such an effect of cGMP has been demonstrated in studies on isolated cardiac myocytes^{24 33} and skinned cardiac fibers.³⁴

Thus, a change in myofilament responsiveness to Ca²⁺ appears to be involved in both the Frank-Starling response and the myocardial effects of nitric oxide. In this study, we have therefore investigated the interaction between nitric oxide and the Frank-Starling response.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Isolated Ejecting Heart Preparation
All procedures conformed with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1985). Adult Dunkin-Hartley guinea pigs (350 to 500 g) of either sex were killed by pentobarbital overdose (60 mg/kg IP). Hearts were excised and immediately immersed in ice-cold Krebs-Henseleit solution (mmol/L: NaCl 118, KCl 4.7, MgSO₄·7H₂O 1.2, NaHCO₃ 24, KH₂PO₄ 1.1, glucose 10, CaCl₂·2H₂O 2.5) gassed with 95% O₂/5% CO₂ and containing indomethacin (1 µmol/L) to inhibit prostanoid effects. Hearts were initially perfused in the Langendorff mode with warmed Krebs-Henseleit solution (37°C) at a constant pressure of 80 cm H₂O. The left atrium was cannulated via the largest pulmonary vein, and after a short period of stabilization, hearts were switched to the recirculating ejecting mode^{22 23} and paced at 10% above their intrinsic rate via a right atrial electrode. Aortic afterload was maintained at 70 cm H₂O, and left atrial filling pressure was set at 10 cm H₂O. The total recirculating volume was 150 mL. Aortic flow was measured with a flotation flowmeter (KDG Mobrey Ltd), and coronary flow was determined from timed collections of pulmonary arterial effluent. Cardiac output was calculated as the sum of aortic and coronary flow, and SV as cardiac output divided by heart rate. High-fidelity LV pressure was monitored by a 2F micromanometer-tipped catheter (Millar Instruments) inserted directly into the LV apex, with care taken to avoid leakage of fluid around the catheter.^{22 23} Pressure was zeroed to atmospheric pressure at the level of the LV apex. Data were sampled at 4 kHz via a Macintosh Maclab system (Analog Digital Instruments).

At least four consecutive LV traces were averaged for each measurement of LVP_max, LV dP/dt_max, and LVEDP. The latter was measured as the pressure at the time of first positive deflection of the LV dP/dt trace. Isovolumic LV relaxation was assessed by calculating T_L for LV pressure points 20 ms after LV dP/dt_min (80 data points), as described previously.^{22 23}

Protocol
Only hearts in which LV pressure, aortic flow, and coronary flow were stable (at least 10 minutes) were included for study. A Frank-Starling curve was generated by measuring LV parameters and cardiac output at left atrial preloads of 10, 15, and 20 cm H₂O, achieved by varying atrial reservoir height. Measurements were made once a stable response to each change in preload had occurred (1.5 to 2 minutes). Delayed or secondary changes in cardiac output after preload elevation contributed <10% of the overall change in cardiac output in this preparation and were not analyzed in detail. After baseline assessment, atrial reservoir height was returned to 10 cm H₂O, and study drugs were added via the gassing chamber into the recirculating buffer (0.15 mL, resulting in a 1000-fold dilution). Frank-Starling curves were regenerated after a stable change in performance (generally 8 to 15 minutes later). We studied (1) 6 hearts treated with 0.15 mL saline and restudied 15 to 30 minutes later, ie, time controls; (2) 10 hearts treated with a specific inhibitor of nitric oxide synthase, L-NMMA, 10 µmol/L; (3) 6 hearts treated with L-NMMA 10 µmol/L together with L-arginine 100 µmol/L, the authentic substrate for nitric oxide synthase; (4) 8 hearts treated with free hemoglobin 1 µmol/L, which scavenges nitric oxide; (5) 6 hearts treated with an exogenous donor of nitric oxide, sodium nitroprusside, 1 µmol/L and 6 hearts with sodium nitroprusside 0.1 µmol/L; and (6) 6 hearts treated with PGF₂ 0.01 µmol/L, used as a nitric oxide–independent coronary vasoconstrictor agent. The dose of L-NMMA (10 µmol/L) was selected as the highest dose that did not markedly reduce baseline coronary flow. Doses of sodium nitroprusside and hemoglobin were chosen on the basis of previous studies with this preparation.^{22 23} The dose of PGF₂ was chosen after pilot studies to determine the dose that reduced coronary flow to an extent similar to that by L-NMMA. In experiments using hemoglobin, one drop of Antifoam A was added to prevent excessive frothing; this did not alter baseline performance.

Statistics
Absolute data are expressed as mean±SEM unless stated otherwise. Comparisons within groups were made by paired Student's t test on absolute data. For comparisons between groups, absolute changes in parameters after intervention were analyzed by two-way ANOVA between preload and treatment.³⁵ Significant differences from the time control group were detected by Duncan's multiple range test. A value of P<.05 was considered statistically significant.

Materials
All chemicals and reagents were obtained from Sigma Chemical Co. Hemoglobin was prepared from human blood.²¹ Stock solutions were prepared in distilled H₂O except for indomethacin, which was dissolved in 100% ethanol and subsequently diluted in distilled H₂O. The final ethanol concentration of 0.01% had no significant effect on contractile parameters. All buffer solutions were prepared fresh each day.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of Preload Elevation Under Baseline Conditions
The Table shows pooled data for the effects of elevation of left atrial filling pressure from 10 to 20 cm H₂O on contractile parameters before addition of drugs. Preload elevation caused significant increases in cardiac output, LVP_max, LVEDP, T_L (ie, prolonged isovolumic relaxation), and coronary flow but no significant change in LV dP/dt_max.

View this table: [in this window] [in a new window]   Table 1. Effects of Preload Elevation in Isolated Ejecting Guinea Pig Hearts

In hearts that were not treated with any additional drugs (ie, time controls), these effects of preload elevation were reproducible for at least 40 minutes. Fig 1 shows the effects of preload elevation in this group at baseline and 15 minutes later (ie, matched to the other groups studied). The pattern of effects of preload elevation was unaltered after 15 minutes, although absolute values of LVP_max and LV dP/dt_max were slightly lower and those of T_L slightly higher (all P<.05).

View larger version (18K): [in this window] [in a new window]   Figure 1. Effects of preload elevation in control (untreated) hearts at baseline () and 15 minutes later (). *Significant change with time at equivalent preloads.

Effects of L-NMMA on the Frank-Starling Response
L-NMMA 10 µmol/L significantly altered the cardiac output response to preload elevation but did not change the response of other parameters to elevation of filling pressure (Fig 2). Two-way ANOVA indicated a significant interaction between treatment and preload for cardiac output (P<.05) but not for any other parameter in the L-NMMA group compared with time controls. L-NMMA attenuated the increase in cardiac output at high preload without reducing cardiac output at low preload. Under baseline conditions (ie, a filling pressure of 10 cm H₂O), L-NMMA had no effect on LVP_max, LV dP/dt_max, LVEDP, or T_L compared with untreated time control hearts. Baseline coronary flow was slightly reduced by L-NMMA (16.5±0.8 to 14.9±0.9 mL/min), but this was not significant compared with time controls. After preload elevation, LVEDP was higher and LVP_max and LV dP/dt_max were lower in the presence of L-NMMA.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effects of preload elevation before () and after () L-NMMA 10 µmol/L. Significant interaction between preload and treatment vs time-control group. *Significant effect of treatment vs time-control group at equivalent preload.

Effects of Combined L-NMMA and L-Arginine
In hearts treated with L-NMMA 10 µmol/L plus L-arginine 100 µmol/L, there were no significant changes in baseline performance at 10 cm H₂O filling pressure (Fig 3). Preload-induced changes in cardiac output, LVP_max, LVEDP, and coronary flow were also similar to the time control group.

View larger version (17K): [in this window] [in a new window]   Figure 3. Effects of preload elevation before () and after () simultaneous L-NMMA 10 µmol/L and L-arginine 100 µmol/L.

Effects of Free Hemoglobin
Hemoglobin 1 µmol/L significantly altered the cardiac output response to preload elevation (Fig 4). Two-way ANOVA indicated a significant interaction between treatment and preload for cardiac output (P<.05) but not for any other parameter after hemoglobin. This effect of hemoglobin was similar to L-NMMA in that preload-induced increases in cardiac output were attenuated at higher filling pressures with no change at the lowest preload. Hemoglobin caused no significant change in baseline LVP_max, LVEDP and coronary flow (Fig 4), or LV dP/dt_max and T_L (not shown) compared with time control hearts. After preload elevation, LVEDP was higher in the presence of hemoglobin. The responses of LVP_max, LV dP/dt_max, and coronary flow to preload elevation were unaltered.

View larger version (17K): [in this window] [in a new window]   Figure 4. Effects of preload elevation before () and after () hemoglobin (Hb) 1 µmol/L. Significant interaction between preload and treatment vs time-control group. *Significant effect of treatment vs time-control group at equivalent preload.

Effects of Sodium Nitroprusside
Sodium nitroprusside 1 µmol/L caused a significant reduction in cardiac output at the lowest preload (Fig 5). Upon subsequent elevation of left atrial filling pressure, absolute cardiac outputs were similar with or without sodium nitroprusside as a result of a greater preload-induced increment in cardiac output in the presence of the drug. However, two-way ANOVA did not reveal a significant interaction between treatment and preload either for cardiac output or for any other parameter. Under baseline conditions (ie, filling pressure 10 cm H₂O), nitroprusside significantly increased coronary flow but had no effect on other parameters. LVEDP was significantly lower in the presence of nitroprusside at a preload of 15 cm H₂O.

View larger version (17K): [in this window] [in a new window]   Figure 5. Effects of preload elevation before () and after () sodium nitroprusside (SNP) 1 µmol/L. *Significant effect of treatment vs time-control group at equivalent preload.

Sodium nitroprusside 0.1 µmol/L had no significant effects on either basal function or the response to preload elevation (data not shown).

Possible Changes in Diastolic Function
Direct investigation of diastolic properties was precluded by the absence of accurate measurements of instantaneous ventricular volume in this preparation. Possible changes in ventricular diastolic function were assessed as follows: if it is assumed that an increase in SV upon elevation of preload in this preparation results predominantly from an increase in LVEDV,³⁶ then this change in SV may be taken to be the increase in LVEDV. The corresponding change in LVEDP is available by direct measurement. Because baseline cardiac output (at 10 cm H₂O) was unaltered by L-NMMA or hemoglobin, we further assumed that LVEDV at the lowest preload was similar before and after these drugs. Fig 6 shows average relationships between preload-induced changes in LVEDP and corresponding changes in SV for the two-step increases in filling pressure. In the absence of drugs, the average LVEDP-SV relation for the 15 to 20 cm H₂O increase in preload was to the left of the corresponding relation for the 10 to 15 cm H₂O increase in preload (eg, Fig 6A), consistent with the expected increase in end-diastolic chamber stiffness as LVEDP increases in the normal heart. These LVEDP-SV relations remained unaltered in time control hearts (Fig 6A). After L-NMMA (Fig 6B), however, the LVEDP-SV relation for the 10 to 15 cm H₂O increment in preload was shifted significantly to the left. That is, a similar increment in LVEDP was associated with a smaller increase in SV, consistent with a shift in the end-diastolic pressure-volume relation toward increased stiffness. A similar shift in the LVEDP-SV relation was observed after treatment with hemoglobin (Fig 6C). By contrast, after treatment with sodium nitroprusside, the LVEDP-SV relation for preload elevation from 10 to 15 cm H₂O was shifted to the right (Fig 6D), consistent with a change in the end-diastolic pressure-volume relationship in the direction of decreased stiffness.

View larger version (15K): [in this window] [in a new window]   Figure 6. Average relations between preload-induced change in LVEDP and corresponding change in SV for increase in filling pressure from 10 to 15 cm H2O ( before and after intervention) and from 15 to 20 cm H2O ( before and after intervention). A, Time controls; B, L-NMMA; C, hemoglobin (Hb); D, sodium nitroprusside (SNP). *P<.05, **P<.01 predrug vs postdrug.

Relationship Between Changes in Coronary Flow and Cardiac Output
Two approaches were taken to address whether the attenuation of preload-induced rises in cardiac output by L-NMMA and hemoglobin might be related to changes in coronary flow induced by these drugs. First, we assessed the relationship between changes in coronary flow and the cardiac output response to preload elevation in hearts treated with L-NMMA or nitroprusside and time control hearts (Fig 7). When absolute data were used (left panel), correlation coefficients for the three groups were L-NMMA, r=-.38; nitroprusside, r=+.69; controls, r=-.36 (all P=NS). When percent data were used (right panel), correlation coefficients were L-NMMA, r=-.61 (P=NS); nitroprusside, r=+.85 (P=.03); controls, r=-.30 (P=NS). Thus, there was no simple relationship between these parameters.

View larger version (13K): [in this window] [in a new window]   Figure 7. Relation between alteration in baseline coronary flow and relative change in Frank-Starling response (cardiac output increment on preload elevation from 10 to 20 cm H2O). , Time-control hearts; , L-NMMA–treated hearts; , nitroprusside (SNP)-treated hearts.

Second, we compared the effects of L-NMMA with those of PGF₂, the latter studied at a dose (0.01 µmol/L) that reduced coronary flow at all preloads to an extent similar to or greater than that withL-NMMA or hemoglobin (Fig 8). PGF₂-induced reduction in coronary flow was not associated with significant changes in cardiac output response to preload elevation (Fig 8), despite significant decreases in LVP_max and LV dP/dt_max (not shown). Two-way ANOVA revealed no interaction between treatment and preload for any parameter. Fig 8 (lower left) shows that in individual hearts treated with PGF₂ or L-NMMA, there was no correlation between changes in coronary flow and the cardiac output response to preload elevation (PGF₂, r=-.04; L-NMMA, r=-.38; both P=NS). The LVEDP-SV relations were also unaffected by PGF₂ (Fig 8).

View larger version (17K): [in this window] [in a new window]   Figure 8. Effect of PGF2 0.01 µmol/L. Top, Effect of preload elevation before () and after () PGF2. * Significant vs time-control group at equivalent preload. Bottom left, Relation between change in baseline coronary flow and change in Frank-Starling response (same format as Fig 7). Bottom right, Relation between preload-induced changes in SV and changes in LVEDP before (, ) and after (, ) PGF2 (same format as Fig 6).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The main finding of the present study is that basal intracardiac production of nitric oxide significantly influences the cardiac output response to preload elevation (ie, the Frank-Starling response) in the isolated ejecting guinea pig heart. Both L-NMMA and free hemoglobin (which inhibit basal nitric oxide production/activity) significantly attenuated preload-induced increases in cardiac output without altering baseline cardiac output at the lower preload. Consistent with a nitric oxide–mediated mechanism, L-NMMA effects were abolished by concurrent treatment with excess L-arginine.

What is the mechanism responsible for these effects of endogenous nitric oxide? One possibility to consider was that the attenuation of preload-induced rises in cardiac output might be related to reduction in coronary flow, because nitric oxide is a potent vasodilator. We consider this unlikely for the following reasons: (1) a relatively low dose of L-NMMA (10 µmol/L) was chosen in the present study to avoid marked reduction in coronary flow and associated myocardial ischemia; indeed, baseline coronary flow was not significantly reduced by either hemoglobin or L-NMMA; (2) preload-induced increases in coronary flow were not significantly altered by either drug; (3) a greater reduction in coronary flow with PGF₂ was not accompanied by attenuation of preload-induced rises in cardiac output; and (4) there was no correlation between changes in coronary flow and changes in cardiac output (Fig 7). Consistent with the present study, Beresewicz and Wozniak³⁷ have reported that a higher dose of L-NMMA (200 µmol/L) also had no effect on preload-induced increases in coronary flow or on lactate release despite significantly reducing baseline coronary flow in isolated ejecting rat hearts.

Another possibility was that endogenous nitric oxide favorably altered ventricular diastolic properties, as suggested by recent studies in human subjects in which both sodium nitroprusside and substance P (an agonist for the release of nitric oxide from endothelial cells) induced a downward shift in the LV end-diastolic pressure-volume relations compatible with reduced stiffness.^{25 26} To address this possibility, the relationship between changes in LVEDP and SV was studied (Fig 6). This relationship was altered by L-NMMA and hemoglobin such that similar increments in LVEDP were associated with smaller increases in SV. By contrast, sodium nitroprusside shifted the relation in the opposite direction. If it is assumed that preload-induced augmentation of SV in this type of isolated heart preparation results largely from increases in LVEDV (as reported by Suga et al³⁶ ), these data would be consistent with the hypothesis that L-NMMA and hemoglobin limited preload-induced rises in LVEDV by increasing diastolic chamber stiffness. A change in end-diastolic stiffness could result from a shift in position or slope of the pressure-volume curve or from a change in the operating position on an unaltered pressure-volume curve. Definitive confirmation of a change in diastolic stiffness was precluded in the present study by the absence of measurements of instantaneous ventricular volume. If there were preload-induced changes in end-systolic LV volume, for example, this would clearly weaken the above assumptions. Studies using isovolumic preparations to address diastolic properties in detail may help to resolve this issue.

It is also possible that systolic dysfunction may have contributed to or accounted for the effects of L-NMMA and hemoglobin on cardiac output. Indeed, in the presence of L-NMMA, LVP_max was depressed at 15 cm H₂O and LV dP/dt_max was depressed at 15 and 20 cm H₂O. However, L-NMMA had no effect on these parameters at the lowest preload, suggesting that systolic dysfunction was induced only at high preloads. In contrast to L-NMMA, hemoglobin had no effect on either parameter at any preload. It is possible that the changes (decrease) in LV dP/dt_max may be secondary to a putative decrease in LVEDV. In vivo, changes in LVEDV are generally paralleled by corresponding small changes in LV dP/dt_max,¹ although we observed no increase in LV dP/dt_max with preload elevation in the absence of drugs. The latter finding is likely to be due to the experimental model studied, consistent with previous data.³⁶

Treatment of isolated ejecting hearts with the exogenous nitric oxide donor sodium nitroprusside induced effects essentially opposite to those of L-NMMA and hemoglobin. Of note, nitroprusside altered cardiac output only at low preload, whereas L-NMMA and hemoglobin affected cardiac output only at high preloads. The reduction in baseline cardiac output with nitroprusside suggests a "negative inotropic" action of exogenous nitric oxide but no such effect of endogenous nitric oxide. A possible explanation for this difference may be the respective myocardial levels of nitric oxide in the two situations. It has been reported that low (submicromolar) doses of nitric oxide exert a small positive or no inotropic effect, whereas higher doses exert negative inotropic effects.^{28 29} In the present study, it is possible that exogenous nitric oxide supplementation resulted in levels sufficiently high to induce a negative inotropic effect. With preload elevation, however, this action would be outweighed by the beneficial effects of nitric oxide on the Frank-Starling response. Another difference between these situations is that treatment with exogenous nitroprusside delivers nitric oxide throughout the myocardium, whereas endogenous nitric oxide is tightly regulated with respect to quantity, timing and site of release, and releasing stimuli.

Several previous studies have investigated the effects of nitric oxide synthase inhibition on basal myocardial function (ie, in the absence of agonist stimulation). No effect has generally been observed in isolated cardiac myocyte or papillary muscle studies (reviewed in Reference 18¹⁸ ). In a study in the isolated ejecting rat heart, L-NMMA 500 µmol/L substantially reduced baseline cardiac output, an effect attributed to myocardial ischemia, because coronary flow was also markedly (40%) decreased.³⁸ However, no effect of L-NMMA 200 µmol/L or nitro-L-arginine methyl ester 3 µmol/L on baseline cardiac output was noted in two other studies in isolated ejecting rat hearts.^{37 39} Intracoronary L-NMMA also had no effect on LV dP/dt_max in human subjects with LV dysfunction.²⁷ In the present study, we observed no significant effect of L-NMMA on baseline LVP_max, LV dP/dt_max, or cardiac output. An effect of L-NMMA on cardiac function was apparent only upon full assessment of the Frank-Starling response.

Subcellular Mechanisms
An increase in myofilament responsiveness to Ca²⁺ is a major factor in the response of cardiac muscle to stretch.^{11 14 15} The concept of length-dependent myofilament activation^{8 10 11} was developed on the basis of observations that the relative slope of the isolated cardiac muscle length-tension relationship was inversely proportional to the degree of potentiation of tension. If muscle tension at shorter lengths was increased by inotropic interventions (eg, an increase in stimulation frequency), a smaller subsequent rise in tension was observed upon increasing muscle length.¹⁰ The present results with L-NMMA and hemoglobin are interesting in that preload-induced elevation of cardiac output was markedly decreased by these agents in the absence of a baseline change in function (ie, effects were restricted predominantly to high preloads). Possible explanations for this observation are (1) that a given action of endogenous nitric oxide (eg, modification of ventricular diastolic properties) affects pump function in a length-dependent manner and/or (2) that muscle length affects the release or action of nitric oxide itself.

The mechanism of possible change in diastolic properties by nitric oxide is speculative. The end-diastolic pressure-volume relationship is influenced by several factors, including myocardial passive elastic properties, changes in diastolic tone, the extent of myocardial relaxation, coronary turgor, geometric factors such as wall thickness, and external factors such as pericardial constraint and ventricular interaction. In the present study, many of these possibilities can be excluded. Pericardial constraint and ventricular interaction are not relevant in the isolated ejecting LV preparation, whereas geometric factors would be important only in the chronic situation. The observed changes in coronary flow (ie, a small reduction with L-NMMA) would, if anything, decrease stiffness through the Salisbury ("garden-hose") effect.⁴⁰ Myocardial relaxation as assessed by T_L was unaltered, indicating that changes in late diastolic properties were not due to incomplete relaxation. The most likely mechanism of reduction in diastolic stiffness may be a nitric oxide–induced change in myocyte diastolic tone.¹⁸ In isolated rat cardiac myocytes, the cGMP analogue 8-bromo-cGMP increases diastolic myocyte length without altering diastolic cytosolic Ca²⁺, compatible with a Ca²⁺-independent reduction in diastolic tone.^{24 33} Alternatively, nitric oxide–induced reduction in Ca²⁺ influx and cytosolic Ca²⁺ secondary to stimulation of cGMP-dependent cAMP phosphodiesterases²⁸ could lead to reduction in Ca²⁺-dependent diastolic tone. This seems less likely, because reduction in Ca²⁺ influx would be expected to depress basal systolic function in normal hearts. Other possibilities include an effect of nitric oxide/cGMP on a protein such as titin, which is believed to be important in determining myocardial passive stiffness and which has phosphorylation sites of unknown function.⁴¹

A potential influence of stretch on the release of nitric oxide is also of interest. Recent studies indicate that NOS-3 is localized in plasmalemmal caveolae, tiny invaginations present on the surface membranes of most cells.^{42 43} Caveolae are postulated to be sites for processing and integration of hormonal and mechanical signals and are the preferred location for many proteins involved in signal transduction, such as G proteins, G protein–linked receptors, and calcium channels.⁴⁴ Caveolar structure and function may be modulated by the cell cytoskeleton, thus providing a mechanism for response to mechanical stimuli.⁴⁵ It is feasible that activation of NOS-3 in the heart may be directly responsive to the mechanical signal of stretch during the cardiac cycle. Stretch activation of NOS-3 could be relevant in both main cardiac cell types that produce nitric oxide, ie, microvascular endothelial cells and cardiac myocytes.^{18 19 20} It is likely that both endothelial and cardiac myocyte NOS-3 were inhibited by L-NMMA in the present study, on the basis of the findings of previous studies using similar protocols (dose and duration).^{27 30} Therefore, no conclusion can be drawn regarding the respective roles of these cell types in the effects observed in the present study.

Potential Physiological and Pathophysiological Relevance
Extrapolation of these data to the in vivo situation is speculative, given that the studies were performed in an isolated buffer-perfused preparation. In vivo, other effects, such as vascular and autonomic reflexes triggered by alterations in vascular afterload as stroke volume increases, are likely to influence the overall changes in cardiac output. Nevertheless, our findings may be of relevance both to normal physiology and pathological states. The Frank-Starling response contributes to the increases in cardiac output associated with changes in posture³ and submaximal exercise,^{4 5} particularly in older subjects in whom the chronotropic response to exercise is impaired or in trained athletes who have less marked chronotropic responses to exercise than sedentary individuals.⁴⁶ It is known that release of nitric oxide increases during acute exercise and that expression of NOS-3 increases with chronic exercise.⁴⁷ An increase in nitric oxide in these settings would be beneficial if it augmented the Frank-Starling response. Conversely, reduced activity of nitric oxide in disease states such as chronic heart failure⁴⁸ could contribute to an impaired Frank-Starling response (in conjunction with other mechanisms such as afterload mismatch). These effects of nitric oxide may be especially important in disease states characterized by LV diastolic dysfunction, such as LV hypertrophy or myocardial ischemia, particularly at high heart rates.⁴⁹

In conclusion, this study shows that basal release of endogenous nitric oxide significantly influences the Frank-Starling response in the isolated ejecting heart. Taken in conjunction with previous findings that nitric oxide modulates the myocardial force-frequency relation, ß-adrenergic inotropic response, myocardial relaxation, and possibly heart rate, these data indicate the potential importance of nitric oxide in the regulation of cardiac contractile function.

	Selected Abbreviations and Acronyms

 

EDV = end-diastolic volume L-NMMA = NG-monomethyl-L-arginine LV = left ventricular LVEDP = LV end-diastolic pressure LVPmax = peak LV pressure NOS-3 = endothelial-type nitric oxide synthase PGF2 = prostaglandin F2 SV = stroke volume TL = time constant of isovolumic relaxation

	Acknowledgments

 
Dr Prendergast was supported by a British Heart Foundation Junior Fellowship, Dr Sagach by a Royal Society Visiting Fellowship, and Dr Shah by a UK Medical Research Council Clinical Senior Fellowship. We are most grateful to J. Wilson for help and advice with statistical analyses.

Received January 9, 1997; revision received February 24, 1997; accepted February 28, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Lakatta EG. Length modulation of muscle performance: Frank-Starling law of the heart. In: Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE, eds. The Heart and Cardiovascular System. New York, NY: Raven Press; 1992:1325-1351.
   
2. Crozatier B. Stretch-induced modifications of myocardial performance: from ventricular function to cellular and molecular mechanisms. Cardiovasc Res. 1996;32:25-37.[Medline] [Order article via Infotrieve]
   
3. Drake-Holland AJ, Mills CJ, Noble MIM, Pugh S. Responses to changes in filling and contractility of indices of human left ventricular mechanical performance. J Physiol. 1990;422:29-39.[Abstract/Free Full Text]
   
4. Plotnick GD, Becker LC, Fisher ML, Gerstenblith G, Renlund DG, Fleg JL, Weisfeldt ML, Lakatta EG. Use of the Frank-Starling mechanism during submaximal versus maximal upright exercise. Am J Physiol. 1986;251:H1101-H1105.
   
5. Higginbotham MB, Morris KG, Sanders Williams R, McHale PA, Coleman RE, Cobb FR. Regulation of stroke volume during submaximal and maximal upright exercise in normal man. Circ Res. 1986;58:281-291.[Abstract/Free Full Text]
   
6. Sato H, Hori M, Ozaki H, Yokoyama H, Imai K, Morikawa M, Takeda H, Inoue M, Kamada T. Exercise-induced upward shift of diastolic left ventricular pressure-volume relation in patients with dilated cardiomyopathy: effects of ß-adrenoceptor blockade. Circulation. 1993;88:2215-2223.[Abstract/Free Full Text]
   
7. Komamura K, Shannon RP, Ihara T, Shen YT, Mirsky I, Bishop SP, Vatner SF. Exhaustion of Frank-Starling mechanism in conscious dogs with heart failure. Am J Physiol. 1993;265:H1119-H1131.[Abstract/Free Full Text]
   
8. Parmley WW, Chuck L. Length-dependent changes in myocardial contractile state. Am J Physiol. 1973;224:1195-1199.
   
9. Fabiato A, Fabiato F. Dependence of the contractile activation of skinned cardiac cells on the sarcomere length. Nature. 1975;256:54-56.[Medline] [Order article via Infotrieve]
   
10. Lakatta EG, Jewell BR. Length-dependent activation: its effect on the length-tension relation in cat ventricular muscle. Circ Res. 1977;40:251-257.[Abstract/Free Full Text]
    
11. Allen DG, Kurihara S. The effects of muscle length on intracellular calcium transients in mammalian cardiac muscle. J Physiol. 1982;327:79-94.[Abstract/Free Full Text]
    
12. Tucci PJF, Bregagnollo EA, Spadaro J, Cicogna AC, Ribiero MCL. Length-dependence of activation studied in the isovolumic blood-perfused dog heart. Circ Res. 1984;55:59-66.[Abstract/Free Full Text]
    
13. Sugiura S, Hunter WC, Sagawa K. Long-term versus intrabeat history of ejection as determinants of canine ventricular end-systolic pressure. Circ Res. 1989;64:255-264.[Abstract/Free Full Text]
    
14. Hibberd MG, Jewell BR. Calcium- and length-dependent force production in rat ventricular muscle. J Physiol. 1982;329:527-540.[Abstract/Free Full Text]
    
15. Hoffman PA, Fuchs F. Effect of length and cross-bridge attachment on Ca²⁺ binding to cardiac troponin C. Am J Physiol. 1987;253:C90-C96.[Abstract/Free Full Text]
    
16. Brutsaert DL, Andries LJ. The endocardial endothelium. Am J Physiol. 1992;263:H985-H1002.[Abstract/Free Full Text]
    
17. Ramaciotti C, McClellan G, Sharkey A, Rose D, Wiseberg A, Winegrad S. Cardiac endothelial cells modulate contractility of rat heart in response to oxygen tension and coronary flow. Circ Res. 1993;72:1044-1064.[Abstract/Free Full Text]
    
18. Shah AM. Paracrine modulation of heart cell function by endothelial cells. Cardiovasc Res. 1996;31:847-867.[Medline] [Order article via Infotrieve]
    
19. Balligand JL, Kelly RA, Marsden PA, Smith TW, Michel T. Control of cardiac muscle function by an endogenous nitric oxide signaling system. Proc Natl Acad Sci U S A. 1993;90:347-351.[Abstract/Free Full Text]
    
20. Balligand JL, 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]
    
21. Smith JA, Shah AM, Lewis MJ. Factors released from the endocardium of the ferret and pig modulate myocardial contraction. J Physiol. 1991;439:1-14.[Abstract/Free Full Text]
    
22. Grocott-Mason R, Fort S, Lewis MJ, Shah AM. Myocardial relaxant effect of exogenous nitric oxide in the isolated ejecting heart. Am J Physiol. 1994;266:H1699-H1705.[Abstract/Free Full Text]
    
23. Grocott-Mason RM, Anning PB, 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]
    
24. Shah AM, Spurgeon H, Sollott SJ, Talo A, Lakatta EG. 8-Bromo cyclic GMP reduces the myofilament response to calcium in intact cardiac myocytes. Circ Res. 1994;74:970-978.[Abstract/Free Full Text]
    
25. Paulus WJ, Vantrimpont PJ, Shah AM. Acute effects of nitric oxide on left ventricular relaxation and diastolic distensibility in man. Circulation. 1994;89:2070-2078.[Abstract/Free Full Text]
    
26. Paulus WJ, Vantrimpont PJ, Shah AM. Paracrine coronary endothelial control of left ventricular function in humans. Circulation. 1995;92:2119-2126.[Abstract/Free Full Text]
    
27. Hare JM, Loh E, Creager MA, Colucci WS. Nitric oxide inhibits the positive inotropic response to ß-adrenergic stimulation in humans with left ventricular dysfunction. Circulation. 1995;92:2198-2203.[Abstract/Free Full Text]
    
28. Mery PF, Pavoine C, Belhassen L, Pecker F, Fischmeister R. Nitric oxide regulates cardiac Ca²⁺ current. J Biol Chem. 1994;268:26286-26295.[Abstract/Free Full Text]
    
29. Mohan P, Brutsaert DL, Paulus WJ, Sys SU. Myocardial contractile response to nitric oxide and cGMP. Circulation. 1996;93:1223-1229.[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. Kaye DM, Wiviott SD, Balligand JL, Simmons WW, Smith TW, Kelly RA. Frequency-dependent activation of a constitutive nitric oxide synthase and regulation of contractile function in adult rat ventricular myocytes. Circ Res. 1996;78:217-224.[Abstract/Free Full Text]
    
32. Shah AM, Lewis MJ, Henderson AH. Effects of 8-bromo-cyclic GMP on contraction and on inotropic response of ferret cardiac muscle. J Mol Cell Cardiol. 1990;23:55-64.
    
33. Shah AM, Silverman HS, Griffith EJ, Spurgeon HA, Lakatta EG. Cyclic GMP prevents delayed relaxation at reoxygenation following brief hypoxia in isolated cardiac myocytes. Am J Physiol. 1995;268:H2396-H2404.[Abstract/Free Full Text]
    
34. Pfitzer G, Rüegg JC, Flockerzi V, Hofmann F. cGMP-dependent protein kinase decreases calcium sensitivity of skinned cardiac fibers. FEBS Lett. 1982;149:171-175.[Medline] [Order article via Infotrieve]
    
35. Ludbrook J. Repeated measurements and multiple comparisons in cardiovascular research. Cardiovasc Res. 1994;28:303-311.[Free Full Text]
    
36. Suga H, Sagawa K, Shoukas AA. Load independence of the instantaneous pressure-volume ratio of the canine left ventricle and effects of norepinephrine and heart rate on the ratio. Circ Res. 1973;32:314-322.[Abstract/Free Full Text]
    
37. Beresewicz A, Wozniak M. Mechanical performance and coronary flow adjustments to changes in workload are not affected by inhibiting nitric oxide production in working rat heart. Pol J Pharmacol. 1993;45:533-548.[Medline] [Order article via Infotrieve]
    
38. Amrani M, O'Shea J, Allen NJ, Harding SE, Jayakumar J, Pepper JR, Moncada S, Yacoub MH. Role of basal release of nitric oxide on coronary flow and mechanical performance of the isolated rat heart. J Physiol. 1992;456:681-687.[Abstract/Free Full Text]
    
39. Schulz R, Panas DL, Catena R, Moncada S, Olley PM, Lopaschuk GD. The role of nitric oxide in cardiac depression induced by interleukin-1ß and tumor necrosis factor-. Br J Pharmacol. 1995;114:27-34.[Medline] [Order article via Infotrieve]
    
40. Salisbury PF, Cross CE, Riebed PA. Influence of coronary pressure on myocardial elasticity. Circ Res. 1960;8:794-800.[Abstract/Free Full Text]
    
41. Keller TCS. Structure and function of titin and nebulin. Curr Opin Cell Biol. 1995;7:32-38.[Medline] [Order article via Infotrieve]
    
42. Shaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna IS, Ying Y, Anderson RGW, Michel T. Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem. 1996;271:6518-6522.[Abstract/Free Full Text]
    
43. Feron O, Belhassen L, Kobzik L, Smith TW, Kelly RA, Michel T. Endothelial nitric oxide synthase targeting to caveolae: specific interactions with caveolin isoforms in cardiac myocytes and endothelial cells. J Biol Chem. 1996;271:22810-22814.[Abstract/Free Full Text]
    
44. Anderson RGW. Caveolae: where incoming and outgoing messengers meet. Proc Natl Acad Sci U S A. 1993;90:10909-10913.[Abstract/Free Full Text]
    
45. Levin KR, Page E. Quantitative studies on plasmalemmal folds and caveolae of rabbit ventricular myocardial cells. Circ Res. 1980;46:244-255.[Abstract/Free Full Text]
    
46. Rodeheffer RJ, Gerstenblith G, Becker LC, Fleg JL, Weisfeldt ML, Lakatta EG. Exercise cardiac output is maintained with increasing age in healthy human subjects: cardiac dilatation and increased stroke volume compensate for a diminished heart rate. Circulation. 1984;69:203-213.[Abstract/Free Full Text]
    
47. Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze TH. Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ Res. 1994;74:349-353.[Abstract/Free Full Text]
    
48. Smith CJ, Sun D, Hoegler C, Roth BS, Zhang X, Zhao G, Xu XB, Kobari Y, Pritchard K, Sessa WC, Hintze TH. Reduced gene expression of vascular endothelial NO synthase and cyclooxygenase-1 in heart failure. Circ Res. 1996;78:58-64.[Abstract/Free Full Text]
    
49. Grossman W. Diastolic dysfunction in congestive heart failure. N Engl J Med. 1991;325:1557-1564.[Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				Y. H. Zhang, M. H. Zhang, C. E. Sears, K. Emanuel, C. Redwood, A. El-Armouche, E. G. Kranias, and B. Casadei Reduced Phospholamban Phosphorylation Is Associated With Impaired Relaxation in Left Ventricular Myocytes From Neuronal NO Synthase-Deficient Mice Circ. Res., February 1, 2008; 102(2): 242 - 249. [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]

				W. J. Paulus and J. G. F. Bronzwaer Nitric oxide's role in the heart: control of beating or breathing? Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H8 - H13. [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]

				Z. Z. Kojic, U. Flogel, J. Schrader, and U. K. M. Decking Endothelial NO formation does not control myocardial O2 consumption in mouse heart Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H392 - H397. [Abstract] [Full Text] [PDF]

				J. G. F. Bronzwaer, C. Heymes, C. A. Visser, and W. J. Paulus Myocardial fibrosis blunts nitric oxide synthase-related preload reserve in human dilated cardiomyopathy Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H10 - H16. [Abstract] [Full Text] [PDF]

				R. Rubio and G. Ceballos Sole activation of three luminal adenosine receptor subtypes in different parts of coronary vasculature Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H204 - H214. [Abstract] [Full Text] [PDF]

				J. Piuhola, I. Szokodi, P. Kinnunen, M. Ilves, R. deChatel, O. Vuolteenaho, and H. Ruskoaho Endothelin-1 Contributes to the Frank-Starling Response in Hypertrophic Rat Hearts Hypertension, January 1, 2003; 41(1): 93 - 98. [Abstract] [Full Text] [PDF]