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(Circulation Research. 2003;92:e52.)
© 2003 American Heart Association, Inc.

UltraRapid Communication

Cardiac Neuronal Nitric Oxide Synthase Isoform Regulates Myocardial Contraction and Calcium Handling

Claire E. Sears, Simon M. Bryant, Euan A. Ashley, Craig A. Lygate, Stevan Rakovic, Helen L. Wallis, Stefan Neubauer, Derek A. Terrar, B. Casadei

From the Department of Cardiovascular Medicine (C.E.S., S.M.B., E.A.A., C.A.L., H.L.W., S.N., B.C.), Oxford University, John Radcliffe Hospital; and the Department of Pharmacology (S.R., D.T.), Oxford University, Oxford, UK.

Correspondence to Dr Claire E. Sears or Dr Barbara Casadei, Department of Cardiovascular Medicine, Oxford University, John Radcliffe Hospital, Oxford, OX3 9DU, UK. E-mail claire.sears{at}cardiov.ox.ac.uk

	Abstract

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A neuronal isoform of nitric oxide synthase (nNOS) has recently been located to the cardiac sarcoplasmic reticulum (SR). Subcellular localization of a constitutive NOS in the proximity of an activating source of Ca²⁺ suggests that cardiac nNOS-derived NO may regulate contraction by exerting a highly specific and localized action on ion channels/transporters involved in Ca²⁺ cycling. To test this hypothesis, we have investigated myocardial Ca²⁺ handling and contractility in nNOS knockout mice (nNOS^-/-) and in control mice (C) after acute nNOS inhibition with 100 µmol/L L-VNIO. nNOS gene disruption or L-VNIO increased basal contraction both in left ventricular (LV) myocytes (steady-state cell shortening 10.3±0.6% in nNOS^-/- versus 8.1±0.5% in C; P<0.05) and in vivo (LV ejection fraction 53.5±2.7 in nNOS^-/- versus 44.9±1.5% in C; P<0.05). nNOS disruption increased I_Ca density (in pA/pF, at 0 mV, -11.4±0.5 in nNOS^-/- versus -9.1±0.5 in C; P<0.05) and prolonged the slow time constant of inactivation of I_Ca by 38% (P<0.05), leading to an increased Ca²⁺ influx and a greater SR load in nNOS^-/- myocytes (in pC/pF, 0.78±0.04 in nNOS^-/- versus 0.64±0.03 in C; P<0.05). Consistent with these data, [Ca²⁺]_i transient (indo-1) peak amplitude was greater in nNOS^-/- myocytes (410/495 ratio 0.34±0.01 in nNOS^-/- versus 0.31±0.01 in C; P<0.05). These findings have uncovered a novel mechanism by which intracellular Ca²⁺ is regulated in LV myocytes and indicate that nNOS is an important determinant of basal contractility in the mammalian myocardium. The full text of this article is available at http://www.circresaha.org.

Key Words: neuronal nitric oxide synthase • ventricular • contraction • calcium

	Introduction

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It is now well-established that nitric oxide (NO) is constitutively generated within the heart, not only by the endothelium but also by the myocytes themselves.¹ However, whether constitutive myocardial NO production regulates basal inotropy and calcium (Ca²⁺) fluxes remains controversial (see reviews^1,2). In particular, some studies have shown that nonisoform specific inhibition of NO synthase (NOS) or targeted disruption of the endothelial NOS isoform (eNOS) has no effect on cell shortening or Ca²⁺ handling.^3,4 Others, however, have indicated that endogenous NO production may tonically inhibit myocardial inotropy^5,6 and the Ca²⁺ current.⁷ These studies assumed that eNOS was the only constitutive isoform involved in the autocrine control of myocardial function. In 1999, however, Xu and collaborators⁸ localized a neuronal-type NOS isoform (nNOS) to murine and human sarcoplasmic reticulum (SR). The subcellular localization of a constitutive NOS isoform in the proximity of an activating source of Ca²⁺ suggests that endogenous NO may exert a specific and localized action on ion channels/transporters involved in Ca²⁺ cycling. In the present study, we report that targeted disruption of the nNOS gene (nNOS^-/-) as well as acute pharmacological nNOS inhibition enhances basal left ventricular (LV) contraction and intracellular Ca²⁺ ([Ca²⁺]_i) transients by increasing Ca²⁺ influx (via the Ca²⁺ current) and SR Ca²⁺ content.

These findings have uncovered a novel mechanism by which [Ca²⁺]_i is regulated in LV myocytes. We suggest that nNOS may provide a negative feedback regulation of Ca²⁺ influx, because increases in [Ca²⁺]_i stimulate nNOS synthesis of NO, which in turn acts to inhibit Ca²⁺ influx. Such mechanisms would contribute to the maintenance of a tight control of [Ca²⁺]_i in physiological conditions and may protect against Ca²⁺ overload in cardiac disease states.

	Materials and Methods

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Animals
Mice homozygous for targeted disruption of the nNOS gene (B6,129-NOS1^tm1plh, nNOS^-/-)⁹ were purchased from Jackson Laboratories (Bar Harbor, Maine) and a colony was established at the John Radcliffe Hospital by backcrossing the nNOS^-/- on a C57BL/6 background. N3 littermate mice homozygous for the nNOS gene (nNOS^+/+) were used as controls in most protocols, in others age-matched C57BL/6 mice were used as wild-type controls as in previous studies.^10,11 The treatment of all animals was in accordance with the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act, 1986 (H.M.S.O.).

In Vivo Measurements of LV Function
Hemodynamic indices were measured in anesthetized mice (isoflurane) using a A 1.4F Millar Mikro-tip catheter (SPR-671) inserted into the LV via the carotid artery. At the end of the experiment, the catheter was withdrawn from the LV to measure aortic pressures. Reported values represent an average of 20 consecutive cardiac cycles. Echocardiography was performed simultaneously using an Agilent Sonos5500 with a 7- to 15-MHz linear-array transducer. Parasternal short-axis images, 2-D and M-mode, were obtained at the level of the papillary muscles and stored digitally.

Myocyte Isolation and Techniques
Single LV myocytes were isolated using a standard enzymatic dispersion technique as described previously.¹² For electrophysiological recordings, myocytes were superfused with a modified Tyrode solution (for composition, see expanded Materials and Methods, available in the online data supplement at http://www. circresaha.org). Membrane current was measured using the whole-cell configuration of the patch-clamp technique (Axopatch 200A, Axon Instruments). Cell length was concurrently monitored with a video-edge detection system (IonOptix Corp), with a temporal resolution of 4.2 ms. Analog signals (current, voltage, and cell length) were digitized (Digidata 1200A, Axon Instruments) and stored on-line to computer for subsequent off-line analysis. Calcium current (I_Ca) and unloaded cell shortening were elicited at 35±1°C as detailed in the expanded Materials and Methods section.

Assessment of SR Ca²⁺ Load
The SR Ca²⁺ content was quantified in voltage-clamped cells by discharging SR Ca²⁺ with a 10 second application of caffeine (10 mmol/L, using a rapid solution-switching device) and integrating the resulting Na⁺-Ca²⁺ exchange (NCX) current, as described previously.¹³ In a cohort of myocytes from each group, the caffeine-induced calcium transient (indo-1) was recorded after exposure to 5 mmol/L nickel for 5 minutes,¹³ and the time constant of decay () was calculated in order to compare the contribution of slow Ca²⁺ extrusion mechanisms in control and nNOS^-/- myocytes.

The contribution of SR Ca²⁺ to excitation-contraction (E-C) coupling in nNOS^-/- myocytes was evaluated by assessing I_Ca and contraction after application of thapsigargin (10 µmol/L). Disabling of the SR by thapsigargin was verified by the absence of an inward current in response to a 10 mmol/L pulse of caffeine (data not shown).

Measurement of [Ca²⁺]_i Transients
Indo-1 fluorescence was monitored from cells preincubated with the acetoxymethyl ester of indo-1 (5 µmol/L, Sigma) for 20 minutes at room temperature. Cells were field-stimulated to contract at 1 Hz at 35°C. In a cohort of cells from each group, calibration of the indo-1 signal was performed using the method of Terracciano and MacLeod.¹⁴ In some experiments, cells were imaged (Fluo-4, 100 µmol/L loaded via the patch pipette) in line-scan mode (2.61 ms per line) using a Leica TCS-NT confocal microscopy system.

Immunoblotting
Western blots were performed on LV membrane subfractions using specific antibodies to the following: -1C subunit of the dihydropyridine calcium channel (Alomone); cardiac ryanodine receptor, calsequestrin, and SERCA2a (Affinity Bioreagents); phospholamban (Cyclacel); NCX (Abcam); and the plasmalemma Ca²⁺-ATPase (PMCA) (Laboratory Vision).

Acute Inhibition of Myocyte nNOS With N⁵-(1-Imino-3-Butenyl)-L-Ornithine (L-VNIO)
L-VNIO is a potent nNOS selective inhibitor. At the concentration used, its K_i for nNOS inhibition is 120-fold lower than for eNOS inhibition.¹⁵ Cohorts of cells taken from the same isolates were either incubated with L-VNIO (100 µmol/L) for 30 minutes (in addition, to ensure intracellular access for the drug, 100 µmol/L L-VNIO was also added to the pipette solution) or were stored under normal conditions and used as control cells. I_Ca, cell shortening, and SR load were assessed as outlined above.

Statistics
Data are expressed as mean±SEM, and n indicates the number of cells used. A two-way ANOVA was used to compare interactions between factors. Point-to-point comparisons were performed with a Student’s t test. Significance was assessed at the P<0.05 level.

	Results

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Contractile Parameters in nNOS^-/- Mice
The role of nNOS in regulating cardiac function was first investigated in vivo in anesthetized mice with homozygous deletion of the nNOS gene (nNOS^-/-) and their littermate controls. LV ejection fraction measured by transthoracic echocardiography was significantly greater in the nNOS^-/- mice than in controls (53.5±2.7% in nNOS^-/- versus 44.9±1.5% in controls; P<0.05; Figure 1A). There was no difference in resting heart rate, LV wall thickness, LV mass to body weight ratio, or aortic blood pressure (Table). Hemodynamic measurements showed a trend for a load-independent measure of LV contractility (maximal rate of rise in LV pressure normalized to instantaneous developed pressure, LV dP/dT_max/IP)¹⁶ to be higher in the nNOS^-/- animals (P=0.059), and for the of LV isovolumetric relaxation to be slower (Table).

View larger version (15K): [in this window] [in a new window]   Figure 1. Contractility is enhanced in vivo and in LV myocytes from nNOS-/- mice. A, Scatter plot to show data for LV ejection fraction (%) in nNOS-/- (open circles) and control (filled triangles) mice. Ejection fraction was significantly greater in the nNOS-/- mice (P=0.01, n=10 for both groups). B, Example records of unloaded cell shortening (expressed as percent resting cell length) elicited by a 200-ms depolarizing step from -40 to 0 mV in control and nNOS-/- myocytes. C, Contraction-voltage relationship shows percent cell shortening is greater in the nNOS-/- myocytes (filled squares) than in control myocytes (open circles) over the voltage range -30 to +60 mV (P<0.05, n=16 and 21, respectively).

View this table: [in this window] [in a new window]   Table 1. Contraction Data From Controls and nNOS-/-

This finding of enhanced LV contraction in the nNOS^-/- mice was confirmed as a single cell phenomenon in isolated myocyte studies. Cell shortening elicited by a step depolarization to 0 mV was significantly greater in nNOS^-/- than in control myocytes over the physiological voltage range (Figures 1B and 1C). Qualitatively similar results were obtained when contraction parameters were assessed at steady state (average of five 200-ms depolarizing steps to 0 mV from -40 mV at 1 Hz; Table). In both protocols, time to 50% relaxation was significantly greater in nNOS^-/- myocytes than in controls. Myocyte length and capacitance did not differ between groups (data not shown).

nNOS-Dependent Regulation of [Ca²⁺]_i
To investigate whether an increased SR load might contribute to the enhanced contraction in nNOS^-/- myocytes, we measured the integral of the caffeine-induced NCX current (which reflects the amount of Ca²⁺ load in the SR).¹³ Both the amplitude (-1.59±0.6 versus -1.68±0.1 pA/pF; Figure 2A) and integral of the NCX current (0.64±0.03 pC/pF in controls versus 0.78±0.04 pC/pF in nNOS^-/-; P<0.05; n=15 and 17, respectively; Figures 2B and 2C) were larger in nNOS^-/- than in controls. The rate of decay of the caffeine-induced current was similar in the two groups (359.2±12.2 ms in controls versus 386.3±18.9 ms in nNOS^-/-), suggesting similar NCX characteristics in both groups. In addition, the time constant of decline of the caffeine-induced Ca²⁺ transient in the presence of Ni²⁺ was similar in control and nNOS^-/- myocytes (, 6.83±1.2 seconds in control versus 5.13±0.7 seconds in nNOS^-/-, n=9 in each group; P=0.25), indicating that there are no significant differences in the slow mechanisms of Ca²⁺ extrusion between the two groups.

View larger version (16K): [in this window] [in a new window]   Figure 2. SR Ca2+ load is enhanced in nNOS-/- myocytes. A, Example records of currents (in pA/pF) elicited in nNOS-/- and control myocytes by a 10-second exposure to 10 mmol/L caffeine. These currents are carried predominantly via the NCX in the Ca2+-extrusion mode. B, Integral of the currents in A in pC/pF. C, Average results of caffeine-induced current integrals, showing a larger charge and therefore greater SR load in nNOS-/- myocytes (*P<0.05, n=15 and 17, respectively).

nNOS-mediated regulation of Ca²⁺ handling was confirmed in indo-1–loaded myocytes. [Ca²⁺]_i transients had significantly greater amplitude in nNOS^-/- myocytes compared with controls. There was no difference in diastolic Ca²⁺, but peak Ca²⁺ was significantly greater (Figure 3A), as was the delta amplitude of the transient (0.087±0.01 in nNOS^-/- versus 0.063±0.01 in controls; P<0.05). [Ca²⁺]_i transients recorded using confocal microscopy in fluo-4–loaded myocytes also had a greater amplitude in nNOS^-/- compared with controls (n=10 and 15, respectively; data not shown). The time to peak of the Ca²⁺ transients recorded with either method did not differ between nNOS^-/- and control myocytes (indo-1 myocytes 29.0±0.9 ms in nNOS^-/- versus 29.4±1.3 ms in controls; fluo-4 26.8±1.9 ms in nNOS^-/- versus 31.3±2.7 ms in controls; P=NS for both).

View larger version (25K): [in this window] [in a new window]   Figure 3. Calcium transients from nNOS-/- myocytes are larger and slower to decay. A, Average raw data trace showing the indo-1 fluorescence ratio (410/495 nm) in control and nNOS-/- myocytes (n=17 and 19, respectively). Transients recorded from nNOS-/- myocytes had greater peak fluorescence. Using the calibration values obtained, average diastolic [Ca2+]i approximated to 97 nmol/L in control and 111 nmol/L in nNOS-/- myocytes, and peak [Ca2+]i to 704 nmol/L in control and to 1.3 µmol/L in nNOS-/-. B, Time course of decay of the Ca2+ transient was significantly slower in the nNOS-/- than in control myocytes (*P<0.05). C, Western blots using antibodies specific to the -subunit of the L-type calcium channel (LTCC), cardiac RyR, phospholamban (PLB), and calsequestrin (Cal) in control and nNOS-/- homogenate. Average results are from 4 hearts per group and are normalized to the GAPDH signal, where appropriate (LTCC and RyR).

A greater SR load and a larger peak [Ca²⁺]_i in nNOS^-/- could reflect tonic inhibition of SR Ca²⁺-ATPase (SERCA) activity by nNOS-derived NO, as suggested by Xu et al.⁸ However, if this were the primary mechanism of action of nNOS, we would expect to see a faster decay of the [Ca²⁺]_i transient and faster relaxation in nNOS^-/- myocytes. Instead, TR₅₀ was prolonged (Table), and the time constant of decay of the [Ca²⁺]_i transient was significantly greater in the nNOS^-/- myocytes (102±11 ms versus 148±11 ms; P<0.05; Figure 3B), suggesting that additional mechanisms may be involved.

Calcium Current in nNOS^-/- Myocytes
Thus, we investigated whether the increased SR load and contraction might result from modulation of another important component of E-C coupling, the Ca²⁺ current. We found that I_Ca density was significantly greater in the nNOS^-/- myocytes at voltages from -30 to +20 mV (Figures 4A and 4B) At 0 mV, I_Ca density was -9.1±0.5 pA/pF in control myocytes and -11.4±0.5 pA/pF in nNOS^-/- myocytes (P<0.01). Steady-state activation curves showed that the voltage at which I_Ca was half-maximally activated was -11.2±0.4 mV in controls and -12.8±0.6 mV in nNOS^-/- myocytes (P=0.04; Figure 4C). The slope of activation was unaltered (5.3±0.1 mV in controls versus 5.2±0.1 mV in nNOS^-/-). Similarly, the voltage at which I_Ca was half-inactivated was similar in both groups (-28.5±0.6 versus -28.7±0.6 mV in nNOS^-/-, slopes 4.7±0.1 and 4.4±0.2 mV, respectively; P=NS for both; Figure 4C).

View larger version (25K): [in this window] [in a new window]   Figure 4. Calcium currents from nNOS-/- myocytes are larger and show slower inactivation. A, Example records of ICa (in pA/pF) elicited by a 200 ms depolarizing step from -40 to 0 mV in control and nNOS-/- myocytes. B, Current-voltage relationship shows ICa density is greater in nNOS-/- myocytes (filled squares) than in controls (open circles) over the voltage range -30 to +20 mV (P<0.05, n=16 and 21, respectively). C, Steady-state activation and inactivation curves for ICa. Activation curves, expressed as relative conductance, show a small negative shift in the voltage of half-maximal activation between nNOS-/- (filled squares) and control myocytes (open triangles). Inactivation curves, expressed as relative current, did not differ between control (open triangles) and nNOS-/- myocytes (filled circles). D, Example records from steady-state ICa recordings during a 200-ms depolarizing step from -40 to 0 mV. To illustrate that deactivation of ICa is slower in nNOS-/- myocytes than in controls, peak current amplitudes for each myocyte have been normalized to -1. E, Decay of ICa was best fitted by a double-exponential function. Average results show that the fast component of decay was not significantly different, whereas the slow time constant was significantly greater in nNOS-/- myocytes than in controls (*P<0.05).

An additional explanation for the increased SR Ca²⁺ load in nNOS^-/- myocytes can be found by looking at the decay characteristics of I_Ca. The decay of steady-state I_Ca is best fitted by a double exponential function, yielding a fast and a slow time constant (Figure 4D). The fast time constant was not significantly different between the two groups (6.08±0.3 ms in controls versus 6.8±0.5 ms in nNOS^-/-), but the slow time constant was approximately 38% greater in the nNOS^-/- myocytes (37.3±21.5 versus 26.9±1.6 ms; Figure 4E). The inward current during slow decay of I_Ca is thought to contribute to Ca²⁺ loading of the SR,¹⁷ and thus its prolongation in nNOS^-/- myocytes may underlie their enhanced SR Ca²⁺ content. Moreover, we found that the steady-state current at the end of the pulse was more inward (-0.32±0.07 versus -0.1±0.04 pA/pF; P<0.05) and the integral of I_Ca over the whole pulse (an overall measure of Ca²⁺ influx) was greater in nNOS^-/- myocytes (-0.2±0.01 versus -0.12±0.01 pC/pF; P<0.05). Both of these findings would contribute toward enhancing SR Ca²⁺ load.

We also examined whether nNOS disruption and the resulting increase in the [Ca²⁺]_i transient were associated with changes in the expression of other Ca²⁺ cycling proteins in the LV myocardium. We found no differences in the protein level of the -subunit of the L-type Ca²⁺ channel (Figure 3C), NCX, SERCA, and PMCA (data not shown). However, expression of both calsequestrin and the ryanodine receptor Ca²⁺ release channel (RyR) were increased in the nNOS^-/-, whereas phospholamban levels were decreased (Figure 3C).

Does the Increase in I_Ca in nNOS^-/- Myocytes Underlie the Increase in Cell Shortening?
To test whether the enhanced cell shortening in nNOS^-/- myocytes was predominantly a function of the increased Ca²⁺ influx via I_Ca, we assessed steady state contraction and I_Ca after disabling the SR with thapsigargin. Cell shortening remained larger in nNOS^-/- myocytes than in controls (4.6±0.5% versus 2.9±0.3%; P<0.05; Figure 5A) in the presence of thapsigargin, but the time to 50% relaxation was no longer prolonged in the nNOS^-/- myocytes (174.8±5.7 ms in control versus 157.4±5.8 ms in nNOS^-/-; P=0.05). Under these conditions, the increase in contraction in nNOS^-/- myocytes may result from either a greater Ca²⁺ influx or an increase in the myofilament sensitivity to Ca²⁺. We found that Ca²⁺ influx, measured by integrating the steady-state I_Ca over the whole pulse was larger in nNOS^-/- than in controls (-0.22±0.02 versus -0.15±0.02 pC/pF; Figure 5B; P<0.05). Moreover, the slow component of I_Ca decay was still prolonged in nNOS^-/- myocytes after thapsigargin (42.5±2.2 ms in controls versus 56.5±5.1 ms in nNOS^-/-; P<0.05), indicating that the difference in I_Ca inactivation kinetics in nNOS^-/- was not dependent on SR Ca²⁺. An index of myofilament sensitivity to Ca²⁺ was estimated by plotting the relationship between cell shortening and the integral of I_Ca in the presence of thapsigargin (Figure 5C). The slope of this relationship did not differ between nNOS^-/- and controls, suggesting that under these conditions an increase in Ca²⁺ influx is mainly responsible for the increased contraction in nNOS^-/- myocytes.

View larger version (15K): [in this window] [in a new window]   Figure 5. Cell shortening is enhanced in nNOS-/- myocytes in the presence of thapsigargin. A, Example records of unloaded cell shortening (expressed as percent resting cell length) elicited by a 200-ms depolarizing step from -40 to 0 mV in control and nNOS-/- myocytes in the presence of thapsigargin. Cell shortening was significantly greater in nNOS-/- myocytes than in controls (n=8 and 9, respectively). B, Example records showing that ICa density recorded concurrently in the presence of thapsigargin was greater in nNOS-/- than in control myocytes. C, Relationship between cell shortening and the integral of ICa under these conditions shows no difference between nNOS-/- and control myocytes.

Effect of Acute nNOS Inhibition With L-VNIO on Cell Shortening, I_Ca, and SR Load
Our findings in nNOS^-/- mice suggest that cardiac nNOS is an important determinant of basal contractility and Ca²⁺ in the mammalian myocardium. However, the potential contribution of compensatory mechanisms secondary to chronic and systemic disruption of nNOS is difficult to assess in this model. To circumvent this problem, we also tested the effect of acute pharmacological inhibition of nNOS with L-VNIO (100 µmol/L)¹⁵ in LV myocytes from control mice. Results were qualitatively similar to those seen in myocytes from nNOS^-/- mice. Steady-state cell shortening was significantly increased in myocytes incubated with L-VNIO compared with control (10.3±0.7% versus 8.2±0.6%; P<0.05; Figures 6A and 6B). The increase in cell shortening was associated with a 23% increase in the density of the steady-state I_Ca (Figures 6C and 6D). Fitting the decay of I_Ca with a double exponential showed an increase in the slow time constant (control 25.9±0.8 ms versus L-VNIO 30.5±1.6 ms; P<0.05), whereas there was no difference in the fast time constant. In addition, the assessment of SR load was significantly greater in L-VNIO exposed cells than in control (Figures 6E and 6F).

View larger version (36K): [in this window] [in a new window]   Figure 6. Cell shortening, SR load, and ICa are enhanced in the presence of L-VNIO. A, Example trace showing unloaded cell shortening (expressed as percent resting cell length) elicited by a 200-ms depolarizing step from -40 to 0 mV in control myocytes and myocytes exposed to the nNOS specific inhibitor, L-VNIO (100 µmol/L). B, Cell shortening was significantly greater in the L-VNIO myocytes than in control (*P<0.05, n=14 and 13, respectively). C, Example records of steady-state ICa (in pA/pF) in control myocytes and myocytes exposed to L-VNIO. D, ICa current density was greater in the L-VNIO myocytes than in control (P<0.05). E, Integral of the currents elicited in control and L-VNIO-treated myocytes by a 10-second exposure to 10 mmol/L caffeine in pC/pF. F, Average results of caffeine-induced current integrals, showing a larger charge and therefore greater SR load in L-VNIO treated than in control myocytes (*P<0.05, n=11 and 10, respectively).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Until recently, eNOS was thought to be the sole isoform constitutively expressed in ventricular myocytes and thus the source of NO involved in the autocrine regulation of myocardial contraction and Ca²⁺ homeostasis.^1,2 Emerging evidence, however, indicates that nNOS is present in the cardiac SR,⁸ suggesting that this isoform may modulate ion channels/transporters involved in Ca²⁺ cycling and contraction. In the present study, we have demonstrated that nNOS gene disruption as well as pharmacological inhibition of nNOS significantly enhances contraction and [Ca²⁺]_i transients in isolated LV myocytes. In addition, we found that LV ejection fraction was significantly increased in nNOS^-/- mice, and there was a trend for LV dP/dt_max to be increased. These data indicate that cardiac nNOS plays a significant role in the autocrine control of cardiac contractility by regulating Ca²⁺ fluxes.

An increased basal LV dP/dt_max/IP in nNOS^-/- mice compared with C57Bl/6 mice has also been reported by Barouch et al,¹¹ although they found no difference in an alternative index of LV systolic performance. These authors, however, did not see a significantly greater basal contraction or [Ca²⁺]_i transients in nNOS^-/- LV myocytes in experiments presumably performed at room temperature. Conversely, both the present data and those of Ashley et al¹⁸ in field-stimulated LV cardiomyocytes show that at 35°C contraction is enhanced in nNOS^-/-.

Several proteins involved in E-C coupling are potential targets for NO modulation. Xu et al⁸ suggested that nNOS-derived NO might inhibit the activity of the SR Ca²⁺-ATPase. The increased SR Ca²⁺ load in nNOS^-/- myocytes and control myocytes after L-VNIO would be consistent with this hypothesis. However, if the main action of cardiac nNOS were to inhibit the SR Ca²⁺-ATPase, we would expect the speed of relaxation and the rate of decay of the [Ca²⁺]_i transient to be increased in nNOS^-/- myocytes, as the re-uptake of Ca²⁺ into the SR would occur more quickly. In addition, a greater peak [Ca²⁺]_i transient is thought to result in faster kinetics of [Ca²⁺]_i decline.¹⁹ On the contrary, we found a slower time to 50% relaxation and a prolonged time-course of decay of the [Ca²⁺]_i transient in nNOS^-/- myocytes, indicating that other mechanisms are likely to be involved.

A potential alternative target for NO is the cardiac ryanodine receptor Ca²⁺ release channel (RyR), which has been shown to be both activated^20,21 and inhibited²² by NO. We found that the time to peak [Ca²⁺]_i was similar in nNOS^-/- and in control LV myocytes, despite the increase in SR Ca²⁺ load and RyR protein expression in the nNOS^-/- myocytes, both of which might be expected to result in a faster time to peak [Ca²⁺]_i. Given the inherent limitations of inferring the function of calcium transport/release systems from the kinetics of calcium transients of differing sizes, it is difficult to draw firm conclusions on this issue.¹⁹ However, our findings are in keeping with the notion that RyR activity could be inhibited in nNOS^-/- myocytes, supporting the model put forward by Xu et al⁸ who proposed that NO may modulate E-C coupling via two discrete mechanisms: (1) a tonic inhibition of I_Ca and (2) an increase in RyR open probability.²⁰ In basal conditions (ie, in the absence of agonists or mechanical stimuli) both effects appear to be dependent on nNOS. Our data also indicate that the nNOS-mediated changes in I_Ca have a greater influence on contraction, in agreement with recent findings by Trafford et al²³ who demonstrated that sustained changes in the open probability of RyR only cause transient effects on [Ca²⁺]_i and cell shortening in rat LV myocytes.

Protein level and activity of the NCX (evaluated by the time course of decay of the caffeine-induced NCX current) did not differ between nNOS^-/- and controls, nor were the protein levels of SERCA different between these two groups. However, we did find an increase in the Ca²⁺ store protein calsequestrin in nNOS^-/- and a reduction in phospholamban. These changes are likely to be compensatory and of uncertain functional significance because acute nNOS inhibition is sufficient to reproduce the nNOS^-/- phenotype.

Because there is close spatial juxtaposition of L-type Ca²⁺ channels and junctional SR,²⁴ nNOS-derived NO from the SR may also affect the L-type calcium channel. There are only a few reports on the effect of NOS inhibition on basal I_Ca in mammalian ventricular myocytes and the findings are inconsistent. In mouse ventricular myocytes at room temperature Vandecasteele et al,⁴ found no effect of L-NMMA on basal I_Ca. However, Gallo et al^7,25 saw a large stimulatory effect of L-NMMA and L-NA on guinea-pig ventricular I_Ca at 35°C, suggesting that NO may exert a tonic inhibition of I_Ca. No difference in basal I_Ca has ever been reported after eNOS gene disruption.^4,26 We found an increased I_Ca density both in nNOS^-/- and after L-VNIO, suggesting that an increase in Ca²⁺ influx may contribute to the enhanced myocyte contraction after nNOS disruption. Experiments showing that I_Ca and contraction remained greater in nNOS^-/- myocytes after disabling the contribution of the SR with thapsigargin further support a predominant effect of nNOS on the Ca²⁺ channel. nNOS-derived NO could be acting via the guanylate cyclase/cyclic GMP second messenger pathway, as suggested by Gallo et al.²⁵ In agreement with these findings, 8-BrcGMP has been shown to inhibit I_Ca in ferret right ventricular myocytes,²⁷ and protein kinase G has been reported to cause a 40% inhibition of I_Ca in rat ventricular myocytes.²⁸ However, it is also possible that the mechanism of action of nNOS-derived NO on I_Ca might involve a change in the redox state of the channel. Hu et al²⁹ showed that S-nitrosothiols decrease I_Ca independent of cGMP in expressed calcium channels, and similar results were obtained with oxidizing agents,³⁰ although Campbell et al²⁷ showed stimulation of I_Ca by nitrosothiol agents in isolated myocytes.

We have also demonstrated that the slow component of the time constant of decay of I_Ca is prolonged in nNOS^-/-. The inward current during slow decay of I_Ca contributes to Ca²⁺ loading of the SR,¹⁷ suggesting that increased Ca²⁺ influx may contribute to the enhanced myocyte contraction in the presence of nNOS gene disruption or inhibition both directly (by increasing the trigger for SR Ca²⁺ release) and indirectly (by enhancing SR Ca²⁺ loading through slowing I_Ca deactivation).^17,31 The mechanism responsible for the effect of nNOS-derived NO on the inactivation of the Ca²⁺ current is unclear but it may involve a change in the phosphorylation state of the Ca²⁺ channel.^32,33 Because the slow component of the time constant of decay is still prolonged in the nNOS^-/- myocytes in the presence of thapsigargin, this effect does not appear to be dependent on SR load and it is likely to reflect NO-mediated regulation of the Ca²⁺ channel itself.

Stimulation of endothelial NO production induces earlier onset of myocardial relaxation with little or no effect on force of contraction in a variety of preparations.² In rat ventricular myocytes, 8-Br-cyclic GMP causes a small reduction in cell shortening and an earlier onset of relaxation in the absence of changes in the intracellular Ca²⁺ transient, suggesting that endogenous NO may reduce myofilament Ca²⁺ sensitivity via cyclic GMP.² We explored this possibility by evaluating the relationship between cell shortening and the integral of I_Ca in the presence of thapsigargin (which disables the SR). Under these conditions, the relationship between Ca²⁺ entry and cell shortening is governed by myofilament sensitivity to Ca²⁺. Our finding that this relationship does not differ between nNOS^-/- and control myocytes suggests that nNOS-derived NO has no significant effect on myofilament Ca²⁺ sensitivity. Disabling the SR also abolished the difference in time to 50% relaxation between nNOS^-/- and control myocytes, suggesting that slower relaxation and prolonged [Ca²⁺]_i transients in nNOS^-/- mice may result from inhibition of SR Ca²⁺ uptake, as has recently been demonstrated by Zhou et al.³⁴

In conclusion, our results demonstrate that nNOS-derived NO regulates myocardial contraction and Ca²⁺ transients by controlling Ca²⁺ handling in LV myocytes. We have shown that gene-disruption or acute inhibition of nNOS increases Ca²⁺ influx both by increasing I_Ca density and by slowing I_Ca inactivation, leading to greater loading of the SR stores and hence greater Ca²⁺-induced Ca²⁺ release. This novel finding challenges the assumption that the only NOS isoform involved in the control of cardiac function is eNOS and as such may stimulate a re-examination of the data in this field. It is conceivable that nNOS plays a negative feedback role in preventing [Ca²⁺]_i overload (particularly in the presence of submaximal ß-adrenergic stimulation¹⁸), as increases in [Ca²⁺]_i stimulate nNOS synthesis of NO, which in turn acts to inhibit Ca²⁺ fluxes. Such mechanisms may contribute to the maintenance of a tight control of intracellular Ca²⁺ in physiological conditions and may protect against the development of triggered arrhythmias in cardiovascular disease.

	Acknowledgments

 
This study was supported by the British Heart Foundation (S.B., C.L., S.R., S.N., D.T.). B.C. is a Senior Fellow of the British Heart Foundation, C.S. is a Dorothy Hodgkin Fellow of the Royal Society, and E.A. and H.W. are Wellcome Trust Research Training Fellows. We should like to thank Dr Nicholas Freestone for his expert assistance with the calcium fluorescence measurements. Dr Sears was awarded the Melvin Marcus Young Investigator Award for the presentation of these data at the 75th Scientific Sessions of the American Heart Association.

	Footnotes

 
Presented in part at the 75th Scientific Sessions of the American Heart Association, Chicago, Ill, November 17–20, 2002, and published in abstract form (Circulation. 2002;106[suppl II]:II-178).

Received October 29, 2002; revision received February 6, 2003; accepted February 20, 2003.

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