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Circulation Research. 2008;102:242-249
Published online before print November 15, 2007, doi: 10.1161/CIRCRESAHA.107.164798
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(Circulation Research. 2008;102:242.)
© 2008 American Heart Association, Inc.

Cellular Biology

Reduced Phospholamban Phosphorylation Is Associated With Impaired Relaxation in Left Ventricular Myocytes From Neuronal NO Synthase–Deficient Mice

Yin Hua Zhang*, Mei Hua Zhang*, Claire E. Sears, Krzysztof Emanuel, Charles Redwood, Ali El-Armouche, Evangelia G. Kranias, Barbara Casadei

From the Department of Cardiovascular Medicine (Y.H.Z., M.H.Z., C.E.S., K.E., C.R., B.C.), University of Oxford, John Radcliffe Hospital, United Kingdom; Institute of Experimental and Clinical Pharmacology (A.E.-A.), University Hospital Eppendorf, Hamburg, Germany; and Department of Pharmacology and Cell Biophysics (E.G.K.), University of Cincinnati College of Medicine, Ohio.

Correspondence to Barbara Casadei, MD, PhD, University Department of Cardiovascular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom. E-mail barbara.casadei{at}cardiov.ox.ac.uk


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
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Stimulation of nitric oxide (NO) release from the coronary endothelium facilitates myocardial relaxation via a cGMP-dependent reduction in myofilament Ca2+ sensitivity. Recent evidence suggests that NO released by a neuronal NO synthase (nNOS) in the myocardium can also hasten left ventricular relaxation; however, the mechanism underlying these findings is uncertain. Here we show that both relaxation (TR50) and the rate of [Ca2+]i transient decay (tau) are significantly prolonged in field-stimulated or voltage-clamped left ventricular myocytes from nNOS–/– mice and in wild-type myocytes (nNOS+/+) after acute nNOS inhibition. Disabling the sarcoplasmic reticulum abolished the differences in TR50 and tau, suggesting that impaired sarcoplasmic reticulum Ca2+ reuptake may account for the slower relaxation in nNOS–/– mice. In line with these findings, disruption of nNOS (but not of endothelial NOS) decreased phospholamban phosphorylation (P-Ser16 PLN), whereas nNOS inhibition had no effect on TR50 or tau in PLN–/– myocytes. Inhibition of cGMP signaling had no effect on relaxation in either group whereas protein kinase A inhibition abolished the difference in relaxation and PLN phosphorylation by decreasing P-Ser16 PLN and prolonging TR50 in nNOS+/+ myocytes. Conversely, inhibition of type 1 or 2A protein phosphatases shortened TR50 and increased P-Ser16 PLN in nNOS–/– but not in nNOS+/+ myocytes, in agreement with data showing increased protein phosphatase activity in nNOS–/– hearts. Taken together, our findings identify a novel mechanism by which myocardial nNOS promotes left ventricular relaxation by regulating the protein kinase A–mediated phosphorylation of PLN and the rate of sarcoplasmic reticulum Ca2+ reuptake via a cGMP-independent effect on protein phosphatase activity.


Key Words: neuronal NOS • nitric oxide • relaxation • phospholamban • phosphatases


*    Introduction
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 up arrowAbstract
 *Introduction
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The facilitatory effects of nitric oxide (NO) on myocardial relaxation and left ventricular (LV) diastolic distensibility are well documented. In animal models and in humans, stimulation of NO release from the coronary endothelium hastens relaxation and enhances LV compliance.1 The paracrine effects of endothelial-derived NO can be reproduced by applying cGMP analogs to isolated LV myocytes2 and have been attributed to a reduction in myofilament Ca2+ sensitivity secondary to troponin I phosphorylation by the cGMP-dependent protein kinase (PK)G.3 Through this mechanism, endogenous NO production may facilitate the Frank–Starling response4 and maintain the LV preload reserve in failing hearts.5 In contrast, the involvement of myocardial NO production in the regulation of relaxation has remained a matter of debate.

An autocrine effect of NO on myocardial relaxation was first suggested in 1999, when inhibition of a neuronal-like NOS (nNOS) localized to the sarcoplasmic reticulum (SR) of LV myocytes was shown to increase the thapsigargin-sensitive Ca2+ uptake from cardiac SR vesicles.6 These data implied that constitutive NO production by myocardial nNOS may impair relaxation by inhibiting the activity of the SR Ca2+ pump (SERCA2a). However, our work and that of others subsequently indicated that nNOS-specific inhibition or targeted deletion of the nNOS gene (nNOS–/–) prolongs myocardial relaxation in vivo and in field-stimulated LV myocytes7,8 and impairs the lusitropic response to high-stimulation frequencies.9 In agreement with these findings, it has recently been reported that SERCA2a activity is impaired in the absence of nNOS.10

Here we demonstrate that nNOS gene deletion or pharmacological inhibition retards relaxation and slows the rate of decay of the [Ca2+]i transient by causing a reduction in phospholamban (PLN) phosphorylation through a cGMP-independent increase in protein phosphatase (PP) activity.


*    Materials and Methods
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 *Materials and Methods
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An expanded Materials and Methods section is in the online data supplement, available at http://circres.ahajournals.org.

Mice (2 to 5 months old) homozygous for targeted disruption of nNOS or endothelial NO synthase (eNOS) gene (nNOS–/–11 and eNOS–/–12) were compared with their age-matched wild-type littermates (nNOS+/+ and eNOS+/+). PLN knockout mice (PLN–/–)13 were also used. Acute inhibition of nNOS was achieved by applying vinyl-L-N-5-(1-imino-3-butenyl)-L-ornithine (L-VNIO) (100 µmol/L) or S-methyl-L-thiocitrulline (SMTC) (100 nmol/L). All protocols were in accordance with the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act, 1986.


*    Results
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 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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Relaxation Is Prolonged and the Decay of the [Ca2+]i Transient Is Slower Both in Field-Stimulated and Voltage-Clamped LV Myocytes From nNOS–/– Mice
Both field-stimulated cell shortening and [Ca2+]i transient amplitude were greater in nNOS–/– myocytes (Figure 1A through 1D). In addition, LV myocytes from nNOS–/– mice showed a significantly prolonged time to 50% relaxation (TR50) and a slower rate of decay (tau) of the [Ca2+]i transient (Figure 1B and 1D), suggesting that Ca2+ reuptake into the SR or extrusion through the sarcolemma may be impaired in nNOS–/– mice. To control for the effects of differences in [Ca2+]i transient amplitude14 and action potential duration on myocyte relaxation and [Ca2+]i decay kinetics, we compared TR50 and tau in voltage clamped (25-ms depolarization pulse from –70 to +20 mV) and fura-2–loaded LV myocytes from nNOS–/– and nNOS+/+ mice after matching the amplitude of the [Ca2+]i transient by lowering the [Ca2+]o concentration to 0.9 mmol/L in the former. Under these controlled conditions (Figure 2), the tau of the [Ca2+]i transient and TR50 remained significantly prolonged in nNOS–/– myocytes, confirming that a slower [Ca2+]i reuptake and/or extrusion accounts for the impaired relaxation of nNOS–/– LV myocytes.


Figure 1
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  		Figure 1. Cell shortening and [Ca2+]i transient amplitude are greater in field-stimulated nNOS–/– myocytes but TR50 and the time constant of decay of the [Ca2+]i transient (tau) are slower. A, Examples of unloaded cell shortening in field-stimulated LV myocytes (3 Hz, 35°C) from nNOS–/– mice and wild-type littermates (nNOS+/+). B, Average values for cell shortening and TR50 (nNOS+/+: open bars, n=14; nNOS–/–: solid bars, n=11; *P=0.001 for cell shortening; *P=0.01 for TR50). C, Examples of the fura-2 fluorescence ratio (after subtraction of the diastolic fluorescence) in nNOS–/– and wild-type myocytes field stimulated at 3 Hz (35°C). D, Average [Ca2+]i transient amplitude and tau in nNOS+/+ (n=30) (open bars) and nNOS–/– myocytes (n=30) (solid bars). *P=0.0001 for the [Ca2+]i transient amplitude; *P=0.005 for tau.


Figure 2
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  		Figure 2. Cell shortening and [Ca2+]i transients in voltage-clamped and fura-2–loaded myocytes from nNOS–/– and nNOS+/+ mice and nNOS+/+ myocytes after acute nNOS inhibition with L-VNIO (100 µmol/L). A and B, Representative raw data traces showing the fura-2 fluorescence ratio and the cell shortening elicited by a 25-ms depolarization pulse from –70 to +20 mV. TR50 and tau were compared after matching the amplitude of the [Ca2+]i transient between groups by decreasing [Ca2+]o to 0.9 mmol/L in nNOS–/– myocytes and after application of L-VNIO. C, Average data show that, under these experimental conditions, the tau of the [Ca2+]i transient and TR50 are still slower in the presence of nNOS gene deletion (nNOS–/– vs nNOS+/+: *P=0.02 for tau, n=10 and 15 myocytes; *P=0.01 for TR50; n=17 and 13 myocytes) or pharmacological inhibition (nNOS+/+ vs nNOS+/+ in the presence of L-VNIO: *P=0.003 for TR50, n=13 and 10 myocytes; *P=0.04 for tau, n=15 and 9 myocytes).

Effect of Acute nNOS Inhibition on LV Myocyte Relaxation and [Ca2+]i Decay
Our findings suggest that nNOS-derived NO may be an important determinant of myocardial relaxation; however, we cannot exclude that compensatory mechanisms resulting from chronic and systemic disruption of nNOS may play a part in the myocardial phenotype of these animals or that absence of PDZ-containing nNOS11 may affect macromolecular complexes involving Ca2+-handling proteins.15 To address these issues, we tested the effect of acute pharmacological inhibition of nNOS with L-VNIO (100 µmol/L) in LV myocytes from nNOS+/+ mice. We have previously shown that intracellular dialysis of L-VNIO in wild-type LV myocytes increases ICa density and cell shortening to a level similar to that seen in nNOS–/– myocytes16; thus, to evaluate the effect of acute nNOS inhibition on TR50 and the rate of [Ca2+]i decay, we compared voltage-clamped and fura-2–loaded nNOS+/+ myocytes after matching the amplitude of [Ca2+]i transient by lowering [Ca2+]o in the presence of L-VNIO, as described above. As shown in Figure 2, nNOS inhibition significantly prolonged both the tau of the [Ca2+]i transient and TR50, confirming that myocardial nNOS-derived NO hastens relaxation by facilitating [Ca2+]i reuptake/extrusion.

Mechanism Underlying Prolonged [Ca2+]i Decay and Relaxation in nNOS–/– Myocytes
The rate of [Ca2+]i decay is determined by the speed at which [Ca2+]i is reuptaken into the SR or extruded through the plasmalemma, predominantly via the Na+/Ca2+ exchanger. To evaluate the contribution of the latter, we compared TR50 and the decay of the [Ca2+]i transient after disrupting SR function by using thapsigargin ([TG] 10 µmol/L) or caffeine (10 mmol/L for 10 s). Cell shortening remained greater in nNOS–/– myocytes after irreversible SERCA2a inhibition with TG (percentage of cell shortening at 3 Hz: 2.8±0.2 in nNOS–/– versus 1.9±0.2 in nNOS+/+; P=0.02, n=15 and 21 cells, respectively); however, under these conditions, TR50 no longer differed between nNOS–/– and nNOS+/+ myocytes (TR50 in ms: 89.5±3.9 versus 97.9±4.0 in nNOS+/+ myocytes; P=0.15). Similarly, the SR Ca2+ content (estimated by the amplitude of the caffeine-induced [Ca2+]i transient) was greater in nNOS–/– myocytes (F365/380 ratio: 0.69±0.04 versus 0.51±0.06 in nNOS+/+ myocytes; P=0.02; n=8 cells in each group), but the decay of the caffeine-induced [Ca2+]i transient was similar in nNOS–/– and nNOS+/+ myocytes (tau in seconds: 1.07±0.2 versus 0.97±0.7 in nNOS+/+ myocytes; P=0.6; Figure I in the online data supplement).

Taken together, these findings indicate that SR-independent regulation of [Ca2+]i decay and relaxation is not impaired in nNOS–/– myocytes and suggest that a decreased SERCA2a activity may underlie these findings. This could result from a reduction in SERCA2a expression or from increased inhibition of SERCA2a activity by PLN; the latter would be relieved on phosphorylation of PLN at a Ser16 and/or Thr17 sites.17

We found that the protein level of SERCA2a was unchanged in nNOS–/– mice (not shown), whereas total PLN was significantly reduced (Figure 3A). Furthermore, we observed a significant reduction in the PLN phosphorylated fraction in LV homogenates (Figure 3A and 3B; n=5 hearts per group) and in LV myocytes from nNOS–/– mice (supplemental Figure IIA), indicating that a greater proportion of the existing PLN exerts an inhibitory action on SERCA2a in the nNOS–/– myocardium.


Figure 3
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  		Figure 3. PLN phosphorylation is significantly reduced in nNOS–/– mice and nNOS+/+ mice after acute nNOS inhibition with SMTC. A, Representative immunoblots using antibodies specific for Ser16- or Thr17-phosphorylated PLN and total PLN (blotted after stripping the membrane) in LV homogenates from nNOS–/– and nNOS+/+ mice. B, Bar graphs showing the average PLN-phosphorylated fraction in both groups (n=5 hearts per group; *P=0.01). C, Effect of the nNOS-specific inhibitor SMTC on total and Ser16- and Thr17-phosphorylated PLN in LV homogenates from nNOS+/+ mice. D, Mean data showing a significant reduction in PLN phosphorylation in the presence of SMTC (n=3 hearts per group, *P=0.001).

Because it is difficult to extrapolate the combined effect on SERCA2a activity of a reduction in total PLN (facilitatory) associated with a lower PLN phosphorylated fraction (inhibitory), we evaluated total and phosphorylated PLN levels in the nNOS+/+ myocardium in the presence or absence of the nNOS inhibitor SMTC (100 nmol/L for 30 minutes). As expected, acute nNOS inhibition did not affect total PLN protein level but caused a significant reduction in PLN phosphorylation (Figure 3C and 3D). Because nNOS inhibition also elicited a significant prolongation of TR50 and tau in nNOS+/+ myocytes (Figure 2), these findings imply that reduced PLN phosphorylation plays an important part in the slower kinetics of [Ca2+]i decay and relaxation in nNOS–/– myocytes. In agreement with this idea, when differences in PLN phosphorylated fraction between groups (nNOS–/– versus nNOS+/+ LV myocytes or nNOS+/+ myocytes in the presence or absence of SMTC; supplemental Figure IIB) were abolished by β-adrenergic receptor stimulation (isoproterenol, 1 µmol/L), we no longer observed a difference in TR50 or tau between groups (Figure 4).


Figure 4
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  		Figure 4. β-Adrenergic stimulation abolished the difference in PLN phosphorylation, cell shortening, and TR50 between nNOS–/– and nNOS+/+ myocytes. A and B, The PLN-phosphorylated fraction was no longer different in nNOS–/– and nNOS+/+ mice in the presence of ISO (1 µmol/L; n=5 in both groups). C and D, ISO (1 µmol/L) significantly increased contraction and shorten relaxation in both nNOS–/– and nNOS+/+ myocytes (n=19 and 26, respectively; P<0.0001 for the effect of ISO). In the presence of ISO, cell shortening and TR50 did not differ between groups. *P<0.05 between nNOS–/– and nNOS+/+ myocytes under control conditions.

Whether modulation of PLN phosphorylation was the sole mechanism by which nNOS controlled myocardial relaxation was further tested by assessing the effect of acute nNOS inhibition (by extracellular application L-VNIO, 100 µmol/L) in field-stimulated LV myocytes from mice with targeted deletion of the PLN gene (PLN–/–). In contrast to our findings in nNOS+/+ myocytes, nNOS inhibition did not prolong TR50 or the rate of [Ca2+]i decay in PLN–/– myocytes (in ms, TR50: 26.1±1.2 in PLN–/– myocytes versus 24.3±1.2 in PLN–/– myocytes in the presence of L-VNIO; P=0.7, n=26 and 32 cells; tau, ms: 56.7±7.7 versus 62.5±6.2 with L-VNIO; P=0.5, n=16 and 18 cells).

Finally, we showed that eNOS gene disruption was not associated with a reduced PLN phosphorylated fraction (supplemental Figure IIC) or with a prolonged TR50,18 indicating that modulation of myocardial relaxation through changes in PLN phosphorylation is a nNOS-specific effect rather than the result of a general reduction in myocardial NO bioavailability.

Intracellular Signaling Underlying the Myocardial Phenotype of nNOS–/– Mice: Role of cGMP-Mediated Signaling and PKA Activity
NO activates soluble guanylyl cyclase (sGC) to produce cGMP, which in turn may signal through the cGMP-dependent PKG or by modulating PKA signaling via the cGMP-modulated phosphodiesterases of cAMP (PDE).19 We found that sGC inhibition by ODQ (oxadiazolo quinoxalin-1-one, 10 µmol/L) had no effect on TR50 in either groups (Figure 5), although it caused a significant increase in contraction in nNOS+/+ myocytes (by 21.3±8.7% in nNOS+/+ versus 1.3±3.8% in nNOS–/–; P=0.03 between groups). Similarly, inhibition of PKG (Rp-8-bromo-cGMP, 100 µmol/L; Figure 5) or of the cGMP-inhibited PDE3 (cilostamide, 1 µmol/L) had no significant effects on relaxation in nNOS–/– myocytes (in ms: 45.3±3.0 versus 46.2±2.9; P=0.8; n=8). Taken together, these findings indicate that cGMP signaling downstream of myocardial NO production has no effect on relaxation in murine LV myocytes.


Figure 5
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  		Figure 5. Inhibition of sGC and PKG does not affect myocardial relaxation in field-stimulated nNOS–/– myocytes. A, Inhibition of sGC with ODQ (10 µmol/L) did not affect relaxation in both nNOS+/+ and nNOS–/– myocytes (P=0.4, n=11 in nNOS+/+; P=0.8, n=14 in nNOS–/–). B, Similar results were obtained after PKG inhibition with Rp-8-bromo-cGMP (RP) (100 µmol/L) (P=0.5 in nNOS+/+; P=0.8 in nNOS–/–; n=18 in each group). TR50 is significantly prolonged in nNOS–/– myocytes before the intervention (*P<0.05 vs nNOS+/+).

In contrast, intracellular dialysis of the specific inhibitor of the PKA catalytic subunit, PKI (1 µmol/L) significantly prolonged TR50 in voltage-clamped nNOS+/+ myocytes but not in nNOS–/– myocytes (Figure 6A), thereby abolishing the differences in relaxation between genotypes. In agreement with these findings, PKA inhibition abolished the differences in PLN phosphorylation between groups by causing a reduction in PLN phosphorylation in nNOS+/+ myocytes only (Figure 6B).


Figure 6
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  		Figure 6. Inhibition of PKA prolongs relaxation and reduces PLN phosphorylation in nNOS+/+ myocytes under voltage-clamped conditions. A, Intracellular perfusion of the PKA inhibitor PKI (1 µmol/L) significantly prolonged TR50 in nNOS+/+ myocytes (P=0.02 for PKI vs control [Ctr]; n=17 and 13) but not in nNOS–/– myocytes (P=0.12; n=16 and 17; P<0.01 for the interaction between the effect of PKI on TR50 and the genotype). B, nNOS+/+ and nNOS–/– myocytes (n=4 hearts each) were incubated for 30 minutes with membrane-permeable PKI (14 to 22 amid, 1.7 µmol/L). PKI greatly reduced PLN phosphorylation in nNOS+/+ but did not affect PLN phosphorylation in nNOS–/– myocytes. *P<0.05.

Phosphorylation of troponin I and ryanodine receptor at PKA-targeted serine residues was also reduced in nNOS–/– mice or in nNOS+/+ mice after nNOS inhibition with SMTC (supplemental Figure III), indicating that nNOS disruption impaired PKA-dependent phosphorylation of several key components of excitation–contraction coupling. Recent evidence indicates that reactive oxygen species can influence the subcellular targeting of PKA in the myocardium.20 Because xanthine oxidoreductase (XOR) activity and oxidative stress have been found to be increased in the nNOS–/– myocardium,21,22 we tested the contribution of this mechanism to the differences in relaxation and PLN phosphorylation signaling between nNOS–/– and nNOS+/+ myocytes. We found that XOR inhibition with oxypurinol (100 µmol/L) did not change PLN phosphorylation in either group but significantly prolonged TR50 in both nNOS–/– and nNOS+/+ myocytes (supplemental Figure IV), indicating that increased XOR activity in the nNOS–/– myocardium is unlikely to account for the differences in PLN phosphorylation and relaxation between nNOS–/– and nNOS+/+ myocytes.

Protein Phosphatase Activity in the nNOS–/– Myocardium
Finally, we tested whether increased PP activity may account for the reduced PKA-mediated phosphorylation in nNOS–/– myocytes. The main PPs in the heart are serine/threonine phosphatases and of these the type1 (PP1) and 2A (PP2A) account for most of the myocardial PP activity.23 Although the protein level of PP1 or PP2A was similar in nNOS–/– and nNOS+/+ hearts (supplemental Figure V), total PP activity was significantly increased in nNOS–/– myocytes (Table). Incubation with okadaic acid (OA) (10 nmol/L, a concentration that preferentially inhibits PP2A23) resulted in a significant reduction in PP activity in both groups. Both the OA-sensitive and residual (largely PP1-dependent) PP activity were greater in nNOS–/– myocytes (Table).


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  		Table 1. Table. Phosphatase Activity in the Cytosolic and Particulate Fraction of nNOS–/– and nNOS+/+ LV Myocytes

To address whether differences in the activity of endogenous inhibitors of PP124 may contribute to the increased PP activity in nNOS–/– myocytes, we compared the PP1-inhibiting capacity of protein extracts from nNOS–/– and wild-type hearts. Our findings showed that PP1 inhibition by nNOS–/– extracts was significantly lower, resulting in a higher recombinant PP1 activity in nNOS–/– after the addition of cardiac protein extracts (in pmol/µg: min: 97.7±0.7 versus 91.5±1.3 in nNOS+/+; n=4 to 5 hearts; P<0.05). These differences were no longer present when cardiac extracts were saturated with PKA (not shown), mirroring our data on PLN phosphorylation and TR50 in the presence of isoproterenol (ISO) (Figure 4).

To investigate the functional impact of these findings, we evaluated the effect of PP inhibitors on PLN phosphorylation and TR50 in nNOS–/– and nNOS+/+ myocytes. Under patch-clamped conditions, PP2A and PP1 inhibition by intracellular perfusion of OA (10 nmol/L) and inhibitor 2 (I-2) (500 nmol/L), respectively, caused a significant reduction in TR50 in nNOS–/– but not in wild-type myocytes (Figure 7A). In agreement with these findings, PP1 and PP2A inhibition with a higher concentration of OA (2 µmol/L) significantly increased PLN phosphorylation in nNOS–/– but not in wild-type myocytes (Figure 7B). After PP1 or PP2A inhibition both speed of relaxation and PLN-phosphorylated fraction became significantly greater in nNOS–/– than in wild-type myocytes, suggesting that PP activity overrides PKA-mediated PLN phosphorylation in nNOS–/– mice.


Figure 7
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  		Figure 7. PP1 and PP2A inhibition shortened relaxation and increased PLN phosphorylation in nNOS–/– mice. A, Intracellular perfusion of OA (10 nmol/L) or I-2 (500 nmol/L) significantly shortened TR50 in nNOS–/– myocytes (P<0.0001 for OA vs control [Ctr], n=17 and 10; P<0.0001 for I-2 vs control, n=17 and 13) but not in nNOS+/+ myocytes (P=0.95 for OA vs control, n=7 and 10; P=0.10 for I-2 vs control, n=12 and 13; P<0.001 for the interaction between the effect of OA or I-2 on TR50 and the genotype). After PP inhibition, TR50 in nNOS–/– myocytes became significantly shorter than in wild-type myocytes. *P<0.05. B, After incubation with OA, the Ser16 PLN-phosphorylated fraction was greatly increased in nNOS–/– myocytes and became significantly higher than in their wild-type littermates (n=4 hearts in each group). *P<0.05.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study indicates that nNOS-derived NO regulates myocardial relaxation and the rate of decay of [Ca2+]i by modulating PKA-dependent phosphorylation of PLN via subcellular targeting of PP activity. This conclusion is supported by a number of novel findings: (1) the prolonged relaxation and slower rate of decay of the [Ca2+]i transient in nNOS–/– myocytes were not accounted for by differences in action potential duration or in SR-independent mechanisms of [Ca2+]i decay and were reproduced by acute nNOS inhibition. In contrast, nNOS-specific inhibitors had no effect on [Ca2+]i decay or relaxation in LV myocytes from PLN–/– mice; (2) both nNOS gene disruption and acute nNOS inhibition caused a significant reduction in the PLN phosphorylated fraction; these effects were specific for nNOS because eNOS gene deletion was not associated with any difference in PLN phosphorylation or TR50; (3) inhibition of sGC, PKG, or PDE3 did not elicit any change in relaxation in both nNOS–/– and nNOS+/+ myocytes, indicating that cGMP-mediated signaling downstream of myocardial NO production is not involved in the regulation of myocardial relaxation; (4) a reduction in PLN phosphorylation may result from either suppressed cAMP-PKA signaling or increased PP activity. We found that PKA inhibition abolished the differences between nNOS–/– and wild-type mice by decreasing PLN phosphorylation and prolonging TR50 only in nNOS+/+ myocytes. Conversely, PP1 or PP2A inhibitors significantly shortened TR50 and increased PLN phosphorylation in nNOS–/– but not in nNOS+/+ myocytes; and (5) PP1 and PP2A activity was significantly higher in nNOS–/– myocytes and the ability of nNOS–/– myocardial extracts to inhibit recombinant PP1 activity was significantly decreased, suggesting that a reduction in the expression or activity of endogenous PP1 inhibitors (eg, of inhibitor 1 and 2) may contribute to the impaired PLN phosphorylation in nNOS–/– mice.

Taken together, our findings identify a novel mechanism by which myocardial nNOS promotes LV relaxation by regulating the PKA-mediated phosphorylation of PLN and the rate of SR Ca reuptake via a cGMP-independent effect on PP activity.

nNOS-Mediated Regulation of SR Ca2+ Transport
Myocardial relaxation is largely initiated by a reduction of cytosolic Ca2+ resulting from the combined activity of SERCA2a and the Na+/Ca2+ exchanger and the plasma membrane Ca2+-ATPase; however, particularly in the mouse myocardium, the speed of myocyte relaxation is mainly determined by the activity of SERCA2a.25 Investigations of the effect of nNOS-derived NO on SERCA2a activity have yielded inconsistent results.6,10 Zhou et al have recently shown that both SR Ca2+-ATPase activity and 45Ca2+ uptake were decreased in SR microvesicles from nNOS–/– or eNOS–/– mice, suggesting that myocardial constitutive NO production may tonically stimulate SERCA2a activity.10 However, LV relaxation and [Ca2+]i decay are not prolonged in eNOS–/– myocytes, casting some doubt on the functional significance of these findings. Our data suggest that basal levels of nNOS-derived NO in the myocardium promote [Ca2+]i decay and relaxation by stimulating SR Ca2+ reuptake and indicate that regulation of SERCA2a activity by nNOS-derived NO is effected through changes in PLN phosphorylation. As shown previously,16 total PLN was significantly reduced in the myocardium of nNOS–/– mice, possibly reflecting an adaptive mechanism compensating for the reduced level of PLN phosphorylation in the nNOS–/– myocardium. As it would be difficult to tease out which of these changes (reduced total PLN or reduced PLN phosphorylated fraction) would have the greatest impact on SERCA2a activity and myocardial relaxation, we repeated these experiments in nNOS+/+ myocytes in the presence or absence of the nNOS-specific inhibitor L-VNIO. Acute nNOS inhibition slowed myocardial relaxation and [Ca2+]i decay and reduced PLN phosphorylation both at the Ser16 and Thr17 sites, in the absence of changes in total PLN. In contrast, L-VNIO did not affect the rate of myocyte relaxation in PLN–/– mice, in agreement with our data indicating that SR-independent mechanisms contributing to the rate of [Ca2+]i decay do not differ between nNOS–/– and wild-type myocytes. Taken together, these findings indicate that decreased PLN phosphorylation, by inhibiting SERCA2a activity, decreases the rate of SR Ca2+ reuptake and impairs relaxation in nNOS–/– myocytes.

It is important to point out that SERCA2a activity, by influencing SR Ca2+ content, is also a key determinant of contraction.17 However, despite reduced PLN phosphorylation and slower uptake of Ca2+ into the SR, LV myocytes from nNOS–/– mice show increased cell shortening and [Ca2+]i transients compared with their wild-type littermates, suggesting that their increased Ca2+ influx via the L-type Ca2+ current16 may override the effect of reduced SERCA2a activity on contraction. Indeed a similar "rescuing" action of an increased Ca2+ current on myocardial contractility has been reported in a mouse model overexpressing a nonphosphorylatable form of PLN.26

PKG-mediated phosphorylation of troponin I, leading to a reduction in myofilament Ca2+ sensitivity, has been shown to be a mechanism through which endothelial-derived NO may facilitate relaxation.2,3 Troponin I phosphorylation was decreased in the myocardium of nNOS–/– mice; however, the functional significance of this finding is unclear because PKG inhibition did not affect TR50 in nNOS–/– or wild-type myocytes. Furthermore, previous studies have indicated that myofilament Ca2+ sensitivity is either unchanged16 or decreased21 in nNOS–/– myocytes, suggesting that this mechanism is unlikely to contribute to impaired myocardial relaxation in these animals. Similarly, the functional significance of a reduced ryanodine receptor phosphorylation in the nNOS–/– mice or after nNOS inhibition is at present unclear, although this phenomenon may contribute to the ability of nNOS–/– mice to maintain their SR Ca2+ load.25

Regulation of PLN Phosphorylation by nNOS-Derived NO
In vivo, PLN can be phosphorylated at two sites, Ser16 and Thr17 by PKA and the Ca2+/calmodulin-dependent protein kinase, respectively.27 Both PP1 and to a lesser extent PP2A have been shown to be capable of dephosphorylating PLN.28 Furthermore, PP1 has been shown to be associated to the SR membrane from where it can regulate PKA-mediated phosphorylation and contribute to targeting the action of the kinase to specific substrates. However, PP2A inhibition, by increasing the activity of inhibitor-1 through phosphorylation of Thr35 has been shown to lead to PP1 inhibition,29 implying that the results we obtained with OA (Figure 7A and 7B) may also reflect a reduction in PP1 activity. Indeed, myocardial PP1 activity is under the control of endogenous inhibitors24; of these, inhibitor-1 is a potent and specific PP1 inhibitor (Ki=1.6 nmol/L) when it is phosphorylated by PKA, whereas I-2 (Ki=3.1 nmol/L) does not need to be phosphorylated to inhibit PP1. Myocardial extract enriched for inhibitor-1 and 230 showed a reduced PP1 inhibiting ability in the absence of nNOS, suggesting that the protein level and/or phosphorylation status of endogenous phosphatase inhibitors may be decreased in nNOS–/– hearts. Because differences in PP1 inhibition between nNOS–/– and wild-type mice were no longer present when cardiac extracts were saturated with PKA, our findings suggest that a reduction in basal inhibitor-1 phosphorylation may account for the increase in PP activity in nNOS–/– hearts.

Emerging evidence suggests that both NO and reactive oxygen species can regulate the activity of several phosphatases. For example, H2O2 has been shown to have a dose-dependent biphasic effect on PP2A activity in the pig cerebral cortex (ie, low concentrations of H2O2 enhance PP2A activity, whereas high concentrations inhibit it).31 As for most NO-mediated actions, the effect of NO on phosphatase activity may differ depending on the subcellular compartment and local redox environment. For instance, inhibition of nNOS in the cerebral cortex has been shown to prevent the hypoxia-induced decrease in phosphatase activity at the cellular membrane while suppressing the increase in phosphatase activity in the cytosol through a cGMP-independent mechanism.32 Although these examples are not directly pertinent to the regulation of PLN phosphorylation in the myocardium, they show that modulation of phosphatase activity by ROS or nNOS-derived NO is possible and may vary in different subcellular compartments. Taken together with recent data indicating that oxidant stress can directly activate PKA and cause the kinase to relocalize to specific subcellular domains,20 these findings indicate that protein phosphorylation can be regulated by the NO/redox state of the myocardium, both through the well-established cGMP-dependent effects on PDE activity19 and through a cGMP-independent regulation of PKA and PP activity leading to subcellular targeting and compartmentalization of cAMP-mediated signaling.

Conclusions
Increasing evidence indicates that nNOS plays an important role in the regulation of basal and β-adrenergic myocardial function in health and disease.33 Our findings reporting that nNOS-derived NO regulates myocardial relaxation and intracellular Ca2+ decay by promoting PKA-mediated PLN phosphorylation uncover a further important target in the downstream signaling pathway of nNOS-derived NO. This mechanism may play an important part in the protective role of myocardial nNOS in infarcted hearts.8,34


*    Acknowledgments
 
We thank Dr Luca Cravello for technical help with some of the experiments.

Sources of Funding

This work was supported by a Programme grant from the British Heart Foundation (to B.C.). A.E.-A. is supported by a grant from DFG-FOR604 and by EUGene Heart.

Disclosures

None.


*    Footnotes
 
Presented in part at the 78th Scientific Sessions of the American Heart Association, Dallas, Tex, November 13–16, 2005, and published in abstract form (Circulation 2005;112[suppl II]:II-308).

*The first two authors contributed equally to this work. Back

Original received April 6, 2006; first resubmission received April 17, 2007; second resubmission received September 24, 2007; accepted November 1, 2007.


*    References
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