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Up-regulation of cardiac nitric oxide synthase 1-derived nitric oxide after myocardial infarction in senescent rats¹

THIBAUD DAMY^*, PHILIPPE RATAJCZAK^*, ESTELLE ROBIDEL^*, JENNIFER K. BENDALL^*, PATRICIA OLIVIÉRO^*, JORGE BOCZKOWSKI, TALIN EBRAHIMIAN, FRANÇOISE MAROTTE^*, JANE-LISE SAMUEL^* and CHRISTOPHE HEYMES^*,2

^* INSERM U572, Hôpital Lariboisière, IFR J. Marrey Paris-7, Université D. Diderot, Paris, France;
INSERM U408, Hôpital Bichat, Paris, France; and
INSERM U541, Hôpital Lariboisière, IFR J. Marrey Paris-7, Université D. Diderot, Paris, France

²Correspondence: INSERM 572, Hôpital Lariboisière, 41 Boulevard de la Chapelle, 75475 Paris Cedex 10, France. E-mail: Christophe.Heymes{at}larib.inserm.fr

SPECIFIC AIMS

Despite much investigation, the source and role of endogenous nitric oxide (NO) production in the regulation of cardiac contractility in the failing heart remain controversial. Although recent evidence indicates that a type 1 NO synthase (NOS1) may modulate myocardial contraction, the regulation and role(s) of NOS1 in this setting remain to be determined. Because myocardial infarction is the most common cause of heart failure in people over 65 years, the aims of the present study were to investigate whether 1) cardiac NOS1 expression is enhanced in an experimental model of myocardial infarction (MI) in senescent rats, 2) its activity is increased, and 3) if this is associated with cardiac contractile dysfunction.

PRINCIPAL FINDINGS

1. NOS1 protein expression and activity are increased after MI in senescent rats
Three months after myocardial infarction in 24-month-old rats (S-MI), NOS2 protein expression and activity were undetectable in all groups of rats. As expected, quantitative Western blot analysis demonstrated a significant decrease in NOS3 protein expression in S-MI rat hearts [P<0.001 vs. age-matched rats (S-Sh), n=14 per group], without any change in its endothelial immunolocalization. However, cardiac NOS activity, assayed by the formation of ³H-L-citrulline, was similar in S-MI and S-Sh groups and inhibited equally by the nonselective NOS inhibitor L-NAME. We hypothesized that the apparent discrepancy between the decreased expression of NOS3 and the maintenance of NOS activity in the S-MI myocardium could be due to the increased expression and/or activity of NOS1. Indeed, Western blot analysis demonstrated a twofold increase in NOS1 expression in S-MI hearts compared with S-Sh animals (P<0.001). Calcium-dependent NOS activity in the S-MI group was dramatically inhibited (>80%) by the preferential NOS1 inhibitor SMTC (P<0.05 vs. S-Sh, Fig. 1 ).

View larger version (14K): [in this window] [in a new window]   Figure 1. Calcium-dependent NOS activity in heart extracts evaluated by the [3H]L-arginine* to [3H]L-citrulline* conversion assay in the presence of either SMTC, the preferential NOS1 inhibitor (A) or L-NAME, a nonselective NOS inhibitor (B). Each inhibitor was used at concentrations ranging from 10-15 to 10-3 mol/L in S-Sh (, n=6) and S-MI (, n=6) groups. Level of activity was expressed as % of the respective basal activity. *P <0.05 vs. S-Sh.

2. Possible role of HSP90 in mediating the increase in NOS1 activity after MI
NOS1 activity has been shown to be regulated post-translationally via its stimulatory interaction with the chaperone protein HSP90 in cultured cells. Western blot analysis showed a significant increase in HSP90 protein expression in LV extracts from S-MI compared with S-Sh hearts (P<0.05, n=6 per group). Moreover, coimmunoprecipitation studies demonstrated an increase in the interaction between HSP90 and NOS1 in Triton X-100 lysates of S-MI hearts compared with the S-Sh group.

3. MI is associated with NOS1 translocation to the sarcolemma
Immunofluorescence revealed a linear intracellular pattern of NOS1 expression in senescent cardiomyocytes, both longitudinal and transverse, consistent with previous data for antigens localized to the longitudinal and junctional sarcoplasmic reticulum (SR) (Fig. 2 A). A unique NOS1 localization appeared at sarcolemma and T-tubule levels in S-MI cardiomyocytes, as corroborated in sections costained for NOS1 and vinculin as a sarcolemmal marker (Fig. 2A , d–f). To further assess NOS1 translocation to the sarcolemma, we investigated the in situ NOS1 physical interaction with its scaffolding protein, caveolin-3 (Cav-3, as described previously in skeletal muscle), using coimmunoprecipitation. As shown in Fig. 2B , Cav-3 from OG lysates of S-Sh and S-MI hearts coprecipitated with NOS1, the amounts of NOS1/Cav-3 complexes being dramatically increased in the S-MI group. The reverse experiment (i.e., coimmunoprecipitation of NOS1 associated with Cav-3 Western blot) confirmed this finding. Taken together, these data demonstrate that cardiac NOS1is translocated to the sarcolemma, where it interacts with Cav-3 after MI in aging rats.

View larger version (111K): [in this window] [in a new window]   Figure 2. A) Immunolocalization of NOS1 (a, d), vinculin (b, e), and merged images of NOS1 and vinculin labels (c, f) in S-Sh (a–c) and S-MI (d–f) heart cryosections. Inserts show transverse sections of cardiomyocytes in S-MI hearts stained respectively with antibodies for NOS1 and vinculin (x300). B) Association between NOS1 and caveolin-3 (Cav-3). LV lysates were immunoprecipitated with a polyclonal antibody against NOS1 or a monoclonal antibody against Cav-3. Immunoprecipitates (IPs) were Western blotted (IB) for NOS1 and Cav-3, respectively. +Positive control.

4. Role of NOS1-derived NO production in post-MI LV dysfunction
In a subset of S-Sh and S-MI rats, an intra-abdominal aortic bolus dose of the preferential NOS1 inhibitor SMTC (0.5 mg/kg) was injected and cardiac pressures were continuously measured. The time constant of ventricular relaxation () was determined in vivo by the derivative method. Inhibition of NOS1 by SMTC significantly increased in the S-MI group (P<0.001, n=3) but induced no change in the S-Sh group (P=0.29, n=3). Similarly, NOS1 inhibition significantly increased peak LV dP/dt_max in the S-MI group (P<0.05, n=3) but caused no change in the S-Sh group (P=0.3, n=3).

CONCLUSIONS AND SIGNIFICANCE

The present study demonstrates that 1) NOS1 is up-regulated within cardiomyocytes and is in part redistributed to the sarcolemma after MI through its interaction with Cav-3, and 2) in vivo inhibition of NOS1 activity enhanced basal post-MI left ventricular dysfunction in senescent rats. These results confirmed and extended previous observations indicating that NOS1 1) is expressed in the cardiac myocytes of both adult and senescent rat hearts and 2) exhibits a major redistribution toward the sarcolemma and T-tubules after MI. Both the expression and specific activity of NOS1 were shown to increase post-MI. On the other hand, NOS3 immunostaining was detected only in the endothelium of myocardial coronary vessels. Although this result agrees with many previous reports, it differs from others showing that cardiac myocytes indeed express NOS3. According to data obtained in dogs and humans, we found that NOS3 expression and activity dramatically decreased in the failing senescent heart. These results are consistent with the pathogenesis of endothelial dysfunction associated with HF.

Our data indicate that myocardial NO derived from NOS3 and NOS2 is not likely to be involved in the deteriorating hemodynamic status of the failing heart either at baseline or after stimulation. Previous evidence is inconclusive: Gyurko et al. found an enhanced LV dP/dt_max in response to ß-adrenergic stimulation in NOS3^-/- mice, whereas Vandecasteele et al. found no change in either calcium current or papillary muscle contractility in the same murine model. Similarly, studies in NOS2^-/- mice produced contradictory results. However, the use of nonspecific NO blockers in humans or isolated guinea pig hearts indicated that cardiac NO altered cardiac contractility. An explanation for these conflicting findings may be related to the discovery of NOS1 on the cardiac SR. Using an in vivo approach, we demonstrated that myocardial contractility is indeed significantly increased when NOS1 activity is blocked by SMTC after MI in senescent rat hearts. Relaxation was mainly affected since significantly increased in post-MI senescent hearts treated with SMTC. These results support and extend those of Ashley et al. in isolated cardiomyocytes incubated with a NOS1 inhibitor or obtained from NOS1^-/- mice. Taken together, it emerges that basal NOS1-derived NO is important in the regulation of ventricular relaxation after MI in aging rats.

Multiple changes in downstream signaling pathways may be involved in the effects of NOS1-derived NO on cardiac contractility. For example, elevated cGMP levels may induce 1) cAMP phosphodiesterase, thereby promoting degradation of cAMP, and/or 2) cGMP-activated protein kinase G, thereby blocking activation of sarcolemmal L-type calcium channels and resulting in decreased calcium influx. NO directly regulates the ryanodine-sensitive calcium channel on the SR. NOS1-derived NO could also affect relaxation in several ways; a tonic inhibition of SERCA2a remains possible, although such an effect should result in a faster relaxation in the presence of NOS1 inhibition.

To conclude, the present data together with findings from our laboratory regarding NOS1 during end-stage human HF (T.D., P.R, J.-L.S., C.H., unpublished results) indicate that NOS1-derived NO in myocardial dysfunction during HF could be of particular physiological relevance.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram of NOS1 location and potential action underlying cardiac myocyte contractile dysfunction in heart failure. NOS1 is translocated to the sarcolemma through its interaction with Cav-3 in the failing myocyte, leading to an increase in intracellular NO production. Dotted lines indicate uncertain data regarding the intracellular pathways mediating NO-induced contractile dysfunction in heart failure.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1208fje; doi: 10.1096/fj.02-1208fje

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