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Circulation Research. 2001;88:97-102

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(Circulation Research. 2001;88:97.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Upregulation of the Nitric Oxide–cGMP Pathway in Aged Myocardium

Physiological Response to l-Arginine

Susan J. Zieman, Gary Gerstenblith, Edward G. Lakatta, Gisele O. Rosas, Koenraad Vandegaer, Kelly M. Ricker, Joshua M. Hare

From the Department of Medicine (S.J.Z., G.G., G.O.R., K.V., K.M.R., J.M.H.), Cardiology Division, Johns Hopkins Medical Institutions, and Gerontology Research Center (E.G.L.), National Institute on Aging, Baltimore, Md.

Correspondence to Joshua M. Hare, MD, Cardiology Division, Johns Hopkins Hospital, 600 N Wolfe St, Carnegie 568, Baltimore, MD 21287-6568. E-mail jhare{at}mail.jhmi.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—Cardiovascular aging is associated with decreased endothelial vasoreactivity and prolonged diastolic relaxation. As diminished NO signaling contributes to age-associated endothelial dysfunction, we tested the hypothesis that impaired NO signaling or bioactivity also contributes to slowed ventricular relaxation with age. Accordingly, we measured myocardial NO synthase (NOS) enzyme activity, protein abundance, and cGMP production in old (22 to 25 months) and young adult (4 to 7 months) male Wistar rats. Both NOS3 protein abundance and calcium-dependent NOS activity were elevated in old compared with young adult hearts (7.2±1.1 versus 4.2±0.6 pmol/mg protein, respectively, P=0.03). However, NOS activity and protein abundance were similar in isolated myocytes, indicating that endothelial NOS likely explains the age difference. Cardiac effluent cGMP (enzyme immunoassay) was 4.8-fold higher (1794±373 fmol/min per mg heart tissue) in older versus younger hearts (P=0.003). To assess NO pathway responsiveness, we administered the NOS substrate l-arginine (100 µm) to isolated perfused rat hearts. Baseline isovolumic relaxation ({tau}) was prolonged in old (42.9±2.5 ms, n=16) versus young hearts (36.0±1.9 ms, n=11, P=0.03). l-Arginine decreased {tau} (P<0.001) and left ventricular end-diastolic pressure in both old and young hearts. Supporting an NO/cGMP-mediating mechanism, the NO donor sodium nitroprusside reduced {tau} (maximal effect, -14±2%, n=5, P<0.001), and this lusitropic effect was attenuated by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3,-a]quinoxalin-1-one (n=7, P<0.001). Thus, the NO-cGMP pathway is upregulated in the endothelial cells of aged hearts. l-Arginine, the NOS precursor, enhances ventricular relaxation in old and young hearts, indicating that the NOS pathway may be exploited to modulate diastolic function in aged myocardium.


Key Words: aging • cGMP • nitric oxide synthase • diastole • l-arginine


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Normal cardiovascular aging is associated with decreased endothelium-mediated vasodilation and slowed and delayed early ventricular relaxation.1 2 3 4 These changes may increase the likelihood for the development and progression of vascular disease and compromise hemodynamics during tachycardic stresses including exercise.4 5 6 The age-associated decrease in vascular responsiveness suggests impaired vascular NO/endothelium-mediated signaling.7 8 9 10 More direct biochemical evidence indicates a significant age-associated decrease in the activity of NO synthase 3 (NOS3), the enzyme that catalyzes the production of NO, in rat aortae.10

In addition to vascular function, NO also influences myocardial function via autocrine11 and/or paracrine effects.12 13 By activating guanylyl cyclase, NO promotes myocyte relaxation via enhanced cGMP production.14 In animal models, increasing NO by endogenous stimulation with substance P or bradykinin or by administering NO donors enhances both active and passive diastolic relaxation.15 16 17 Paulus et al12 18 reported that intracoronary sodium nitroprusside (SNP) or substance P administration promotes left ventricular (LV) diastolic relaxation and end-diastolic distensibility in humans. Shah et al14 19 achieved similar lusitropic effects with cGMP analogues, further supporting an intermediary role of this nucleotide in mediating myocardial relaxation.

We hypothesized that impaired myocardial NO/NOS bioactivity or signaling contributes to the age-associated slowing of myocardial relaxation. To test this hypothesis, we measured the NOS activity, NOS3 protein abundance, and cGMP concentrations in the myocardium of Wistar rats, a well-established model for cardiac aging.4 5 Moreover, we located the source of cardiac NOS by measuring the activity and protein abundance of this enzyme in myocytes isolated from old and young rat hearts. The results demonstrate that, counter to our hypothesis, NO-cGMP signaling is substantially elevated in aged myocardium. We further tested whether the NO-cGMP pathway could be used to enhance diastolic performance by measuring the influence of l-arginine, the NOS substrate, on isovolumic relaxation in old and young adult rat myocardium.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Animal Model
Wistar male rats, a well-established model of cardiovascular aging demonstrating age-associated changes in vascular and myocardial function similar to those in humans,4 5 were bred at the Gerontology Research Center of the National Institute on Aging. We studied aged animals (n=42), defined as 22 to 25 months old, the age at which there is 50% mortality of the colony, and young adults (n=33) 4 to 7 months old and having reached sexual maturity.4 Additional physiological studies were performed in adult male Wistar rats that were 10 to 11 months old (n=16).

Measurement of Myocardial NOS Enzyme Activity and Protein Abundance
NOS activity was measured in pulverized hearts from old (n=9) and young adult (n=7) rats by l-[14C]arginine–to–l-[14C]citrulline conversion assay as previously described.20 21 NOS3 protein abundance was measured by Western blot as described in total heart tissue from old and young (n=2 each) Wistar rats using monoclonal NOS3 antibody (Transduction Laboratories).20 To further delineate the location of the cardiac NOS3 protein, NOS activity was measured by l-[14C]arginine–to–l-[14C]citrulline conversion assay in myocytes isolated by collagenase digestion from hearts of old and young rats (n=4 each).22 23 In addition, NOS3 protein abundance was measured by Western blotting as described above in both the cytosolic and particulate (membrane) fractions of the isolated myocytes, separated by centrifugation at 100g for 60 minutes, from old and young adult hearts (n=2 each).

Cardiac Effluent and Tissue cGMP Levels
Quantitative assays for cGMP from both isolated heart effluent and cardiac tissue were performed using a commercial enzyme immunoassay kit (Amersham Pharmacia Biotech, Piscataway, NJ). Effluent solution was collected from isolated retrograde-perfused rat hearts (n=11 old and n=10 young). For myocardial cGMP content, frozen heart tissue (n=6 old and n=5 young) was homogenized in 6% trichloroacetic acid (1 mL trichloroacetic acid/100 mg tissue), centrifuged, and extracted with water-saturated diethyl ether as previously described.24 The aqueous layer was vacuum dried at -60°C and resuspended in sodium acetate buffer.

Physiological Assessment: Isovolumic Relaxation
The physiological impact of the cardiac NO/NOS signaling pathway on isovolumic relaxation, measured as {tau},25 was assessed in isolated retrograde perfused hearts from old (n=16) and young (n=11) rats by infusing l-arginine (100 µmol/L), the NOS substrate.26 This concentration of l-arginine is similar to that shown to improve endothelial function and symptoms of angina pectoris in humans.27 The specificity of l-arginine was tested by infusing d-arginine (100 µmol/L) in additional identically prepared hearts from adult animals (10 to 11 months old, n=4). Excised hearts were perfused with fixed coronary blood flow to avoid performance changes related to the Gregg effect28 and paced at constant heart rate as previously described.29 Care was taken to assure that each isolated heart was functioning within 90% of its peak Frank-Starling curve by titration of balloon volume. The coronary perfusion pressure and LV pressures were monitored continuously. Digitized data were analyzed to determine the rate of rise of LV pressure (peak +dP/dt) and the time course of isovolumic relaxation ({tau}; method of Weiss et al25 ). Hearts were allowed to stabilize for 15 minutes before baseline physiological parameters were recorded. Then, after a 15-minute infusion of the NOS substrate l-arginine (100 µmol/L) or its inactive isomer d-arginine (100 µmol/L), baseline parameters were rerecorded.

To further explore the hypothesis that observed lusitropic responses were related to NO/cGMP signaling, we infused the NO donor SNP (10-10 to 10-7 mol/L) to additional isolated perfused rat hearts from male animals (10 to 11 months old, n=5). SNP infusions (10-8 to 10-6 mol/L) were also performed in hearts pretreated with the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3,-a]quinoxalin-1-one (ODQ, 10 µmol/L, n=7). The Johns Hopkins University School of Medicine Animal Care and Use Committee approved all animal protocols.

Statistical Analysis
Comparisons between old and young adult rats were made using unpaired Student t tests, whereas comparisons before and during l-arginine and d-arginine were made using paired Student t tests. Concentration-effect responses to SNP with and without ODQ were analyzed using 2-way ANOVA. Results are presented as the mean±SEM.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
NOS Activity: Biochemical Assessment
Calcium-dependent NOS activity was 7.2±1.1 pmol/mg heart protein in old (n=9) compared with 4.2±0.6 in young (n=7, P=0.03, Figure 1ADown) adult rat hearts. The increased NOS activity with age was associated with a 40% increase in NOS3 protein abundance in heart extract from old compared with young adult rats (Figure 1BDown, n=2 each). In contrast to whole-heart extracts, there was no difference in calcium-dependent NOS activity from isolated, purified myocytes (0.52±0.23 pmol/mg protein in old versus 0.53±0.19 in young adult rats [n=4 each, P=NS]). Furthermore, there were no differences in NOS3 protein abundance (detected only in the membrane fraction) in isolated myocytes from hearts of the 2 age groups (Figure 1CDown), suggesting that the age-associated increased NOS3 is of endothelial, rather than of myocyte, origin. Whereas calcium-independent NOS (NOS2) activity was not detectable in old hearts, the activity was 0.42±0.09 pmol/mg protein in young adult hearts.



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  		Figure 1. Calcium-dependent NO activity and NOS3 protein abundance are elevated in old compared with young adult myocardium. A, Calcium-dependent myocardial NOS activity in hearts from old (22 to 25 months, n=9, solid bars) and young adult (4 to 6 months, n=7, hatched bars) Wistar rats. Results are expressed as mean±SEM per mg of heart extract protein, *P<0.05. B, Western blot demonstrating myocardial NOS3 protein abundance from heart extracts of old and young adult (n=2 each) Wistar rats using monoclonal NOS3 antibody. NOS3 protein abundance is increased in heart extracts from the older animals. C, Western blot of membrane and cytosolic fractions of myocytes isolated from a heart of an old (24 months) and a young adult (6 months) Wistar rat demonstrating that myocardial NOS3 protein abundance is restricted to the membrane fraction (Memb) and does not appear in the cytoplasmic fraction (Cyt). + Represents positive control.

Consistent with the age-associated increase in NOS3 activity, the level of cGMP in cardiac effluent was 1794±373 fmol/min per mg tissue in hearts from old animals (n=11) and 375±58 in hearts from young animals (n=10, P=0.003, Figure 2Down). Finally, concentrations of cGMP within heart tissue extracts were higher in the old animals (9.7±1.1 pmol/g, n=6) than in the young animals (6.7±0.7 pmol/g, n=5, P=0.05).



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  		Figure 2. Tissue and heart effluent cGMP concentrations are elevated in old compared with young adult rats. A, Levels of cardiac effluent cGMP from hearts isolated from old (n=11, solid bars) and young adult (n=9, hatched bars) Wistar rats. B, Tissue cGMP levels of hearts isolated from old (n=6, solid bars) and young adult (n=5, hatched bars) Wistar rats. Results are expressed as mean±SEM. *P<0.05.

NOS Pathway Activity: Physiological Response to l-Arginine
Despite no age differences in mean body weight (537±19 g in the older and 507±19 g in the younger rats), the mean heart weight of older rats was 2.39±0.12 g compared with a mean of 1.61±0.04 g in those from the young adults (P<0.0005). This increase in heart weight–to–body weight ratio with increasing age is consistent with previous reports of age-associated cardiac hypertrophy in this model.5 Additionally, the mean ventricular balloon was 0.44±0.03 mL in hearts from aged animals compared with 0.34±0.02 mL in young adults (P<0.005).

Table 1Down depicts baseline physiological parameters of isolated hearts from old (n=16) and young adult (n=11) rats in the absence and presence of l-arginine. {tau} was 42.9±2.5 ms in old hearts and 36.0±1.9 ms in young adult hearts (P=0.03, Figure 3Down) at matched LV end-diastolic pressures (LVEDPs). During the infusion of l-arginine, {tau} shortened to 33.0±2.3 ms in old (P<0.0001) and to 29±2.4 ms in young (P=0.005) adult hearts (Figure 3Down, Table 1Down). In the presence of l-arginine, {tau} was no longer elevated in old compared with young hearts. LVEDP, which was similar at baseline in old and young adult hearts (Table 1Down), was also reduced by l-arginine despite fixed LV end-diastolic balloon volume. The magnitudes of the reduction from baseline, 7.9±1.9 mm Hg in old (P<0.01) and 6.6±2.1 in young adult (P<0.01) hearts, were similar in the 2 age groups (Table 1Down). Peak +dP/dt was lower in older than in younger adult hearts and was not altered by l-arginine in either age group (Table 1Down). In contrast to l-arginine, infusion of d-arginine did not change {tau} (39.5±0.5 and 39.2±0.4 ms, before and after d-arginine, respectively, P=NS) or LVEDP (24.7±0.3 and 25.0±0.7 mm Hg before and after d-arginine, respectively, P=NS) when infused into hearts isolated from adult rats (10 to 11 months old, n=4).


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  		Table 1. Baseline Physiological Parameters From Isolated Retrograde Perfused Hearts From Old (22 to 24 Months) and Young Adult (4 to 6 Months) Rats in the Absence and Presence of L-Arginine



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  		Figure 3. Positive lusitropic effects of l-arginine in old and young hearts. Time constant of isovolumic relaxation, {tau}, in retrograde-perfused hearts isolated from old (n=16, •) and young adult (n=11, {blacksquare}) rats expressed as mean±SEM. *P<0.05, old vs young. Myocardial relaxation is shown at baseline in the absence (-) of l-arginine and then during l-arginine infusion (+) in hearts from old and young adult animals. {dagger}P<0.05, -l-arginine vs +l-arginine.

Similar to the response to l-arginine, the NO donor SNP (10-10 to 10-7 mol/L) shortened {tau} and reduced LVEDP (Table 2Down and Figure 4Down). In contrast, after pretreatment with the guanylyl cyclase inhibitor ODQ, the lusitropic effect of SNP, reflected by the change in both {tau} and LVEDP, was attenuated (Figure 3Up). These data provide additional evidence that the lusitropic response to l-arginine is likely due to activation of the NO and/or cGMP pathways.


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  		Table 2. Cardiac Response to SNP (10-7 mol/L) Alone and in the Presence of ODQ (10-5 mol/L) in Isolated Perfused Rat Hearts



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  		Figure 4. Impact of SNP on {tau} in the presence and absence of guanylyl cyclase inhibition. Shown is percentage change in {tau} in response to increasing concentration of SNP infusion ({blacksquare}, n=5, *P=0.05 vs baseline). Lusitropic response to SNP is attenuated by pretreatment with infusion of the guanylyl cylase inhibitor ODQ (•, n=7). *P=0.05 vs baseline, {dagger}P=0.02 with and without ODQ. Data are mean±SEM.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study, we sought to extend observations regarding aging vasculature to ventricular myocardial function. In the vasculature, reduced NOS activity likely contributes to decreased endothelium-dependent vasoreactivity with increasing age.7 8 9 10 Accordingly, we tested the hypothesis that diminished NO contributes to prolonged diastolic relaxation in aged myocardium. Our results demonstrate that rat myocardial NOS activity is not only intact, but upregulated with age. Specifically, calcium-dependent NOS activity and protein abundance were elevated in hearts from older animals, and these increases were of endovascular, rather than of myocyte, origin. Furthermore, cGMP concentrations in both myocardial tissue and cardiac effluent were increased with age. The NOS precursor l-arginine induced a potent lusitropic effect in both old and young adult rat hearts, eliminating the baseline age-related prolongation in isovolumic relaxation. Thus, cardiac NOS activity is elevated with increasing age and provides a potential target for modulating ventricular relaxation.

Cardiac NOS Abundance and Activity
Our biochemical findings of elevated cardiac NOS are consistent with those of Cernadas et al,10 who reported an age-associated increase in the expression of NOS2 and NOS3 and cGMP levels in the aortae of Wistar rats. Yet, despite this increased NOS expression, both NOS3 activity and the vasodilator response to NO-dependent agonists were diminished in their study, possibly explaining the age-associated decrease in endothelial function. In our study, NOS3 protein abundance and enzyme activity were upregulated in aging hearts. Other settings in which increased myocardial NOS expression and activity are reported are heart failure and sepsis.30 31 32 However, an important difference relates to the upregulated NOS isoform; NOS2 activity33 and expression32 are increased in heart failure and sepsis.

The mechanism for increased cardiac NOS3 in aging is unknown. Unlike NOS2, which is activated by cytokines and other inflammatory factors, NOS3 expression can be enhanced by vascular shear stress,34 35 which could be elevated in aging as a result of increased vascular stiffening and/or raised pulse-pressure.6 In aging, increased NOS3 expression may also reflect the known age-associated decrease in cAMP, a nucleotide that can suppress NOS3 gene expression.36 In terms of the elevated concentrations of cGMP measured in both effluent and myocardial tissue, this likely reflects the observed increase in NOS activity. There are, however, other possible contributory factors, including the previously described increased levels of atrial natriuretic peptide, an agonist of receptor guanylyl cyclase.37

Our finding that elevated NOS resides primarily in the endothelium of aged hearts is consistent with paracrine NO signaling in the heart.12 Similarly, Gyurko et al38 have reported that the majority of cardiac NO signaling originates from endothelial cells. However, the upregulation of endothelial cell NO-cGMP may serve roles in addition to cardiac paracrine signaling. For example, NO has been shown to inhibit endothelial cell apoptosis by activating telomerase.39

NO and Diastolic Performance
As l-arginine has been shown to improve endothelial reactivity in aged vasculature,8 we sought to determine whether this NOS precursor could promote cardiac lusitropy. l-Arginine enhanced early ventricular relaxation in both old and young hearts, as demonstrated by a reduction in {tau}, and increased chamber compliance as indicated by reduced LVEDP at constant LV end-diastolic volume. In contrast, no change in {tau} or LVEDP was present after infusion of the inactive isomer d-arginine. The mechanism by which l-arginine enhances NO signaling despite adequate intracellular levels of this substrate remains controversial40 but has been widely documented in the setting of endothelial reactivity (see Reference 2727 for example). The specific contribution of the NO-cGMP signaling pathway to enhanced lusitropy is further supported by our findings that the NO donor SNP, like l-arginine, accelerates isovolumic relaxation and enhances LV diastolic compliance and that these responses are blunted by the soluble guanylyl cyclase inhibitor ODQ.

In the current study, we did not investigate potential alterations in l-arginine availability that could lead to decreased NO production. Among the possibilities are changes in l-arginine transport41 and/or an increase in levels of endogenous NOS inhibitors with aging, which can be overcome with increased l-arginine.26 Consistent with the latter theory, Miyazaki et al42 reported an age-associated increase in plasma levels of one such inhibitor, asymmetrical dimethylarginine, in humans. Another important consideration is that certain NO actions may be inhibited by oxidative inactivation, as reported in cholesterol-exposed vessels.43 Yet, regardless of any age-associated alterations in precursor availability/metabolism or downstream NO inactivation, l-arginine administration appears to overcome them. Thus, the response to l-arginine may have important therapeutic implications, as enhanced early diastolic relaxation may increase older individuals’ ability to exercise and may lessen the hemodynamic consequences of tachycardic and hypertensive stresses.6

Initially, we considered the possibility that alterations in NO-cGMP signaling contribute to age-associated changes in early diastolic relaxation, as NO has positive lusitropic properties.12 15 16 17 18 44 However, the fact that aged myocardium has delayed diastolic relaxation in the presence of upregulation of the NOS-cGMP pathway suggests that other factors are responsible for the prolongation of isovolumic relaxation, measured as {tau}. In this regard, prolonged intracellular calcium currents, due in part to downregulation of the sarcoplasmic reticulum Ca2+-ATPase,45 likely contribute.4 5 Increased cGMP may decrease the functional consequences of this age effect by reducing myofilament sensitivity to calcium and/or by antagonizing the stimulatory effects of cAMP on L-type calcium channels.13 14 46 47 48 It is attractive to speculate that early diastolic relaxation in aged myocardium would be even slower in the absence of elevated NOS activity and that increased NOS activity in aged myocardium serves an adaptive function.

NO in the Aging Cardiovascular System
NO may have other adaptive roles in the aging cardiovascular system. Cardiac output in older individuals during exercise stress is more dependent on increased stroke volume mediated by increased LV end-diastolic volume than in younger subjects,49 50 51 and NO is known to enhance the Frank-Starling response.52 The findings of Pinsky et al53 that cardiac NO levels rise in response to increases in ventricular preload further supports a physiological link between NO and the Frank-Starling mechanism.

There are some limitations of our study that warrant mention. First, as our studies were performed in male animals, important gender-related alterations could not be assessed. Future studies will address the potential effects of estrogen (or lack thereof) on NOS signaling with age.54 Second, our experiments were performed in a crystalloid perfused preparation that has the potential to either enhance or diminish NO-related signaling55 ; further studies will be required to confirm the response to l-arginine in blood-perfused models. Additionally, future investigations will focus on the histopathological assessment of vasculature and perivascular and interstitial regions, which are likely to be altered in older humans and/or disease states and thus may limit the potential benefit of l-arginine.56

In conclusion, we demonstrate that adult rats of advanced age exhibit increased myocardial NOS-cGMP signaling associated with increased NOS3 protein abundance. This upregulation, primarily present in cardiac endothelial cells, may be physiologically modulated to enhance ventricular relaxation by the administration of l-arginine. This response may have both physiological and therapeutic implications in the management of older individuals with disease processes associated with increased ventricular stiffness.


*    Acknowledgments
 
This work was supported by NIH Grant K08 HL-03238 and a Grant-in-Aid from the American Heart Association (to J.M.H.) and by the National Institute on Aging Intramural Research Program. J.M.H. is a recipient of a Paul Beeson Physician Faculty Scholar in Aging Research Award. S.J.Z. is supported by Grant NIH-T32-HL-07227-24, and G.G is supported by Grant NIH-AG-98-07. We are indebted to Dr Harold Spurgeon (Gerontology Research Center, National Institute on Aging) for animal handling and technical advice.


*    Footnotes
 
Original received June 14, 2000; revision received November 7, 2000; accepted November 7, 2000.

This manuscript was sent to Richard A. Walsh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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