© 2001 American Heart Association, Inc.
Integrative Physiology |
Upregulation of the Nitric Oxide–cGMP Pathway in Aged Myocardium
Physiological Response to l-Arginine
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
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Abstract |
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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 (
) 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
(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
(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
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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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
,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
(; 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.
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Results |
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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 1A
) 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 1B, 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 1C), 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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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 2). 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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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 1 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. was
42.9±2.5 ms in old hearts and 36.0±1.9 ms in young adult hearts
(P=0.03,
Figure 3) at matched LV end-diastolic pressures (LVEDPs).
During the infusion of
l-arginine, 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 3, Table 1). In the presence of
l-arginine, was no
longer elevated in old compared with young hearts. LVEDP, which was
similar at baseline in old and young adult hearts
(Table 1), 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 1). Peak +dP/dt was lower in older than in younger
adult hearts and was not altered by
l-arginine in either age
group
(Table 1). In contrast to
l-arginine, infusion of
d-arginine did not change
(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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) 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. 
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 and reduced LVEDP
(Table 2 and
Figure 4). In contrast, after pretreatment with the guanylyl
cyclase inhibitor ODQ, the lusitropic effect of SNP, reflected by the
change in both and LVEDP, was attenuated
(Figure 3
). 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 , and increased chamber compliance as indicated by
reduced LVEDP at constant LV end-diastolic volume. In contrast, no
change in 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 . 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.
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Acknowledgments |
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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.
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Footnotes |
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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.
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