Tetrahydrobiopterin Restores Diastolic Function and Attenuates Superoxide Production in Ovariectomized mRen2.Lewis Rats

Chronic BH₄ supplementation improves diastolic function, attenuates cardiac remodeling, and reduces oxidative stress in female mRen2.Lewis rats following the loss of ovarian hormones.

Abstract

After oophorectomy, mRen2.Lewis rats exhibit diastolic dysfunction associated with elevated superoxide, increased cardiac neuronal nitric oxide synthase (nNOS) expression, and diminished myocardial tetrahydrobiopterin (BH₄) content, effects that are attenuated with selective nNOS inhibition. BH₄ is an essential cofactor of nNOS catalytic activity leading to nitric oxide production. Therefore, we assessed the effect of 4 wk BH₄ supplementation on diastolic function and left ventricular (LV) remodeling in oophorectomized mRen2.Lewis rats compared with sham-operated controls. Female mRen2.Lewis rats underwent either bilateral ovariectomy (OVX) (n = 19) or sham operation (n = 13) at 4 wk of age. Beginning at 11 wk of age, OVX rats were randomized to receive either BH₄ (10 mg/kg · d) or saline, whereas the sham rats received saline via sc mini-pumps. Loss of ovarian hormones reduced cardiac BH₄ when compared with control hearts; this was associated with impaired myocardial relaxation, augmented filling pressures, increased collagen deposition, and thickened LV walls. Additionally, superoxide production increased and nitric oxide decreased in hearts from OVX compared with sham rats. Chronic BH₄ supplementation after OVX improved diastolic function and attenuated LV remodeling while restoring myocardial nitric oxide release and preventing reactive oxygen species generation. These data indicate that BH₄ supplementation protects against the adverse effects of ovarian hormonal loss on diastolic function and cardiac structure in mRen2.Lewis rats by restoring myocardial NO release and mitigating myocardial O₂⁻ generation. Whether BH₄ supplementation is a therapeutic option for the management of diastolic dysfunction in postmenopausal women will require direct testing in humans.

Cardiovascular disease in women has gained attention in recent years in light of the recognized sex differences between males and females (1–4). Before menopause, the incidence of heart disease is significantly lower in women compared with men (5). However, both the risk and the prevalence of heart failure dramatically increases in women after the onset of menopause (6). Unlike their male counterparts, women predominantly exhibit diastolic impairment and eventual diastolic heart failure rather than systolic dysfunction (7, 8). The interplay between age and declining estrogens were deemed the common denominators in women's heart disease and led to robust examination of hormone replacement therapy in the cardioprotection of postmenopausal women. However, conflicting results failed to show cardioprotective benefit with replacement of various estrogen therapies (9), underscoring the need to gain further insight into the mechanisms that influence the development of heart disease in women.

Using the female mRen2.Lewis rat, an angiotensin-II-dependent model of hypertension, we show diastolic functional abnormalities and remodeling of the cardiac interstitium after early bilateral oophorectomy, a cardiac phenotype that bears a resemblance to the cardiac and vascular changes resulting from surgically induced or natural menopause (10). In this same rodent model, the loss of ovarian hormones exacerbates the increases in systolic blood pressure, which can be corrected with 17β-estradiol replacement (11). In a recent study, we further show that the pathogenesis of diastolic dysfunction in the female mRen2.Lewis rat involves a critical interaction between neuronal nitric oxide synthase (nNOS) and ovarian hormones, particularly estrogens (12). That is, selective nNOS inhibition with N5-(1-Imino-3-butenyl)-1-ornithine (L-VNIO) attenuates diastolic dysfunction and cardiac remodeling in ovariectomized (OVX) mRen2.Lewis females, presumably due to the ability of L-VNIO to mitigate myocardial reactive oxygen species (ROS) generation. Taken together, these findings suggest that a dynamic shift occurs in the nNOS pathway within the female heart such that the loss of estrogens leads to uncoupling of nNOS and the subsequent generation of superoxide (O₂⁻) rather than nitric oxide (NO) (12).

The effects of nNOS on diastolic function based upon the bioavailability of NO are also documented (13). Additional data show that estrogens can modulate the production of nNOS (14–17) as well as regulate guanosine triphosphate cyclohydrolase I (GTPCH) (18–20). GTPCH is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin (BH₄), the necessary cofactor responsible for maintaining nNOS in a coupled state, which allows the enzyme's catalytic activity to generate NO (21). In our previous study, diminished BH₄ levels within the heart tissue of oophorectomized rats was associated with compromised ventricular relaxation and cardiac structural alterations including increased collagen deposition and increased relative wall thickness (12). Theorizing that the loss of estrogens and consequent depletion of BH₄ may lead to nNOS uncoupling and the release of reactive free radicals instead of NO, we hypothesized that BH₄ supplementation would exert beneficial effects on diastolic function in terms of improving myocardial relaxation, attenuating left ventricular (LV) remodeling, and decreasing the generation of ROS in the heart of the OVX mRen2.Lewis rat.

Materials and Methods

Experimental model

Congenic female mRen2.Lewis rats obtained from the transgenic rat colony of the Hypertension and Vascular Research Center of Wake Forest University School of Medicine were weaned at 3 wk of age and allowed to acclimate in a temperature-controlled, Association for Assessment and Accreditation of Laboratory Animal-approved facility (12-h light, 12-h dark cycle) with ad libitum food and water. The experimental procedures were approved by Wake Forest University School of Medicine's Animal Care and Use Committee. Rats were randomly divided into two groups at 4 wk of age: bilateral oophorectomized (OVX; n = 17) or sham-operated (n = 13), performed under 2% isoflurane anesthesia, as previously described (10, 12). Once the rats reached 11 wk of age, the OVX group was randomly divided to receive (6R)-5,6,7,8-tetrahydro-l-biopterin dihydrochloride (BH₄; OVX+BH₄; n = 8; Schricks Laboratories, Jona, Switzerland) administered via an osmotic minipump (Alzet model 2ML4, 2.5 μl/h, 4 wk; Durect Corp., Cupertino, CA) at a targeted dose of 10 mg/kg · d or saline (2.5 μl/h) as a vehicle control (OVX; n = 9). This dose of BH₄ has been shown to be therapeutically effective in previous reports by other laboratories (22–24). Weekly tail-cuff plethysmography (NIBP-LE5001; Panlab, Barcelona, Spain) was used to measure blood pressure from 5–15 wk of age. Oophorectomy and depletion of circulating estradiol was confirmed using a serum estradiol assay (5 pg/ml detection limit; Polymedco, Cortlandt Manor, NY).

Echocardiographic evaluation

Cardiac function and structure were assessed before the protocol termination in anesthetized (ketamine/xylazine, 80/12 mg/kg) animals using a Philips 5500 echocardiograph (Philips Medical Systems, Andover, MA) and a 12-MHz phased array probe as previously described (10). Relative wall thickness (RWT) was calculated as: 2 × posterior wall thickness/LV end diastolic dimension (LVEDD).

Measurement of total biopterin and BH₄ levels

Cardiac tissue BH₄ levels were measured by HPLC with fluorescence detection as previously documented (25). Total biopterins [BH₄, dihydrobiopterin (BH₂), and oxidized biopterin (B)] were measured after acid oxidation of the deproteinated cardiac supernatant. BH₂ plus B was determined by alkali oxidation, followed by iodine reduction and acidification. The resulting samples were centrifuged and small aliquots injected into a 250-mm long, 4.6-mm inner diameter Spherisorb ODS-1 column (5-μm particle size; Alltech Associates, Inc., Deerfield, IL). Fluorescence (350 nm excitation, 450 nm emission) was detected by a fluorescence detector (RF10AXL; Shimadzu Co., Columbia, MD). BH₄ concentration was calculated by subtracting BH₂ and B from total biopterins.

Nitrite measurements

Nitrite levels of were determined using a chemiluminescence-based nitric oxide analyzer (Sievers, Inc., GE Analytical Instruments, Boulder, CO) as previously described (26). Standard curves were obtained and used for quantitative measurements.

Morphometry and histopathology

Collagen deposition within the heart was evaluated using picrosirius red-stained, 4-μm, 4% paraformaldehyde-fixed, paraffin-embedded sections as documented previously (12). Additional sections stained with hematoxylin and eosin were used to measure myocyte cross-sectional area in approximately 100 cardiomyocytes from two sections per rat (×400 magnification) as previously described (12). Histological analysis was completed using a Leica DM4000B microscope (Bannockburn, IL) with a tandem Leica DFC digital camera and Simple PCI version 6.0 software as well as an Olympus polarizing microscope system (Center Valley, PA) equipped with a Digital SPOT RT, 3-pass capture, thermoelectrically cooled charge-coupled camera (Sterling Heights, MI) and SPOT Advanced software. Adobe Photoshop Creative Suite 3 (Adobe Inc., San Jose, CA) was used to calculate the ratios of collagen-positive stained pixels to unstained pixels in each photomicrograph (four randomized quadrant field images per rat, magnified ×200).

In situ ROS production

ROS was determined using dihydroethidium (DHE) as previously described (12). The resulting images were analyzed at ×400 using a fluorescence microscope (Leica DM4000B; excitation = 510–550 nm, emission = 590 nm for DHE; excitation = 330–380 nm, emission = 420 nm for 4′,6-diamidino-2-phenylindole) connected to a Leica DFC digital camera. The fluorescent signal intensities were analyzed within the heart using Simple PCI version 6.0 software. All data are presented as average gray-scale intensities ± sem.

Statistical analysis

All values are expressed as mean ± sem. For all endpoints, one-way ANOVA evaluated significant effects among the groups. Significant interactions between the groups were further characterized using Bonferroni post hoc tests. Analyses were performed using GraphPad Prism version 5 (GraphPad, San Diego, CA). Differences were considered significant at P < 0.05.

Results

Cardiac biopterin and BH₄ levels

Figure 1 depicts total cardiac biopterin and BH₄ levels from sham, OVX, and OVX BH₄-treated rats. Supplementation with BH₄ (OVX+BH₄) was associated with more than a 3-fold increase in myocardial biopterin concentrations compared with untreated sham and OVX littermates (P < 0.001). This confirmed that the administered BH₄ reached the heart. As expected, estrogen depletion by surgical oophorectomy was associated with a 3.2-fold reduction in cardiac BH₄ when compared with levels in sham-operated littermates (P < 0.05). Exogenous BH₄ supplementation attenuated this OVX-associated fall in cardiac BH₄ concentration but did not restore levels to those measured in sham rats.

Fig. 1.

Cardiac concentrations of total biopterins are shown as white bars, and BH₄ values are shown as black bars from sham (n = 8), OVX (n = 8), and OVX+BH₄ (n = 7) rats. Data are mean ± sem. *, P < 0.001 compared with sham; #, P < 0.001 compared with OVX.

Physical characteristics and hemodynamics

The effects of OVX and BH₄ supplementation on body and heart weight are shown in Table 1. Before BH₄ or vehicle treatment, there were no differences in body weight or systolic blood pressure (SBP) among the three cohorts. Serum estradiol levels were significantly less in OVX rats compared with the sham group (sham 34.8 ± 3.4 pg/ml vs. OVX 5.5 ± 0.5 pg/ml; P < 0.001). At the end of the study, the OVX and OVX+BH₄ rats were 13 and 16% heavier, respectively, than sham-operated controls (P < 0.001 for each), corroborating previous reports from this and other laboratories that deprivation of estrogens leads to increased body mass (10, 12, 27, 28). Likewise, the heart weights from the OVX rats were substantially higher compared with the sham group (sham 830 ± 17 mg vs. OVX 939 ± 35 mg; P < 0.01). BH₄ had no effect on the OVX-induced heart weight increase. Normalized heart weights (heart weight/body weight) were similar among the three groups.

Table 1.

Body and heart weight values

Variables	Sham (n = 13)	OVX (n = 9)	OVX + BH4 (n = 10)
Body weight (g)	239 ± 2	269 ± 7a	277 ± 5a
Heart weight (mg)	830 ± 17	939 ± 35b	905 ± 20
Heart weight/body weight (mg/g)	3.47 ± 0.07	3.50 ± 0.13	3.27 ± 0.05

Data are expressed as mean ± sem. P values are compared with sham.

^aP < 0.001.

^bP < 0.01.

Progressive increases in SBP occurred in sham and OVX rats from 5–11 wk (Fig. 2). Similar to earlier findings (11, 12), substantial rises in blood pressure were observed by wk 6 in OVX rats compared with intact littermates (P < 0.0001), and this larger increase in SBP continued throughout the duration of the 15-wk study. Although there was a tendency for BH₄ administration to limit the exacerbated hypertension, there were no significant differences between the OVX groups during the 4-wk supplementation period.

Fig. 2.

Tail-cuff systolic blood pressures in sham (n = 13), OVX (n = 9), and OVX+BH₄ (n = 10) conscious mRen2.Lewis rats. Values are means ± sem. *, P < 0.01 compared with sham.

Heart rate and diastolic function

Table 2 shows the effects of ovarian hormonal loss and BH₄ supplementation on heart rate and the conventional Doppler indices of diastolic function. Although heart rate and the more load-dependent, conventional measures of diastolic function were similar among groups (Table 2), the loss of ovarian hormones led to modest reductions in ventricular relaxation, (or e′) and elevations in LV filling pressure (or E/e′) (P < 0.001) when compared with Doppler indices from intact-littermates (Fig. 3). Importantly, 4 wk of BH₄ treatment restored ventricular lusitropy and reduced filling pressures to that of sham-operated littermates. Specifically, BH₄ increased mitral annular velocity (e′) by 27% and reduced Doppler-derived filling pressures (E/e′) by 20% when compared with untreated OVX rats.

Table 2.

Echocardiographic indices of diastolic function

Variables	Sham (n = 13)	OVX (n = 9)	OVX + BH4 (n = 10)
Emax (cm/sec)	63 ± 2	64 ± 4	64 ± 3
Amax (cm/sec)	42 ± 2	40 ± 3	40 ± 2
E/A	1.5 ± 0.1	1.6 ± 0.1	1.7 ± 0.1
Edec time (sec)	0.057 ± 0.002	0.062 ± 0.003	0.060 ± 0.003

Data are expressed as mean ± sem. P values are compared with sham. E_max, Maximum early transmitral filling velocity; A_max, maximum late transmitral filling velocity; E/A, early-to-late transmitral filling ratio; E_dec time, early-filling deceleration time.

Fig. 3.

A, Myocardial relaxation indicated by early mitral annular velocity (e′) in sham (n = 13), OVX (n = 9), and OVX+BH₄ (n = 10) mRen2.Lewis rats. Data are mean ± sem. #, P < 0.05 compared with OVX. B, Ratio of early transmitral filling velocity to mitral annular velocity (E/e′) in sham (n = 13), OVX (n = 9), and OVX+BH₄ (n = 10) rats. Data are expressed as mean ± sem. *, P < 0.05 compared with sham; #, P < 0.05 compared with OVX.

LV structural characteristics and systolic function

Figure 4A demonstrates a 1.6-fold increase in perivascular fibrosis in OVX mRen2.Lewis rats compared with their estrogens-intact littermates (P < 0.005). OVX had no effect on myocyte cross-sectional area (Fig. 4B). Chronic BH₄ supplementation in OVX mRen2.Lewis rats reversed or prevented the excess collagen deposition (37%, P < 0.001) (Fig. 4A), without altering myocyte size (Fig. 4B).

Fig. 4.

A, Quantification and representative picrosirius red staining revealing perivascular collagen in the hearts of sham (n = 9), OVX (n = 9), and OVX+BH₄ (n = 10) rats. Values are mean ± sem. *, P < 0.05 compared with sham; #, P < 0.05 compared with OVX. Magnification, ×200. B, Myocyte cross-sectional areas of cardiac myocytes from sham (n = 9), OVX (n = 9), and OVX+BH₄ (n = 10) mRen2.Lewis hearts. Data are mean ± sem. C, RWT of sham (n = 13), OVX (n = 9), and OVX+BH₄ (n = 10) mRen2.Lewis rats. Data are mean ± sem. *, P < 0.05 compared with sham; #, P < 0.05 compared with OVX.

M-mode measurements of LV dimensions and wall thicknesses are summarized in Table 3. The loss of ovarian hormones led to a 25% increase in posterior wall thickness (P < 0.01) (Table 3), which, combined with no alterations in the LVEDD, yielded a robust 2-fold increase in RWT compared with sham-operated rats (P < 0.001) (Fig. 4C). Treatment with BH₄ reversed this effect because the RWT of the OVX+BH₄ compared with OVX was 0.51 ± 0.04 vs. 0.71 ± 0.05 cm, respectively (P < 0.01) (Fig. 3C). Systolic function indicated as the percent fractional shortening was similar among the three groups (Table 3).

Table 3.

M-mode-derived measures of LV dimension, wall thickness, and systolic function

Variables	Sham (n = 13)	OVX (n = 9)	OVX + BH4 (n = 10)
Heart rate (beats/min)	251 ± 8	245 ± 12	237 ± 6
LVEDD (cm)	0.68 ± 0.02	0.68 ± 0.01	0.67 ± 0.02
LVESD (cm)	0.35 ± 0.02	0.37 ± 0.02	0.39 ± 0.02
RWTed (cm)	0.37 ± 0.03	0.71 ± 0.05a	0.51 ± 0.04a,b
PWTed (cm)	0.18 ± 0.01	0.23 ± 0.01a	0.20 ± 0.01
AWTed (cm)	0.17 ± 0.01	0.20 ± 0.01	0.20 ± 0.02
% FS	47 ± 1	46 ± 1	43 ± 2

Data are expressed as mean ± sem. LVESD, LV end-systolic dimension; RWTed, RWT at end diastole; PWTed, posterior wall thickness at end diastole; AWTed, anterior wall thickness at end diastole; FS, fractional shortening.

^aP < 0.0001 compared with sham.

^bP < 0.05 compared with OVX.

ROS and nitrite profile

Similar to our earlier findings, (12), DHE staining of the cardiac tissue illustrated abundant O₂⁻ production within the myocytes of OVX rats compared with the estrogens-intact mRen2.Lewis rats (Fig. 5A). This increase in ROS generation was associated with a 33% reduction in cardiac tissue nitrite availability, or NO release, in the OVX group (P < 0.05) (Fig. 5B). In hearts from OVX+BH₄ rats, the intensity of the fluorescent product, 2-hydroxyethidium, was diminished to a level found in hearts from SHAM rats (Fig. 5A). The decreased O₂⁻ production in the OVX+BH₄ group corresponded to a 1.7-fold increase in available nitrite content (Fig. 4B).

Fig. 5.

A, Quantification and representative images illustrating the effect of OVX and BH₄ supplementation on cardiac levels of fluorescent 2-hydroxyethidium from DHE staining in sham (n = 8), OVX (n = 8), and OVX+BH₄ (n = 8) rats. Fluorescent images are shown at ×400 magnification where red indicates DHE staining and blue indicates nuclei. Data are mean ± sem. *, P < 0.05 compared with sham; #, P < 0.05 compared with OVX. B, Cardiac concentrations of nitrite as an indicator of nitric oxide availability from sham (n = 8), OVX (n = 8), and OVX+BH₄ (n = 8) rat hearts. Data are mean ± sem. *, P < 0.05 compared with sham; #, P < 0.05 compared with OVX.

Discussion

The major findings of this study are 1) loss of estrogens by surgical OVX results in myocardial BH₄ deficiency in mRen2.Lewis rats; 2) this deficiency is associated with diastolic dysfunction, adverse LV remodeling, increases in cardiac O₂⁻ production, and reduced cardiac NO release; and 3) BH₄ supplementation reverses all these changes except the elevated heart weight induced by OVX. The observation that the loss of estrogens is accompanied by increased body weight explains the presence of a compensatory increase in cardiac mass likely due to excessive lengthening of cardiac myocytes and ventricular dilation, (29) and/or from the loss of estrogen-stimulated cGMP activity (30). Collectively, this study supports our original hypothesis that diminished BH₄ availability after the loss of ovarian hormones, and specifically estrogens, leads to nNOS uncoupling within the heart of the OVX mRen2.Lewis rat. Under this situation, O₂⁻ rather than NO becomes the product of nNOS activity, ultimately contributing to the diastolic dysfunction phenotype (12). It is therefore plausible that BH₄ supplementation may represent a rational approach to the management of diastolic dysfunction in women due to either surgically induced or natural menopause. The benefits of BH₄ supplementation may be due to suppression of ROS generation alone or in combination with enhanced NO release (12) and/or a direct antioxidant action (31).

Extensive strides have been made to establish the cardioprotective role of endothelial nitric oxide synthase (eNOS) in the regulation of cardiac function (18, 32–35). However, efforts targeting equivocal appreciation of cardiac (36, 37) and vascular nNOS (38) have been far more limited. It is important to note that nNOS compared with the other NOS isoforms is more likely to produce superoxide when the enzyme is uncoupled (39). In an earlier report by Yamaleyeva et al. (40), renal eNOS decreased after OVX in mRen2.Lewis rats, whereas nNOS protein expression increased. In their study, selective inhibition of nNOS attenuated the exacerbated increase in SBP characteristic of this model. Likewise, we observed overexpression of cardiac nNOS in OVX mRen2.Lewis rats compared with estrogens-intact littermates but no difference in cardiac eNOS between groups (12). Intrigued by the possibility that the excess nNOS had a functional role in the model, we administered the specific nNOS inhibitor L-VNIO. The L-VNIO treatment led to cardioprotection in the OVX mRen2.Lewis model, effects we proposed were due to blockade of the uncoupled nNOS activity, because ROS generation was decreased, whereas NO increased in the heart (12). Taken together with the finding that cardiac BH₄ levels were suppressed in OVX rats compared with littermates with intact estrogens (12) and that deficiency of estrogens has been reported to cause decreased synthesis of BH₄ by the de novo pathway (41) provides the rational basis for the use of BH₄ supplementation in our model. BH₄ is not only an essential NOS cofactor, it has an important role in maintaining the structure of all NOS isoforms by stabilizing the active or coupled form of the enzyme, which leads to the production of NO rather than superoxide, the end-product of nNOS uncoupling. These current data complement and extend these previous observations by showing that BH₄ supplementation halts the functional and structural changes in the OVX mRen2.Lewis hearts, even in the presence of exacerbated increases in blood pressure induced by OVX. Although the lack of an overt change in blood pressure by BH₄ might raise questions, others report similar findings. In a clinically relevant canine model of cardiopulmonary bypass with hypothermic cardiac arrest, a 30-min infusion of BH₄ before reperfusion resulted in better systolic and diastolic functional recovery compared with saline vehicle-treated dogs, and these effects occurred without overt changes in systemic blood pressure (42). Moreover, in a small, randomized, double-blind placebo control trial in hypercholesterolemia patients, 4 wk of oral BH₄ treatment restored the forearm blood flow response to acetylcholine and decreased plasma markers of oxidative stress compared with placebo, and these effects were independent of blood pressure (43). Whether BH₄ affected endothelial function in our model is not known. Nonetheless, the observed cardioprotective effects indicate that BH₄ availability is important for maintaining cardiac NO synthesis and low superoxide production.

Although the heart data are limited, the link between estrogens and BH₄ is highlighted in a report by Lam et al. (44) that showed decreased bioavailability of BH₄ in the rat vasculature after OVX. Another study has shown that estrogens can directly increase BH₄ through up-regulation of GTPCH mRNA and activity in cultured endothelial cells (18). Moreover, 17β-estradiol therapy normalized aortic BH₄ levels and suppressed O₂⁻ generation (41). Although it is not known whether the decreased concentration of cardiac BH₄ in hearts from the OVX mRen2.Lewis rats was due to the withdrawal of the regulation of estrogens on GTPCH, resetting the nNOS system through BH₄ supplementation was a sufficient means by which to normalize diastolic function, attenuate remodeling, and restore myocardial antioxidant capacity to that of littermates with intact estrogens.

Independent of the status of estrogens, equivalent doses of BH₄ as used in the present study have repeatedly been shown to preserve cardiac function and prevent remodeling after myocardial infarction (45, 46). In isolated hearts from Dahl salt-sensitive rats, administration of a BH₄ donor increased LV developed pressure and recovery as measures of ischemia resistance (47). Additionally, in a pressure-overload mouse model, BH₄ reversed cardiac hypertrophy and improved heart function as documented in improved sarcomere activity and calcium handling (48). The benefit of BH₄ extends beyond in vivo and in vitro experiments. In a pilot study designed to establish a dose-response curve and determine the duration of oral BH₄ therapy in uncontrolled human hypertensive patients, BH₄ significantly reduced systolic blood pressure and improved flow-mediated vasodilation (49).

The first limitation of the present study is that these studies were conducted in a rodent oophorectomized model, which is not an identical mimetic of naturally occurring menopause. However, the phenotypic characteristics of the mRen2.Lewis after depletion of ovarian hormones, including LV hypertrophy and exacerbated hypertension, are similar to the pathophysiological changes occurring in postmenopausal women (50, 51).

Second, the actual accumulated levels of BH₄ detected within the myocardial tissue of the BH₄-treated OVX rats were not significantly increased compared with OVX. These observations are likely due to the dual regulation of BH₄ metabolism that can lead to rapid oxidation of BH₄ to BH₂ and simultaneous recycling of BH₄ from BH₂ by the enzyme dihydrofolate reductase (52, 53). Channon and colleagues (54) eloquently described this phenomenon within the vasculature where they suggest that the overall availability of BH₄ within the vascular tissue is the summation of BH₄ synthesis, BH₄ oxidation, and finally, its recycling from BH₂. In our studies, the HPLC biopterin assay cannot determine the rate of oxidation.

Finally, this study focused on proposed nNOS uncoupling as the source of ROS generation leading to diastolic dysfunction and LV remodeling. Oxidative stress can also occur via alternative processes, including xanthine oxidase, reduced nicotinamide adenine dinucleotide phosphate oxidases, or cytochrome P450. However, our rationale to explore the nNOS uncoupling phenomenon in this model was based on the paradoxical increase in cardiac nNOS expression after oophorectomy (12). This is an important consideration given the fact that until recently, it had been assumed that nNOS was a positive contributor to cardiovascular function through the production of NO as opposed to the maladaptive characteristics (increased ROS and decreased NO) now demonstrated in this study. Targeting individual components of the pathway allows for the progressive understanding of this complex system and the extrapolation of potential clinical applications.

In summary, we show that BH₄ supplementation led to significant improvements in diastolic function, attenuation of cardiac remodeling, and reductions in oxidative stress in the mRen2.Lewis female after ovarian hormonal loss. We propose that the restorative effects are due to BH₄ therapy recoupling the NOS activity, which shifts the maladaptive pathway of ROS generation back to coupled, adaptive catalytic activity. In light of the controversy surrounding estrogen replacement therapy, it is plausible that BH₄ may be a viable therapeutic option for diastolic patients because it is currently used clinically for other syndromes such as phenylketonuria. Phenylketonuria patients who have either phenylalanine hydroxylase mutations or are BH₄ deficient, therapeutically receive BH₄, the necessary cofactor for phenylalanine hydroxylase activity, just as it is for NOS activity. In those patients that are responsive to BH₄, the level of circulating phenylalanine is significantly diminished, sparing the individuals from developmental disorders, cognitive impairment, and seizures (55–57). Importantly, these patients exhibit minimal side effects (58), providing hope that BH₄ supplementation could provide beneficial effects in patients with diastolic dysfunction.