BH4 Increases nNOS Activity and Preserves Left Ventricular Function in Diabetes

RATIONALE: In diabetic patients, heart failure with predominant left ventricular (LV) diastolic dysfunction is a common complication for which there is no effective treatment. Oxidation of the NOS (nitric oxide synthase) cofactor tetrahydrobiopterin (BH4) and dysfunctional NOS activity have been implicated in the pathogenesis of the diabetic vascular and cardiomyopathic phenotype. OBJECTIVE: Using mice models and human myocardial samples, we evaluated whether and by which mechanism increasing myocardial BH4 availability prevented or reversed LV dysfunction induced by diabetes. METHODS AND RESULTS: In contrast to the vascular endothelium, BH4 levels, superoxide production, and NOS activity (by liquid chromatography) did not differ in the LV myocardium of diabetic mice or in atrial tissue from diabetic patients. Nevertheless, the impairment in both cardiomyocyte relaxation and [Ca 2+ ]i (intracellular calcium) decay and in vivo LV function (echocardiography and tissue Doppler) that developed in wild-type mice 12 weeks post–diabetes induction (streptozotocin, 42–45 mg/kg) was prevented in mGCH1-Tg (mice with elevated myocardial BH4 content secondary to trangenic overexpression of GTP-cyclohydrolase 1) and reversed in wild-type mice receiving oral BH4 supplementation from the 12th to the 18th week after diabetes induction. The protective effect of BH4 was abolished by CRISPR/Cas9-mediated knockout of nNOS (the neuronal NOS isoform) in mGCH1-Tg. In HEK (human embryonic kidney) cells, S-nitrosoglutathione led to a PKG (protein kinase G)-dependent increase in plasmalemmal density of the insulin-independent glucose transporter GLUT-1 (glucose transporter-1). In cardiomyocytes, mGCH1 overexpression induced a NO/sGC (soluble guanylate cyclase)/PKG–dependent increase in glucose uptake via GLUT-1, which was instrumental in preserving mitochondrial creatine kinase activity, oxygen consumption rate, LV energetics (by 31 phosphorous magnetic resonance spectroscopy), and myocardial function. CONCLUSIONS: We uncovered a novel mechanism whereby myocardial BH4 prevents and reverses LV diastolic and systolic dysfunction associated with diabetes via an nNOS-mediated increase in insulin-independent myocardial glucose uptake and utilization. These findings highlight the potential of GCH1/BH4–based therapeutics in human diabetic cardiomyopathy. GRAPHIC ABSTRACT: A graphic abstract is available for this article. mechanisms underpinning LV dysfunction in diabetic patients are not completely understood. Here we show that diabetic mice develop LV dysfunction despite maintaining normal NO synthesis and myocardial BH4 levels, suggesting that NOS dysfunction and oxidative stress are not required for the development of diabetic cardiomyopathic phenotype. Nevertheless, increasing intracellular BH4 in cardiomyocytes, prevented and reversed LV dysfunction via an nNOS-mediated increase in insulin-inde-pendent glucose uptake and utilization. Our findings suggest that BH4 supplementation may both prevent and ameliorate LV dysfunction in patients with diabetes.

D iabetes mellitus (DM) is a major cause of death and disability and a large economic burden on healthcare systems across the world. 1 Globally, 1 in 12 deaths in adults has been attributed to DM and its complications 2 ; among which, the proportion of heart failure cases is substantial both in type I and type II DM and persisting after adjustment for differences in coronary artery disease or other relevant risk factors. 3,4 Together with postmortem findings demonstrating left ventricular (LV) dysfunction in diabetic patients in the absence of coronary artery disease or hypertension, 5 epidemiological data suggest that DM may in itself give rise to a specific cardiomyopathy characterized by predominant LV diastolic dysfunction, leading to heart failure with preserved or mildly impaired LV ejection fraction. 6 Several factors, including mitochondrial dysfunction, oxidative stress, impaired calcium handling, dysfunctional NOS (nitric oxide synthase) activity, and remodeling of the extracellular matrix, have been advocated in the pathogenesis of diabetic cardiomyopathy 6 ; however, a unifying mechanism upstream of the observed LV functional changes is still lacking.
Constitutive NO production regulates LV compliance and relaxation through its action on myofilament Ca 2+ sensitivity and intracellular Ca 2+ handling. 7 Under physiological conditions, tetrahydrobiopterin (BH4) is a limiting factor in myocardial NO synthesis by the nNOS (neuronal NOS) isoform. 8 Increasing cardiomyocyte BH4 content by myocardial-specific overexpression of the first enzyme involved in its synthesis, GCH1 (GTP-cyclohydrolase 1), enhances nNOS activity and hastens the rate of intracellular Ca 2+ reuptake and myocardial relaxation in healthy mice by increasing the PLB (phospholamban) phosphorylated fraction. 8,9 In the presence of DM, increasing BH4 content by myocardial GCH1 overexpression or inhibition of GCH1 protein degradation has been shown to attenuate the increase in myocardial superoxide production and maintain nNOS in its dimeric form. 10 Impaired NO signaling, due to BH4 oxidation and dysfunctional eNOS (endothelial NOS) activity, accounts for the endothelial dysfunction reported in diabetic patients and animal models. [11][12][13] Similar changes in the myocardium would be expected to lead to LV diastolic

Novelty and Significance
What Is Known?
• Nitric oxide (NO) regulates cardiac contractility and relaxation. • Myocardial nNOS (neuronal NO synthase) activity and relaxation can be enhanced by increasing the intracellular level of the NOS cofactor, tetrahydrobiopterin (BH4). • Diabetes reduces NO synthesis in the vascular endothelium by oxidizing BH4.
What New Information Does This Article Contribute?
• Left ventricular (LV) dysfunction associated with diabetes can occur in the absence of BH4 oxidation, increased superoxide production, or reduced NOS activity both in humans and in mice. • Nevertheless, the diabetic cardiomyopathic phenotype is prevented by raising BH4 content in cardiomyocytes through the overexpression of GCH1 (GTP-cyclohydrolase 1) and reversed by oral administration of BH4. • BH4-mediated preservation of myocardial function and energetics is abolished by nNOS gene deletion or GLUT (glucose transporter)-1 inhibition.
The exact mechanisms underpinning LV dysfunction in diabetic patients are not completely understood.
Here we show that diabetic mice develop LV dysfunction despite maintaining normal NO synthesis and myocardial BH4 levels, suggesting that NOS dysfunction and oxidative stress are not required for the development of diabetic cardiomyopathic phenotype. Nevertheless, increasing intracellular BH4 in cardiomyocytes, prevented and reversed LV dysfunction via an nNOS-mediated increase in insulin-independent glucose uptake and utilization. Our findings suggest that BH4 supplementation may both prevent and ameliorate LV dysfunction in patients with diabetes.
dysfunction and increased oxidative stress 9,14,15 ; however, whether dysfunctional NOS activity and altered nitroso-redox balance are key factors in the pathogenesis of diabetic cardiomyopathy remains to be established. Likewise, the extent to which endothelial dysfunction induced by DM contributes to the cardiomyopathic phenotype is unclear.
Here, we show that increasing myocardial BH4 and nNOS activity by transgenic overexpression of GCH1 does not preserve endothelial-mediated vasodilatation but prevents LV dysfunction in diabetic mice-not by averting NOS dysfunction, maintaining PLB phosphorylation, or reducing oxidative stress-but by preserving myocardial energetics via an nNOS-mediated increase in glucose uptake through GLUT-1 (the insulin-independent transporter-1). Importantly, oral BH4 supplementation is able to reverse the cardiomyopathic phenotype in diabetic wild-type (WT) mice, indicating that GCH1/BH4-based therapeutics may be used to treat as well as prevent diabetic cardiomyopathy.

Data Availability
The authors declare that all data and methods supporting the findings of this study are available in the Data Supplement or from the corresponding authors on reasonable request. Please see expanded methods and the Major Resources Table in

Human Samples
Samples of the right atrial appendage were collected from patients undergoing on-pump cardiac surgery for coronary revascularization and stored at −80 °C. Investigations were approved by the Research Ethics Committee; all patients gave informed written consent.

Diabetes Induction
DM was induced by low doses (42-45 mg/kg) of streptozotocin dissolved in citrate buffer and injected intraperitoneal daily for 5 consecutive days. Control mice were injected in parallel with buffer only. Mice with glucose levels <15 mmol/L after 2 weeks of streptozotocin injection were excluded. Studies were carried out and data analyzed with the operator blind of the genotype or treatment allocation.

BH4 Supplementation
WT mice were allocated to normal chow (Teklad global 16% protein diet, Harlan Laboratories) or BH4-supplemented chow (200 mg/kg per day for 6 weeks) beginning at week 12 poststreptozotocin. Data were collected and analyzed with the operator blind of treatment allocation. Randomization was performed by cage.
Representative images were selected as reflecting either the mean or the median (in case of non-normal data) of their respective data series.

DM-Induced LV Dysfunction Is Prevented by Myocardial GCH1 Overexpression and Reversed by Oral BH4 Supplementation
Streptozotocin decreased plasma insulin levels and body weight and increased plasma glucose similarly in WT and mGCH1-Tg (mice with elevated myocardial BH4 content secondary to overexpression of GTP-cyclohydrolase 1) at 4 and 12 weeks post-DM induction ( Table I in Figure 1). At the latter time point, aortas from both diabetic WT and mGCH1-Tg displayed an enhanced contractile response to phenylephrine and impaired vasodilatation in response to acetylcholine or the peptide activator of the SLIGRL (proteaseactivated receptor-2) compared with shaminjected nondiabetic littermates ( Figure 1B through 1D), whereas endothelial-independent vasodilatation in response to the NO donor, sodium nitroprusside, was preserved in all groups ( Figure 1E). Preincubation of aortic rings with the NOS inhibitor, N-nitro-L-arginine methyl ester (100 µmol/L), abolished all differences between diabetic and nondiabetic mice ( Figure 1F  At 12 weeks post-DM induction, nondiabetic groups showed similar function as at 4 weeks. However, LV diastolic function was significantly impaired in diabetic WT, as indicated by a higher ratio between the peak early mitral filling velocity (E) and the tissue Doppler-derived peak early diastolic velocity at the mitral annulus (E′; P<0.001 versus WT non-DM controls and diabetic mGCH1-Tg mice, Figure 2C). Echocardiographic examination showed no differences in LV end-diastolic volume between genotypes in the presence or absence of DM whereas LV end-systolic volume increased in diabetic WT (Table II in the Data Supplement), leading to a significant reduction in LV ejection fraction and fractional shortening when compared with both WT nondiabetic controls and diabetic mGCH1-Tg mice ( Figure 2D and Table II in the Data Supplement). By contrast, LV diastolic and systolic function were unaltered in mGCH1-Tg mice at 12 weeks post-DM induction (Figure 2A through 2D and Table II in the Data Supplement). These findings were mirrored by concordant changes in the myocardial performance index ( Table II in the Data Supplement), confirming that impairment in this heart rate/arterial pressure-independent   Table II in the Data Supplement. Scatterplots for these data are shown in Figure Table III in the Data Supplement. Scatterplots for these data are shown in Figure VI in the Data Supplement. BH4 indicates tetrahydrobiopterin; DM, diabetes; and LV, left ventricular. measurement of overall LV function was prevented in diabetic mGCH1-Tg.
LV mass was not different between groups at all timepoints (Table II in the Data Supplement). Heart rate was lower in mGCH1-Tg compared with their WT littermates and did not change significantly after DM induction in either genotype (Table II in the Data Supplement).
We have previously shown that GCH1 overexpression and raised BH4 content significantly augment myocardial nNOS activity. 8 However, BH4 is also an antioxidant molecule and a cofactor for the formation of biogenic amines and serotonin. 16 To evaluate to which extent nNOS-derived NO was responsible for preserving LV function in the presence of DM, we performed these experiments in mGCH1-Tg in which nNOS was knocked out ( Figure IIIG in the Data Supplement) using CRISPR-Cas9-mediated gene editing. As shown in Figure 2A through 2D and Table II in the Data Supplement, the protective effect of myocardial GCH1 overexpression was lost in diabetic mice lacking nNOS, consistent with an essential role of this NOS isoform in mediating the cardioprotective effects of BH4 in the presence of DM.
We then tested whether LV function in diabetic WT mice could be restored by supplementing their diet with BH4 (200 mg/kg per day for 6 weeks, beginning at week 12 post-DM induction).
At 12 weeks and before BH4 was introduced in the protocol, both groups of WT DM mice showed reduced LV diastolic and systolic function ( Figure 2E through 2H, Figure VI and Table III in the Data Supplement).
After 18 weeks, DM was associated with a significant impairment in LV diastolic and systolic function in WT fed normal chow, as indicated by a lower LV ejection fraction and a higher E/E′ ratio (ratio between early mitral inflow velocity and mitral annular early diastolic velocity) and myocardial performance index (versus nondiabetic WT); by contrast, LV diastolic and systolic dysfunction was completely reversed in diabetic WT receiving BH4-supplementation ( Figure 2E through 2H, Table III  To establish whether the changes in LV function observed in WT diabetic mice in vivo reflect altered cardiomyocyte dynamics and Ca 2+ handling, we undertook these measurements in field-stimulated LV myocytes (3 Hz, 35±0.5 °C; Figure 3A through 3F).  8 there were significant genotype differences in the rate of myocyte relaxation and [Ca 2+ ] i reuptake in nondiabetic mice, which were preserved in the presence of DM. By contrast, at 12 weeks post-DM, both relaxation velocity and the rate of decay of the [Ca 2+ ] i transient were slower in diabetic WT myocytes compared to nondiabetic controls, whereas myocardial overexpression of GCH1 prevented the adverse effect of DM on both parameters ( Figure 3C and 3F). Both at 4 and 12 weeks post-DM induction, cell shortening was significantly higher in Tg compared with WT, but the amplitude of the [Ca 2+ ] i transient did not differ between groups ( Figure 3B and 3E).
Oral BH4 supplementation for 6 weeks beginning at week 12 poststreptozotocin injection reversed the effect of DM on both relaxation velocity and rate of [Ca 2+ ] i decay in isolated LV myocytes but did not affect cell shortening or the amplitude of the [Ca 2+ ] i transient significantly As reported previously, 8 PLB protein content was lower in the LV myocardium of mGCH1-Tg, which also showed a significantly higher PLB Ser16 phosphorylated fraction compared to WT ( Figure 5A). There were no significant differences in the PLB Thr17 phosphorylated fraction or in SERCA2A (sarcoplasmic reticulum calcium ATPase 2A) between genotypes ( Figure 5A and 5B).
DM did not affect the LV content or phosphorylation status of any of these proteins in either genotype but significantly increased the LV content of hydroxyproline in both WT and mGCH1-Tg and led to a comparable nonsignificant increase in collagen staining in both genotypes ( Figure 5C and 5D).

LV Dysfunction in DM Is Independent of BH4 Oxidation and Dysfunctional NOS Activity
In the aortic endothelium, BH4 and superoxide production in sham-injected mGCH1-Tg did not differ significantly from WT. DM induction lowered the ratio between BH4 and its oxidized products to a similar extent in both genotypes and increased superoxide production ( Figure 6A and 6B). By contrast, in the LV myocardium of nondiabetic GCH1-Tg, BH4 content and NOS activity were significantly higher compared with WT ( Figure 6C and 6D), in the absence of differences in the protein content of NOS isoforms between genotypes ( Figure IB in the Data Supplement). Surprisingly, none of these parameters was altered at 12 weeks after DM induction in either genotype. Total and reduced myocardial glutathione was significantly elevated in mGCH1-Tg hearts compared to WT but, again, both measurements were unaltered by DM ( Figure 6E). In line with these findings, neither total nor   The inactive isomer D-NAME was used as a control; (G) LV superoxide levels were also determined by HPLC (n=9-11 hearts per group). Normally distributed data in A, B, and G are shown as means±SEM and were compared using 2-way ANOVA with Bonferroni correction. Non-normally distributed data in D, E, and F (D' Agostino-Pearson test, P=0.011, 0.002, and 0.005, respectively) were compared using Kruskal-Wallis 1-way ANOVA, followed by the Dunn test. n denotes number of aortas or hearts. L-NAME indicates N-nitro-L-arginine methyl ester. *P<0.05, **P<0.01, ***P<0.001. AUC indicates area under the curve; BH4, tetrahydrobiopterin; DM, diabetes; D-NAME, N-nitro-D-argining methyl ester; HPLC, high performance liquid chromatography; L-NAME, N-nitro-L-arginine methyl ester; LV, left ventricular; NOS, nitric oxide synthase; and RLU, relative light units. NOS-derived (ie, N-nitro-L-arginine methyl ester inhibitable) myocardial superoxide production was raised in diabetic mice from either genotype ( Figure 6F and 6G). As already mentioned, oral BH4 supplementation led to a significant increase in BH4 content and total NOS activity in the myocardium of diabetic WT but, even at 18 weeks post-DM induction, myocardial BH4 level (5.6±0.6 versus 5.8±0.8 pmol/mg protein in WT group) and NOS activity were unchanged in WT DM mice (% citrulline conversion, 0.3±0.08 versus 0.2±0.05 in WT group), suggesting that, in contrast to the vascular endothelium, myocardial BH4 oxidation and NOS dysfunction are not an early hallmark of DM nor are they required to induce the cardiomyopathic phenotype.

ORIGINAL RESEARCH
To establish whether these unexpected findings were also pertinent to the myocardium of diabetic patients, we measured myocardial biopterins in samples of the right atrial appendage from 17 diabetic patients and 19 matched nondiabetic controls undergoing coronary revascularization (Table IV in the Data Supplement). LV ejection fraction was significantly lower in diabetic patients compared with their matched nondiabetic controls; nevertheless, myocardial BH4 content and the ratio between BH4 and its oxidized products were similar between groups and so was total and NOS-derived superoxide production ( Figure II in the Data Supplement), indicating that, in agreement with our findings in diabetic mice, oxidant stress is not increased and NOS activity is not uncoupled in the myocardium of diabetic patients.

mGCH1 Overexpression Preserves Myocardial Energetics in DM by Increasing Myocardial Glucose Uptake and Utilization via a NO/sGC/ Protein Kinase G-Dependent Mechanism
In the presence of DM, myocardial glucose transport and glycolysis are compromised and fatty acids (FA) become the exclusive source of ATP generation leading to an increase in oxygen consumption and a reduction in cardiac efficiency. 17 Accordingly, the myocardial expression of Pparα (peroxisome proliferator-activated receptor α) which promotes FA uptake and utilization, was significantly elevated in diabetic WT, but not in mGCH1-Tg, compared with sham-injected controls ( Figure 7A). Similarly, the LV content of the mitochondrial UCP3 (uncoupling protein-3), a downstream target of PPARα, 18 was 50% higher in diabetic WT at 4 weeks post-DM induction ( Figure IIIA in the Data Supplement) in keeping with an early switch to FA metabolism. However, the increase in myocardial UCP3 was much greater in WT by 12 weeks post-DM induction ( Figure 7B) as was that of PDK4 (pyruvate dehydrogenase kinase 4; Figure IIIB in the Data Supplement), implying that myocardial metabolism was increasingly compromised as the duration of DM increased.
Diabetic WT hearts also displayed a reduction in the activity of mitochondrial CK (creatine kinase; Figure 7C), indicating reduced capacity for high-energy phosphate shuttling out of the mitochondria. There was no difference in cytoplasmic CK activity ( Figure 7D) or in markers of mitochondrial cell density, as evaluated by citrate synthase activity and VDAC (mitochondrial voltage dependent ion channels) expression ( Figure 7E and  7F). Despite similar levels of plasma glucose and insulin in both genotypes ( Table I in the Data Supplement), PPARα expression, UCP3 protein level, and mitochondrial CK activity were unaltered in the heart of diabetic mGCH1-Tg ( Figure 7A through 7C).
To evaluate whether myocardial energetics differed between genotypes, we performed 31 phosphorous magnetic resonance spectroscopy in isolated perfused hearts. In sham-injected mice, there was no difference in the LV phosphocreatine-to-ATP ratio (PCr/ATP) between genotypes ( Figure 7G). As observed in diabetic patients, 19,20 the PCr/ATP ratio was significantly reduced in the LV myocardium of diabetic WT mice but was unchanged in diabetic mGCH1-Tg ( Figure 7G), suggesting that myocardial glucose uptake or utilization may be less affected by DM in mGCH-Tg. Indeed, myocardial 2-deoxyglucose uptake was significantly higher in LV myocytes from diabetic mGCH1-Tg compared with diabetic WT ( Figure 8A). Selective inhibition of the insulin-independent GLUT-1 (glucose transporter-1), with STF-31 (10 µmol/L), significantly reduced 2-deoxyglucose uptake in diabetic mGCH1-Tg and abolished differences between genotypes ( Figure 8A).
We determined whether higher myocardial glucose transport in mGCH1-Tg mice was accompanied by increased glucose oxidation by measuring oxygen consumption rate (OCR) in intact isolated LV myocytes from diabetic and sham-injected mice from both genotypes ( Figure 8B). OCR was significantly higher in LV myocytes from mGCH1-Tg, irrespective of diabetic status. Preincubation of LV myocytes with the GLUT-1 inhibitor, STF-31 (10 µmol/L), significantly decreased OCR both in WT and mGCH1-Tg myocytes and abolished the difference between genotypes ( Figure 8C). OCR was also measured after blocking NOS (N-nitro-L-arginine methyl ester, 1 mmol/L) or PKG activity (Rp-8-pCPT-PET-cGMPS, 10 µmol/L) and, again, under these conditions, OCR no longer differed between genotypes ( Figure IV in the Data Supplement), indicating that mGCH1 overexpression dynamically increases myocardial glucose uptake via the GLUT-1 transporter in a NO/PKG-dependent manner. In agreement with these findings, incubation with the NO donor S-nitrosoglutathione (1 mmol/L) was associated with a 2-fold increase in the density of GLUT-1 on the cell surface of HEK (human embryonic kidney)-293 cells when compared with control cells. Inhibition of PKG significantly reduced GLUT-1 mobilization under these conditions ( Figure IV in the Data Supplement).
The functional impact of genotype differences in glucose uptake via GLUT-1 was evaluated in LV myocytes from diabetic WT and mGCH1-Tg. As shown in Figure 8D through 8G, GLUT-1 inhibition with STF-31 had no effect in myocytes from diabetic WT mice but abolished the advantage conferred by mGCH1 overexpression in the presence of DM by slowing the rate of [Ca 2+ ] i decay and reducing both relaxation velocity and cell shortening in diabetic mGCH1-Tg, again without affecting the amplitude of the [Ca 2+ ]i transient.

DISCUSSION
We have shown that increasing myocardial BH4 levels can prevent or reverse the LV dysfunction induced by DM and uncovered a novel mechanism by which BH4 exerts its beneficial effects in the myocardium via nNOS (see Graphical abstract). In contrast to previous reports where prevention of diabetic vascular endothelial dysfunction by Tie2-driven GCH1 overexpression was mediated by preservation of eNOS coupled activity, 13 we demonstrate that an increase in insulinindependent myocardial glucose uptake accounts for the maintenance of myocardial relaxation in diabetic mGCH1-Tg in the absence of NOS uncoupling.
DM-induced disruption of myocardial nitroso-redox balance has been reported in association with cardiac dysfunction and proposed as an important determinant of the diabetic cardiomyopathy phenotype. 10,21 Other studies, however, have shown no difference or a reduction in myocardial superoxide generation in diabetic mice. 22,23 We did not observe a significant difference in myocardial total superoxide production, glutathione or BH4 oxidation, and NOS activity in the myocardium of diabetic mice with LV dysfunction. Similar findings were obtained in samples of the right atrial myocardium from diabetic patients with reduced LV ejection fraction undergoing myocardial revascularization.
Although additional beneficial effects of mGCH1/ BH4 overexpression on myocardial oxidative stress may occur in more advanced stages of cardiomyopathy, as reported by Wu et al 10 and a subtle localized increase in mitochondrial reactive oxygen species cannot be categorically excluded by our experiments, our findings demonstrate that LV dysfunction associated with DM can occur in the absence of reduced NOS activity or PLB phosphorylation and that augmenting myocardial BH4 content prevents diabetic cardiomyopathy and increases glucose transport and utilization in the absence of BH4 oxidation.
Prevention of the diabetic myocardial phenotype by myocardial GCH1 overexpression was abolished by nNOS gene deletion or GLUT-1 inhibition, indicating that BH4 exerts its protective effect via an nNOS-mediated increase in glucose availability and utilization. A dynamic NO/PKG-dependent translocation of the insulin-independent GLUT-1 to the sarcolemmal membrane may underpin enhanced glucose transport in the presence of increased levels of myocardial BH4, in line with previous reports showing that NO induces GLUT-1 membrane translocation and a PKG-dependent increase in insulinindependent glucose uptake in rat ovarian cells. 24 Our data indicate that higher glucose uptake via GLUT-1 in cardiomyocytes from diabetic mGCH-Tg is mediated by NO via cGMP-PKG signaling, as it is abolished by both sGC and PKG inhibition. Stimulation of myocardial PKG signaling by kinase oxidation 25 seems unlikely as, at difference with the aorta, we did not observe an increase in oxidative markers or superoxide production in diabetic hearts of either genotype.
DM lowered LV mitochondrial CK activity and PCr/ATP ratio, as assessed by 31 phosphorous cardiac magnetic resonance spectroscopy, in WT but not in mGCH1-Tg, where elevation of PPARα and UCP3 was also prevented. Both proteins have been found to be raised in rodent models of type 1 and type 2 DM 26 where they support FA utilization, enhance the FA inhibition of glucose oxidation, and promote oxygen wasting for noncontractile purposes. 27 Impairment in myocardial energetics is increasingly regarded as an important player in the pathogenesis of diabetic cardiomyopathy and other conditions leading to LV dysfunction. [28][29][30] Limitations to effective energy supply to the heart can adversely impact the ATP-dependent Ca 2+ reuptake during each cardiac cycle and impact diastolic function. 31 Indeed, impaired respiratory capacity precedes the development of LV dysfunction in type 1 DM, 23 and a reduced PCr/ATP ratio, proportional to the degree of LV diastolic dysfunction, has also been reported in patients with type 1 or type 2 DM. 20,32,33 Although insulin and glycemic levels did not differ between 4 and 12 weeks of DM induction, impairment of LV function in WT mice was only observed at 12 weeks in association with significantly higher myocardial levels of UCP3 and PDK4, suggesting that DM caused a time-dependent substrate switch from glucose oxidation toward lactate production and increased fatty acid metabolism leading to LV dysfunction. These findings are in keeping with the lack of effect of acute hyperglycemia on human myocardial function and the development of insulin resistance and reduced ability to oxidize fatty acids observed in type 1 DM over time. 34,35 Our study has revealed important differences between the vascular endothelium and the myocardium in the response to hyperglycemia. Whereas LV dysfunction did not develop until 12 weeks following diabetes induction, endothelial dysfunction was already evident at 4 weeks. As reported previously, 13 endothelial dysfunction was associated with increased superoxide production, significant BH4 oxidation, and impaired NO signaling. These findings highlight the relative vulnerability of the endothelium to hyperglycemia but also indicate that the development of significant endothelial dysfunction, as measured in the aorta, is not sufficient to induce the cardiomyopathic phenotype in diabetic mice in the early stages of the disease. We have previously reported that myocardial spillover of biopterins in mGCH1-Tg is minimal or absent and that BH4 levels are not increased in the heart nonmyocyte cellular component, 8 accordingly, there was no evidence of a protective effect on the vascular endothelium of diabetic mGCH1-Tg nor was there evidence of reduced myocardial fibrosis, despite which mGCH1 overexpression was still able to prevent the development of LV dysfunction.
Taken together, our findings open the possibility that BH4 supplementation may have beneficial effects in patients with diabetic cardiomyopathy. In a previous study in patients with ischemic heart disease, administration of a synthetic formulation of the active 6R-isomer of BH4, sapropterin, 2 to 6 weeks before coronary artery bypass surgery failed to enhance vascular NOS activity or improve endothelial-mediated vasodilatation. 36 Our current findings indicate that NOS recoupling may not be the best surrogate end-point for gauging the efficacy of BH4 supplementation, at least in myocardial disease states. In contrast with findings in human vessels, 36 BH4 content (both in absolute terms and relative to its oxidized products) increased in a dose-dependent manner in myocardial samples collected from patients treated with sapropterin (unpublished results), confirming that oral BH4 supplementation increases myocardial BH4 content in humans.

Limitations
We did not investigate the mechanisms by which oral BH4 supplementation improved LV function in diabetic WT mice. In contrast with myocardial-specific GCH1 overexpression, oral BH4 supplementation is also known to improve endothelial function and prevent inflammation 37,38 ; to which extent these additional effects contributed to recovering LV function in WT diabetic mice remains to be established. Investigation of the effect of BH4 supplementation in diabetic mGCH-Tg lacking nNOS may provide important information on the contribution of the extra-myocardial additional effect of BH4 of the diabetic cardiomyopathic phenotype.
Streptozotocin injection results in loss of pancreatic β-cell activity, leading to hyperglycemia secondary to insulin deficiency that resembles human type 1 DM. Although type II DM is more common, patients with type 1 DM have a high risk of developing heart failure that is dependent on glycemic control and associated with higher fatality 3,39,40 even in the absence of factors, such as hypertension and obesity, which may confound the pathophysiology of LV dysfunction associated with type II DM. To this end, rodent models streptozotocin-induced DM are well suited to evaluate the toxic effects of hyperglycemia and impaired glucose utilization in the myocardium. Male rodents are more susceptible to the diabetogenic action of streptozotocin than females; for this reason, our study was conducted in male mice. Although this is an important limitation, there is no evidence indicating that development of heart failure requiring hospitalization in patients with type I DM is different between men and women. 3 Although offtarget adverse effects of streptozotocin delivered as a highdose bolus have been reported, these can be minimized by the administration of low-dose streptozotocin delivered by the multiple low-dose streptozotocin injections protocol, used in our article. 41 31 Phosphorous magnetic resonance spectroscopy experiments showing a reduction in PCr/ATP ratio in diabetic WT hearts but not in Tg were conducted in the absence of FA supplementation, raising the possibility that provision of this source of energy may have attenuated the impact of DM on myocardial energetics. Nevertheless, significant genotype differences were observed in hearts exposed to the same conditions and similar changes in myocardial PCr/ATP ratio have been reported in vivo in patients with type 1 or type 2 DM. 20,32,33 Finally, since at difference with the in vivo data, measurements in isolated myocytes could not be obtained sequentially in the same mice, we have not compared these data over time but between groups at 4 and 12 weeks. We think this is the appropriate way of comparing data obtained using this study design, as in an experiment of long duration variations due to equipment refurbishment and different batches of mice may affect longitudinal measurements but not crosssectional comparisons. It should be noted that changes in unloaded cell shortening brought about by DM were not associated with differences in the amplitude of the [Ca 2+ ]i transient, in keeping with data from human myocardial biopsies showing depressed cardiac myofilament function and Ca 2+ responsiveness in the presence of diabetes. 42

Conclusions
Our work provides original insights into the management and prevention of early metabolic triggers of diabetic