Metallothionein Abrogates GTP Cyclohydrolase I Inhibition–Induced Cardiac Contractile and Morphological Defects
Role of Mitochondrial Biogenesis
One key mechanism for endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O2•− rather than NO because of deficient eNOS cofactor tetrahydrobiopterin (BH4). This study was designed to examine the effect of BH4 deficiency on cardiac morphology and function, as well as the impact of metallothionein (MT) on BH4 deficiency–induced abnormalities, if any. Friend virus B (FVB) and cardiac-specific MT transgenic mice were exposed to 2,4-diamino-6-hydroxy-pyrimidine (DAHP; 10 mmol/L, 3 weeks), an inhibitor of the BH4 synthetic enzyme GTP cyclohydrolase I. DAHP reduced plasma BH4 levels by 85% and elevated blood pressure in both FVB and MT mice. Echocardiography found decreased fractional shortening and increased end-systolic diameter in DAHP-treated FVB mice. Cardiomyocytes from DAHP-treated FVB mice displayed enhanced O2•− production, contractile and intracellular Ca2+ defects including depressed peak shortening and maximal velocity of shortening/relengthening, prolonged duration of relengthening, reduced intracellular Ca2+ rise, and clearance. DAHP triggered mitochondrial swelling/myocardial filament aberrations and mitochondrial O2•− accumulation, assessed by transmission electron microscopy and MitoSOX Red fluorescence, respectively. DAHP also promoted the NG-nitro-l-arginine methyl ester–inhibitable O2•− production and eNOS phosphorylation at Thr497. Although MT had little effect on cardiac mechanics and ultrastructure, it attenuated DAHP-induced defects in cardiac function, morphology, O2•− production, and eNOS phosphorylation (Thr497). The DAHP-induced cardiomyocyte mechanical responses were alleviated by in vitro BH4 treatment. DAHP inhibited mitochondrial biogenesis, mitochondrial uncoupling protein 2, and chaperone heat shock protein 90, and all but uncoupling protein 2 were rescued by MT. Our data suggest a role for BH4 deficiency in cardiac dysfunction and the therapeutic potential of antioxidants against eNOS uncoupling in the heart.
Nitric oxide is an essential regulator of cardiac contraction, although a consensus as to the main cardiac physiological function of NO has only recently emerged by means of genetic deletion or overexpression of all 3 NO synthase (NOS) isoforms in cardiomyocytes.1,2 Despite their apparent promiscuity, each NOS isozyme appears to correlate with specific signaling events, partially because of their subcellular compartmentation with colocalized effectors and limited NO diffusibility. Endothelial NOS (eNOS) is believed to sustain normal cardiac excitation-contraction coupling,3 which is consistent with our data showing that eNOS gene transfer improves cardiomyocyte contractile and intracellular Ca2+ properties via a phosphatidylinositol 3-kinase–mediated mechanism.4 An ample amount of experimental evidence has indicated that eNOS can be uncoupled because of enhanced oxidative stress under certain disease states, eg, hypertension, atherosclerosis, and diabetes mellitus.5–7 Functional eNOS oxidizes l-arginine to l-citrulline and NO in the presence of the essential NOS cofactor tetrahydrobiopterin (BH4).8,9 In disease state, BH4 is oxidized by high peroxynitrite formed by the NO-superoxide (O2•−) reaction, rendering eNOS to produce O2•− rather than NO in the vasculature (a phenomena known as “eNOS uncoupling”).9,10 Although eNOS uncoupling plays an important role for the pathogenesis of vascular diseases, eg, endothelial dysfunction and hypertension,10,11 its impact on the pathophysiology of heart diseases remains poorly understood. We hypothesized that eNOS uncoupling may contribute to the pathogenesis of cardiac dysfunction, whereas “eNOS recoupling” through the preserved redox balance is permissive to the maintenance of physiological cardiac function. To this end, the present study was designed to examine the effect of eNOS uncoupling through inhibition of the rate-limiting BH4 synthetic enzyme GTP cyclohydrolase I on murine myocardial contractile function and ultrastructure. Given the prominent role of free radical accumulation and antioxidant reserve in the development of eNOS uncoupling,7,10 the impact of GTP cyclohydrolase I inhibition was also examined in transgenic mice with overexpression of the thiol-rich metallothionein (MT), a heavy metal scavenger known to preserve the hearts against environmental toxic insults and oxidative damage.12,13 2,4-Diamino-6-hydroxy-pyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I, was used to block BH4 synthesis.14 In an effort to elucidate the cellular mechanisms involved in DAHP and MT-induced myocardial alterations, if any, special attention was drawn to O2•− production and the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α)–regulated mitochondrial biogenesis. Mitochondrial uncoupling protein 2 (UCP2), a novel member of the mitochondrial anion carrier family, and heat shock protein 90 (HSP90), an essential mitochondrial chaperone, are both involved in the control of mitochondrial function and reactive oxygen species generation.15,16 Expression of both proteins in association with mitochondrial O2•− accumulation was evaluated in myocardium from DAHP-treated wild-type Friend virus B (FVB) and MT transgenic mice.
Materials and Methods
See the Methods section in the online data supplement for additional details (http://hyper.ahajournals.org).
Experimental Animals and DAHP Treatment
The experimental protocols have been approved by the animal use and care committee at the University of Wyoming. Adult male wild-type FVB and cardiac-specific MT overexpression transgenic mice (5 months of age) were fed DAHP (10 mmol/L) in drinking water for 3 weeks.14 Because DAHP affects neurotransmitter release, eg, serotonin and catecholamine, which may alter glucose metabolism,17 serum glucose levels were monitored.
Measurement of Total Biopterin and BH4 Levels
Plasma BH4 and total biopterin (BH4; dihydropterin [BH2], and oxidized biopterin [B]) levels were measured by high-performance liquid chromatography (HPLC) with florescence detection, as described previously.18
Cardiac geometry and function were evaluated using a 2D guided M-mode echocardiography equipped with a 15- to 16-MHz linear transducer. Fractional shortening was calculated from end-diastolic diameter (EDD) and end-systolic diameter (ESD) using the following equation: (EDD−ESD)/EDD.19
Intracellular Fluorescence Measurement of O2•−
Myocytes loaded with dihydroethidium (5 μmol/L) were sampled using an Olympus BX-51 microscope equipped with an Olympus MagnaFire SP digital camera and ImagePro image analysis software.20
HPLC Fluorescence Detection of O2•−
2-Hydroxyethidium (2-OH-E+), a product formed from the reaction of O2•− and hydroethidine, is considered a more suitable probe for intracellular O2•−.21 Hydroethidine, ethidium, and 2-OH-E+ were separated by an HPLC system equipped with a fluorescence detector.21
MitoSOX Red Fluorescence Measurement of Mitochondrial O2•−
Cells loaded with MitoSOX Red (2 μmol/L) were rinsed, and MitoSOX Red fluorescence intensity was captured at 510/580 nm using an Olympus BX51 microscope equipped with a digital cooled charged-coupled device camera.22
Cardiomyocyte Isolation and Mechanics
Mouse cardiomyocytes were isolated as described.12 Mechanical properties of myocytes were assessed using an IonOptix soft-edge system.12 Cell shortening and relengthening were assessed using the following indices: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), and maximal velocities of shortening/relengthening (±dL/dt).
Intracellular Ca2+ Transients and Sacroplasmic Reticulum Ca2+ Release
Myocytes loaded with fura-2-acetoxymethyl ester (0.5 μmol/L) were exposed to lights emitted through either a 360- or a 380-nm filter. Fluorescence emissions were detected between 480 and 520 nm.12 Sacroplasmic reticulum (SR) Ca2+ release was assessed by a rapid puff of caffeine (10 mmol/L)-induced intracellular Ca2+ transients in fura-2-loaded myocytes.23
After perfusion fixation, left ventricular and interventricular septal tissues were minced to 1 mm3 followed by fixation and postfixation. Tissue blocks were embedded in Epon/Araldite and cured for 48 hours at 60°C. Thin sections were collected on naked copper (300-mesh) grids, stained with lead citrate and uranyl acetate, and imaged with a Hitachi 7500 transmission electron microscope.19
Western Blot Analysis
Membrane proteins were separated on sodium dodecyl sulfate–polyacrylamide gels and were transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% milk and then incubated overnight with specific antibodies. The antigens were detected by the luminescence method.19
Total RNA Extraction, cDNA Synthesis, Reverse Transcription, and Real-Time PCR
Total RNA extraction, cDNA synthesis, and real-time PCR were carried out as described previously.12
Data are shown as the mean±SEM. Statistical significance (P<0.05) was estimated by ANOVA followed by a Tukey’s test for posthoc analysis where appropriate.
General Morphometric and Echocardiographic Characteristics
As shown in the Table, DAHP and MT transgenes failed to elicit any effects on the body, heart, liver or kidney weights, organ size (normalized to body weight), heart rate, EDD, left ventricular mass (absolute and normalized), and blood glucose. DAHP moderately but significantly elevated both systolic and diastolic blood pressures, the effect of which was unaffected by MT. DAHP treatment significantly increased ESD but reduced wall thickness and fractional shortening, which were significantly attenuated by MT transgene. MT itself did not affect myocardial geometry and fractional shortening. As expected, blockade of GTP cyclohydrolase I with DAHP significantly reduced total biopterin (by 64%) and BH4 (by 85%) levels regardless of MT overexpression. Measurement of O2•− levels using dihydroethidium fluorescence revealed enhanced O2•− levels in cardiomyocytes from DAHP-treated FVB but not MT mice. DAHP-elicited increase of O2•− production was significantly attenuated by the NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 100 μmol/L), indicating the presence of eNOS uncoupling (Figure 1A through 1C). Furthermore, DAHP promoted eNOS phosphorylation at the Thr497 residue without any effect on total eNOS expression, which is another hallmark of eNOS uncoupling.9 Consistent with its effect on the DAHP-induced O2•− production, MT nullified the DAHP-elicited phosphorylation of eNOS at Thr497 (Figure 1D). Given that oxidation of hydroethidine to 2-OH-E+ was used as a more suitable measurement to quantify the production of O2•−,21 HPLC fluorescence detection was performed for 2-OH-E+ levels. Our data revealed that DAHP stimulated the superoxide dismutase–inhibitable O2•− production in FVB but not MT mice. Our result also indicated that the MT transgene did not have any effect on 2-OH-E+ production (Figure 2).
Effect of DAHP and BH4 on Cardiomyocyte Mechanical and Fluorescent Properties
DAHP treatment had no overt effect on cell phenotype (data not shown). The resting cell length of cardiomyocytes was comparable between the FVB and MT groups, regardless of DAHP treatment. Cardiomyocytes from the FVB-DAHP group exhibited significantly depressed PS, reduced ±dL/dt, and prolonged TR90 associated with comparable TPS. Interestingly, cardiac overexpression of MT abrogated the DAHP-induced changes in PS, ±dL/dt, and TR90 without eliciting any significant effect by itself. To further examine the role of BH4 deficiency in these mechanical aberrations, cardiomyocytes from DAHP-treated FVB mice were incubated in vitro with BH4 (10 μmol/L) for 4 hours before mechanical assessment. Our results depicted that BH4 significantly alleviated the DAHP-induced cardiomyocyte mechanical dysfunction in a manner reminiscent of the MT transgene (Figure 3). To explore the mechanism of action behind DAHP-, MT-, and BH4-induced mechanical responsiveness, intracellular Ca2+ transients were recorded using fura-2 fluorescence in cardiomyocytes from FVB and MT mice with or without DAHP treatment. Figure 4 displays that DAHP depressed the electrically stimulated rise in fura-2 fluorescent intensity (ΔFFI), SR Ca2+ release, and prolonged intracellular Ca2+ decay without affecting baseline intracellular Ca2+ FFI. Although MT itself did not affect any of these intracellular Ca2+ properties, it prevented the DAHP-induced depression in ΔFFI, SR Ca2+ release, and intracellular Ca2+ clearance. Consistent with the mechanical data, in vitro BH4 incubation significantly attenuated the DAHP-induced dysregulation in intracellular Ca2+ homeostasis.
Electron Microscopy in Myocardium From FVB and MT Mice After DAHP Treatment
Without DAHP treatment, little ultrastructural difference was noticeable in myocardial samples between FVB and MT groups. DAHP treatment triggered extensive focal damage in left ventricular and intraventricular septal (data not shown) myocardial tissue sections in the FVB group. This was evidenced by cytoarchitectural damage, including mitochondrial swelling and overtly disrupted sarcomeres and myofilament array. Consistent with the mechanical observations, MT alleviated the DAHP-induced cardiac ultrastructural damage (Figure 5). Myocardial tissues from MT-DAHP mice were ultrastructurally indistinguishable from control samples.
Mitochondrial Biogenesis: PGC-1α, Its Downstream Factors, and Mitochondrial DNA Copy Number
Given the above ultrastructural finding of mitochondrial anomalies in response to DAHP treatment and the proven association between eNOS uncoupling and mitochondrial damage in endothelial cells,24 the impact of DAHP and MT on the expression of the mitochondrial biogenesis regulator PGC-1α and its downstream factors, including nuclear respiratory factor (NRF) 1 and 2, mitochondrial transcription factor A (mtTFA), and mitochondrial DNA copy number,25 was examined. Our data revealed that DAHP significantly downregulated PGC-1α by ≈20% associated with marked reductions in mRNA levels of NRF1, NRF2, and mtTFA. MT effectively alleviated the DAHP-induced depression of PGC-1α and its downstream nuclear factors with little effect by itself. The mitochondrial DNA copy number, a key index for oxidative mitochondrial damage and mitochondrial biogenesis, was also downregulated after DAHP treatment, the effect of which was reversed by MT (Figures 6 and S1).
Western Blot Analysis for UCP2, HSP90, and Mitochondrial O2•− Production
Immunoblot analysis exhibited that DAHP treatment significantly reduced the expression of mitochondrial UCP2 and mitochondrial chaperone HSP90, both of which are closely associated with mitochondrial dysfunction, oxidative stress, and heart failure.15,16 Although the MT transgene itself did not alter protein expression of UCP2 and HSP90, it attenuated the DAHP-induced decline in protein expression of HSP90 but not UCP2 (see Figure S1). To further evaluate mitochondrial damage and its potential contribution to the enhanced intracellular O2•− generation, mitochondrial O2•− production was detected using the MitoSOX Red probe. DAHP significantly promoted mitochondrial O2•− production in cardiomyocytes from FVB but not MT mice. MT itself did not elicit any significant effect on mitochondrial O2•− production (see Figure S2).
The major findings of our study are that BH4 deficiency with DAHP treatment directly leads to moderate elevation in blood pressure, cardiac contractile dysfunction, intracellular Ca2+ dysregulation, deranged myocardial ultrastructure, loss of mitochondrial biogenesis, and reduced expression of UCP2 and HSP90, as well as enhanced intracellular/mitochondrial O2•− levels and threonine 497 phosphorylation of eNOS, all of which, with the exception of elevated blood pressure and downregulated UCP2, were alleviated by the free radical scavenger MT. Our study demonstrated for the first time myocardial contractile and intracellular Ca2+ dysfunctions associated with the morphological aberration associated with BH4 deficiency and eNOS uncoupling. The deranged cardiac ultrastructural integrity, including mitochondrial swelling and anomalies in contractile filament, further confirmed that the myopathy is focal to cardiomyocytes and mitochondria. Our observation that MT abrogates the DAHP-induced changes in cardiac mechanical function and ultrastructural integrity indicates the therapeutic potential of antioxidants in eNOS uncoupling–induced cardiac injury.
Inhibition of GTP cyclohydrolase I is considered a rather selective tool to assess the role of de novo synthesis of BH4 in a given biological system.14,17 To this end, DAHP has been widely used as the prototypical GTP cyclohydrolase I inhibitor.14,17,26 Our present study revealed that specific GTP cyclohydrolase I inhibition leads to elevated blood pressure, which is not affected by cardiac overexpression of MT, thus not favoring a role of blood pressure regulation in the DAHP- and MT-induced responses on myocardial function and ultrastructure. DAHP markedly reduced levels of both total biopterin and BH4, depressed peak shortening, depressed maximal velocities of shortening/relengthening, prolonged relengthening duration and intracellular Ca2+ clearance, and reduced intracellular Ca2+ recruitment and SR Ca2+ release capacity. These observations indicated the presence of compromised cardiac contractile function, consistent with our previous report of cardiac excitation-contraction dysfunction under oxidant injury.19 The deleterious effects of DAHP may be explained by increased O2•− because of the uncoupled eNOS. BH4 depletion is known to prompt eNOS to produce O2•− instead of NO as a result of the threonine 497 rather than serine 1179 phosphorylation of eNOS,9 consistent with our finding of enhanced Thr497 phosphorylation. Our observation that l-NAME is capable of inhibiting the DAHP-elicited O2•− production further consolidated the presence of eNOS uncoupling in our current experimental setting. O2•− accumulation has been demonstrated to result in myocardial contractile dysfunction, rigor, and cytosolic Ca2+ overload.27,28 In addition, O2•− and other free radicals may disrupt intracellular Ca2+ homeostasis through inhibition of L-type Ca2+ currents, SR Ca2+ load, Na+-Ca2+ exchange, and SR Ca2+ uptake,27,28 en route to compromised cardiac contractility, contraction, and relaxation duration consistent with the reduced fractional shortening, PS, ±dL/dt, prolonged TR90, and intracellular Ca2+ decay found in our study. TPS and resting intracellular Ca2+ were spared by DAHP treatment, indicating possible disparity in the sensitivity of cardiac contractile/Ca2+ regulating proteins to myocardial eNOS uncoupling. Prolonged duration of contraction and relaxation, delayed intracellular Ca2+ clearance, and dampened intracellular Ca2+ release are considered hallmarks of cardiomyopathy in a number of disease states, eg, heart failure, hypertension, obesity, diabetes mellitus, and oxidative stress.12,13,19,23,28,29 The alterations in mechanical and intracellular Ca2+ properties observed in our study, although relatively small (≈20%), were consistent with the degree of change from failing human hearts29 and were further supported by the apparent cardiac remodeling (changes in wall thickness and ESD) and focal structural damage (mitochondrial swelling and displacement of cardiac contractile filaments) after chronic DAHP treatment. Mitochondria, a main target for O2•−, exhibit mitochondrial permeability transition leading to mitochondrial swelling, cytochrome c release, loss of biogenesis, and function in response to sustained O2•− challenge.24 This is consolidated by our data on lost mitochondrial biogenesis, UCP2, and HSP90, as well as mitochondrial O2•− generation after DAHP treatment. Collectively, our data favored an essential role of BH4 and functional eNOS in the maintenance of cardiac functional and ultrastructural integrity under physiological conditions.
Perhaps the most interesting finding of our study is that cardiac-specific overexpression of MT prevents cardiac functional, ultrastructural, and O2•− production and protein disarray associated with DAHP treatment. MT failed to elicit any effect on the loss of total biopterin and BH4, although it mimicked BH4 supplementation-elicited protection against DAHP-induced detrimental effects on cardiomyocyte mechanical and intracellular Ca2+ properties. Given that MT alleviated DAHP-induced l-NAME–inhibitable intracellular O2•− production and threonine 497 phosphorylation of eNOS, it is plausible to speculate that the MT transgene may protect against DAHP-induced cardiac injury through counteracting eNOS uncoupling. Despite the low plasma BH4 levels, MT may efficiently scavenge the O2•− and other free radicals in cardiomyocytes, thus prompting the eNOS recoupling. Administration of antioxidants, eg, l-ascorbic acid or the eNOS coupler folic acid, has been shown to improve endothelial function through “eNOS dimerization or eNOS recoupling.”10,30,31 Furthermore, our data revealed that MT abrogated the DAHP-induced mitochondrial damage, mitochondrial O2•− production, loss of mitochondrial DNA, and downregulation of mitochondrial biogenesis factor PGC-1α and its downstream nuclear factors. Reduced mitochondrial biogenesis and downregulation of genes required for mitochondrial oxidative phosphorylation have been implicated in insulin resistance and type 2 diabetes mellitus.12,32 PGC-1α promotes mitochondrial genes by enhancing levels of NRF1 and NRF2, alternatively called GA-binding protein, and facilitates GA-binding protein binding to regulatory regions of the mtTFA promoter.25 It is reasonable to speculate that eNOS uncoupling–induced downregulation of PGC-1α, NRF1, NRF2, and mtTFA contributes to the loss of mitochondrial DNA and biogenesis. PGC-1α is crucial for normal heart function, because PGC-1α knockout or deficiency results in compromised cardiac function.25 The precise mechanism responsible for loss of mitochondrial biogenesis under eNOS uncoupling is unknown, although certain posttranslational oxidative modification may play a role.25 Our findings demonstrated the ability of MT to compensate for the DAHP-induced downregulation of HSP90. Reduction of the mitochondrial chaperone HSP90 has been implicated as an event secondary to the uncoupling of eNOS, leading to mitochondrial dysfunction and dysregulated endothelial function associated with cardiovascular diseases.16 Our results suggest that the antioxidant protection against downregulated HSP90 but not mitochondrial UCP2 may contribute to the beneficial effect of MT on mitochondrial integrity. Last but not least, neuronal NOS and inducible NOS are constitutively expressed or pathologically induced, respectively, in cardiomyocytes. Both NOS isoforms participate in the modulation of myocardial functions, including inotropy, relaxation, β-adrenergic responsiveness, and force-frequency relationship.33 Additional study is warranted to elucidate the role of other NOS isoforms in the antioxidant-offered beneficial effects in myocardial function in the state of eNOS uncoupling.
Our present work has provided evidence that eNOS uncoupling directly triggers cardiac ultrastructural and functional anomalies associated with mitochondrial oxidative and biogenesis damage. These results have consolidated a role of functional eNOS in the maintenance of cardiac physiology and, more importantly, the therapeutic value of antioxidants against the eNOS uncoupling–induced cardiac pathology. Our data also indicated the therapeutic value of BH4 and GTP cyclohydrolase I in the management of cardiovascular dysfunction.10,34 Furthermore, it prompts the speculation that eNOS uncoupling seen in the clinical conditions of vascular injuries, eg, hypertension and atherosclerosis, may account, at least in part, for the increased cardiac events under these conditions. Therefore, eNOS recoupling may benefit both vascular anomalies and cardiac pathologies.
The authors greatly appreciated the technical assistance of Dr Kurt Dolence and Bonnie Zhao from University of Wyoming College of Health Sciences, as well as Donna Laturnus and Janice Audette from the University of North Dakota Department of Anatomy and Cell Biology. MT transgenic line was kindly provided by Dr Paul N. Epstein from University of Louisville.
Sources of Funding
This work was supported in part by grants from the American Diabetes Association (7-08-RA-130), the American Heart Association Pacific Mountain Affiliate (0355521Z), and the National Institutes of Health/National Center for Research Resources University of Wyoming North Rockies Regional IDeA Networks of Biomedical Research Excellence (P20 RR016474) to J.R.; the American Diabetes Association (7-05-CD-02) to L.C.; and NIH R01 GM077352, the American Diabetes Association (7-08-RA-23), and the American Heart Association (0855601G) to A.F.C. S.-J.L. was an awardee of the American Heart Association Postdoctoral Fellowship 0720118Z.
A.F.C.-I. and K.K.G. contributed equally to this work.
- Received September 21, 2008.
- Revision received October 12, 2008.
- Accepted March 26, 2009.
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