Metallothionein abrogates GTP cyclohydrolase I inhibition-induced cardiac contractile and morphological defects: role of mitochondrial biogenesis

Abstract

One key mechanism for endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O(2)(*-) 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 O(2)(*-) production, contractile and intracellular Ca(2+) defects including depressed peak shortening and maximal velocity of shortening/relengthening, prolonged duration of relengthening, reduced intracellular Ca(2+) rise, and clearance. DAHP triggered mitochondrial swelling/myocardial filament aberrations and mitochondrial O(2)(*-) accumulation, assessed by transmission electron microscopy and MitoSOX Red fluorescence, respectively. DAHP also promoted the N(G)-nitro-l-arginine methyl ester-inhibitable O(2)(*-) 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, O(2)(*-) 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.

Fig. 1

Effect of DAHP on content of total biopterin (panel A), BH4 (panel B), O₂*^_ levels in the absence or presence of the NOS inhibitor L-NAME (100 μmol/l) (panel C) and p-eNOS (Thr497) (panel D) in FVB and MT mice. Inset: Representative gel blots of p-eNOS and total eNOS. Mean ± SEM, n = 5–7, * p<0.05 vs. FVB, # p<0.05 vs. FVB-DAHP group.

Fig. 2

HPLC identification of 2-OH-E⁺ from the O₂^•_/HE reaction in cardiomyocytes. Panels A – D display the HPLC chromatograms from FVB (A), FVB-DAHP (B), MT (C) and MT-DAHP (D) groups. Panel E: Standard HPLC peak intensity of authentic 2-OH-E⁺. Panel F: Levels of 2-OH-E⁺ in FVB, FVB-DAHP, MT and MT-DAHP groups with or without copper zinc SOD (100 units/ml). 2-OH-E⁺: 2-Hydroxyethidium (labeled peak 1); E⁺: Ethidium (labeled peak 2). Mean ± SEM, n = 100–120 cells per group, * p<0.05 vs. FVB, # p<0.05 vs. respective FVB-DAHP group, ** p<0.05 vs. FVB-DAHP group in the absence of SOD.

Fig. 3

Effect of DAHP-induced response on cardiomyocyte contractile function in FVB and MT mice. A cohort of cardiomyocytes from FVB and FVB-DAHP groups was incubated with BH4 (10 μmol/l) for 4 hrs prior to mechanical assessment. (A): Resting cell length; (B): Peak cell shortening; (C): + dL/dt; (D): − dL/dt; (E): TPS; and (F): TR₉₀. Mean ± SEM, n = 132–133 cells from 6 mice per group, * p<0.05 vs. FVB, # p<0.05 vs. FVB-DAHP.

Fig. 4

Effect of DAHP on intracellular Ca2+ transients and SR Ca²⁺ release in cardiomyocytes from FVB and MT mice. A cohort of cardiomyocytes from FVB and FVB-DAHP groups was incubated with BH4 (10 μmol/l) for 4 hrs prior to intracellular Ca²⁺ assessment. (A): Baseline intracellular Ca²⁺ fura-2 fluorescent intensity (FFI); (B): Change of fura-2 fluorescence intensity in response to electrical stimulation (ΔFFI); (C): Intracellular Ca²⁺ fluorescence decay rate; and (D): SR Ca²⁺ release. Mean ± SEM, n = 68–70 cells (7 cells for SR Ca²⁺ release) per group, * p<0.05 vs. FVB; # p<0.05 vs. FVB-DAHP.

Fig. 5

Transmission electron microscopic micrographs of left ventricular heart tissues from FVB and MT mice with or without DAHP treatment. (A): FVB; (B): FVB-DAHP; (C): MT; and (D): MT-DAHP. Myocardial tissues in A, C and D appear normal with myofibrils composed of regular and uninterrupted sarcomeres separated by continuous rows of normal mitochondria. In the myocardium from FVB-DAHP mice, irregularly shaped mitochondria and myelin figures are randomly distributed between highly disrupted myofibrils.

Fig. 6

RT-PCR measurement of (A) nuclear respiratory factor 1 (NRF1), (B) nuclear respiratory factor 2 (NRF2), (C) mitochondrial transcription factor A (mtTFA) and (D) mtDNA copy number in left ventricles from FVB and MT mice with or without DAHP treatment. Mean ± SEM, n = 5–6, * p<0.05 vs. FVB, # p<0.05 vs. FVB-DAHP.