Tetrahydrobiopterin, Endothelial Nitric Oxide Synthase, and Mitochondrial Function in the Heart
Tetrahydrobiopterin (BH4) is an essential cofactor for the normal enzymatic function of endothelial NO synthase (eNOS) to produce NO, because it is involved in the catalytic process of l-arginine oxidation. Insufficient BH4 availability impairs this process, and the free radical superoxide anion (O2·−) is released rather than NO, a condition termed “eNOS uncoupling.”1 BH4, eNOS, and NO levels physiologically increase in parallel, as seen in response to shear stress, the most important physiological stimulus for endothelial NO production. Shear stress increases BH4 through a casein kinase 2–dependent phosphorylation of GTP cyclohydrolase-1, the enzyme catalyzing the first step in BH4 synthesis. Blockade of GTP cyclohydrolase-1 by the specific inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) results in eNOS uncoupling in endothelial cells exposed to shear stress.2 In transgenic mice overexpressing eNOS, a large portion of the enzyme is uncoupled because of BH4 deficiency, leading to excessive endothelial formation of O2·−, which reacts rapidly to form peroxynitrite.3 Because BH4 is highly sensitive to oxidation by the particular aggressive reactive oxygen species (ROS) peroxynitrite, in most cardiovascular disease states, BH4 is depleted.4 Compelling evidence exists that eNOS uncoupling contributes to endothelial dysfunction in diabetes mellitus, atherosclerosis, and hypertension. NO is also an essential regulator of cardiac structure and function; however, only a few studies have investigated the role of BH4 in this regard. Takimoto et al5 revealed the importance of BH4 depletion for eNOS uncoupling and subsequent left ventricular hypertrophy in mice subjected to pressure overload. Exogenous BH4 was able to recouple eNOS and reverse pre-established advanced hypertrophy.6 In addition, depletion of BH4 and subsequent uncoupling of cardiac NO synthase appear to contribute to the development of diastolic dysfunction.
In the present issue of Hypertension, Ceylan-Isik et al7 report that eNOS uncoupling by BH4 depletion through treatment with the GTP cyclohydrolase-1 inhibitor DAHP impaired myocyte and mitochondrial function in the heart (Figure). Their data clearly show that BH4 depletion and subsequent O2·− formation disrupt calcium handling and cardiomyocyte function. The data of Ceylan-Isik et al7 further indicate a crosstalk between ROS produced from uncoupled eNOS and the mitochondria, leading to increased mitochondrial ROS production and mitochondrial dysfunction. The importance of mitochondrial ROS formation has also been highlighted recently in endothelial cells in response to angiotensin II, which induced mitochondrial dysfunction via protein kinase C–dependent activation of NADPH oxidase, and a crosstalk between mitochondrial- and NADPH-oxidase–derived ROS was also found in nitroglycerin-triggered vascular dysfunction.8,9
Cardiomyocyte-restricted overexpression of the ROS scavenger metallothionein prevented the DAHP-induced increase in O2·− formation and preserved cardiomyocyte calcium handling and cardiac function. Because elevated blood pressure during DAHP treatment was not normalized by metallothionein overexpression, BH4 depletion appears to impair cardiac structure and function independent of blood pressure changes. Metallothionein overexpression also prevented eNOS phosphorylation at threonine497 associated with eNOS uncoupling during BH4 deficiency. An open question remains the relative contribution of other NO synthase isoforms affected by BH4 deficiency. Interestingly, uncoupling of a putative mitochondrial NO synthase in cardiomyocytes was described recently to stimulate mitochondrial ROS production.10
In summary, by BH4 depletion using GTP cyclohydrolase-1 blockade, Ceylan-Isik et al7 clearly demonstrate that BH4 deficiency and subsequent eNOS uncoupling disrupt cardiac structure and function, which is linked to mitochondrial dysfunction, indicating a crosstalk between uncoupled NO synthase and the mitochondria. Scavenging O2·− through cardiomyocyte-restricted overexpression of metallothionein prevented most sequelae of BH4 deficiency.
Sources of Funding
Original research of J.B. and J.D.W. was funded by the Deutsche Forschungsgemeinschaft and the Deutsche Akademie der Naturforscher Leopoldina.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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