Impact of glucose-6-phosphate dehydrogenase deficiency on the pathophysiology of cardiovascular disease

Animal model of G6PD deficiency.

Complete genetic deletion of G6PD produces embryonic lethality (58). However, the generation of G6PD-deficient (G6PDX) mice that recapitulate key aspects of clinical deficiency was reported in 1988 (84). This was done by treating male mice with a DNA ethylating agent 1-ethyl-1-nitrosourea to induce DNA mutations, and G6PD deficiency was identified in subsequent offspring. G6PDX mice have an A:T mutation in the 5′-untranslated region at the splice site of the 3′-end of exon 1 (93). This mutation results in decreased translation of G6PD and leads to ∼20–40% residual G6PD activity (class III deficiency) in G6PDX mice compared with wild-type (WT) littermate control mice. G6PDX mice mature normally but have been reported to develop modest cardiac hypertrophy at 9 mo of age (44). Overall, G6PDX mice provide a clinically relevant model of G6PD deficiency that reflects the extent of limited G6PD activity commonly found in human patients with G6PD deficiency (6, 12).

Statin-like effect.

A protective cardiovascular role for G6PD deficiency is conceivable in light of the effects of G6PD deficiency on cholesterol synthesis (71). The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase is rate determining in the cholesterol synthesis pathway and is dependent on NADPH to catalyze its reaction. Decreased cholesterol synthesis was found in peripheral lymphomononuclear cells obtained from G6PD-deficient subjects (5), and evidence suggests that G6PD deficiency may decrease the overall cholesterol content in the blood of deficient subjects (71). We also found evidence for decreased cholesterol synthesis in G6PDX mice (88). Furthermore, in an apolipoprotein E-deficient mouse model of atherosclerosis, G6PD deficiency decreased cholesterol and the development of vascular lesions (63). Thus G6PD deficiency may provide a statin-like cholesterol-lowering effect and thereby protect against atherosclerosis (57, 70, 71, 94).

Reductive stress.

In addition to decreasing cholesterol synthesis, G6PD deficiency may be protective under conditions of reductive stress (85). Reductive stress refers to an increase in NADPH and GSH and may result in adverse cellular events such as protein misfolding and aggregation, mitochondrial dysfunction, pathological gene expression, and increased susceptibility to apoptosis (11, 14, 60, 98, 106, 112, 114). Evidence for a role of reductive stress in heart failure was found in a mouse model of desmin-related cardiomyopathy induced by overexpression of mutant human αB-crystallin (hR120GCryAB) in mice (CryAB mice) (85–87). This cardiomyopathy model results in protein aggregation within the cell, hypertrophy, and failure of the ventricle (29, 64, 85). Support for the role of reductive stress in the cardiomyopathy observed in CryAB mice was provided by the observation that G6PD and glutathione reductase activities were increased with a concomitant increase in GSH and the GSH-to-GSSG ratio (GSH/GSSG) (85). An increase in GSH contrasts with the typically reported decrease in GSH/GSSG that is associated with oxidative stress (86). When CryAB mice were crossed with G6PDX mice, there was a decrease in protein aggregation and cardiac hypertrophy, thus supporting a protective role for G6PD deficiency in reducing reductive stress in protein aggregation-associated cardiomyopathy (85). Overall, the data suggest that decreasing G6PD activity may alleviate reductive stress (17).

G6PD fuels superoxide production.

Although G6PD supplies the antioxidant glutathione system with NADPH, the NADPH produced by G6PD is also used to produce superoxide via NADPH oxidase (Nox), uncoupled nitric oxide synthase, and xanthine oxidase (Fig. 2) (3, 83, 107, 110). These reactions are all implicated in heart failure, and decreasing the production of ROS by these enzymes may exert beneficial effects on cardiac hypertrophy and dysfunction (18, 37, 41, 42, 49, 110). An upregulation of G6PD expression may fuel these superoxide-producing enzymes in failing myocardium (32, 35). Specifically, G6PD expression and activity were increased in failing myocardium in dogs and humans (Fig. 4) (32, 35). The increase in G6PD corresponded with an increase in NADPH levels and superoxide production, and pharmacological inhibition of G6PD in vitro decreases NADPH levels and ROS production.

Similar evidence for a role of G6PD in deriving NADPH-dependent ROS was observed in a number of other experimental models with different cell and tissue types (2, 36, 51, 78–80). These include a decrease in aortic superoxide production in apolipoprotein E-deficient atherosclerotic mice that are crossed with G6PDX mice (63). G6PD deficiency also decreased aortic dihydroethidium and nitrotyrosine and lowered the hypertensive response to angiotensin-II infusion (62). Superoxide production is increased in the aorta, heart, and liver in genetically obese Zucker rats, and in vitro pharmacological inhibition of G6PD decreased the generation of superoxide in these tissues (31, 97). Recently, G6PD was reported to decrease superoxide production by Nox4 in isolated liver nuclei (100). Overall, the production of superoxide may be increased when G6PD activity is increased or decreased when there is limited G6PD activity.

Suppression of myocardial ROS generation in response to pharmacological G6PD inhibition suggests that inhibiting G6PD may also decrease the generation of ROS in vivo (32, 35) and thus suggests a potential therapeutic approach for treating heart failure patients (33, 34). Agents that inhibit G6PD include 6-aminonicotinamide and dehydroepiandrosterone (DHEA). 6-Aminonicotinamide is not amenable to in vivo use due to toxic side effects. DHEA is a naturally occurring hormone with pleiotropic effects such as an inhibition of growth that may be unrelated to the inhibition of G6PD (8, 33, 73, 81). Although DHEA inhibits G6PD activity, it may also increase the transcription of G6PD that confounds its inhibitory effect (35, 43, 73) and makes it impossible to attribute and observed effects to G6PD inhibition. Taken together, current pharmacological inhibitors of G6PD are inadequate for evaluating the long-term cardiovascular effects of G6PD inhibition.

It is important to note that most of the studies that showed decreased superoxide production in response to G6PD inhibition did not examine the balance between decreased superoxide production and GSH capacity. This is important because although G6PD deficiency could be beneficial if it decreases excessive superoxide production (18, 37, 41, 42, 49, 110), G6PD deficiency could still have adverse cardiac effects if it lowers antioxidant defenses (39, 44, 45). Furthermore, it should be kept in mind that an increase in superoxide production does not always adversely affect cardiac function (38, 66). In fact, superoxide is a signaling molecule that is necessary for vascular function, and Nox4 deficiency, which decreases superoxide levels, decreased angiogenesis and accelerated the development of heart failure (1, 26, 77, 95, 113). In light of the recent finding that Nox4-derived superoxide is necessary for vascular growth (89, 95, 113), others found that G6PD may fuel Nox4-derived superoxide production (100). Furthermore, we found decreased superoxide and capillary density in failing G6PD-deficient myocardium (39). Overall, the finding that limiting G6PD activity also decreases superoxide production does not necessarily mean that this is a beneficial effect of G6PD deficiency.

G6PD deficiency and cardiac ischemia-reperfusion.

Because G6PD deficiency decreases myocardial antioxidant capacity, it may exacerbate the adverse cardiac effects of acute oxidative stress such as with ischemia-reperfusion injury (92). Isolated perfused hearts from G6PDX mice displayed a greater impairment in relaxation and pressure development after injury compared with WT mice (Fig. 5) (45). In these experiments, acute ischemia-reperfusion injury increased G6PD activity in WT mice, whereas G6PD-deficient myocardium was unable to compensate for the increased oxidative stress as indicated by a decreased GSH/GSSG ratio in these hearts (45). The exacerbated impairments in relaxation and pressure development in G6PDX mice were rescued by antioxidant treatment, but not by ribose supplementation, demonstrating that the deficit was due to a deficiency in antioxidant defenses rather than a ribose deficiency resultant from the nonoxidative phase of the pentose phosphate pathway. Thus G6PD deficiency increases the sensitivity of the myocardium for ROS and exacerbates contractile dysfunction in the face of oxidative injury.

Heart failure and G6PD deficiency.

We recently tested the effects of G6PD deficiency on the development of heart failure and found that G6PD deficiency exacerbated cardiac remodeling after chronic stress (39). G6PDX mice were subjected to 6 wk of pressure overload or to myocardial infarction with 12 wk of follow-up. In these experiments, G6PD deficiency worsened left ventricular dilation (Fig. 6) without affecting the overall heart mass or diastolic or systolic function. We further stressed pressure-overloaded mice with a high fructose diet and found that in this case, G6PD deficiency increased systolic dysfunction, fetal gene expression, and hypertrophy among high fructose-fed mice. Thus G6PD deficiency adversely affected the development of left ventricular chamber expansion and heart failure.

Interestingly, in these studies NADPH levels were increased in G6PD-deficient myocardium in response to either infarction or pressure overload (39), suggesting that there is a compensatory mechanism that increases NADPH levels in G6PD deficiency. This compensatory increase in NADPH levels could be due to an increase in mitochondrial NADPH-producing enzymes and may not affect the cytoplasmic pool of NADPH levels. In this regard, superoxide production was decreased in G6PDX mice, possibly due to decreased cytoplasmic availability of NADPH for use by Nox. However, despite the compensatory increase in NADPH levels and the decrease in superoxide production, G6PD-deficient myocardium had increased oxidative stress in response to pressure overload as indicated by a decrease in GSH, indicating an adverse effect of G6PD deficiency.

In the myocardial infarction model of heart failure described above, there was no effect of G6PD deficiency on superoxide production, but there was an increase in lipid peroxidation products in G6PDX mice, indicating increased oxidative stress in this heart failure model as well (39). A possible explanation for the increased oxidative stress despite there being an increase in NADPH levels and no overall change in superoxide generation in this case is that G6PD-deficient myocardium are unable to fully alleviate oxidative stress. Here it should be considered that the measured level of NADPH does not necessarily reflect the flux through G6PD or the rate of production of NADPH but simply reflects the concentration of NADPH at equilibrium. Also, measures of G6PD activity are generally reflective only of the amount of enzyme, rather than the activity within the cell, because at physiological concentrations of NADPH, G6PD is almost completely inhibited (22). Thus activation of G6PD is a matter of de-repression of the enzyme. In the 1970s, Eggleston and Krebs found that GSSG de-represses G6PD through the activation of some cofactor (22, 76). Although their data have been challenged, the activation of G6PD by GSSG remains an intriguing possibility (56). If GSSG activates G6PD, then it may take high levels of GSSG to sufficiently activate G6PD to counterbalance oxidative stress in G6PD-deficient myocardium. This would result in greater oxidative stress as observed by a decreased GSH/GSSG ratio, but not necessarily an increase in the production of ROS. This change in the levels of GSH/GSSG (and NADPH) may result in aberrant signaling and the adverse changes that we observed with G6PD deficiency with heart failure (11, 14, 16, 60, 85, 106).

Beneficial effects of pharmacologic G6PD activation in heart failure.

Benfotiamine is a vitamin B₁ analog that activates the pentose phosphate pathway by increasing the activity of G6PD and transketolase (46). A recent investigation in a mouse model of myocardial infarction found administration of benfotiamine for 4 wk before coronary ligation increased G6PD activity; decreased infarct size and oxidative stress; and improved survival, capillary density, blood flow, and functional parameters in the heart (46). Other studies found benfotiamine reduced diastolic dysfunction associated with diabetes (13, 46, 47). In cardiomyocytes exposed to hypoxia, silencing of G6PD prevented antiapoptotic signaling in response to benfotiamine, indicating that the beneficial effects of benfotiamine on cardiomyocytes may be due to increased activation of G6PD. Overall, these studies show that the activation of G6PD may be beneficial in heart failure.

G6PD deficiency and vascular dysfunction.

The pro- and antioxidant effects of G6PD also play an important role in vascular redox homeostasis and vascular reactivity. In the vascular endothelium, G6PD deficiency increases oxidant stress and decreases bioavailable nitric oxide (52). In vitro studies in human coronary artery endothelial cells and in vivo studies in G6PDX mice have shown that this occurs as a result of uncoupling of endothelial nitric oxide synthase (eNOS) to increase ROS formation and is associated with a decrease in GSH levels and the GSH/GSSG ratio (52, 53). G6PD deficiency is also associated with a decrease in nitric oxide levels owing to increased consumption by ROS as well as a decrease in eNOS activity owing to limited NADPH stores (52). In contrast, overexpression of G6PD limits endothelial oxidant stress and increases nitric oxide levels (55). With the use of intravital videomicroscopy, G6PDX mice were shown to have an impaired vasodilator response to the endothelium-dependent vasodilator acetylcholine (53). These findings were supported in a small study of G6PD-deficient individuals that examined endothelial function using brachial artery vascular reactivity. When compared with age-matched controls, G6PD-deficient subjects demonstrated impaired forearm blood flow responses, and this was associated with increased levels of 8-isoprostanes, a marker of systemic oxidant stress (25).

Elevated levels of aldosterone, which is a steroid hormone that is structurally similar to DHEA, decrease endothelial G6PD activity leading to endothelial dysfunction (53). This acquired G6PD-deficient state is associated with eNOS uncoupling and eNOS-derived ROS formation, decreased GSH levels and GSH/GSSG ratio, and an increase in peroxynitrite formation. Aldosterone decreased endothelium-dependent vasodilation in WT mice but had no further effect on G6PDX mice and G6PD activity, and vasodilator responses were restored with vascular gene transfer of G6PD (53). The increase in ROS associated with this acquired G6PD-deficient state was also found to inhibit cGMP formation through a mechanism that involved oxidative posttranslational modification of guanylyl cyclase (61). Taken together, these findings indicate that G6PD deficiency has important consequences for nitric oxide-mediated vasodilator responses.

Vascular contraction with potassium chloride or amphotericin B leads to an increase in G6PD activity that occurs in a protein kinase Cδ-dependent manner (30). Under these conditions, inhibition of G6PD decreased ROS and contractility. Aortas from G6PDX mice were also found to have a decrease in potassium chloride-evoked force compared with WT mice (30). Further work revealed that G6PD inhibition also modulates calcium stores to promote precontracted vascular smooth muscle relaxation by decreasing calcium influx and increasing sequestration as well as inhibiting Rho kinase (2). Thus, in the contractile state, G6PD deficiency decreases oxidant stress and promotes vascular relaxation.