Mitochondrial NAD+/NADH Redox State and Diabetic Cardiomyopathy

Abstract

Significance: Diabetic cardiomyopathy (DCM) is a frequent complication occurring even in well-controlled asymptomatic diabetic patients, and it may advance to heart failure (HF). Recent Advances: The diabetic heart is characterized by a state of "metabolic rigidity" involving enhanced rates of fatty acid uptake and mitochondrial oxidation as the predominant energy source, and it exhibits mitochondrial electron transport chain defects. These alterations promote redox state changes evidenced by a decreased NAD⁺/NADH ratio associated with an increase in acetyl-CoA/CoA ratio. NAD⁺ is a co-substrate for deacetylases, sirtuins, and a critical molecule in metabolism and redox signaling; whereas acetyl-CoA promotes protein lysine acetylation, affecting mitochondrial integrity and causing epigenetic changes. Critical Issues: DCM lacks specific therapies with treatment only in later disease stages using standard, palliative HF interventions. Traditional therapy targeting neurohormonal signaling and hemodynamics failed to improve mortality rates. Though mitochondrial redox state changes occur in the heart with obesity and diabetes, how the mitochondrial NAD⁺/NADH redox couple connects the remodeled energy metabolism with mitochondrial and cytosolic antioxidant defense and nuclear epigenetic changes remains to be determined. Mitochondrial therapies targeting the mitochondrial NAD⁺/NADH redox ratio may alleviate cardiac dysfunction. Future Directions: Specific therapies must be supported by an optimal understanding of changes in mitochondrial redox state and how it influences other cellular compartments; this field has begun to surface as a therapeutic target for the diabetic heart. We propose an approach based on an alternate mitochondrial electron transport that normalizes the mitochondrial redox state and improves cardiac function in diabetes.

Keywords: NAD; NADH; cardiomyopathy; diabetes; mitochondria; redox balance.

FIG. 1.

Morphological and metabolic features of diabetic cardiomyopathy. Diabetic cardiomyopathy is defined by the presence of an initial decreased myocardial diastolic dysfunction [heart failure with preserved ejection fraction (55)] in the absence of diabetes-induced standard cardiac risk factors, and may evolve to systolic dysfunction (heart failure with reduced ejection fraction) and congestive heart failure. These functional abnormalities are progressively induced by thickness and stiffness of the ventricular wall (defect in cardiomyocyte relaxation, interstitial fibrosis, and cardiomyocyte hypertrophy), cardiomyocyte death, and eventually contractile dysfunction. The heart relies on a large and constant energy supplied by oxidative metabolism. The normal heart is free to switch fuel substrates for oxidation and ATP synthesis to respond to energy demands, whereas the diabetic heart is insulin resistant and metabolically inflexible experiencing a decrease in glucose oxidation, and an increase in FA oxidation. Diabetic cardiomyopathy is considered a disease of the myocardial energetic metabolism characterized by a decreased efficiency of oxygen utilization induced by mitochondrial dysfunction. Healthy mitochondria release minimal ROS levels that are compatible with the physiological signaling, whereas the ROS flux released by diabetic cardiac mitochondria is reported to be increased by most studies. Other critical processes that are interrelated with mitochondrial failure are changes in the redox environment (described in this review) and Ca²⁺ handling (not shown in the picture). FA, fatty acids; LV, left ventricle; ROS, reactive oxygen species.

FIG. 2.

Metabolism of fuel substrates drives the levels of reduced equivalents (NADH and NADPH) in normal cardiomyocytes. Normal adult heart obtains 40% of energy from metabolism of glucose, lactate, and ketones (mainly βHB), with the remaining 60% delivered from FA oxidation (55). Glucose uptake into cardiomyocytes is insulin dependent, whereas FA and βHB uptake is not hormonally regulated, and is driven by their bloodstream availability (FA_circ, βHB_circ) (17, 147). Glucose enters cardiomyocytes predominantly via the insulin-dependent glucose transporter 4 (GLUT4), and it follows multiple metabolic pathways including glycolysis, glycogen synthesis, and polyol pathway (with sorbitol and fructose formation), or is shuttled into the hexosamine biosynthetic or pentose phosphate pathways (with 6-Phosphogluconate, 6-PG, and ribulose 5-phosphate formation). Pyruvate is either converted to lactate or transported into mitochondria via the MPC, and converted by PDH to acetyl-CoA (AcCoA) for the TCA cycle. PDH is inhibited by pyruvate kinase (PDK4) that is activated by an excess of AcCoA and NADH. For simplicity, fluxes through glycogen synthesis and hexosamine pathways are not shown. After entry into cardiomyocytes (via CD36 and FA translocase, FAT), long chain FA are activated to FA-CoA that is either esterified as triacylglycerol (stored in the cytosol, not shown) or enters the mitochondria via carnitine palmitoyltransferases (CPT1 and 2), and they are oxidized via FA β-oxidation. The end products of each FA β-oxidation cycle are NADH, FADH₂, and acetyl-CoA, which are further oxidized by ETC complexes or TCA, respectively, ultimately leading to ATP synthesis via mitochondrial oxidative phosphorylation. FA β-oxidation is controlled at different steps, including the inhibitory effect of malonylCoA (formed from AcCoA via AcCoA carboxylase, ACC), FADH₂/FAD⁺ and NADH/NAD⁺ redox ratios, and acetyl-CoA/CoA ratio, all of which are unfavorable to FA oxidation. MalonylCoA is degraded by MCD, thus releasing its inhibitory effect on CPT1. The control of FA β-oxidation provided by post-translational modifications of β-oxidation enzymes and their transcriptional regulation is not depicted in the figure. βHB is the main ketone body utilized by the heart as an energy fuel. Produced by the liver at rates that are proportional to FA oxidation and NADH/NAD⁺ ratio, βHB's cardiomyocyte uptake is facilitated by the monocarboxylate transporter 1 (MCT1). Within mitochondria, βHB is oxidized by mitochondrial βHB dehydrogenase (BDH1) to acetoacetate (AcAc) that is converted to acetoacetyl-CoA (AcAc-CoA) by the enzyme succinyl-CoA:3 oxoacid-CoA transferase (SCOT). AcAc-CoA is then converted to acetyl-CoA for TCA cycle. Mitochondrial NADH/NAD⁺ redox is unfavorable to the βHB oxidative flux (147). Cardiac mitochondria can also fully metabolize branched chain amino acids (leucine, isoleucine, and valine), providing acetyl-CoA for the TCA cycle and succinyl-CoA for anaplerosis (not depicted in the figure). TCA cycle is a source of reducing equivalents in the form of NADH and NADPH. The figure depicts other sources of reducing equivalents. GDH converts glutamate to α-ketoglutarate that is coupled to either NAD⁺ or NADP⁺ reduction. Mitochondrial isoforms of MEs also can reduce NADP⁺ to NADPH. Mitochondrial oxidative phosphorylation provides more than 95% of the cardiac ATP (55), with the remainder derived from glycolysis. Electrons are transferred from the reducing equivalents, NADH and FADH2, to oxygen by the ETC complexes, whereas an electrochemical gradient is built across the mitochondrial IM, which is used by the ATP synthase (complex V) to form ATP. Mitochondrial-generated ATP is transferred to the cytosol by the mitochondrial and cytosolic creatine kinases (CK) for contractile apparatus, sarcoplasmic reticulum Ca^2+-ATPase, and other ion pumps. Components of the contractile apparatus and calcium handling are affected by oxidative damage (9, 10), supporting a close link between mitochondrial bioenergetics, redox state, and myocardial contraction. The inset represents an electron micrograph of cardiac muscle showing mouse interfibrillar mitochondria. For simplicity, the nicotinamide nucleotide transhydrogenase, a mitochondrial IM enzyme that reduces NADPH⁺ by oxidizing NADH and using the mitochondrial proton motive force, is not shown in this figure. The reduced NADH and NADPH are shown in red. βHB, β-hydroxybutyrate; CPT1, carnitine palmitoyltransferase 1; ETC, electron transport chain; GDH, glutamate dehydrogenase; IM, inner membrane; MCD, malonylCoA decarboxylase; ME, malic enzyme; MPC, mitochondrial pyruvate carrier; PDH, pyruvate dehydrogenase; TCA, tricarboxylic acid cycle. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

The four main redox couples governing the redox balance in cardiac mitochondria (NAD⁺/NADH, NADP⁺/NADPH, GSH/GSSG, and TrxSH2/TrxSS) are hinged by NNT. Normal cardiomyocytes maintain a constant NAD pool mostly by converting biosynthetic precursors via the salvage pathway rather than de novo synthesis [not shown in the figure, revised in (83)]. Mitochondrial NAD content is increased by the import of cytosolic NAD through hypothetical transport mechanisms (35) that were identified in many species but not in mammals. However, it is believed that a bi-directional mitochondrial-cytosol transport system exists for both NAD and its precursors because exogenously added NAD leads to a greater accumulation in mitochondria than the cytosol (145). NAD⁺ is both a coenzyme for redox enzymes and an enzyme substrate for nonredox reactions in which the adenine diphosphate ribose moiety of NAD⁺ is cleaved, leading to the depletion of the NAD pool (not shown). Nicotinamide adenine nucleotide (NAD⁺) is a phosphate acceptor being converted to the phosphorylated form, NADP, via the enzyme NAD kinase (120). Only the cytosolic isoform of the NAD kinase is depicted in the figure. Therefore, NAD⁺ is a precursor for NADP. Both NAD⁺ and NADP⁺ are hybrid acceptors, and are converted to the reduced forms, NADH and NADPH. NADH transfers reducing equivalents that are derived from fuel oxidation, and, therefore, the NAD⁺/NADH couple is critical for energy generation. The NADPH/NADP⁺ redox couple is central to anabolism and antioxidant defense by donating electrons to glutathione (GSH/GSSG) and thioredoxin [Trx(SH)2/TrxSS)] systems, both of which are critical in scavenging H₂O₂ (generated from superoxide, O^•₂, by dismutation) via the enzymes GR, GPx, and thioredoxin reductase-Prx, respectively. Mitochondrial antioxidant system is mirrored by a similar scavenging mechanism in the cytosol. In these reactions, the reduced and oxidized members of the redox couples interconvert but are not consumed. Unlike these antioxidant mechanisms, catalase, which also scavenges H₂O₂, does not require reducing equivalents from NADPH for its function. Mitochondrial NADH/NAD⁺ and NADPH/NADP⁺ redox couples are linked by the enzyme NNT that reduces NADP⁺ at the expense of NADH oxidation and utilizing the mitochondrial IM proton motive force to drive this process. NNT maintains mitochondrial matrix NADPH/NADP⁺ pool in a reduced form, and it is a physiologically relevant source of NADPH to drive the enzymatic degradation of H₂O₂. The figure shows that the mitochondrial redox state of the NADH/NAD⁺ and NADPH/NADP⁺ redox couples are maintained different as the nucleotides have different metabolic roles. The NADH/NAD⁺ pool supports the divergent transfer of electrons from fuel substrates to both the ETC and antioxidant system via NNT, and thus is only partially reduced in comparison to NADPH/NADP⁺. NNT maintains the NADPH/NADP⁺ ratio several fold higher than the NADH/NAD⁺ (161). Cytosolic NADPH is regenerated via the pentose phosphate pathway, and redox reactions are catalyzed by isocitrate dehydrogenase, malic enzyme, aldehyde dehydrogenase, and methylene tetrahydropholate dehydrogenase (115). The cytosolic NADH is imported in mitochondria by redox shuttles, most commonly the M-A and glycerol 3 phosphate shuttles (G3P). GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, glutathione disulfide; H₂O₂, hydrogen peroxide; NNT, nicotinamide nucleotide transhydrogenase; M-A, malate-aspartate; Prx, peroxiredoxin. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Maintenance of mitochondrial and extramitochondrial NAD pools. NAD occurs as either oxidized (NAD⁺) or reduced (NADH) forms. In the extramitochondrial compartment, NAD is a co-substrate for enzymes, including PARPs, sirtuins (SIRT 1, 2, 5, 6, 7), and cyclic ADP-ribose (cADPR) synthases (CD38, CD157). These enzymes decrease the extramitochondrial NAD pool by continuously degrading NAD to NAM. The major mitochondrial NAD consumers are sirtuins 3, 4, and 5. The NAD biosynthetic pathway maintains a stable NAD subcellular concentration, and it relies on precursors such as dietary NA, tryptophan, NAM, and NR. The latter two may be also administered exogenously. NAD salvage pathway recycles the NAM generated as a by-product of the NAD-consuming enzyme activities. Transformation of NAM to NMN is catalyzed by nicotinamide phosphoribosyltransferases (NAMPT 1, 2, and 3), which are rate limiting in the salvage pathway. Conversion of NMN to NAD is then catalyzed by NMN adenylyltransferases (NMNAT 1, 2, and 3). As the mitochondrial membrane is impermeable for NADH, the reducing equivalents generated in the cytosol are transferred to the mitochondria via redox shuttles. Although the NMNAT3 isoform is present in mitochondria, the mitochondrial NAD salvage pathway has not been fully elucidated. Exogenous NAD increases mitochondrial function, but a mammalian NAD transporter has yet to be found. NNT reduces NADP⁺ to NADPH at the expense of NADH oxidation. NA, nicotinic acid; NAM, nicotinamide; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside; PARP, poly(ADP-ribose) polymerase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

Excess of glucose and fatty acids changes the NAD⁺/NADH ratio in cardiomyocytes in different compartments. Glucose is taken up by cardiomyocytes via GLUT1 and GLUT4, with the latter being responsible for the insulin-dependent glucose uptake. As insulin activity is decreased and GLUT-1 is responsible for the bulk of basal glucose uptake, circulating hyperglycemia leads to an increase in the GLUT1-mediated insulin-independent glucose uptake by the diabetic heart. There is a metabolic shift away from glycolysis and pyruvate oxidation (164), and, therefore, these pathways are not responsible for changes in the mitochondrial acetyl-CoA/CoA and NAD⁺/NADH ratios in diabetes. In the cytosol, glucose follows alternative non-ATP-producing pathways such as polyol and advanced glycation end products formation (131, 156, 187), and activation of protein kinase C (193) and hexosamine pathways (101, 149). Glucose conversion to sorbitol (via aldose reductase, AR, with NADPH oxidation) and then to fructose (via sorbitol dehydrogenase, SD, with NAD⁺ reduction), although extensively studied in microvascular complications, has no established role in the diabetic myocardium. Diversion of the glycolytic intermediates to pathways others than full oxidation and ATP production is favored by the inhibition of the glycolytic enzyme GAPDH by oxidation (60), NADH (31), and polyADP-ribosylation (172), thus creating an amplification loop, depleting the cytosolic NAD pool, and inhibiting cytosolic NAD⁺-dependent SIRT1. In mitochondria, increased FA oxidation and ETC defects (i.e., complex I defect) cause an increase in NADH and acetyl-CoA, which favor the decreased activity of mitochondrial SIRT3. The cytosolic and mitochondrial NAD pools communicate via a potentially bi-directional NAD transporter on the mitochondrial IM as the OM is freely permeable for small molecules. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OM, outer membrane. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Potential therapeutic targets to normalize the NAD⁺/NADH ratio in the heart during excess energetic supply. The irreversible inhibition (X) of CPT1 with etomoxir proved efficacious in DM patients but was stopped due to side effects (87). MalonylCoA inhibits CPT1, but it is degraded by MCD. MCD inhibitors reduce FA oxidation, increase glucose oxidation, and improve insulin sensitivity (179). Further, MCD-deficient mice were protected against insulin resistance on HFD (190), suggesting that MCD is an important therapeutic target to balance cardiac metabolism in DM, but its benefit has not been evaluated clinically. Trimetazidine, an inhibitor of 3-ketoacyl-CoA thiolase, the last enzymatic step in FA oxidation, increases glucose oxidation (119) and is cardioprotective in diet-induced obese (191) and DM (116) mice, in addition to improving cardiac function in diabetic patients with cardiomyopathy (204). A novel therapeutic approach in diabetes is the use of mitochondrial alternative electron carriers to normalize the NAD⁺/NADH redox ratio, decrease lysine acetylation, and alleviate the metabolic rigidity and cardiac dysfunction of the diabetic heart (21). The figure shows a proposed mechanism for the lysine deacetylating effect of methylene blue (MB). Diabetic-induced complex I defect causes a decrease in NADH oxidation. In experimental models of rotenone-induced complex I defect, MB accepts electrons from catalytic subunits of complex I and becomes reduced (MBH2) whereas cytochrome c reoxidizes MBH2 to MB [20]. Therefore, MB provides an alternative electron route within the diabetic complex I-deficient cardiac mitochondria and favors NADH oxidation, thus increasing NAD⁺ and SIRT3 activity. The administration of exogenous NAD or precursors (+) improved the mitochondrial NAD pool and cardiac function (discussed in the main text). DM, diabetes mellitus; HFD, high fat diet. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars