Fumarate is cardioprotective via activation of the Nrf2 antioxidant pathway

Abstract

The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.

Figure 1

A Summary of Metabolic Pathways Relating to the CAC Carbons derived from amino acids contribute to citric acid cycle (CAC) flux (anaplerosis), reducing NAD+ to NADH and ultimately yielding ATP. To maintain this energy-yielding flow, especially in the context of CAC interruption and during hypoxia/ischemia, carbon moieties must exit the CAC, e.g., through succinate and succinyl CoA.

Figure 2

Fh1 KO Hearts Are Anatomically and Physiologically Comparable to Controls (A) Fh1^fl/fl mice (Pollard et al., 2007) were crossed with mice expressing Cre recombinase under the promoter of myosin light chain, (MLC2v)-Cre (Chen et al., 1998), to generate heterozygous and homozygous knockout (KO) mice. (B) Assessed at 5–6 weeks of age, Fh1 KO hearts had substantially reduced Fh1 protein levels as demonstrated by the representative immunoblots and densitometric analysis (29% of controls). (C) Isolated cardiomyocytes in Fh1 KO had complete depletion of Fh1 protein. (D and E) Fh1 KO hearts were comparable to controls, with no evidence of cellular hypertrophy as assessed by wheat germ agglutinin (WGA) and gross hypertrophy. (F) Systolic cardiac function as assessed by echocardiographic left ventricular ejection fraction was comparable between KO and control hearts. (G and H) Invasive assessment of blood pressure and of contractile function revealed that Fh1 KO hearts were comparable to controls except with respect to LVEDP (H), which decreased in Fh1 KO hearts with increasing doses of dobutamine. ○, controls; x, HET KO; ●, Fh1 KO. (I) Fh1 KO hearts exhibited comparable baseline energetics to controls as assessed by ³¹P-MRS. Values are mean ± SEM. ^∗p < 0.05 versus Fh11^fl/fl mice.

Figure 3

Cardiac Fh1 Deficiency Confers Cardioprotection (A) Fh1-KO exhibited significant attenuation of myocardial necrosis (determined with TTC staining) compared with control hearts when perfused with 40 min of no-flow ischemia followed by 120 min of reperfusion. (B) This was accompanied by substantially better recovery in coronary flow. (C and D) This reduction in myocardial necrosis was confirmed by a significant reduction in markers of cardiac injury (cardiac troponin I, TnI; and creatine kinase, CK). (E) Functional recovery as assessed by the rate pressure product was comparable in the Fh1-KOs and controls. (F–K) Myocardial microdialysis coupled to an ultraperformance liquid chromatography/electrospray-tandem mass spectrometry approach demonstrated differences in the interstitial concentrations of succinate, glutamate, and adenosine during stabilization, ischemia, and reperfusion between Fh1-KO and control hearts. Fh1-KO (●) and controls (○). Values are mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01 versus control; ^∗∗∗p < 0.001 versus control.

Figure 4

Fh1 Deficiency Confers Cardioprotection through Both Increasing Carbon Flux and Upregulating Heme Oxygenase 1 by Nrf2 (A) In silico modeling of optimal metabolic flux distributions for maximal ATP production in Fh1-deficient hearts. In contrast to the normal heart (Figure S3), reconstituting the CAC around absent Fh1 requires amino acids (e.g., glutamate and branched chain amino acids) to be fed into the first span of the CAC and aspartate, via the malate-asparate shuttle, into the second span of the CAC. This anaplerotic carbon flux is balanced by cataplerosis through succinate export and porphyrin synthesis-heme degradation channeled through Hmox1. (B) Nrf2 target genes and the genes coding for the related antioxidant enzymes of one-carbon metabolism are significantly upregulated in Fh1-KO hearts as assessed by Taqman qRT-PCR. White bars, control; black bars, Fh1-KO. (C) Representative immunoblots and densitometric analysis of Fh1-KO hearts demonstrating the downregulation of Keap1 and upregulation of Nrf2, Hmox1, Nqo1, Mthfdh2, Gsta3, and Gsta1. (D) Fh1-KO hearts exhibit significantly increased levels of succination compared to WT. (E) Hmox1 inhibition by ZnBG abrogates cardioprotection in Fh1-KO hearts as assessed by the extent of necrosis resulting from ex vivo cardiac I/R. Values are mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01; and ^∗∗∗p < 0.001 versus control.

Figure 5

Administration of Fumarate Attenuates Ischemia-Reperfusion Injury (A) Representative immunoblots and densitometric analysis of murine hearts gavaged with dimethylfumarate (DMF) at a dose of 15 mg/kg twice daily for 5 days, demonstrating the upregulation of Nrf2 (left). DMF (10 μM) for 6 hr promoted nuclear translocation and localization of Nrf2 in HL-1 cells as assessed by confocal microscopy (right). (B) Oral DMF upregulates Nrf2 target genes as assessed by Taqman qPCR. (C) Representative immunoblots (upper panel) and densitometric analysis (lower panel) of protein products of Nrf2 targets. (D and E) Oral DMF treatment significantly attenuated myocardial necrosis resulting from ex vivo cardiac I/R and (E) was accompanied by an improved recovery in coronary flow. (F) To assess the cardioprotective effect of fumarate in vivo and the pertinence of Nrf2 to this protection, a coronary artery ligation model of acute MI was applied to WT and Nrf2-KO mice both treated with DMF and vehicle (n = 12 in each of the four groups). The average surgical mortality of coronary ligation surgery was 24% and did not differ significantly across the groups. WT and Nrf2-KO mice treated with vehicle (WT/V and Nrf2-KO/V, respectively) or 5 days of DMF (15 mg/kg) twice daily (WT/F and Nrf2-KO/F, respectively) via oral gavage as above. Representative sections of the myocardium from each group are presented stained with 4% TTC, with the necrotic area represented in white, the area at risk (AAR) in red, and the nonischemic area in blue. There was no significant difference in the size of the underperfused area (AAR/LV) for each group (data not shown). While DMF reduced MI size in the WT animals treated with DMF compared to vehicle-treated controls (MI/AAR by ANOVA with Bonferroni post hoc comparison to vehicle-treated WT mice), there was no difference in the MI size in Nrf2-KO-treated DMF and vehicle-treated control mice, nor was there a difference in MI size as a percentage of the LV area. Values are mean ± SEM. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.