Alpha-ketoglutarate ameliorates pressure overload-induced chronic cardiac dysfunction in mice

Abstract

Increasing evidence indicates the involvement of myocardial oxidative injury and mitochondrial dysfunction in the pathophysiology of heart failure (HF). Alpha-ketoglutarate (AKG) is an intermediate metabolite of the tricarboxylic acid (TCA) cycle that participates in different cellular metabolic and regulatory pathways. The circulating concentration of AKG was found to decrease with ageing and is elevated after acute exercise and resistance exercise and in HF. Recent studies in experimental models have shown that dietary AKG reduces reactive oxygen species (ROS) production and systemic inflammatory cytokine levels, regulates metabolism, extends lifespan and delays the occurrence of age-related decline. However, the effects of AKG on HF remain unclear. In the present study, we explored the effects of AKG on left ventricular (LV) systolic function, the myocardial ROS content and mitophagy in mice with transverse aortic constriction (TAC). AKG supplementation inhibited pressure overload-induced myocardial hypertrophy and fibrosis and improved cardiac systolic dysfunction; in vitro, AKG decreased the Ang II-induced upregulation of β-MHC and ANP, reduced ROS production and cardiomyocyte apoptosis, and repaired Ang II-mediated injury to the mitochondrial membrane potential (MMP). These benefits of AKG in the TAC mice may have been obtained by enhanced mitophagy, which cleared damaged mitochondria. In summary, our study suggests that AKG improves myocardial hypertrophy remodelling, fibrosis and LV systolic dysfunction in the pressure-overloaded heart by promoting mitophagy to clear damaged mitochondria and reduce ROS production; thus, AKG may have therapeutic potential for HF.

Keywords: Alpha-ketoglutarate; Cardiac insufficiency; Mitophagy; Myocardial hypertrophy; Transverse aortic constriction.

Graphical abstract

Fig. 1

AKG attenuates cardiac hypertrophy in TAC mice. (A) The time-line diagram of AKG administration in TAC mice. (B) Heart weight-to-lung weight ratio(HW/LW). (C) Heart weight normalized to body weight(HW/BW). (D) Heart weight normalized to tibia length(HW/TL). (E) Circulating AKG concentration of mice. *: P＜0.05 vs sham, **: P＜0.01 vs sham, ***: P＜0.001 vs sham, #: P＜0.05 vs TAC, ##: P＜0.01 vs TAC, ###: P＜0.001 vs TAC. n = 10 each group.

Fig. 2

AKG alleviates myocardial hypertrophy induced by pressure overload. Myocardial hypertrophy reflected by (A) Heart photos, (B) HE staining in global and (C) in details, as well as (D) WGA staining(n = 9). (E)Protein expression of ANP and β-MHC in heart tissue(n = 5). (F) mRNA expression of Nppa, Nppb and Myh7 in heart tissue(n = 9); *: P＜0.05 vs sham, **: P＜0.01 vs sham, ***: P＜0.001 vs sham, #: P＜0.05 vs TAC, ##: P＜0.01 vs TAC, ###: P＜0.001 vs TAC.

Fig. 3

AKG improves pressure overload-induced LV remodelling and cardiac dysfunction. The echocardiography(ECG) was examined at 6w(A) and 10w(B) after surgery: (C)LV mass, (D)left ventricular anterior wall in diastole(LVAWd), (E)left ventricular posterior wall in diastole(LVPWd), (F)left ventricular internal dimension in diastole(LVIDd), (G) left ventricular internal dimension in systole(LVIDs), (H)fractional shortening(FS), (I)left ventricular diastolic volume(LVdVol), (J) left ventricular systolic volume(LVsVol), (K)ejection fraction(EF); *: P＜0.05 vs sham, **: P＜0.01 vs sham, ***: P＜0.001 vs sham, #: P＜0.05 vs TAC, ##: P＜0.01 vs TAC, ###: P＜0.001 vs TAC. n = 10 each group.

Fig. 4

AKG improves pressure overload-induced LV strain decline. The LV endocardial strain in long axis(A) and short axis(B) at 10w after surgery measured by Speckle tracking echocardiography: (C) LV endocardial radial strain(RS), longitudinal strain(LS) and maximum opposing wall delay in long axis, (D) LV endocardial radial strain(RS), circumferential strain(CS) and maximum opposing wall delay in short axis; *: P＜0.05 vs sham, **: P＜0.01 vs sham, ***: P＜0.001 vs sham, #: P＜0.05 vs TAC, ##: P＜0.01 vs TAC, ###: P＜0.001 vs TAC. n = 6 each group.

Fig. 5

AKG inhibits myocardial fibrosis in TAC mice. Collagen quantification was performed on Masson staining (A)in global and (B)in detail(n = 9), and (C)Sirius Red staining(n = 9). (D)Protein(n = 5) and (E)mRNA(n = 9) expression of TGF-β1; *: P＜0.05 vs sham, **: P＜0.01 vs sham, ***: P＜0.001 vs sham, #: P＜0.05 vs TAC, ##: P＜0.01 vs TAC, ###: P＜0.001 vs TAC. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Effect of AKG on myocardial ROS content and cellular apoptosis in TAC mice. (A)Dihydroethidium (DHE) staining to reflect myocardial ROS production(n = 9). (B)The apoptosis rate of cardiomyocytes by TUNEL assay(n = 9). (C)Effect of AKG on mRNA expression of PINK1, Parkin, caspase-3 and Bcl-2(n = 9). (D) Relative protein expression of PINK1, Parkin, caspase-3 and Bcl-2(n = 5); *: P＜0.05 vs sham, **: P＜0.01 vs sham, ***: P＜0.001 vs sham, #: P＜0.05 vs TAC, ##: P＜0.01 vs TAC, ###: P＜0.001 vs TAC. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Effect of AKG on mitophagy, MMP and ROS production in vitro. (A)TUNEL staining(n = 5). (B) Western blotting of β-MHC, PINK1, Parkin, caspase-3, Bcl-2 and ANP(n = 5). (C) mRNA expression levels of Nppa, Nppb, Myh7, Bcl-2, caspase-3, PINK1 and Parkin(n = 9). (D) Colocalization of Mitochondria and Lysosomes(n = 5). (E)TEM showing the effect of 2 mM AKG supplementation on mitochondrial morphology in cardiomyocytes. (F) DCFH-DA staining showing the effect of 2 mM AKG supplementation on ROS level(n = 5). (G)Effect of 2 mM AKG supplementation on MMP(n = 5); *: P＜0.05 vs sham, **: P＜0.01 vs sham, ***: P＜0.001 vs sham, #: P＜0.05 vs TAC, ##: P＜0.01 vs TAC, ###: P＜0.001 vs TAC. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

AKG supplementation promotes mitophagy to clear damaged mitochondria, reduces oxidative damage caused by ROS, attenuates myocardial hypertrophy and fibrosis caused by pressure overload, and improves cardiac insufficiency.