Cardiac-specific deletion of acetyl CoA carboxylase 2 prevents metabolic remodeling during pressure-overload hypertrophy

Abstract

Rationale: Decreased fatty acid oxidation (FAO) with increased reliance on glucose are hallmarks of metabolic remodeling that occurs in pathological cardiac hypertrophy and is associated with decreased myocardial energetics and impaired cardiac function. To date, it has not been tested whether prevention of the metabolic switch that occurs during the development of cardiac hypertrophy has unequivocal benefits on cardiac function and energetics.

Objective: Because malonyl CoA production via acetyl CoA carboxylase 2 (ACC2) inhibits the entry of long chain fatty acids into the mitochondria, we hypothesized that mice with a cardiac-specific deletion of ACC2 (ACC2H-/-) would maintain cardiac FAO and improve function and energetics during the development of pressure-overload hypertrophy.

Methods and results: ACC2 deletion led to a significant reduction in cardiac malonyl CoA levels. In isolated perfused heart experiments, left ventricular function and oxygen consumption were similar in ACC2H-/- mice despite an ≈60% increase in FAO compared with controls (CON). After 8 weeks of pressure overload via transverse aortic constriction (TAC), ACC2H-/- mice exhibited a substrate utilization profile similar to sham animals, whereas CON-TAC hearts had decreased FAO with increased glycolysis and anaplerosis. Myocardial energetics, assessed by 31P nuclear magnetic resonance spectroscopy, and cardiac function were maintained in ACC2H-/- after 8 weeks of TAC. Furthermore, ACC2H-/--TAC demonstrated an attenuation of cardiac hypertrophy with a significant reduction in fibrosis relative to CON-TAC.

Conclusions: These data suggest that reversion to the fetal metabolic profile in chronic pathological hypertrophy is associated with impaired myocardial function and energetics and maintenance of the inherent cardiac metabolic profile and mitochondrial oxidative capacity is a viable therapeutic strategy.

Figure 1. ACC2 Deletion is Cardiac-Specific and Leads to Decreased Malonyl CoA Levels

(A) Western blotting of ACC2 in heart in heterozygous and homozygous ACC2 floxed mice that are Cre⁻ (f/WT, f/f) or Cre⁺(−/+, −/−). C57BL6/J (C57) included as control. (B) Western blotting analysis of heart, gastrocnemius, and liver tissue in ACC2^f/f (CON) and ACC2H^−/−. (C) ACC1 mRNA levels. Values expressed as fold change over CON, n = 3 each group. (D) Malonyl CoA levels assessed by LCMS. *P < 0.05, n = 5 each group.

Figure 2. Effects of ACC2 Deletion on Cardiac Metabolism

(A) Relative contribution of fatty acids, glucose, and other substrates (lactate, endogenous) to the TCA cycle in hearts perfused with ¹³C-labeled substrates, n= 4–5 each group. (B) Cardiac triglyceride (TAG) content normalized to tissue weight, n = 5–7 each group. (C) Glycogen content normalized to tissue weight, n = 8–9 each group. (D) Fatty acylcarnitine species from heart extracts of 2 month old mice assessed by LCMS, n = 5 each group. (E) Expression of genes involved in glucose metabolism, lipid metabolism, and mitochondrial biogenesis reported as fold change over CON, n = 3–5 each group. *P < 0.05 vs. ACC2H^−/−.

Figure 3. Cardiac Fatty Acid Metabolism is Maintained with Normal Function and Morphology at 12 months of Age

(A) Relative contribution of glucose and fatty acids to TCA cycle in hearts perfused with ¹³C-labeled substrates. *P < 0.05 vs. CON, n= 4–5. (B,C,D,E) Fractional shortening; left ventricular wall thickness in diastole (LVPW;d); left ventricular internal diameter in diastole (LVID;d); and heart rate assessed by echocardiography in CON and ACC2H^−/− at 2 months (n=28–33 each group), 6 months (n=6–7 each group), and 12 months (n=8–9 each group) of age.

Figure 4. Myocardial Energetics and Cardiac Function of ACC2H^−/−

(A) Myocardial oxygen consumption assessed in isolated perfused hearts. Hearts were perfused with a buffer containing glucose and pyruvate followed by a buffer containing fatty acids, glucose, lactate, and insulin (mixed substrate). Serial measures were made in each heart, n = 3 each group. (B) Oxygen efficiency for contractile function, estimated as amount of oxygen used per unit of contractile performance (MVO₂/RPP), during perfusion with glucose and pyruvate or mixed substrate buffer. Serial measures were made in each heart, n=3 each group. (C,E,G) Phosphocreatine (PCr), ATP, and inorganic phosphate (Pi) by 31P NMR spectroscopy in isolated perfused hearts during baseline conditions and high workload conditions. *P < 0.05 vs. respective baseline, +P<0.05 vs. control at high workload, n = 6–10 each group. (D, E, F) LV developed pressure (LVDevP), heart rate (HR), and end-diastolic pressure (EDP) response of isolated perfused hearts subjected to a high workload challenge with 4mM Ca²⁺ for 20 min. **P < 0.05 vs. CON, n = 6 each group.

Figure 5. Metabolic Remodeling in Pressure-Overload Hypertrophy

(A) Relative contribution of glucose, fatty acids, and other substrates (lactate, endogenous) to TCA cycle in TAC hearts perfused with ¹³C-labeled substrates (n= 8–10). (B) Enrichment of ¹³C3-alanine of hearts perfused with ¹³C labeled glucose. Data presented as ratio of ¹³C3-alanine peak area to ¹³C1-glucose peak area (n = 8–10). (C) Enrichment of ¹³C3-lactate of hearts perfused with ¹³C labeled glucose. Data presented as ratio of ¹³C3-lactate peak area to ¹³C1-glucose peak area (n = 8–10). (D) Total anaplerosis determined from isotopomer analysis of C4 and C3 glutamate peaks in hearts perfused with ¹³C labeled substrates. Data presented as percent anaplerosis divided by TCA cycle flux (n = 8–10). (E) Heat map represents metabolites from glucose metabolism, TCA cycle intermediates, amino acids, and other significantly different metabolites in CON and ACC2H^−/− hearts 4 wks after TAC or sham surgery. Color coding for each metabolite was assigned using a log2 fold change versus the mean value of CON (n = 6 each group). *P < 0.05 vs. CON-sham; **P < 0.05 vs. CON-TAC,

Figure 6. Myocardial Energetics and Cardiac Function after Pressure-Overload Hypertrophy

(A) Phosphocreatine to ATP ratio (PCr/ATP) assessed by ³¹P NMR spectroscopy in isolated perfusion hearts (n = 8–10). (B) Rate pressure product (RPP, the product of LVDevP and heart rate) during isolated heart perfusion experiments (n= 8–10). (C) Fractional shortening (FS) % assessed by echocardiography before (BL) and at 4 and 8 weeks post-TAC (n=8–10). *P < 0.05 vs. CON-sham,

Figure 7. Reduced Hypertrophy and Fibrosis in ACC2H^−/− After 4 Weeks of Pressure-Overload

(A) Brain natriuretic peptide (BNP) mRNA values normalized to 18s and reported as fold change from CON-sham. *P < 0.05 vs. respective shams, n=5–8. (B) Heart weight normalized to tibial length (HW:TL) of CON and ACC2H^−/− undergoing sham or TAC surgery (n=5–8). (C) Myocyte cross sectional area in histological sections stained with Wheat Germ Agglutinin (WGA) to assess myocyte hypertrophy (n=2–3). (D) Percentage of fibrosis in histological sections stained with Masson’s Trichrome (n=2–3). (E) Representative images of WGA staining. (F) Representative images of Masson’s trichrome staining. *P < 0.05 vs. CON-sham, **P < 0.05 vs. CON-TAC.