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. 2012 Feb;32(4):740-50.
doi: 10.1128/MCB.06470-11. Epub 2011 Dec 12.

Myocardial ATGL Overexpression Decreases the Reliance on Fatty Acid Oxidation and Protects Against Pressure Overload-Induced Cardiac Dysfunction

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Myocardial ATGL Overexpression Decreases the Reliance on Fatty Acid Oxidation and Protects Against Pressure Overload-Induced Cardiac Dysfunction

Petra C Kienesberger et al. Mol Cell Biol. .
Free PMC article

Abstract

Alterations in myocardial triacylglycerol content have been associated with poor left ventricular function, suggesting that enzymes involved in myocardial triacylglycerol metabolism play an important role in regulating contractile function. Myocardial triacylglycerol catabolism is mediated by adipose triglyceride lipase (ATGL), which is rate limiting for triacylglycerol hydrolysis. To address the influence of triacylglycerol hydrolysis on myocardial energy metabolism and function, we utilized mice with cardiomyocyte-specific ATGL overexpression (MHC-ATGL). Biochemical examination of MHC-ATGL hearts revealed chronically reduced myocardial triacylglycerol content but unchanged levels of long-chain acyl coenzyme A esters, ceramides, and diacylglycerols. Surprisingly, fatty acid oxidation rates were decreased in ex vivo perfused working hearts from MHC-ATGL mice, which was compensated by increased rates of glucose oxidation. Interestingly, reduced myocardial triacylglycerol content was associated with moderately enhanced in vivo systolic function in MHC-ATGL mice and increased isoproterenol-induced cell shortening of isolated primary cardiomyocytes. Most importantly, MHC-ATGL mice were protected from pressure overload-induced systolic dysfunction and detrimental structural remodeling following transverse aortic constriction. Overall, this study shows that ATGL overexpression is sufficient to alter myocardial energy metabolism and improve cardiac function.

Figures

Fig 1
Fig 1
Biochemical and histological characterization of WT and MHC-ATGL hearts. (A) Cardiac Pnpla2 (Atgl) mRNA expression in 4- to 5-h-fasted male mice (11 to 13 weeks old, n = 7; ***, P < 1 × 10−8). (B) Immunoblots showing ATGL protein expression in the heart and soleus muscle as well as PLIN5 expression in the heart from 4- to 5-h-fasted male mice (35 to 36 weeks old). The p85 subunit of PI3K (p85PI3K) served as the loading control. (C) Cytoplasmic TG hydrolase activity in heart homogenates from fed mice (mixed gender, 15 weeks old, n = 4; ***, P < 0.0001). (D) Myocardial TG content in fed and 16-h-fasted mice (mixed gender, 13 to 25 weeks old, n = 7 or 8 for fed, n = 3 for fasted; ***, P < 0.001 versus WT; #, P < 0.001 versus fed). (E) TG content in isolated ex vivo perfused working hearts from male mice (18 weeks old, n = 6 to 8; ***, P < 0.0001). Hearts were perfused for 60 min with 0.8 mmol/liter oleate and 5 mmol/liter glucose in the presence of 50 μU/ml insulin. (F) DG content in hearts from 5-h-fasted female mice (20 to 21 weeks old, n = 4 or 5). Myocardial ceramide content (G) and long-chain acyl-CoA content (H) of male 4- to 5-h-fasted mice (35 to 36 weeks old, n = 5). (I) Representative histological images of apical heart sections stained with hematoxylin and eosin (H&E) and Masson's trichrome (M.T.) stain at ×400 magnification. Scale bars indicate 30 μm.
Fig 2
Fig 2
Baseline in vivo cardiac function in WT and MHC-ATGL mice. (A to G) Functional parameters obtained by transthoracic echocardiography in male mice (31 to 33 weeks old, n = 6; *, P < 0.05; **, P < 0.01). (A) Representative M-mode images; (B) ejection fraction; (C) velocity of circumferential fiber shortening (Vcf); left ventricular internal diameter in diastole (LVIDd) (D) and in systole (LVIDs) (E); (F) interventricular septal thickness in diastole (IVSd); (G) left ventricular posterior wall thickness in diastole (LVPWd); (H) ratio of heart ventricle weight to tibia length in male mice (35 to 36 weeks old, n = 5).
Fig 3
Fig 3
Analysis of cardiomyocyte shortening and calcium homeostasis. (A and B) Cell shortening of isolated cardiomyocytes from female mice (21 to 26 weeks old, n = 8 to 13 cardiomyocytes from 3 mice per genotype). Cell shortening in the absence of isoproterenol (A) and percent increase in cell shortening when comparing stimulation with 100 nmol/liter isoproterenol to incubation without isoproterenol (B) (*, P < 0.05); insets show representative tracings of cell shortening. (C) Percent increase in calcium transients when comparing stimulation with 100 nmol/liter isoproterenol (+ISO) to incubation without isoproterenol (−ISO) (n = 7 or 8 cardiomyocytes from 2 female mice per genotype, 36 to 41 weeks old); insets show representative calcium tracings (ΔF, change in fluorescence intensity). (D) Immunoblots and densitometric analysis showing protein expression of SERCA2 and phospholamban (PLN) as well as phospholamban phosphorylation (P-PLN; ratio of phosphorylated PLN to total PLN) in hearts from male 4- to 5-h-fasted mice (35 to 36 weeks old). Ran GTPase served as the loading control.
Fig 4
Fig 4
Treadmill test and ex vivo work jump perfusion. (A) Maximum run time during treadmill stress test (10- to 12-week-old males, n = 5 or 6 mice; *, P < 0.05). (B and C) Hearts from female mice (13 to 15 weeks old, n = 4 or 5) were perfused in the working mode with 1.2 mmol/liter oleate, 5 mmol/liter glucose, and 50 μU/ml insulin for 30 min at 50 mmHg afterload pressure (normal workload). Subsequently, hearts were perfused with an increased afterload of 80 mmHg and perfusate containing isoproterenol (300 nmol/liter) for an additional 30 min (high workload). (B) Heart rate during normal and high workload perfusion (***, P < 0.001 versus normal workload); (C) peak systolic pressure (PSP) at the end of the high workload perfusion. The percentage of hearts that produced aortic outflow until the end of perfusion is indicated.
Fig 5
Fig 5
FA and glucose metabolism in WT and MHC-ATGL hearts. (A) Cardiac power; (B) palmitate oxidation rates; (C) glucose oxidation rates; (D) Krebs cycle acetyl-CoA production in ex vivo perfused hearts from male 33- to 35-week-old mice (n = 8; *, P < 0.05); (E) ATP content; (F) ATP-to-AMP ratio; (G) phosphocreatine-to-ATP ratio in ex vivo perfused hearts from male mice (17 to 18 weeks old, n = 5 to 8); (H) FA uptake in ex vivo perfused hearts from female mice (22 to 28 weeks old, n = 4; *, P < 0.05); (I) myocardial CD36 protein expression in male mice (13 to 16 weeks old, n = 5; **, P < 0.01); (J) myocardial mRNA expression of genes involved in FA metabolism (4- to 5-h-fasted, 11- to 13-week-old male mice, n = 7; *, P < 0.05; **, P < 0.01; ***, P < 0.001); (K) immunoblots and densitometric analysis of myocardial OXPHOS protein expression (4- to 5-h-fasted, 35- to 36-week-old male mice, n = 5).
Fig 6
Fig 6
In vivo heart function and cardiac morphometry following 5 weeks of TAC. (A) Ratio of ventricle weight to tibia length (n = 5 mice for sham, n = 6 to 10 mice for TAC; *, P < 0.05; **, P < 0.01). (B) Representative apical heart sections stained with hematoxylin and eosin visualized at ×400 magnification. Scale bars indicate 30 μm. (C) Cardiomyocyte cross-sectional area of 223 ± 9 myocytes from 4 to 6 mice per group was determined using hematoxylin- and eosin-stained heart sections (*, P < 0.05; ***, P < 0.001). (D) Left ventricular posterior wall thickness in diastole (LVPWd); (E) body weight; (F) heart rate; (G) ejection fraction; left ventricular internal diameter in systole (LVIDs) (H) and diastole (LVIDd) (I); (J) mRNA expression of hypertrophy marker genes; (K) myocardial TG content; (L) immunoblot analysis of STAT3 phosphorylation (ratio of phosphorylated STAT3 to total STAT3); (M) immunoblot analysis of SERCA2 and phospholamban (PLN) protein expression and PLN phosphorylation (ratio of phosphorylated PLN to total PLN) (n = 5 mice for sham, n = 6 to 10 mice for TAC; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

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