Cholesterol lowering attenuates pressure overload-induced heart failure in mice with mild hypercholesterolemia

Normalisation of plasma lipoprotein cholesterol levels following AAV8-LDLr gene transfer in mice with mild hypercholesterolemia

Total and non-HDL, VLDL, IDL, LDL, and HDL plasma cholesterol (mg/dl) at day 10 following AAV8-null (controls) or AAV8-LDLr gene transfer in female C57BL/6 LDLr^-/- mice fed standard chow diet are summarized in Figure 1. AAV8-LDLr gene transfer decreased plasma cholesterol levels by 65.9% (p<0.0001) compared to controls. Non-HDL, VLDL, IDL, and LDL cholesterol levels were reduced by 77.4% (p<0.0001), 68.4% (p<0.0001), 80.7% (p<0.0001), and 77.8% (p<0.0001) in AAV8-LDLr mice in comparison to control mice. Compared to controls, HDL cholesterol levels were 22.4% (p<0.01) lower in AAV8-LDLr mice (Figure 1). Plasma cholesterol levels in control mice and in AAV8-LDLr mice remained unchanged for the full duration of the experiment (data not shown).

Decreased mortality in AAV8-LDLr TAC mice compared to control TAC mice

The TAC operation was executed at the age of 17 weeks. Mortality rate in AAV8-LDLr TAC mice during a follow-up period of 8 weeks was significantly lower than in control TAC mice (hazard ratio for mortality 0.423, 95% confidence interval 0.199 to 0.901) (Figure 2A). No mortality was observed in mice undergoing the sham procedure (data not shown).

Cholesterol lowering AAV8-LDLr gene transfer potently inhibits cardiac hypertrophy and reduces lung congestion after TAC

Body weight (Figure 2B) and tibia length (Figure 2C) were not significantly different between sham and TAC groups. Cardiac weight was increased by 2.29-fold (p<0.001) and by 2.14-fold (p<0.001) in control TAC mice and AAV8-LDLr TAC mice, respectively, compared to respective sham groups (Figure 2D). Heart weight was 14.2% (p<0.01) lower in AAV8-LDLr TAC mice than in control TAC mice (Figure 2D), indicating a marked reduction of the degree of cardiac hypertrophy induced by cholesterol lowering gene therapy. Hypertrophy of the left ventricle was attenuated in AAV8-LDLr TAC mice as evidenced by an 11.2% (p<0.05) reduction of left ventricular wall area (Figure 2E) and a significant decrease of the anterior wall thickness (p<0.05) (Figure 2F). Figure 3 contains representative Sirius Red-stained cross-sections of sham hearts and TAC hearts, which clearly illustrate that cardiac hypertrophy following pressure overload was attenuated in AAV8-LDLr TAC mice compared to control TAC mice. At the microscopic level, a significant increase of cardiomyocyte cross-sectional area was observed in TAC groups compared to respective sham groups (Figure 2G). Myocardial vessel density was decreased by 34.4% (p<0.001) and by 28.1% (p<0.001) in control TAC mice and in AAV8-LDLr TAC mice, respectively, compared to respective sham groups (Figure 2H). Figure 4 comprises representative photomicrographs of laminin-stained cardiomyocytes and of CD31-positive capillaries, which illustrate cardiomyocyte hypertrophy and capillary rarefaction after TAC. Cholesterol lowering gene therapy diminished lung congestion as evidenced by the 24.5% (p<0.01) reduction of lung weight in AAV8-LDLr TAC mice compared to control TAC mice (Figure 2I). Atherosclerosis was undetectable in the left anterior descending coronary artery of sham and TAC mice. Taken together, these data indicate that cholesterol lowering AAV8-LDLr gene transfer in C57BL/6 LDLr^-/- mice with mild hypercholesterolemia inhibits ventricular hypertrophy and counteracts left ventricular failure after TAC.

AAV8-LDLr gene therapy potently reduces pro-hypertrophic myocardial proteins in TAC mice

To investigate the anti-hypertrophic effects of cholesterol lowering gene therapy at the biochemical level, pro-hypertrophic myocardial proteins were quantified in TAC mice. Myocardial Akt (Figure 5A) and p-Akt (Figure 5B) protein levels were decreased by 26.7% (p<0.001) and by 34.1% (p<0.001), respectively, in AAV8-LDLr TAC mice compared to control TAC mice. Mammalian or mechanistic target of rapamycin (mTOR) (Figure 5C) and p-mTOR (Figure 5D) levels were reduced by 34.4% (p<0.001) and by 49.1% (p<0.001), respectively, in AAV8-LDLr TAC mice compared to control TAC mice. Myocardial protein levels of p38 mitogen-activated protein kinase (p38 MAPK) (Figure 5E) and of p-p38 MAPK (Figure 5F) were 24.7% (p<0.001) and 39.9% (p<0.001) lower, respectively, in AAV8-LDLr TAC mice than in control TAC mice. To verify that the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a reliable reference for normalizing protein levels, GAPDH and ß-tubulin protein expression levels were directly compared (Figure 5G). GAPDH/ß-tubulin ratios were nearly identical in the four different groups (Figure 5G). Representative western blot images are provided in Figure 5H.

Cholesterol lowering decreases apoptosis and reduces interstitial and perivascular myocardial fibrosis in mice subjected to pressure overload

Apoptosis in the myocardium of C57BL/6 LDLr^-/- mice was quantified using immunohistochemical staining of cleaved caspase-3. Cleaved caspase-3-positive cells were undetectable in myocardial tissue of sham mice (Figure 6A). Compared to control TAC mice, myocardial content of cleaved caspase-3-positive cells was decreased by 17.7% (p<0.05) in AAV8-LDLr TAC mice (Figure 6A). The extent of interstitial myocardial fibrosis (Figure 6B) and of perivascular myocardial fibrosis (Figure 6C) was reduced by 31.0% (p<0.001) and by 29.8% (p<0.001) lower, respectively, in AAV8-LDLr TAC mice, compared to control TAC mice. Figure 6D contains representative photomicrographs of Sirius Red-stained interstitial collagen viewed under polarized light illustrating reduced myocardial fibrosis following cholesterol lowering gene therapy.

The 25 kD isoform of transforming growth factor (TGF)-ß1 was reduced by 37.5% (p<0.001) in AAV8-LDLr TAC mice compared to control TAC mice (Figure 6E). Cholesterol lowering gene therapy in TAC mice decreased Smad2/3 (Figure 6F) and Smad4 (Figure 6G) by 38.7% (p<0.001) and by 37.2% (p<0.001), respectively. These data are illustrated by representative images of western blots in Figure 6H.

Cholesterol lowering gene therapy in C57BL/6 LDLr^-/- mice with mild hypercholesterolemia ameliorates cardiac function in the presence of pressure overload

Systolic cardiac function and diastolic cardiac function were not significantly dissimilar in AAV8-LDLr sham mice than in control sham mice (Table 1). Systolic cardiac function in AAV8-LDLr TAC mice was enhanced compared to control TAC mice as evidenced by a 1.18-fold (p<0.05) elevation of the peak rate of isovolumetric contraction (dP/dt_max). Cholesterol lowering gene therapy ameliorated diastolic function in TAC mice as evidenced by a 1.18-fold (p<0.05) increment of the absolute value of isovolumetric relaxation (dP/dt_min) and a 15.9% (p<0.05) decrease of the time constant of isovolumetric relaxation (Table 1).

AAV8-LDLr gene therapy in TAC mice reduces pro-oxidative enzymes, increases anti-oxidant defense systems, and decreases nitro-oxidative stress in the myocardium

Thiobarbituric acid reactive substances (TBARS) were 1.42-fold (p<0.05) increased in plasma of control TAC mice compared to control sham mice (Figure 7A). In contrast, no significant elevation of TBARS was discovered in AAV8-LDLr TAC mice compared to AAV8-LDLr sham mice. Xanthine oxidase activity in plasma was increased by 5.64-fold (p<0.001) and by 2.65-fold (p<0.001) in control TAC mice and in AAV8-LDLr TAC mice, respectively, compared to respective sham groups (Figure 7B). Xanthine oxidase activity was 53.1% (p<0.001) lower in AAV8-LDLr TAC mice than in control TAC mice. Myocardial NADPH oxidase 2 (Figure 7C) and NADPH oxidase 4 (Figure 7D) protein levels were reduced by 43.8% (p<0.001) and 44.8% (p<0.001), respectively, in AAV8-LDLr TAC mice compared to control TAC mice. Myocardial dismutase protein levels (Figure 7E) and plasma concentration of myocardial dismutase (Figure 7F) were 1.61-fold (p<0.01) and 1.43-fold (p<0.01) higher, respectively, in AAV8-LDLr TAC mice than in control TAC mice. Compared to respective sham groups, myocardial 3-nitrotyrosine-positive area (%) was increased by 4.45-fold (p<0.001) and by 2.03-fold (p<0.05) in control TAC mice and in AAV8-LDLr TAC mice, respectively. The 3-nitrotyrosine-positive area was 53.7% (p<0.01) lower in AAV8-LDLr TAC mice than in control TAC mice (Figure 7G), consistent with decreased nitro-oxidative stress.

Cholesterol lowering AAV8-LDLr gene therapy counteracts metabolic remodelling induced by pressure overload

Capillary glucose (Figure 8A) and plasma insulin (Figure 8B) levels were 23.1% (p<0.05) and 33.7% (p<0.05) lower, respectively, in control TAC mice than in control sham mice. Compared to control TAC mice, capillary glucose and plasma insulin levels were increased by 23.3% (p<0.05) and by 64.8% (p<0.05), respectively, in AAV8-LDLr TAC mice.

Myocardial uptake of glucose uptake was determined by micro-PET imaging using the tracer [¹⁸F]-fluorodeoxyglucose (FDG). Glucose uptake in the myocardium was significantly increased following TAC. The maximal standardized uptake value (SUV) (Figure 8C), the average SUV in voxels with a value above 50% of the maximal SUV (SUV 50%), and the average SUV in voxels with a value above 75% of the maximal SUV (SUV 75%) were significantly increased in control TAC mice (p<0.001) and in AAV8-LDLr TAC mice (p<0.05) compared to respective sham groups (Table 2). The total myocardial uptake of glucose was increased by 3.29-fold (p<0.001) and by 2.02-fold (p<0.05) in control TAC mice and in AAV8-LDLr TAC mice, respectively, compared to respective sham groups (Figure 8D). Glucose uptake in control TAC mice and in AAV8-LDLr TAC mice were highly similar. Differences in glucose uptake are illustrated by representative micro-PET images in Figure 8E.

To further evaluate the impact of cholesterol lowering on metabolic remodelling after TAC, metabolic myocardial proteins were quantified by western blot (Table 3). Cholesterol lowering AAV8-LDLr gene transfer in TAC mice decreased GLUT4 protein levels by 28.0% (p<0.001) and pyruvate dehydrogenase (PDH) levels by 30.0% (p<0.01) compared to control TAC mice. Myocardial protein levels of PDH kinase (PDHK), which leads to an inactivation of PDH, were decreased by 53.5% (p<0.01) in control TAC mice compared to control sham mice. PDHK levels were 2.10-fold (p<0.01) higher in AAV8-LDLr TAC mice than in control TAC mice (Table 3). Taken together, these data suggest that glucose oxidation may be lower in following cholesterol lowering AAV8-LDLr gene transfer in mice with pressure overload.

Myocardial protein levels of AMP-activated protein kinase (AMPK) and of p-AMPK were reduced by 36.2% (p<0.05) and by 42.7% (p<0.05), respectively, in control TAC mice compared to control sham mice (Table 3). Myocardial protein content of AMPK and p-AMPK was 1.76-fold (p<0.001) and 2.19-fold (p<0.001) higher, respectively, in AAV8-LDLr TAC mice than in control TAC mice. Acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC) protein level in the myocardium was reduced by 19.4 % (p<0.05) in AAV8-LDLr TAC mice compared to control TAC mice. Myocardial protein content of p-ACC, which makes up the inactive form of the enzyme, was increased by 1.48-fold (p<0.001) in AAV8-LDLr TAC mice compared to control TAC mice. Peroxisome proliferator-activated receptor (PPAR)-α was 1.78-fold (p<0.001) higher in AAV8-LDLr TAC mice than in control TAC mice. Myocardial protein content of carnitine palmitoyltransferase IB (CPT1B) was increased by 1.60-fold (p<0.001) in AAV8-LDLr TAC mice compared to control TAC mice (Table 3). Taken together, data on ACC, p-ACC, PPAR-α, and CPT1B are consistent with a preserved capacity for myocardial fatty acid oxidation in AAV8-LDLr TAC mice and a reduced capacity for fatty acid oxidation in control TAC mice. Myocardial protein levels of liver X receptor (LXR)-α and of LXR-ß were 2.28-fold (p<0.001) and 2.63-fold (p<0.01) higher, respectively, in in AAV8-LDLr TAC mice than in control TAC mice. Figure 9 contains representative images of western blots illustrating the quantitative data of Table 3.

Limitations and future directions

The phenotype following TAC is modified by genetic background and by sex of the animals [38, 39]. It is reasonable to assume that there is no interaction between AAV8-LDLr gene transfer and genetic background/sex on the cardiovascular phenotype and heart failure in TAC mice. A second limitation is that cholesterol AAV8-LDLr lowering gene transfer was performed prior to TAC. Therefore, this experimental investigation is not equivalent to a clinical intervention trial in patients with established cardiac failure. Whether cholesterol lowering gene therapy ameliorates established non-ischemic cardiac failure induced by TAC represents an area for future research.

Construction, generation, and production of gene transfer vectors

Cholesterol lowering gene therapy was performed using an adeno-associated viral (AAV) serotype 8 vector containing a hepatocyte-specific expression cassette to induce expression of the murine low-density lipoprotein receptor (LDLr) (AAV8-LDLr). The expression cassette of this vector consists of the 1272 bp DC172 promoter, comprising an 890 bp α₁-antitrypsin promoter fused together with 2 copies of the 160 bp α₁-microglobulin enhancer, upstream of the human A-I 5’UTR containing the first intron (247 bp) followed by the murine LDLr cDNA sequence (2598 bp), and the rabbit ß-globin polyadenylation signal (127 bp) [12, 40, 41]. The control vector AAV8-null contains the transcriptional regulatory sequences but no insert. AAV vector production was performed as described [42].

In vivo experiments

All experimental procedures in animals were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee of the Catholic University of Leuven (Approval number: P154/2013). Female C57BL/6 LDLr^-/- mice, originally purchased from Jackson Laboratories (Bar Harbor, ME, USA), were fed standard chow diet (Sniff Spezialdiäten GMBH, Soest, Germany) following weaning. Gene transfer in C57BL/6 LDLr^-/- mice was performed at the age of 15 weeks by tail vein injection of 2 x 10¹² genome copies/kg of AAV8-LDLr. Control mice were treated with an equivalent dose of AAV8-null. To induce pressure overload, transverse aortic constriction (TAC) was performed two weeks later [43]. TAC initially leads to compensatory hypertrophy of the heart, but over time, the response to chronic hemodynamic overload becomes maladaptive and results in cardiac dilatation and heart failure. Briefly, anaesthesia was performed with a single intraperitoneal injection of sodium pentobarbital (Nembutal^®, Ceva Sante Animale, Brussels, Belgium) at a dose of 40-70 mg/kg. Mice were put in supine position and temperature was maintained at 37°C with a heating pad. A horizontal skin incision of 0.5 cm to 1 cm in length was made at the level of the suprasternal notch. A 2 mm to 3 mm longitudinal cut was performed in the proximal portion of the sternum and the thymus gland was retracted. This allowed visualization of the aortic arch under low-power magnification. A wire with a snare at the end was passed under the aorta between the origin of the right innominate artery and the left common carotid artery. A 7-0 silk suture (Ethicon, Johnson & Johnson, Livingston, Scotland) was snared with the wire and pulled back around the aorta. Subsequently, a bent 27-gauge needle (BD Microlance^®, BD, Franklin Lakes, New Jersey) was placed next to the aortic arch and the suture was snugly tied around the needle and the aorta. Afterwards, the needle was quickly removed. The skin was closed and mice were allowed to recover on a warming pad until they were fully awake. The sham procedure was identical except that no constriction on the aorta was applied. Buprenorphine (Temgesic®) (Reckitt Benckiser Healthcare Ltd., Hull, UK) was administered at a dose of 0.1 mg/kg body weight subcutaneously for peri-operative pain relief. Postoperative analgesia was applied immediately following the intervention. Euthanasia of mice at the end of the experiment was performed by an intraperitoneal injection of sodium pentobarbital (200 mg/kg) and cervical dislocation.

Group assignment at the start of the study was performed at random. In the first experimental layer, mice were assigned for hemodynamic quantification and morphometric and histological analysis. The second experimental layer consisted of mice that did not undergo perfusion fixation and that were used for quantification of organ weights and for quantification of protein and mRNA levels. At the end of the study, data of all surviving mice were included in the analysis. Investigators who performed endpoint analyses were blinded to group allocation. Unblinding of animal numbers corresponding to specific allocation groups was performed at completion of measurements. All randomized mice were included in the analyses.

In vivo hemodynamic measurements

Invasive hemodynamic measurements were performed 8 weeks after TAC or after sham operation as described [12, 43, 44]. Mice were anesthetized by intraperitoneal administration of 1.4 g/kg urethane (Sigma, Steinheim, Germany). Body temperature was maintained with a heating pad and monitored with a rectal probe. An incision in the right carotid artery was made with a 26-gauge needle between a distal and proximal non-occlusive ligation of the artery. A 1.0 French Millar pressure catheter (SPR-67/NR; Millar instruments, Houston, Texas, USA) was inserted and advanced to the left ventricle (LV). After stabilisation of the catheter, heart rate, maximal systolic LV pressure, minimal diastolic LV pressure, the peak rate of isovolumetric LV contraction (dP/dt_max), and the peak rate of isovolumetric LV relaxation (dP/dt_min) were measured. The end-diastolic LV pressure was calculated manually from the pressure in function of time curves. The time constant of isovolumetric LV pressure fall (tau) was calculated using the method of Weiss et al. [45]. Arterial blood pressure measurements were obtained after withdrawal of the catheter from the LV to the ascending aorta. Data were registered with Powerlab Bridge Amplifier and Chart Software (sampling rate 2000 Hz; ADInstruments Ltd, Oxford, United Kingdom).

Blood sampling

Blood was obtained by puncture of the vena cava inferior at the end of the experiment just before euthanasia. Anticoagulation was performed with 0.1 volume of 136 mmol/L trisodium citrate and plasma was immediately isolated by centrifugation at 1100 x g for 10 minutes and stored at −80 °C.

Plasma lipoprotein analysis

Mouse lipoproteins were separated by density gradient ultracentrifugation in a swing-out rotor as described before [46]. Fractions were stored at -20°C until analysis. Non-HDL cholesterol was determined as the sum of cholesterol within very low-density lipoproteins (VLDL) (0.95 < d < 1.006 g/ml), intermediate-density lipoproteins (IDL) (1.006 < d < 1.019 g/ml), and low-density lipoproteins (LDL) (1.019 < d < 1.05 g/ml) lipoprotein fractions. The cut-off value (d=1.05 g/ml) between LDL and high-density lipoproteins (HDL) for murine samples was chosen based on previous work by Camus, Chapman et al. [47]. Cholesterol in plasma and lipoprotein fractions was determined with commercially available enzymes (Roche Diagnostics, Basel, Switzerland). Precipath L (Roche Diagnostics) was used as a standard.

Analysis of lipid peroxidation in plasma

Measurement of Thiobarbituric Acid Reactive Substances (TBARS) used for quantification of lipid peroxidation was performed according to the instructions of the manufacturer (Cayman Chemical, Ann Arbor, MI, USA).

Quantification of myocardial protein levels by western blot

Myocardial tissue samples were isolated 56 days after sham operation or TAC and immediately frozen in liquid nitrogen and stored at -80°C. Tissues were placed in lysing matrix tubes (QBiogene/MP Biomedicals, Solon, OH, USA), mixed with 1 ml of protein extraction buffer containing 10 mM imidazole, 300 mM sucrose, 1 mM dithiotreitol, 1mM sodium metabisulfite, 25 mM sodium fluoride, 5 mM sodium ethylenediaminetetraacetic acid, 5 mM sodium pyrophosphate, 0.3 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Roche Diagnostics Belgium, Vilvoorde, Belgium), and homogenised in the FastPrep24 instrument (MP Biomedicals). Protein concentration was quantified using the Pierce BCA Protein Assay kit (Pierce Biotechnology Inc., Rockford, IL, USA). Equal amounts of proteins were separated on 4-20 % Tris-Glycine gradient gels (Bio-Rad Laboratories N.V., Temse, Belgium) and blotted onto polyvinylidene difluoride membranes (Bio-Rad Laboratories N.V.). Membranes were incubated with primary antibodies against Akt, phospho (p)-Akt (Ser/Thr), p38 MAPK, p-p38 MAPK (Thr180/Tyr182), mammalian or mechanistic target of rapamycin (mTOR), p-mTOR (Ser2481), acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), p-ACC (Ser79), AMP-activated protein kinase (AMPK)α, p-AMPKα (Thr172), Smad 2/3, Smad4, GLUT 4, pyruvate dehydrogenase (PDH), PDH kinase, transforming growth factor (TGF)-β1, ß-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all prior antibodies from Cell Signalling Technologies, Beverly, MA, USA), NADPH oxidase 2, NADPH oxidase 4, peroxisome proliferator-activated receptor (PPAR-α), carnitine palmitoyltransferase IB (CPT1B), liver X receptor (LXR)-α and LXR-ß (Abcam, Cambridge, UK). Protein expression was detected with Super signal west pico chemilumninescent reagents (Thermo Scientific, Rockford, IL, USA) and quantified using Image lab TM Analyzer software (Bio-Rad laboratories N.V.). All protein levels were normalized to the GAPDH protein level.

Histological and morphometric analysis

Histological and morphometric analyses were executed as described [44]. After hemodynamic analysis, mice were perfused via the abdominal aorta with phosphate-buffered saline (PBS) and hearts were arrested in diastole by KCl (100 μL; 0.1 mol/L), followed by perfusion fixation with 1% paraformaldehyde in phosphate buffered saline. After dissection, hearts were post-fixated overnight in 1% paraformaldehyde, embedded in paraffin, and 6 μm thick cross-sections at 130 μm spaced intervals were made extending from the apex to the basal part of the left ventricle. Left ventricle (LV) remodelling was assessed by morphometric analysis on mosaic images of Sirius red-stained heart cross-sections using Axiovision 4.6 software (Zeiss, Zaventem, Belgium). Anterior wall thickness and septal wall thickness were determined. All geometric measurements were computed in a blinded fashion from representative tissue sections of 4 separate regions and the average value was used to represent that animal for statistical purposes [48, 49].

To measure collagen content in the interstitium, Sirius Red staining was performed as described by Junqueira et al. [50]. Sirius Red polarization microscopy on a Leica RBE microscope with KS300 software (Zeiss) was used to quantify thick tightly packed mature collagen fibers as orange-red birefringent and loosely packed less cross-linked and immature collagen fibers as yellow-green birefringent. Collagen positive area was normalized to the LV remote area and was expressed as percentage. Any perivascular fibrosis was excluded from this analysis. Perivascular fibrosis was quantified as the ratio of the fibrosis area surrounding the vessel to the total vessel area. Two mid-ventricular sections were studied per animal [5].

Cardiomyocyte hypertrophy was analysed on paraffin sections stained with rabbit anti-mouse laminin (Sigma; 1/50) by measuring the cardiomyocyte cross-sectional area (μm²) of at least 200 randomly selected cardiomyocytes in the LV myocardium. The capillary density in the myocardium was determined on CD31-stained sections using rat anti-mouse CD31 antibodies (BD; 1/500). Two mid-ventricular cross-sections were analysed per mouse [48, 49].

Immunostaining for 3-nitrotyrosine was performed with rabbit anti-nitrotyrosine antibodies (Merck Millipore, Overijse, Belgium; dilution 1/250).

Apoptosis was quantified on deparaffinised tissue sections using SignalStain^® cleaved caspase-3 IHC detection kit (Cell Signaling Technologies, Beverly, MA, USA), which utilizes a polyclonal rabbit antibody to the neoepitope peptide at the end of cleaved caspase-3 [43].

Evaluation of cardiac glucose metabolism by micro-positron emission tomography (micro-PET)

Glucose uptake in the myocardium and in the skeletal muscle was quantified by micro-PET using [¹⁸]F-fluorodeoxyglucose (FDG) as a tracer (309 ± 22 μCi) [12]. Imaging was performed 60 min after tracer administration. Animals were anesthetized by inhalation of 2% isoflurane in 100% oxygen and underwent static imaging for 10 minutes on a micro-PET Focus 220 scanner (Concorde Microsystems, Knoxville, TN, USA). Images were reconstructed with ordered subset expectation maximization algorithm with 6 iterations (OSEM3D 6i) and analysed with PMOD v.3.4 (Pmod Technologies, Zurich, Switzerland). To exclude any effect of diurnal variability, micro-PET data acquisition was consistently performed within the same 2 hours’ time frame of the day. The standardized uptake value (SUV) is the ratio between the uptake in a specific volume of interest versus the average uptake in the whole body. The simultaneous quantification of skeletal SUVs was performed since myocardial glucose metabolism is not always parallel to skeletal and whole-body glucose metabolism [51].

Quantification of superoxide dismutase concentration and xanthine oxidase activity in plasma

Superoxide dismutase plasma protein concentration was quantified using the Superoxide Dismutase Assay kit (Cayman Chemical, Ann Arbor, MI, USA). Xanthine oxidase activity were assayed using commercially available kits (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.

Statistical analysis

All data are expressed as means ± standard error of the means (SEM). Parameters between four groups were compared by one-way analysis of variance followed by Bonferroni multiple comparisons post-test for comparing sham groups, TAC groups, and sham versus respective TAC groups using GraphPad Instat (GraphPad Software, San Diego, USA). When indicated, a logarithmic transformation or a square root transformation or a non-parametric test was performed. Parameters between two groups were compared using Student’s t test. When indicated, a logarithmic transformation, a square root transformation, or a non-parametric Mann-Whitney test was performed. The assumption of Gaussian distribution was tested using the method Kolmogorov and Smirnov. Kaplan-Meier survival curves were analysed by log-rank test using Prism4 (GraphPad Software). A two-sided p-value of less than 0.05 was considered statistically significant.