Five-week old male C57BL/6J mice (Harlan, Spain) were housed (five per cage) under a 12 h light/12 h dark cycle, in a temperature-controlled room (22°C) with food and water ad libitum. Animals were divided in two groups with similar average BW and assigned either to a control or to a high-fat diet (HFD). Control diet (D12450B, 10 kcal% fat, 70 kcal% carbohydrates, and 20 kcal% protein; 3.85 kcal/g) and HFD (D12451, 45 kcal% fat, 35 kcal% carbohydrates, and 20 kcal% protein; 4.73 kcal/g) were supplied by Test Diet Limited BCM IPS Ltd (UK). After 32 week dietary treatment, groups of animals were separated for biochemical and functional studies. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85–23, revised 1996). This study was approved by the ethics committee of the University CEU-San Pablo (SAF 2009-09714).

Animals were killed by decapitation between 9 and 10 a.m., blood collected in chilled EDTA-coated polypropylene tubes and left ventricle dissected and frozen. Plasma samples were stored at −80°C until assay. For enzymatic activities, tissues were immediately processed and prepared for assay. Proteins, lactate, and glutathione were determined in frozen tissues. Plasma leptin (Linco Research, USA; 4.9% intra-assay variation, 3.3% inter-assay variation) and insulin (Mercodia, Denmark; 2.2% intra-assay variation, 4.9% inter-assay variation) were quantified by specific radioimmunoassay and ELISA, respectively.

Triglyceride content in heart was determined as previously described (8). Briefly, 20 mg of wet tissue were homogenized in a mixture containing 40 ml of 2 mM NaCl/20 mM EDTA/50 mM sodium phosphate buffer (pH 7.4) 40 ml of tert-butanol, and 20 ml of Triton X-100/methanol (1/1). Triglycerides were measured with a Sigma diagnostic kit (USA).

We quantified uncoupling protein-1, 2, and 3 (UCP1, UCP2, UCP3, respectively) Mn- and Cu/Zn-superoxide dismutase (Mn-SOD, Cu/Zn-SOD), and catalase. Western-blotting was performed as previously described (8, 9). Briefly, primary antibodies against UCP1, UCP3 (Affinity BioReagent, USA), UCP2 (Santa Cruz Biothecnology, USA), and Mn-SOD, Zn-SOD, and catalase (Sigma, USA) were applied overnight at 4°C at the appropriate dilution. After washing and incubation with appropriate IgG-peroxidase complexes, blots were incubated in commercial enhanced chemiluminescence reagents (ECL, Amersham Bioscence, UK) and exposed to autoradiographic film. Films were scanned using a GS-800 Calibrated Densitometer (Bio-Rad, Spain) and quantified using Quantity One software (Bio-Rad, Spain). Values were normalized with β-actin to account for variations in gel loading.

Lactate dehydrogenase, CPT, CS, and pyruvate dehydrogenase activities were measured by means of colorimetric methods as described in the literature (8, 9, 23, 24). Glucose-6-phosphate dehydrogenase (G6PD) activity was determined by measuring Δ absorbance at 340 nm (Versamax, Molecular Devices, USA) due to the conversion of NADP⁺ to NADPH by G6PD and 6-phosphogluconate-dehydrogenase (6PGD). For total activity (G6PD and 6PGD), samples (1 mg/ml protein) were incubated in 96-well microplates containing assay buffer (50 mM Tris pH = 8.1, 1 mM MgCl2, 0.2 mM glucose-6-phosphate, 0.2 mM 6-phosphogluconate, and 0.1 mM NADP⁺). For 6PGD activity determination, samples were incubated in absence of glucose-6-phosphate and then G6PD activity theoretically estimated by subtracting 6PGD activity from total activity. Results were expressed as Δ Abs/min/mg protein (25).

Production of superoxide anion (O2−) was determined in fresh samples by measuring lucigenin-enhanced chemiluminescence (26, 27). Briefly, heart explants were incubated for 30 min at 37°C in oxygenated 20 mM HEPES buffer (pH = 7.4, 99 mM NaCl, 4.7 mM KCl, 1.9 mM CaCl₂, 25 mM NaHCO₃, 1 mM KH₂PO₄, 1.2 mM MgSO₄, 11 mM glucose). Explants, NADPH (1 mmol/l) and lucigenin (5 μmol/l) were then added to tubes containing 1 ml buffer. O2− production was expressed in arbitrary units/mg protein. Lactate concentration was measured as described (8). For glutathione assay, samples were homogenized in phosphate buffer (pH = 6–7) containing EDTA (1 mM) and centrifuged for 15 min (10,000 g; 4°C). Supernatants were collected and kept at −20°C until assay. Both reduced (GSH) and oxidized glutathione (GSSG) concentrations were quantified by colorimetric assay by using a commercial kit (Cayman Chemical, USA) following the manufacturer instructions. Data were expressed as (total glutathione)/(GSSG).

Mice were heparinized (1 IU/kg i.p.) and anesthetized with pentobarbital (50 mg/kg). Hearts were rapidly dissected, mounted on a modified Langendorff apparatus and retrogradely perfused through the aorta (2–3 min, 36–37°C) with Ca²⁺-free Tyrode solution (TS; 130 mM NaCl, 5.4 mM KCl, 0.4 mM NaH₂PO₄, 0.5 mM MgCl₂, 25 mM HEPES, 5 mM NaHCO₃, 22 mM glucose; pH adjusted to 7.4 with NaOH) containing 0.2 mM EGTA, then 3–4 min with TS containing 251 IU/ml type II collagenase (Worthington, USA), and 0.1 mM CaCl₂. After perfusion, hearts were chopped in small pieces, and gently stirred in TS containing 1 mg/ml bovine serum albumin (BSA, Sigma, USA). Isolated cells were filtered, centrifuged and suspended in TS containing 2 mg/ml BSA and 0.5 mM CaCl₂. After centrifugation, cells were suspended in a solution containing 1 mM CaCl₂ and 2 mg/ml BSA and stored at room temperature until electrophysiological assays (within 5 h of the isolation).

Electrophysiological experiments were performed at room temperature (24–26°C) on Ca²⁺-tolerant rod-shaped myocytes. Whole-cell configuration of the patch-clamp technique was employed to measure the L-type Ca²⁺ current (I_CaL), the transient outward K⁺ current (Ito_total), the ultra-rapid delayed rectifier K⁺ current (I_Kur), and the steady-state outward K⁺ current (I_ss). The voltage-clamp circuit was provided by an Axopatch-200B amplifier controlled by a computer equipped with a pClamp 6.0 and interfaced to the amplifier with a Digidata 1322A (Axon Instruments, USA). Recording pipettes were made from 1.5 mm-OD soft-glass capillary tubing by using a microprocessor-based patch-clamp puller (P97/PC, Sutter Instruments, USA). Tip resistances after filling with the internal solution ranged between 0.9–2 MΩ. Whole-cell currents were expressed as current densities, which were calculated from the current amplitude normalized by the membrane capacitance. Membrane capacitance (C_m) was calculated according to the equation C_m = τ_c I₀/ΔE_m [1 − (I_∞/I₀)] (τ_c, time constant of the membrane capacitance; I₀ maximum capacitance current value; ΔE_m, amplitude of the voltage step; I∞, amplitude of the steady-state current). Cm was elicited by applying ±10 mV voltage steps from a holding potential of −60 mV. Current-voltage (I − V) relationship for the I_CaL was obtained from a holding potential of –50 mV and cell depolarization (300 ms, 0.2 Hz) in 10 mV increments from −40 to +60 mV. Current-voltage (I − V) relationship for Ito_total was constructed from 500 ms series test potentials varying from −70 to +50 mV in 10 mV increments, from a holding potential of −80 mV at a frequency rate of 0.1 Hz. The same protocol was applied but now preceded by a 100 ms inactivating pre-pulse (−40 mV) in order to suppress the transient portion of the Ito_total (28). The remaining current (I_Kslow) is composed of both a I_Kur (4-aminopyridine-sensitive component) and a I_ss (4-aminopyridine-resistant component). For I_ss recording, an inactivating pre-pulse was applied in presence of 250 μM 4-AP. I_Kur was calculated by subtracting I_ss from the current recorded in the absence 4-AP. Current densities were determined at the peak current. Action potentials (AP) were elicited at 10 s intervals by 1.5-fold excitation threshold current pulses of 2.5 ms in duration. After stabilization of the records, 10 successive APs were recorded. The parameters of the APs for each cell corresponded to the mean of these 10 APs. The APD was measured at 20, 50, and 90% repolarization. The solution for I_CaL recording contained (in mM): 140 NaCl, 1.1 MgCl₂, 5.4 CsCl, 10 glucose, 5 HEPES, 1.8 CaCl₂; pH adjusted to 7.4 with NaOH. The intracellular recording pipette solution for whole-cell experiments contained (in mM): 100 CsCl, 20 triethylamine, 5 EGTA, 10 HEPES, 5 Na₂ATP, 0.4 Na₂GTP, 5 Na₂creatine phosphate, 0.06 CaCl₂; pH adjusted to 7.4 with CsOH. The solution for K⁺ current recordings contained (in mM): 135 NaCl, 10 glucose, 10 HEPES, 1 MgCl₂, 1 CaCl₂, 5.4 KCl, 1 CoCl₂, 1 BaCl₂ (250 μM 4-AP), pH adjusted to 7.4 with NaOH. The solution for AP recordings contained (in mM): 140 NaCl, 4 KCl, 1.1 MgCl₂, 1.8 CaCl₂, 10 glucose, 10 HEPES, pH adjusted to 7.4 with NaOH. The intracellular recording pipette solution for both Ito and AP contained (in mM): 135 KCl, 4 MgCl₂, 5 EGTA, 10 HEPES, 10 glucose, 5 Na₂ATP, 3-phosphocreatine, pH adjusted to 7.2 with KOH.

Mice were anesthetized with sodium pentobarbital (60 mg/kg). Thirty two-gauge needle steel electrodes were inserted subcutaneously, according to a standard three-lead ECG scheme. Acquisition was performed during 8–10 min following anesthesia and data collected for 8–15 consecutive min at a rate of 4 kHz by using a ML136 Animal Bio amplifier. Parameters for each individual animal were calculated from 40 to 45 consecutive traces (LabChart 47.0 Pro, ADInstruments, UK). QTc interval was calculated using a modified Bazzett’z correction formula [QTc = QT/(RR/100)^1/2 (29)].

All parameters were analyzed by a one-way ANOVA, followed by Newman-Keuls post hoc test. Statistical significance was set at p < 0.05.

As summarized in Table 1, HFD animals displayed a 40% increase BW, compared to control animals (p < 0.001). Heart weight was increased approx. about 7% in HFD mice (p < 0.05), although cardiac TGs were not different between control and HFD groups. Plasma insulin (p < 0.001) and leptin (p < 0.05) were also increased by HFD treatment.

We observed that both lactate dehydrogenase [F_(1,21) = 5.717; p < 0.05; Figure 1A] and pyruvate dehydrogenase activities [F_(1,8) = 12.479; p < 0.001; Figure 1B] were reduced in HFD mice. This suggests that the contribution of lactate and pyruvate to cardiac energetics would be reduced in HFD mice. Moreover, as previously reported (9), 1-way ANOVA revealed an increase of total CPT activity in HFD mice [F_(1,18) = 15,417; p < 0.05; Figure 1C], suggesting that mitochondrial uptake of FA is up-regulated by HFD treatment.

The ability of cardiac mitochondria to incorporate acetyl-CoA to the tricarboxylic acid cycle can be estimated by measuring CS activity, which accounts for the condensation of acetyl-CoA and oxaloacetic acid to yield citric acid. Figure 1D illustrates the effect of HFD on CS activity, which was significantly increased in HFD mice [1-ANOVA F_(1,10) = 5,349; p < 0.05]. This up-regulation corresponds to almost 30% increase of CS activity. We determined the amount of UCP1, UCP2 (data not shown), and UCP3 and only UCP3 (Figure 2) appeared to be up-regulated in HFD mice [1-ANOVA F_(1,12) = 5,327; p < 0.05].

Cu/Zn-SOD, Mn-SOD, and catalase were increased in the HFD group [1-ANOVA F_(1,11) = 5.593, p < 0.05; 1-ANOVA F_(1,10) = 5.017, p < 0.05; 1-ANOVA F_(1,10) = 5.141, p < 0.05 respectively; see Figures 3A–C. (Total glutathione)/(Oxidized glutathione) ratios were not different between control (7.69 ± 1.21) and HFD animals (7.76 ± 1.69). O2− production appeared to be slightly increased (p = 0.08) in HFD mice (Figure 3D). Finally, we observed a reduction of glucose-6-phosphate dehydrogenase activity (Figure 3E). This enzyme is a main source of NADPH, a co-factor necessary for maintaining redox equilibrium.

To analyze the influence of HFD in the cellular electrophysiological properties of ventricular myocytes, patch-clamp experiments were carried-out. Figure 4A shows that mean APD values, measured at 20, 50, and 90% repolarization, were identical in control and HFD ventricular myocytes. Figure 4B shows current density-voltage relationship for I_CaL obtained in myocytes isolated from control and HFD mice. The maximum value of I_CaL density was −7.8 ± 0.7 pA/pF at 0 mV (n = 10) for control and −7.0 ± 0.7 pA/pF at 0 mV (n = 9) for HFD mice. Figure 4C illustrates current density-voltage relationships for K⁺ currents (Ito_total, I_kur, and I_ss). Mean density values and voltage-dependence were similar between control and HFD myocytes. Ito_total at +60 mV was 23.4 ± 4.6 pA/pF (n = 10) for control and 22.0 ± 3.1 pA/pF (n = 10) for HFD. I_kur at +60 mV was 15.1 ± 4.2 pA/pF (n = 9) for control and 15.3 ± 3.6 pA/pF (n = 7) for HFD. I_ss at +60 mV was 10.5 ± 0.9 pA/pF (n = 9) for control and 10.1 ± 0.9 (n = 7) for HFD. Taken together these data indicate that HFD did not induce changes in the cellular electrophysiological properties of ventricular myocytes.

Figure 5 illustrates typical examples of ECG recordings in control and HFD mice. Heart rates were similar between groups. In addition, QRS complexes, QT, and T wave duration were similar between groups, reflecting a normal velocity of ventricular depolarization/repolarization. PR intervals were slightly shorter (p < 0.05) in HFD than in control animals.