Cardiac magnetic resonance imaging

After 3 weeks of feeding, anaesthesia was induced in one group of control (n = 5) and high fat-fed (n = 5) rats using 4% isoflurane in 100% oxygen. Rats were placed in a purpose built cradle for subsequent vertical positioning in the magnet bore [48]. Maintenance anaesthesia was 1.5–2% isoflurane at 1 l min⁻¹ oxygen flow. Heart electrical activity and respiration were monitored, using an in-house electrocardiography and respiratory gating device, via subcutaneous needle electrodes inserted into the fore limbs, and a motion loop on the abdomen, respectively.

As described previously [48], magnetic resonance imaging was carried out on an 11.7 T (500 MHz) vertical bore (123-mm internal diameter) system (Magnex Scientific, Oxon, UK), using a Bruker Avance console (Bruker Medical, Ettlingen, Germany) with a linear transmit and receive, 500 MHz birdcage coil with an inner diameter of 60 mm (constructed in-house).

Long- and short-axis scout images of the heart, using a cardiac- and respiration-gated segmented fast low angle shot sequence, were used to position 7–8 contiguous, short-axis slices (slice thickness 1.5 mm) covering the entire heart. The sequence was electrocardiogram and respiration gated and between 20 and 30 frames per heart cycle were acquired, depending on the heart rate. The field of view was 5.12 × 5.12 cm with a matrix size of 256, TE/TR = 1.43/4.6 ms. One phase-encoding step was acquired per heart beat and four or eight phase encoded steps acquired per respiration cycle, resulting in a total experimental time of approximately 1 h.

Image reconstruction was performed using purpose-written software in Matlab (Mathworks, Natick, Maryland, USA). Raw data were isotropically zero-filled by a factor of two and filtered (modified third-order Butterworth filter) before Fourier transformation, resulting in an in-plane voxel size of 100 × 100 μM. Image data were exported into Scion Image (Scion Corporation, Frederick, Maryland, USA). End-diastolic and end-systolic frames were selected according to maximal and minimal ventricular volumes. In both frames, the epicardial border and left ventricular cavity were outlined and left ventricular mass was obtained by multiplying the volume by the specific gravity of myocardial tissue (1.05 g/cm³). Based on the end-systolic volumes (ESV) and end-diastolic volumes (EDV), all parameters characterising cardiac function, such as stroke volume (SV = EDV − ESV), ejection fraction (EF = SV/EDV) and cardiac output (CO = SV × heart rate) were calculated.

Isolated working heart perfusion

Another group of control (n = 7) and high fat-fed (n = 7) rats were anaesthetised with a 0.8 ml i.p. injection of 60 mg ml⁻¹ sodium pentobarbitone (Sagatal, Rhône Mèrieux, Dublin, Ireland). Following the cessation of peripheral nervous function, hearts were excised and arrested using ice-cold modified Krebs–Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.75 mM CaCl₂, 0.5 mM Na₂EDTA, 25 mM NaHCO₃, 1.2 KH₂PO₄). Hearts were weighed before perfusion, and then perfused in Langendorff mode for 10 min with modified Krebs–Henseleit buffer containing 11 mM glucose, 0.2 mM pyruvate and 0.5 mM lactate as substrates. After left atrial cannulation, hearts were switched to the working mode for 20 min with 200 ml of recirculating buffer. Preload was 15 mmHg and afterload was 80 mmHg. Hearts were then perfused with buffer containing 0.2 mM palmitate pre-bound to essentially fatty acid free 1.5% wt/vol bovine serum albumin (BSA) for 20 min. This was followed by 20 min perfusion with buffer containing 1 mM palmitate pre-bound to 1.5% BSA. In four experiments, hearts were perfused with buffer containing 1 mM palmitate, then 0.2 mM palmitate to ensure that the order of buffer change did not affect the results. Aortic and coronary flows were measured every 10 min and, using online measurement of aortic developed pressure, cardiac power was calculated [3]. Atrial venous oxygen concentration differences were determined using online chemifluorescence methods, which simultaneously monitored temperature-compensated oxygen partial pressure in the atrial inflow line, and from the pulmonary trunk. The oxygen content of buffer flowing in the left atrium was maintained above 550 mmHg throughout all experiments. Heart temperature was maintained at 37.4 ± 0.2°C. Myocardial oxygen consumption was calculated using the arterio-venous differences and coronary flows. Cardiac efficiency was calculated from cardiac power and myocardial oxygen consumption [3]. Hearts were snap-frozen in liquid nitrogen for subsequent biochemical analyses.

Isolation of mitochondria

A further group of control (n = 6) and high fat-fed (n = 6) rats were anaesthetised as described in the perfusion study. After the cessation of peripheral nervous function, hearts were quickly excised and arrested in ice-cold mitochondrial isolation solution (100 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM MgSO₄.7H₂O, 1 mM ATP, pH 7.4). A sample of whole left ventricle (~80 mg) was washed in mitochondrial isolation solution, frozen using pre-cooled Wollenberger clamps under liquid nitrogen and stored at −80°C for later analysis of enzyme activity. The remaining left ventricular tissue was used for mitochondrial isolation. Epididymal fat pads were removed from the carcass and weighed, as an indicator of whole body adiposity [52].

Interfibrillar mitochondria were isolated from rat hearts as previously described [33] except that a modified Chappell–Perry buffer (containing 100 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM MgSO₄.7H₂O, and 1 mM ATP, pH 7.4 at 4°C) was used. The final mitochondrial suspension was immediately used for measurement of mitochondrial respiratory parameters, and the remaining preparation later stored at −80°C for production of protein lysates for immunoblotting.

Measurement of mitochondrial respiratory parameters

Respiratory rates of rat heart mitochondria were measured using a Clark-type oxygen electrode (Strathkelvin Instruments Ltd, Glasgow, UK). Respiration rates were recorded and quantified using 782 Oxygen System v3.0 software (Strathkelvin Instruments). Oxygen concentrations were measured in 0.5 ml respiration medium (100 mM KCl, 50 mM MOPS, 1.0 mM KH₂PO₄, 1.0 mg/ml defatted BSA, pH 7.4) in two respiration chambers maintained at 30°C containing magnetic stirrers [25]. The chambers were treated identically throughout the experiment, except that one contained 1 μmol GDP (dissolved in 10 μl of H₂O, pH 7.4), added to the respiratory medium at the start of the experiment (i.e. before mitochondria were added) and allowed to mix. GDP is an inhibitor of the mitochondrial uncoupling proteins [53], such that differences between the two chambers indicate the influence of UCP3 on oxygen utilisation. Oxygen concentrations were first recorded in the absence of mitochondria, and after the addition of approximately 0.5 mg mitochondrial protein, assayed using a protein assay kit (Pierce, Rockford IL, USA). Glutamate (20 mM), malate (5 mM) plus pyruvate (0.5 mM) or malate (5 mM) plus palmitoyl carnitine (0.04 mM), were added as substrates, before State III respiration was stimulated by the addition of 100 nmol of ADP. State IV respiration, which occurred when all the ADP added to the respiration medium had been phosphorylated, was subsequently measured. State III and state IV measurements were repeated after addition of a further 100 nmol of ADP. Acceptor control ratios (ACRs) were calculated as an indication of mitochondrial integrity by dividing state III respiration rates by state IV respiration rates. ADP/O ratios (the ratios of molecules of ADP phosphorylated for each oxygen atom consumed) were calculated as described by Estabrook [16]. ADP/O ratios are independent of the rates of oxygen consumption and give a more sensitive and precise measure of mitochondrial respiratory coupling than can be inferred from respiration rates alone.

Enzyme analysis

Whole snap-frozen heart tissue was crushed under liquid nitrogen and activities of the mitochondrial β-oxidation enzyme MCAD and citrate synthase, a measure of mitochondrial content, were measured using methods described previously [20].

Immunoblotting

Levels of cardiac UCP3 protein were measured in mitochondrial preparations by immunoblotting, as described previously [30], using a polyclonal rabbit anti-UCP3 antibody (Alpha Diagnostic International, San Antonio, TX) at a concentration of 1:1,000 in 5% milk TBS–Tween. MTE-1 protein levels were measured in mitochondrial preparations as described previously [26], using a polyclonal rabbit anti-MTE-1 antibody (gift of Dr Stefan Alexson, Karolinska Institutet, Stockholm, Sweden) at a concentration of 1:2,000 in 5% milk TBS–Tween. Levels of ANT protein were determined using a polyclonal goat anti-ANT antibody (Santa Cruz Biotechnology, CA, USA) as described previously [29]. Levels of F₀F₁ ATP synthase α-subunit protein were determined using a polyclonal mouse anti-α subunit F₀F₁ ATP synthase antibody (Mitosciences Inc, Eugene, USA) at a concentration of 1:2,000 in 5% milk TBS–Tween. Total cellular GLUT 4 protein content was determined using a monoclonal anti-COOH-terminal GLUT4 antibody (kind gift, G.D. Holman, Bath), as described previously [34]. The secondary antibody used, in all cases, was the appropriate horseradish peroxidase conjugate polyclonal antibody (Autogen Bioclear, Wiltshire, UK) at a concentration of 1:3,500 in 5% milk TBS–Tween. Even protein loading and transfer were confirmed by Ponceau staining, and protein levels were related to internal standards to ensure homogeneity between samples and gels. Bands were quantified using UN-SCAN-IT gel software (Silk Scientific, USA), and all samples were run in duplicate on separate gels to confirm results.

To measure oxidative modification of mitochondrial proteins, protein carbonylation was measured using an OxyBlot kit (Millipore, MA, USA). In brief, carbonyl groups were derivatised to 2,4-dinitrophenylhydrazone (DNP) by 2,4-dinitrophenylhydrazine, and proteins were then separated on a gel, using western blotting techniques described above. Blots were incubated with a DNP-specific antibody contained in the kit, and the sum of the separated protein bands was quantified as total protein carbonylation in the sample.

Blood plasma analyses

At the point of heart excision, blood was taken from the chest cavity, rapidly centrifuged at 1,000×g at 4°C, and the plasma frozen at −80°C for biochemical analysis. Lipoprotein lipase inhibitor (2%) was added to an aliquot of plasma for non-esterified FFA analysis. Plasma levels of glucose, triglycerides, total cholesterol, HDL cholesterol and LDL cholesterol were measured using an ABX Pentra Clinical Chemistry bench-top analyser (Horiba ABX, Montpellier, France). Plasma levels of FFAs were measured using an NEFA assay kit (Wako Chemicals GmbH, Neuss, Germany).

Fasting blood plasma metabolites

All rats had similar fasting plasma levels of glucose and triglycerides (Table 1). Fasting plasma levels of free-fatty acids were elevated by 52% to 0.38 mM in the high fat-fed rats when compared with 0.25 mM in the control rats (p < 0.05). Total blood cholesterol levels were 25% higher in the high fat-fed rats at 2 mM when compared with 1.6 mM in controls (p < 0.05), with an increased LDL cholesterol fraction of 0.19 mM when compared with 0.04 mM in controls (p < 0.0001), and no difference in the HDL cholesterol fraction.

Isolated working heart

Consistent with in vivo measurement of cardiac function, aortic flows were the same for all rat hearts at all palmitate concentrations (Fig. 2). Coronary flows were 15% greater in high fat-fed rat hearts under all perfusion conditions (p < 0.04), but cardiac outputs were the same for all hearts. Therefore, high fat feeding had no significant effect on cardiac function, whether measured in vivo (Table 1) or in the isolated heart (Fig. 2).

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Fig. 2

Isolated working heart aortic flows, coronary flows and cardiac outputs (left to right) following 3 weeks of high fat feeding. Hearts were perfused with three buffers, all of which contained 11 mM glucose, 0.2 mM pyruvate and 0.5 mM lactate as substrates (clear bars), and the addition of 0.2 mM palmitate (shaded bars), or 1.0 mM palmitate (hatched bars). *p < 0.05 versus chow fed

Oxygen consumption in chow-fed rat hearts was significantly increased on perfusion with 1 mM palmitate, when compared with perfusion with no palmitate, or with 0.2 mM palmitate (Fig. 3, p < 0.02). Overall, oxygen consumption in high fat-fed rat hearts was 19% higher than chow-fed rat hearts (p < 0.01), but was independent of palmitate concentration. Consistent with measurements of in vivo function, cardiac power in high fat-fed rat hearts (8.8 ± 0.7 mW) was the same as chow-fed rat hearts (9.4 ± 0.6 mW) under all perfusion conditions. Cardiac efficiency was higher in chow-fed rat hearts when perfused without palmitate than with either 0.2 mM (p < 0.01) or 1.0 mM palmitate (p < 0.02). Cardiac efficiency was on average 21% lower in high fat-fed rat hearts than in chow-fed rat hearts (Fig. 3), as a consequence of the increased oxygen consumption. High fat-fed rat hearts had 43% lower efficiency as compared to chow-fed rat hearts when perfused without palmitate (p < 0.01), and 28% lower efficiency when perfused with 1.0 mM palmitate (p < 0.05).

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Fig. 3

Oxygen consumption (left), cardiac power (middle) and cardiac efficiency (right) in isolated rat hearts following high fat feeding. Hearts were perfused with three buffers, all of which contained 11 mM glucose, 0.2 mM pyruvate and 0.5 mM lactate as substrates (clear bars), and the addition of 0.2 mM palmitate (shaded bars), or 1.0 mM palmitate (hatched bars). *p < 0.05 versus chow-fed rat hearts perfused without palmitate, ^† p < 0.05 versus chow-fed rat hearts perfused with 0.2 mM palmitate

Respiration in isolated mitochondria

State 3 respiration rates were higher in isolated cardiac mitochondria from high fat-fed rats than chow-fed rats (p < 0.05) in the presence of palmitoyl carnitine and malate (Fig. 4), but were unchanged with pyruvate and malate or glutamate as substrates. The addition of the UCP3 inhibitor, GDP, did not significantly change state 3 respiration rates. With all substrates, state 4 respiration rates (Fig. 4) were not significantly different in cardiac mitochondria from high fat-fed when compared with chow-fed animals, and were unchanged with the addition of GDP. ADP/O ratios were lower in cardiac mitochondria from high fat-fed rats as compared to those from chow-fed animals, irrespective of substrate (p < 0.01). The addition of GDP did not alter ADP/O ratios of mitochondria from chow-fed rat hearts (Fig. 4), but normalised the ADP/O ratio in high fat-fed rat heart mitochondria (p < 0.01) with all substrates.

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Fig. 4

State 3 respiration rates and ADP/O ratios in rat left ventricular mitochondria with palmitoyl-carnitine plus malate (upper), pyruvate plus malate (middle) or glutamate (lower) as substrates. In addition to the control condition (clear bars), mitochondria were incubated with the UCP3 inhibitor GDP (shaded bars). *p < 0.05 versus chow fed, ^† p < 0.05 versus high fat-fed without GDP

MCAD and citrate synthase activity and GLUT 4 protein expression

Cardiac MCAD activity (Fig. 5) was 23% higher in high fat-fed rat hearts (p < 0.05). Citrate synthase activity was the same for all hearts (119 ± 4 μmol gww⁻¹ min⁻¹ after chow feeding and 124 ± 6 μmol gww⁻¹ min⁻¹ after high fat feeding), indicating higher cardiac β-oxidation in high fat-fed animals with unchanged cardiac mitochondrial content. MCAD activity correlated positively (p < 0.001) with oxygen consumption in hearts perfused with 0.2 mM palmitate (Fig. 5, p < 0.001). Total cardiac protein levels of the insulin-sensitive glucose transporter, GLUT 4, were unchanged with high fat feeding (1.0 ± 0.06 after chow feeding, 0.93 ± 0.05 after high fat feeding).

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Fig. 5

Cardiac medium-chain acyl-CoA dehydrogenase (MCAD) activity (left) in chow-fed (clear bars) and high fat-fed rats (shaded bars) *p < 0.05 versus chow fed. Correlation between cardiac MCAD activity and oxygen consumption (right) in isolated perfused working heart from chow (open symbols) and high fat-fed rats (closed symbols)

Cardiac insulin resistance

We set out in this study to examine cardiac metabolism in a pre-diabetic model of early dietary-induced obesity, so there is a possibility that animals were insulin resistant after 3 weeks of high fat feeding. 4 weeks of feeding a 34% fat diet elevated plasma insulin [17], and 6–8 weeks of a 60% fat diet produced systemic glucose intolerance [12, 42]. However, cardiac insulin sensitivity was normal after 8 weeks of feeding a 60% fat diet, a diet similar to that in the present study [12]. The heart appears to maintain a normal insulin response, despite systemic glucose intolerance, for a protracted period. For example, the obese Zucker rat had not developed cardiac insulin resistance by 6 months of age, and only had mild cardiac insulin resistance at 12 months [44]. Therefore, short-term high fat feeding has only a low likelihood of inducing cardiac insulin resistance.

Cardiac function and efficiency

Mechanical factors may have contributed to increased cardiac oxygen consumption in our rat hearts. Increased myocardial oxygen consumption has been closely linked to increased left ventricular wall stress [24, 46], however in vivo MRI measurements of cardiac function and morphology showed no abnormalities in end-diastolic volume or wall thickness in our high fat-fed rat hearts. In another study [43], high fat feeding increased systolic blood pressure by ~40%, which would increase wall stress, but afterload and hydraulic resistance were constant in our isolated working heart preparation, so the effects of blood pressure were not relevant. These results indicated to us that the increase in oxygen consumption was due to changes in cardiac metabolism, rather than mechanical factors. High fat-fed rats had normal cardiac function when measured in vivo or when hearts were perfused in working mode, and thus it seems that ATP synthesis was maintained. However, oxygen consumption was increased, with the result that cardiac efficiency was decreased in high fat-fed rat hearts.

Fatty acid oxidation

We found that high fat-fed rat hearts had increased mitochondrial fatty acid oxidation when compared with control rat hearts. Mitochondria from high fat-fed rat hearts had increased state 3 respiration only in the presence of palmitoyl-carnitine, whereas respiration was normal with pyruvate or glutamate. It is likely, therefore, that the increased respiration rate was associated with the upregulation of enzymes of fat metabolism, such as MCAD (Fig. 1), which increases in heart following PPARα activation [19]. MCAD activity was elevated by 23% in high fat-fed rat hearts, similar to the increase in mitochondrial fatty acid oxidation, but citrate synthase activity and protein levels of F₀F₁ ATP synthase and ANT were unaltered, therefore an increase in the number of mitochondria could not account for these differences.

Our finding of increased fatty acid oxidation and MCAD activity is consistent with long-term high fat feeding studies [32, 37]. Increased fatty acid oxidation has been reported in isolated, perfused working rat hearts after 4 weeks of feeding a 60% saturated fat diet, but this was not associated with decreased cardiac efficiency [51]. Fatty acid oxidation is unaltered in rats bred for low aerobic capacity with moderate obesity [41]. In the present study, a switch in substrate preference away from more oxygen efficient glucose oxidation, towards fatty acid oxidation with high fat feeding [5] may have contributed to an increase in oxygen consumption and decrease in cardiac efficiency. This effect would be relatively minor, as the maximum difference in oxygen consumption between glucose oxidation alone to palmitate oxidation alone is 10–12% [5, 49]. Therefore, increased fatty acid oxidation would have played a minor role in decreasing cardiac efficiency with high fat feeding.

High FFAs increased oxygen consumption by up to 40% in either unloaded or arrested isolated hearts on perfusion with >1 mM fatty acids [2, 11], and in vivo infusion of 10% intra-lipid increased oxygen consumption by 26% [28]. In our study, there was a 20% increase in oxygen consumption on perfusion with 1 mM fatty acids, and in proportion to the exogenous fatty acid supply (Fig. 3). The mechanisms responsible for such increases in oxygen consumption in the normal heart remain unclear, but may be related to direct action of FFAs on mitochondrial uncoupling [36]. We found that mitochondrial ADP/O ratios from chow-fed rat hearts were not affected by GDP, indicating that uncoupling proteins were not responsible for the increased oxygen consumption in hearts from chow-fed animals. However, this does not discount the possibility that direct action of FFAs cause uncoupling [36] in the absence of changes in mitochondrial proteins. In contrast to chow-fed rat hearts, high fat-fed rat hearts had significantly elevated oxygen consumption even when no exogenous FFA was present, with reduced cardiac efficiency, which cannot be explained by the mechanisms described above [2, 36, 38]. This, therefore, points to abnormalities in oxidative metabolism downstream of substrate supply (Fig. 1).

We thank Emma Carter for her invaluable technical assistance, and Dr Charles L Hoppel and his research group for their generous help in teaching us their mitochondrial isolation and respiration protocols. Stefan Alexson is thanked for his generous gift of the MTE-1 antibody. Sara McAleese thanks the Wellcome Trust for her Undergraduate Vacation Studentship. This study was supported by the British Heart Foundation, grant number RG/02/009/13631.