Impairments in mitochondrial palmitoyl-CoA respiratory kinetics that precede development of diabetic cardiomyopathy are prevented by resveratrol in ZDF rats

Abstract

Alterations in lipid metabolism within the heart may have a causal role in the establishment of diabetic cardiomyopathy; however, this remains equivocal. Therefore, in the current study we determined cardiac mitochondrial bioenergetics in ZDF rats before overt type 2 diabetes and diabetic cardiomyopathy developed. In addition, we utilized resveratrol, a compound previously shown to improve, prevent or reverse cardiac dysfunction in high-fat-fed rodents, as a tool to potentially recover dysfunctions within mitochondria. Fasting blood glucose and invasive left ventricular haemodynamic analysis confirmed the absence of type 2 diabetes and diabetic cardiomyopathy. However, fibrosis was already increased (P < 0.05) ∼70% in ZDF rats at this early stage in disease progression. Assessments of mitochondrial ADP and pyruvate respiratory kinetics in permeabilized fibres from the left ventricle revealed normal electron transport chain function and content. In contrast, the apparent Km to palmitoyl-CoA (P-CoA) was increased (P < 0.05) ∼60%, which was associated with an accumulation of intracellular triacylgycerol, diacylglycerol and ceramide species. In addition, the capacity for mitochondrial reactive oxygen species emission was increased (P < 0.05) ∼3-fold in ZDF rats. The provision of resveratrol reduced fibrosis, P-CoA respiratory sensitivity, reactive lipid accumulation and mitochondrial reactive oxygen species emission rates. Altogether the current data support the supposition that a chronic dysfunction within mitochondrial lipid-supported bioenergetics contributes to the development of diabetic cardiomyopathy, as this was present before overt diabetes or cardiac dysfunction. In addition, we show that resveratrol supplementation prevents these changes, supporting the belief that resveratrol is a potent therapeutic approach for preventing diabetic cardiomyopathy.

Figure 1. Left ventricular pressure characteristics were determined in lean (LC), Zucker diabetic fatty rats (ZDF) and ZDF rats supplemented with resveratrol (Z-Resv)

Maximal left ventricular pressure (A; LVP max); end-diastolic pressure (B; EDP); maximal rate of pressure development (C; +dP/dt) and maximal rate of pressure decline (D; −dP/dt) were determined in 11-week-old animals. Scatter plots depict the individual values as well as the means ± SEM. n = 11–12. *Significantly (P < 0.05) different from LC.

Figure 2. Left ventricular fibrosis in LC, ZDF and Z-Resv rats

Representative images are shown at ×4.2 magnification, and the black bar represents 30 μm. Values represent means ± SEM. n = 4–5. *Significantly (P < 0.05) different from LC. †Significantly (P < 0.05) different from ZDF.

Figure 3. ADP respiratory kinetics in permeabilized muscle fibres from the left ventricle of LC, ZDF and Z-Resv rats

ADP titrated in the presence of 10 mm pyruvate and 2 mm malate revealed typical Michaelis–Menten kinetics (A). Maximal respiration (B; V_max) and the apparent K_m (C) were not different in any group. Values represent means ± SEM. n = 12–13 for determination of the apparent K_m. A few fibres were not adequately recovered to determine maximal respiration normalized to dry weight, and therefore n = 8 for V_max. JO₂ = rate of oxygen consumption.

Figure 4. Pyruvate respiratory kinetics in permeabilized muscle fibres from the left ventricle of LC, ZDF and Z-Resv rats

Pyruvate was titrated in the presence of 5 mm ADP and 2 mm malate revealed typical Michaelis–Menten kinetics (A). Maximal respiration (B; V_max) and the apparent K_m (C) were not different in any group. Values represent means ± SEM. n = 9–12 for determination of the apparent K_m. A few fibres were not adequately recovered to determine maximal respiration normalized to dry weight, and therefore n = 6–7 for V_max.

Figure 5. Markers of electron transport chain protein content in the left ventricle of LC, ZDF and Z-Resv rats

Complex I subunit NDUFB8 (A), complex II subunit 30 kDa (B), complex III subunit core 2 (C), complex IV subunit I (D), ATP synthase subunit α (E) were analysed and no significant differences were found. A representative OXPHOS blot, as well as α-tubulin which was used as a loading control, is shown in F. Values represent means ± SEM, n = 7 for all conditions, and 5 μg was loaded for all samples.

Figure 6. Palmitoyl-CoA (P-CoA) respiratory kinetics in permeabilized muscle fibres from the left ventricle of LC, ZDF and Z-Resv rats

P-CoA titrated in the presence of 5 mm ADP, 2 mm malate and 2 mm l-carnitine revealed typical Michaelis–Menten kinetics (A). Maximal respiration (B; V_max) and the apparent K_m (C) were not different in any group. Values represent means ± SEM. n = 6–7 for determination of the apparent K_m and V_max. *Significantly (P < 0.05) different from LC. †Significantly (P < 0.05) different from ZDF.

Figure 7. Left ventricular triacylglycerol (TAG), diacylglycerol (DAG) and ceramide contents in LC, ZDF and Z-Resv rats

Total and various lipid species of TAG (A and B), DAG (C and D) and ceramides (E and F) were determined using gas chromatography. Values represent means ± SEM. n = 8 for all analyses. *Significantly (P < 0.05) different from LC. †Significantly (P < 0.05) different from ZDF.

Figure 8. Rates of mitochondrial hydrogen peroxide emission (), the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) and antioxidant enzyme expression were determined in the left ventricle of LC, ZDF and Z-Resv rats

Rates of hydrogen peroxide were determine in permeabilized muscle fibres (A), while rapidly frozen whole muscle samples were used to determine GSH/GSSG (B) as well as the protein content of superoxide dismutase 2 (SOD2; C) and catalase (CAT; D) (OD, optical density). Values represent means ± SEM. n = 5–7. *Significantly (P < 0.05) different from LC. †Significantly (P < 0.05) different from ZDF.