NADPH Oxidase 2 Mediates Myocardial Oxygen Wasting in Obesity

Abstract

Obesity and diabetes are independent risk factors for cardiovascular diseases, and they are associated with the development of a specific cardiomyopathy with elevated myocardial oxygen consumption (MVO₂) and impaired cardiac efficiency. Although the pathophysiology of this cardiomyopathy is multifactorial and complex, reactive oxygen species (ROS) may play an important role. One of the major ROS-generating enzymes in the cardiomyocytes is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), and many potential systemic activators of NOX2 are elevated in obesity and diabetes. We hypothesized that NOX2 activity would influence cardiac energetics and/or the progression of ventricular dysfunction following obesity. Myocardial ROS content and mechanoenergetics were measured in the hearts from diet-induced-obese wild type (DIO_WT) and global NOK2 knock-out mice (DIO_KO) and in diet-induced obese C57BL/6J mice given normal water (DIO) or water supplemented with the NOX2-inhibitor apocynin (DIO_APO). Mitochondrial function and ROS production were also assessed in DIO and DIO_APO mice. This study demonstrated that ablation and pharmacological inhibition of NOX2 both improved mechanical efficiency and reduced MVO₂ for non-mechanical cardiac work. Mitochondrial ROS production was also reduced following NOX2 inhibition, while cardiac mitochondrial function was not markedly altered by apocynin-treatment. Therefore, these results indicate a link between obesity-induced myocardial oxygen wasting, NOX2 activation, and mitochondrial ROS.

Keywords: NADPH-oxidase; ROS; cardiac efficiency; metabolism; myocardial oxygen consumption; obesity.

Figure 1

Reactive oxygen species-products hydroxyethidium (EOH) and ethidium (E) per dihydroethidium (DHE) consumed in cardiac tissue from lean controls (CON_WT and CON_KO) and obese (DIO_WT and DIO_KO) wild type and NOX2 KO mice fed a high fat diet for 28 weeks (A,C). Also shown in lean (CON), obese (DIO), and apocynin-treated obese (DIO_APO) C57BL/6J mice fed a western diet for 28 weeks (B,D) n = 4–8 in each group. Values are normalized to EOH in lean controls. Single values and means ± SEM. * p < 0.05 CON vs. DIO within same genotype, ^# p < 0.05 DIO_WT vs. DIO_KO and DIO vs. DIO_APO.

Figure 2

Steady state left ventricular end-diastolic volumes (LVEDV) and pressures (LVEDP) at three different workloads (preload: 4,6 and 8 mmHg and afterload: 50mmHg) from lean controls (CON_WT and CON_KO) and obese (DIO_WT and DIO_KO) wild type and NOX2 KO mice fed a high fat diet for 28 weeks (A), as well as lean (CON), obese (DIO) and apocynin-treated obese (DIO_APO) C57BL/6J mice fed a western diet for 18 (B) and 28 weeks (C). n = 5–10 per group, the values are mean ± SEM.

Figure 3

Left ventricular mechanical efficiency (A–C) expressed as stroke-work (SW, G–I) relative to myocardial oxygen consumption (D–F) in hearts from lean controls (CON_WT and CON_KO) and diet-induced obese (DIO) wild type and NOX2 KO mice (DIO_WT and DIO_KO) fed a high fat diet for 28 weeks (A,D,G) as well as lean controls (CON) and untreated and apocynin-treated obese C57BL/6J mice (DIO and DIO_APO) fed a western diet for 18 weeks (B,E,H) or 28 weeks (C,F,I). Single values and means ± SEM. * p < 0.05 CON vs. DIO within same genotype, ^# p < 0.05 DIO_WT vs. DIO_KO and DIO vs. DIO_APO.

Figure 4

Myocardial oxygen consumption in mechanically unloaded hearts (MVO_2unloaded, A–C) and for processes associated with excitation-contraction coupling (MVO_2ECC, D–F) and basal metabolism (MVO_2BM, G–I) that was obtained from lean controls wild-type (CON_WT) and NOX2-KO mice (CON_KO) and obese mice fed a high fat diet for 28 weeks (DIO_WT and DIO_KO). (A,D,G). Also shown in lean controls (CON) and untreated and apocynin-treated obese C57BL/6J mice (DIO and DIO_APO) that were fed a western diet for 18 weeks (B,E,H) or 28 weeks (C,F,I). Single values and means ± SEM. * p < 0.05 within the same genotype, ^# p < 0.05 between genotypes within the same diet.

Figure 5

Glucose (A) and palmitate (B) oxidation rates measured in isolated working hearts from lean controls (CON), diet-induced obese (DIO), and obese apocynin-treated (DIO_APO) C57BL/6J mice fed an obesogenic western diet for 18 weeks. Single values and mean ± SEM, * p < 0.05 vs. DIO.

Figure 6

Oxygen fluxes (leak and maximal mitochondrial respiration capacity, OXPHOS) and respiratory coupling ratio (RCR) in isolated cardiac mitochondria using pyruvate and glutamate (PG, A–C) or palmityol-carnitine as substrates (PC, D–F). Mitochondria were obtained from lean controls (CON), untreated and apocynin-treated obese C57BL/6J mice (DIO and DIO_APO) fed a western diet for 28 weeks. Single values and mean ± SEM, ^# p < 0.05 DIO vs. DIO_APO.

Figure 7

Mass-specific and flux-specific H₂O₂- release from isolated cardiac mitochondria using either pyruvate and glutamate (PG, panel A and C) or palmityol-carnitine as substrates (PC, panel B and D) as substrates. Mitochondria were obtained from lean controls (CON) and untreated and apocynin-treated obese C57BL/6J mice (DIO and DIO_APO) fed a western diet for 28 weeks. Single values and means ± SEM, * p < 0.05 CON versus DIO, ^# p < 0.05 DIO vs. DIO_APO.