Hyperbaric oxygen therapy effectively alleviates D-galactose-induced-age-related cardiac dysfunction via attenuating mitochondrial dysfunction in pre-diabetic rats

Abstract

Currently, the prevalence of obesity in aging populations is fast growing worldwide. Aging induced by D-galactose (D-gal) is proven to cause the worsening of cardiac dysfunction in pre-diabetic rats via deteriorating cardiac mitochondrial function. Hyperbaric oxygen therapy (HBOT) has been shown to attenuate D-gal-induced cognitive deterioration through decreased inflammation and apoptosis. We tested the hypothesis that HBOT alleviates D-gal induced cardiac dysfunction via improving mitochondrial function in pre-diabetic rats. Wistar rats (n=56) were fed normal diet or high-fat diet for 12 weeks. For subsequent 8 weeks, they were subcutaneously injected either vehicle (0.9% normal saline) or D-gal (150mg/kg/day). Rats were randomly subdivided into 7 groups at week 21: sham-treated (normal diet fed rats with vehicle (NDV), high-fat diet fed rats with vehicle (HFV), normal diet fed rats with D-gal (NDDg), high-fat diet fed rats with D-gal (HFDg)) and HBOT-treated (HFV, NDDg, HFDg). Sham rats received ambient pressure of oxygen while HBOT-treated ones received 100% oxygen given once daily for 60 minutes at 2 atmosphere absolute. HBOT reduced metabolic impairments, mitochondrial dysfunction and increased autophagy, resulting in an improvement of cardiac function in aged pre-diabetic rats.

Keywords: D-galactose; aging; heart; hyperbaric oxygen therapy; mirochondria.

Figure 1

Effect of HBOT on senescence marker expression in left ventricular cardiomyocytes of pre-diabetic rats after induction of aging by D-gal. (A) Result of SA-β-gal staining. (B) Representative figures of SA-β-gal staining. NDV, normal diet fed rats with vehicle; NDDg, normal diet fed rats with D-gal; HFV, high-fat diet fed rats with vehicle; HFDg, high-fat diet fed rats with D-gal; SA-β-gal, senescence associated β galactosidase; HBOT, hyperbaric oxygen therapy. (n = 3/group). ^*P < 0.05.

Figure 2

Effect of HBOT on cardiac function in pre-diabetic rats after induction of aging by D-gal. (A) Ejection fraction. (B) Fractional shortening. (C–H) P-V loop Analysis. NDV, normal diet fed rats with vehicle; NDDg, normal diet fed rats with D-gal; HFV, high-fat diet fed rats with vehicle; HFDg, high-fat diet fed rats with D-gal; EF, ejection fraction; FS, Fractional shortening; HBOT, hyperbaric oxygen therapy. (n = 8/group). ^*P < 0.05.

Figure 3

Effect of HBOT on typical P-V loop tracings in pre-diabetic rats after induction of aging by D-gal. (A) A typical P-V loop tracing of sham-treated NDV rat. (B) A typical P-V loop tracing of sham-treated HFV rat. (C) A typical P-V loop tracing of sham-treated NDDg rat. (D) A typical P-V loop tracing of sham-treated HFDg rat. (E) A typical P-V loop tracing of HBOT-treated HFV rat. (F) A typical P-V loop tracing of HBOT-treated NDDg rat. (G) A typical P-V loop tracing of HBOT-treated HFDg rat. NDV, normal diet fed rats with vehicle; NDDg, normal diet fed rats with D-gal; HFV, high-fat diet fed rats with vehicle; HFDg, high-fat diet fed rats with D-gal.

Figure 4

Effect of HBOT on heart rate variability and blood pressure in pre-diabetic rats after induction of aging by D-gal. (A) Heart rate variability. (B) Systolic blood pressure. (C) Diastolic blood pressure. (D) Mean arterial blood pressure. NDV, normal diet fed rats with vehicle; NDDg, normal diet fed rats with D-gal; HFV, high-fat diet fed rats with vehicle; HFDg, high-fat diet fed rats with D-gal; LF/HF, low frequency/high frequency ratio; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure; HBOT, hyperbaric oxygen therapy. (n = 8/group). ^*P < 0.05.

Figure 5

Effect of HBOT on apoptosis using TUNEL staining in cardiomyocytes of pre-diabetic rats after induction of aging by D-gal. (A) Representative figure of TUNEL positive cells in sham-treated rats. (B) Representative figure of TUNEL positive cells in HBOT-treated rats. (C) Percentage of apoptotic index. NDV, normal diet fed rats with vehicle; NDDg, normal diet fed rats with D-gal; HFV, high-fat diet fed rats with vehicle; HFDg, high-fat diet fed rats with D-gal; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; HBOT, hyperbaric oxygen therapy. (n = 3/group). ^*P < 0.05, ^**P < 0.01 compared to other groups.

Figure 6

Effect of HBOT on cardiomyocytes apoptosis, inflammation and oxidative stress in pre-diabetic rats after induction of aging by D-gal. (A) Bax/Bcl-2 ratio. (B) Cleaved caspase-3/caspase-3 ratio. (C) Inflammation. (D) Oxidative stress. NDV, normal diet fed rats with vehicle; NDDg, normal diet fed rats with D-gal; HFV, high-fat diet fed rats with vehicle; HFDg, high-fat diet fed rats with D-gal; Bax, B-cell lymphoma 2 associated X protein; Bcl-2, B-cell lymphoma 2; TNF-α, tumor necrosis factor alpha; MDA, malondialdehyde; HBOT, hyperbaric oxygen therapy. (n = 5/group). ^*P < 0.05.

Figure 7

Effect of HBOT on mitochondrial functions in cardiomyocytes of pre-diabetic rats after induction of aging by D-gal. (A) Cardiac mitochondrial ROS production. (B) Cardiac mitochondrial membrane potential. (C) Cardiac mitochondrial swelling. (D) TEM representative images of cardiac mitochondria. NDV, normal diet fed rats with vehicle; NDDg, normal diet fed rats with D-gal; HFV, high-fat diet fed rats with vehicle; HFDg, high-fat diet fed rats with D-gal; ROS, reactive oxygen species; TEM, transmission electron microscopy; HBOT, hyperbaric oxygen therapy. (n = 8/group). ^*P < 0.05.

Figure 8

Effect of HBOT on mitochondrial dynamics parameters in cardiomyocytes of pre-diabetic rats after induction of aging by D-gal. (A) Mitochondrial MFN1 level. (B) Mitochondrial MFN2 level. (C) Phosphorylated Drp1 at serine 616 in cytosol. (D) Mitochondrial Drp1 level. NDV, normal diet fed rats with vehicle; NDDg, normal diet fed rats with D-gal; HFV, high-fat diet fed rats with vehicle; HFDg, high-fat diet fed rats with D-gal; MFN1, mitofusin 1; MFN2, mitofusin 2; Drp1, dynamin-related protein 1; VDAC, voltage-dependent anion channels; HBOT, hyperbaric oxygen therapy. (n = 5/group). ^*P < 0.05 compared to other groups.

Figure 9

Effect of HBOT on autophagy in cardiomyocytes of pre-diabetic rats after induction of aging by D-gal. (A) Beclin-1 expression. (B) p62 expression. (C) LC3II expression. (D) Representative images of western blotting bands. NDV, normal diet fed rats with vehicle; NDDg, normal diet fed rats with D-gal; HFV, high-fat diet fed rats with vehicle; HFDg, high-fat diet fed rats with D-gal; p62, Sequestosome-1 (ubiquitin-binding protein); LC3II, microtubule associated light chain 3II; HBOT, hyperbaric oxygen therapy. (n = 5/group). ^*P < 0.05.

Figure 10

Study design. Fifty-six Wistar rats were fed normal diet or high-fat diet for 12 weeks. For subsequent 8 weeks, they were subcutaneously injected either vehicle (0.9% normal saline) or D-gal (150mg/kg/day). Rats were randomly subdivided into 7 groups at week 21: sham-treated (normal diet fed rats with vehicle (NDV), high-fat diet fed rats with vehicle (HFV), normal diet fed rats with D-gal (NDDg), high-fat diet fed rats with D-gal (HFDg)) and HBOT-treated (HFV, NDDg, HFDg). Sham rats received ambient pressure of oxygen while HBOT-treated ones received 100% oxygen given once daily for 60 minutes at 2 ATA. ND, normal diet; HFD, high-fat diet; SC, subcutaneous; NSS, normal saline; NDV, normal diet fed rats with vehicle; NDDg, normal diet fed rats with D-gal; HFV, high-fat diet fed rats with vehicle; HFDg, high-fat diet fed rats with D-gal, ATA, atmosphere absolute; HBOT, hyperbaric oxygen therapy.