Long-Term Sleep Deprivation-Induced Myocardial Remodeling and Mitochondrial Dysfunction in Mice Were Attenuated by Lipoic Acid and N-Acetylcysteine

Abstract

The impact of long-term sleep deprivation on the heart and its underlying mechanisms are poorly understood. The present study aimed to investigate the impact of chronic sleep deprivation (CSD) on the heart and mitochondrial function and explore an effective drug for treating CSD-induced heart dysfunction. We used a modified method to induce CSD in mice; lipoic acid (LA) and N-acetylcysteine (NAC) were used to treat CSD mice. Echocardiography, hematoxylin-eosin (H&E) staining, Sirius red staining, and immunohistochemistry were used to determine heart function and cardiac fibrosis. The serum levels of brain natriuretic peptide (BNP), superoxide Dismutase (SOD), micro malondialdehyde (MDA), and glutathione (GSH) were measured to determine cardiovascular and oxidative stress-related damage. Transmission electron microscopy was used to investigate mitochondrial damage. RNA-seq and Western blotting were used to explore related pathways. We found that the left ventricular ejection fraction (LVEF) and fraction shortening (LVFS) values were significantly decreased and myocardial hypertrophy was induced, accompanied by damaged mitochondria, elevated reactive oxygen species (ROS), and reduced SOD levels. RNA-sequence analysis of the heart tissue showed that various differentially expressed genes in the metabolic pathway were enriched. Sirtuin 1 (Sirt1) and Glutathione S-transferase A3 (Gsta3) may be responsible for CSD-induced heart and mitochondrial dysfunction. Pharmacological inhibition of ROS by treating CSD mice with LA and NAC effectively reduced heart damage and mitochondrial dysfunction by regulating Sirt1 and Gsta3 expression. Our data contribute to understanding the pathways of CSD-induced heart dysfunction, and pharmacological targeting to ROS may represent a strategy to prevent CSD-induced heart damage.

Keywords: N-acetylcysteine; lipoic acid; mitochondria; myocardial remodeling; reactive oxygen species; sleep deprivation.

Figure 1

CSD led to myocardial hypertrophy and heart failure in adult mice. (A) Representative echocardiogram and analysis of (B) left ventricular (LV) Eject Fraction (EF), (C) Fraction Shortening (FS), (D) left ventricle end-systolic diameter (LVESd), and (E) left ventricle end-diastolic diameter (LVEDd). (F) Representative anatomic images of control (NC) and chronic sleep deprivation (CSD) mice (scale bar = 2 mm). (G) Hematoxylin & eosin (H&E) images of crosscut section heart of control and CSD mice (scale bar = 1 mm or 100 μm). (H) The heart weight (HW) ratio to tibia length (TL) with or without four months of CSD. (I) Representative image and analyzed data of immunostaining of WGA indicating the cardiomyocyte size of NC and CSD. (J) Representative images and analyzed data of Sirius red-stained hearts showed increased collagen deposition in CSD hearts. The fibrotic regions (Red) in six different fields per heart (four hearts per group) were measured by ImageJ (scale bar = 100 μm). Serum levels of GSH (K) and BNP (L) in NC and CSD mice.

Figure 2

CSD induced mitochondrial impairment and elevated oxidative stress in the heart. (A) DHE staining and analysis (B) unravels increased lipid peroxidation in the CSD heart, indicating elevated oxidative stress. DHE fluorescent intensity was measured by ImageJ (scale bar = 125 μm). (C) Electron micrographs showing mitochondria in heart tissue obtained from control (NC) and chronic sleep deprivation (CSD) mice (scale bar = 5 μm, 2 μm, 1 μm). Serum levels of SOD (D) and MDA (E) in NC and CSD mice. (F) Representative Western blot image and quantification of SOD1 and SOD2 in NC and CSD hearts. (G) Representative Western blot image and quantification of HO-1 and Nrf2 in NC and CSD hearts.

Figure 3

RNA sequencing revealed the Sirt1−Gsta3 axis is involved in CSD−regulated heart dysfunction. (A) RNA sequencing heat map clustered in hierarchy volcano map (B) shows up− or down−regulated genes in NC and CSD hearts. (C) Signal pathway analysis of the differential expression (DE) gene of RNA−seq shows the top 10 pathways related to CSD treatment. (D) Heat map clustered in hierarchy shows that Sirt1 of the Nicotinate and nicotinamide metabolism pathway (Left panel) and Gsta3 of the metabolic pathway were downregulated in the CSD heart. (E) Representative Western blot images and quantification of Sirt1 and Gsta3 in NC and CSD hearts. (F) Representative Western blot images and quantification of phosphorylation or total of AMPK and Akt in NC and CSD hearts.

Figure 4

Antioxidants significantly rescue CSD-induced heart failure and myocardial hypertrophy. Representative echocardiography (A) and quantification of EF (B), FS (C), LVEDd (D), and LVESd (E) of the four indicating groups. (F) Representative anatomic images of four indicating groups (scale bar = 1 mm). (G) The ratio of heart weight (HW) to tibia length (heart/tibia) of four indicated groups. (H) Hematoxylin & eosin (HE) images of crosscut section heart of four displaying groups. Representative image (I) and quantification (J) of immunostaining of WGA indicating the cardiomyocyte size of four indicating groups (scale bar = 75 μm). Representative images (K) and analyzed data (L) of Sirius red-stained hearts of four indicating groups (scale bar = 50 μm).

Figure 5

Antioxidants alleviated CSD-induced impaired mitochondria. (A) DHE staining images and analyzed data (B) unravel increased lipid peroxidation heart of four indicating groups (scale bar = 125 μm). (C) Representative transmission electron microscopy (TEM) images of the heart of four indicated groups (scale bar = 5 μm, 2 μm, 1 μm). Serum levels of BNP (D), GSH (E), SOD (F), and MDA (G) in four indicating groups.

Figure 6

Antioxidants regulated Sirt1/Gsta3/SODs signaling pathway in CSD-induced heart failure. Representative western blot images and quantification of SOD1 (A,B), SOD2 (A,C), Nrf2 (D,E), HO1 (D,F), Sirt1 (G,H), Gsta3 (G,I), and the phosphorylation of AMPK (J,K) and Akt (J,L) in four indicating hearts.