Cardiac and respiratory muscle responses to dietary N-acetylcysteine in rats consuming a high-saturated fat, high-sucrose diet

Abstract

New findings: What is the central question of this study? This study addresses whether a high-fat, high-sucrose diet causes cardiac and diaphragm muscle abnormalities in male rats and whether supplementation with the antioxidant N-acetylcysteine reverses diet-induced dysfunction. What is the main finding and its importance? N-Acetylcysteine attenuated the effects of high-fat, high-sucrose diet on markers of cardiac hypertrophy and diastolic dysfunction, but neither high-fat, high-sucrose diet nor N-acetylcysteine affected the diaphragm. These results support the use of N-acetylcysteine to attenuate cardiovascular dysfunction induced by a 'Western' diet.

Abstract: Individuals with overweight or obesity display respiratory and cardiovascular dysfunction, and oxidative stress is a causative factor in the general aetiology of obesity and of skeletal and cardiac muscle pathology. Thus, this preclinical study aimed to define diaphragmatic and cardiac morphological and functional alterations in response to an obesogenic diet in rats and the therapeutic potential of an antioxidant supplement, N-acetylcysteine (NAC). Young male Wistar rats consumed ad libitum a 'lean' or high-saturated fat, high-sucrose (HFHS) diet for ∼22 weeks and were randomized to control or NAC (2 mg/ml in the drinking water) for the last 8 weeks of the dietary intervention. We then evaluated diaphragmatic and cardiac morphology and function. Neither HFHS diet nor NAC supplementation affected diaphragm-specific force, peak power or morphology. Right ventricular weight normalized to estimated body surface area, left ventricular fractional shortening and posterior wall maximal shortening velocity were higher in HFHS compared with lean control animals and not restored by NAC. In HFHS rats, the elevated deceleration rate of early transmitral diastolic velocity was prevented by NAC. Our data showed that the HFHS diet did not compromise diaphragmatic muscle morphology or in vitro function, suggesting other possible contributors to breathing abnormalities in obesity (e.g., abnormalities of neuromuscular transmission). However, the HFHS diet resulted in cardiac functional and morphological changes suggestive of hypercontractility and diastolic dysfunction. Supplementation with NAC did not affect diaphragm morphology or function but attenuated some of the cardiac abnormalities in the rats receiving the HFHS diet.

Keywords: diaphragm; diastolic function; hypertrophy; oxidants.

Figure 1.. Body weight, energy intake, and water consumption.

Lean = lean control diet, HFHS = high-saturated fat, high sucrose (HFHS) diet, N-acetylcysteine = NAC in drinking water. (A) Weight gain with age. The dotted line indicates the start of NAC treatment, which was ~14 weeks after start of assigned diets. (B) Terminal body weights. (C) Terminal tibia length as a marker of body size. (D) BW/TL ratio as a marker of adiposity. (E) Average apparent daily energy intake. (F) Average apparent daily water consumption. Data are mean ± SD in panel A. In panels B-D, data are shown as scatter plots and mean bars. In panels E-F, data are shown as mean scatter plot data per animal, and bars are group averages of these means. Comparisons among 3 groups were conducted using One-Way ANOVA. Individual p values for post hoc tests (Dunnett’s) are shown when feasible. * = p<0.05 Lean vs HFHS, Φ = p <0.05 HFHS vs. HFHS+NAC. BW = body weight, TL = tibia length

Figure 2.. Glucose handling.

(A) Glucose tolerance test. (B) Fasting blood glucose (6-hour fast). (C) Glucose tolerance test area under the curve (AUC) calculations. Data are mean ± SD in panel A. In panels B-C, data are shown as scatter plot and mean bars. Comparisons among 3 groups were conducted using One-Way ANOVA. Individual p values for post hoc tests (Dunnett’s) are shown when feasible.

Figure 3.. Diaphragm contractile function.

(A) Twitch (1 Hz), (B) submaximal (40 Hz), and (C) maximal (120 Hz) isometric specific force of diaphragm bundle. (D) Power of diaphragm bundle using a clamped load of 30%–35% maximal force. Comparisons among 3 groups were conducted using One-Way ANOVA. No comparisons surpassed the threshold for statistical significance (P < 0.05).

Figure 4.. Diaphragm fiber cross-sectional area.

Diaphragm fiber cross-sectional area and myosin heavy chain isoform distribution. (A) Sample images of diaphragm immunohistochemistry for fiber typing and cross-sectional area. Colors represent specific MHC isoforms (blue = type I, green = type IIa, black = type IIb/x). (B) Cross-sectional area for each fiber type in the diaphragm. Immunohistochemistry data are shown as scatter plots of the average value of individual fibers measured for each animal and bars representing group means. Comparisons among 3 groups were conducted using One-Way ANOVA. No comparisons surpassed the threshold for statistical significance (P < 0.05).

Figure 5.. Diaphragm passive tension, stiffness, and fibrosis.

(A) Sample images of diaphragm Masson’s Trichrome stain for quantification of fibrotic tissue. (B) Quantification of % fibrosis in diaphragm shown as scatter plots of the average value for each animal and bars representing group means. (C) Relationship between passive tension (N/cm²) and strain (normalized to optimal length) in diaphragm bundles. (D) Young’s elastic modulus of diaphragm bundles was calculated as the change in passive tension normalized to strain (~3% from optimal length). Data are shown as scatter plots and mean bars. Comparisons among 3 groups were conducted using One-Way ANOVA. No comparisons surpassed the threshold for statistical significance (P < 0.05).

Figure 6.. Terminal cardiac size and function.

(A) Heart weight normalized to estimated body surface area (BSA). (B) Left ventricular weight/BSA, (C) right ventricular weight/BSA, (D) Fractional shortening (%), (E) posterior wall shortening velocity, and (F) E wave deceleration rate at the level of the mitral valve. (G) Representative 2D M-mode images. Data are shown as scatter plots and mean bars. Comparisons among 3 groups were conducted using One-Way ANOVA. Individual p values for post hoc tests (Dunnett’s) are shown. E = E wave, DT = deceleration time

Figure 7.. Effects of NAC treatment on diastolic function.

E wave deceleration rate (E/DT) at the level of the mitral valve prior to control or NAC treatment (pre) and after approximately 8-weeks of treatment (post) for lean (A), HFHS (B), and (C) HFHS+NAC rats. Data are shown as scatter plots with lines connecting paired values per animal. Pre and Post values were compared within each group using a paired Student t-test. (D) Change in E/DT shown as scatter plot and mean bars with comparison among the 3 groups conducted using One-Way ANOVA. Individual p values for post hoc tests (Dunnett’s) are shown. E = E wave, DT = deceleration time