Chronic Dietary Stress Is Associated with Early Cardiac Dysfunction and Progressive MASH Accompanied by Systemic Inflammation and Fibrosis

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  Figure 1. Chronic dietary stress induces early cardiac dysfunction and cardiomyocyte hypertrophy. (A) Experimental design illustrating two groups of mice fed either normal chow (NC) or the GAN diet (GD) for 6 months. Cardiac function and metabolic parameters were assessed every 2 months. (B,C) Progressive reductions in ejection fraction (EF) and fractional shortening (FS) in GD-fed mice at 2, 4, and 6 months indicate impaired systolic function. (D) Representative longitudinal M-mode echocardiographic images. (E,F) Increased E/A and E/E′ ratios over time demonstrate the development of diastolic dysfunction in GD-fed mice. (G) Representative Doppler images of transmitral inflow and tissue Doppler waveforms obtained from the left ventricular posterior wall, used to assess left ventricular diastolic function and regional wall motion abnormalities, respectively. (H) Increased body weight (BW) in GD-fed mice. (I) Reduced heart weight normalized to body weight (HW/BW) in GD-fed mice. (J) No significant difference in heart weight normalized to tibial length (HW/TL) between groups (n = 8 mice). (K) Representative wheat germ agglutinin (WGA) immunofluorescence staining and (L) quantitative analysis…

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  Figure 2. Chronic dietary stress induces cardiac fibrosis and inflammation. (A) Representative Masson’s trichrome-stained cardiac sections and (B) quantitative analysis showing increased myocardial fibrotic area in GD-fed mice compared with NC controls (n = 7 mice/group; eight sections/mouse). (C) Representative Sirius Red-stained cardiac sections and (D) quantitative analysis showing increased collagen deposition in GD-fed mice compared with NC controls (n = 7 mice/group; four sections/mouse). Scale bars = 200 μm. (E,G) Representative CD68 and F4/80 immunostaining, respectively, and (F,H) corresponding quantitative analyses showing increased macrophage infiltration in GD-fed mice (n = 6 mice/group; eight sections/mouse). Scale bars = 20 μm. (I) Increased Ctgf and Tgfβ expression supports activation of profibrotic signaling in GD hearts. (J) Increased Il6 and Tnfα expression indicates enhanced cardiac inflammatory signaling in GD-fed mice. (K) Increased expression of Myh7, Nppb (BNP), and Nppa (ANF) is consistent with pathological cardiac remodeling in GD-fed mice (n = 4 biologically independent experiments). (L) Representative Western blot showing increased BNP protein abundance in…

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  Figure 3. Chronic dietary stress promotes metabolic dysfunction, hypercholesterolemia, and hepatic injury. (A) Progressive increase in body weight (BW) in GD-fed mice, indicative of sustained metabolic stress. (B) Elevated total cholesterol (TC) levels from 2 to 6 months of dietary intervention, demonstrating the development of hypercholesterolemia. (C) No significant differences in circulating triglyceride (TG) levels between groups. (D–F) Increased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), indicating hepatocellular injury and liver dysfunction in GD-fed mice. (G) Increased liver weight normalized to body weight (LW/BW), consistent with hepatic enlargement and injury. (H) Increased liver weight as well as inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) mass in GD-fed mice, reflecting enhanced adiposity and metabolic remodeling. (I) Oral glucose tolerance test (OGTT) demonstrating a transient increase in blood glucose in GD-fed mice at 30 min after glucose administration. Data are presented as mean ± SEM; n = 8 mice in each group. Longitudinal body weight (A) and glucose measurements…

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  Figure 4. GAN diet is associated with increased adiposity, altered metabolic parameters, and reduced physical activity. (A–C) GD-fed mice exhibited increased body weight, fat mass, and fat mass normalized to body weight (fat mass/BW), indicating increased adiposity. (D) No significant difference was observed in lean mass between groups. (E) Lean mass normalized to body weight (lean mass/BW) was significantly reduced in GD-fed mice compared with NC controls. (F) Respiratory exchange ratio (RER) was lower in GD-fed mice during the dark cycle, consistent with a relative shift toward greater lipid utilization. (G,H) Oxygen consumption (VO2) and carbon dioxide production (VCO2) were lower in GD-fed mice during the dark cycle. (I) Total water intake was comparable between groups. (J) Energy expenditure, measured as heat production (kcal/min), differed between groups during the light cycle but not during the dark cycle. (K,L) Decreased ambulatory activity (XTOT counts) and total movement (ZTOT counts) indicate reduced locomotor and overall physical activity in GD-fed mice. (M) No significant difference in food intake was observed between NC and GD groups. Data are presented as mean ± SEM, n = 5…

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  Figure 5. GAN diet promotes an advanced MASH phenotype and fibrotic remodeling. (A) Representative Masson’s trichrome staining and (B) quantitative analysis demonstrating marked hepatic fibrosis in GD-fed mice compared with NC controls (n = 6 mice/group; eight sections per mouse). Scale bar = 100 μm. (C–F) Representative photomicrographs and quantitative analyses of CD68 and F4/80 immunostaining showing increased macrophage infiltration and hepatic inflammation in GD-fed mice. Scale bar = 20 μm. (G) Histopathological assessment of steatosis, hepatocellular ballooning, and lobular inflammation in normal chow NC- and GAN diet GD-fed mice. The NAFLD Activity Score (NAS) was calculated as the sum of the steatosis, ballooning, and inflammation scores. GD-fed mice showed significantly higher steatosis, ballooning, and inflammatory scores, resulting in a higher composite NAS compared with NC-fed mice. Values are presented as mean ± SEM (n = 6 mice/group). Individual mouse-level data are provided in Supplementary Table S2. Statistical comparisons were performed using an exact two-sided Mann–Whitney U test; the U statistic was 0 and the exact p value was 0.0022 for each comparison. Holm correction…

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  Figure 6. Experimental design and graphical summary illustrating the progression of chronic dietary stress-induced cardiometabolic dysfunction. The schematic depicts the experimental timeline and the sequential effects of GAN diet feeding, highlighting the development of obesity, hypercholesterolemia, impaired glucose homeostasis, and systemic inflammation. These metabolic disturbances promote hepatic steatosis, inflammation, fibrosis, and progression to a MASH-like phenotype, while simultaneously driving cardiac inflammation, cardiomyocyte hypertrophy, fibrotic remodeling, and both systolic and diastolic dysfunction. Together, these findings demonstrate how chronic dietary stress induces coordinated pathological changes in the liver and heart, contributing to progressive cardiometabolic disease. Gubra Amylin NASH (GAN); ejection fraction (EF); fractional shortening (FS); total cholesterol (TC); alanine aminotransferase (ALT); metabolic dysfunction-associated steatohepatitis (MASH).