Early-Stage Alcoholic Cardiomyopathy Highlighted by Metabolic Remodeling, Oxidative Stress, and Cardiac Myosin Dysfunction in Male Rats

Abstract

Chronic ethanol use can lead to alcoholic cardiomyopathy (ACM), while the impact on the molecular and cellular aspects of the myocardium is unclear. Accordingly, male Sprague-Dawley rats were exposed to an ethanol-containing diet for 16 weeks and compared with a control group that was fed an isocaloric diet. Histological measurements from H&E slides revealed no significant differences in cell size. A proteomic approach revealed that alcohol exposure leads to enhanced mitochondrial lipid metabolism, and electron microscopy revealed impairments in mitochondrial morphology/density. Cardiac myosin purified from the hearts of ethanol-exposed animals demonstrated a 15% reduction in high-salt ATPase activity, with no significant changes in the in vitro motility and low-salt ATPase or formation of the super-relaxed (SRX) state. A protein carbonyl assay indicated a 20% increase in carbonyl incorporation, suggesting that alcohol may impact cardiac myosin through oxidative stress mechanisms. In vitro oxidation of healthy cardiac myosin revealed a dramatic decline in ATPase activity and in vitro motility, demonstrating a link between myosin protein oxidation and myosin mechanochemistry. Collectively, this study suggests alcohol-induced metabolic remodeling may be the initial insult that eventually leads to defects in the contractile machinery in the myocardium of ACM hearts.

Keywords: actin; alcohol; heart failure; mitochondria; muscle contraction; myosin.

Figure 1

Workflow overview. A schematic representation of the project workflow is illustrated highlighting the key experimental steps: heart dissections from control and ethanol-consuming (EtOH) groups followed by cardiac histological analysis, mass spectrometry, myosin extraction, in vitro motility, assembly of synthetic thick filaments, measurements of steady-state ATPase activity and single ATP turnover rate, and protein oxidation experiments.

Figure 2

Histological images. Representative images of hematoxylin and eosin (H&E, A,B) and Masson’s Trichrome (C,D) staining from the control (A,C) and EtOH (B,D) groups at 20× magnification (insets at 1×).

Figure 3

Electron microscopy (EM) images. Representative EM images highlight the ultrastructural differences observed between the control and EtOH groups presented at three different magnifications: 10,000× (A,B), 25,000× (C,D), and 50,000× (E,F). Quantitative analysis of mitochondrial area (G) and electron lucency (H) is illustrated using a SuperPlot, where color-coded transparent dots represent individual mitochondrial measurements collected from different animals. Each solid dot indicates the mean for each animal (n = 6 per group). The black line denotes the group mean ± SD. Statistical significance was evaluated using an unpaired t-test. Corresponding numerical data and p-values are provided in Table 2.

Figure 4

Gene Ontology (GO) Enrichment Analysis. GO enrichment analysis filtered through three primary categories: biological process (green), molecular function (red), and cellular component (blue). Results highlight that differentially expressed proteins are involved in fatty acid β-oxidation through specific multienzyme complexes within the mitochondria.

Figure 5

In vitro motility assay. Actin sliding velocities from the unloaded in vitro motility assay are represented as a SuperPlot. Each triangle corresponds to an individual experiment (sample mean) from separate myosin preparations (n = 8). The color-coded transparent dots illustrate the individual velocities associated with each triangle, while the black line represents the mean of the sample means.

Figure 6

Mechanochemical characterization. Biochemical analyses of myosin properties in the control and EtOH groups, including low-salt ATPase activity (A), high-salt ATPase activity (B), fraction of the super-relaxed state (SRX) determined by single ATP turnover (C), SRX rates (D), DRX rates (E), and carbonyl intensity (F). Data are presented as mean ± SD (n = 5 per group). Statistical significance was evaluated using an unpaired t-test.

Figure 7

In vitro oxidation assay. Comparison of high-salt ATPase activity (A) and in vitro motility (B) across three conditions: untreated, mild oxidative treatment (1 mm H₂O₂), and high oxidative treatment (100 mM H₂O₂, 10 μM FeCl₃, 1 mM ascorbic acid). Data are presented as mean ± SD (n = 3 per group). Statistical significance was evaluated using an unpaired t-test.