Lethal Arrhythmogenic Role of Left Ventricular Myocardial Interstitial Fibrosis in Apolipoprotein E/Low-Density Lipoprotein Receptor Double-Knockout Mice with Metabolic Dysfunction-Associated Steatohepatitis

Abstract

The combination of alcohol and a low-carbohydrate, high-protein, high-fat atherogenic diet (AD) increases the risk of lethal arrhythmias in apolipoprotein E/low-density lipoprotein receptor double-knockout (AL) mice with metabolic dysfunction-associated steatotic liver disease (MASLD). This study investigates whether left ventricular (LV) myocardial interstitial fibrosis (MIF), formed during the progression of metabolic dysfunction-associated steatohepatitis (MASH), contributes to this increased risk. Male AL mice were fed an AD with or without ethanol for 16 weeks, while age-matched AL and wild-type mice served as controls. Liver and heart tissues were analyzed, and susceptibility to lethal arrhythmias was assessed through histopathology, fluorescence immunohistochemistry, RNA-Seq, RT-PCR, and lethal arrhythmia-evoked test. Ethanol combined with an AD significantly induced LV MIF in MASH-affected AL mice, as shown by increased fibrosis-related gene expression, Sirius-Red staining, and elevated collagen 1a1 and 3a1 mRNA levels, alongside a higher incidence of lethal arrhythmias. Cardiac myofibroblasts exhibited sympathetic activation and produced elevated levels of fibrosis-promoting factors. This study highlights the role of cardiac myofibroblasts in LV MIF, contributing to an increased incidence of lethal arrhythmias in MASH-affected AL mice fed ethanol and AD, even after the alcohol was fully metabolized on the day of consumption.

Keywords: alcohol; high-fat atherogenic diet; high-protein; left ventricular myocardial interstitial fibrosis; lethal arrhythmia; low-carbohydrate; metabolic dysfunction-associated steatotic liver disease.

Figure 1

The key characteristics of the animals. Representative results for body weight (BW) (A), liver weight (B), and LV weight (C). The data are presented as mean values with standard deviations (SDs). Statistical significance (* p < 0.05 vs. WT+SCD, Et (−); ^† p < 0.05 vs. AL+SCD, EtOH (−); ^# p < 0.05 vs. AL+AD, EtOH (−)) was determined using ANOVA followed by Bonferroni–Dunn post hoc test.

Figure 2

The effects of a co-diet of ethanol and AD on hepatic fat accumulation, inflammation, and fibrosis. Examples of Oil-Red-O staining, CD68 immunostaining, and Sirius-Red staining of liver sections (A). The scale bars are 200 μm for Oil-Red-O and Sirius-Red staining and 30 μm for CD68 immunostaining. Representative results of hepatic Oil-Red-O content (B), CD68-positive area (C), Cd68 mRNA expression (D), Sirius-Red content (E), and Col 1a1 mRNA expression (F). The data are presented as mean values with standard deviations (SDs). Statistical significance (* p < 0.05 vs. WT+SCD, Et (−); ^† p < 0.05 vs. AL+SCD, EtOH (−); ^# p < 0.05 vs. AL+AD, EtOH (−)) was determined using ANOVA followed by Bonferroni–Dunn post hoc test.

Figure 3

Sudden cardiac death resulting from the lethal arrhythmia-evoked test. Shown are representative electrocardiograms (ECGs) depicting the occurrence of complete atrioventricular block (CAVB), ventricular tachycardia (VT), and asystole induced by acute restraint stress (ARS) in AL mice fed with a co-diet of ethanol and AD. Each panel displays a 2 min ECG and 15 s zoomed-in lethal arrhythmia-evoked test, with an arrow indicating the onset of ARS.

Figure 4

Ethanol and AD changed gene expression related to fibrosis in LV. Cellular components of the genes significantly changed in the gene ontology (GO) enrichment analysis: to assess the results of the RNA-Seq analysis in terms of cellular components, the genes that changed in mice LV myocardium were matched to the genes related to each GO term, and the 10 terms with the lowest p-value were listed (A). The heatmap shows the gene expression changes related to fibrosis in the LV, ranked by the lowest p-values as determined by RNA-Seq analysis (B).

Figure 5

LV MIF resulting from the co-diet of ethanol and AD. Examples of LV sections stained with Sirius-Red (A), with a scale bar of 100 μm. Representative data of LV Sirius-Red content (B), LV Col 1a1 mRNA expression (C), and LV Col 3a1 mRNA expression (D). The data are presented as mean values with standard deviations (SDs). Statistical significance (* p < 0.05 vs. WT+SCD, Et (−); ^† p < 0.05 vs. AL+SCD, EtOH (−); ^# p < 0.05 vs. AL+AD, EtOH (−)) was determined using ANOVA followed by Bonferroni–Dunn post hoc test.

Figure 6

The combination diet of ethanol and AD increased the co-localization of TH (sympathetic activation marker) and α-SMA (activated cardiac myofibroblast marker) and the co-localization of α-SMA and TGF-β1 (associated with fibrotic diseases) in the immunostained LV sections. Examples merging nuclear (DAPI, blue), TH, and α-SMA appear as pink and merging nuclear (DAPI, blue), α-SMA, and TGF-β1 appear as yellow (A) alongside measurements of the LV-TH-positive area (B), α-SMA-positive area (C), and TGF-β1-positive area (D) within the same visual field of immunostained LV sections. The scale bar denotes 30 μm. Results are presented as mean ± SD. Statistical significance was determined as * p < 0.05 compared to WT+SCD, Et (−), ^† p < 0.05 compared to AL+SCD, EtOH (−), and ^# p < 0.05 compared to AL+AD, EtOH (−) using ANOVA followed by Bonferroni–Dunn post hoc test.

Figure 7

The impact of a co-diet of ethanol and AD on the mRNA expressions in the LV as determined by RT-PCR. The mRNA levels of Npy (A), Acta2 (B), and Tgfb1 (C) in mice subjected to the lethal arrhythmia-evoked test. The data are presented as mean values with standard deviations (SDs). Statistical significance (* p < 0.05 vs. WT+SCD, Et (−); ^† p < 0.05 vs. AL+SCD, EtOH (−); ^# p < 0.05 vs. AL+AD, EtOH (−)) was determined using ANOVA followed by Bonferroni–Dunn post hoc test.