Proteomic Profiling Reveals Roles of Stress Response, Ca²⁺ Transient Dysregulation, and Novel Signaling Pathways in Alcohol‐Induced Cardiotoxicity

Abstract

Background: Alcohol use in pregnancy increases the risk of abnormal cardiac development, and excessive alcohol consumption in adults can induce cardiomyopathy, contractile dysfunction, and arrhythmias. Understanding molecular mechanisms underlying alcohol-induced cardiac toxicity could provide guidance in the development of therapeutic strategies. Methods: We have performed proteomic and bioinformatic analysis to examine protein alterations globally and quantitatively in cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) treated with ethanol (EtOH). Proteins in both cell lysates and extracellular culture media were systematically quantitated. Results: Treatment with EtOH caused severe detrimental effects on hiPSC-CMs as indicated by significant cell death and deranged Ca 2+ handling. Treatment of hiPSC-CMs with EtOH significantly affected proteins responsible for stress response (e.g., GPX1 and HSPs), ion channel-related proteins (e.g. ATP1A2), myofibril structure proteins (e.g., MYL2/3), and those involved in focal adhesion and extracellular matrix (e.g., ILK and PXN). Proteins involved in the TNF receptor-associated factor 2 signaling (e.g., CPNE1 and TNIK) were also affected by EtOH treatment. Conclusions: The observed changes in protein expression highlight the involvement of oxidative stress and dysregulation of Ca 2+ handling and contraction while also implicating potential novel targets in alcohol-induced cardiotoxicity. These findings facilitate further exploration of potential mechanisms, discovery of novel biomarkers, and development of targeted therapeutics against EtOH-induced cardiotoxicity.

Multi-Omics Approach Reveals Dysregulation of Protein Phosphorylation Correlated with Lipid Metabolism in Mouse Non-Alcoholic Fatty Liver

“…We then performed a Student’s t -test to unveil proteins that were significantly regulated after DIO. A volcano plot representation of our filtered data shows that the distribution of upregulated and downregulated proteins was remarkably symmetrical, with 312 proteins above the q < 0.05 and 0.38 |log 2 (fold-change)| cutoffs [ 21 , 22 , 23 , 24 , 25 ], set as the minimum to consider proteins as “significantly regulated” ( Figure 3 B). Specifically, 141 proteins were downregulated and 171 were upregulated.…”

Carfilzomib Treatment Causes Molecular and Functional Alterations of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes

“…Proteins were extracted from 3 to 4×10 6 hiPSC‐CMs per sample by suspending the cells in the lysis buffer as described previously. 30 Proteins were purified through methanol‐chloroform precipitation. For the secretome analysis, the media was passed through a filter (0.45 µm) and then concentrated by centrifugation (molecular weight 3 kDa cutoff).…”

Chronic Ethanol Exposure Induces Deleterious Changes in Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells

“…Every three days, cardiac spheres were collected and resuspended in fresh culture medium, the same volume of 2 × EtOH-working solutions were added in the dishes. Mineral oil was overlaid on top of the medium to prevent EtOH evaporation for both EtOH-treated and untreated groups [30]. Cardiac spheres were treated with EtOH at 0, 17, and 50 mM for up to 5 weeks.…”

“…After digestion, peptides were purified using tC18 Sep-Pak cartridges. Tandem mass tag (TMT) labeling and fractionation, LC-MS/MS analysis, database search, data filtering, peptide quantification, and bioinformatic analysis were conducted as described previously [30]. Proteins were considered being up-or down-regulated when the abundance changed by > 1.2-fold between two groups.…”

“…hiPSC-CMs have been adopted broadly in cardiac disease modeling, including hypertrophic cardiomyopathy [25], dystrophy cardiomyopathy [26], and dilated cardiomyopathy [27]. hiPSC-CMs have also been used to study cardiotoxicity, including those induced by chemotherapeutic drug [28,29], short-term EtOH exposure [13,30], and iron overload [31].…”