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. Proteins in both cell lysates and extracellular culture media were systematically quantitated.

Results:

Treatment with ethanol caused severe detrimental effects on hiPSC-CMs as indicated by significant cell death and deranged Ca²⁺ handling. Treatment of hiPSC-CMs with ethanol 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 TRAF2 signaling (e.g. CPNE1 and TNIK) were also affected by ethanol treatment.

Conclusions:

The observed changes in protein expression highlight the involvement of oxidative stress and dysregulation of Ca²⁺ 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 ethanol-induced cardiotoxicity.

INTRODUCTION

Excessive alcohol consumption continues to be a major cause of mortality and morbidity in the US and the relevance of alcohol abuse for public health is well established (GBD 2016 Alcohol Collaborators, 2018; White et al., 2020). In a recent study, the National Institute on Alcohol Abuse and Alcoholism (NIAAA) reported that nearly 1 million people died from alcohol-related causes between 1999 and 2017 and that in 2017 alcohol played a role in 2.6% of all deaths in the US (White et al., 2020). Excessive alcohol consumption is known to elicit adverse effects on multiple organs and systems, including the heart.

With respect to the heart, alcohol can induce cardiomyopathy, contractile dysfunction and arrhythmias, leading to heart failure, myocardial infarction (MI), and sudden cardiac death (Whitman et al., 2017). Heavy alcohol consumption in a binge pattern can cause an acute cardiac rhythm and/or conduction disturbance even in those with normal heart function. In this type of severe arrhythmia, atrial fibrillation is the most common, followed by atrial flutter, junctional tachycardia, premature ventricular and atrial complexes, paroxysmal atrial tachycardia, and ventricular tachycardia (Day and Rudd, 2019). In addition, alcohol exposure during pregnancy is known to disrupt fetal development and increase the risk of fetal alcohol spectrum disorder (FASD) (Roozen et al., 2016). Nearly 1 out of 3 FASD children are diagnosed with congenital heart defects and each year there are approximately 10,000 cases of alcohol-induced congenital heart defects in the US, indicating that parental alcohol consumption confers detriment effect on cardiac development in a large number of the children (Zhang et al., 2020). Unfortunately, without effective therapies further heart complications may develop later in life.

Understanding pathophysiological mechanisms underlying alcohol-induced cardiac toxicity could provide guidance in the development of therapeutic strategies. Studies in several model systems have provided significant insights into the mechanisms of ethanol-induced damage to cardiomyocyte (CM) (Tan et al., 2012; Guo et al., 2012; Maiuolo et al., 2018). For instance, oxidative stress mediated mitochondria dysfunction was found to contribute to alcohol-induced heart failure in mice (Brandt et al., 2016). Sarcoplasmic reticulum Ca²⁺-leak and disordered excitation-contraction coupling were found to play an important role in the arrhythmogenic effects of ethanol on murine and human CMs (Mustroph et al., 2018). More recently, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used to study alcohol-induced cardiotoxicity (Rampoldi et al., 2019)—hiPSC-CMs are a physiologically relevant model to human fetal cardiomyocytes because of their immature nature and they can overcome the limitations of human primary CM samples which are difficult to acquire and maintain in vitro (Pinheiro et al., 2019). In this model, treatment of the cells dose-dependently increased the production of reactive oxygen species, cell death, and abnormal Ca²⁺ transients and CM contraction (Rampoldi et al., 2019).

Comparative gene expression profiling could help uncover genes and pathways that play important roles in disease processes. Previous studies have provided data on transcriptome alterations in ethanol-induced cardiotoxicity (Rampoldi et al., 2019). However, global protein changes upon the treatment of the CMs with ethanol have not been reported. Modern mass spectrometry (MS)-based proteomics technique enables global protein identification and quantification in complex biological samples (Xiao et al., 2015; Aebersold and Mann, 2016; Mann, 2016; Orlando and Aebersold, 2019), and it has been employed to unmask molecular mechanisms such as doxorubicin-induced cardiotoxicity, dilated cardiomyopathy, and MI (Isserlin et al., 2010; Peng et al., 2014; Tomlinson et al., 2019).

In this study, we have performed proteomic analysis to examine protein alterations globally and quantitatively in hiPSC-CMs treated with ethanol. Proteins in both cell lysates and extracellular culture media were systematically quantitated. Here we report the proteins with significant changes caused by ethanol treatment as well as the biological processes and pathways associated with the affected proteins. These proteins are involved in stress response, ion channels, myofibril structure, focal adhesion, and extracellular matrix (ECM). Our study also reveals other novel targets including TNF receptor-associated factor 2 (TRAF2) signaling in alcohol-induced cardiotoxicity. We provide a unique resource: a human proteomic dataset for ethanol-induced cardiotoxicity that aids in a better understanding of the molecular mechanisms of the alcohol-induced cardiotoxicity.

MATERIALS AND METHODS

Culture of hiPSCs and cardiomyocyte differentiation

Undifferentiated hiPSCs (SCVI-273 line, obtained from Stanford Cardiovascular Institute) were fed daily on Matrigel-coated plates with mTeSR™ medium and passaged using Versene when compact colonies reached 90% confluence. For CM differentiation, hiPSCs were induced using a small molecule-guided differentiation protocol (Burridge et al., 2014). At the day of induction (day 0), medium was replaced with RPMI 1640 medium supplemented with 2% B27 minus insulin containing 6 μM CHIR99021. After induction for 48 h, the medium was replaced with RPMI supplemented with 2% B27 minus insulin. After another 24 h, the medium was replaced with fresh RPMI supplemented with 2% B27 minus insulin containing 5 μM IWR1. After a further 48 h, the medium was replaced with RPMI supplemented with 2% B27 with insulin. At day 6, the cells were dissociated and re-seeded into AggreWell™400 plates to acquire cardiospheres (CSs) (Jha et al., 2016). After 24 h, the CSs formed were transferred to low-adhesion dishes for suspension culture. The medium was changed every other day. CSs typically started beating spontaneously by day 8 to 10. Information about the vendors and catalog numbers for major reagents are included in Table S1.

Immunocytochemistry

For the immunocytochemical staining, differentiation cultures were dissociated and reseeded onto 96-well plates. Cells were fixed in 4% paraformaldehyde for 15 min and permeabilized using ice-cold methanol for 2 min at room temperature (RT). The cells were then incubated with 5% normal goat serum (NGS) in phosphate-buffered saline (PBS) at RT for 1 h and then incubated with primary antibodies (Table S2) in 3% NGS overnight at 4°C. After washing, the cells were incubated with the corresponding secondary antibodies at RT for 1 h in dark followed by counterstaining the nuclei with 7 μM Hoechst33342. Imaging was performed using an inverted microscope (Axio Vert.A1).

Cardiomyocyte purity assay

Differentiation cultures were analyzed for CM purity using antibodies against NKX2–5, a cardiac specific transcription factor. Following immunocytochemistry, images of Hoechst and NKX2–5-positive cells were acquired and quantitatively analyzed using ArrayScan™ XTI Live High Content Platform (Thermo Fisher Scientific). Acquisition Software Cellomics Scan was used to capture images via a 10× objective camera. Each well was sectioned to 7×7 fields in total and 20 fields in the center were selected. Images were analyzed using Cellomics View Software with mask modifiers for Hoechst and NKX2–5 restricted to the nucleus. The percentage of cells positive for NKX2–5 was calculated as CM purity (Rampoldi et al., 2019).

Ethanol treatment

For the ethanol treatment experiments, a working solution of 200 mM ethanol was freshly prepared by diluting pure ethyl alcohol in the culture medium. Prior to ethanol treatment, medium in cell culture wells was replaced with 1 volume of medium. An equal volume of the 200 mM ethanol working solution was then added to the wells. The final concentration of ethanol in the cell culture was 100 mM. Mineral oil was overlaid on top of the medium to prevent ethanol evaporation. In addition, ethanol-containing medium was refreshed every other day.

Cell viability assay

Cell viability was measured using the CellTiter-Blue® Cell Viability Assay, which is a fluorometric method for estimating the number of viable cells based on the metabolic capacity of cells. On the day of analyzing cell viability, CellTiter-Blue reagent was added into culture wells at a ratio of 1:5 to the medium, and the plates were incubated for 2 to 4 h at 37°C in dark. After the incubation, fluorescence was measured using the Synergy 2 Microplate Reader (BioTek) and Gen5 3.03 Microplate Reader and Imager Software with an excitation wavelength of 530 nm and an emission wavelength of 590 nm.

Ca²⁺ transient assay

Live cell imaging of intracellular Ca²⁺ transients was performed using cell permeant dye Fluo-4 AM. hiPSC-CMs at a low density were incubated with 5μM Fluo-4 AM for 30 min at 37℃ in dark followed by a 5 min wash with warm 1× normal Tyrode solution (148 mM NaCl, 4 mM KCl, 0.5 mM MgCl₂·6H₂O, 0.3 mM NaPH₂O₄·H₂O, 5 mM HEPES, 10 mM D-Glucose, 1.8 mM CaCl₂·H₂O, pH adjusted to 7.4 with NaOH). Fluorescent images were recorded using the ImageXpress Micro XLS System (Molecular Devices) at a frequency of 5 Hz for 12–32 s with 20× magnification. The fluorescence intensities over time for individual cells were analyzed through MetaXpress software (Molecular Devices) by measurements in the region of interest.

Proteomic analysis

Cell lysis and protein digestion.

Proteins were extracted from 3~4×10⁶ hiPSC-CMs per sample by suspending the cells in the lysis buffer (50 mM HEPES pH = 7.4, 150 mM NaCl, 0.5% SDC, 10 units/mL benzonase, and 1 tablet/10 mL protease inhibitor) at 4℃ for 45 min. Protein concentration was determined by the BCA assay, and proteins in all samples were then normalized based on their measured concentrations. Proteins were reduced with 5 mM DTT (56°C, 30 min), followed by alkylation with 14 mM iodoacetamide (RT, 30 min in the dark). They were purified through methanol-chloroform protein precipitation. For secretome analysis, the media was first passed through a filter (0.45 μm) and then concentrated by centrifugation (3 kDa cutoff). The other steps were the same as described for cell lysates. The isolated proteins were digested with trypsin in a buffer containing 50 mM HEPES (pH 8.5), 1.6 M urea at 37°C overnight. After the digestion, peptides were purified using tC18 Sep-Pak cartridges.

TMT labeling and fractionation.

Purified peptides from each sample were labeled with each channel of the six-plexed tandem mass tag (TMT) reagents according to the manufacturer’s protocol. The labeled peptides from all six samples were combined and desalted using a tC18 Sep-Pak cartridge. For peptides from the cell lysates, they were further separated into 20 fractions by high pH reversed-phase high-performance liquid chromatography (HPLC) using a 40 min gradient of 5–55% ACN in 10 mM ammonium acetate (pH = 10). Each fraction was purified again by the stage tip method. For secretome analysis, peptides were separated into six fractions during the stage tip.

LC-MS/MS.

Purified and dried peptide samples were dissolved in the loading buffer (5% ACN and 4% formic acid), and 2 μl was injected onto a microcapillary column packed with C18 beads using a WPS-3000TPLRS autosampler (UltiMate 3000). After being separated by reversed-phase HPLC, eluted peptides were directly detected in a hybrid dual-cell quadrupole linear ion trap-orbitrap mass spectrometer (LTQ Orbitrap Elite, Thermo Scientific) using a data-dependent Top15 method. Full MS scan (resolution: 60,000) was recorded in the Orbitrap at 10⁶ AGC target. Peptides were fragmented using higher-energy collision dissociation (HCD) with 40% normalized energy and then fragments were detected in the Orbitrap cell with high resolution and high mass accuracy. Ions with a single or unassigned charge were excluded for further sequencing.

Database search, data filtering and quantification.

The raw files were converted to an mzXML format and then searched against the database containing sequences of all human proteins (Homo sapiens) downloaded from the UniProt with the SEQUEST algorithm (version 28) (Eng et al., 1994). The following parameters were used during the search: 20 ppm precursor mass tolerance; 0.025 Da product ion mass tolerance; fully digested with trypsin; up to three missed cleavages; fixed modifications: carbamidomethylation of cysteine (+57.0214); TMT tag of lysine (+229.1629) and peptide N-terminus (+229.1629); variable modifications: oxidation of methionine (+15.9949). The target-decoy method was used to evaluate the false discovery rates (FDRs) of peptide and protein identifications, and linear discriminant analysis (LDA), which integrates several parameters including XCorr, precursor mass error, and charge state, was employed to control the quality of peptide identifications (Elias and Gygi, 2007; Kall et al., 2007). Peptides with fewer than seven amino acid residues were removed. Peptide spectral matches and proteins were both filtered to be <1% FDR. The TMT reporter ion intensities in the tandem mass spectra were used to quantify identified peptides and the TMT intensity for one protein was calculated from the median TMT intensity of all peptides from this protein.

Bioinformatic analysis

Differentially expressed proteins were recognized when the abundance changed by >1.3-fold (log₂(1.3) = 0.38) compared to the control group and the P-value was <0.05. Herein P-values were calculated using Perseus (Tyanova et al., 2016), in which a one-sample t-test (S0 = 0) was performed. This threshold allowed the selection of a larger number of differentially expressed proteins than if more stringent threshold was used for a broad GO-term and pathway analyses, which should be taken into consideration in the interpretation of these analyses although protein changes at this threshold could have significant biological consequence. Protein functional annotation including Gene Ontology (GO) analysis and KEGG pathway analysis was performed with Database for Annotation, Visualization and Integrated Discovery (DAVID) (Huang et al., 2008). Protein network was constructed through STRING database (interaction score ≥ 0.7) and visualized by Cytoscape (Szklarczyk et al., 2014; Shannon et al., 2003).

Statistical methods

Data were analyzed in Excel or R and graphed in GraphPad Prism 7.04 or OriginPro 2020. Data were presented as mean ± SD. Comparisons were conducted via Student’s t-test or two-sided Chi-square test with significant differences defined by P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****). Sample sizes were given for each experiment.

Proteomics datasets

Proteomics datasets from this study can be found on the PeptideAtlas (Identifier: PASS01620; Password: CY5836iy).

RESULTS

Ethanol treatment induces hiPSC-CM death and Ca²⁺ handling defect

Ethanol treatment induces cardiotoxicity in hiPSC-CMs at the cellular and functional levels (Rampoldi et al., 2019). To further understand ethanol-induced changes in proteins and biological processes, we first generated enriched hiPSC-CMs and treated them with 0 and 100 mM of ethanol for 5 days as we previously reported (Rampoldi et al., 2019) (Fig. 1A, Fig. S1). The concentration of 100 mM ethanol corresponds to the blood alcohol level of ~6 times legally permissible limits and causes confusion. As shown in Fig. 1B, based on CellTiter-Blue Cell Viability Assay, cell viability decreased by 20% when cells were treated with ethanol compared with untreated control.

Fig. 1.

Ethanol treatment induces the death of hiPSC-CMs and defect in Ca²⁺ handling. (A) Representative images of immunocytochemistry showing the majority of the cells in culture were positive for cardiac markers at day 17. (B) Normalized cell viability in hiPSC-CMs exposed to 0 and 100 mM of ethanol for 5 days (n = 4). (C) Representative traces showing intracellular Ca²⁺ transients in hiPSC-CMs. Type i: normal Ca²⁺ transients; Types ii to vi: abnormal Ca²⁺ transients. (D) Proportions of hiPSC-CMs exhibiting normal (blue) and abnormal Ca²⁺ transients (red) following the treatment with 0 and 100 mM of ethanol for 5 days are shown with the larger pie graphs. The distribution of the abnormal Ca²⁺ transient subtypes are shown with the smaller pie graphs. The number of cells analyzed were 85 for 100 mM and 24 for untreated. ***, P-value < 0.001; ****, P-value < 0.0001.

We also assessed the effect of ethanol treatment on the intracellular Ca²⁺ transients in hiPSC-CMs, a critical index of CM function since Ca²⁺ acts as a bridge between electrical excitation and mechanical contraction. We counted the numbers of cells exhibiting normal or abnormal Ca²⁺ transients and calculated the proportion of each category for each culture condition according to the following criteria. Specifically, cells were categorized as normal if the Ca²⁺ transients had mostly consistent amplitudes and rhythmicity, typical cardiac Ca²⁺ transient morphology (i.e. rapid upstroke and decay kinetics), and no obvious spontaneous Ca²⁺ release between transients (Fig. 1C–i). Cells were categorized as abnormal if they exhibited sluggish upstroke or decay morphology (Fig. 1C–ii), oscillations of the diastolic Ca²⁺ signal (Fig. 1C–iii and Fig. 1C–iv), unrecognizable single transient morphology (Fig. 1C–v), or notable inconsistent amplitudes or beat periods (Fig. 1C–vi). In the control group, the majority of the cells exhibited normal Ca²⁺ transients, whereas in hiPSC-CMs treated with ethanol the percentage of cells exhibiting abnormal Ca²⁺ transients increased to 69% (Fig. 1D). In addition, among the observed abnormal Ca²⁺ transients, only two types occurred in the control group (type iv: 33.3%, type vi: 66.7%), whereas all the five were observed in the treatment group types (type ii: 6.8%, type iii: 16.9%, type iv: 57.6%, type vi: 6.8%, type vi: 11.9%) (Fig. 1D). These observations indicate that exposure of hiPSC-CMs to ethanol results in cell death and dysfunction of intracellular Ca²⁺ handling.

Proteomic analysis identifies differentially expressed proteins caused by ethanol treatment

To characterize protein expression changes caused by ethanol treatment, we collected cell lysates and culture media separately from the triplicates of hiPSC-CM 3D-cultures that had been treated with 0 mM or 100 mM ethanol for 5 days. After protein extraction and digestion, peptides were labeled with the TMT reagents for protein quantitation. The TMT-labeled samples (6 for cell lysates and 6 for media) were then mixed and fractionated. Each fraction was analyzed by an online LC-MS system. In total, we detected 4,538 proteins in the cell lysate samples and 384 proteins in the media samples. The experimental procedure is shown in Fig. 2A.

Fig. 2.

Quantitative analysis of the proteome and secretome of hiPSC-CMs treated with ethanol. (A) The workflow to characterize the alteration of protein expression in cell lysates and media caused by ethanol exposure. (B) Reproducibility of the biological triplicates from each cell lysate sample. The number represents Pearson’s correlation coefficient (r). (C) Volcano plots illustrating proteins in cell lysates and media with statistically significant difference in their abundance in ethanol-treated vs. untreated hiPSC-CMs. (D) Heatmap presenting the overlapped DEGs identified by both of proteomics and RNA-Seq analyses of the cell lysates. The squares of red and blue colors represented the log₂(fold change). FC, fold change.

To examine the reproducibility of the biological triplicates, we calculated pairwise Pearson’s correlation coefficients for quantitated proteins among the triplicated cell lysate samples. As shown in Fig. 2B, the Pearson’s correlation coefficients were high for all comparisons within the ethanol-treated group and within the control group, suggesting high reproducibility among the triplicates in each group.

To identify proteins affected by ethanol treatment, we systematically quantitated proteins in ethanol-treated and untreated cells. In the cell lysate samples, 201 proteins were found to be significantly different between the ethanol-treated group and the control group (P < 0.05, fold change > 1.3; see Fig. 2C). Among the 201 differentially expressed proteins, 3 proteins (BST2, CORIN, and VTN) were up-regulated and 198 protein were down-regulated in the ethanol-treated group compared with the control group (Fig. 2C). Table 1 lists the top 30 differentially expressed proteins including 3 up-regulated and 27 down-regulated ones. Among them, 7 were related to muscle system process (CRYAB, FLNA, SORBS2, MYL2, ANXA6, HSPB6, and SLMAP) and 13 involved in focal adhesion (VTN, CD59, FLNA, XIRP1, SORBS2, FHL1, AJUBA, FERMT2, LMO7, NEXN, ANXA6, SH3KBP1, and MYL2). Several proteins associated with the TNF receptor-associated factor 2 (TRAF2) pathway were among the significantly down-regulated proteins: CPNE1 (Table 1) and TNIK (expression was reduced by ethanol treatment to 73% of the untreated control, P = 0.0015).

Table 1.

Top up-regulated and down-regulated proteins in hiPSC-CMs treated with ethanol compared with no treatment

Regulation	Symbol	Description	Fold Change	P-value
Up	BST2	Bone marrow stromal cell antigen 2	1.42	0.0030
	CORIN	Corin, serine peptidase	1.35	0.0332
	VTN	Vitronectin	1.30	0.0212
Down	DMXL2	DmX-like protein 2	0.33	0.0103
	CRYAB	Alpha-crystallin B chain	0.42	0.0007
	H1–3	Histone H1.3	0.48	0.0027
	H1–2	Histone H1.2	0.50	0.0040
	FN3KRP	Ketosamine-3-kinase	0.53	0.0118
	H1–0	Histone H1.0	0.53	0.0069
	HSPB6	Heat shock protein beta-6	0.56	0.0015
	SORBS2	Sorbin and SH3 domain-containing protein 2	0.56	0.0094
	MYL2	Myosin regulatory light chain 2	0.56	0.0027
	ANXA3	Annexin A3	0.57	0.0121
	CPNE1	Copine-1	0.58	0.0295
	CD59	CD59 glycoprotein	0.58	0.0227
	SLMAP	Sarcolemmal membrane-associated protein	0.58	0.0371
	NEXN	Nexilin	0.60	0.0110
	ANXA6	Annexin A6	0.60	0.0464
	FLNA	Filamin-A	0.60	0.0133
	CAVIN4	Caveolae-associated protein 4	0.60	0.0076
	XIRP1	Xin actin-binding repeat-containing protein 1	0.60	0.0086
	AKAP2	A-kinase anchor protein 2	0.60	0.0262
	H1–4	Histone H1.4	0.60	0.0109
	AJUBA	LIM domain-containing protein ajuba	0.60	0.0238
	OR1D4	Olfactory receptor 1D4	0.60	0.0415
	LMO7	LIM domain only protein 7	0.60	0.0252
	DNAJC6	Putative tyrosine-protein phosphatase auxilin	0.61	0.0025
	SH3KBP1	SH3 domain-containing kinase-binding protein 1	0.61	0.0124
	FHL1	Four and a half LIM domains protein 1	0.61	0.0100
	FERMT2	Fermitin family homolog 2	0.61	0.0443

In the media samples, 5 proteins were found to be significantly different in their levels between the ethanol-treated group and the control group (Fig. 2C). Among the 5 differentially expressed proteins, 4 proteins (BANF1, S100A13, RPS21, and HSPB1) were up-regulated and one (VADC1) was down-regulated in the ethanol-treated group compared with the control group (Fig. 2C).

To help evaluate if ethanol treatment indeed caused changes of protein levels in cell lysates, we examined whether ethanol treatment also caused changes in the RNA level of the corresponding genes. As shown in Fig. 2D, the mRNA level of BST2 was upregulated in ethanol-treated hiPSC-CMs based on an RNA-Seq analysis (data available at GEO database, Accession: GSE125917 (Rampoldi et al., 2019)) (fold change = 1.31, P-value = 0.0096), which was consistent with the proteomic analysis in this study (fold change = 1.42, P-value = 0.0030). Several other differentially expressed proteins were also found to be differentially expressed at the RNA level (Fig 2D), including those involved in focal adhesion (FHL1, MCAM, AJUBA, and ZYX) and relevant to myofibril (CSRP3, MYOZ2, CRYAB, and MYL2).

Ethanol treatment alters multiple biological processes and molecular functions

To determine the biological processes and molecular functions affected by ethanol treatment, we performed GO analysis among the 198 down-regulated proteins in the ethanol-treated hiPSC-CM lysate samples. This analysis resulted in 466 GO terms that were significantly enriched (P < 0.05). Of these terms, 283 terms belonged to biological process, 117 terms to cellular component, and 66 terms to molecular function. To focus on more valuable GO terms, we excluded duplicate enriched GO terms, which had the same corresponding protein lists, and filtered all the remaining GO terms with the following more stringent criteria: P < 0.01, fold enrichment > 2, and the number of involved proteins ≥ 5. As shown in Fig. 3A, 159 GO terms met these criteria. The number of terms associated with biological process, cellular component and molecular function was 97, 43 and 19, respectively. Specifically, as shown in Fig. 3A and Table 2, ethanol exposure strikingly down-regulated the expression of proteins associated with the regulation of the force of heart contraction (5 proteins), myofibril assembly (8 proteins), striated muscle cell development (15 proteins), muscle system process (27 proteins), actin filament-based process (33 proteins), cytoskeleton organization (46 proteins), sarcomere (24 proteins), and adherens junction (44 proteins).

Fig. 3.

GO enrichment analysis of the down-regulated proteins in ethanol-treated vs. untreated cell lysates identified by MS-based proteomics. (A) Bubble plot showing GO terms with P-value < 0.01, fold enrichment > 2, and protein count ≥ 5. The area of bubbles indicates the number of involved proteins in each GO term. BP, biological process; CC, cellular component; MF, molecular function. (B) Chord diagram showing connections between the down-regulated proteins and interested GO terms of biological process. GO terms were presented on the right, and proteins contributing to these enrichments were drawn on the left. The log₂(fold change) for each gene is indicated with the color (yellow, green, and purple) in the rectangles next to the gene name, and the key to the color is provided near the bottom of the chord diagram.

Table 2.

Top down-regulated GO terms in hiPSC-CMs treated with ethanol compared with no treatment

ID	GO Term Description	Count	Fold Enrichment	P-value
GO:0005912	Adherens junction	44	6.53	4.41E-23
GO:0070161	Anchoring junction	44	6.37	1.13E-22
GO:0030054	Cell junction	56	4.18	1.57E-20
GO:0008092	Cytoskeletal protein binding	46	5.04	1.08E-19
GO:0030017	Sarcomere	24	13.14	4.80E-19
GO:0030016	Myofibril	25	12.14	4.94E-19
GO:0043228	Non-membrane-bounded organelle	93	2.38	1.12E-18
GO:0043232	Intracellular non-membrane-bounded organelle	93	2.38	1.12E-18
GO:0031674	I band	21	16.51	1.24E-18
GO:0015629	Actin cytoskeleton	33	7.28	1.70E-18
GO:0043292	Contractile fiber	25	11.49	1.80E-18
GO:0003779	Actin binding	32	7.48	2.85E-18
GO:0044449	Contractile fiber part	24	12.05	3.43E-18
GO:0005856	Cytoskeleton	64	3.21	6.86E-18
GO:0005925	Focal adhesion	30	7.90	9.69E-18
GO:0005924	Cell-substrate adherens junction	30	7.84	1.19E-17
GO:0030055	Cell-substrate junction	30	7.74	1.67E-17
GO:0030018	Z disc	18	15.71	1.55E-15
GO:0007010	Cytoskeleton organization	46	3.75	8.83E-15
GO:0005737	Cytoplasm	151	1.44	4.12E-14
GO:0003012	Muscle system process	27	6.22	1.85E-13
GO:0030029	Actin filament-based process	33	4.58	8.67E-13
GO:0005829	Cytosol	74	2.24	1.64E-12
GO:0005911	Cell-cell junction	30	4.80	4.18E-12
GO:0044822	Poly(A) RNA binding	43	3.33	4.61E-12
GO:0005515	Protein binding	158	1.36	4.66E-12
GO:0030036	Actin cytoskeleton organization	29	4.89	6.50E-12
GO:0005913	Cell-cell adherens junction	22	6.82	9.88E-12
GO:0050839	Cell adhesion molecule binding	26	5.23	2.6E-11
GO:0005198	Structural molecule activity	33	3.91	5.52E-11

In Fig. 3B, we present a detailed map showing the GO terms linked with specific differentially expressed proteins that were associated with biological processes. In this analysis, individual proteins could be linked with more than one GO terms. For example, integrin-linked kinase (ILK) is known to contribute to wound healing, cell-cell adhesion, extracellular matrix organization, heart development, and myofibril; caveolin 1 (CAV1) is involved in wound healing, response to transforming growth factor β (TGF-β), response to oxygen-containing compound, cell-cell adhesion, and regulation of metal ion transport; actinin alpha 2 (ACTN2) is involved in response to oxygen-containing compound, regulation of metal ion transport, heart development, and myofibril; and filamin A (FLNA) is known to be involved in wound healing, cell-cell adhesion, regulation of metal ion transport, and myofibril.

To further explore the potential underlying signaling pathways affected under ethanol treatment, we performed KEGG pathway analysis on the 198 down-regulated proteins in the ethanol-treated cell lysate samples. In this analysis, three of the top five significantly enriched pathways had connection to the GO terms listed in Fig. 3B, including focal adhesion, ECM-receptor interaction, and regulation of actin cytoskeleton (Fig. 4A). In addition, as a large number of differentially expressed proteins function as a part of focal adhesion, we also delineated the protein-protein interaction among these proteins, which further implicates their contributions to ethanol-induced cardiotoxicity in hiPSC-CMs (Fig. 4B). Notably, paxillin (PXN), zyxin (ZYX), and actinin alpha 1 (ACTN1) interacted with multiple focal adhesion related proteins: 11 for PXN, 7 for ZYX and 6 for ACTN1.

Fig. 4.

KEGG enrichment and protein network analyses of the down-regulated proteins in ethanol-treated vs. untreated cell lysates identified in the proteomics experiment. (A) Chord plot showing the down-regulated proteins and associated KEGG clusters. KEGG pathways were presented on the right, and proteins contributing to these enrichments were drawn on the left. (B) Interaction network of down-regulated proteins involved in focal adhesion. The log₂(fold change) for each gene is indicated with the color (yellow, green, and purple) in the oval surrounding the gene name, and the key to the color is provided. The protein list of focal adhesion was the same as in the KEGG pathway except 2 proteins that did not show any interaction at the interaction score above 0.7.

DISCUSSION

Treatment with ethanol causes severe detrimental effects on hiPSC-CMs as indicated by significant cell death and defective Ca²⁺ handling. By performing the proteomic and bioinformatic analysis, we have identified proteins and pathways affected by ethanol treatment which aids in a better understanding of the underlying molecular mechanisms.

Among the proteins that were significantly altered in ethanol-treated hiPSC-CMs, several are known to contribute or respond to oxidative stress. This finding is consistent with our previous study demonstrating that oxidative stress plays a central role in ethanol-induced cardiotoxicity at the cellular level (Rampoldi et al., 2019). Specifically, glutathione peroxidase 1 (GPX1), a powerful ubiquitous enzyme that can reduce cytosolic and mitochondrial reactive oxygen species (ROS), was 27% lower in ethanol-treated hiPSC-CMs compared with untreated control which could disturb the balance between ROS and antioxidants and result in oxidative stress (Espinosa-Diez et al., 2015). A second protein, poly(ADP-ribose) polymerase-1 (PARP-1), was reduced by 24% following ethanol treatment. PARP-1 is the major isoform of an expanding family of poly(ADP-ribosyl)ation enzymes which primarily functions as a DNA damage sensor in the nucleus to modulate DNA repair and maintain genomic integrity (Virag and Szabo, 2002). It is generally recognized that PARP-1 activation can exacerbate the deleterious pathophysiological effects in CMs under oxidative stress, mainly through induction of mitochondrial dysfunction and promotion of mitochondrial cell death pathways (Chen et al., 2004). A third protein, voltage-dependent anion-selective channel protein (VDAC), was reduced by 48% under ethanol treatment. VDAC1 is the most abundant isoform in the heart (Tian et al., 2019) and functions as a positive regulator of mitochondria-mediated apoptosis since it is a target for the pro-apoptotic protein BAX and is involved in the release of apoptotic proteins located in the intermembrane space of mitochondria (Li et al., 2016). Another protein, Corin (a transmembrane serine protease identified in the heart), was increased by 35% in hiPSC-CMs treated with ethanol. Increased expression of Corin in hiPSC-CMs probably reflects its potential role in counter-acting ethanol cardiotoxicity because Corin is reported to protect CMs under oxidative stress via diminishing apoptosis (Sullivan et al., 2020).

Stress-related proteins that we found to be dysregulated in ethanol-treated hiPSC-CMs also include four small heat shock proteins (sHSPs). The human family of sHSPs (also refers to HSPBs) is constituted by 10 proteins (HSPB1–10), which has been shown to be crucial in maintaining the function and integrity of a wide range of cell types and tissues under stress conditions (Kampinga and Garrido, 2012). Specifically, by their expression level, HSPB6 (also refers to HSP20) was decreased by 44%, HSPB2 was decreased by 31%, HSPB5 (also refers to CRYAB) was decreased by 58%, and HSPB1 (also refers to HSP27) was increased by 39%. These four HSPBs are all recognized to have cardio-protective effects in the heart against stress-induced injuries including MI and cardiac hypertrophy, mainly through modulating apoptotic process (Wang et al., 2019; Mitra et al., 2013). Particularly, studies have shown that HSPB1 is an essential regulator on alleviating inflammation in myocardial repair after MI and maintaining cardiac function against atrial fibrillation (Wang et al., 2019; Brundel et al., 2006). Taken all together, ethanol can suppress as well as evoke the defense system against oxidative stress related apoptosis in hiPSC-CMs. These proteins could be promising targets to reduce ethanol-induced cardiotoxicity.

Alteration in the expression of ion channel related proteins represents another category of the differentially expressed proteins in hiPSC-CMs treated with ethanol. This finding is consistent with the observation that treatment of hiPSC-CMs with ethanol triggered Ca²⁺ transient abnormality as well as the growing evidence that ethanol exposure increases the risk of arrhythmias and negative inotropic effects via Ca²⁺ handling defects and disordered excitation-contraction coupling (Voskoboinik et al., 2016; Mustroph et al., 2019; Mustroph et al., 2018). Since the ion transporting requires the assistance of certain ion channels and exchangers, the down-regulation of several proteins related to regulation of metal ion channels in ethanol-treated hiPSC-CMs could be partially responsible for the CM dysfunction. For example, annexin 6 (ANXA6) is one of the abundant annexins in myocardium which has been shown to regulate Ca²⁺ influx via modulating the activity of the L-type Ca²⁺ channel and to regulate Ca²⁺ release via modulating the activity of the ryanodine receptor 2 (RyR2) and Na⁺/Ca²⁺ exchanger (Matteo and Moravec, 2000). ATPase Na⁺/K⁺ transporting subunit α2 (ATP1A2) composes a part of the Na⁺/K⁺ pump which can move Na⁺ from the cytoplasm to the extracellular space in order to maintain Na⁺ homeostasis (Shattock et al., 2015). Studies have also shown that ATP1A2 can regulate intracellular Ca²⁺ signaling, contractility and pathological hypertrophy (Liu et al., 2016). In this work, we found that the ANXA6 level was decreased by 40% and that ATP1A2 level was decreased by 29%.

Other differentially expressed proteins in ethanol-treated hiPSC-CMs that could affect contractility of the cells include some myofibril structure proteins. These myofibril structure proteins were significantly down-regulated in hiPSC-CMs exposed to ethanol: myosin light chain 2/3 (MYL2/3), Sorbin and SH3 domain-containing protein 2 (SORBS2), myozenin 2 (MYOZ2), cysteine and glycine rich protein 3 (CSRP3), and ACTN1/2. Dysregulation of any of these proteins can impact cardiac contraction or even lead to the development of dilated or hypertrophic cardiomyopathy and heart failure (Pauly, 2011; Li et al., 2019; Bang, 2017; Frey et al., 2004; Wang et al., 2016; Chiu et al., 2010). Hence, the alteration in the expression of ion channels and contractile fiber structure-related proteins could be connected to the corresponding molecular mechanisms of the occurrence of arrhythmias and dysfunctional contraction induced by ethanol.

Our proteomic analysis also reveals the down-regulation of focal adhesion proteins in hiPSC-CMs treated with ethanol. Focal adhesions are complexes of a variety of transmembrane, cytoplasmic proteins, and enzymes that link ECM to the cytoskeleton (Swaminathan et al., 2017). Focal adhesions mediate signaling from ECM to different cellular responses, including proliferation and cell (Vitillo et al., 2016). Considering the role of focal adhesion proteins in cell death, their decreased abundances can imply possible role of focal adhesion proteins in ethanol-induced cardiotoxicity. Among those focal adhesion proteins affected by ethanol treatment, ILK is a multifunctional protein that regulates cell–matrix–cytoskeletal interactions and plays a critical role in ECM-mediated signaling pathway, contributing to various cellular phenomena including growth, repair, and contractility (Hannigan et al., 2007). ILK is reported to modulate the spontaneous beating rate and Ca²⁺ uptake in hiPSC-CMs—it can mediate CM force transduction through regulation of the Ca²⁺ regulatory protein sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) (Traister et al., 2014). In addition, ILK has cardio-protective property on CMs during dilated cardiomyopathy and heart failure (White et al., 2006), whereas inhibition of ILK induces cell apoptosis (McDonald et al., 2008). In line with the findings of increased cell death, defective Ca²⁺ handling, and decreased contractility in ethanol-treated hiPSC-CMs, ILK could serve as a pivotal upstream regulator in all of these toxic events.

Several other focal adhesion proteins were down-regulated by ethanol treatment of hiPSC-CMs. PXN is a focal adhesion-associated adaptor protein that recruits diverse signaling proteins to focal adhesions to guarantee regular signal reception and transduction (Schaller, 2001). It is responsible to modulate the expression of stretch-responsive genes via interacting with ILK, Parvin, and PINCH, the deficiency of which can lead to contractile disability and even heart failure (Hirth et al., 2016). Herein, the expression levels of PXN and Parvin β (PARVB) were decreased in ethanol-treated hiPSC-CMs, which could be one of the reasons for ethanol-induced contractility reduction. In addition, the other down-regulated proteins, ZYX, FLNA, talin 2 (TLN2), and ACTN1/2, are crucial to intracellular contractile force transduction (Santoro et al., 2019). Furthermore, CAV1 is a versatile focal adhesion protein that is also known to regulate TGF-β signaling pathway, Ca²⁺ signaling, and oxidative stress (Shihata et al., 2017). The decreased level of CAV1 in hiPSC-CMs exposed to ethanol could therefore play multiple roles in TGF-β-mediated apoptosis and fibrosis and increased risk of arrhythmias. Together, dysregulation of focal adhesion proteins could be a prominent mechanism underlying ethanol-induced cardiotoxicity.

In addition, among the differentially expressed proteins identified in this study, some were also found to be differentially expressed at the RNA level. Intriguingly, 4 were involved in focal adhesion (FHL1, MCAM, AJUBA, and ZYX) and 4 were relevant to myofibril (CSRP3, MYOZ2, CRYAB, and MYL2). These findings suggest the important role that focal adhesion and myofibril related proteins play in ethanol-induced cardiotoxicity.

Finally, the current results also reveal potential involvement of other signaling pathways in ethanol-induced cardiotoxicity. For example, two proteins associated with the TRAF2 signaling were significantly down-regulated by ethanol treatment. One of them, CPNE1, is a Ca²⁺-dependent lipid-binding protein and CPNE1 overexpression upregulates TRAF2 expression (Liang et al., 2017). The other one, TNIK, is a TRAF2 and NCK interacting kinase and functions as an activator of the Wnt signaling pathway.

A limitation of this study is that our bioinformatics analyses were performed in a human cardiomyocyte cell model. Consequently, physiological translation of our findings in either rodent or human tissues remains to be established. Nevertheless, given that hiPSC-CMs are physiologically relevant to human CMs, our findings provide interesting leads for further studies in vivo.

In conclusion, we have systemically and quantitatively investigated the expression changes of proteins in the lysates and media of hiPSC-CMs following the treatment with ethanol. Consistent with the cell-level assessments including decreased cell survival and impaired CM function, many dysregulated proteins have been found to be involved in wound healing, apoptosis, oxidative stress, heart contraction, and regulation of metal ion transport. Expression of focal adhesion and ECM related proteins is also down-regulated, including ILK, PXN, and CAV1. Furthermore, the current study reveals a novel role for proteins associated with the TRAF2 signaling in cardiotoxicity induced by ethanol treatment.

Supplementary Material

Sup S1

Fig. S1. Schematic diagrams of the experimental design

Table S1. Information of major reagents

Table S2. Antibodies for immunocytochemistry