Inhibition of Cardiac p38 Highlights the Role of the Phosphoproteome in Heart Failure Progression

Abstract

Heart failure (HF) is a complex condition. Among altered signal transduction pathways associated with HF pathogenesis, the stress-activated p38 mitogen-activated protein kinase (Mapk) pathway has attracted attention for its role in HF progression and cardiac hypertrophy. However, the mechanisms by which p38-Mapk influences HF remain unclear. Addressing knowledge gaps may provide insight into why p38 inhibition has yielded inconsistent outcomes in clinical trials. Here, we investigate the effects of p38-Mapk inhibition via SB203580 on cardiac remodeling in a guinea pig model of HF and sudden cardiac death. Using an HF model with ascending aortic constriction and daily isoproterenol (ACi) administration, we assessed three groups: sham-operated controls, untreated ACi, and ACi treated with SB203580 (ACiSB). Cardiac function was evaluated by M-mode echocardiography. Proteome and phosphoproteome profiles were analyzed using multiplexed Tandem Mass Tag labeling and LC–MS/MS. Our findings demonstrate that SB203580 treatment protects against cardiac dysfunction in HF. Proteomic data indicate that SB203580 exerts broad protection of the cardiac phosphoproteome, inhibiting maladaptive p38-dependent phosphorylation, extending to Pka and Ampk networks, ultimately protecting the phosphorylation status of critical myofibrillar and Ca²⁺-handling proteins. Though SB203580 had a limited impact on widespread protein changes in HF, its biosignature revealed preserved mitochondrial energetics and reduced oxidative and inflammatory stress.

Introduction

Heart failure (HF) is a complex condition that brings substantial risks to health and life. With over 64 million individuals worldwide affected by HF, it has emerged as a pressing concern, and reducing its impact has thus become a principal goal. The pathophysiology of HF is characterized by a multifaceted interplay of cellular mechanisms, including altered cyclic AMP, cyclic GMP, and Ca²⁺/calmodulin-dependent signaling; Ca²⁺ handling impairment; and mitochondrial oxidative stress. Stress-activated kinases of the mitogen-activated protein kinase (Mapk) family have also garnered significant attention. , Triggered by osmotic, mechanical, or oxidative stress, Mapk signaling cascades have been implicated as regulators of both cardiac hypertrophy and HF progression. , Mapks are a group of highly conserved serine/threonine protein kinases that transmit signals through a multilevel kinase cascade. Four primary subgroups of the Mapk signaling pathway have been recognized: Erk, c-Jnk, p38/Mapk, and Erk5. , These kinases regulate key physiological and pathological processes, including apoptosis and inflammation, as well as proliferation, growth, and differentiation of cardiac resident cells such as cardiomyocytes, fibroblasts, endothelial cells, and macrophages.

There are four P38 Mapks (α, β, γ, and δ) encoded by genes Mapk14, Mapk11, Mapk12, and Mapk13, respectively. They are activated in response to cellular stressors including oxidative stress, DNA damage, and cytokine receptor stimulation. Activation leads to phosphorylation of downstream targets including Mapk Apk2/3, Msk1 Hsp27, and several important cardiac transcription factors (e.g., Atf2, Myc, Stat1, Mef2, Nfat, Creb1, and Pgc1α). , p38 Mapk has been shown to contribute to the growth response of cultured cardiomyocytes to hypertrophic agonists. Of the four p38s, p38α and β have received the greatest scrutiny for their role in HF pathogenesis. Evidence garnered from mouse knockout and overexpression models would indicate that maladaptive p38α activation via phosphorylation by dual-specificity kinases, MKK3 or MKK6, contributes to cardiomyocyte cell death and contractile dysfunction in settings of both chronic pressure overload , and ischemia, though it may also play an adaptive role in response to acute changes in afterload. P38β expression in the heart is comparatively low, although it may have distinct functional roles, for example, in the estrogen-dependent modulation of mitochondrial reactive oxygen species.

Targeted P38 α/β inhibition appears to have therapeutic potential. Both enzymes share high sequence homology and conserved key functional residues within their kinase domains and have similar pharmacological inhibition profiles. One such inhibitor, SB203580 ([4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole]), exhibits IC50s ranging from 50 to 500 nM, depending on the cell types, which may differ in the relative amount of α and β forms. − Inhibition of p38 Mapk has been proposed as a treatment to inhibit HF pathogenesis. ,

Clinical evaluation of p38 inhibition for several conditions (recovery from myocardial infarction (MI), COPD, or depression) has been pursued, thus far, with limited success. Losmapimod, a novel inhibitor of p38 Mapk, was well tolerated upon oral administration and demonstrated efficacy in improving the prognosis of MI patients in a phase II clinical trial. However, a larger phase III trial (LATITUDE-TIMI 60) showed that, while losmapimod effectively reduced the inflammatory response post-MI compared to placebo, it did not mitigate the risk of major ischemic cardiovascular events. Similarly, despite early positive results, a different p38 Mapk inhibitor was recently terminated because it was not expected to meet the primary end point in a global phase 3 trial (REALM-DCM) in patients with symptomatic dilated cardiomyopathy (DCM) due to a mutation of the gene encoding the lamin A/C protein (LMNA). These findings indicate that although p38 Mapk inhibition holds promise in improving cardiac function in the context of HF, further investigation of p38 Mapks cellular targets and effects is warranted to ascertain the best strategy to optimize interventions that could improve cardiovascular outcomes.

Here, we examine the outcome of p38 Mapk inhibition with SB203580 (SB) on cardiac HF remodeling in a guinea pig model of HF and sudden cardiac death. Our objective is to understand how inhibiting p38 Mapk affects the progression of HF, focusing on proteome remodeling and alterations in protein phosphorylation associated with HF. We find that p38 Mapk inhibition protects against cardiac decompensation by impacting select classes of HF-associated protein changes while exerting broader protection of the cardiac phosphoproteome.

Methods

Animal Model

Hartley guinea pigs (∼300 g; HillTop Lab Animals) were housed in the animal facility at Johns Hopkins University. This study conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the Johns Hopkins Animal Care and Use Committee. Male animals were anesthetized with 4% isoflurane in a closed box for 4 min and then intubated. Animals were ventilated with oxygen and 2% isoflurane. Ascending aortic constriction (AC) was produced by tying a suture around the ascending aorta using an 18-gauge needle as a spacer, which was then removed. Sham operation was performed following the same procedure without the ligation. Daily bolus of Isoproterenol (ISO) was administered via a programmable iPRECIO pump (SMP-200, Data Science International, St. Paul MN) that was implanted in the peritoneal cavity. The pump was programmed for 1 h daily delivery of Isoproterenol for a dose of 1 mg/kg/day. For the treatment of SB203580, an osmotic pump (Alzet Osmotic pump, model 2004, Cupertino, CA) was implanted in the abdominal cavity. SB203580 was delivered continuously at 0.5 mg/kg/day of DA. A vehicle-filled osmotic pump was implanted for control in the ACi group. The following treatment groups were studied: 1. Sham-operated, serving as controls; 2. ACi (AC + isoproterenol treatment until end point); and 3. ACi-SB (AC + isoproterenol treatment + SB203580 treatment). All personnel involved in data collection and analysis were blinded to the treatment and nontreatment groups. Both groups received similar incisions and thus could not be distinguished based on these interventions. Each animal was assigned a unique computer-generated numeric ID.

Echocardiography

Transthoracic echocardiography was performed on conscious nonanesthetized guinea pigs by using a Vevo 2100 high-resolution in vivo imaging system with a 24 MHz transducer (VisualSonics, Toronto, ON, Canada) and analyzed with the Advanced Cardiovascular Package software (VisualSonics).

Data and Statistical Analysis

For heart weight, lung weight, and FS % analysis between groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis was used.

Proteomic Studies

Protein Extraction and Immunoblotting

Ventricles were harvested. Tissues were rinsed in cold PBS, rapidly heat-stabilized (Stabilizor, Denator, Inc.), snap-frozen in liquid nitrogen, and stored in a −80 freezer. To extract protein, stabilized tissues were homogenized with RIPA buffer in the presence of 2% SDS, solubilized, and boiled in 1× LDS sample buffer for SDS-PAGE. The protein mixture was separated on a 4–12% NuPAGE gel (1 mm, Invitrogen). Samples were run at room temperature for 35 min at 200 V. Proteins were transferred to nitrocellulose membranes with iBlot (Invitrogen, Inc.) using program 3 for 7 min. Membranes were stained with a Ponceau S solution (Sigma-Aldrich) to evaluate the transfer efficiency. Membranes were blocked for 1 h using Odyssey blocking buffer (Li-Cor Biosciences) and incubated with primary antibody overnight at 4 °C. Antibody binding was visualized with an infrared imaging system using IRDye secondary antibodies, and quantification of band intensity was performed using the Odyssey Application Software 3.0 (see Table ).

1. Antibody Information Table.

antibodies	brand	cat #	species	type	dilution
p38 Mapk antibody	Cell Signaling Technology	9212	rabbit	primary	1:1000
Phospho-p38 Mapk (Thr180/Tyr182) (D3F9) XP rabbit mAb	Cell Signaling Technology	4511	rabbit	primary	1:1000
IRDye 800CW goat anti-rabbit IgG secondary antibody	Li-Cor	926–32,211	goat	secondary	1:10,000

Densitometric analysis of p38 and phosphorylated p38 (p-p38) signals in three distinct groups was conducted using ImageJ software, with data derived from digitized .tif image files. These signals were subsequently normalized to the total protein levels detected via Ponceau S staining. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, to evaluate the comparison of p-p38/p38 ratios across the different groups.

Sample Preparation, Proteolysis, and Tandem Mass Tag (TMT) Labeling

The experiment compared 3 experimental groups: (1) Sham-operated controls, (2) ACi failing hearts (at 4 weeks), and (3) ACi animals treated concomitantly with SB203580 (0.5 mg/kg/day).

Hearts were harvested and washed with ice cold phosphate-buffered saline. The left ventricle free wall was isolated and immediately subjected to heat denaturation to abolish all enzyme activity using the Denator Stabilizor System (Denator) according to the manufacturer’s instructions. Denatured samples were then stored at −80 °C until the next step. The left ventricle of each guinea pig heart was homogenized with a hand-held Polytron tissue disrupter in filtered and deionized, Tris-buffered 9 M urea (5 mL), pH 7.5. Samples were allowed to solubilize for 30 min at room temperature. Soluble homogenates were subjected to methanol/chloroform/water extraction and protein precipitation by the method of Wessel and Flugge. Samples were dried under nitrogen gas to remove residual chloroform before resolubilizing for 30 min in 9 M urea. Aggregates were disrupted by brief bursts of sonication (<30 s total). Peptides were diluted 6-fold into 60 mM HEPES and 0.6 mM DTT, pH 7.5 such that the final reaction buffer contained 50 mM HEPES, 1.5 M urea, 0.5 mM DTT, pH 7.5. All samples were diluted further with reaction buffer to a common final protein concentration of 1 mg/mL. Samples (1 mg) were subjected to proteolytic digestion with proteomics grade Trypsin (1 μg/100 μg protein; Promega) at room temperature, overnight. The following morning, samples were supplemented with Trypsin (1 μg/100 μg protein; Promega). At that time, DTT was added to samples at a concentration of 5 mM, and the digest was allowed to proceed for 1 h prior to peptide alkylation by addition of iodoacetamide to a final concentration of 15 mM. Alkylation was allowed to proceed 1 h at room temperature in the dark. Peptides were subsequently acidified by addition of trifluoroacetic acid to a final concentration 0.5% (v/v) and purified by solid-phase extraction using Sep-Pak tC18 cartridges (Waters) on a vacuum manifold. Purified peptides were eluted with 60% (v/v) acetonitrile in aqueous 0.1% (v/v) formic acid. Peptides were evaporated to dryness on an Eppendorf Vacufuge. Peptide samples were resolubilized in triethylammonium bicarbonate (TEAB) pH 8.5 and labeled with TMT reagents according to the manufacturer’s instructions.

Chromatography and Mass Spectrometry

Following TMT labeling of individual samples, peptides were pooled and subjected to high-pH reversed-phase liquid chromatography (bRP-HPLC), as detailed by Foster et al. Briefly, samples were fractionated at 250 μL/min on a Waters BEH C18 column with a gradient running from 10 mM TEAB to 90% Acetonitrile and 10 mM TEAB over 105 min. The fractions were concatenated into 24 fractions, from which 20% of each was taken for the expression proteome determination. The remaining 80% was combined into 11 fractions, and TiO₂ enrichment (Top Tip Titanium Dioxide, MTIO0010.1 g; Glygen Corporation, Columbia, MD, USA, with a particle size of 10 μm) was performed to assess the phosphoproteome.

Following concatenation, samples were analyzed using a nanoAquity nanoLC system (Waters) interfaced with an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific). Peptides were injected onto a 2 cm trap column at 5 μL/min for 6 min before being eluted onto a 75 μm × 15 cm in-house packed column (Michrom Magic C18AQ, 5 μm, 100 Å) operating at 300 nL/min. Each sample was run on a 90 min gradient. Data were acquired using a 3 s cycle between MS1 scans in FT–FT acquisition mode. The survey full-scan MS (400–1600 Th) was performed at a resolution 120,000 with an automatic gain control target ion intensity of 4 × 10⁵, while MS2 scans were performed at a resolution of 50,000 with a target value of 1.25 × 10⁵. Mass tolerances were ±10 ppm for MS1. Maximum injection times were set to “Auto” for MS1 and 86 ms for MS². A 0.7 Da isolation window was used, and HCD fragmentation was performed with a normalized collision energy of 36. Internal calibration was set to Easy-IC. Spectra whose charge was unassigned or +1 were not tabulated. Dynamic exclusion was set to a repeat count of 1 with a 15 s exclusion time.

Protein Identification

The .Raw files for both the enriched and unenriched (12 + 24 respectively) were searched against a guinea pig database of predicted protein sequences (NCBI RefSeq, taxonomy: Cavia porcellus, date: 01/20/2021, FASTA format, 37727 sequences) using Mascot Version: 2.8.0 (Matrix Science) interfaced through Proteome Discoverer 2.4.0.305 (Thermo). Peaks were filtered at a signal-to-noise ratio of 3, deisotoped, and searched with a parent ion mass tolerance of 5 ppm and an MS2 mass tolerance of 0.02 Da. Trypsin was specified as the enzyme, and 1 missed cleavage was allowed. N-terminal labeling with TMTpro reagent and carbamidomethyl was specified as a fixed modification, and dynamic modifications included deamidated NQ, oxidized M, phosphorylation on STY, and TMTpro on lysine. All searches were conducted with the reversed database search mode engaged. Percolator software was used for peptide FDR (q-value) calculations. Mascot output files (.dat) tabulated were in Proteome Discoverer. Only high confidence peptides (q < 0.01) were used for protein identification.

Spectral Inclusion Criteria for Quantitation

Analysis was confined to uniquely and unambiguously assigned spectra (1% peptide FDR). Missingness of ion intensities for a single spectrum across reporter channels was low (<2% of unique spectra with intensities missing from one or more channels), indicative of efficient TMT labeling and fragmentation.

Protein Quantification by TMT and Statistical Analysis

Reporter ion intensities were integrated over 20 ppm using the most confident centroid method and corrected for purity in Proteome Discoverer 2.5 (Thermo Fisher Scientific). Spectral TMT signals were quantified using the median sweep algorithm originally described by Herbrich et al. essentially as implemented recently by Foster et al. with a minor modification. TMT reporter ion intensities were (1) logarithmically transformed (base 2), (2) median-centered within each channel prior to (3) median-centering each individual spectrum across channels, and (4) determining protein abundance by taking the median value of the logarithmically transformed median-centered intensities for all spectra belonging to that protein in a given channel (median summarization). For phosphopeptide analysis, all spectra (phosphorylated and unphosphorylated) median-centered, as above, and median-summarized at the peptide level. Unphosphorylated peptides were filtered from the final table.

Following the median sweep, differential protein abundance between experimental groups was assessed using a Bayesian statistical framework, specifically, linear modeling of microarrays (LIMMA) with multigroup comparison. Pairwise contrast was also performed. The resulting moderated p-values were used to assess the positive false discovery rate (q-value) method of Storey , The code used for the median sweep procedure and statistical analysis can be found at https://github.com/Frostman300/p38-upload. P-Values derived from pairwise-comparisons were utilized to determine specific differences between the groups, as demonstrated in the boxplots presented in the figures.

Retrieval of Homologous Human Peptides

An Excel macro was written to find human peptides homologous to the detected guinea pig peptides using the command line version of Protein–protein BLAST (2.15.0+). In brief, for each guinea pig peptide detected, blastp was used to compare the full-length sequence of the corresponding guinea pig protein with a fasta format database of all Homo sapiens refseq proteins downloaded from the NCBI protein database. Blastp was then used to align the guinea pig peptide with the corresponding highest scoring human protein target. The best homologous peptide match was then output to the spreadsheet. The macro code is available at https://github.com/Frostman300/p38-upload.

Ingenuity Pathways Core Analysis Search Parameters

For Ingenuity Pathway Analysis (Qiagen IPA) of the proteome, uploaded data fields included the gene name and log2­(ACi/ACiSB) value. Data were compared to the Ingenuity Knowledge Base reference set, which included genes only. The search space was limited to consideration of database relationships derived from primary tissues; data arising from cancer cell lines was excluded from analysis. Both direct (transcriptional) and indirect (signaling) relationships were probed. Highlighted pathways were among those with Benjamin–Hochberg-corrected p-values of <0.05. Pathways are colored according to their z-score. The z-score integrates, not only gene over-representation, but the degree of concordance between supplied data (direction of change, magnitude, and p-value) and a database of curated relationships compiled from scientific literature and publicly available data sets. IPA provides an inference about whether upstream gene regulatory and signaling pathways may be activated or inhibited. Higher scores, whether positive or negative, reflect the strength of the inference.

Network Analysis

Functional protein association/interaction networks were constructed by loading the gene identifiers of up- and downregulated proteins into stringApp 2.0.3 embedded in Cytoscape 3.10.2. The default association/interaction threshold (STRING score >0.4) was used to map relationships between proteins. Network modularity was assessed with the Markov clustering function in the clusterMaker2 app (v.2.3.4) using the STRING score (>0.6) for edge weighting. The granularity parameter (inflation value) was set empirically. Modules were rearranged for clarity and named consistent with STRING’s multipathway enrichment terms (e.g., Gene Ontology, Reactome, KEGG, among others). Singletons that were ontologically consistent with a module were grouped with it. The phosphorylation association network was further annotated using Omics Visualizer 1.3.1. embedded in Cytoscape.

Results

P38 Mapk Inhibition Attenuates HF Progression in Guinea Pigs

We employed a guinea pig model of HF that combines ascending AC with administration of the β-adrenergic agonist isoproterenol once a day (1 mg/kg/day) via an implanted programmable pump. This model has been validated previously. , P38 Mapk, as demonstrated in Figure A,B, is activated by phosphorylation at Thr180/Tyr182 in guinea pig HF, and treatment with SB prevented its activation. The following treatment groups were studied: (1) Sham-operated, serving as Controls; (2) ACi (AC + isoproterenol); and (3) ACi-SB (ACi + SB treatment via implanted osmotic pump; 0.5 mg/kg/day).

1.

p38 inhibition attenuates HF progression in guinea pigs. (A) The p38 is activated by phosphorylation at Thr180/Tyr182 in guinea pig HF. Treatment with SB203580 prevents activation. (B) Ensemble analysis of p38 activation normalized for p38 expression. SB attenuates the decline in fractional shortening (C) and prevents cardiac hypertrophy (D) and pulmonary edema (E). F. Experimental designCreated in BioRender. Sedighi, S. (2025) https://BioRender.com/vt73yx4. To illustrate peptide-level detection of post-translational modifications, Myl2 was chosen as a representative example. Mass spectra corresponding to its phosphorylated and nonphosphorylated peptides are displayed.

An appreciable decline in FS was noted in the ACi group compared to its respective Control group (ACi: 30.5 ± 2.9%, n = 8; Control: 44.9 ± 1.2%, n = 7, p < 0.0001), further confirming the validity of our HF model. SB treatment effectively abrogated 61% of the decline in FS seen in the ACi-4w group (i.e., 39.3 ± 5.6%, n = 8 vs 30.5 ± 2.9%, n = 8; p-value = 0.0005) (Figure C). ACi-induced hypertrophy was also blunted by SB treatment. Specifically, heart weight/tibia length was decreased from 0.7 ± 0.06 g/mm (n = 8) in the ACi group to 0.6 ± 0.05 g/mm (n = 8) in the ACiSB group (p-value <0.001) (Figure D). Additionally, lung weight/tibia length decreased significantly, from 1.5 ± 0.4 g/mm (n = 8) to 0.8 ± 0.2 g/mm (n = 8) with SB treatment, indicating a reduction in pulmonary edema (p-value = 0.0003; Figure E).

To elucidate the impact of P38 Mapk inhibition on the HF proteome, we conducted a comprehensive 16-plex TMT analysis across the three experimental groups. The experimental design is summarized in Figure F for Control, ACi-, and ACiSB-treated guinea pigs. These samples were extracted, digested, and analyzed as detailed in the Methods. Briefly, we used a 2D-LC-MS/MS strategy. TMT-labeled peptides were pooled prior to high-pH reversed-phase liquid chromatography (bRP-HPLC). The bulk of each concatenated bRP-HPLC fraction (80%) was subjected to titanium dioxide (TiO₂) phosphopeptide enrichment. Both enriched and unenriched fractions were subjected to RP-LC-MS/MS. Data analysis consisted of median-sweep scaling, followed by statistical analysis using LIMMA, as we have reported previously. ,,

P38 Mapk Inhibition Partially Mitigates Protein Changes Associated with HF

Consistent with our prior work, the ACi protocol elicits substantial proteome remodeling after 4 weeks. Fully, 2480 of 5016 quantified proteins (i.e., 49%) were differentially expressed in the ACi group (p < 0.05 ACi vs Control by LIMMA with post hoc pairwise contrast). We defined expression as SB-responsive if protein abundance differed significantly between the ACiSB and ACi groups (p < 0.05). We found 292 proteins differed between the groups, irrespective of whether they changed significantly between ACi and Control. Thus, 227 (of 2480) proteins whose expression differed from control in the ACi group (i.e., 9%) were deemed SB-responsive. These results are summarized in the Venn diagram of Figure A. Complete tabulated protein levels and their statistical analyses are provided in Table S1. Figure B depicts a PCA biplot of the statistically SB-responsive subset proteins, showing that, even within that subset, the variance of the ACiSB group was distinct from Control, lying between Control and ACi groups. This trend is illustrated more explicitly in the heatmap depicted in Figure C, where the protein levels of the SB-responsive group lie between those of the Control and ACi groups. Thus, while select proteins were more SB-responsive than others, on aggregate, SB treatment only partially offset ACi-induced differential protein expression. The volcano plot in Figure D highlights some of the proteins whose abundance was most impacted by the SB treatment. Additionally, specific examples of proteins that differ between ACi and Control group are presented in Figure S1.

2.

p38 inhibition mitigates protein changes associated with HF. (A) Venn diagram illustrates that fully 2480 of the 5016 quantified proteins change abundance in HF, but only about 1/10 of these proteins are impacted by SB treatment. SB also modulated the expression of 65 proteins that were not otherwise changing in HF. (B) The PCA biplot of proteins changing significantly between ACi and ACiSB. (C) Hierarchical clustering of proteins that differed between ACi and ACiSB. (D) Volcano plot highlighting specific examples of proteins responsive to SB treatment. (E) Functional association network of proteins in D Blue nodes indicates proteins that were downregulated in ACi relative to ACiSB, while yellow nodes indicate proteins that were upregulated in relative to ACiSB. (F) Pathway analysis of proteins from (D). Highlighted pathways were among those with Benjamin–Hochberg-corrected p-values of <0.05. Ochre indicates pathways inferred to be augmented by SB and blue indicates pathways inferred to be attenuated. G. Inferred activation or inhibition of transcription factor activity based on observed protein changes. The color scheme is the same as for (F).

For further insights, we subjected 292 proteins to both network- and pathway-based annotation enrichment analyses. The significantly changing proteins in Figure D were queried using the STRINGdb functional annotation network. Modules of interest were revealed by Markov clustering in Figure E. As in Figure D, blue nodes represent proteins downregulated in ACi that were SB-responsive, while yellow nodes indicate responsive upregulated proteins. The 33 modules depicted encompass 214 of the 292 SB-responsive proteins and summarize the major features of the data set. Among downregulated, yet SB-responsive proteins, several modules correspond to mitochondrial processes or pathways, including oxidative phosphorylation, mitochondrial translation, mitochondrial iron–sulfur cluster biogenesis, mitochondrial protein import, and nicotinamide metabolism. Proteins of the respiratory complexes were particularly SB-responsive (see also Figure ).

4.

Expression of many proteins involved in mitochondrial bioenergetics are changing significantly and are responsive to SB: Global significance (p < 0.05) established via F-test, with p-values derived from LIMMA contrast matrices for intergroup comparisons. P-Values <0.05 are indicated by faceted brackets.

Among the upregulated proteins, major nodes implicate extracellular and acute phase response proteins, ER and endosomal proteins, and proteins of the cytoskeleton. Acute phase response proteins were among the most SB-responsive (also see Figure ).

5.

Expression of many acute phase proteins are significantly increased with ACi: Out of those that are changing Hp, Serpina1, Iqgap2, and Vwf are SB-responsive. Global significance (p < 0.05) established via F-test, with p-values derived from LIMMA contrast matrices for intergroup comparisons. P-Values <0.05 are indicated by faceted brackets.

Complementary Ingenuity Pathway Analysis (Figure F) is consistent with network-based annotation, implicating both oxidative phosphorylation and acute phase response signaling, whose dysregulation in the ACi group was ameliorated by treatment with SB. Finally, Ingenuity upstream regulator analysis (URA) provides a set of candidate transcription factors whose activity might explain the changes in observed protein levels arising from SB treatment (Figure G). URA strongly implicates Tead1, whose activity could explain the coordinate expression of 20 respiratory complex proteins, particularly from complex I (see Figure S2). Prior work has shown that Tead1 deletion decreases phospholamban phosphorylation, Serca2a expression, and mitochondrial gene expression, resulting in cardiomyopathy in mice. Here, the inferred involvement of Tead1 is consistent with reports that stress-induced activation of p38 Mapk results in its interaction with, and phosphorylation of, Tead1 in the cytoplasm, inhibiting its nuclear function as a transcription factor in the Yap/Taz pathway. The Tead1 transcriptional program partially overlaps with the Rb1 and Pgc1α programs. Together Tead1, Rb1, and Pgc1α regulation likely account for most of the mitochondrial protein downregulation in ACi that responds to SB (Figure S2).

With respect to the mechanisms of chromatin remodeling, Kdm5a, a lysine-specific demethylase involved in the regulation of gene expression, is inferred to be strongly inhibited by SB treatment (p = 1.3 × 10^–11, z-score = −4.1). Kdm5a has previously been identified as a key regulator of cardiac fibrosis and is upregulated in fibroblasts from patients with dilated cardiomyopathy (DCM) via the angiotensin II and PI3K/AKT signaling pathways.

Expression of Mapk Cascade Proteins Change in HF but Are Largely Unresponsive to SB

Mapk cascade proteins including Mapk14 (p38a), Mapk9 (Jnk2), and Mapk1 (Erk2), along with their upstream activators, Map2k1, Map2k2, Map2k3, and Map4k5 (Khs1), exhibited alterations in their abundances in ACi but showed no significant responsiveness to SB treatment (Figure A–I). An exception to the trend, Map3k17 (Taok2), which is an upstream kinase in the p38 Mapk cascade, did not change in ACi but was decreased with SB treatment. Notably, while the abundance of Mapk14 (p38α) and Map4k5 (Khs1) did not undergo significant changes, their phosphorylation was markedly altered, as depicted in Figure and discussed hereinafter. Several Mapk cascade proteinsincluding Map2k4, Map2k7, Map3k4, and Map4k4remained unchanged in ACi and were unresponsive to SB treatment (Figure S3). With respect to known p38 Mapk substrates, the expression of most remained unchanged in ACi. Among the substrates of p38, only Gys1, Mef2d, and Spag9 and Lsp1 exhibit altered abundances in ACi, and furthermore, only Lsp1 was responsive to SB treatment (Figure S4).

3.

Several Mapk cascade proteins are differentially expressed in HF but largely unresponsive to SB: Global significance (p < 0.05) established via F-test, with p-values derived from LIMMA contrast matrices for intergroup comparisons. P-Values <0.05 are indicated by faceted brackets.

7.

Phosphorylation of p38a and several upstream kinases are responsive to SB: global significance (p < 0.05) established via F-test, with p-values derived from LIMMA contrast matrices for intergroup comparisons. P-Values <0.05 are marked with brackets.

SB203580 Curbs Changes in Mitochondrial and Acute Phase Response Proteins

Downregulation of mitochondrial proteins is a consistent biosignature of HF and correlates with mitochondrial dysfunction. The mitochondrial network modules highlighted in Figure E are consistent with prior studies. Figure A–P specifically shows that several subunits of respiratory complexes I (Ndufa8, Ndufaf7, Ndufb1, Ndufc1, Ndufs1, and Ndufv1) and IV (Cox6b1, Cox7C, Cox11, and Cox19) were downregulated in the ACi group. Figure also shows that their decline is substantively withdrawn in the ACiSB group. Atp5f1d, a subunit of ATP synthase, and Uqcrb, from complex III, are likewise SB responsive. The Pdks, or pyruvate dehydrogenase kinases, are key regulators of pyruvate metabolism to acetyl-CoA via phosphorylation of the pyruvate dehydrogenase complex. In HF, Pdk1 levels typically decline while Pdk4 levels rise. This observation holds in the ACi model. SB treatment had a mild, though significant, impact on Pdk1, although there was no significant effect on Pdk4. Taken together, this suggests that SB treatment might offset impaired mitochondrial function in HF. However, not all mitochondrial proteins were affected; a few examples of unchanged proteins are provided in Figure S5.

Like mitochondrial dysfunction, inflammation and activation of the acute phase response is a hallmark of human HF and recapitulated here in the guinea pig ACi Model. Several proteins associated with innate immunity were significantly upregulated in ACi (Figure ), and all these trended toward mitigation of the response in the ACiSB group. However, owing to high variation in the response for this class of proteins, only 4 of these showed statistically significant inhibition by SB treatment; specifically, Hp, Serpina1, lqgap2, and Vwf (Figure ).

Characteristics of the Cardiac Phosphoproteome

Our study identified 4310 unique high confidence phosphopeptides (1% FDR), encompassing 3844 unique phosphorylation sites. 3482 phosphopeptides (80%) could be linked to a quantified protein. Of the 5016 proteins quantified, phosphorylation sites were detected for 1129 (22%) of them. Phosphoproteome analysis of the failing heart is complicated by the fact that nearly half of all quantified proteins in our expression proteome are differentially expressed between ACi and Control groups. As Figure A illustrates, the observed change in phosphorylation levels strongly correlates with changes in the levels of underlying protein between the Control and ACi groups (Pearson r = 0.72). More explicitly, the R² value of 0.52 indicates that half of the variance in measured phosphorylation can be attributed to changes in underlying protein abundance. To discriminate between changes in bona fide phosphosite occupancy from changes arising from differential phosphoprotein abundance, phosphopeptides were normalized to the underlying protein abundance for each TMT channel. Briefly, the TiO₂-enriched peptide relative abundances were compiled by row-wise median-centering of the logged peptide-spectral match intensities across all TMT channels and then median-centering the PSM intensities within each TMT channel before median-summarizing them to yield a regularized matrix of relative peptide abundances. Relative protein abundance levels from the non-TiO₂-enriched data set were compiled using the same “median sweep” algorithm. Just as one might normalize nonlogged phosphopeptide abundances to the underlying protein levels by dividing the phosphopeptide abundances by the protein abundances, on log-transformed data, the logged protein abundance is simply subtracted from the logged phosphopeptide abundance for each TMT channel, i.e., the logged protein abundance matrix is subtracted from the logged phosphopeptide matrix. This step accounts for variations in total protein expression and provides a relative measure of phosphorylation levels for each biological replicate. Following normalization, changes in phosphopeptide abundance showed only a mild residual inverse dependence on changes in protein abundance (R ² = 0.07; Figures S6 and S7).

6.

p38 inhibition substantially attenuates changes in phosphorylation associated with HF. (A) Scatterplot reveals the correlation between phosphosite changes in HF relative to changes in underlying protein abundance. Just over 50% of the variance in phosphorylation can be explained by changes in relative protein levels. (B) Venn analysis reveals that of the 1613 unique phosphosites changing between control and ACi; 32% were impacted by SB treatment. A further 231 phosphosites differed between ACiSB and ACi, despite no significant change in ACi relative to controls. (C) PCA biplot analysis of the 525 unique phosphopsites indicates the character of the SB-significant phosphoproteome is closer to that of control hearts than failing hearts. (D) Hierarchical cluster highlights that about the largest impact of SB on the phosphoproteome was by inhibiting phosphorylation that otherwise increases in failing hearts. (E) Depicts the z-scored phosphosite signals, superimposed on a functional annotation network. Functional modules are revealed through network Markov clustering using the String score for edge-weighting. A legend of network node attributes is provided (bottom right).

p38 Mapk Inhibition Impacts a Major Portion of HF-Associated Phosphorylation Changes

Statistical analysis was conducted on all phosphopeptides. 1613 changed significantly between Control and ACi groups, of which 525 (33%) were significantly impacted by SB treatment. A further 231 phosphopeptides differed between ACiSB and ACi, irrespective of whether they changed in ACi relative to Controls (Figure B). PCA biplot analysis of the SB-responsive phosphosites is illustrated in Figure C (p < 0.05, ACi vs ACiSB). This is illustrated explicitly in the hierarchically clustered heat map in Figure D, where three major SB-dependent trends can be distinguished. First, there is a set of sites that become hyperphosphorylated in ACi that are largely prevented by SB treatment. Second, there are phosphorylated sites that become hypophosphorylated in ACi but which SB-preserves at Control levels, perhaps through indirect impact on intermediary phosphatases. Third, SB inhibits phosphorylation of a unique set of phosphorylation sites that are phosphorylated in both Control and ACi conditions.

To extract greater insight into processes impacted by SB, the quantitative information from the heatmap in Figure D was superimposed onto a Markov-clustered STRINGdb functional annotation network to visualize changes in relative phosphosite occupancy in the context of ontologically enriched modules (Figure E). As depicted in the legend (6E, bottom right), the center of the node denotes Control levels of phosphorylation, the outer ring represents phosphorylation levels in ACi, and the middle ring denotes phosphorylation in the ACiSB group.

Among phosphoproteins, cytostructural proteins constitute a major proportion. Large modules include the myofibrils, cell junction, actin cytoskeleton, and microtubules. Additional modules consist of cytoskeletal regulatory proteins, such as the Rho GTPases and Rho GTPase effectors. A second broad category of phosphoproteins encompasses membrane-associated or membrane-trafficking ontologies including Golgi vesicle transport, dynein/dynactin complexes, kinesins, and nuclear envelope proteins. Channels and transporters are encompassed by calcium signaling and transmembrane transport modules. Finally, a third major category represented in Figure E are proteins involved in phosphorylation-mediated signal transduction. Notable modules include Protein kinase A (Pka) signaling, Amp Kinase (Ampk) signaling, Vascular endothelial growth factor (VEGF) signaling, assorted kinases and phosphatases, and SH3 domains/binding.

Phosphorylation of p38α, Upstream Kinases, and Downstream Substrates Is Responsive to SB

Mapk family signaling is characterized by extensive crosstalk and feedback regulation by both direct phosphorylation and through indirect phosphorylation/dephosphorylation through intermediary kinases and phosphatases as well as indirect effects on the gene regulation of intermediary phosphatases. Accordingly, one might expect SB to affect the phospho-status of upstream regulators of p38 Mapk, Erks, and Jnks, as well as their known substrates, p38 Mapk substrates. Figure Indicates that Mapk14 (p38α) showed a significant decrease in phosphorylation upon SB treatment, as shown earlier by Western blot (Figure A). Map2k4­(Mek4), Map3k7­(Tak1), Map4k5­(Khs1), and Map4k6­(Mink1) displayed increased phosphorylation in the ACi group, which, for Map2k4, was significantly attenuated by SB treatment. Conversely, Map3k2 (Mekk2) showed a significant decrease in phosphorylation in the ACi group. Additional Mapk cascade proteins that did not exhibit significant changes across groups are presented in Figure S8.

With respect to known protein substrates of p38 Mapk, the phosphorylation levels of Spag9 (Ser 584) and Nelfe (Ser S115) increased with HF and exhibited significant responsiveness to SB treatment. Additionally, Hspb6 (Ser 16; Hsp20) stood out as being SB sensitive. This site increased by ∼2.5-fold in the ACi group compared to Controls, which was mitigated by SB treatment (Figure S9). Figure S10 further illustrates the phosphorylation of kinases responsive to ACi and SB treatment.

Phosphorylation of Select Ion Transport Proteins Sensitive to SB

Phosphorylation status of several ion transport proteins, including Atp1a2, Cacna1c, Cacnb2, Kcnh2, Kcnq1, Trpm7, Piezo1, and Clcc1, were also altered in the context HF; Only Trpm7, Piezo1, and Clcc1 were responsive to SB treatment (Figure A–E).

8.

Phosphorylation of select proteins of the ion transport is sensitive to SB: Global significance (p < 0.05) established via F-test, with p-values derived from LIMMA contrast matrices for intergroup comparisons. p-Values <0.05 are marked with brackets. Only a subset of results are shown in the figure.

Discussion

In this study, we showed that inhibition of p38 Mapk with SB203580 offered considerable efficacy in protecting against experimental HF, offsetting the bulk of the decline in fractional shortening as well as alleviating both pulmonary edema and cardiac hypertrophy. Our proteomic studies indicate that this protection is characterized by a substantial impact on the observable phosphoproteome. The scope of the impact on the proteome was modest by comparison, although the impacted processes, most notably mitochondrial function and inflammation, are key determinants of cardiac function. Taken together, notwithstanding the broad protein expression changes in HF, the data are consistent with a contributing role for p38 Mapk-driven phosphoproteome modifications in cardiac decompensation. The significance of targeting p38 Mapk in HF is further supported by findings in HFpEF models, where treatment with Doramapimod reduced cardiac and pulmonary hypertrophy and lowered pathological markers such as GAL-3, LDHA, and BNP. Additionally, Erk1/2-targeted therapies, including herbal compounds (e.g., oxymatrine, scutellarin, and salidroside) and nonpharmacologic approaches like Follistatin and MicroRNA-26b agomir, have been reported to suppress p38 activity as part of broader antifibrotic effects. Unlike these multitargeted strategies, our use of SB203580 enables direct interrogation of p38’s specific role in myocardial remodeling and supports its utility as a focused therapeutic target in HF. These collective insights suggest that p38 inhibition may complement or enhance broader therapeutic strategies aimed at attenuating myocardial fibrosis and dysfunction in HF. We discuss the impact of SB203580 on the proteome and phosphoproteome in turn.

p38 Mapk Inhibition Offsets the Impact of ACi on the Mitochondrial Proteome

Though scarcely 10% of differentially expressed proteins in HF were deemed SB-responsive, many were associated with mitochondrial pathways. The protein-level data suggest that SB treatment maintains mitochondrial functional integrity by preserving the stoichiometry of respiratory oxidative phosphorylation (oxphos) complexes. Not only does SB prevent the decline in the levels of over 30 complex subunits but it also offsets declines in the mitochondrial protein translation machinery that makes the mitochondrially encoded subunits and normalizes the expression of proteins involved in iron–sulfur biogenesis and complex assembly. SB further impacts the levels of the MICOS complex members responsible for cristae formation, thereby maximizing bioenergetic efficiency. By maintaining respiratory chain integrity, SB may serve to optimize ATP production, while minimizing mitochondrial ROS generation, a principal driver of HF pathogenesis. We further note that key antioxidant defense proteins, including thioredoxin reductase 2 (TrxnRd2) and ferredoxin reductase, were also SB-responsive. Finally, SB may help to preserve proper mitochondrial substrate utilization by ameliorating HF-induced changes in fatty acid oxidation enzymes and perhaps forestalling the switch in pyruvate dehydrogenase kinase activity. Recent research suggests Pdk4 inhibition as a promising strategy for HFrEF therapy. Targeting Pdk4 could offer a novel adjunctive therapy for HF, especially for patients resistant to conventional treatments.

Potential mechanisms for preserving mitochondrial integrity and fatty acid oxidation are suggested by the URA analysis, which implicated the lysine demethylase Kdm5a and the transcription factors Tead1, Pgc1α, and Rb1. The network diagram in Figure S2 shows that these four transcriptional regulators could reasonably account for the coordinate expression of mitochondrial and metabolic proteins. Tead1 plays a vital role as a central transcriptional hub, autonomously regulating a broad network of genes associated with mitochondrial function and biogenesis. Ablating Tead1 in mice causes cardiomyopathy. Perhaps, its best documented role is as one of the end-effector transcription factors of the HIPPO-Yap/Taz pathway, where it is activated by the nuclear translocation of dephosphorylated Yap/Taz. However, p38 has also been shown to downregulate Tead1 activity directly, independently of Yap/Taz. Specifically, p38 phosphorylates Tead1 and prevents its shuttling from the cytoplasm to the nucleus. , Applied here, p38 hyperactivation would then be expected to inhibit the Tead1 bioenergetic program, leading to mitochondrial dysfunction. SB-treatment, by inhibiting Tead1 phosphorylation, could ensure there is no brake on nuclear shuttling and thus preserve Tead1-mediated transcription.

SB203580 Curbs the Acute Phase Protein Response in HF

The P38 Mapk pathway regulates inflammatory cytokine expression, immune cell functions, and cardiac healing. Acute inflammation is crucial for cardiac protection, but unresolved inflammation can lead to HF. In our study, SB treatment reduced acute phase proteins and inflammation in HF, likely contributing to improved cardiac function. Because the acute phase proteins identified are typically the ones secreted into the circulation by the liver in response to inflammatory cytokines, part of the SB beneficial effect in HF is likely to be linked to the inhibition of systemic inflammation.

SB203580 Broadly Impacts the HF Phosphoproteome

We identified over 800 phosphopeptides that were responsive to SB treatment. Notwithstanding the specificity of the inhibitor, the set of SB-responsive phosphosites extends to peptides that do not conform to the canonical Mapk family consensus site characterized by a proline residue immediately C-terminally adjacent to the phosphosite (S/T-Pro). Presumably, the 4 week administration of SB influenced the phosphostatus of both direct substrates, as well as indirect targets, by influencing the expression and/or activity of other kinases and phosphatases. This was keenly apparent in Figure E, where several modules within the SB-responsive phospho-network were associated with phosphorylation pathway signaling by other kinases and phosphatases (e.g., VEGF, Pka, and Ampk substrates).

The impact of SB treatment on Pka signaling is noteworthy, given that HF is characterized by a loss of β-adrenergic responsiveness, manifested as a reduction in cardiac contractile power and slowed ventricular relaxation. A hallmark of the Pka signaling deficit is the progressive dephosphorylation of the thin filament regulatory protein, cardiac Troponin I (cTnI) at serines 23 and 24 (database numbering). , Dephosphorylation aberrantly increases the Ca²⁺ sensitivity of myofibril contraction and slows myofibril relaxation rate. Here, we show that SB treatment indirectly preserves the cTnI Ser23 phosphorylation. cTnI is also phosphorylated at Ser150 (Ser151 herein) by Ampk, which has been shown to blunt Pka phosphorylation at Ser 23/24. We also demonstrate that Ser151 phosphorylation is elevated in HF but is blunted by the SB treatment. Thus, an unexpected or emergent consequence of chronic SB treatment in HF is that it preserves β-adrenergic signaling to thin filaments. Besides cTnI, SB treatment impacts phosphorylation of over 25 myofibrillar proteins such as titin on nearly 100 unique phosphopeptides, the majority of which have not been characterized. In addition to contractile proteins, the myofibrillar substrates include Z-disk proteins and structural links to the costameres. We therefore speculate that SB treatment could also modulate the mechanotransduction from the sarcolemma to the myofibrils and the nucleus.

How p38 inhibition preserves β-adrenergic signaling homeostasis is unclear, although we observed substantially altered phosphorylation among the A-kinase anchoring proteins or Akaps, including Akap1, Akap5, Akap6, Akap9, and Akap13 that localize Pka and/or PKC signaling to discrete nanodomains. The AKAPs are particularly notable, as several have documented roles in Ca²⁺ handling, serving as docking points for Pka, which in turn phosphorylate and modulate the activity of ion channels. Examples include the role of Akap5 in the regulation of Ca²⁺-influx through the T-tubular voltage-gated Ca²⁺ channel (Cav1.2) and the role of Akap6 in the regulation of sarcoplasmic reticulum Ca²⁺ release through Ryr2. Coincidentally, we note that SB normalizes phosphorylation of the Cav1.2 regulatory subunit (Cacna1b), as well as two mechanosensitive sarcolemmal divalent cation channels, TrpM7 and Piezo1. SB likewise preserved the phosphorylation state of Ryr2 and phospholamban.

Apart from Pka signaling, we also note that SB treatment impacted Ampk signaling and Ca²⁺-Calmodulin-activated kinase (CamkII) phosphorylation. Specifically, SB abrogated the ACi-induced hyperphosphorylation of the Ampka1 catalytic subunit (Prkaa1) at Ser351 (guinea pig numbering; equivalent to Ser496 in the mouse) within its AMP sensor domain. SB similarly prevents Ampk beta subunit2 (Prkab2) hyperphosphosphorylation at Ser108, within its glycogen-binding domain, which would be predicted to impair glycogen binding. Hyperactivation of the Ca²⁺-Calmodulin-activated kinases, particularly Camk2D, is well documented in HF. The activation process is mediated, in part, through autophosphorylation at multiple sites, some of which are better characterized than the others. Here, we show that phosphorylation at Thr337, which has previously been shown to increase kinase activity, is blunted by SB treatment.

Comparing the Impact of SB203580 and the Antioxidant mitoTEMPO on HF Progression

Our previous research highlighted the significance of targeting mitochondria as a therapeutic approach for HF. Employing mitochondrially targeted antioxidant MitoTEMPO normalized cellular ROS levels. Additionally, administering MitoTEMPO to HF animals in vivo prevented and reversed HF, mitigated the risk of sudden cardiac death by reducing repolarization dispersion and ventricular arrhythmias, attenuated the chronic HF-induced remodeling of proteome expression, and prevented specific alterations in the phosphoproteome. Furthermore, the Mapk kinase pathway emerged as a pathway sensitive to mitochondrial ROS (mROS), known to be activated by it and exhibit altered signaling in HF models. Notably, the activation of Mapk was evident in the subset of proteins displaying changes in the expression proteome of failing hearts, a phenomenon moderated by MitoTEMPO treatment.

Even though MitoTEMPO’s effect on protein remodeling in HF was significantly greater than that of SB and had a wider influence, comparing the effects of MitoTEMPO and SB on the HF proteome highlights the diverse remodeling pathways triggered by mitochondrial ROS, including the activation of p38 Mapk. It is noteworthy that p38, downstream of mitochondrial ROS, plays a role in oxidative phosphorylation and mitochondrial processes, suggesting that its inhibition could yield beneficial effects.

Limitations of the Study

We have captured a snapshot of how SB treatments impact the HF phosphoproteome in HF pathogenesis. Therefore, it is a challenge to discern how many of the SB-responsive phosphopeptides are bona fide p38 Mapk substrates. While the presence of a proline at P+1 is a defining feature of the p38 Mapk consensus sequence, assigning substrates is complicated by the fact that the consensus sites of other “proline-directed” kinases (Mapks, Cdks) are highly similar. Moreover, though a subset of proline-containing SB-responsive sites represent p38 substrates, the experimental design (i.e., chronic SB treatment) makes it difficult to parse the direct p38-mediated impact of SB on substrate phosphorylation. Only a time-course of SB-mediated p38 inhibition in cardiac cell types could help address the issue. Finally, we are using whole heart lysates, which do not distinguish between effects on different cell types, such as fibroblasts, whose activation state is known to be modulated by p38 Mapk. This would require additional cell-type-specific studies.

Conclusion

In conclusion, chronic SB treatment elicits substantial protection against HF by exerting the effects on the phosphoproteome that percolate beyond direct inhibition of p38 to influence the broader web of cardiac kinase signaling from Pka to Ampk and CamkII, ultimately ameliorating the phospho-status of key myofibrillar and Ca²⁺-handling substrates. The impact of SB on the underlying HF proteome, while not expansive, is consistent with preserved energetics as well as reduced levels of oxidative and inflammatory stress. Further research and clinical investigation are warranted to unravel the full potential of targeting p38 Mapk as a part of therapeutic strategies aimed at improving outcomes in HF management.

The MS proteomics raw data (.raw), complete search results (.msf), and spectra (.mzidentML) have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org/;) via the PRIDE partner repository with the data set identifier PXD058012 and 10.6019/PXD058012. The R code used for data analysis along with macros used for finding homologous human peptides can be found at https://github.com/Frostman300/p38-upload.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03687.

• Volcano plot highlighting specific examples of proteins changing between ACi and Control; network diagram of transcriptional regulators, including Tead1, Kdm5a, Ppargc1a, and Rb1; expression analysis of Mapk cascade proteins; expression analysis of p38 Mapk substrates; expression analysis of selected mitochondrial bioenergetics proteins; impact of correcting phosphorylation signals for protein abundance on phosphorylation-protein dynamics; PCA and scree plot on phosphoproteome uncorrected for protein abundance; phosphorylation of selected Mapk cascade proteins; phosphorylation of known p38 Mapk substrates and targets; and ACiSB-responsive kinase phosphorylation (PDF)

• Comprehensive summary of protein quantification, phosphopeptide quantification, and unique phosphosites (XLSX)

This project was supported by the National Heart Lung and Blood Institute (NHLBI) of the NIH, grants R01HL134821 (DBF and BO’R), and R01HL164478 (DBF), U.S. Army Medical Research Acquisition Activity USAMRAA HT94252410277 (DBF), as well as American Heart Association grants AHA965158 (BO’R) and Transformational Project Award 18TPA34170575 (DBF).

The authors declare no competing financial interest.