Fast skeletal myosin binding protein-C expression exacerbates dysfunction in heart failure.

During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences that may affect cardiac function. However, it is unknown if the expression of fMyBP-C expression in the heart is a pathological or compensatory response. We aim to elucidate the cardiac consequence of either increased or knockout of fMyBP-C expression. To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the complete absence of cMyBP-C. Finally, fMyBP-C null mice underwent transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. We confirmed the upregulation of fMyBP-C in several models of cardiac disease, including the use of lineage tracing. Low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. Exclusive expression of fMyBP-C in a heart failure model further exacerbated cardiac pathology. Following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the differential regulation of the myosin super-relaxed state. These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure. Significance Statement: Recently, the sarcomere - the machinery that controls heart and muscle contraction - has emerged as a central target for development of cardiac therapeutics. However, there remains much to understand about how the sarcomere is modified in response to disease. We recently discovered that a protein normally expressed in skeletal muscle, is present in the heart in certain settings of heart disease. How this skeletal muscle protein affects the function of the heart remained unknown. Using genetically engineered mouse models to modulate expression of this skeletal muscle protein, we determined that expression of this skeletal muscle protein in the heart negatively affects cardiac performance. Importantly, deletion of this protein from the heart could improve heart function suggesting a possible therapeutic avenue.

Oxidative Post-translational Protein Modifications upon Ischemia/Reperfusion Injury.

While reperfusion, or restoration of coronary blood flow in acute myocardial infarction, is a requisite for myocardial salvage, it can paradoxically induce a specific damage known as ischemia/reperfusion (I/R) injury. Our understanding of the precise pathophysiological molecular alterations leading to I/R remains limited. In this study, we conducted a comprehensive and unbiased time-course analysis of post-translational modifications (PTMs) in the post-reperfused myocardium of two different animal models (pig and mouse) and evaluated the effect of two different cardioprotective therapies (ischemic preconditioning and neutrophil depletion). In pigs, a first wave of irreversible oxidative damage was observed at the earliest reperfusion time (20 min), impacting proteins essential for cardiac contraction. A second wave, characterized by irreversible oxidation on different residues and reversible Cys oxidation, occurred at late stages (6-12 h), affecting mitochondrial, sarcomere, and inflammation-related proteins. Ischemic preconditioning mitigated the I/R damage caused by the late oxidative wave. In the mouse model, the two-phase pattern of oxidative damage was replicated, and neutrophil depletion mitigated the late wave of I/R-related damage by preventing both Cys reversible oxidation and irreversible oxidation. Altogether, these data identify protein PTMs occurring late after reperfusion as an actionable therapeutic target to reduce the impact of I/R injury.

High-Throughput Single-Cell Proteomic Analysis of Organ-Derived Heterogeneous Cell Populations by Nanoflow Dual-Trap Single-Column Liquid Chromatography.

Identification and proteomic characterization of rare cell types within complex organ-derived cell mixtures is best accomplished by label-free quantitative mass spectrometry. High throughput is required to rapidly survey hundreds to thousands of individual cells to adequately represent rare populations. Here we present parallelized nanoflow dual-trap single-column liquid chromatography (nanoDTSC) operating at 15 min of total run time per cell with peptides quantified over 11.5 min using standard commercial components, thus offering an accessible and efficient LC solution to analyze 96 single cells per day. At this throughput, nanoDTSC quantified over 1000 proteins in individual cardiomyocytes and heterogeneous populations of single cells from the aorta.

High-Field Asymmetric Waveform Ion Mobility Spectrometry: Practical Alternative for Cardiac Proteome Sample Processing.

Heart tissue sample preparation for mass spectrometry (MS) analysis that includes prefractionation reduces the cellular protein dynamic range and increases the relative abundance of nonsarcomeric proteins. We previously described "IN-Sequence" (IN-Seq) where heart tissue lysate is sequentially partitioned into three subcellular fractions to increase the proteome coverage more than a single direct tissue analysis by mass spectrometry. Here, we report an adaptation of the high-field asymmetric ion mobility spectrometry (FAIMS) coupled to mass spectrometry, and the establishment of a simple one step sample preparation coupled with gas-phase fractionation. The FAIMS approach substantially reduces manual sample handling, significantly shortens the MS instrument processing time, and produces unique protein identification and quantification approximating the commonly used IN-Seq method in less time.

Hyperphosphorylation of hepatic proteome characterizes nonalcoholic fatty liver disease in S-adenosylmethionine deficiency.

Methionine adenosyltransferase 1a (MAT1A) is responsible for hepatic S-adenosyl-L-methionine (SAMe) biosynthesis. Mat1a -/- mice have hepatic SAMe depletion, develop nonalcoholic steatohepatitis (NASH) which is reversed with SAMe administration. We examined temporal alterations in the proteome/phosphoproteome in pre-disease and NASH Mat1a -/- mice, effects of SAMe administration, and compared to human nonalcoholic fatty liver disease (NAFLD). Mitochondrial and peroxisomal lipid metabolism proteins were altered in pre-disease mice and persisted in NASH Mat1a -/- mice, which exhibited more progressive alterations in cytoplasmic ribosomes, ER, and nuclear proteins. A common mechanism found in both pre-disease and NASH livers was a hyperphosphorylation signature consistent with casein kinase 2α (CK2α) and AKT1 activation, which was normalized by SAMe administration. This was mimicked in human NAFLD with a metabolomic signature (M-subtype) resembling Mat1a -/- mice. In conclusion, we have identified a common proteome/phosphoproteome signature between Mat1a -/- mice and human NAFLD M-subtype that may have pathophysiological and therapeutic implications.

High Throughput Single Cell Proteomic Analysis of Organ Derived Heterogeneous Cell Populations by Nanoflow Dual Trap Single Column Liquid Chromatography.

Identification and proteomic characterization of rare cell types within complex organ derived cell mixtures is best accomplished by label-free quantitative mass spectrometry. High throughput is required to rapidly survey hundreds to thousands of individual cells to adequately represent rare populations. Here we present parallelized nanoflow dual-trap single-column liquid chromatography (nanoDTSC) operating at 15 minutes of total run time per cell with peptides quantified over 11.5 minutes using standard commercial components, thus offering an accessible and efficient LC solution to analyze 96 single-cells per day. At this throughput, nanoDTSC quantified over 1,000 proteins in individual cardiomyocytes and heterogenous populations of single cells from aorta.

BIRCH: An Automated Workflow for Evaluation, Correction, and Visualization of Batch Effect in Bottom-Up Mass Spectrometry-Based Proteomics Data.

Recent surges in large-scale mass spectrometry (MS)-based proteomics studies demand a concurrent rise in methods to facilitate reliable and reproducible data analysis. Quantification of proteins in MS analysis can be affected by variations in technical factors such as sample preparation and data acquisition conditions leading to batch effects, which adds to noise in the data set. This may in turn affect the effectiveness of any biological conclusions derived from the data. Here we present Batch-effect Identification, Representation, and Correction of Heterogeneous data (BIRCH), a workflow for analysis and correction of batch effect through an automated, versatile, and easy to use web-based tool with the goal of eliminating technical variation. BIRCH also supports diagnosis of the data to check for the presence of batch effects, feasibility of batch correction, and imputation to deal with missing values in the data set. To illustrate the relevance of the tool, we explore two case studies, including an iPSC-derived cell study and a Covid vaccine study to show different context-specific use cases. Ultimately this tool can be used as an extremely powerful approach for eliminating technical bias while retaining biological bias, toward understanding disease mechanisms and potential therapeutics.

Parallelization with Dual-Trap Single-Column Configuration Maximizes Throughput of Proteomic Analysis.

Proteomic analysis on the scale that captures population and biological heterogeneity over hundreds to thousands of samples requires rapid mass spectrometry methods, which maximize instrument utilization (IU) and proteome coverage while maintaining precise and reproducible quantification. To achieve this, a short liquid chromatography gradient paired to rapid mass spectrometry data acquisition can be used to reproducibly quantify a moderate set of analytes. High-throughput profiling at a limited depth is becoming an increasingly utilized strategy for tackling large sample sets but the time spent on loading the sample, flushing the column(s), and re-equilibrating the system reduces the ratio of meaningful data acquired to total operation time and IU. The dual-trap single-column configuration (DTSC) presented here maximizes IU in rapid analysis (15 min per sample) of blood and cell lysates by parallelizing trap column cleaning and sample loading and desalting with the analysis of the previous sample. We achieved 90% IU in low microflow (9.5 µL/min) analysis of blood while reproducibly quantifying 300-400 proteins and over 6000 precursor ions. The same IU was achieved for cell lysates and over 4000 proteins (3000 at CV below 20%) and 40,000 precursor ions were quantified at a rate of 15 min/sample. Thus, DTSC enables high-throughput epidemiological blood-based biomarker cohort studies and cell-based perturbation screening.

US Severe Acute Respiratory Syndrome Coronavirus 2 Epsilon Variant: Highly Transmissible but With an Adjusted Muted Host T-Cell Response.

BACKGROUND: The multiple mutations comprising the epsilon variant demonstrate the independent convergent evolution of severe acute respiratory syndrome coronavirus (SARS-CoV-2), with its spike protein mutation L452R present in the delta (L452R), kappa (L452R), and lambda (L452Q) variants. METHODS: Coronavirus disease 2019 (COVID-19) variants were detected in 1017 patients using whole-genome sequencing and were assessed for outcome and severity. The mechanistic effects of the epsilon versus non-epsilon variants were investigated using a multiomic approach including cellular response assays and paired cell and host transcriptomic and proteomic profiling. RESULTS: We found that patients carrying the epsilon variant had increased mortality risk but not increased hospitalizations (P < .02). Cells infected with live epsilon compared with non-epsilon virus displayed increased sensitivity to neutralization antibodies in all patients but a slightly protective response in vaccinated individuals (P < .001). That the epsilon SARS-CoV-2 variant is more infectious but less virulent is supported mechanistically in the down-regulation of viral processing pathways seen by multiomic analyses. Importantly, this paired transcriptomics and proteomic profiling of host cellular response to live virus revealed an altered leukocyte response and metabolic messenger RNA processing with the epsilon variant. To ascertain host response to SARS-CoV-2 infection, primary COVID-19-positive nasopharyngeal samples were transcriptomically profiled and revealed a differential innate immune response (P < .001) and an adjusted T-cell response in patients carrying the epsilon variant (P < .002). In fact, patients infected with SARS-CoV-2 and those vaccinated with the BNT162b2 vaccine have comparable CD4+/CD8+ T-cell immune responses to the epsilon variant (P < .05). CONCLUSIONS: While the epsilon variant is more infectious, by altering viral processing, we showed that patients with COVID-19 have adapted their innate immune response to this fitter variant. A protective T-cell response molecular signature is generated by this more transmissible variant in both vaccinated and unvaccinated patients.

Sexual Dimorphism in Cardiovascular Biomarkers: Clinical and Research Implications.

Sex-based differences in cardiovascular disease presentation, diagnosis, and response to therapies are well established, but mechanistic understanding and translation to clinical applications are limited. Blood-based biomarkers have become an important tool for interrogating biologic pathways. Understanding sexual dimorphism in the relationship between biomarkers and cardiovascular disease will enhance our insights into cardiovascular disease pathogenesis in women, with potential to translate to improved individualized care for men and women with or at risk for cardiovascular disease. In this review, we examine how biologic sex associates with differential levels of blood-based biomarkers and influences the effect of biomarkers on disease outcomes. We further summarize key differences in blood-based cardiovascular biomarkers along central biologic pathways, including myocardial stretch/injury, inflammation, adipose tissue metabolism, and fibrosis pathways in men versus women. Finally, we present recommendations for leveraging our current knowledge of sex differences in blood-based biomarkers for future research and clinical innovation.

Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease.

MATα1 catalyzes the synthesis of S-adenosylmethionine, the principal biological methyl donor. Lower MATα1 activity and mitochondrial dysfunction occur in alcohol-associated liver disease. Besides cytosol and nucleus, MATα1 also targets the mitochondria of hepatocytes to regulate their function. Here, we show that mitochondrial MATα1 is selectively depleted in alcohol-associated liver disease through a mechanism that involves the isomerase PIN1 and the kinase CK2. Alcohol activates CK2, which phosphorylates MATα1 at Ser114 facilitating interaction with PIN1, thereby inhibiting its mitochondrial localization. Blocking PIN1-MATα1 interaction increased mitochondrial MATα1 levels and protected against alcohol-induced mitochondrial dysfunction and fat accumulation. Normally, MATα1 interacts with mitochondrial proteins involved in TCA cycle, oxidative phosphorylation, and fatty acid ß-oxidation. Preserving mitochondrial MATα1 content correlates with higher methylation and expression of mitochondrial proteins. Our study demonstrates a role of CK2 and PIN1 in reducing mitochondrial MATα1 content leading to mitochondrial dysfunction in alcohol-associated liver disease.

S-adenosylmethionine inhibits la ribonucleoprotein domain family member 1 in murine liver and human liver cancer cells.

BACKGROUND AND AIMS: Methionine adenosyltransferase 1A (MAT1A) is responsible for S-adenosylmethionine (SAMe) biosynthesis in the liver. Mice lacking Mat1a have hepatic SAMe depletion and develop NASH and HCC spontaneously. Several kinases are activated in Mat1a knockout (KO) mice livers. However, characterizing the phospho-proteome and determining whether they contribute to liver pathology remain open for study. Our study aimed to provide this knowledge. APPROACH AND RESULTS: We performed phospho-proteomics in Mat1a KO mice livers with and without SAMe treatment to identify SAMe-dependent changes that may contribute to liver pathology. Our studies used Mat1a KO mice at different ages treated with and without SAMe, cell lines, in vitro translation and kinase assays, and human liver specimens. We found that the most striking change was hyperphosphorylation and increased content of La-related protein 1 (LARP1), which, in the unphosphorylated form, negatively regulates translation of 5'-terminal oligopyrimidine (TOP)-containing mRNAs. Consistently, multiple TOP proteins are induced in KO livers. Translation of TOP mRNAs ribosomal protein S3 and ribosomal protein L18 was enhanced by LARP1 overexpression in liver cancer cells. We identified LARP1-T449 as a SAMe-sensitive phospho-site of cyclin-dependent kinase 2 (CDK2). Knocking down CDK2 lowered LARP1 phosphorylation and prevented LARP1-overexpression-mediated increase in translation. LARP1-T449 phosphorylation induced global translation, cell growth, migration, invasion, and expression of oncogenic TOP-ribosomal proteins in HCC cells. LARP1 expression is increased in human NASH and HCC. CONCLUSIONS: Our results reveal a SAMe-sensitive mechanism of LARP1 phosphorylation that may be involved in the progression of NASH to HCC.

Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids.

BACKGROUND: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. METHODS: Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. RESULTS: Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV <30%. The mid-throughput workflow had 67% (plasma), 90% (depleted), and 78% (dried blood) of peptides display an inter-day CV <30%. Lower limits of detection and quantification were determined for peptides and proteins observed in each biofluid and workflow. Based on each protein and peptide's analytical performance, we could describe the observable, reliable, reproducible, and quantifiable proteomes for each biofluid and workflow. CONCLUSION: The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.

Discovery Proteomics for COVID-19: Where We Are Now.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the pandemic coronavirus disease 2019 (COVID-19), which has had a devastating impact on society. Here, we summarize proteomic research that has helped elucidate hallmark proteins associated with the disease with respect to both short- and long-term diagnosis and prognosis. Additionally, we review the highly variable humoral response associated with COVID-19 and the increased risk of autoimmunity.

The autoimmune signature of hyperinflammatory multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) manifests as a severe and uncontrolled inflammatory response with multiorgan involvement, occurring weeks after SARS-CoV-2 infection. Here, we utilized proteomics, RNA sequencing, autoantibody arrays, and B cell receptor (BCR) repertoire analysis to characterize MIS-C immunopathogenesis and identify factors contributing to severe manifestations and intensive care unit admission. Inflammation markers, humoral immune responses, neutrophil activation, and complement and coagulation pathways were highly enriched in MIS-C patient serum, with a more hyperinflammatory profile in severe than in mild MIS-C cases. We identified a strong autoimmune signature in MIS-C, with autoantibodies targeted to both ubiquitously expressed and tissue-specific antigens, suggesting autoantigen release and excessive antigenic drive may result from systemic tissue damage. We further identified a cluster of patients with enhanced neutrophil responses as well as high anti-Spike IgG and autoantibody titers. BCR sequencing of these patients identified a strong imprint of antigenic drive with substantial BCR sequence connectivity and usage of autoimmunity-associated immunoglobulin heavy chain variable region (IGHV) genes. This cluster was linked to a TRBV11-2 expanded T cell receptor (TCR) repertoire, consistent with previous studies indicating a superantigen-driven pathogenic process. Overall, we identify a combination of pathogenic pathways that culminate in MIS-C and may inform treatment.

Seroprevalence of antibodies to SARS-CoV-2 in healthcare workers: a cross-sectional study.

OBJECTIVE: We sought to determine the extent of SARS-CoV-2 seroprevalence and the factors associated with seroprevalence across a diverse cohort of healthcare workers. DESIGN: Observational cohort study of healthcare workers, including SARS-CoV-2 serology testing and participant questionnaires. SETTINGS: A multisite healthcare delivery system located in Los Angeles County. PARTICIPANTS: A diverse and unselected population of adults (n=6062) employed in a multisite healthcare delivery system located in Los Angeles County, including individuals with direct patient contact and others with non-patient-oriented work functions. MAIN OUTCOMES: Using Bayesian and multivariate analyses, we estimated seroprevalence and factors associated with seropositivity and antibody levels, including pre-existing demographic and clinical characteristics; potential COVID-19 illness-related exposures; and symptoms consistent with COVID-19 infection. RESULTS: We observed a seroprevalence rate of 4.1%, with anosmia as the most prominently associated self-reported symptom (OR 11.04, p<0.001) in addition to fever (OR 2.02, p=0.002) and myalgias (OR 1.65, p=0.035). After adjusting for potential confounders, seroprevalence was also associated with Hispanic ethnicity (OR 1.98, p=0.001) and African-American race (OR 2.02, p=0.027) as well as contact with a COVID-19-diagnosed individual in the household (OR 5.73, p<0.001) or clinical work setting (OR 1.76, p=0.002). Importantly, African-American race and Hispanic ethnicity were associated with antibody positivity even after adjusting for personal COVID-19 diagnosis status, suggesting the contribution of unmeasured structural or societal factors. CONCLUSION AND RELEVANCE: The demographic factors associated with SARS-CoV-2 seroprevalence among our healthcare workers underscore the importance of exposure sources beyond the workplace. The size and diversity of our study population, combined with robust survey and modelling techniques, provide a vibrant picture of the demographic factors, exposures and symptoms that can identify individuals with susceptibility as well as potential to mount an immune response to COVID-19.

Lysine and Arginine Protein Post-translational Modifications by Enhanced DIA Libraries: Quantification in Murine Liver Disease.

Proteoforms containing post-translational modifications (PTMs) represent a degree of functional diversity only harnessed through analytically precise simultaneous quantification of multiple PTMs. Here we present a method to accurately differentiate an unmodified peptide from its PTM-containing counterpart through data-independent acquisition-mass spectrometry, leveraging small precursor mass windows to physically separate modified peptidoforms from each other during MS2 acquisition. We utilize a lysine and arginine PTM-enriched peptide assay library and site localization algorithm to simultaneously localize and quantify seven PTMs including mono-, di-, and trimethylation, acetylation, and succinylation in addition to total protein quantification in a single MS run without the need to enrich experimental samples. To evaluate biological relevance, this method was applied to liver lysate from differentially methylated nonalcoholic steatohepatitis (NASH) mouse models. We report that altered methylation and acetylation together with total protein changes drive the novel hypothesis of a regulatory function of PTMs in protein synthesis and mRNA stability in NASH.

Flow Cytometry Has a Significant Impact on the Cellular Metabolome.

The characterization of specialized cell subpopulations in a heterogeneous tissue is essential for understanding organ function in health and disease. A popular method of cell isolation is fluorescence-activated cell sorting (FACS) based on probes that bind surface or intracellular markers. In this study, we analyze the impact of FACS on the cell metabolome of mouse peritoneal macrophages. Compared with directly pelleted macrophages, FACS-treated cells had an altered content of metabolites related to the plasma membrane, activating a mechanosensory signaling cascade causing inflammation-like stress. The procedure also triggered alterations related to energy consumption and cell damage. The observed changes mostly derive from the physical impact on cells during their passage through the instrument. These findings provide evidence of FACS-induced biochemical changes, which should be taken into account in the design of robust metabolic assays of cells separated by flow cytometry.

Proteomic footprint of myocardial ischemia/reperfusion injury: Longitudinal study of the at-risk and remote regions in the pig model.

Reperfusion alters post-myocardial infarction (MI) healing; however, very few systematic studies report the early molecular changes following ischemia/reperfusion (I/R). Alterations in the remote myocardium have also been neglected, disregarding its contribution to post-MI heart failure (HF) development. This study characterizes protein dynamics and contractile abnormalities in the ischemic and remote myocardium during one week after MI. Closed-chest 40 min I/R was performed in 20 pigs sacrificed at 120 min, 24 hours, 4days, and 7days after reperfusion (n = 5 per group). Myocardial contractility was followed up by cardiac magnetic resonance (CMR) and tissue samples were analyzed by multiplexed quantitative proteomics. At early reperfusion (120 min), the ischemic area showed a coordinated upregulation of inflammatory processes, whereas interstitial proteins, angiogenesis and cardio-renal signaling processes increased at later reperfusion (day 4 and 7). Remote myocardium showed decreased contractility at 120 min- and 24 h-CMR accompanied by transient alterations in contractile and mitochondrial proteins. Subsequent recovery of regional contractility was associated with edema formation on CMR and increases in inflammation and wound healing proteins on post-MI day 7. Our results establish for the first time the altered protein signatures in the ischemic and remote myocardium early after I/R and might have implications for new therapeutic targets to improve early post-MI remodeling.