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Variants in RNA binding motif protein 20 (RBM20) are causative in a severe form of dilated cardiomyopathy referred to as RBM20 cardiomyopathy, yet the mechanisms are unclear. Moreover, the reason(s) for phenotypic heterogeneity in carriers with different pathogenic variants are similarly opaque. To gain insight, we carried out multi-omics analysis, including the first analysis of gene expression changes at the protein level, of mice carrying two different pathogenic variants in the RBM20 nuclear localization signal (NLS). Direct comparison of the phenotypes confirmed greater premature morality in S639G variant carrying mice compared to mice with the S637A variant despite similar cardiac remodeling and dysfunction. Analysis of differentially spliced genes uncovered alterations in the splicing of both RBM20 target genes and non-target genes, including several genes previously implicated in arrhythmia. Global proteomics analysis found that a greater number of proteins were differentially expressed in the hearts of Rbm20 S639G mice relative to WT than in Rbm20 S637A versus WT. Gene ontology analysis suggested greater mitochondrial dysfunction in Rbm20 S639G mice, although direct comparison of protein expression in the hearts of Rbm20 S639G versus Rbm20 S637A mice failed to identify any significant differences. Similarly, few differences were found by direct comparison of gene expression at the transcript level in Rbm20 S639G and Rbm20 S637A despite greater coverage. Our data provide a comprehensive overview of gene splicing and expression differences associated with pathogenic variants in RBM20, as well as insights into the molecular underpinnings of phenotypic heterogeneity associated with different dilated cardiomyopathy-associated variants., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Ltd.)

The giant protein titin is an essential component of muscle sarcomeres. A single titin molecule spans half a sarcomere and mediates diverse functions along its length by virtue of its unique domains. The A-band of titin functions as a molecular blueprint that defines the length of the thick filaments, the I-band constitutes a molecular spring that determines cell-based passive stiffness, and various domains, including the Z-disk, I-band, and M-line, serve as scaffolds for stretch-sensing signaling pathways that mediate mechanotransduction. This review aims to discuss recent insights into titin's functional roles and their relationship to cardiac function. The role of titin in heart diseases, such as dilated cardiomyopathy and heart failure with preserved ejection fraction, as well as its potential as a therapeutic target, is also discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Stroik, Gregorich, Raza, Ge and Guo.)

RBM20 (RNA-binding motif protein 20) is a vertebrate- and muscle-specific RNA-binding protein that belongs to the serine-arginine-rich family of splicing factors. The RBM20 gene was first identified as a dilated cardiomyopathy-linked gene over a decade ago. Early studies in Rbm20 knockout rodents implicated disrupted splicing of RBM20 target genes as a causative mechanism. Clinical studies show that pathogenic variants in RBM20 are linked to aggressive dilated cardiomyopathy with early onset heart failure and high mortality. Subsequent studies employing pathogenic variant knock-in animal models revealed that variants in a specific portion of the arginine-serine-rich domain in RBM20 not only disrupt splicing but also hinder nucleocytoplasmic transport and lead to the formation of RBM20 biomolecular condensates in the sarcoplasm. Conversely, mice harboring a disease-associated variant in the RRM (RNA recognition motif) do not show evidence of adverse remodeling or exhibit sudden death despite disrupted splicing of RBM20 target genes. Thus, whether disrupted splicing, biomolecular condensates, or both contribute to dilated cardiomyopathy is under debate. Beyond this, additional questions remain, such as whether there is sexual dimorphism in the presentation of RBM20 cardiomyopathy. What are the clinical features of RBM20 cardiomyopathy and why do some individuals develop more severe disease than others? In this review, we summarize the reported observations and discuss potential mechanisms of RBM20 cardiomyopathy derived from studies employing in vivo animal models and in vitro human-induced pluripotent stem cell-derived cardiomyocytes. Potential therapeutic strategies to treat RBM20 cardiomyopathy are also discussed., Competing Interests: Disclosures None.

Myosin functions as the "molecular motor" of the sarcomere and generates the contractile force necessary for cardiac muscle contraction. Myosin light chains 1 and 2 (MLC-1 and -2) play important functional roles in regulating the structure of the hexameric myosin molecule. Each of these light chains has an 'atrial' and 'ventricular' isoform, so called because they are believed to exhibit chamber-restricted expression in the heart. However, recently the chamber-specific expression of MLC isoforms in the human heart has been questioned. Herein, we analyzed the expression of MLC-1 and -2 atrial and ventricular isoforms in each of the four cardiac chambers in adult non-failing donor hearts using top-down mass spectrometry (MS)-based proteomics. Strikingly, we detected an isoform thought to be ventricular, MLC-2v (gene: MYL2), in the atria and confirmed the protein sequence using tandem MS (MS/MS). For the first time, a putative deamidation post-translation modification (PTM) located on MLC-2v in atrial tissue was localized to amino acid N13. MLC-1v (MYL3) and MLC-2a (MYL7) were the only MLC isoforms exhibiting chamber-restricted expression patterns across all donor hearts. Importantly, our results unambiguously show that MLC-1v, not MLC-2v, is ventricle-specific in adult human hearts. Moreover, we found elevated MLC-2 phosphorylation in male hearts compared to female hearts across each cardiac chamber. Overall, top-down proteomics allowed an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs., Competing Interests: Declaration of Competing Interest T.J. Kamp is a consultant for Fujifilm Cellular Dynamics Incorporated., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knockin (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We showed that Rbm20ΔRS mice manifested DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 was mis-localized to the sarcoplasm in Rbm20ΔRS mouse hearts and formed RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif showed similar mis-splicing of major RBM20 target genes but did not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrated that only DCM-associated mutations in the RS domain facilitated RBM20 nucleocytoplasmic transport and promoted granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain of RBM20. Mutation analysis of phosphorylation sites in the RS domain suggested that this modification may be dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.

Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications. Herein, we have developed a highly sensitive functionally integrated top-down proteomics method for the comprehensive analysis of proteoforms from single cells. We applied this method to single muscle fibers (SMFs) to resolve their heterogeneous functional and proteomic properties at the single-cell level. Notably, we have detected single-cell heterogeneity in large proteoforms (>200 kDa) from the SMFs. Using SMFs obtained from three functionally distinct muscles, we found fiber-to-fiber heterogeneity among the sarcomeric proteoforms which can be related to the functional heterogeneity. Importantly, we detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high reproducibility to enable the classification of individual fiber types. This study reveals single muscle cell heterogeneity in large proteoforms and establishes a direct relationship between sarcomeric proteoforms and muscle fiber types, highlighting the potential of top-down proteomics for uncovering the molecular underpinnings of cell-to-cell variation in complex systems.

Rationale: Flask-shaped invaginations of the cardiomyocyte sarcolemma called caveolae require the structural protein caveolin-3 (Cav-3) and host a variety of ion channels, transporters, and signaling molecules. Reduced Cav-3 expression has been reported in models of heart failure, and variants in CAV3 have been associated with the inherited long-QT arrhythmia syndrome. Yet, it remains unclear whether alterations in Cav-3 levels alone are sufficient to drive aberrant repolarization and increased arrhythmia risk., Objective: To determine the impact of cardiac-specific Cav-3 ablation on the electrophysiological properties of the adult mouse heart., Methods and Results: Cardiac-specific, inducible Cav3 homozygous knockout (Cav-3KO) mice demonstrated a marked reduction in Cav-3 expression by Western blot and loss of caveolae by electron microscopy. However, there was no change in macroscopic cardiac structure or contractile function. The QT c interval was increased in Cav-3KO mice, and there was an increased propensity for ventricular arrhythmias. Ventricular myocytes isolated from Cav-3KO mice exhibited a prolonged action potential duration (APD) that was due to reductions in outward potassium currents (I to , I ss ) and changes in inward currents including slowed inactivation of I Ca,L and increased I Na,L . Mathematical modeling demonstrated that the changes in the studied ionic currents were adequate to explain the prolongation of the mouse ventricular action potential. Results from human iPSC-derived cardiomyocytes showed that shRNA knockdown of Cav-3 similarly prolonged APD., Conclusion: We demonstrate that Cav-3 and caveolae regulate cardiac repolarization and arrhythmia risk via the integrated modulation of multiple ionic currents., Competing Interests: Declaration of Competing Interest TJK is a consultant for Fujifilm Cellular Dynamics Incorporated., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

(1) Background: RNA binding motif 20 (RBM20) regulates mRNA splicing specifically in muscle tissues. Missense mutations in the arginine/serine (RS) domain of RBM20 lead to abnormal gene splicing and have been linked to severe dilated cardiomyopathy (DCM) in human patients and animal models. Interestingly, many of the reported DCM-linked missense mutations in RBM20 are in a highly conserved RSRSP stretch within the RS domain. Recently, it was found that the two Ser residues within this stretch are constitutively phosphorylated, yet the identity of the kinase(s) responsible for phosphorylating these residues, as well as the function of RSRSP phosphorylation, remains unknown. (2) Methods: The ability of three known SR protein kinases (SRPK1, CLK1, and AKT2) to phosphorylate the RBM20 RSRSP stretch and regulate target gene splicing was evaluated by using both in vitro and in vivo approaches. (3) Results: We found that all three kinases phosphorylated S638 and S640 in the RSRSP stretch and regulated RBM20 target gene splicing. While SRPK1 and CLK1 were both capable of directly phosphorylating the RS domain in RBM20, whether AKT2-mediated control of the RS domain phosphorylation is direct or indirect could not be determined. (4) Conclusions: Our results indicate that SR protein kinases regulate the splicing of a cardiomyopathy-relevant gene by modulating phosphorylation of the RSRSP stretch in RBM20. These findings suggest that SR protein kinases may be potential targets for the treatment of RBM20 cardiomyopathy.

Dilated cardiomyopathy (DCM) is a major risk factor for developing heart failure and is often associated with an increased risk for life-threatening arrhythmia. Although numerous causal genes for DCM have been identified, RNA binding motif protein 20 ( Rbm20 ) remains one of the few splicing factors that, when mutated or genetically ablated, leads to the development of DCM. In this study we sought to identify changes in the cardiac proteome in Rbm20 knockout (KO) rat hearts using global quantitative proteomics to gain insight into the molecular mechanisms precipitating the development of DCM in these rats. Our analysis identified changes in titin-interacting proteins involved in mechanical stretch-based signaling, as well as mitochondrial enzymes, which suggests that activation of pathological hypertrophy and altered mitochondrial metabolism and/or dysfunction, among other changes, contribute to the development of DCM in Rbm20 KO rats. Collectively, our findings provide the first report on changes in the cardiac proteome associated with genetic ablation of Rbm20 .

Research and therapeutic applications using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require robust differentiation strategies. Efforts to improve hPSC-CM differentiation have largely overlooked the role of extracellular matrix (ECM). The present study investigates the ability of defined ECM proteins to promote hPSC cardiac differentiation. Fibronectin (FN), laminin-111, and laminin-521 enabled hPSCs to attach and expand. However, only addition of FN promoted cardiac differentiation in response to growth factors Activin A, BMP4, and bFGF in contrast to the inhibition produced by laminin-111 or laminin-521. hPSCs in culture produced endogenous FN which accumulated in the ECM to a critical level necessary for effective cardiac differentiation. Inducible shRNA knockdown of FN prevented Brachyury + mesoderm formation and subsequent hPSC-CM generation. Antibodies blocking FN binding integrins α4β1 or αVβ1, but not α5β1, inhibited cardiac differentiation. Furthermore, inhibition of integrin-linked kinase led to a decrease in phosphorylated AKT, which was associated with increased apoptosis and inhibition of cardiac differentiation. These results provide new insights into defined matrices for culture of hPSCs that enable production of FN-enriched ECM which is essential for mesoderm formation and efficient cardiac differentiation., Competing Interests: JZ, ZG, RT, GK, PL, JC, YM, DM, SP No competing interests declared, TK is a consultant for Fujifilm Cellular Dynamics Incorporated, a stem cell company, (© 2022, Zhang et al.)

Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry-based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction ( n = 16) compared to nonfailing donor hearts ( n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease., Competing Interests: Competing interest statement: T.J.K. is a consultant for Fujifilm Cellular Dynamics Incorporated., (Copyright © 2020 the Author(s). Published by PNAS.)

Heterogeneity in skeletal muscle contraction time, peak power output, and resistance to fatigue, among others, is necessary to accommodate the wide range of functional demands imposed on the body. Underlying this functional heterogeneity are a myriad of differences in the myofilament protein isoform expression and post-translational modifications; yet, characterizing this heterogeneity remains challenging. Herein, we have utilized top-down liquid chromatography (LC)-mass spectrometry (MS)-based proteomics to characterize myofilament proteoform heterogeneity in seven rat skeletal muscle tissues including vastus lateralis, vastus medialis, vastus intermedius, rectus femoris, soleus, gastrocnemius, and plantaris. Top-down proteomics revealed that myofilament proteoforms varied greatly across the seven different rat skeletal muscle tissues. Subsequently, we quantified and characterized myofilament proteoforms using online LC-MS. We have comprehensively characterized the fast and slow skeletal troponin I isoforms, which demonstrates the ability of top-down MS to decipher isoforms with high sequence homology. Taken together, we have shown that top-down proteomics can be used as a robust and high-throughput method to characterize the molecular heterogeneity of myofilament proteoforms from various skeletal muscle tissues.

Rationale: Human pluripotent stem cell (hPSC)-derived cardiomyocytes exhibit the properties of fetal cardiomyocytes, which limits their applications. Various methods have been used to promote maturation of hPSC-cardiomyocytes; however, there is a lack of an unbiased and comprehensive method for accurate assessment of the maturity of hPSC-cardiomyocytes., Objective: We aim to develop an unbiased proteomics strategy integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for the accurate and comprehensive assessment of hPSC-cardiomyocyte maturation., Methods and Results: Utilizing hPSC-cardiomyocytes from early- and late-stage 2-dimensional monolayer culture and 3-dimensional engineered cardiac tissue, we demonstrated the high reproducibility and reliability of a top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expression and associated post-translational modifications. This method allowed for the detection of known maturation-associated contractile protein alterations and, for the first time, identified contractile protein post-translational modifications as promising new markers of hPSC-cardiomyocytes maturation. Most notably, decreased phosphorylation of α-tropomyosin was found to be associated with hPSC-cardiomyocyte maturation. By employing a bottom-up global proteomics strategy, we identified candidate maturation-associated markers important for sarcomere organization, cardiac excitability, and Ca 2+ homeostasis. In particular, upregulation of myomesin 1 and transmembrane 65 was associated with hPSC-cardiomyocyte maturation and validated in cardiac development, making these promising markers for assessing maturity of hPSC-cardiomyocytes. We have further validated α-actinin isoforms, phospholamban, dystrophin, αB-crystallin, and calsequestrin 2 as novel maturation-associated markers, in the developing mouse cardiac ventricles., Conclusions: We established an unbiased proteomics method that can provide accurate and specific assessment of the maturity of hPSC-cardiomyocytes and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering the molecular pathways involved in cardiac development and disease using hPSC-cardiomyocytes.

Enigma Homologue (ENH) is a component of the Z-disc, a structure that anchors actin filaments in the contractile unit of muscle, the sarcomere. Cardiac-specific ablation of ENH protein expression causes contractile dysfunction that ultimately culminates in dilated cardiomyopathy. However, whether ENH is involved in the regulation of myocardial contractility is unknown. To determine if ENH is required for the mechanical activity of cardiac muscle, we analyze muscle mechanics of isolated trabeculae from the hearts of ENH +/+ and ENH -/- mice. We detected no differences in steady-state mechanical properties but show that when muscle fibers are allowed to relax and then are restretched, the rate at which tension redevelops is depressed in ENH -/- mouse myocardium relative to that in ENH +/+ myocardium. SDS-PAGE analysis demonstrated that the expression of β-myosin heavy chain is increased in ENH -/- mouse myocardium, which could partially, but not completely, account for the depression in tension redevelopment kinetics. Using top-down proteomics analysis, we found that the expression of other thin/thick filament regulatory proteins is unaltered, although the phosphorylation of a cardiac troponin T isoform, cardiac troponin I, and myosin regulatory light chain is decreased in ENH -/- mouse myocardium. Nevertheless, these alterations are very small and thus insufficient to explain slowed tension redevelopment kinetics in ENH -/- mouse myocardium. These data suggest that the ENH protein influences tension redevelopment kinetics in mouse myocardium, possibly by affecting cross-bridge cycling kinetics. Previous studies also indicate that ablation of specific Z-disc proteins in myocardium slows contraction kinetics, which could also be a contributing factor in this study., (© 2019 Gregorich et al.)

Despite advancements in symptom management for heart failure (HF), this devastating clinical syndrome remains the leading cause of death in the developed world. Studies using animal models have greatly advanced our understanding of the molecular mechanisms underlying HF; however, differences in cardiac physiology and the manifestation of HF between animals, particularly rodents, and humans necessitates the direct interrogation of human heart tissue samples. Nevertheless, an ever-present concern when examining human heart tissue samples is the potential for artefactual changes related to temperature changes during tissue shipment or sample processing. Herein, we examined the effects of temperature on the post-translational modifications (PTMs) of sarcomeric proteins, the proteins responsible for muscle contraction, under conditions mimicking those that might occur during tissue shipment or sample processing. Using a powerful top-down proteomics method, we found that sarcomeric protein PTMs were differentially affected by temperature. Specifically, cardiac troponin I and enigma homolog isoform 2 showed robust increases in phosphorylation when tissue was incubated at either 4 °C or 22 °C. The observed increase is likely due to increased cyclic AMP levels and activation of protein kinase A in the tissue. On the contrary, cardiac troponin T and myosin regulatory light chain phosphorylation decreased when tissue was incubated at 4 °C or 22 °C. Furthermore, significant protein degradation was also observed after incubation at 4 °C or 22 °C. Overall, these results indicate that temperature exerts various effects on sarcomeric protein PTMs and careful tissue handling is critical for studies involving human heart samples. Moreover, these findings highlight the power of top-down proteomics for examining the integrity of cardiac tissue samples., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cardiac troponin T (cTnT) regulates the Ca 2+ -mediated interaction between myosin thick filaments and actin thin filaments during cardiac contraction and relaxation. cTnT is released into the blood following injury, and increased serum levels of the protein are used clinically as a biomarker for myocardial infarction. Moreover, mutations in cTnT are causative in a number of familial cardiomyopathies. With the increasing use of large animal (swine) model to recapitulate human diseases, it is essential to characterize species-dependent protein sequence variants, alternative RNA splicing, and post-translational modifications (PTMs), but challenges remain due to the incomplete database and lack of validation of the predicted splicing isoforms. Herein, we integrated top-down mass spectrometry (MS) with online liquid chromatography (LC) and immunoaffinity purification to comprehensively characterize miniature swine cTnT proteoforms, including those arising from alternative RNA splicing and PTMs. A total of seven alternative splicing isoforms of cTnT were identified by LC/MS from swine left ventricular tissue, with each isoform containing un-phosphorylated and mono-phosphorylated proteoforms. The phosphorylation site was localized to Ser1 for the mono-phosphorylated proteoforms of cTnT1, 3, 4, and 6 by online MS/MS combining collisionally activated dissociation (CAD) and electron transfer dissociation (ETD). Offline MS/MS on Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer with CAD and electron capture dissociation (ECD) was then utilized to achieve deep sequencing of mono-phosphorylated cTnT1 (35.2 kDa) with a high sequence coverage of 87%. Taken together, this study demonstrated the unique advantage of top-down MS in the comprehensive characterization of protein alternative splicing isoforms together with PTMs. Graphical Abstract ᅟ.

Background: Here, we generated human cardiac muscle patches (hCMPs) of clinically relevant dimensions (4 cm × 2 cm × 1.25 mm) by suspending cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human induced-pluripotent stem cells in a fibrin scaffold and then culturing the construct on a dynamic (rocking) platform., Methods: In vitro assessments of hCMPs suggest maturation in response to dynamic culture stimulation. In vivo assessments were conducted in a porcine model of myocardial infarction (MI). Animal groups included: MI hearts treated with 2 hCMPs (MI+hCMP, n=13), MI hearts treated with 2 cell-free open fibrin patches (n=14), or MI hearts with neither experimental patch (n=15); a fourth group of animals underwent sham surgery (Sham, n=8). Cardiac function and infarct size were evaluated by MRI, arrhythmia incidence by implanted loop recorders, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Additional studies examined the myocardial protein expression profile changes and potential mechanisms of action that related to exosomes from the cell patch., Results: The hCMPs began to beat synchronously within 1 day of fabrication, and after 7 days of dynamic culture stimulation, in vitro assessments indicated the mechanisms related to the improvements in electronic mechanical coupling, calcium-handling, and force generation, suggesting a maturation process during the dynamic culture. The engraftment rate was 10.9±1.8% at 4 weeks after the transplantation. The hCMP transplantation was associated with significant improvements in left ventricular function, infarct size, myocardial wall stress, myocardial hypertrophy, and reduced apoptosis in the periscar boarder zone myocardium. hCMP transplantation also reversed some MI-associated changes in sarcomeric regulatory protein phosphorylation. The exosomes released from the hCMP appeared to have cytoprotective properties that improved cardiomyocyte survival., Conclusions: We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity., (© 2017 American Heart Association, Inc.)

Protein phosphorylation is a ubiquitous and critical post-translational modification (PTM) involved in numerous cellular processes. Mass spectrometry (MS)-based proteomics has emerged as the preferred technology for protein identification, characterization, and quantification. Whereas ionization/detection efficiency of peptides in electrospray ionization (ESI)-MS are markedly influenced by the presence of phosphorylation, the physicochemical properties of intact proteins are assumed not to vary significantly due to the relatively smaller modification on large intact proteins. Thus, the ionization/detection efficiency of intact phosphoprotein is hypothesized not to alter appreciably for subsequent MS quantification. However, this hypothesis has never been rigorously tested. Herein, we systematically investigated the impact of phosphorylation on ESI-MS quantification of mono- and multiply phosphorylated proteins. We verified that a single phosphorylation did not appreciably affect the ESI-MS quantification of phosphoproteins as demonstrated in the enigma homolog isoform 2 (28 kDa) with monophosphorylation. Moreover, different ionization and desolvation parameters did not impact phosphoprotein quantification. In contrast to monophosphorylation, multiphosphorylation noticeably affected ESI-MS quantification of phosphoproteins likely due to differential ionization/detection efficiency between unphosphorylated and phosphorylated proteoforms as shown in the pentakis-phosphorylated β-casein (24 kDa).

Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins-the proteins that constitute the contractile apparatuses. Top-down quantitative proteomics identified significant changes in the post-translational modifications (PTMs) of critical myofilament proteins in the fast-twitch skeletal muscles of aging rats, in accordance with the vulnerability of fast-twitch muscles to sarcopenia. Surprisingly, age-related alterations in the phosphorylation of Cypher isoforms, proteins that localize to the Z-discs in striated muscles, were also noted in the fast-twitch skeletal muscle of aging rats. This represents the first report of changes in the phosphorylation of Z-disc proteins in skeletal muscle during aging. In addition, increased glutathionylation of slow skeletal troponin I, a novel modification that may help protect against oxidative damage, was observed in slow-twitch skeletal muscles. Furthermore, we have identified and characterized novel muscle type-specific proteoforms of myofilament proteins and Z-disc proteins, including a novel isoform of the Z-disc protein Enigma. The finding that the phosphorylation of Z-disc proteins is altered in response to aging in the fast-twitch skeletal muscles of aging rats opens new avenues for the investigation of the role of Z-discs in age-related muscle dysfunction., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Myosin is the principal component of the thick filaments that, through interactions with the actin thin filaments, mediates force production during muscle contraction. Myosin is a hexamer, consisting of two heavy chains, each associated with an essential (ELC) and a regulatory (RLC) light chain, which bind the lever-arm of the heavy chain and play important modulatory roles in striated muscle contraction. Nevertheless, a comprehensive assessment of the sequences of the ELC and RLC isoforms, as well as their post-translational modifications, in the heart remains lacking. Herein, utilizing top-down high-resolution mass spectrometry (MS), we have comprehensively characterized the sequences and N-terminal modifications of the atrial and ventricular isoforms of the myosin light chains from human and swine hearts, as well as the sites of phosphorylation in the swine proteins. In addition to the correction of disparities in the database sequences of the swine proteins, we show for the first time that, whereas the ventricular isoforms of the ELC and RLC are methylated at their N-termini, which is consistent with previous studies, the atrial isoforms of the ELC and RLC from both human and swine are N α -methylated and N α -acetylated, respectively. Furthermore, top-down MS with electron capture dissociation enabled localization of the sites of phosphorylation in swine RLC isoforms from the ventricles and atria to Ser14 and Ser22, respectively. Collectively, these results provide new insights into the sequences and modifications of myosin light chain isoforms in the human and swine hearts, which will pave the way for a better understanding of their functional roles in cardiac physiology and pathophysiology., (Copyright © 2017. Published by Elsevier Ltd.)

Postinfarction remodeling and expansion of the peri-infarct border zone (BZ) directly correlate with mortality following myocardial infarction (MI); however, the cellular and molecular mechanisms underlying remodeling processes in the BZ remain unclear. Herein, we utilized a label-free quantitative proteomics approach in combination with immunohistochemical analyses to gain a better understanding of processes contributing to postinfarction remodeling of the peri-infarct BZ in a swine model of MI with reperfusion. Our analysis uncovered a significant down-regulation of proteins involved in energy metabolism, indicating impaired myocardial energetics and possibly mitochondrial dysfunction, in the peri-scar BZ. An increase in endothelial and vascular smooth muscles cells, as well as up-regulation of proteins implicated in vascular endothelial growth factor (VEGF) signaling and marked changes in the expression of extracellular matrix and subendothelial basement membrane proteins, is indicative of active angiogenesis in the infarct BZ. A pronounced increase in macrophages in the peri-infarct BZ was also observed, and proteomic analysis uncovered evidence of persistent inflammation in this tissue. Additional evidence suggested an increase in cellular proliferation that, concomitant with increased nestin expression, indicates potential turnover of endogenous stem cells in the BZ. A marked up-regulation of pro-apoptotic proteins, as well as the down-regulation of proteins important for adaptation to mechanical, metabolic, and oxidative stress, likely contributes to increased apoptosis in the peri-infarct BZ. The cellular processes and molecular pathways identified herein may have clinical utility for therapeutic intervention aimed at limiting remodeling and expansion of the BZ myocardium and preventing the development of heart failure post-MI.

Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia.

Introduction: Heart diseases are a leading cause of morbidity and mortality for both men and women worldwide, and impose significant economic burdens on the healthcare systems. Despite substantial effort over the last several decades, the molecular mechanisms underlying diseases of the heart remain poorly understood., Areas Covered: Altered protein post-translational modifications (PTMs) and protein isoform switching are increasingly recognized as important disease mechanisms. Top-down high-resolution mass spectrometry (MS)-based proteomics has emerged as the most powerful method for the comprehensive analysis of PTMs and protein isoforms. Here, we will review recent technology developments in the field of top-down proteomics, as well as highlight recent studies utilizing top-down proteomics to decipher the cardiac proteome for the understanding of the molecular mechanisms underlying diseases of the heart. Expert commentary: Top-down proteomics is a premier method for the global and comprehensive study of protein isoforms and their PTMs, enabling the identification of novel protein isoforms and PTMs, characterization of sequence variations, and quantification of disease-associated alterations. Despite significant challenges, continuous development of top-down proteomics technology will greatly aid the dissection of the molecular mechanisms underlying diseases of the hearts for the identification of novel biomarkers and therapeutic targets., Competing Interests: Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.

Top-down mass spectrometry (MS)-based proteomics is arguably a disruptive technology for the comprehensive analysis of all proteoforms arising from genetic variation, alternative splicing, and posttranslational modifications (PTMs). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis. In contrast to the well-developed software packages available for data analysis in bottom-up proteomics, the data analysis tools in top-down proteomics remain underdeveloped. Moreover, despite recent efforts to develop algorithms and tools for the deconvolution of top-down high-resolution mass spectra and the identification of proteins from complex mixtures, a multifunctional software platform, which allows for the identification, quantitation, and characterization of proteoforms with visual validation, is still lacking. Herein, we have developed MASH Suite Pro, a comprehensive software tool for top-down proteomics with multifaceted functionality. MASH Suite Pro is capable of processing high-resolution MS and tandem MS (MS/MS) data using two deconvolution algorithms to optimize protein identification results. In addition, MASH Suite Pro allows for the characterization of PTMs and sequence variations, as well as the relative quantitation of multiple proteoforms in different experimental conditions. The program also provides visualization components for validation and correction of the computational outputs. Furthermore, MASH Suite Pro facilitates data reporting and presentation via direct output of the graphics. Thus, MASH Suite Pro significantly simplifies and speeds up the interpretation of high-resolution top-down proteomics data by integrating tools for protein identification, quantitation, characterization, and visual validation into a customizable and user-friendly interface. We envision that MASH Suite Pro will play an integral role in advancing the burgeoning field of top-down proteomics., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The heart is characterized by a remarkable degree of heterogeneity, the basis of which is a subject of active investigation. Myofilament protein post-translational modifications (PTMs) represent a critical mechanism regulating cardiac contractility, and emerging evidence shows that pathological cardiac conditions induce contractile heterogeneity that correlates with transmural variations in the modification status of myofilament proteins. Nevertheless, whether there exists basal heterogeneity in myofilament protein PTMs in the heart remains unclear. Here we have systematically assessed chamber-specific and transmural variations in myofilament protein PTMs, specifically, the phosphorylation of cardiac troponin I (cTnI), cardiac troponin T (cTnT), tropomyosin (Tpm), and myosin light chain 2 (MLC2). We show that the phosphorylation of cTnI and αTm vary in the different chambers of the heart, whereas the phosphorylation of MLC2 and cTnT does not. In contrast, no significant transmural differences were observed in the phosphorylation of any of the myofilament proteins analyzed. These results highlight the importance of appropriate tissue sampling-particularly for studies aimed at elucidating disease mechanisms and biomarker discovery-in order to minimize potential variations arising from basal heterogeneity in myofilament PTMs in the heart., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Tissue proteomics is increasingly recognized for its role in biomarker discovery and disease mechanism investigation. However, protein solubility remains a significant challenge in mass spectrometry (MS)-based tissue proteomics. Conventional surfactants such as sodium dodecyl sulfate (SDS), the preferred surfactant for protein solubilization, are not compatible with MS. Herein, we have screened a library of surfactant-like compounds and discovered an MS-compatible degradable surfactant (MaSDeS) for tissue proteomics that solubilizes all categories of proteins with performance comparable to SDS. The use of MaSDeS in the tissue extraction significantly improves the total number of protein identifications from commonly used tissues, including tissue from the heart, liver, and lung. Notably, MaSDeS significantly enriches membrane proteins, which are often under-represented in proteomics studies. The acid degradable nature of MaSDeS makes it amenable for high-throughput MS-based proteomics. In addition, the thermostability of MaSDeS allows for its use in experiments requiring high temperature to facilitate protein extraction and solubilization. Furthermore, we have shown that MaSDeS outperforms the other MS-compatible surfactants in terms of overall protein solubility and the total number of identified proteins in tissue proteomics. Thus, the use of MaSDeS will greatly advance tissue proteomics and realize its potential in basic biomedical and clinical research. MaSDeS could be utilized in a variety of proteomics studies as well as general biochemical and biological experiments that employ surfactants for protein solubilization.

Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which requires effective enrichment of phosphoproteins. Here we have developed superparamagnetic nanoparticles (NPs) whose surface is functionalized by multivalent ligand molecules that specifically bind to the phosphate groups on any phosphoproteins. These NPs enrich phosphoproteins from complex cell and tissue lysates with high specificity as confirmed by SDS-PAGE analysis with a phosphoprotein-specific stain and mass spectrometry analysis of the enriched phosphoproteins. This method enables universal and effective capture, enrichment, and detection of intact phosphoproteins toward a comprehensive analysis of the phosphoproteome.

To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics.

Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography-mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The pathophysiology of heart failure (HF) is diverse, owing to multiple etiologies and aberrations in a number of cellular processes. Therefore, it is essential to understand how defects in the molecular pathways that mediate cellular responses to internal and external stressors function as a system to drive the HF phenotype. Mass spectrometry (MS)-based proteomics strategies have great potential for advancing our understanding of disease mechanisms at the systems level because proteins are the effector molecules for all cell functions and, thus, are directly responsible for determining cell phenotype. Two MS-based proteomics strategies exist: peptide-based bottom-up and protein-based top-down proteomics--each with its own unique strengths and weaknesses for interrogating the proteome. In this review, we will discuss the advantages and disadvantages of bottom-up and top-down MS for protein identification, quantification, and analysis of post-translational modifications, as well as highlight how both of these strategies have contributed to our understanding of the molecular and cellular mechanisms underlying HF. Additionally, the challenges associated with both proteomics approaches will be discussed and insights will be offered regarding the future of MS-based proteomics in HF research.

Proteomics is essential for deciphering how molecules interact as a system and for understanding the functions of cellular systems in human disease; however, the unique characteristics of the human proteome, which include a high dynamic range of protein expression and extreme complexity due to a plethora of PTMs and sequence variations, make such analyses challenging. An emerging "top-down" MS-based proteomics approach, which provides a "bird's eye" view of all proteoforms, has unique advantages for the assessment of PTMs and sequence variations. Recently, a number of studies have showcased the potential of top-down proteomics for the unraveling of disease mechanisms and discovery of new biomarkers. Nevertheless, the top-down approach still faces significant challenges in terms of protein solubility, separation, and the detection of large intact proteins, as well as underdeveloped data analysis tools. Consequently, new technological developments are urgently needed to advance the field of top-down proteomics. Herein, we intend to provide an overview of the recent applications of top-down proteomics in biomedical research. Moreover, we will outline the challenges and opportunities facing top-down proteomics strategies aimed at understanding and diagnosing human diseases., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The rapid advancements in mass spectrometry (MS) instrumentation, particularly in Fourier transform (FT) MS, have made the acquisition of high-resolution and high-accuracy mass measurements routine. However, the software tools for the interpretation of high-resolution MS data are underdeveloped. Although several algorithms for the automatic processing of high-resolution MS data are available, there is still an urgent need for a user-friendly interface with functions that allow users to visualize and validate the computational output. Therefore, we have developed MASH Suite, a user-friendly and versatile software interface for processing high-resolution MS data. MASH Suite contains a wide range of features that allow users to easily navigate through data analysis, visualize complex high-resolution MS data, and manually validate automatically processed results. Furthermore, it provides easy, fast, and reliable interpretation of top-down, middle-down, and bottom-up MS data. MASH Suite is convenient, easily operated, and freely available. It can greatly facilitate the comprehensive interpretation and validation of high-resolution MS data with high accuracy and reliability.

The formation of disulfide bonds between cysteine residues is crucial for the stabilization of native protein structures and, thus, determination of disulfide linkages is an important facet of protein structural characterization. Nonetheless, the identification of disulfide bond linkages remains a significant analytical challenge, particularly in large proteins with complex disulfide patterns. Herein, we have developed a new LC/MS strategy for rapid screening of disulfides in an intact protein mixture after a straightforward reduction step with tris(2-carboxyethyl)phosphine. LC/MS analysis of reduced and nonreduced protein mixtures quickly revealed disulfide-containing proteins owing to a 2 Da mass increase per disulfide reduction and, subsequently, the total number of disulfide bonds in the intact proteins could be determined. We have demonstrated the effectiveness of this method in a protein mixture composed of both disulfide-containing and disulfide-free proteins. Our method is simple (no need for proteolytic digestion, alkylation, or the removal of reducing agents prior to MS analysis), high throughput (fast on-line LC/MS analysis), and reliable (no S-S scrambling), underscoring its potential as a rapid disulfide screening method for proteomics applications., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)