Titin: roles in cardiac function and diseases.

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.

Mechanisms of RBM20 Cardiomyopathy: Insights From Model Systems.

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.

Top-down proteomics of myosin light chain isoforms define chamber-specific expression in the human heart.

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.

High sensitivity top-down proteomics captures single muscle cell heterogeneity in large proteoforms.

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.

Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy.

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.

Top-down Proteomics of Myosin Light Chain Isoforms Define Chamber-Specific Expression in the Human Heart.

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, 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 and MLC-2a 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. Overall, top-down proteomics allowed us an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs.

Caveolin-3 and Caveolae regulate ventricular repolarization.

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 QTc 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 (Ito, Iss) and changes in inward currents including slowed inactivation of ICa,L and increased INa,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.

SR Protein Kinases Regulate the Splicing of Cardiomyopathy-Relevant Genes via Phosphorylation of the RSRSP Stretch in RBM20.

(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.

Rbm20 ablation is associated with changes in the expression of titin-interacting and metabolic proteins.

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.

Cardiac differentiation of human pluripotent stem cells using defined extracellular matrix proteins reveals essential role of fibronectin.

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 α4ß1 or αVß1, but not α5ß1, 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.

Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics.

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.

Top-Down Proteomics Reveals Myofilament Proteoform Heterogeneity among Various Rat Skeletal Muscle Tissues.

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.

An Unbiased Proteomics Method to Assess the Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

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 Ca2+ 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.

Deletion of Enigma Homologue from the Z-disc slows tension development kinetics in mouse myocardium.

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 ß-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.

Temperature-sensitive sarcomeric protein post-translational modifications revealed by top-down proteomics.

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.

Comprehensive Characterization of Swine Cardiac Troponin T Proteoforms by Top-Down Mass Spectrometry.

Cardiac troponin T (cTnT) regulates the Ca2+-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.

Impact of Phosphorylation on the Mass Spectrometry Quantification of Intact Phosphoproteins.

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 ß-casein (24 kDa).

Large Cardiac Muscle Patches Engineered From Human Induced-Pluripotent Stem Cell-Derived Cardiac Cells Improve Recovery From Myocardial Infarction in Swine.

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.

Novel Sarcopenia-related Alterations in Sarcomeric Protein Post-translational Modifications (PTMs) in Skeletal Muscles Identified by Top-down Proteomics.

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.

Distinct sequences and post-translational modifications in cardiac atrial and ventricular myosin light chains revealed by top-down mass spectrometry.

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 Nα-methylated and Nα-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.