Biology subjects

Sadayappan, S.

Publications and source records attributed to Sadayappan, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Rewiring of 3D Enhancer-Promoter Interactome Underlies Diabetic Endothelial Dysfunction

BackgroundDiabetic vascular complications are driven by endothelial dysfunction, yet the role of 3D genome organization in this process is unknown. We sought to define the alterations in chromatin architecture in diabetic endothelium and identify the key regulators involved. MethodsWe generated a high-resolution 3D epigenomic atlas of diabetic endothelial cells from mouse models and human subjects using H3K27ac HiChIP, complemented by ChIP-seq, ATAC-seq, and RNA-seq. A human cohort was used to assess protein expression in diabetic versus non-diabetic endothelial cells. To identify JUNB-interacting proteins, we performed rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME), with protein-protein interaction validated by co-immunoprecipitation. Functional validation was performed using in vitro, ex vivo, and in vivo approaches, including endothelial-specific knockdown in a diabetic hindlimb ischemia model. ResultsMulti-omics profiling revealed extensive enhancer reprogramming in diabetic endothelium, with AP-1 binding motifs being consistently and selectively enriched in downregulated enhancers across three distinct diabetic models. Analysis of a human cohort confirmed significantly reduced JUNB protein levels in diabetic endothelial cells. We identified widespread disruption of JUNB-anchored enhancer-promoter interactions, which underlies transcriptional repression of key endothelial genes. RIME and co-immunoprecipitation established the E3 ubiquitin ligase RBBP6 as a direct JUNB interactor that promotes its polyubiquitination and proteasomal degradation in response to hyperglycemia. Human cohort analysis further showed reciprocal elevation of RBBP6 in diabetic endothelial cells. Either JUNB overexpression or RBBP6 knockdown restored enhancer-promoter connectivity, reactivated vasoprotective transcriptional programs, and rescued endothelial function. Critically, endothelial-specific knockdown of Rbbp6 in diabetic mice restored endothelium-dependent vasorelaxation and improved perfusion recovery after hindlimb ischemia, independent of systemic glucose levels. ConclusionsOur study unveils a novel mechanism whereby hyperglycemia induces enhancer reprogramming and disrupts endothelial 3D genome architecture through RBBP6-mediated degradation of JUNB. The RBBP6-JUNB axis is established as a crucial link between metabolic stress and epigenomic reprogramming in vascular disease, presenting a promising therapeutic target for diabetic vasculopathy.

genomics↗

bioRxiv2025

Modulating Cardiac-Gut Microbiome Interaction Post-Myocardial Infarction with Engineered Bacteria

The gut microbiome plays a critical role in the pathophysiology of acute myocardial infarction (MI). MI events significantly impact intestinal integrity which results in leakage of bacterial products into the systemic circulation. We demonstrate that MI not only compromises intestinal integrity, leading to systemic leakage of bacterial products like LPS, but also results in the translocation and colonization of live, intact gut bacteria in the MI heart - a novel aspect of the heart-gut axis. Our initial findings with natural murine gut microbiome were substantiated using orally administered E. coli Nissle 1917 (EcN), as a tracer bacterium. Furthermore, we engineered EcN to express the microbial anti-inflammatory molecule (MAM) derived from the probiotic Faecalibacterium prausnitzii. Treatment with this engineered strain, EcN-MAM, led to significantly improved survival and cardiac function in MI mice. This was attributed to enhanced gut barrier integrity, resulting in reduced systemic bacterial permeation and subsequent inflammation. These findings shed light on a previously unrecognized dimension of the heart-gut axis and highlight the potential of microbiome-based interventions in MI management.

synthetic biology↗

bioRxiv2024

In-silico heart model phantom to validate cardiac strain imaging

The quantification of cardiac strains as structural indices of cardiac function has a growing prevalence in clinical diagnosis. However, the highly heterogeneous four-dimensional (4D) cardiac motion challenges accurate "regional" strain quantification and leads to sizable differences in the estimated strains depending on the imaging modality and post-processing algorithm, limiting the translational potential of strains as incremental biomarkers of cardiac dysfunction. There remains a crucial need for a feasible benchmark that successfully replicates complex 4D cardiac kinematics to determine the reliability of strain calculation algorithms. In this study, we propose an in-silico heart phantom derived from finite element (FE) simulations to validate the quantification of 4D regional strains. First, as a proof-of-concept exercise, we created synthetic magnetic resonance (MR) images for a hollow thick-walled cylinder under pure torsion with an exact solution and demonstrated that "ground-truth" values can be recovered for the twist angle, which is also a key kinematic index in the heart. Next, we used mouse-specific FE simulations of cardiac kinematics to synthesize dynamic MR images by sampling various sectional planes of the left ventricle (LV). Strains were calculated using our recently developed non-rigid image registration (NRIR) framework in both problems. Moreover, we studied the effects of image quality on distorting regional strain calculations by conducting in-silico experiments for various LV configurations. Our studies offer a rigorous and feasible tool to standardize regional strain calculations to improve their clinical impact as incremental biomarkers.

bioengineering↗

bioRxiv2024

A Multi-Omics Approach to Defining Target Organ Injury in Youth with Primary Hypertension

BACKGROUNDPrimary hypertension in childhood tracks into adulthood and may be associated with increased cardiovascular risk. Studies conducted in children and adolescents provide an opportunity to explore the early cardiovascular target organ injury (CV-TOI) in a population free from many of the co-morbid cardiovascular disease risk factors that confound studies in adults. METHODSYouths (n=132, mean age 15.8 years) were stratified by blood pressure (BP) as low, elevated, and high-BP and by left ventricular mass index (LVMI) as low- and high-LVMI. Systemic circulating RNA, miRNA, and methylation profiles in peripheral blood mononuclear cells and deep proteome profiles in serum were determined using high-throughput sequencing techniques. RESULTSVASH1 gene expression was elevated in youths with high-BP with and without high-LVMI. VASH1 expression levels positively correlated with systolic BP (r=0.3143, p=0.0034). The expression of hsa-miR-335-5p, one of the VASH1-predicted miRNAs, was downregulated in high-BP with high-LVMI youths and was inversely correlated with systolic BP (r=-0.1891, p=0.0489). GSE1 hypermethylation, circulating PROZ upregulation (log2FC=0.61, p=0.0049 and log2FC=0.62, p=0.0064), and SOD3 downregulation (log2FC=-0.70, p=0.0042 and log2FC=-0.64, p=0.010) were observed in youths with elevated BP and high-BP with high-LVMI. Comparing the transcriptomic and proteomic profiles revealed elevated HYAL1 levels in youths displaying high-BP and high-LVMI. CONCLUSIONSThe findings are compatible with a novel blood pressure-associated mechanism that may occur through impaired angiogenesis and extracellular matrix degradation through dysregulation of Vasohibin-1 and Hyaluronidase1 was identified as a possible mediator of CV-TOI in youth with high-BP and suggests strategies for ameliorating TOI in adult-onset primary hypertension.

genomics↗

bioRxiv2024

Complete spatiotemporal quantification of cardiac motion in mice through enhanced acquisition and super-resolution reconstruction

The quantification of cardiac motion using cardiac magnetic resonance imaging (CMR) has shown promise as an early-stage marker for cardiovascular diseases. Despite the growing popularity of CMR-based myocardial strain calculations, measures of complete spatiotemporal strains (i.e., three-dimensional strains over the cardiac cycle) remain elusive. Complete spatiotemporal strain calculations are primarily hampered by poor spatial resolution, with the rapid motion of the cardiac wall also challenging the reproducibility of such strains. We hypothesize that a super-resolution reconstruction (SRR) framework that leverages combined image acquisitions at multiple orientations will enhance the reproducibility of complete spatiotemporal strain estimation. Two sets of CMR acquisitions were obtained for five wild-type mice, combining short-axis scans with radial and orthogonal long-axis scans. Super-resolution reconstruction, integrated with tissue classification, was performed to generate full four-dimensional (4D) images. The resulting enhanced and full 4D images enabled complete quantification of the motion in terms of 4D myocardial strains. Additionally, the effects of SRR in improving accurate strain measurements were evaluated using an in-silico heart phantom. The SRR framework revealed near isotropic spatial resolution, high structural similarity, and minimal loss of contrast, which led to overall improvements in strain accuracy. In essence, a comprehensive methodology was generated to quantify complete and reproducible myocardial deformation, aiding in the much-needed standardization of complete spatiotemporal strain calculations.

bioengineering↗

bioRxiv2024

MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids

BACKGROUNDMYBPC3, encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). METHODSThe hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro. RESULTSConfocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS(R)) in MYBPC3D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONSConceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIMYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. C_LIO_LID389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. C_LIO_LIThe molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined. C_LI What New Information Does This Article Contribute?O_LIThe authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. C_LIO_LIThe MYBPC3D389V-mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. C_LIO_LIOur data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS(R)), a myosin inhibitor. C_LIO_LIIn addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m), highlighting a possible early adaptive response to primary sarcomeric changes. C_LIO_LIEarly treatment of MYBPC3D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. C_LI GRAPHICAL ABSTRACTA graphical abstract is available for this article. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/596463v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1489301org.highwire.dtl.DTLVardef@1ab60c1org.highwire.dtl.DTLVardef@5d49d1org.highwire.dtl.DTLVardef@9944d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

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 StatementRecently, 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.

physiology↗

bioRxiv2023

Unlocking the Role of sMyBP-C: A Key Player in Skeletal Muscle Development and Growth

Skeletal muscle is the largest organ in the body, responsible for gross movement and metabolic regulation. Recently, variants in the MYBPC1 gene have been implicated in a variety of developmental muscle diseases, such as distal arthrogryposis. How MYBPC1 variants cause disease is not well understood. Here, through a collection of novel gene-edited mouse models, we define a critical role for slow myosin binding protein-C (sMyBP-C), encoded by MYBPC1, across muscle development, growth, and maintenance during prenatal, perinatal, postnatal and adult stages. Specifically, Mybpc1 knockout mice exhibited early postnatal lethality and impaired skeletal muscle formation and structure, skeletal deformity, and respiratory failure. Moreover, a conditional knockout of Mybpc1 in perinatal, postnatal and adult stages demonstrates impaired postnatal muscle growth and function secondary to disrupted actomyosin interaction and sarcomere structural integrity. These findings confirm the essential role of sMyBP-C in skeletal muscle and reveal specific functions in both prenatal embryonic musculoskeletal development and postnatal muscle growth and function.

physiology↗

bioRxiv2023

Fast myosin binding protein C knockout in skeletal muscle alters length-dependent activation and myofilament structure.

In striated muscle, some sarcomere proteins regulate crossbridge cycling by varying the propensity of myosin heads to interact with actin. Myosin-binding protein C (MyBP-C) is bound to the myosin thick filament and is predicted to interact and stabilize myosin heads in a docked position against the thick filament and limit crossbridge formation, the so-called OFF state. Via an unknown mechanism, MyBP-C is thought to release heads into the so-called ON state, where they are more likely to form crossbridges. To study this proposed mechanism, we used the C2-/- mouse line to knock down fast-isoform MyBP-C completely and total MyBP-C by [~]24%, and conducted mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers presented deficits in force production and reduced calcium sensitivity. Structurally, passive C2-/- fibers presented altered SL-independent and SL-dependent regulation of myosin head ON/OFF states, with a shift of myosin heads towards the ON state. Unexpectedly, at shorter sarcomere lengths, the thin filament was axially extended in C2-/- vs. non-transgenic controls, which we postulate is due to increased low-level crossbridge formation arising from relatively more ON myosins in the passive muscle that elongates the thin filament. The downstream effect of increasing crossbridge formation in a passive muscle on contraction performance is not known. Such widespread structural changes to sarcomere proteins provide testable mechanisms to explain the etiology of debilitating MyBP-C-associated diseases.

physiology↗

bioRxiv2023

The structural OFF and ON states of myosin can be decoupled from the biochemical super-relaxed and disordered-relaxed states.

There is a growing awareness that both thick filament and classical thin filament regulation play central roles in modulating muscle contraction. Myosin ATPase assays have demonstrated that under relaxed conditions, myosin may reside in either a high energy-consuming disordered-relaxed (DRX) state available for binding actin to generate force, or in an energy-sparing super-relaxed (SRX) state unavailable for actin binding. X-ray diffraction studies have shown the majority of myosin heads are in a quasi-helically ordered OFF state in a resting muscle and that this helical ordering is lost when myosin heads are turned ON for contraction. It has been assumed that myosin heads in SRX and DRX states are equivalent to the OFF and ON state respectively and the terms have been used interchangeably. Here, we use X-ray diffraction and ATP turnover assays to track the structural and biochemical transitions of myosin heads respectively induced with either omecamtiv mecarbil (OM) or piperine in relaxed porcine myocardium. We find that while OM and piperine induce dramatic shifts of myosin heads from the OFF to ON states, there are no appreciable changes in the population of myosin heads in the SRX and DRX states in both unloaded and loaded preparations. Our results show that biochemically defined SRX and DRX can be decoupled from structurally-defined OFF and ON states. In summary, while SRX/DRX and OFF/ON transitions can be correlated in some cases, these two phenomena are measured using different approaches, do not necessarily reflect the same properties of the thick filament and should be investigated and interpreted separately. SignificanceMyosin based thick filament regulation is now known to be critical for muscle contraction with myosin dysregulation found in hypertrophic and dilated cardiomyopathies. While previously thought to be synonymous, this study finds that biochemical and structural thick filament disengagement are distinct properties and should be investigated as independent phenomena. Understanding the details of thick filament regulation will be of great relevance to defining sarcomere-level dysfunction in myopathies and understanding and better designing and testing sarcomere therapies aimed at reversing them for treatment of cardiomyopathy.

biophysics↗

bioRxiv2023

BRD9-SMAD2/3 orchestrates stemness and tumorigenesis in pancreatic ductal adenocarcinoma

The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodelling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGF{beta}/Activin-SMAD2/3 signalling pathway. Inhibition and genetic ablation of BDR9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumours from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

cancer biology↗

bioRxiv2022

Myosin-Binding Protein C Stabilizes, But Is Not the Sole Determinant of SRX Myosin in Cardiac Muscle

The myosin Super Relaxed (SRX) state is central to striated muscle metabolic and functional regulation. In skeletal muscle, SRX myosin are predominantly colocalized with Myosin-Binding Protein C (MyBP-C) in the C-zone, proximal to the sarcomere center. To define how MyBP-C and its specific domains contribute to stabilizing the SRX state in cardiac muscle, we took advantage of transgenic MyBP-C null mice and those expressing MyBP-C with a 271 residue N-terminal truncation. Utilizing super-resolution microscopy, we determined the lifetime and sub-sarcomeric location of individual fluorescent-ATP turnover events within isolated cardiac myofibrils. The proportion of SRX myosin was highest in the C-zone (71{+/-}6%) and lower in the D-zone (45{+/-}10%) which lies farther from the sarcomere center and lacks MyBP-C, suggesting a possible role for MyBP-C in stabilizing the SRX state within the C-zone. However, myofibrils from MyBP-C null mice demonstrated an ~40% SRX reduction, not only within the now MyBP-C-free C-zone (49{+/-}9% SRX), but also within the D-zone (22{+/-}5% SRX). These data suggest that the influence of MyBP-C on the SRX state is not limited to the C-zone, but extends along the thick filament. Interestingly, myofibrils with N-terminal truncated MyBP-C had an SRX content and spatial gradient similar to the MyBP-C null, indicating that the N terminus of cardiac MyBP-C is necessary for MyBP-Cs role in establishing SRX along the entire thick filament. Given that SRX myosin are enriched in the C-zone, even in the absence of MyBP-C or its N-terminus, an inherent bias must exist in the structure of the thick filament to stabilize the SRX state. One candidate may be a differential in the super repeats of titin that interact with MyBP-C and myosin and which template the thick filament.

physiology↗

bioRxiv2022

Knockout of Sorbs2 in Cardiomyocytes Leads to Dilated Cardiomyopathy in Mice

2.RationaleSorbs2 is a cardiomyocyte-enriched, cytoskeletal adaptor protein, and given some evidence for its dysregulated expression in failing hearts, there is growing interest in understanding its roles in cardiac biology and disease. While Sorbs2 global knockout mice display lethal cardiomyopathy with severe arrhythmias, the underlying mechanisms remain unclear, and whether this results from intrinsic loss of Sorbs2 in cardiomyocytes is unknown, as Sorbs2 is also well-expressed in the nervous system and vasculature. In addition, the potential relevance of Sorbs2 in human cardiomyopathy remains underexplored. ObjectiveTo characterize the effects and potential underlying mechanisms of cardiomyocyte- specific deletion of Sorbs2 on cardiac structure and function in mice, and to further examine Sorbs2 dysregulation in failing hearts and explore potential links between Sorbs2 genetic variations and human cardiovascular disease phenotypes. Methods and ResultsWe report that myocardial Sorbs2 expression is consistently upregulated in humans with ischemic and idiopathic cardiomyopathies, and in experimental animal models of heart failure (HF). We generated mice with cardiomyocyte-specific loss of Sorbs2 (Sorbs2-cKO) and found early atrial and ventricular conduction abnormalities, despite unaltered expression of primary action potential ion channels and gap junction proteins. At mid-life, Sorbs2-cKO mice exhibit impaired cardiac contractility with cardiomyofibers failing to maintain adequate mechanical tension. As a result, these mice develop progressive diastolic and systolic dysfunction, enlarged cardiac chambers, and die with congestive HF at approximately one year of age. Comprehensive survey of potential underlying mechanisms revealed that Sorbs2-cKO hearts exhibit defective microtubule polymerization and compensatory upregulation of structural proteins desmin, vinculin, and tubulins. Finally, consistent with our observations in mice, we identified suggestive links between Sorbs2 genetic variants and related human cardiac phenotypes, including conduction abnormalities, atrial enlargement, and dilated cardiomyopathy. ConclusionsOur studies show that Sorbs2 is essential for maintaining cytoskeletal structural integrity in cardiomyocytes likely through strengthening the interactions between microtubules and other structural proteins at crosslink sites. Overall, this study provides key insights into the critical role for Sorbs2 in cardiomyocytes and likely other cell types in maintaining normal cardiac structure and function and highlights its potential clinical relevance.

physiology↗

bioRxiv2020

Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis

Recent interest in understanding cardiomyocyte cell-cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow abscission of daughter cardiomyocytes. Here we identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. /{gamma}-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phosphoforms of -adducin in-vitro and in-vivo identified Thr445/Thr480 phosphorylation of a short isoform of adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this -adducin variant along with {gamma}-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with -actinin. These results highlight an important mechanism for coordination of cytoskeletal morphological changes during cardiomyocyte mitosis.

cell biology↗

bioRxiv2019

SKELETAL MyBP-C ISOFORMS TUNE THE MOLECULAR CONTRACTILITY OF DIVERGENT SKELETAL MUSCLE SYSTEMS

Skeletal muscle myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein; localized through its C terminus to distinct regions (C-zones) of the sarcomere. MyBP-C modulates muscle contractility, presumably through its N terminus extending from the thick filament and interacting with either the myosin head region and/or the actin thin filament. Two isoforms of MyBP-C (fast- and slow-type) are expressed in a muscle-type specific manner. Are the expression, localization, and Ca2+-dependent modulatory capacities of these isoforms different in fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles derived from Sprague-Dawley rats? By mass spectrometry, four MyBP-C isoforms (one fast-type MyBP-C and three N-terminally spliced slow-type MyBP-C) were expressed in EDL but only the three slow-type MyBP-C isoforms in SOL. Using EDL and SOL native thick filaments in which the MyBP-C stoichiometry and localization are preserved, native thin filament sliding over these thick filaments showed that only in the C-zone, MyBP-C Ca2+-sensitizes the thin filament and slows thin filament velocity. These modulatory properties depended on MyBP-Cs N-terminus, as N-terminal proteolysis attenuated MyBP-Cs functional capacities. To determine each MyBP-C isoforms contribution to thin filament Ca2+-sensitization and slowing in the C-zone, we used a combination of in vitro motility assays using expressed recombinant N-terminal fragments and in silico mechanistic modeling. Our results suggest that each skeletal MyBP-C isoforms N terminus is functionally distinct and has modulatory capacities that depend on the muscle-type in which they are expressed, providing the potential for molecular tuning of skeletal muscle performance through differential MyBP-C expression.\n\nSIGNIFICANCEMyosin-binding protein C (MyBP-C) is a critical component of the skeletal muscle sarcomere, muscles smallest contractile unit. MyBP-Cs importance is evident by genetic mutations leading to human myopathies, such as distal arthrogryposis (i.e. club foot). However, the molecular basis of MyBP-Cs functional impact on skeletal muscle contractility is far from certain. Complicating matters further is the expression of fast- and slow-type MyBP-C isoforms that depend on whether the muscle is fast- or slow-twitch. Using multi-scale proteomic, biophysical and mathematical modeling approaches, we define the expression, localization, and modulatory capacities of these distinct skeletal MyBP-C isoforms in rat skeletal muscles. Each MyBP-C isoform appears to modulate muscle contractility differentially; providing the capacity to fine-tune muscles mechanical performance as physiological demands arise.

physiology↗

bioRxiv2018

An acute immune response underlies the benefit of cardiac adult stem cell therapy

Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day1-3 despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biologic effect4-6. The rationale for these cell therapy trials is derived from animal studies that show a modest but reproducible improvement in cardiac function in models of cardiac ischemic injury7-9. Here we examined the mechanistic basis for cell therapy in mice after ischemia/reperfusion (I/R) injury, and while heart function was enhanced, it was not associated with new cardiomyocyte production. Cell therapy improved heart function through an acute sterile immune response characterized by the temporal and regional induction of CCR2+ and CX3CR1+ macrophages. Here we observed that intra-cardiac injection of 2 distinct types of progenitor cells, freeze/thaw-killed cells or a chemical inducer of the innate immune response similarly induced regional CCR2+ and CX3CR1+ macrophage accumulation and provided functional rejuvenation to the I/R-injured heart. Mechanistically, this selective macrophage response altered cardiac fibroblast activity and reduced border zone extracellular matrix (ECM) content and enhanced the mechanical properties of the injured area. The functional benefit of cardiac cell therapy is thus due to an acute inflammatory-based wound healing response that rejuvenates the mechanical properties of the infarcted area of the heart. Such results suggest a re-evaluation of strategies underlying cardiac cell therapy in current and planned human clinical trials.

cell biology↗

bioRxiv2018

Mitogen-Activated Protein Kinase-Dependent Fiber-Type Regulation in Skeletal Muscle

Mitogen-activated protein kinases (MAPK) are conserved protein kinases that regulate a diverse array of cellular activities. Stress or mitogenic signals activate three primary branches of the greater MAPK cascade, each of which consists of a phosphorylation-dependent array of successively acting kinases. The extracellular signal-regulated kinase 1/2 (ERK1/2) branch is regulated by growth factory signaling at the cell membrane, leading to phosphorylation of the dual-specificity kinase MEK1, which is dedicated to ERK1/2 phosphorylation. Previous studies have established a link between MAPK activation and endurance exercise, but whether a single MAPK is responsible for establishing muscle metabolic fate is unclear. Using mouse genetics we observed that muscle-specific expression of a constitutively active MEK1 promotes greater ERK1/2 signaling that mediates fiber-type switching in mouse skeletal muscle to a slow, oxidative phenotype with type I myosin heavy chain expression. Induced expression of the activated MEK1 mutant using either a MyoD-Cre or myosin light chain-Cre strategy equally increased the number of type I fibers in skeletal muscle with significantly reduced size compared to controls. Moreover, activation of MEK1 in mature myofibers of an adult mouse using a transgene containing a tamoxifen inducible MerCreMer cDNA under the control of a skeletal -actin promoter produced a similar phenotype of switching towards a slow-oxidative program. Physiologic assessment of mice with greater skeletal muscle slow-oxidative fibers showed enhanced metabolic activity and oxygen consumption with greater fatigue resistance of individual muscles. In summary, these results show that sustained MEK1-ERK1/2 activity in skeletal muscle produces a fast-to-slow fiber-type switch, suggesting that modulation of this signaling pathway may represent a therapeutic approach to enhance the long-term metabolic effectiveness of muscle in vivo.

developmental biology↗

bioRxiv2018

Krüppel like factor 2 - deficient myeloid cells promote skeletal muscle regeneration after injury

Regeneration of adult skeletal muscle after injury is coordinated by complex interactions between the injured muscle and the innate immune system. Myeloid lineage cells predominate in this process. This study examined the role of Kruppel - like factor 2 (KLF2), a zinc-finger transcription factor that regulates myeloid cell activation state, in muscle regeneration. Gastrocnemius muscles of wild-type and myeKlf2-/- mice, which lack KLF2 in all myeloid cells, were subjected to cardiotoxin injury and followed for 21 days. Injured muscles of myeKlf2-/- contained more infiltrating, inflammatory Ly6C+ monocytes, with elevated expression of inflammatory mediators. Infiltrating monocytes matured earlier into pro-inflammatory macrophages with phenotype Ly6C+, CD11b+, F4/80+. Inflammation resolved earlier and progressed to myogenesis, marked by an earlier decline of Ly6C+ macrophages and their replacement with anti-inflammatory Ly6C- populations, in association with elevated expression of factors that resolve inflammation and promote myogenesis. Overall, regeneration was completed earlier. These findings identify myeloid KLF2 as a central regulator of the innate immune response to acute skeletal muscle injury. Manipulating myeloid KLF2 levels may be a useful strategy for accelerating regeneration.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY SynopsisThe zinc-finger transcription factor, KLF2, is a central regulator of the innate immune response to skeletal muscle injury. Targeted deletion of KLF2 in myeloid lineage cells of mice (myeKlf2-/-) enhances the immune response and, notably, accelerates muscle repair. O_LIInjured muscles of myeKlf2-/- mice recruit greater numbers of Ly6C+ (inflammatory) monocytes from the circulation.\nC_LIO_LIThese mature in situ into pro-inflammatory macrophages which phagocytose necrotic tissue and prepare the environment for muscle regeneration.\nC_LIO_LISubsequently, Ly6C+ macrophages decline and are replaced by anti-inflammatory (Ly6C-) macrophages that promote myogenesis of new fibers.\nC_LIO_LIInjured muscles of myeKlf2-/- mice complete regeneration earlier, with phenotypically adult fibers.\nC_LI\n\nC_FIG_DISPLAY

immunology↗