Adducin Regulates Sarcomere Disassembly During Cardiomyocyte Mitosis.

BACKGROUND: 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 the abscission of daughter cardiomyocytes. METHODS: Here, we use a proteomics screen to identify adducin, an actin capping protein previously not studied in cardiomyocytes, as a regulator of sarcomere disassembly. We generated many adeno-associated viruses and cardiomyocyte-specific genetic gain-of-function models to examine the role of adducin in neonatal and adult cardiomyocytes in vitro and in vivo. RESULTS: We identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phospho-isoforms of α-adducin in identified Thr445/Thr480 phosphorylation of a short isoform of α-adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin. CONCLUSIONS: These results highlight an important mechanism for coordinating cytoskeletal morphological changes during cardiomyocyte mitosis.

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

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

Macrophage-enriched Sectm1a promotes efficient efferocytosis to attenuate ischemia/reperfusion-induced cardiac injury.

Efficient clearance and degradation of apoptotic cardiomyocytes by macrophages (collectively termed efferocytosis) is critical for inflammation resolution and restoration of cardiac function after myocardial ischemia/reperfusion (I/R). Here, we define secreted and transmembrane protein 1a (Sectm1a), a cardiac macrophage-enriched gene, as a modulator of macrophage efferocytosis in I/R-injured hearts. Upon myocardial I/R, Sectm1a-KO mice exhibited impaired macrophage efferocytosis, leading to massive accumulation of apoptotic cardiomyocytes, cardiac inflammation, fibrosis, and consequently, exaggerated cardiac dysfunction. By contrast, therapeutic administration of recombinant SECTM1A protein significantly enhanced macrophage efferocytosis and improved cardiac function. Mechanistically, SECTM1A could elicit autocrine effects on the activation of glucocorticoid-induced TNF receptor (GITR) at the surface of macrophages, leading to the upregulation of liver X receptor α (LXRα) and its downstream efferocytosis-related genes and lysosomal enzyme genes. Our study suggests that Sectm1a-mediated activation of the Gitr/LXRα axis could be a promising approach to enhance macrophage efferocytosis for the treatment of myocardial I/R injury.

Hypertrophic cardiomyopathy in MYBPC3 carriers in aging.

Hypertrophic cardiomyopathy (HCM) is characterized by abnormal thickening of the myocardium, leading to arrhythmias, heart failure, and elevated risk of sudden cardiac death, particularly among the young. This inherited disease is predominantly caused by mutations in sarcomeric genes, among which those in the cardiac myosin binding protein-C3 (MYBPC3) gene are major contributors. HCM associated with MYBPC3 mutations usually presents in the elderly and ranges from asymptomatic to symptomatic forms, affecting numerous cardiac functions and presenting significant health risks with a spectrum of clinical manifestations. Regulation of MYBPC3 expression involves various transcriptional and translational mechanisms, yet the destiny of mutant MYBPC3 mRNA and protein in late-onset HCM remains unclear. Pathogenesis related to MYBPC3 mutations includes nonsense-mediated decay, alternative splicing, and ubiquitin-proteasome system events, leading to allelic imbalance and haploinsufficiency. Aging further exacerbates the severity of HCM in carriers of MYBPC3 mutations. Advancements in high-throughput omics techniques have identified crucial molecular events and regulatory disruptions in cardiomyocytes expressing MYBPC3 variants. This review assesses the pathogenic mechanisms that promote late-onset HCM through the lens of transcriptional, post-transcriptional, and post-translational modulation of MYBPC3, underscoring its significance in HCM across carriers. The review also evaluates the influence of aging on these processes and MYBPC3 levels during HCM pathogenesis in the elderly. While pinpointing targets for novel medical interventions to conserve cardiac function remains challenging, the emergence of personalized omics offers promising avenues for future HCM treatments, particularly for late-onset cases.

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

There is a growing awareness that both thick-filament and classical thin-filament regulations play central roles in modulating muscle contraction. Myosin ATPase assays have demonstrated that under relaxed conditions, myosin may reside either in 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 that 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 states, respectively, and the terms have been used interchangeably. In this study, 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 the ON state, 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, reflect different properties of the thick filament, and should be investigated and interpreted separately.

Dynamic changes in mitral valve extracellular matrix, tissue mechanics and function in a mouse model of Marfan syndrome.

OBJECTIVE: Mouse models of Marfan syndrome (MFS) with Fibrillin 1 (Fbn1) variant C1041G exhibit cardiovascular abnormalities, including myxomatous valve disease (MVD) and aortic aneurism, with structural extracellular matrix (ECM) dysregulation. In this study, we examine the structure-function-mechanics relations of the mitral valve related to specific transitions in ECM composition and organization in progressive MVD in MFS mice from Postnatal day (P)7 to 1 year-of-age. APPROACH AND RESULTS: Mechanistic links between mechanical forces and biological changes in MVD progression were examined in Fbn1C1041G/+ MFS mice. By echocardiography, mitral valve dysfunction is prevalent at 2 months with a decrease in cardiac function at 6 months, followed by a preserved cardiac function at 12 months. Mitral valve (MV) regurgitation occurs in a subset of mice at 2-6 months, while progressive dilatation of the aorta occurs from 2 to 12 months. Mitral valve tissue mechanical assessments using a uniaxial Permeabilizable Fiber System demonstrate decreased stiffness of MFS MVs at all stages. Histological and microscopic analysis of ECM content, structure, and fiber orientation demonstrate that alterations in ECM mechanics, composition, and organization precede functional abnormalities in Fbn1C1041G/+MFS MVs. At 2 months, ECM abnormalities are detected with an increase in proteoglycans and decreased stiffness of the mitral valve. By 6-12 months, collagen fiber remodeling is increased with abnormal fiber organization in MFS mitral valve leaflets. At the same time, matrifibrocyte gene expression characteristic of collagen-rich connective tissue is increased, as detected by RNA in situ hybridization and qPCR. Together, these studies demonstrate early prevalence of proteoglycans at 2 months followed by upregulation of collagen structure and organization with age in MVs of MFS mice. CONCLUSIONS: Altogether, our data indicate dynamic regulation of mitral valve structure, tissue mechanics, and function that reflect changes in ECM composition, organization, and gene expression in progressive MVD. Notably, increased collagen fiber organization and orientation, potentially dependent on increased matrifibrocyte cell activity, is apparent with altered mitral valve mechanics and function in aging MFS mice.

BRD9-SMAD2/3 Orchestrates Stemness and Tumorigenesis in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: 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. The epigenetic mechanisms regulating CSCs are currently insufficiently understood, which hampers the development of novel strategies for eliminating CSCs. METHODS: By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodeling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signaling pathway. RESULTS: Inhibition and genetic ablation of BRD9 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 tumors from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. CONCLUSIONS: Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

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.

IL-1ß-mediated adaptive reprogramming of endogenous human cardiac fibroblasts to cells with immune features during fibrotic remodeling.

The source and roles of fibroblasts and T-cells during maladaptive remodeling and myocardial fibrosis in the setting of pulmonary arterial hypertension (PAH) have been long debated. We demonstrate, using single-cell mass cytometry, a subpopulation of endogenous human cardiac fibroblasts expressing increased levels of CD4, a helper T-cell marker, in addition to myofibroblast markers distributed in human fibrotic RV tissue, interstitial and perivascular lesions in SUGEN/Hypoxia (SuHx) rats, and fibroblasts labeled with pdgfrα CreERt2/+ in R26R-tdTomato mice. Recombinant IL-1ß increases IL-1R, CCR2 receptor expression, modifies the secretome, and differentiates cardiac fibroblasts to form CD68-positive cell clusters. IL-1ß also activates stemness markers, such as NANOG and SOX2, and genes involved in dedifferentiation, lymphoid cell function and metabolic reprogramming. IL-1ß induction of lineage traced primary mouse cardiac fibroblasts causes these cells to lose their fibroblast identity and acquire an immune phenotype. Our results identify IL-1ß induced immune-competency in human cardiac fibroblasts and suggest that fibroblast secretome modulation may constitute a therapeutic approach to PAH and other diseases typified by inflammation and fibrotic remodeling.

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.

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.

On the possibility of estimating myocardial fiber architecture from cardiac strains.

The myocardium is composed of a complex network of contractile myofibers that are organized in such a way as to produce efficient contraction and relaxation of the heart. The myofiber architecture in the myocardium is a key determinant of cardiac motion and the global or organ-level function of the heart. Reports of architectural remodeling in cardiac diseases, such as pulmonary hypertension and myocardial infarction, potentially contributing to cardiac dysfunction call for the inclusion of an architectural marker for an improved assessment of cardiac function. However, the in-vivo quantification of three-dimensional myo-architecture has proven challenging. In this work, we examine the sensitivity of cardiac strains to varying myofiber orientation using a multiscale finite-element model of the LV. Additionally, we present an inverse modeling approach to predict the myocardium fiber structure from cardiac strains. Our results indicate a strong correlation between fiber orientation and LV kinematics, corroborating that the fiber structure is a principal determinant of LV contractile behavior. Our inverse model was capable of accurately predicting the myocardial fiber range and regional fiber angles from strain measures. A concrete understanding of the link between LV myofiber structure and motion, and the development of non-invasive and feasible means of characterizing the myocardium architecture is expected to lead to advanced LV functional metrics and improved prognostic assessment of structural heart disease.

Lipocalin 10 is essential for protection against inflammation-triggered vascular leakage by activating LDL receptor-related protein 2-slingshot homologue 1 signalling pathway.

AIMS: Systemic inflammation occurs commonly during many human disease settings and increases vascular permeability, leading to organ failure, and lethal outcomes. Lipocalin 10 (Lcn10), a poorly characterized member of the lipocalin family, is remarkably altered in the cardiovascular system of human patients with inflammatory conditions. Nonetheless, whether Lcn10 regulates inflammation-induced endothelial permeability remains unknown. METHODS AND RESULTS: Systemic inflammation models were induced using mice by injection of endotoxin lipopolysaccharide (LPS) or caecal ligation and puncture (CLP) surgery. We observed that the expression of Lcn10 was dynamically altered only in endothelial cells (ECs), but not in either fibroblasts or cardiomyocytes isolated from mouse hearts following the LPS challenge or CLP surgery. Using in vitro gain- and loss-of-function approaches and an in vivo global knockout mouse model, we discovered that Lcn10 negatively regulated endothelial permeability upon inflammatory stimuli. Loss of Lcn10 augmented vascular leakage, leading to severe organ damage and higher mortality following LPS challenge, compared to wild-type controls. By contrast, overexpression of Lcn10 in ECs displayed opposite effects. A mechanistic analysis revealed that both endogenous and exogenous elevation of Lcn10 in ECs could activate slingshot homologue 1 (Ssh1)-Cofilin signalling cascade, a key axis known to control actin filament dynamics. Accordingly, a reduced formation of stress fibre and increased generation of cortical actin band were exhibited in Lcn10-ECs, when compared to controls upon endotoxin insults. Furthermore, we identified that Lcn10 interacted with LDL receptor-related protein 2 (LRP2) in ECs, which acted as an upstream factor of the Ssh1-Confilin signalling. Finally, injection of recombinant Lcn10 protein into endotoxic mice showed therapeutic effects against inflammation-induced vascular leakage. CONCLUSION: This study identifies Lcn10 as a novel regulator of EC function and illustrates a new link in the Lcn10-LRP2-Ssh1 axis to controlling endothelial barrier integrity. Our findings may provide novel strategies for the treatment of inflammation-related diseases.

Machine learning-based classification of cardiac relaxation impairment using sarcomere length and intracellular calcium transients.

Impaired relaxation of cardiomyocytes leads to diastolic dysfunction in the left ventricle. Relaxation velocity is regulated in part by intracellular calcium (Ca2+) cycling, and slower outflux of Ca2+ during diastole translates to reduced relaxation velocity of sarcomeres. Sarcomere length transient and intracellular calcium kinetics are integral parts of characterizing the relaxation behavior of the myocardium. However, a classifier tool that can separate normal cells from cells with impaired relaxation using sarcomere length transient and/or calcium kinetics remains to be developed. In this work, we employed nine different classifiers to classify normal and impaired cells, using ex-vivo measurements of sarcomere kinematics and intracellular calcium kinetics data. The cells were isolated from wild-type mice (referred to as normal) and transgenic mice expressing impaired left ventricular relaxation (referred to as impaired). We utilized sarcomere length transient data with a total of n = 126 cells (n = 60 normal cells and n = 66 impaired cells) and intracellular calcium cycling measurements with a total of n = 116 cells (n = 57 normal cells and n = 59 impaired cells) from normal and impaired cardiomyocytes as inputs to machine learning (ML) models for classification. We trained all ML classifiers with cross-validation method separately using both sets of input features, and compared their performance metrics. The performance of classifiers on test data showed that our soft voting classifier outperformed all other individual classifiers on both sets of input features, with 0.94 and 0.95 area under the receiver operating characteristic curves for sarcomere length transient and calcium transient, respectively, while multilayer perceptron achieved comparable scores of 0.93 and 0.95, respectively. However, the performance of decision tree, and extreme gradient boosting was found to be dependent on the set of input features used for training. Our findings highlight the importance of selecting appropriate input features and classifiers for the accurate classification of normal and impaired cells. Layer-wise relevance propagation (LRP) analysis demonstrated that the time to 50% contraction of the sarcomere had the highest relevance score for sarcomere length transient, whereas time to 50% decay of calcium had the highest relevance score for calcium transient input features. Despite the limited dataset, our study demonstrated satisfactory accuracy, suggesting that the algorithm can be used to classify relaxation behavior in cardiomyocytes when the potential relaxation impairment of the cells is unknown.

Mutation Spectrum of ß-Thalassemia in Some Ethnic Groups of North Maharashtra, India.

Beta-thalassemia is the most common inherited single-gene disorder in the world, caused by more than 200 known mutations in the HBB gene. In India, the average prevalence of ß-thalassemia carriers is 3-4%. Several ethnic groups have a much higher prevalence, about 8% in the tribal groups, according to the 2011 census. The study's main goal is to identify common ß-thalassemia mutations and the frequencies of different haplotypes in various communities in North Maharashtra. Nashik district had the highest prevalence of ß-thalassemia (34%), followed by Ahmednagar (29%), Jalgaon (16%), Dhule (14%), and Nandurbar (7.0%). Prevalence of ß-thalassemia was highest in the schedule caste community (SC) (48%), followed by (17%) in Muslims, (14%) in other backward classes (OBC), (13%) in Schedule Tribe (ST), and (8.0%) in the general population The six most common ß-thalassemia mutations detected in this study are IVS 1 > 5 (G→C), Cd 15(G→A), Cd 41/41 (-TCTT), Cd 8/9(+G), IVS 1 > 1(G→T) and Cap + 1(A > G). Among these mutations, IVS 1 > 5 (G > C) was the most common type of mutation found in ß-thalassemia patients in the North Maharashtra population. Type-I haplotype was the most prevalent among all communities. Nashik and Ahmednagar districts were highly affected by ß-thalassemia. Among different ethnic groups, the SC and Muslim communities were the worst affected with a higher proportion of ß-thalassemia and increased frequency of mutations.

Research Initiative Supporting Excellence at the University of Cincinnati (RISE-UC): A Program to Develop and Support Research-Active Faculty Members.

A combination of forces have markedly increased challenges to research-active faculty achieving sustained success. This article describes how one department at the University of Cincinnati College of Medicine (UCCOM) implemented a strategic plan, the Research Initiative Supporting Excellence at the University of Cincinnati (RISE-UC), to promote the research activity of its research-active faculty, fiscal year (FY) 2011-FY 2021. RISE-UC was implemented and regularly updated to address evolving needs. RISE-UC supported faculty members pursuing research via fiscal and administrative services to grow a critical mass of investigators; establish a shared governance model; create pathways for developing physician-scientists; develop discrete and targeted internal research funding; establish an Academic Research Service (ARS) unit (as infrastructure to support research); enhance faculty member mentorship; and recognize, celebrate, and reward research success. RISE-UC was informed by shared governance and resulted in substantial increases in total size of the faculty and external funding. More than 50% of Physician-Scientist Training Program graduates are active researchers at UCCOM. The internal awards program realized a return on investment of ~16.4-fold, and total external direct cost research funds increased from ~$55,400,000 (FY 2015) to ~$114,500,000 (FY 2021). The ARS assisted in the submission of 57 grant proposals and provided services faculty members generally found very helpful or helpful. The peer-mentoring group for early-career faculty members resulted in 12 of 23 participants receiving major grant funding (≥ $100,000; spring 2017-spring 2021) from sources including National Institutes of Health awards, Department of Defense funding, Veterans Affairs funding, and foundation awards. Research recognition included ~$77,000/year in incentive payments to faculty members for grant submissions and grants awarded. RISE-UC is an example of a comprehensive approach to promote research faculty member success and may serve as a model for other institutions with similar aspirations.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

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 TGFß/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.

Multiscale characterization of left ventricle active behavior in the mouse.

The myocardium possesses an intricately designed microarchitecture to produce an optimal cardiac contraction. The contractile behavior of the heart is generated at the sarcomere level and travels across several length scales to manifest as the systolic function at the organ level. While passive myocardial behavior has been studied extensively, the translation of active tension produced at the fiber level to the organ-level function is not well understood. Alterations in cardiac systolic function are often key sequelae in structural heart diseases, such as myocardial infarction and systolic heart failure; thus, characterization of the contractile behavior of the heart across multiple length scales is essential to improve our understanding of mechanisms collectively leading to depressed systolic function. In this study, we present a methodology to characterize the active behavior of left ventricle free wall (LVFW) myocardial tissues in mice. Combined with active tests in papillary muscle fibers and conventional in vivo contractility measurement at the organ level in an animal-specific manner, we establish a multiscale active characterization of the heart from fiber to organ. In addition, we quantified myocardial architecture from histology to shed light on the directionality of the contractility at the tissue level. The LVFW tissue activation-relaxation behavior under isometric conditions was qualitatively similar to that of the papillary muscle fiber bundle. However, the maximum stress developed in the LVFW tissue was an order of magnitude lower than that developed by a fiber bundle, and the time taken for active forces to plateau was 2-3 orders of magnitude longer. Although the LVFW tissue exhibited a slightly stiffer passive response in the circumferential direction, the tissues produced significantly larger active stresses in the longitudinal direction during active testing. Also, contrary to passive viscoelastic stress relaxation, active stresses relaxed faster in the direction with larger peak stresses. The multiscale experimental pipeline presented in this work is expected to provide crucial insight into the contractile adaptation mechanisms of the heart with impaired systolic function. STATEMENT OF SIGNIFICANCE: Heart failure cause significant alterations to the contractile-relaxation behavior of the yocardium. Multiscale characterization of the contractile behavior of the myocardium is essential to advance our understanding of how contractility translates from fiber to organ and to identify the multiscale mechanisms leading to impaired cardiac function. While passive myocardial behavior has been studied extensively, the investigation of tissue-level contractile behavior remains critically scarce in the literature. To the best of our knowledge, our study here is the first to investigate the contractile behavior of the left ventricle at multiple length scales in small animals. Our results indicate that the active myocardial wall is a function of transmural depth and relaxes faster in the direction with larger peak stresses.

Etiology of genetic muscle disorders induced by mutations in fast and slow skeletal MyBP-C paralogs.

Skeletal muscle, a highly complex muscle type in the eukaryotic system, is characterized by different muscle subtypes and functions associated with specific myosin isoforms. As a result, skeletal muscle is the target of numerous diseases, including distal arthrogryposes (DAs). Clinically, DAs are a distinct disorder characterized by variation in the presence of contractures in two or more distal limb joints without neurological issues. DAs are inherited, and up to 40% of patients with this condition have mutations in genes that encode sarcomeric protein, including myosin heavy chains, troponins, and tropomyosin, as well as myosin binding protein-C (MYBPC). Our research group and others are actively studying the specific role of MYBPC in skeletal muscles. The MYBPC family of proteins plays a critical role in the contraction of striated muscles. More specifically, three paralogs of the MYBPC gene exist, and these are named after their predominant expression in slow-skeletal, fast-skeletal, and cardiac muscle as sMyBP-C, fMyBP-C, and cMyBP-C, respectively, and encoded by the MYBPC1, MYBPC2, and MYBPC3 genes, respectively. Although the physiology of various types of skeletal muscle diseases is well defined, the molecular mechanism underlying the pathological regulation of DAs remains to be elucidated. In this review article, we aim to highlight recent discoveries involving the role of skeletal muscle-specific sMyBP-C and fMyBP-C as well as their expression profile, localization in the sarcomere, and potential role(s) in regulating muscle contractility. Thus, this review provides an overall summary of MYBPC skeletal paralogs, their potential roles in skeletal muscle function, and future research directions.