"I realized I was not alone": A mixed-methods investigation of the implementation of Ubuntu groups to reduce burnout and social isolation in an allopathic medical School in the Southeastern United States.

PURPOSE/BACKGROUND: Healthcare providers experience higher rates of workplace burnout, a reality highlighted by the COVID-19 pandemic. In response, small groups, inspired by South African philosophy, Ubuntu, were introduced to decrease burnout and social isolation and build community and belonging. This study examines how participation in these groups can impact these measures. METHODS: In this mixed-methods study, trained facilitators led small groups that utilized story-sharing to foster connections within the group and broader community. Quantitative and qualitative data were analyzed separately and merged to identify convergence. RESULTS: Three main qualitative themes emerged: 1) seeking and building connections and community, 2) curiosity, learning, and growing, and 3) open-hearted and thriving. These themes were linked to quantitative outcomes, showing a statistically significant decrease in social isolation among staff/faculty and students. Furthermore, faculty/staff exhibited reduced burnout compared to students, while students reported increased feelings of belonging. CONCLUSION: Participation in Ubuntu groups positively influenced students' sense of belonging, reduced faculty/staff burnout, and alleviated social isolation for all participants. Future research should explore the potential of this intervention to further promote wellness on medical campuses. Programs emphasizing the well-being of individuals, including faculty, staff, and students, are crucial for supporting the overall health of medical communities and the wider society.

HuR-dependent expression of Wisp1 is necessary for TGFß-induced cardiac myofibroblast activity.

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGFß-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGFß-induced myofibroblast activation upon HuR inhibition. We identified twenty-four mRNA transcripts that were enriched for HuR binding following TGFß treatment via photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Eleven of these HuR-bound mRNAs also showed significant co-expression correlation with HuR, αSMA, and periostin in primary fibroblasts isolated from the ischemic-zone of infarcted mouse hearts. Of these, WNT1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 partially rescued myofibroblast-induced collagen gel contraction following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGFß. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGFß, in part through post-transcriptional regulation of Wisp1.

Allosteric regulation within the highly interconnected structural scaffold of AraC/XylS homologs tolerates a wide range of amino acid changes.

To create bacterial transcription "circuits" for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coli AraC transcriptional activator, for which binding to DNA is modulated by binding L-arabinose. Here, we engineered chimeric AraC/XylS transcription activators that recognized ara DNA binding sites and responded to varied effector ligands. The first step, identifying domain boundaries in the natural homologs, was challenging because (i) no full-length, dimeric structures were available and (ii) extremely low sequence identities (≤10%) among homologs precluded traditional assemblies of sequence alignments. Thus, to identify domains, we built and aligned structural models of the natural proteins. The designed chimeric activators were assessed for function, which was then further improved by random mutagenesis. Several mutational variants were identified for an XylS•AraC chimera that responded to benzoate; two enhanced activation to near that of wild-type AraC. For an RhaR•AraC chimera, a variant with five additional substitutions enabled transcriptional activation in response to rhamnose. These five changes were dispersed across the protein structure, and combinatorial experiments testing subsets of substitutions showed significant non-additivity. Combined, the structure modeling and epistasis suggest that the common AraC/XylS structural scaffold is highly interconnected, with complex intra-protein and inter-domain communication pathways enabling allosteric regulation. At the same time, the observed epistasis and the low sequence identities of the natural homologs suggest that the structural scaffold and function of transcriptional regulation are nevertheless highly accommodating of amino acid changes.

Evaluation of BCL6 and SIRT1 as Non-Invasive Diagnostic Markers of Endometriosis.

(1) Background: Endometriosis is characterized by the presence of endometrial glands and stroma outside of the uterus and is often associated with severe pelvic pain and infertility. Our study explored the utilization of B-Cell Lymphoma 6 (BCL6) and Sirtuin 1 (SIRT1) as potential biomarkers in serum, plasma, urine, and cervical mucus for a non-invasive diagnostic test for endometriosis. BCL6 was chosen based on its previously reported elevated expression in endometrial biopsies, and SIRT1 is co-expressed and upregulated in the endometrium of women with endometriosis. (2) Methods: BCL6 and SIRT1 levels were measured using enzyme-linked immunoassay (ELISA) in samples from 20 women with endometriosis (ten with stages I/II and ten with stages III/IV) and ten women without endometriosis. (3) Results: Levels of SIRT1 in sera showed a statistically significant elevation in advanced stages III/IV compared to controls and stages I/II. No significant differences were found in other bodily fluids for SIRT1 or any bodily fluids tested for BCL6. (4) Conclusions: These results suggest some potential of SIRT1 expression within serum as a predictor of advanced asymptomatic stages of endometriosis. Using immunohistochemistry (IHC) staining and H-SCORE values for the elevated BCL6 (and potentially SIRT1) levels in endometrial biopsy samples seems to have higher diagnostic potential based on the previously published studies.

Adipocyte-specific deletion of HuR induces spontaneous cardiac hypertrophy and fibrosis.

Adipose tissue homeostasis plays a central role in cardiovascular physiology, and the presence of thermogenically active brown adipose tissue (BAT) has recently been associated with cardiometabolic health. We have previously shown that adipose tissue-specific deletion of HuR (Adipo-HuR-/-) reduces BAT-mediated adaptive thermogenesis, and the goal of this work was to identify the cardiovascular impacts of Adipo-HuR-/-. We found that Adipo-HuR-/- mice exhibit a hypercontractile phenotype that is accompanied by increased left ventricle wall thickness and hypertrophic gene expression. Furthermore, hearts from Adipo-HuR-/- mice display increased fibrosis via picrosirius red staining and periostin expression. To identify underlying mechanisms, we applied both RNA-seq and weighted gene coexpression network analysis (WGCNA) across both cardiac and adipose tissue to define HuR-dependent changes in gene expression as well as significant relationships between adipose tissue gene expression and cardiac fibrosis. RNA-seq results demonstrated a significant increase in proinflammatory gene expression in both cardiac and subcutaneous white adipose tissue (scWAT) from Adipo-HuR-/- mice that is accompanied by an increase in serum levels of both TNF-α and IL-6. In addition to inflammation-related genes, WGCNA identified a significant enrichment in extracellular vesicle-mediated transport and exosome-associated genes in scWAT, whose expression most significantly associated with the degree of cardiac fibrosis observed in Adipo-HuR-/- mice, implicating these processes as a likely adipose-to-cardiac paracrine mechanism. These results are significant in that they demonstrate the spontaneous onset of cardiovascular pathology in an adipose tissue-specific gene deletion model and contribute to our understanding of how disruptions in adipose tissue homeostasis may mediate cardiovascular disease.NEW & NOTEWORTHY The presence of functional brown adipose tissue in humans is known to be associated with cardiovascular health. Here, we show that adipocyte-specific deletion of the RNA binding protein HuR, which we have previously shown to reduce BAT-mediated thermogenesis, is sufficient to mediate a spontaneous development of cardiac hypertrophy and fibrosis. These results may have implications on the mechanisms by which BAT function and adipose tissue homeostasis directly mediate cardiovascular disease.

l-Arginine Synthesis from l-Citrulline in Myeloid Cells Drives Host Defense against Mycobacteria In Vivo.

Immunonutrition as a therapeutic approach is rapidly gaining interest in the fight against infection. Targeting l-arginine metabolism is intriguing, considering this amino acid is the substrate for antimicrobial NO production by macrophages. The importance of l-arginine during infection is supported by the finding that inhibiting its synthesis from its precursor l-citrulline blunts host defense. During the first few weeks following pulmonary mycobacterial infection, we found a drastic increase in l-citrulline in the lung, even though serum concentrations were unaltered. This correlated with increased gene expression of the l-citrulline-generating (i.e., iNOS) and l-citrulline-using (i.e., Ass1) enzymes in key myeloid populations. Eliminating l-arginine synthesis from l-citrulline in myeloid cells via conditional deletion of either Ass1 or Asl resulted in increased Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis H37Rv burden in the lungs compared with controls. Our data illustrate the necessity of l-citrulline metabolism for myeloid defense against mycobacterial infection and highlight the potential for host-directed therapy against mycobacterial disease targeting this nutrient and/or its metabolic pathway.

Extracorporeal life support in the multidisciplinary management of cardiogenic shock complicating acute myocardial infarction.

OBJECTIVES: To analyze the results of extracorporeal life support (ECLS) for cardiogenic shock complicating acute myocardial infarction (AMI) in a single-center experience. BACKGROUND: Cardiogenic shock is still a leading cause of death for AMI. Conventional management carries mortality rates exceeding 50%. ECLS may be considered as a bridge to decision in the setting of AMI complicated by cardiogenic shock not responsive to standard management. METHODS: We performed an observational analysis of our local database. The primary end-point was survival to hospital discharge. All variables were compared between survivors and nonsurvivors. RESULTS: Between January 2007 and December 2017, 56 patients were supported for cardiogenic shock complicating AMI. The mean age was 56.7 years and 89.3% were males. Baseline characteristics were comparable between both groups. Of the 50 primary percutaneous coronary interventions that were attempted, 44 (88.0%) were successful. Twenty-three (41.1%) patients died during ECLS support. The complications' rate during ECLS support was comparable between both groups. Twenty-eight (50%) patients were successfully weaned from ECLS after a mean support of 8.7 days. Eight (14.3%) patients eventually died after weaning before hospital discharge. Five (8.9%) patients could not be weaned from ECLS and were switched to a long-term mechanical circulatory support. Overall survival to hospital discharge was 41.1% (n = 23). Eighteen (32.1%) patients were alive after a mean follow-up of 38.0 ± 29.9 (range, 4.2-95.4) months. CONCLUSIONS: ECLS should be considered as a therapeutic solution in the management of AMI-related cardiogenic shock with a satisfactory short- and long-term survival.

Electronic health record year and country of birth testing and patient navigation to increase diagnosis of chronic viral hepatitis.

The United States Preventive Services Task Force recommends hepatitis C testing people born from 1945 to 1965, "birth cohort" as well as hepatitis C and hepatitis B testing people from countries of birth with endemic infection risk. We automated the hospital electronic health record system to test birth cohort and those born in countries with endemic infection risk. A script is launched searching the laboratory database upon registration for any hepatitis C antibody, hepatitis C RNA and/or hepatitis B surface antigen result. If no positive result was found, a hepatitis C antibody/reflex RNA and/or hepatitis B surface antigen were ordered. A patient navigator received weekly results and assisted patients with positive serology to schedule an appointment with their primary care provider or treatment specialist. A total of 10 726 participants were hepatitis C antibody tested, with 6.9% antibody positive. Monthly hepatitis C testing from January to July 2016 compared to August 2016-August 2017 increased 342% as a result of "birth cohort" testing. Following country of birth testing, monthly hepatitis B and hepatitis C testing increased 91%, and 44%, respectively, during June-August 2017 compared to September 2017-March 2018. 67% of hepatitis C-positive patients were linked to care. If the navigator contacted the patient, 92% were linked to care, and 32% were treated. Of hepatitis B surface antigen-positive patients, 43% were linked to care, 5% were on treatment, and 15% started treatment. Automated electronic health record ordering of hepatitis C and/or hepatitis B testing is feasible and increases testing. In the population tested, much improvement is needed with linkage to care and treatment.

A survey of bolus tube feeding prevalence and practice in adult patients requiring home enteral tube feeding.

Anecdotal evidence suggests the use of bolus tube feeding is increasing in the long-term home enteral tube feed (HETF) patients. A cross-sectional survey to assess the prevalence of bolus tube feeding and to characterise these patients was undertaken. Dietitians from ten centres across the UK collected data on all adult HETF patients on the dietetic caseload receiving bolus tube feeding (n 604, 60 % male, age 58 years). Demographic data, reasons for tube and bolus feeding, tube and equipment types, feeding method and patients' complete tube feeding regimens were recorded. Over a third of patients receiving HETF used bolus feeding (37 %). Patients were long-term tube fed (4·1 years tube feeding, 3·5 years bolus tube feeding), living at home (71 %) and sedentary (70 %). The majority were head and neck cancer patients (22 %) who were significantly more active (79 %) and lived at home (97 %), while those with cerebral palsy (12 %) were typically younger (age 31 years) but sedentary (94 %). Most patients used bolus feeding as their sole feeding method (46 %), because it was quick and easy to use, as a top-up to oral diet or to mimic mealtimes. Importantly, oral nutritional supplements (ONS) were used for bolus feeding in 85 % of patients, with 51 % of these being compact-style ONS (2·4 kcal (10·0 kJ)/ml, 125 ml). This survey shows that bolus tube feeding is common among UK HETF patients, is used by a wide variety of patient groups and can be adapted to meet the needs of a variety of patients, clinical conditions, nutritional requirements and lifestyles.

Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms.

BACKGROUND: Here we present an in-depth characterization of the mechanism of sequencer-induced sample contamination due to the phenomenon of index swapping that impacts Illumina sequencers employing patterned flow cells with Exclusion Amplification (ExAmp) chemistry (HiSeqX, HiSeq4000, and NovaSeq). We also present a remediation method that minimizes the impact of such swaps. RESULTS: Leveraging data collected over a two-year period, we demonstrate the widespread prevalence of index swapping in patterned flow cell data. We calculate mean swap rates across multiple sample preparation methods and sequencer models, demonstrating that different library methods can have vastly different swapping rates and that even non-ExAmp chemistry instruments display trace levels of index swapping. We provide methods for eliminating sample data cross contamination by utilizing non-redundant dual indexing for complete filtering of index swapped reads, and share the sequences for 96 non-combinatorial dual indexes we have validated across various library preparation methods and sequencer models. Finally, using computational methods we provide a greater insight into the mechanism of index swapping. CONCLUSIONS: Index swapping in pooled libraries is a prevalent phenomenon that we observe at a rate of 0.2 to 6% in all sequencing runs on HiSeqX, HiSeq 4000/3000, and NovaSeq. Utilizing non-redundant dual indexing allows for the removal (flagging/filtering) of these swapped reads and eliminates swapping induced sample contamination, which is critical for sensitive applications such as RNA-seq, single cell, blood biopsy using circulating tumor DNA, or clinical sequencing.

Order parameters and time evolution of mesophases in the lyotropic chromonic liquid crystal Sunset Yellow FCF by DNMR.

Uniaxial order parameters of the nematic and columnar mesophases in the lyotropic chromonic liquid crystal Sunset Yellow FCF have been determined from deuteron nuclear magnetic resonance, where random confinement of the system by the dispersion of aerosil nanoparticles has been performed to help obtain the angular dependent spectra. The long-time evolution study of the order parameters shows that the system requires tens of hours to stabilize after a deep change in temperature, in contrast with the very fast assembly process of the aggregates. Finally, the degree of order of the water molecules, forced by the uniaxial environment, has been determined.

Tranilast Blunts the Hypertrophic and Fibrotic Response to Increased Afterload Independent of Cardiomyocyte Transient Receptor Potential Vanilloid 2 Channels.

Tranilast is clinically indicated for the treatment of allergic disorders and is also a nonselective blocker of the transient receptor potential vanilloid 2 (TRPV2) channel. Previous studies have found that it has protective effects in various animal models of cardiac disease. Our laboratory has found that genetic deletion of TRPV2 results in a blunted hypertrophic response to increased afterload; thus, this study tested the hypothesis that tranilast through cardiomyocyte TRPV2 blockade can inhibit the hypertrophic response to pressure overload in vivo through transverse aortic constriction and ex vivo through isolated myocyte studies. The in vivo studies demonstrated that tranilast blunted the fibrotic response to increased afterload and, to a lesser extent, the hypertrophic response. After 4 weeks, this blunting was associated with improved cardiac function, although at 8 weeks, the cardiac function deteriorated similarly to the control group. Finally, the in vitro studies demonstrated that tranilast was not inhibiting these responses at the cardiomyocyte level. In conclusion, we demonstrated that tranilast blunting of the fibrotic and hypertrophic response occurs independently of cardiac TRPV2 channels and may be cardioprotective in the short term but not after prolonged administration.

Educational Intervention for Nutrition Education in Patients Attending an Outpatient Wound Care Clinic: A Feasibility Study.

PURPOSE: The purpose of this study was to determine whether nurses could use a structured intervention to educate patients with wounds about foods that promote healing and whether this educational intervention could be provided in a cost-effective manner. DESIGN: Cross-sectional survey. SUBJECTS AND SETTING: The study was conducted at an outpatient wound care center located on a hospital campus in the Southern United States; 3 full-time nurses and 2 nurses employed on part-time status delivered the intervention. METHODS: A nutrition education intervention was developed through collaborative efforts of a registered dietitian and a nurse. A cross-sectional survey design was used to (1) evaluate nurses' perceptions of the intervention and (2) identify barriers to implementation of the intervention. Direct costs related to materials and nursing time required to deliver the intervention were calculated. RESULTS: Participants indicated they were competent to deliver the structured intervention, and all were willing to continue its use. Survey results indicated that nurses believed the intervention was beneficial to their patients and they indicated that patients were responsive to the intervention. The intervention was found to be low cost ($8.00 per teaching session); no barriers to implementation of the intervention were identified. CONCLUSION: The results of this exploratory study suggest that a structured nutrition education intervention can be provided by nurses in outpatient wound clinics at low cost. Further study is needed to determine the impact of this intervention on nutritional intake and wound healing.

Translational readthrough-promoting drugs enhance pseudoknot-mediated suppression of the stop codon at the Moloney murine leukemia virus gag­pol junction.

Translational readthrough-promoting drugs enhance the incorporation of amino acids at stop codons and can thus bypass premature termination during protein synthesis. The polymerase (Pol) proteins of Moloney murine leukemia virus (MoMLV) are synthesized as a large Gag­Pol fusion protein, formed by the readthrough of a stop codon at the end of the gag ORF. The downstream pol ORF lacks its own start codon, and Pol protein synthesis is wholly dependent on translation of the upstream gag gene and the readthrough event for expression. Here, we explored the effects of readthrough-promoting drugs ­ aminoglycoside antibiotics and the small molecule ataluren ­ on the efficiency of readthrough of the stop codon in the context of the MoMLV genome. We showed that these compounds increased readthrough of the stop codon at the MoMLV gag­pol junction in vivo above the already high basal level and that the resulting elevated gag­pol readthrough had deleterious effects on virus replication. We also showed that readthrough efficiency could be driven to even higher levels in vitro, and that the combination of the small molecules and the RNA structure at the MoMLV stop codon could achieve extremely high readthrough efficiencies.

Transcatheter aortic valve implantation using the left transcarotid approach in patients with previous ipsilateral carotid endarterectomy.

OBJECTIVES: To assess the feasibility and safety of transcatheter aortic valve implantation (TAVI) through a left transcarotid approach in patients previously operated on for ipsilateral carotid endarterectomy (CEA). BACKGROUND: The healthcare impact of extracranial carotid artery disease is essential as stroke is the third-leading cause of death in industrialized nations and CEA is often present in the history of patients awaiting TAVI. METHODS: The primary endpoint was to evaluate 30-day mortality and freedom from major TAVI-related complications in an observational analysis. RESULTS: From December 2011 to February 2014, we performed 9 TAVI. The mean age was 84.6 years. The procedure was performed without any technical complication or vascular injury in every patient. There was neither intraoperative mortality nor intraoperative major complications. One (11.1%) patient experienced spatial-temporal disorientation but cerebral computed tomography did not show any sign of stroke. Two (22.2%) patients needed the implantation of a pacemaker due to third-degree atrioventricular block appearance. Three (33.3%) patients were transfused with packed red blood cells and 1 (11.1%) patient developed a groin hematoma. Only 1 (11.1%) patient showed a residual paravalvular regurgitation ≥ 2. At 30-day follow-up there was neither mortality nor other TAVI-related complications and echocardiography parameters remained stable. CONCLUSIONS: TAVI through a left transcarotid approach in patients previously operated on for ipsilateral CEA is feasible and safe. The presence of a previous ipsilateral CEA represents no more a limitation to the utilization of this promising access route. At short-term follow-up, mortality and major complications rates are low.

Whole genome deep sequencing of HIV-1 reveals the impact of early minor variants upon immune recognition during acute infection.

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.

Redefining the relevance of established cancer cell lines to the study of mechanisms of clinical anti-cancer drug resistance.

Although in vitro models have been a cornerstone of anti-cancer drug development, their direct applicability to clinical cancer research has been uncertain. Using a state-of-the-art Taqman-based quantitative RT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of six cancer types, in established cancer cell lines (grown in monolayer, 3D scaffold, or in xenograft) and clinical samples, either containing >75% tumor cells or microdissected. The MDR transcriptome was determined a priori based on an extensive curation of the literature published during the last three decades, which led to the enumeration of 380 genes. No correlation was found between clinical samples and established cancer cell lines. As expected, we found up-regulation of genes that would facilitate survival across all cultured cancer cell lines evaluated. More troubling, however, were data showing that all of the cell lines, grown either in vitro or in vivo, bear more resemblance to each other, regardless of the tissue of origin, than to the clinical samples they are supposed to model. Although cultured cells can be used to study many aspects of cancer biology and response of cells to drugs, this study emphasizes the necessity for new in vitro cancer models and the use of primary tumor models in which gene expression can be manipulated and small molecules tested in a setting that more closely mimics the in vivo cancer microenvironment so as to avoid radical changes in gene expression profiles brought on by extended periods of cell culture.

MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids.

BACKGROUND: MYBPC3 , 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). METHODS: The hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3 D389V 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 . RESULTS: Confocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3 D389V 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 MYBPC3 D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3 D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential (ΔΨm). 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®) in MYBPC3 D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3 D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONS: Conceptually, 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 MYBPC3 D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and Significance: What Is Known?: MYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. D389V 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. The molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined.What New Information Does This Article Contribute ?: The 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. The MYBPC3 D389V -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. Our 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®), a myosin inhibitor. In addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential (ΔΨm), highlighting a possible early adaptive response to primary sarcomeric changes. Early treatment of MYBPC3 D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. GRAPHICAL ABSTRACT: A graphical abstract is available for this article.

Roles of cMyBP-C phosphorylation on cardiac contractile dysfunction in db/db mice.

Type 2 diabetes mellitus (T2DM) is a metabolic disease and comorbidity associated with several conditions, including cardiac dysfunction leading to heart failure with preserved ejection fraction (HFpEF), in turn resulting in T2DM-induced cardiomyopathy (T2DM-CM). However, the molecular mechanisms underlying the development of T2DM-CM are poorly understood. It is hypothesized that molecular alterations in myopathic genes induced by diabetes promote the development of HFpEF, whereas cardiac myosin inhibitors can rescue the resultant T2DM-mediated cardiomyopathy. To test this hypothesis, a Leptin receptor-deficient db/db homozygous (Lepr db/db) mouse model was used to define the pathogenesis of T2DM-CM. Echocardiographic studies at 4 and 6 months revealed that Lepr db/db hearts started developing cardiac dysfunction by four months, and left ventricular hypertrophy with diastolic dysfunction was evident at 6 months. RNA-seq data analysis, followed by functional enrichment, revealed the differential regulation of genes related to cardiac dysfunction in Lepr db/db heart tissues. Strikingly, the level of cardiac myosin binding protein-C phosphorylation was significantly increased in Lepr db/db mouse hearts. Finally, using isolated skinned papillary muscles and freshly isolated cardiomyocytes, CAMZYOS ® (mavacamten, MYK-461), a prescription heart medicine used for symptomatic obstructive hypertrophic cardiomyopathy treatment, was tested for its ability to rescue T2DM-CM. Compared with controls, MYK-461 significantly reduced force generation in papillary muscle fibers and cardiomyocyte contractility in the db/db group. This line of evidence shows that 1) T2DM-CM is associated with hyperphosphorylation of cardiac myosin binding protein-C and 2) MYK-461 significantly lessened disease progression in vitro, suggesting its promise as a treatment for HFpEF.