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Among the many academic challenges faced by dual-degree MD-PhD students is access to professional support networks designed to overcome the unique academic and personal barriers to physician-scientist training. In the current study, we hypothesized that regional access to a student MD-PhD conference, termed the Southeastern Medical Scientist Symposium (SEMSS), would enhance medical and/or graduate training by fostering such relationships between physician-scientist trainees, doing so by discussing both the challenges of physician-scientist training and effective strategies to overcome them. In the current study, we used a mixed-methods approach to evaluate the overall usefulness of SEMSS over a ten-year period (2010-2020) to identify key areas of particular benefit to trainees. The authors used conference registration data to compile self-reported demographic and regional attendance, followed by a post-conference survey to gauge attendee satisfaction. Over the reporting period, SEMSS was attended by equivalent proportions of MD-PhD and undergraduate students, among which were a high-percentage of students from underrepresented minority (URM) groups relative to the national MD-PhD applicant pool; nearly one-third of URM students attendees later matriculated into MD-PhD programs, far exceeding the national MD-PhD matriculation rate. Among the benefits reported by students were “opportunities to network with peers” and opportunities to learn about the physician-scientist career track. Therefore, we therefore propose regional MD-PhD conferences as an effective model to promote diversity within the physician-scientist training pipeline.

Precision-based molecular phenotyping of heart failure must overcome limited access to cardiac tissue. Although epigenetic alterations have been found to underlie pathological cardiac gene dysregulation, the clinical utility of myocardial epigenomics remains narrow owing to limited clinical access to tissue. Therefore, the current study determined whether patient plasma confers indirect phenotypic, transcriptional, and/or epigenetic alterations to ex vivo cardiomyocytes to mirror the failing human myocardium. Neonatal rat ventricular myocytes (NRVMs) and single-origin human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and were treated with blood plasma samples from patients with dilated cardiomyopathy (DCM) and donor subjects lacking history of cardiovascular disease. Following plasma treatments, NRVMs and hiPSC-CMs underwent significant hypertrophy relative to non-failing controls, as determined via automated high-content screening. Array-based DNA methylation analysis of plasma-treated hiPSC-CMs and cardiac biopsies uncovered robust, and conserved, alterations in cardiac DNA methylation, from which 100 sites were validated using an independent cohort. Among the CpG sites identified, hypo-methylation of the ATG promoter was identified as a diagnostic marker of HF, wherein cg03800765 methylation (AUC = 0.986, P

Although recent advances in the treatment of acute coronary heart disease have reduced mortality rates, few therapeutic strategies exist to mitigate the progressive loss of cardiac function that manifests as heart failure. Nuclear factor, erythroid 2 like 2 (Nfe2l2, Nrf2) is a transcriptional regulator that is known to confer transient myocardial cytoprotection following acute ischemic insult; however, its sustained activation paradoxically causes a reductive environment characterized by excessive antioxidant activity. We previously identified a subset of 16 microRNAs (miRNA) significantly diminished in Nrf2-ablated (Nrf2−/−) mouse hearts, leading to the hypothesis that increasing levels of Nrf2 activation augments miRNA induction and post-transcriptional dysregulation. Here, we report the identification of distinct miRNA signatures (i.e. “reductomiRs”) associated with Nrf2 overexpression in a cardiac-specific and constitutively active Nrf2 transgenic (caNrf2-Tg) mice expressing low (TgL) and high (TgH) levels. We also found several Nrf2 dose-responsive miRNAs harboring proximal antioxidant response elements (AREs), implicating these “reductomiRs” as putative meditators of Nrf2-dependent post-transcriptional regulation. Analysis of mRNA-sequencing identified a complex network of miRNAs and effector mRNAs encoding known pathological hallmarks of cardiac stress-response. Altogether, these data support Nrf2 as a putative regulator of cardiac miRNA expression and provide novel candidates for future mechanistic investigation to understand the relationship between myocardial reductive stress and cardiac pathophysiology.

Heart failure (HF) is a multifactorial syndrome that remains a leading cause of worldwide morbidity. Despite its high prevalence, only half of patients with HF respond to guideline-directed medical management, prompting therapeutic efforts to confront the molecular underpinnings of its heterogeneity. In the current study, we examined epigenetics as a yet unexplored source of heterogeneity among patients with end-stage HF. Specifically, a multicohort-based study was designed to quantify cardiac genome-wide cytosine-p-guanine (CpG) methylation of cardiac biopsies from male patients undergoing left ventricular assist device (LVAD) implantation. In both pilot (n = 11) and testing (n = 31) cohorts, unsupervised multidimensional scaling of genome-wide myocardial DNA methylation exhibited a bimodal distribution of CpG methylation found largely to occur in the promoter regions of metabolic genes. Among the available patient attributes, only categorical self-identified patient race could delineate this methylation signature, with African American (AA) and Caucasian American (CA) samples clustering separately. Because race is a social construct, and thus a poor proxy of human physiology, extensive review of medical records was conducted, but ultimately failed to identify covariates of race at the time of LVAD surgery. By contrast, retrospective analysis exposed a higher all-cause mortality among AA (56.3%) relative to CA (16.7%) patients at 2 yr following LVAD placement (P = 0.03). Geocoding-based approximation of patient demographics uncovered disparities in income levels among AA relative to CA patients. Although additional studies are needed, the current analysis implicates cardiac DNA methylation as a previously unrecognized indicator of socioeconomic disparity in human heart failure outcomes. NEW & NOTEWORTHY A bimodal signature of cardiac DNA methylation in heart failure corresponds with racial differences in all-cause mortality following mechanical circulatory support. Racial differences in promoter methylation disproportionately affect metabolic signaling pathways. Socioeconomic factors are associated with racial differences in the cardiac methylome among men with end-stage heart failure. Listen to this article’s corresponding podcast at https://ajpheart.podbean.com/e/racial-socioeconomic-determinants-of-the-cardiac-epigenome/.

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear whether these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy, we generated transgenic mice with inducible cardiomyocyte-specific expression of the GLUT4. We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in nondiabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest that reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

Background Epigenetic mechanisms are critical for hippocampus-dependent memory formation. Building on previous studies that implicate the N-lysine methyltransferase SETD6 in the activation of nuclear factor-κB RELA (also known as transcription factor p65) as an epigenetic recruiter, we hypothesized that SETD6 is a key player in the epigenetic control of long-term memory. Methods Using a series of molecular, biochemical, imaging, electrophysiological, and behavioral experiments, we interrogated the effects of short interfering RNA–mediated knockdown of Setd6 in the rat dorsal hippocampus during memory consolidation. Results Our findings demonstrate that SETD6 is necessary for memory-related nuclear factor-κB RELA methylation at lysine 310 and associated increases in H3K9me2 (histone H3 lysine 9 dimethylation) in the dorsal hippocampus and that SETD6 knockdown interferes with memory consolidation, alters gene expression patterns, and disrupts spine morphology. Conclusions Together, these findings suggest that SETD6 plays a critical role in memory formation and may act as an upstream initiator of H3K9me2 changes in the hippocampus during memory consolidation.

African Americans (AA) face a greater burden of cardiovascular diseases, including ischemic cardiomyopathy (ICM), compared to Caucasian Americans (CA). Our recent work supports that epigenetics is likely a unifying mechanism between health disparities of socioeconomic inequalities and heart failure severity; whereby environmental factors are incorporated as stable DNA modifications to confer susceptibility to heart failure. We hypothesized that ICM possesses racially distinct methylome signatures. Cardiac biopsies from male age- and disease-matched AA (n = 14) and CA (n = 15) heart failure patients with ICM and non-ICM (NICM) were subjected to expression analysis using RNA-seq ( P < 0.05, ±1.5-fold) and differential methylation analysis using Illumina EPIC array ( P < 0.05, ±5%) using generalized linear models with a focus on changes corresponding to ICM and racial differences. Comparing ICM vs NICM, both AA and CA patients showed racially distinct differentially expressed genes (594/366 within AA; 439/487 within CA; up/down respectively). Similarly, pathway analysis using the NIH’s BioPlanet database found a significant change ( Q < 0.1) in AA for adipogenesis (25/133 overlap) and in CA for complement cascade (18/77 overlap). In contrast, racially distinct DNA methylation changes were more prominent for CA (29703/22703; up/down) compared to AA (2904/2897; up/down). Upon combining both datasets, known heart failure marker NPPA , which encodes Atrial Natriuretic Peptide, was found to be downregulated in ICM compared to NICM only for AA ( Q = 0, 18.9-fold change), corresponding to hypermethylation of 6 CpG sites ( P < 0.05, 6.4 - 11.3%). NPPA inhibits cardiac hypertrophy, fibrosis, and decreases inflammation. It is also used as a diagnostic biomarker in ICM. But recent trials found that AA have lower levels of NPPA in blood compared to CA, even in healthy condition. This patient cohort also indicated a racially diverse pattern of NPPA expression and methylation. Given its role in adipogenesis, NPPA may also explain the pathway enrichment specific to AA-ICM patients. Our data indicate that NPPA may connect the socioeconomic effects through DNA methylation with clinical diversity of ICM. It also underscores the need for a better prognostic biomarker for ICM.

Introduction: Ca 2+ /calmodulin-dependent protein kinase II (CaMK2) is a recognized driver of pathological cardiac remodeling and heart failure, but its role in the metabolic reprogramming of heart failure remains unclear, as do the molecular processes that trigger this pathologic switch. Hypothesis: Myocardial CaMKIIδ drives the pathological remodeling of cardiac metabolic substrate preference. Methods and Results: Adeno-associated viral (ssAAV9) overexpression of constitutively-active cytosolic (CaMK2δC) and nuclear (CaMK2δB) CaMK2δ isoforms, as well as the catalytic PKA subunit (PKAca), were created under the control of the cardiac troponin-T promoter and introduced via tail-vein injection into 12-week old mice (1 x 10 12 v.g., n = 5). In vivo metabolic phenotyping via 18-fluorodeoxyglucose ( 18 FDG)-based positron emission tomography (PET) revealed a 3.4-fold (P = 0.02) higher glucose uptake among mice receiving ssAAV9-TnnT-CaMK2δC commensurate with cardiac CaMK2δC expression. In contrast, no differences were seen in glucose uptake following AAV overexpression of cardiac CaMK2δB or PKAca relative to luciferase control vector. Echocardiography subsequently revealed a 14.6% decrease in left ventricular ejection fraction at 6 weeks following CaMK2δC overexpression (P = 0.047), whereas no changes were seen following CaMK2δB or PKAca overexpression in systolic or diastolic cardiac parameters. Conversely, transcriptomic analysis of hearts from mice lacking cardiac CaMK2δC expression revealed disproportionate up-regulation of fatty acid oxidative genes (P < 0.001). Conclusion: Taken together, these preliminary findings implicate CaMK2δC as a crucial regulator of the cardiac metabolic reprogramming that likely precedes the hemodynamic manifestations of cardiac dysfunction in heart failure.

Prompt recognition and treatment for thrombotic thrombocytopenic purpura (TTP) are critical to prevent the irreversible manifestations of this rare and quickly fatal hematologic disorder. Untreated TTP is typically a rapid‐onset disease with mortality exceeding 90% within days in the absence of appropriate treatment. In the current report, we describe a case of immune‐mediated TTP (iTTP) in a 62‐year‐old man manifesting as longstanding thrombocytopenia, recurrent cardioembolic strokes, and valvular thrombogenesis over a period of 3 years. We provide correlative evidence to support the potential contribution of adalimumab, a TNFα inhibitor, to the development of iTTP. We offer several educational insights regarding the identification of atypical presentations of iTTP owing to the longstanding disease course and numerous clinical comorbidities seen in this patient., We report an atypical manifestation of immune‐mediated thrombotic thrombocytopenic purpura (TTP) that we hypothesize represents a longstanding, or “smoldering,” process. As typified by this case, the prompt recognition and treatment for immune‐mediated TTP are often confounded by clinical comorbidities. Thus, TTP should be considered in cases of unremitting thrombocytopenia of unknown origin in patients with stroke or other thrombosis.

Heart failure (HF) is a leading cause of morbidity and mortality in the United States and worldwide. As a multifactorial syndrome with unpredictable clinical outcomes, identifying the common molecular underpinnings that drive HF pathogenesis remains a major focus of investigation. Disruption of cardiac gene expression has been shown to mediate a common final cascade of pathological hallmarks wherein the heart reactivates numerous developmental pathways. Although the central regulatory mechanisms that drive this cardiac transcriptional reprogramming remain unknown, epigenetic contributions are likely. In the current study, we examined whether the epigenome, specifically DNA methylation, is reprogrammed in HF to potentiate a pathological shift in cardiac gene expression. To accomplish this, we used paired-end whole genome bisulfite sequencing and next-generation RNA sequencing of left ventricle tissue obtained from seven patients with end-stage HF and three nonfailing donor hearts. We found that differential methylation was localized to promoter-associated cytosine-phosphate-guanine islands, which are established regulatory regions of downstream genes. Hypermethylated promoters were associated with genes involved in oxidative metabolism, whereas promoter hypomethylation enriched glycolytic pathways. Overexpression of plasmid-derived DNA methyltransferase 3A in vitro was sufficient to lower the expression of numerous oxidative metabolic genes in H9c2 rat cardiomyoblasts, further supporting the importance of epigenetic factors in the regulation of cardiac metabolism. Last, we identified binding-site competition via hypermethylation of the nuclear respiratory factor 1 (NRF1) motif, an established upstream regulator of mitochondrial biogenesis. These preliminary observations are the first to uncover an etiology-independent shift in cardiac DNA methylation that corresponds with altered metabolic gene expression in HF. NEW & NOTEWORTHY The failing heart undergoes profound metabolic changes because of alterations in cardiac gene expression, reactivating glycolytic genes and suppressing oxidative metabolic genes. In the current study, we discover that alterations to cardiac DNA methylation encode this fetal-like metabolic gene reprogramming. We also identify novel epigenetic interference of nuclear respiratory factor 1 via hypermethylation of its downstream promoter targets, further supporting a novel contribution of DNA methylation in the metabolic remodeling of heart failure.

Pancreatic β-cells undergo profound hyperplasia during pregnancy to maintain maternal euglycemia. Failure to reprogram β-cells into a more replicative state has been found to underlie susceptibility to gestational diabetes mellitus (GDM). We recently identified a requirement for prolactin receptor (PRLR) signaling in the metabolic adaptations to pregnancy, where β-cell-specific PRLR knockout (βPRLRKO) mice exhibit a metabolic phenotype consistent with GDM. However, the underlying transcriptional program that is responsible for the PRLR-dependent metabolic adaptations during gestation remains incompletely understood. To identify PRLR signaling gene regulatory networks and target genes within β-cells during pregnancy, we performed a transcriptomic analysis of pancreatic islets isolated from either βPRLRKO mice or littermate controls in late gestation. Gene set enrichment analysis identified forkhead box protein M1 and polycomb repressor complex 2 subunits, Suz12 and enhancer of zeste homolog 2 (Ezh2), as novel candidate regulators of PRLR-dependent β-cell adaptation. Gene ontology term pathway enrichment revealed both established and novel PRLR signaling target genes that together promote a state of increased cellular metabolism and/or proliferation. In contrast to the requirement for β-cell PRLR signaling in maintaining euglycemia during pregnancy, PRLR target genes were not induced following high-fat diet feeding. Collectively, the current study expands our understanding of which transcriptional regulators and networks mediate gene expression required for islet adaptation during pregnancy. The current work also supports the presence of pregnancy-specific adaptive mechanisms distinct from those activated by nutritional stress.

Ischemic cardiomyopathy (ICM) is the clinical endpoint of coronary heart disease and a leading cause of heart failure. Despite growing demands to develop personalized approaches to treat ICM, progress is limited by inadequate knowledge of its pathogenesis. Since epigenetics has been implicated in the development of other chronic diseases, the current study was designed to determine whether transcriptional and/or epigenetic changes are sufficient to distinguish ICM from other etiologies of heart failure. Specifically, we hypothesize that genome-wide DNA methylation encodes transcriptional reprogramming in ICM. RNA-sequencing analysis was performed on human ischemic left ventricular tissue obtained from patients with end-stage heart failure, which enriched known targets of the polycomb methyltransferase EZH2 compared to non-ischemic hearts. Combined RNA sequencing and genome-wide DNA methylation analysis revealed a robust gene expression pattern consistent with suppression of oxidative metabolism, induced anaerobic glycolysis, and altered cellular remodeling. Lastly, KLF15 was identified as a putative upstream regulator of metabolic gene expression that was itself regulated by EZH2 in a SET domain-dependent manner. Our observations therefore define a novel role of DNA methylation in the metabolic reprogramming of ICM. Furthermore, we identify EZH2 as an epigenetic regulator of KLF15 along with DNA hypermethylation, and we propose a novel mechanism through which coronary heart disease reprograms the expression of both intermediate enzymes and upstream regulators of cardiac metabolism such as KLF15.

Background Glycemic control is a strong predictor of long-term cardiovascular risk in patients with diabetes mellitus, and poor glycemic control influences long-term risk of cardiovascular disease even decades after optimal medical management. This phenomenon, termed glycemic memory, has been proposed to occur due to stable programs of cardiac and endothelial cell gene expression. This transcriptional remodeling has been shown to occur in the vascular endothelium through a yet undefined mechanism of cellular reprogramming. Methods In the current study, we quantified genome-wide DNA methylation of cultured human endothelial aortic cells (HAECs) via reduced-representation bisulfite sequencing (RRBS) following exposure to diabetic (250 mg/dL), pre-diabetic (125 mg/dL), or euglycemic (100 mg/dL) glucose concentrations for 72 h (n = 2). Results We discovered glucose-dependent methylation of genomic regions (DMRs) encompassing 2199 genes, with a disproportionate number found among genes associated with angiogenesis and nitric oxide (NO) signaling-related pathways. Multi-omics analysis revealed differential methylation and gene expression of VEGF (↑5.6% DMR, ↑3.6-fold expression), and NOS3 (↓20.3% DMR, ↓1.6-fold expression), nodal regulators of angiogenesis and NO signaling, respectively. Conclusion In the current exploratory study, we examine glucose-dependent and dose-responsive alterations in endothelial DNA methylation to examine a putative epigenetic mechanism underlying diabetic vasculopathy. Specifically, we uncover the disproportionate glucose-dependent methylation and gene expression of VEGF and NO signaling cascades, a physiologic imbalance known to cause endothelial dysfunction in diabetes. We therefore hypothesize that epigenetic mechanisms encode a glycemic memory within endothelial cells.

Background Perturbations in myocardial substrate utilization have been proposed to contribute to the pathogenesis of cardiac dysfunction in diabetic subjects. The failing heart in nondiabetics tends to decrease reliance on fatty acid and glucose oxidation, and increases reliance on ketone body oxidation. In contrast, little is known regarding the mechanisms mediating this shift among all 3 substrates in diabetes mellitus. Therefore, we tested the hypothesis that changes in myocardial glucose utilization directly influence ketone body catabolism. Methods and Results We examined ventricular‐cardiac tissue from the following murine models: (1) streptozotocin‐induced type 1 diabetes mellitus; (2) high‐fat‐diet–induced glucose intolerance; and transgenic inducible cardiac‐restricted expression of (3) glucose transporter 4 (transgenic inducible cardiac restricted expression of glucose transporter 4); or (4) dominant negative O ‐GlcNAcase. Elevated blood glucose (type 1 diabetes mellitus and high‐fat diet mice) was associated with reduced cardiac expression of β‐hydroxybutyrate‐dehydrogenase and succinyl‐CoA:3‐oxoacid CoA transferase. Increased myocardial β‐hydroxybutyrate levels were also observed in type 1 diabetes mellitus mice, suggesting a mismatch between ketone body availability and utilization. Increased cellular glucose delivery in transgenic inducible cardiac restricted expression of glucose transporter 4 mice attenuated cardiac expression of both Bdh1 and Oxct1 and reduced rates of myocardial BDH1 activity and β‐hydroxybutyrate oxidation. Moreover, elevated cardiac protein O ‐GlcNAcylation (a glucose‐derived posttranslational modification) by dominant negative O ‐GlcNAcase suppressed β‐hydroxybutyrate dehydrogenase expression. Consistent with the mouse models, transcriptomic analysis confirmed suppression of BDH1 and OXCT1 in patients with type 2 diabetes mellitus and heart failure compared with nondiabetic patients. Conclusions Our results provide evidence that increased glucose leads to suppression of cardiac ketolytic capacity through multiple mechanisms and identifies a potential crosstalk between glucose and ketone body metabolism in the diabetic myocardium.

African Americans (AA) have an elevated risk for cardiovascular diseases compared to Caucasian Americans (CA), including heart failure (HF). Type 2 diabetes (T2D) is a major risk factor for HF that also disproportionately affects AA. These health disparities and others reduce life expectancy ~3.5 y for AA compared to CA. While prior studies have explored the connection between diabetes and heart failure, the current understanding of HF pathogenesis is based almost exclusively from studies of CA, whereas those considering race have been either epidemiologic or narrow in focus. The purpose of this study was to examine things from a different angle through the use of genome-wide RNA-sequencing to uncover how diabetes differentially or similarly affects end-stage heart failure in AA vs CA. To accomplish this, human biopsy samples were obtained from 32 age and diabetes status (T2D or non-diabetic (ND)) matched male patients undergoing left ventricle assist device surgeries (n = 8: CA-ND, CA-T2D, AA-ND, AA-T2D). Differential expression analysis was then performed using generalized linear modeling to control for clinical covariates including hypertension and coronary artery disease. Results of T2D vs ND in AA patients showed a greater number of differentially expressed genes (DEGs, P < 0.05

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear if these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy we generated transgenic mice with inducible cardiomyocyte-specific expression of the glucose transporter (GLUT4). We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in non-diabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset, exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

A longstanding challenge facing MD-PhD students and other dual-degree medical trainees is the loss of clinical knowledge that occurs during the non medical phases of training. Academic medical institutions nationwide have developed continued clinical training and exposure to maintain clinical competence; however, quantitative assessment of their usefulness remains largely unexplored. The current study therefore sought to both implement and optimize an online game platform to support MD-PhD students throughout their research training. Sixty three current MD-PhD students completing the PhD research phase of training were enrolled in an institutionally-developed online game platform for 2 preliminary and 4 competition rounds of 3-4 weeks each. During preliminary game rounds, we found that participation, though initially high, declined precipitously throughout the duration of each round, with 37 students participating to some extent. Daily reminders were implemented in subsequent rounds, which markedly improved player participation. Average participation in competition rounds exceeded 35% (23/63) active participants each round, with trending improvement in scores throughout the duration of PhD training. Both player participation and progress through the research phase of the MD-PhD program correlated positively with game performance and therefore knowledge retention and/or acquisition. Coupled with positive survey-based feedback from participants, our data therefore suggest that gamification is an effective tool for MD-PhD programs to combat loss of clinical knowledge during research training.

Objective Breakthroughs in HIV treatment, especially combination antiretroviral therapy (ART), have massively reduced AIDS-associated mortality. However, ART administration amplifies the risk of non-AIDS defining illnesses including obesity, diabetes, and cardiovascular disease, collectively known as metabolic syndrome. Initial reports suggest that ART-associated risk of metabolic syndrome correlates with socioeconomic status, a multifaceted finding that encompasses income, race, education, and diet. Therefore, determination of causal relationships is extremely challenging due to the complex interplay between viral infection, ART, and the many environmental factors. Methods In the current study, we employed a mouse model to specifically examine interactions between ART and diet that impacts energy balance and glucose metabolism. Previous studies have shown that high-fat feeding induces persistent low-grade systemic and adipose tissue inflammation contributing to insulin resistance and metabolic dysregulation via adipose-infiltrating macrophages. Studies herein test the hypothesis that ART potentiates the inflammatory effects of a high-fat diet (HFD). C57Bl/6J mice on a HFD or standard chow containing ART or vehicle, were subjected to functional metabolic testing, RNA-sequencing of epididymal white adipose tissue (eWAT), and array-based kinomic analysis of eWAT-infiltrating macrophages. Results ART-treated mice on a HFD displayed increased fat mass accumulation, impaired glucose tolerance, and potentiated insulin resistance. Gene set enrichment and kinomic array analyses revealed a pro-inflammatory transcriptional signature depicting granulocyte migration and activation. Conclusion The current study reveals a HFD-ART interaction that increases inflammatory transcriptional pathways and impairs glucose metabolism, energy balance, and metabolic dysfunction., Graphical abstract Image 1, Highlights • Antiretroviral therapy (ART) exacerbates high-fat diet induced obesity and dysregulation of glucose homeostasis. • Transcriptomic and Kinomic analyses identify increased pro-inflammatory, adipose-tissue macrophages after ART-treatment. • ART and high-fat diet synergistically induce the G-protein coupled receptor, Gpr50, in white adipose tissue.

Purpose The authors examined the usefulness of a commercially available Step 1 question bank as a formative academic support tool throughout organ-based modules in an integrated preclinical medical curriculum. The authors also determined the extent to which correlation between question bank utilization and academic metrics varied with Medical College Admission Test (MCAT) scores. Method In 2015, a cohort of 185 first-year medical students at University of Alabama School of Medicine were provided with 18-month full access to a commercially available Step 1 question bank of over 2,100 items throughout organ-based modules, although there were no requirements for use. Data on student use of the question bank were collected via an online administrative portal. Relationships between question bank utilization and academic outcomes including exams, module grades, and United States Medical Licensing Examination (USMLE) Step 1 were determined using multiple linear regression. Results MCAT scores and number of items attempted in the question bank significantly predicted all academic measures, with question bank utilization as the stronger predictor. The association between question bank utilization and academic outcome was stronger for individuals with lower MCAT scores. Conclusions The findings elucidate a novel academic support mechanism that, for some programs, may help bridge the gap between holistic and mission-based admissions practices and a residency match process that places a premium on USMLE exam scores. Distributed formative use of USMLE Step 1 practice questions may be of value as an academic support tool that benefits all students, but particularly those entering with lower MCAT scores.

Lack of the adiponectin target receptor adiponectin receptor 1 (AdipoR1) impairs gene expression of mitochondrial OXPHOS proteins due to impaired AMPK-SIRT1-PGC-1α signaling. Since decreased adiponectin serum levels in diabetes mellitus should thus compromise AdipoR1 signaling, we hypothesized that impaired AdipoR1 signaling may causally contribute to typically observed mitochondrial defects in diabetes complications. Thus, we performed comparative proteomics in cardiac, renal and hepatic tissue of AdipoR1−/− mice using LC-MS/MS. Using principal component analysis, heatmapping and hierarchical clustering, a significant separation of genotypes was observed across tissues. Enrichment analysis of differentially expressed proteins revealed disproportionate representation of proteins involved in oxidative phosphorylation, TCA cycle and fatty acid oxidation in all tissues. While 121 or 98 or 78 proteins were differentially regulated in cardiac or renal or hepatic tissue, respectively, only 15 proteins were regulated in the same direction across all tissues. Pathway analysis identified HNF4, NRF1, LONP, RICTOR, SURF1, insulin receptor and PGC-1α as most likely upstream regulators. Importantly, we found a dramatic downregulation of AdipoR1 expression in heart (−70%), liver (−90%) and kidney (−80%; all p

Human heart failure (HF) is accompanied by changes in cardiac gene expression; however, the impact of diabetes mellitus on this transcriptional regulation remains unclear. Our laboratory studies the role of epigenetics in HF and focuses on DNA methylation, commonly accepted as a negative regulator of gene expression when present in a gene’s promoter. In the current study, we hypothesized that diabetes alters cardiac DNA methylation and the transcriptome. We performed an integrated analysis of RNA-sequencing and DNA methylation using human left ventricle biopsies from HF patients to determine the effect of diabetes on this reprogramming. Our analysis identified global changes in cardiac DNA methylation as well as mRNA expression sufficient to distinguish diabetic from non-diabetic patients. Specifically, we identified significant regulation of 1,133 genes (|1.5| fold-change, P

Pancreatic β-cells undergo profound hyperplasia during pregnancy to maintain maternal euglycemia. Failure to reprogram β-cells into a more replicative state has been found to underlie susceptibility to gestational diabetes (GDM). Our laboratory has recently identified a requirement for prolactin receptor (PRLR) signaling in the metabolic adaptations to pregnancy, where mice lacking β-cell PRLR (βPRLRKO) exhibit a metabolic phenotype consistent with GDM. However, the underlying transcriptional program that is responsible for the PRLR-dependent metabolic adaptations during gestation remains incompletely understood. To identify PRLR signaling gene regulatory networks and target genes within β-cells during pregnancy, we performed a transcriptomic analysis ofpancreatic islets isolated from either βPRLRKO mice or littermate controls in late gestation.Gene set enrichment analysis identified Forkhead box protein M1 (Foxm1) and polycomb repressor complex 2 (PRC2) subunitsSuz12 andEzh2 as novel candidate regulators of PRLR-dependent down-regulated and up-regulated genes, respectively. GO-term pathway enrichment revealed both established and novel PRLR signaling target genes that together describe a state of increased cellular metabolism and/or proliferation. In contrast to the requirement forβ-cell PRLR signaling in maintaining euglycemia during pregnancy, PRLR target genes were not induced following high-fat-diet feeding. Altogether the current study develops our understanding of genes and transcriptional regulators mediating islet adaptation during pregnancy and suggests pregnancy-specific adaptive mechanisms distinct from those activated bynutritional stress.

Acute kidney injury (AKI) is a devastating clinical condition affecting at least two-thirds of critically ill patients, and, among these patients, it is associated with a greater than 60% risk of mortality. Kidney mononuclear phagocytes (MPs) are implicated in pathogenesis and healing in mouse models of AKI and, thus, have been the subject of investigation as potential targets for clinical intervention. We have determined that, after injury, F4/80(hi)-expressing kidney-resident macrophages (KRMs) are a distinct cellular subpopulation that does not differentiate from nonresident infiltrating MPs. However, if KRMs are depleted using polyinosinic/polycytidylic acid (poly I:C), they can be reconstituted from bone marrow–derived precursors. Further, KRMs lack major histocompatibility complex class II (MHCII) expression before P7 but upregulate it over the next 14 days. This MHCII(–) KRM phenotype reappears after injury. RNA sequencing shows that injury causes transcriptional reprogramming of KRMs such that they more closely resemble that found at P7. KRMs after injury are also enriched in Wingless-type MMTV integration site family (Wnt) signaling, indicating that a pathway vital for mouse and human kidney development is active. These data indicate that mechanisms involved in kidney development may be functioning after injury in KRMs.

Background: Transient activation of nuclear factor, erythroid 2 like 2 (Nfe2l2/Nrf2), a master regulator of antioxidant transcription, protects from oxidative insult. However, sustained Nrf2 signaling causes proteotoxic reductive stress (RS). Using Nrf2 - / - mice, we recently uncovered several cardiac microRNAs (miRNAs) potentially expressed in an Nrf2-dependent manner. Here, we utilized cardiac-specific constitutively active Nrf2 transgenic (caNrf2-Tg) mice to elucidate necessary and sufficient miRNAs, and hypothesized that miRNA dysregulation underlies RS. Methods: Next-generation RNA sequencing (RNAseq) was used to compare mRNA and miRNA transcriptomes of 6-month low (TgL) and high (TgH) transgene-expressing caNrf2-Tg mice to non-transgenic (NTg) controls (n=3-4/group). Differentially expressed genes (DEGs) were run through Ingenuity Pathway Analysis (IPA). Real-time qPCR validated RNAseq results (n=4-6/group). In silico target prediction correlated miRNA changes with corresponding mRNA levels. Results: Principal component and hierarchal clustering analyses revealed distinct TgL and TgH transcriptomes. Relative to NTg, TgL mice exhibited 246 DEGs, 214 of which were consistent in TgH hearts. Strikingly, TgH mice displayed 1031 DEGs. As expected, IPA of DEGs indicated enhanced free radical scavenging; however, caNrf2 expression also dose-dependently enriched hypertrophic signaling, protein ubiquitination and the unfolded protein response. While 16 miRNAs were significantly altered in TgL hearts, a total of 101 miRNAs were detected TgH mice, several of which were predicted to account for mRNA changes in RS pathways. Notably, miR-671-3p and miR-455-5p, two miRNAs decreased in Nrf2 - / - mice, were reciprocally increased in caNrf2-Tg mice suggesting that Nrf2 is necessary and sufficient for their function in the heart. Conclusion: While RS underlies human mutant protein aggregation cardiomyopathy, molecular determinants of pathogenesis downstream of Nrf2 remain unknown. Here, we identified key transcriptional responses and pathways associated with RS. These results link post-transcriptional regulation with redox imbalances in the heart, and highlight novel miRNAs for future mechanistic studies.

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss. We have identified that the hepatic Farnesol X Receptor (FXR) plays an important role in an anti-obesogenic effect of glucagon receptor (GcgR) agonism. Chronic administration of IUB288, a potent GcgR agonist, significantly reduced diet-induced obesity (DIO) in wild type (WT, 17%) mice compared to (FXR∆liver, 6%) liver-specific FXR knock-out mice. Weight loss was associated with increased energy expenditure (p Disclosure T. Kim: None. S. Nason: None. J.P. Antipenko: None. C.S. Vestri: None. K. Smith-Johnston: None. M.E. Pepin: None. A.R. Wende: None. B. Finan: Employee; Self; Novo Nordisk Inc. R. DiMarchi: Employee; Self; Novo Nordisk Inc. D. Perez-Tilve: Research Support; Self; Novo Nordisk A/S, Cohbar. D.R. Moellering: None. K.M. Habegger: Consultant; Self; Glyscend, Inc.. Research Support; Self; Glyscend, Inc.. Consultant; Self; Intarcia Therapeutics, Inc..

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss, in part through potentiation of fibroblast growth factor 21 (FGF21) secretion. However, FGF21 is only a partial mediator of metabolic actions ensuing from glucagon receptor (GCGR) activation, prompting us to search for additional pathways. Intriguingly, chronic GCGR agonism increases plasma bile acid levels. We hypothesized that GCGR agonism regulates energy metabolism, at least in part, through farnesoid X receptor (FXR). To test this hypothesis, we studied whole-body and liver-specific FXR-knockout (Fxr∆liver) mice. Chronic GCGR agonist (IUB288) administration in diet-induced obese (DIO) Gcgr, Fgf21, and Fxr whole-body or liver-specific knockout (∆liver) mice failed to reduce body weight when compared with wild-type (WT) mice. IUB288 increased energy expenditure and respiration in DIO WT mice, but not Fxr∆liver mice. GCGR agonism increased [14C]palmitate oxidation in hepatocytes isolated from WT mice in a dose-dependent manner, an effect blunted in hepatocytes from Fxr∆liver mice. Our data clearly demonstrate that control of whole-body energy expenditure by GCGR agonism requires intact FXR signaling in the liver. This heretofore-unappreciated aspect of glucagon biology has implications for the use of GCGR agonism in the therapy of metabolic disorders.

To identify the role of glucose in the development of diabetic cardiomyopathy, we had directly assessed glucose delivery to the intact heart on alterations of DNA methylation and gene expression using both an inducible heart-specific transgene (glucose transporter 4; mG4H) and streptozotocin-induced diabetes (STZ) mouse models. We aimed to determine whether long-lasting diabetic complications arise from prior transient exposure to hyperglycemia via a process termed “glycemic memory.” We had identified DNA methylation changes associated with significant gene expression regulation. Comparing our results from STZ, mG4H, and the modifications which persist following transgene silencing, we now provide evidence for cardiac DNA methylation as a persistent epigenetic mark contributing to glycemic memory. To begin to determine which changes contribute to human heart failure, we measured both RNA transcript levels and whole-genome DNA methylation in heart failure biopsy samples (n = 12) from male patients collected at left ventricular assist device placement using RNA-sequencing and Methylation450 assay, respectively. We hypothesized that epigenetic changes such as DNA methylation distinguish between heart failure etiologies. Our findings demonstrated that type 2 diabetic heart failure patients (n = 6) had an overall signature of hypomethylation, whereas patients listed as ischemic (n = 5) had a distinct hypermethylation signature for regulated transcripts. The focus of this initial analysis was on promoter-associated CpG islands with inverse changes in gene transcript levels, from which diabetes (14 genes; e.g. IGFBP4) and ischemic (12 genes; e.g. PFKFB3) specific targets emerged with significant regulation of both measures. By combining our mouse and human molecular analyses, we provide evidence that diabetes mellitus governs direct regulation of cellular function by DNA methylation and the corresponding gene expression in diabetic mouse and human hearts. Importantly, many of the changes seen in either mouse type 1 diabetes or human type 2 diabetes were similar supporting a consistent mechanism of regulation. These studies are some of the first steps at defining mechanisms of epigenetic regulation in diabetic cardiomyopathy.

Pathological cardiac hypertrophy may be associated with reduced expression of glucose transporter 4 (GLUT4) in contrast to exercise-induced cardiac hypertrophy, where GLUT4 levels are increased. However, mice with cardiac-specific deletion of GLUT4 (G4H−/−) have normal cardiac function in the unstressed state. This study tested the hypothesis that cardiac GLUT4 is required for myocardial adaptations to hemodynamic demands. G4H−/−and control littermates were subjected to either a pathological model of left ventricular pressure overload [transverse aortic constriction (TAC)] or a physiological model of endurance exercise (swim training). As predicted after TAC, G4H−/−mice developed significantly greater hypertrophy and more severe contractile dysfunction. Somewhat surprisingly, after exercise training, G4H−/−mice developed increased fibrosis and apoptosis that was associated with dephosphorylation of the prosurvival kinase Akt in concert with an increase in protein levels of the upstream phosphatase protein phosphatase 2A (PP2A). Exercise has been shown to decrease levels of ceramide; G4H−/−hearts failed to decrease myocardial ceramide in response to exercise. Furthermore, G4H−/−hearts have reduced levels of the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1, lower carnitine palmitoyl-transferase activity, and reduced hydroxyacyl-CoA dehydrogenase activity. These basal changes may also contribute to the impaired ability of G4H−/−hearts to adapt to hemodynamic stresses. In conclusion, GLUT4 is required for the maintenance of cardiac structure and function in response to physiological or pathological processes that increase energy demands, in part through secondary changes in mitochondrial metabolism and cellular stress survival pathways such as Akt.NEW & NOTEWORTHY Glucose transporter 4 (GLUT4) is required for myocardial adaptations to exercise, and its absence accelerates heart dysfunction after pressure overload. The requirement for GLUT4 may extend beyond glucose uptake to include defects in mitochondrial metabolism and survival signaling pathways that develop in its absence. Therefore, GLUT4 is critical for responses to hemodynamic stresses.

A thermally responsive copolymer is designed to modulate the properties of an electrolyte solution. The copolymer is prepared using pNIPAM, which governs the thermal properties, and acrylic acid, which provides the electrolyte ions. As the polymer undergoes a thermally activated phase transition, the local environment around the acid groups is reversibly switched, decreasing ion concentration and conductivity. The responsive electrolyte is used to control the activity of redox electrodes with temperature.

Myostatin is a TGFβ family member and negative regulator of muscle size. Due to the complexity of the molecular pathway between myostatin mRNA/protein and changes in transcription, it has been difficult to understand whether myostatin plays a role in resistance exercise-induced skeletal muscle hypertrophy. To circumvent this problem, we determined the expression of a unique myostatin target gene, Mighty, following resistance exercise. Mighty mRNA increased by 6 h (82.9 ± 24.21%) and remained high out to 48 h (56.5 ± 19.67%) after resistance exercise. Further examination of the soleus, plantaris and tibialis anterior muscles showed that the change in Mighty mRNA at 6 h correlated with the increase in muscle size associated with this protocol (R(2) = 0.9996). The increase in Mighty mRNA occurred both independent of Smad2 phosphorylation and in spite of an increase in myostatin mRNA (341.8 ± 147.14% at 3 h). The myostatin inhibitor SKI remained unchanged. However, activated Notch, another potential inhibitor of TGFβ signaling, increased immediately following resistance exercise (83 ± 11.2%) and stayed elevated out to 6 h (78 ± 16.6%). Electroportion of the Notch intracellular domain into the tibialis anterior resulted in an increase in Mighty mRNA (63 ± 13.4%) that was equivalent to the canonical Notch target HES-1 (94.4 ± 7.32%). These data suggest that acute resistance exercise decreases myostatin signaling through the activation of the TGFβ inhibitor Notch resulting in a decrease in myostatin transcriptional activity that correlates well with muscle hypertrophy.