Susceptibility to innate immune activation in genetically-mediated myocarditis.

BACKGROUND: Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment for myocarditis is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis with biomarkers and pathological features indistinguishable from other forms of myocarditis. DSP-associated myocarditis can progress to dilated cardiomyopathy with heightened arrhythmia risk. METHODS: To model the cardiomyocyte aspects of DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients and gene-edited healthy control hiPSC lines. Homozygous and heterozygous DSP disrupted EHTs were generated to contain 90% hiPSC-CMs and 10% healthy control human cardiac fibroblasts. We measured innate immune activation and function at baseline and in response to Toll-like receptor (TLR) stimulation in EHTs. RESULTS: At baseline, DSP-/- EHTs displayed a transcriptomic signature of immune activation which was mirrored by EHT cytokine release. Importantly, DSP-/- EHTs were hypersensitive to TLR stimulation demonstrating greater contractile function impairment compared to isogenic controls. Compared to homozygous DSP-/- EHTs, heterozygous DSP patient-derived EHTs had less functionally impairment but also displayed heightened sensitivity to TLR stimulation. When subjected to strain, heterozygous DSP EHTs developed greater functional deficit indicating reduced contractile reserve compared to healthy control. Colchicine or NFΚB inhibitors improved baseline force production and strain-induced force deficits in DSP EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. CONCLUSIONS: Genetic reduction of DSP renders cardiomyocytes susceptible to innate immune activation and strain-dependent contractile deficits. EHTs replicate electrical and contractile phenotypes seen in human myocarditis implicating cytokine release as a key part of the myogenic susceptibility to inflammation. This heightened innate immune activation and sensitivity is a target for clinical intervention.

The impact of underrepresented minority or marginalized identity status on training outcomes of MD-PhD students.

Dual-degree MD-PhD programs have historically lacked diversity of race, ethnicity, gender, sexual orientation, and other facets of identity. Like MD- and PhD-granting programs, MD-PhD program training environments are also marked by structural barriers that negatively impact measurable academic outcomes of underrepresented and/or marginalized students in academic medicine (racial and ethnic minority groups considered underrepresented by the National Institute of Health, sexual and gender minorities, individuals with disabilities, and individuals of low socioeconomic status). In this article, we review the existing literature on MD-PhD program disparities affecting students from these groups and provide recommendations grounded on the reviewed evidence. Our literature review identified four generalizable barriers that can impact the training outcomes of students from these marginalized and/or underrepresented groups: 1) discrimination and bias, 2) impostor syndrome and stereotype threat, 3) lack of identity-similar mentors, and 4) suboptimal institutional policies and procedures. We propose goal-oriented interventions that may begin to ameliorate the disparities present in MD-PhD program training environments that affect students from marginalized and/or underrepresented groups in academic medicine.

Virtual Pathology Elective Provides Uninterrupted Medical Education and Impactful Pathology Education During the COVID-19 Pandemic.

As students do not qualify as essential health care workers, medical education faced severe disruptions during the COVID-19 pandemic including initial suspension of all in-person lectures and on-site rotations. Our Pathology Department was among the first at Northwestern to offer a completely virtual rotation with the goals of: (1) providing a comprehensive introduction to the practice of anatomic and clinical pathology, (2) emphasizing uninterrupted and continued excellence in education, and (3) minimizing exposure risk during the pandemic. The innovative 2-week curriculum incorporated diverse teaching modalities including live and recorded lectures; live and recorded video demonstrations; interactive small group discussions; interactive virtual sign-outs; and written and multimedia assignments, quizzes, and projects. The virtual elective ran from March to July 2020 with 52 total participating medical students. On post-rotation evaluations, students rated the pathology virtual elective 4.7/5.0 compared to other virtual rotations and 4.0/5.0 compared to all rotations (including in-person and virtual). Furthermore, continual improvements were made to the established framework based on rotation feedback such that curriculum content was more abundant and more favorably rated by the last cohort when compared to the first. Finally, although students identified interest in over 10 different medical specialties, all participants expressed increased interest in choosing pathology as a specialty and better understanding of pathology's role in patient care. We hope our detailed description of creating and evaluating a completely virtual elective rotation serves as a model for other departments to improve pathology education and visibility.

Epigenetic reprogramming to prevent genetic cardiomyopathy.

Mutations in the gene that codes for lamin A/C (LMNA) are a common cause of adult-onset cardiomyopathy and heart failure. In this issue of the JCI, Guénantin and Jebeniani et al. identify impaired cardiomyocyte development and maturation as a prenatal feature in a model of laminopathy. Cardiomyocytes carrying the Lmna point mutation H222P misexpressed genes involved in the epithelial-mesenchymal transition and showed decreased methylation at the fourth lysine of histone H3 (H3K4). Notably, inhibiting lysine-specific demethylase 1 in the LMNA H222P mouse model treated this congenital form of cardiomyopathy and improved survival in utero. These data highlight early epigenomic modifications in lamin A/C-mediated pathology and indicate a unique therapeutic strategy for cardiomyopathy.

Gut intraepithelial T cells calibrate metabolism and accelerate cardiovascular disease.

The biochemical response to food intake must be precisely regulated. Because ingested sugars and fats can feed into many anabolic and catabolic pathways1, how our bodies handle nutrients depends on strategically positioned metabolic sensors that link the intrinsic nutritional value of a meal with intermediary metabolism. Here we describe a subset of immune cells-integrin ß7+ natural gut intraepithelial T lymphocytes (natural IELs)-that is dispersed throughout the enterocyte layer of the small intestine and that modulates systemic metabolism. Integrin ß7- mice that lack natural IELs are metabolically hyperactive and, when fed a high-fat and high-sugar diet, are resistant to obesity, hypercholesterolaemia, hypertension, diabetes and atherosclerosis. Furthermore, we show that protection from cardiovascular disease in the absence of natural IELs depends on the enteroendocrine-derived incretin GLP-12, which is normally controlled by IELs through expression of the GLP-1 receptor. In this metabolic control system, IELs modulate enteroendocrine activity by acting as gatekeepers that limit the bioavailability of GLP-1. Although the function of IELs may prove advantageous when food is scarce, present-day overabundance of diets high in fat and sugar renders this metabolic checkpoint detrimental to health.

Protein scaffold-activated protein trans-splicing in mammalian cells.

Conditional protein splicing is a powerful biotechnological tool that can be used to rapidly and post-translationally control the activity of a given protein. Here we demonstrate a novel conditional splicing system in which a genetically encoded protein scaffold induces the splicing and activation of an enzyme in mammalian cells. In this system the protein scaffold binds to two inactive split intein/enzyme extein protein fragments leading to intein fragment complementation, splicing, and activation of the firefly luciferase enzyme. We first demonstrate the ability of antiparallel coiled-coils (CCs) to mediate splicing between two intein fragments, effectively creating two new split inteins. We then generate and test two versions of the scaffold-induced splicing system using two pairs of CCs. Finally, we optimize the linker lengths of the proteins in the system and demonstrate 13-fold activation of luciferase by the scaffold compared to the activity of negative controls. Our protein scaffold-triggered conditional splicing system is an effective strategy to control enzyme activity using a protein input, enabling enhanced genetic control over protein splicing and the potential creation of splicing-based protein sensors and autoregulatory systems.