Sarcopenia and aortic valve disease.

Valvular heart disease, including calcific or degenerative aortic stenosis (AS), is increasingly prevalent among the older adult population. Over the last few decades, treatment of severe AS has been revolutionised following the development of transcatheter aortic valve replacement (TAVR). Despite improvements in outcomes, older adults with competing comorbidities and geriatric syndromes have suboptimal quality of life outcomes, highlighting the cumulative vulnerability that persists despite valve replacement. Sarcopenia, characterised by loss of muscle strength, mass and function, affects 21%-70% of older adults with AS. Sarcopenia is an independent predictor of short-term and long-term outcomes after TAVR and should be incorporated as a prognostic marker in preprocedural planning. Early diagnosis and treatment of sarcopenia may reduce morbidity and mortality and improve quality of life following TAVR. The adverse effects of sarcopenia can be mitigated through resistance training and optimisation of nutritional status. This is most efficacious when administered before sarcopenia has progressed to advanced stages. Management should be individualised based on the patient's wishes/preferences, care goals and physical capability. Exercise during the preoperative waiting period may be safe and effective in most patients with severe AS. However, future studies are needed to establish the benefits of prehabilitation in improving quality of life outcomes after TAVR procedures.

CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants.

BACKGROUND: TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity. METHODS: We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy-associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation. RESULTS: CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts. CONCLUSIONS: TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.

Cross-species single-cell comparison of systemic and cardiac inflammatory responses after cardiac injury.

The immune system coordinates the response to cardiac injury and is known to control regenerative and fibrotic scar outcomes in the heart and subsequent chronic low-grade inflammation associated with heart failure. Here we profiled the inflammatory response to heart injury using single cell transcriptomics to compare and contrast two experimental models with disparate outcomes. We used adult mice, which like humans lack the ability to fully recover and zebrafish which spontaneously regenerate after heart injury. The extracardiac reaction to cardiomyocyte necrosis was also interrogated to assess the specific peripheral tissue and immune cell reaction to chronic stress. Cardiac macrophages are known to play a critical role in determining tissue homeostasis by healing versus scarring. We identified distinct transcriptional clusters of monocytes/macrophages in each species and found analogous pairs in zebrafish and mice. However, the reaction to myocardial injury was largely disparate between mice and zebrafish. The dichotomous response to heart damage between the mammalian and zebrafish monocytes/macrophages may underlie the impaired regenerative process in mice, representing a future therapeutic target.

Development of an improved and specific inhibitor of NADPH oxidase 2 to treat traumatic brain injury.

NADPH oxidases (NOX's), and the reactive oxygen species (ROS) they produce, play an important role in host defense, thyroid hormone synthesis, apoptosis, gene regulation, angiogenesis and other processes. However, overproduction of ROS by these enzymes is associated with cardiovascular disease, fibrosis, traumatic brain injury (TBI) and other diseases. Structural similarities between NOX's have complicated development of specific inhibitors. Here, we report development of NCATS-SM7270, a small molecule optimized from GSK2795039, that inhibited NOX2 in primary human and mouse granulocytes. NCATS-SM7270 specifically inhibited NOX2 and had reduced inhibitory activity against xanthine oxidase in vitro. We also studied the role of several NOX isoforms during mild TBI (mTBI) and demonstrated that NOX2 and, to a lesser extent, NOX1 deficient mice are protected from mTBI pathology, whereas injury is exacerbated in NOX4 knockouts. Given the pathogenic role played by NOX2 in mTBI, we treated mice transcranially with NCATS-SM7270 after injury and revealed a dose-dependent reduction in mTBI induced cortical cell death. This inhibitor also partially reversed cortical damage observed in NOX4 deficient mice following mTBI. These data demonstrate that NCATS-SM7270 is an improved and specific inhibitor of NOX2 capable of protecting mice from NOX2-dependent cell death associated with mTBI.

Spontaneous Coronary Artery Dissection Following SARS-CoV-2-Associated Multisystem Inflammatory Syndrome.

Spontaneous coronary artery dissection (SCAD) is an underdiagnosed cause of acute coronary syndrome, myocardial infarction, and sudden cardiac death. During the coronavirus disease 2019 (COVID-19) pandemic, a multisystem inflammatory syndrome (MIS) emerged that is incompletely understood. While the involvement of numerous organ systems has been described, the potential cardiovascular manifestations, such as myocarditis, arterial thrombosis, or SCAD, are particularly worrisome. Here, we present a case of MIS that was preceded by an unremarkable case of COVID-19 and followed by the development of SCAD. This case highlights the importance of furthering our understanding of the potential sequelae of COVID-19 and of the potential relationship between SCAD and MIS.

The Cardiac Sarcomere and Cell Cycle.

PURPOSE OF REVIEW: The lack of adult human cardiomyocyte proliferative capacity impairs cardiac regeneration such as after myocardial injury. The sarcomere, a specialized actin cytoskeletal structure that is essential for twitch contraction in cardiomyocytes, has been considered a critical factor limiting adult human cardiomyocyte proliferation through incompletely understood mechanisms. RECENT FINDINGS: This review summarizes known and emerging regulatory mechanisms connecting the human cardiomyocyte sarcomere to cell cycle regulation including structural and signaling mechanisms. Cardiac regeneration could be augmented through targeting the inhibitory effects of the sarcomere on cardiomyocyte proliferation.

Medical student residency preferences and motivational factors: a longitudinal, single-institution perspective.

BACKGROUND: A high proportion of medical school graduates pursue specialties different from those declared at matriculation. While these choices influence the career paths, satisfaction, and potential regret students will experience, they also impact the supply and demand ratio of the shorthanded physician workforce across many specialties. In this study, we investigate how the choice of medical specialty and the factors motivating those choices change between the beginning and end of medical school training. METHODS: A questionnaire was administered annually from 2017 to 2020 to a cohort of medical students at the University of Connecticut to determine longitudinal preferences regarding residency choice, motivational factors influencing residency choice, future career path, and demographic information. RESULTS: The questionnaire respondent totals were as follows: n = 76 (Year 1), n = 54 (Year 2), n = 31 (Year 3), and n = 65 (Year 4). Amongst newly matriculated students, 25.0% were interested in primary care, which increased ~ 1.4-fold to 35.4% in the final year of medical school. In contrast, 38.2% of matriculated students expressed interest in surgical specialties, which decreased ~ 2.5-fold to 15.4% in the final year. Specialty choices in the final year that exhibited the largest absolute change from matriculation were orthopedic surgery (- 9.9%), family medicine (+ 8.1%), radiology (+ 7.9%), general surgery (- 7.2%), and anesthesiology (+ 6.2%). Newly matriculated students interested in primary care demonstrated no differences in their ranking of motivational factors compared to students interested in surgery, but many of these factors significantly deviated between the two career paths in the final year. Specifically, students interested in surgical specialties were more motivated by the rewards of salary and prestige compared to primary care students, who more highly ranked match confidence and family/location factors. CONCLUSIONS: We identified how residency choices change from the beginning to the end of medical school, how certain motivational factors change with time, how these results diverge between primary care and surgery specialty choice, and propose a new theory based on risk-reward balance regarding residency choice. Our study promotes awareness of student preferences and may help guide school curricula in developing more student-tailored training approaches. This could foster positive long-term changes regarding career satisfaction and the physician workforce.

Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions.

BACKGROUND: Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, titin (TTN) functions, and therapeutic targets. METHODS: We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Last, we engineered a full-length TTN protein reporter assay and used next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. RESULTS: An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome editing using SpCas9 and a TTNtv-specific guide RNA restored the TTN protein reading frame, which increased full-length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. CONCLUSIONS: An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. Although both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a one-and-done genome editing strategy to target ≈30% of DCM-associated TTNtvs.

Actinin BioID reveals sarcomere crosstalk with oxidative metabolism through interactions with IGF2BP2.

Actinins are strain-sensing actin cross-linkers that are ubiquitously expressed and harbor mutations in human diseases. We utilize CRISPR, pluripotent stem cells, and BioID to study actinin interactomes in human cardiomyocytes. We identify 324 actinin proximity partners, including those that are dependent on sarcomere assembly. We confirm 19 known interactors and identify a network of RNA-binding proteins, including those with RNA localization functions. In vivo and biochemical interaction studies support that IGF2BP2 localizes electron transport chain transcripts to actinin neighborhoods through interactions between its K homology (KH) domain and actinin's rod domain. We combine alanine scanning mutagenesis and metabolic assays to disrupt and functionally interrogate actinin-IGF2BP2 interactions, which reveal an essential role in metabolic responses to pathological sarcomere activation using a hypertrophic cardiomyopathy model. This study expands our functional knowledge of actinin, uncovers sarcomere interaction partners, and reveals sarcomere crosstalk with IGF2BP2 for metabolic adaptation relevant to human disease.

Sarcomere function activates a p53-dependent DNA damage response that promotes polyploidization and limits in vivo cell engraftment.

Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse imaging, in vitro cardiomyocyte replication patterns recapitulate the progressive mononuclear polyploidization and replicative arrest observed in vivo. Single-cell transcriptomics and chromatin state analyses reveal that polyploidization is preceded by sarcomere assembly, enhanced oxidative metabolism, a DNA damage response, and p53 activation. CRISPR knockout screening reveals p53 as a driver of cell-cycle arrest and polyploidization. Inhibiting sarcomere function, or scavenging ROS, inhibits cell-cycle arrest and polyploidization. Finally, we show that cardiomyocyte engraftment in infarcted rat hearts is enhanced 4-fold by the increased proliferation of troponin-knockout cardiomyocytes. Thus, the sarcomere inhibits cell division through a DNA damage response that can be targeted to improve cardiomyocyte replacement strategies.

Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human TNNT2 Variants.

BACKGROUND: Pathogenic TNNT2 variants are a cause of hypertrophic and dilated cardiomyopathies, which promote heart failure by incompletely understood mechanisms. The precise functional significance for 87% of TNNT2 variants remains undetermined, in part, because of a lack of functional genomics studies. The knowledge of which and how TNNT2 variants cause hypertrophic and dilated cardiomyopathies could improve heart failure risk determination, treatment efficacy, and therapeutic discovery, and provide new insights into cardiomyopathy pathogenesis, as well. METHODS: We created a toolkit of human induced pluripotent stem cell models and functional assays using CRISPR/Cas9 to study TNNT2 variant pathogenicity and pathophysiology. Using human induced pluripotent stem cell-derived cardiomyocytes in cardiac microtissue and single-cell assays, we functionally interrogated 51 TNNT2 variants, including 30 pathogenic/likely pathogenic variants and 21 variants of uncertain significance. We used RNA sequencing to determine the transcriptomic consequences of pathogenic TNNT2 variants and adapted CRISPR/Cas9 to engineer a transcriptional reporter assay to assist prediction of TNNT2 variant pathogenicity. We also studied variant-specific pathophysiology using a thin filament-directed calcium reporter to monitor changes in myofilament calcium affinity. RESULTS: Hypertrophic cardiomyopathy-associated TNNT2 variants caused increased cardiac microtissue contraction, whereas dilated cardiomyopathy-associated variants decreased contraction. TNNT2 variant-dependent changes in sarcomere contractile function induced graded regulation of 101 gene transcripts, including MAPK (mitogen-activated protein kinase) signaling targets, HOPX, and NPPB. We distinguished pathogenic TNNT2 variants from wildtype controls using a sarcomere functional reporter engineered by inserting tdTomato into the endogenous NPPB locus. On the basis of a combination of NPPB reporter activity and cardiac microtissue contraction, our study provides experimental support for the reclassification of 2 pathogenic/likely pathogenic variants and 2 variants of uncertain significance. CONCLUSIONS: Our study found that hypertrophic cardiomyopathy-associated TNNT2 variants increased cardiac microtissue contraction, whereas dilated cardiomyopathy-associated variants decreased contraction, both of which paralleled changes in myofilament calcium affinity. Transcriptomic changes, including NPPB levels, directly correlated with sarcomere function and can be used to predict TNNT2 variant pathogenicity.

A Contraction Stress Model of Hypertrophic Cardiomyopathy due to Sarcomere Mutations.

Thick-filament sarcomere mutations are a common cause of hypertrophic cardiomyopathy (HCM), a disorder of heart muscle thickening associated with sudden cardiac death and heart failure, with unclear mechanisms. We engineered four isogenic induced pluripotent stem cell (iPSC) models of ß-myosin heavy chain and myosin-binding protein C3 mutations, and studied iPSC-derived cardiomyocytes in cardiac microtissue assays that resemble cardiac architecture and biomechanics. All HCM mutations resulted in hypercontractility with prolonged relaxation kinetics in proportion to mutation pathogenicity, but not changes in calcium handling. RNA sequencing and expression studies of HCM models identified p53 activation, oxidative stress, and cytotoxicity induced by metabolic stress that can be reversed by p53 genetic ablation. Our findings implicate hypercontractility as a direct consequence of thick-filament mutations, irrespective of mutation localization, and the p53 pathway as a molecular marker of contraction stress and candidate therapeutic target for HCM patients.

Simultaneous Host-Pathogen Transcriptome Analysis during Granulibacter bethesdensis Infection of Neutrophils from Healthy Subjects and Patients with Chronic Granulomatous Disease.

Polymorphonuclear leukocytes (PMN) from patients with chronic granulomatous disease (CGD) fail to produce microbicidal concentrations of reactive oxygen species (ROS) due to mutations in NOX2. Patients with CGD suffer from severe, life-threatening infections and inflammatory complications. Granulibacter bethesdensis is an emerging Gram-negative pathogen in CGD that resists killing by PMN of CGD patients (CGD PMN) and inhibits PMN apoptosis through unknown mechanisms. Microarray analysis was used to study mRNA expression in PMN from healthy subjects (normal PMN) and CGD PMN during incubation with G. bethesdensis and, simultaneously, in G. bethesdensis with normal and CGD PMN. We detected upregulation of antiapoptotic genes (e.g., XIAP and GADD45B) and downregulation of proapoptotic genes (e.g., CASP8 and APAF1) in infected PMN. Transcript and protein levels of inflammation- and immunity-related genes were also altered. Upon interaction with PMN, G. bethesdensis altered the expression of ROS resistance genes in the presence of normal but not CGD PMN. Levels of bacterial stress response genes, including the ClpB gene, increased during phagocytosis by both normal and CGD PMN demonstrating responses to oxygen-independent PMN antimicrobial systems. Antisense knockdown demonstrated that ClpB is dispensable for extracellular growth but is essential for bacterial resistance to both normal and CGD PMN. Metabolic adaptation of Granulibacter growth in PMN included the upregulation of pyruvate dehydrogenase. Pharmacological inhibition of pyruvate dehydrogenase by triphenylbismuthdichloride was lethal to Granulibacter. This study expands knowledge of microbial pathogenesis of Granulibacter in cells from permissive (CGD) and nonpermissive (normal) hosts and identifies potentially druggable microbial factors, such as pyruvate dehydrogenase and ClpB, to help combat this antibiotic-resistant pathogen.