Transcriptomic and spatial dissection of human ex vivo right atrial tissue reveals proinflammatory microvascular changes in ischemic heart disease.

Cardiovascular disease plays a central role in the electrical and structural remodeling of the right atrium, predisposing to arrhythmias, heart failure, and sudden death. Here, we dissect with single-nuclei RNA sequencing (snRNA-seq) and spatial transcriptomics the gene expression changes in the human ex vivo right atrial tissue and pericardial fluid in ischemic heart disease, myocardial infarction, and ischemic and non-ischemic heart failure using asymptomatic patients with valvular disease who undergo preventive surgery as the control group. We reveal substantial differences in disease-associated gene expression in all cell types, collectively suggesting inflammatory microvascular dysfunction and changes in the right atrial tissue composition as the valvular and vascular diseases progress into heart failure. The data collectively suggest that investigation of human cardiovascular disease should expand to all functionally important parts of the heart, which may help us to identify mechanisms promoting more severe types of the disease.

Artificial intelligence-enabled prediction of chemotherapy-induced cardiotoxicity from baseline electrocardiograms.

Anthracyclines can cause cancer therapy-related cardiac dysfunction (CTRCD) that adversely affects prognosis. Despite guideline recommendations, only half of the patients undergo surveillance echocardiograms. An AI model detecting reduced left ventricular ejection fraction from 12-lead electrocardiograms (ECG) (AI-EF model) suggests ECG features reflect left ventricular pathophysiology. We hypothesized that AI could predict CTRCD from baseline ECG, leveraging the AI-EF model's insights, and developed the AI-CTRCD model using transfer learning on the AI-EF model. In 1011 anthracycline-treated patients, 8.7% experienced CTRCD. High AI-CTRCD scores indicated elevated CTRCD risk (hazard ratio (HR), 2.66; 95% CI 1.73-4.10; log-rank p < 0.001). This remained consistent after adjusting for risk factors (adjusted HR, 2.57; 95% CI 1.62-4.10; p < 0.001). AI-CTRCD score enhanced prediction beyond known factors (time-dependent AUC for 2 years: 0.78 with AI-CTRCD score vs. 0.74 without; p = 0.005). In conclusion, the AI model robustly stratified CTRCD risk from baseline ECG.

Prdm16 mutation determines sex-specific cardiac metabolism and identifies two novel cardiac metabolic regulators.

AIMS: Mutation of the PRDM16 gene causes human dilated and non-compaction cardiomyopathy. The PRDM16 protein is a transcriptional regulator that affects cardiac development via Tbx5 and Hand1, thus regulating myocardial structure. The biallelic inactivation of Prdm16 induces severe cardiac dysfunction with post-natal lethality and hypertrophy in mice. The early pathological events that occur upon Prdm16 inactivation have not been explored. METHODS AND RESULTS: This study performed in-depth pathophysiological and molecular analyses of male and female Prdm16csp1/wt mice that carry systemic, monoallelic Prdm16 gene inactivation. We systematically assessed early molecular changes through transcriptomics, proteomics, and metabolomics. Kinetic modelling of cardiac metabolism was performed in silico with CARDIOKIN. Prdm16csp1/wt mice are viable up to 8 months, develop hypoplastic hearts, and diminished systolic performance that is more pronounced in female mice. Prdm16csp1/wt cardiac tissue of both sexes showed reductions in metabolites associated with amino acid as well as glycerol metabolism, glycolysis, and the tricarboxylic acid cycle. Prdm16csp1/wt cardiac tissue revealed diminished glutathione (GSH) and increased inosine monophosphate (IMP) levels indicating oxidative stress and a dysregulated energetics, respectively. An accumulation of triacylglycerides exclusively in male Prdm16csp1/wt hearts suggests a sex-specific metabolic adaptation. Metabolic modelling using CARDIOKIN identified a reduction in fatty acid utilization in males as well as lower glucose utilization in female Prdm16csp1/wt cardiac tissue. On the level of transcripts and protein expression, Prdm16csp1/wt hearts demonstrate an up-regulation of pyridine nucleotide-disulphide oxidoreductase domain 2 (Pyroxd2) and the transcriptional regulator pre-B-cell leukaemia transcription factor interacting protein 1 (Pbxip1). The strongest concordant transcriptional up-regulation was detected for Prdm16 itself, probably through an autoregulatory mechanism. CONCLUSIONS: Monoallelic, global Prdm16 mutation diminishes cardiac performance in Prdm16csp1/wt mice. Metabolic alterations and transcriptional dysregulation in Prdm16csp1/wt affect cardiac tissue. Female Prdm16csp1/wt mice develop a more pronounced phenotype, indicating sexual dimorphism at this early pathological window. This study suggests that metabolic dysregulation is an early event in the PRDM16 associated cardiac pathology.

Identification of Platinum(II) Sulfide Complexes Suitable as Intramuscular Cyanide Countermeasures.

The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide's in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum-sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl5-DMSO and Na (NH3)2PtCl-DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.

Multinational Federated Learning Approach to Train ECG and Echocardiogram Models for Hypertrophic Cardiomyopathy Detection.

BACKGROUND: Novel targeted treatments increase the need for prompt hypertrophic cardiomyopathy (HCM) detection. However, its low prevalence (0.5%) and resemblance to common diseases present challenges that may benefit from automated machine learning-based approaches. We aimed to develop machine learning models to detect HCM and to differentiate it from other cardiac conditions using ECGs and echocardiograms, with robust generalizability across multiple cohorts. METHODS: Single-institution HCM ECG models were trained and validated on external data. Multi-institution models for ECG and echocardiogram were trained on data from 3 academic medical centers in the United States and Japan using a federated learning approach, which enables training on distributed data without data sharing. Models were validated on held-out test sets for each institution and from a fourth academic medical center and were further evaluated for discrimination of HCM from aortic stenosis, hypertension, and cardiac amyloidosis. Last, automated detection was compared with manual interpretation by 3 cardiologists on a data set with a realistic HCM prevalence. RESULTS: We identified 74 376 ECGs for 56 129 patients and 8392 echocardiograms for 6825 patients at the 4 academic medical centers. Although ECG models trained on data from each institution displayed excellent discrimination of HCM on internal test data (C statistics, 0.88-0.93), the generalizability was limited, most notably for a model trained in Japan and tested in the United States (C statistic, 0.79-0.82). When trained in a federated manner, discrimination of HCM was excellent across all institutions (C statistics, 0.90-0.96 and 0.90-0.96 for ECG and echocardiogram model, respectively), including for phenotypic subgroups. The models further discriminated HCM from hypertension, aortic stenosis, and cardiac amyloidosis (C statistics, 0.84, 0.83, and 0.88, respectively, for ECG and 0.93, 0.94, 0.85, respectively, for echocardiogram). Analysis of electrocardiography-echocardiography paired data from 11 823 patients from an external institution indicated a higher sensitivity of automated HCM detection at a given positive predictive value compared with cardiologists (0.98 versus 0.81 at a positive predictive value of 0.01 for ECG and 0.78 versus 0.59 at a positive predictive value of 0.24 for echocardiogram). CONCLUSIONS: Federated learning improved the generalizability of models that use ECGs and echocardiograms to detect and differentiate HCM from other causes of hypertrophy compared with training within a single institution.

Single Cell Biology: Exploring Somatic Cell Behaviors, Competition and Selection in Chronic Disease.

The full range of cell functions is under-determined in most human diseases. The evidence that somatic cell competition and clonal imbalance play a role in non-neoplastic chronic disease reveal a need for a dedicated effort to explore single cell function if we are to understand the mechanisms by which cell population behaviors influence disease. It will be vital to document not only the prevalent pathologic behaviors but also those beneficial functions eliminated or suppressed by competition. An improved mechanistic understanding of the role of somatic cell biology will help to stratify chronic disease, define more precisely at an individual level the role of environmental factors and establish principles for prevention and potential intervention throughout the life course and across the trajectory from wellness to disease.

Wnt Signaling Interactor WTIP (Wilms Tumor Interacting Protein) Underlies Novel Mechanism for Cardiac Hypertrophy.

BACKGROUND: The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. METHODS: In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell-derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. RESULTS: WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio. Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell-derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. CONCLUSIONS: We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.

PIEZO1 mediates a mechanothrombotic pathway in diabetes.

Thrombosis is the leading complication of common human disorders including diabetes, coronary heart disease, and infection and remains a global health burden. Current anticoagulant therapies that target the general clotting cascade are associated with unpredictable adverse bleeding effects, because understanding of hemostasis remains incomplete. Here, using perturbational screening of patient peripheral blood samples for latent phenotypes, we identified dysregulation of the major mechanosensory ion channel Piezo1 in multiple blood lineages in patients with type 2 diabetes mellitus (T2DM). Hyperglycemia activated PIEZO1 transcription in mature blood cells and selected high Piezo1­expressing hematopoietic stem cell clones. Elevated Piezo1 activity in platelets, red blood cells, and neutrophils in T2DM triggered discrete prothrombotic cellular responses. Inhibition of Piezo1 protected against thrombosis both in human blood and in zebrafish genetic models, particularly in hyperglycemia. Our findings identify a candidate target to precisely modulate mechanically induced thrombosis in T2DM and a potential screening method to predict patient-specific risk. Ongoing remodeling of cell lineages in hematopoiesis is an integral component of thrombotic risk in T2DM, and related mechanisms may have a broader role in chronic disease.

Evaluation of the Usage and Dosing of Guideline-Directed Medical Therapy for Heart Failure With Reduced Ejection Fraction Patients in Clinical Practice.

BACKGROUND: Although strategies for optimization of pharmacologic therapy in patients with heart failure with reduced ejection fraction (HFrEF) are scripted by guidelines, data from HF registries suggests that guideline-directed medical therapies (GDMT) are underutilized among eligible patients. Whether this discrepancy reflects medication intolerance, contraindications, or a quality of care issue remains unclear. OBJECTIVE: The objective of this initiative was to identify reasons for underutilization and under-dosing of HFrEF therapy in patients at a large, academic medical center. METHODS: Among 500 patients with HFrEF enrolled in a quality improvement project at a tertiary center, we evaluated usage and dosing of 4 categories of GDMT: ACE inhibitors/Angiotensin Receptor Blockers (ACE-i/ARB), Angiotensin Receptor-Neprilysin Inhibitors (ARNi), beta blockers, and Mineralocorticoid Receptor Antagonists (MRA). Reasons for nonprescription and usage of suboptimal doses were abstracted from notes in the chart and from telephone review of previous medication trials with the patient. RESULTS: Of 500 patients identified, 472 subjects had complete data for analysis. Among eligible patients, ACE-i/ARB were prescribed in 81.4% (293 of 360) and beta blockers in 94.4% (442 of 468). Of these patients, 10.6% were prescribed target doses of ACE-i/ARB and 12.4% were prescribed target doses of beta blockers. Utilization of other categories of GDMT was lower, with 54% of eligible patients prescribed MRAs and 27% prescribed an ARNi. In most cases, the reasons for nonprescription or under-dosing of GDMT were not apparent on review of the health record or discussion with the patient. CONCLUSION: Clear rationale for nonprescription and under-dosing of GDMT often cannot be ascertained from detailed review and is only rarely related to documented medication intolerance or contraindications, suggesting an opportunity for quality improvement.

Noninvasive Scale Measurement of Stroke Volume and Cardiac Output Compared With the Direct Fick Method: A Feasibility Study.

Background Objective markers of cardiac function are limited in the outpatient setting and may be beneficial for monitoring patients with chronic cardiac conditions. We assess the accuracy of a scale, with the ability to capture ballistocardiography, electrocardiography, and impedance plethysmography signals from a patient's feet while standing on the scale, in measuring stroke volume and cardiac output compared with the gold-standard direct Fick method. Methods and Results Thirty-two patients with unexplained dyspnea undergoing level 3 invasive cardiopulmonary exercise test at a tertiary medical center were included in the final analysis. We obtained scale and direct Fick measurements of stroke volume and cardiac output before and immediately after invasive cardiopulmonary exercise test. Stroke volume and cardiac output from a cardiac scale and the direct Fick method correlated with r=0.81 and r=0.85, respectively (P<0.001 each). The mean absolute error of the scale estimated stroke volume was -1.58 mL, with a 95% limits of agreement of -21.97 to 18.81 mL. The mean error for the scale estimated cardiac output was -0.31 L/min, with a 95% limits of agreement of -2.62 to 2.00 L/min. The changes in stroke volume and cardiac output before and after exercise were 78.9% and 96.7% concordant, respectively, between the 2 measuring methods. Conclusions In a proof-of-concept study, this novel scale with cardiac monitoring abilities may allow for noninvasive, longitudinal measures of cardiac function. Using the widely accepted form factor of a bathroom scale, this method of monitoring can be easily integrated into a patient's lifestyle.

Artificial intelligence-enabled fully automated detection of cardiac amyloidosis using electrocardiograms and echocardiograms.

Patients with rare conditions such as cardiac amyloidosis (CA) are difficult to identify, given the similarity of disease manifestations to more prevalent disorders. The deployment of approved therapies for CA has been limited by delayed diagnosis of this disease. Artificial intelligence (AI) could enable detection of rare diseases. Here we present a pipeline for CA detection using AI models with electrocardiograms (ECG) or echocardiograms as inputs. These models, trained and validated on 3 and 5 academic medical centers (AMC) respectively, detect CA with C-statistics of 0.85-0.91 for ECG and 0.89-1.00 for echocardiography. Simulating deployment on 2 AMCs indicated a positive predictive value (PPV) for the ECG model of 3-4% at 52-71% recall. Pre-screening with ECG enhance the echocardiography model performance at 67% recall from PPV of 33% to PPV of 74-77%. In conclusion, we developed an automated strategy to augment CA detection, which should be generalizable to other rare cardiac diseases.

The endosomal trafficking regulator LITAF controls the cardiac Nav1.5 channel via the ubiquitin ligase NEDD4-2.

The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current INa and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on INa We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.

Moving Genomics to Routine Care: An Initial Pilot in Acute Cardiovascular Disease.

BACKGROUND: Whole-genome sequencing (WGS) costs are falling, yet, outside oncology, this information is seldom used in adult clinics. We piloted a rapid WGS (rWGS) workflow, focusing initially on estimating power for a feasibility study of introducing genome information into acute cardiovascular care. METHODS: A prospective implementation study was conducted to test the feasibility and clinical utility of rWGS in acute cardiovascular care. rWGS was performed on 50 adult patients with acute cardiovascular events and cardiac arrest survivors, testing for primary and secondary disease-causing variants, cardiovascular-related pharmacogenomics, and carrier status for recessive diseases. The impact of returning rWGS results on short-term clinical care of participants was investigated. The utility of polygenic risk scores to stratify coronary artery disease was also assessed. RESULTS: Pathogenic variants, typically secondary findings, were identified in 20% (95% CI, 11.7-34.3). About 60% (95% CI, 46.2-72.4) of participants were carriers for one or more recessive traits, most commonly in HFE and SERPINA1 genes. Although 64% (95% CI, 50.1-75.9) of participants carried at least one pharmacogenetic variant of cardiovascular relevance, these were actionable in only 14% (95% CI, 7-26.2). Coronary artery disease prevalence among participants at the 95th percentile of polygenic risk score was 88.2% (95% CI, 71.8-95.7). CONCLUSIONS: We demonstrated the feasibility of rWGS integration into the inpatient management of adults with acute cardiovascular events. Our pilot identified pathogenic variants in one out of 5 acute vascular patients. Integrating rWGS in clinical care will progressively increase actionability.

Cyp1 Inhibition Prevents Doxorubicin-Induced Cardiomyopathy in a Zebrafish Heart-Failure Model.

Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, a zebrafish model of doxorubicin-induced cardiomyopathy was previously established for small-molecule screening. Using this model, several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish and in mouse models have previously been identified. In this study, exploration of doxorubicin cardiotoxicity is expanded by screening 2271 small molecules from a proprietary, target-annotated tool compound collection. It is found that 120 small molecules can prevent doxorubicin-induced cardiotoxicity, including 7 highly effective compounds. Of these, all seven exhibited inhibitory activity towards cytochrome P450 family 1 (CYP1). These results are consistent with previous findings, in which visnagin, a CYP1 inhibitor, also prevents doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection.

The uptake of family screening in hypertrophic cardiomyopathy and an online video intervention to facilitate family communication.

BACKGROUND: Individuals with hypertrophic cardiomyopathy (HCM), even when asymptomatic, are at-risk for sudden cardiac death and stroke from arrhythmias, making it imperative to identify individuals affected by this familial disorder. Consensus guidelines recommend that first-degree relatives (FDRs) of a person with HCM undergo serial cardiovascular evaluations. METHODS: We determined the uptake of family screening in patients with HCM and developed an online video intervention to facilitate family communication and screening. Family screening and genetic testing data were collected through a prospective quality improvement initiative, a standardized clinical assessment and management plan (SCAMP), utilized in an established cardiovascular genetics clinic. Patients were prescribed an online video if screening of their FDRs was incomplete and a pilot study on video utilization and family communication was conducted. RESULTS: Two-hundred and sixteen probands with HCM were enrolled in SCAMP Phase I and 190 were enrolled in SCAMP Phase II. In both phases, probands reported that 51% of FDRs had been screened (382/749 in Phase I, 258/504 in Phase II). Twenty patients participated in a pilot study on video utilization and family communication. Nine participants reported watching the video and six participants reported sharing the video with relatives; however only one participant reported sharing the video with relatives who were not yet aware of the diagnosis of HCM in the family. CONCLUSION: Despite care in a specialized cardiovascular genetics clinic, approximately one half of FDRs of patients with HCM remained unscreened. Online interventions and videos may serve as supplemental tools for patients communicating genetic risk information to relatives.

LITAF (Lipopolysaccharide-Induced Tumor Necrosis Factor) Regulates Cardiac L-Type Calcium Channels by Modulating NEDD (Neural Precursor Cell Expressed Developmentally Downregulated Protein) 4-1 Ubiquitin Ligase.

BACKGROUND: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. METHODS: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (ICa,L) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. RESULTS: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. CONCLUSIONS: LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.

Closing the 'phenotype gap' in precision medicine: improving what we measure to understand complex disease mechanisms.

The central concept underlying precision medicine is a mechanistic understanding of each disease and its response to therapy sufficient to direct a specific intervention. To execute on this vision requires parsing incompletely defined disease syndromes into discrete mechanistic subsets and developing interventions to precisely address each of these etiologically distinct entities. This will require substantial adjustment of traditional paradigms which have tended to aggregate high-level phenotypes with very different etiologies. In the current environment, where diagnoses are not mechanistic, drug development has become so expensive that it is now impractical to imagine the cost-effective creation of new interventions for many prevalent chronic conditions. The vision of precision medicine also argues for a much more seamless integration of research and development with clinical care, where shared taxonomies will enable every clinical interaction to inform our collective understanding of disease mechanisms and drug responses. Ideally, this would be executed in ways that drive real-time and real-world discovery, innovation, translation, and implementation. Only in oncology, where at least some of the biology is accessible through surgical excision of the diseased tissue or liquid biopsy, has "co-clinical" modeling proven feasible. In most common germline disorders, while genetics often reveal the causal mutations, there still remain substantial barriers to efficient disease modeling. Aggregation of similar disorders under single diagnostic labels has directly contributed to the paucity of etiologic and mechanistic understanding by directly reducing the resolution of any subsequent studies. Existing clinical phenotypes are typically anatomic, physiologic, or histologic, and result in a substantial mismatch in information content between the phenomes in humans or in animal 'models' and the variation in the genome. This lack of one-to-one mapping of discrete mechanisms between disease and animal models causes a failure of translation and is one form of 'phenotype gap.' In this review, we will focus on the origins of the phenotyping deficit and approaches that may be considered to bridge the gap, creating shared taxonomies between human diseases and relevant models, using cardiovascular examples.

Zebrafish model of amyloid light chain cardiotoxicity: regeneration versus degeneration.

Cardiac dysfunction is the most frequent cause of morbidity and mortality in amyloid light chain (AL) amyloidosis caused by a clonal immunoglobulin light chain (LC). Previously published transgenic animal models of AL amyloidosis have not recapitulated the key phenotype of cardiac dysfunction seen in AL amyloidosis, which has limited our understanding of the disease mechanisms in vivo, as well as the development of targeted AL therapeutics. We have developed a transgenic zebrafish model in which a λ LC derived from a patient with AL amyloidosis is conditionally expressed in the liver under the control of the Gal4 upstream activation sequence enhancer system. Circulating LC levels of 125 µg/ml in these transgenic zebrafish are comparable to median pathological serum LC levels. Functional analysis links abnormal contractile function with evidence of cellular and molecular proteotoxicity in the heart, including increased cell death and autophagy. However, despite pathological and functional phenotypes analogous to human AL, the lifespan of the transgenic fish is comparable to control fish without the expressed AL-LC transgene. Nuclear labeling experiments suggest increased cardiac proliferation in the transgenic fish, which can be counteracted by treatment with a small molecule proliferation inhibitor leading to increased zebrafish mortality because of cardiac apoptosis and functional deterioration. This transgenic zebrafish model provides a platform to study underlying AL disease mechanisms in vivo further. NEW & NOTEWORTHY Heart failure is a major cause of mortality in amyloid light (AL) amyloidosis, yet it has been difficult to model in animals. We report the generation of a transgenic zebrafish model for AL amyloidosis with pathological concentration of circulating human light chain protein that results in cardiac dysfunction. The light chain toxicity triggers regeneration in the zebrafish heart resulting in functional compensation early in life, but with age develops into cardiac dysfunction.

Trafficking of the human ether-a-go-go-related gene (hERG) potassium channel is regulated by the ubiquitin ligase rififylin (RFFL).

The QT interval is an important diagnostic feature on surface electrocardiograms because it reflects the duration of the ventricular action potential. A previous genome-wide association study has reported a significant linkage between a single-nucleotide polymorphism ∼11.7 kb downstream of the gene encoding the RING finger ubiquitin ligase rififylin (RFFL) and variability in the QT interval. This, along with results in animal studies, suggests that RFFL may have effects on cardiac repolarization. Here, we sought to determine the role of RFFL in cardiac electrophysiology. Adult rabbit cardiomyocytes with adenovirus-expressed RFFL exhibited reduced rapid delayed rectifier current (IKr). Neonatal rabbit cardiomyocytes transduced with RFFL-expressing adenovirus exhibited reduced total expression of the potassium channel ether-a-go-go-related gene (rbERG). Using transfections of 293A cells and Western blotting experiments, we observed that RFFL and the core-glycosylated form of the human ether-a-go-go-related gene (hERG) potassium channel interact. Furthermore, RFFL overexpression led to increased polyubiquitination and proteasomal degradation of hERG protein and to an almost complete disappearance of IKr, which depended on the intact RING domain of RFFL. Blocking the ER-associated degradation (ERAD) pathway with a dominant-negative form of the ERAD core component, valosin-containing protein (VCP), in 293A cells partially abolished RFFL-mediated hERG degradation. We further substantiated the link between RFFL and ERAD by showing an interaction between RFFL and VCP in vitro We conclude that RFFL is an important regulator of voltage-gated hERG potassium channel activity and therefore cardiac repolarization and that this ubiquitination-mediated regulation requires parts of the ERAD pathway.

Genetics of Atrial Fibrillation.

Background Atrial fibrillation ( AF ) is a common arrhythmia seen in clinical practice. Occasionally, no common risk factors are present in patients with this arrhythmia. This suggests the potential underlying role of genetic factors associated with predisposition to developing AF . Methods and Results We conducted a comprehensive review of the literature through large online libraries, including PubMed. Many different potassium and sodium channel mutations have been discussed in their relation to AF . There have also been non-ion channel mutations that have been linked to AF . Genome-wide association studies have helped in identifying potential links between single-nucleotide polymorphisms and AF . Ancestry studies have also highlighted a role of genetics in AF . Blacks with a higher percentage of European ancestry are at higher risk of developing AF . The emerging field of ablatogenomics involves the use of genetic profiles in their relation to recurrence of AF after catheter ablation. Conclusions The evidence for the underlying role of genetics in AF continues to expand. Ultimately, the role of genetics in risk stratification of AF and its recurrence is of significant interest. No established risk scores that are useful in clinical practice are present to date.