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1.
Circulation ; 148(21): 1691-1704, 2023 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-37850394

RESUMO

BACKGROUND: Hypercontractility and arrhythmia are key pathophysiologic features of hypertrophic cardiomyopathy (HCM), the most common inherited heart disease. ß-Adrenergic receptor antagonists (ß-blockers) are the first-line therapy for HCM. However, ß-blockers commonly selected for this disease are often poorly tolerated in patients, where heart-rate reduction and noncardiac effects can lead to reduced cardiac output and fatigue. Mavacamten, myosin ATPase inhibitor recently approved by the US Food and Drug Administration, has demonstrated the ability to ameliorate hypercontractility without lowering heart rate, but its benefits are so far limited to patients with left ventricular (LV) outflow tract obstruction, and its effect on arrhythmia is unknown. METHODS: We screened 21 ß-blockers for their impact on myocyte contractility and evaluated the antiarrhythmic properties of the most promising drug in a ventricular myocyte arrhythmia model. We then examined its in vivo effect on LV function by hemodynamic pressure-volume loop analysis. The efficacy of the drug was tested in vitro and in vivo compared with current therapeutic options (metoprolol, verapamil, and mavacamten) for HCM in an established mouse model of HCM (Myh6R403Q/+ and induced pluripotent stem cell (iPSC)-derived cardiomyocytes from patients with HCM (MYH7R403Q/+). RESULTS: We identified that carvedilol, a ß-blocker not commonly used in HCM, suppresses contractile function and arrhythmia by inhibiting RyR2 (ryanodine receptor type 2). Unlike metoprolol (a ß1-blocker), carvedilol markedly reduced LV contractility through RyR2 inhibition, while maintaining stroke volume through α1-adrenergic receptor inhibition in vivo. Clinically available carvedilol is a racemic mixture, and the R-enantiomer, devoid of ß-blocking effect, retains the ability to inhibit both α1-receptor and RyR2, thereby suppressing contractile function and arrhythmias without lowering heart rate and cardiac output. In Myh6R403Q/+ mice, R-carvedilol normalized hyperdynamic contraction, suppressed arrhythmia, and increased cardiac output better than metoprolol, verapamil, and mavacamten. The ability of R-carvedilol to suppress contractile function was well retained in MYH7R403Q/+ iPSC-derived cardiomyocytes. CONCLUSIONS: R-enantiomer carvedilol attenuates hyperdynamic contraction, suppresses arrhythmia, and at the same time, improves cardiac output without lowering heart rate by dual blockade of α1-adrenergic receptor and RyR2 in mouse and human models of HCM. This combination of therapeutic effects is unique among current therapeutic options for HCM and may particularly benefit patients without LV outflow tract obstruction.


Assuntos
Cardiomiopatia Hipertrófica , Metoprolol , Humanos , Camundongos , Animais , Carvedilol/farmacologia , Carvedilol/uso terapêutico , Metoprolol/uso terapêutico , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Cardiomiopatia Hipertrófica/complicações , Cardiomiopatia Hipertrófica/tratamento farmacológico , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Miócitos Cardíacos/metabolismo , Verapamil/uso terapêutico , Receptores Adrenérgicos/metabolismo
2.
Biophys J ; 118(5): 1165-1176, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32023435

RESUMO

All medications have adverse effects. Among the most serious of these are cardiac arrhythmias. Current paradigms for drug safety evaluation are costly, lengthy, conservative, and impede efficient drug development. Here, we combine multiscale experiment and simulation, high-performance computing, and machine learning to create a risk estimator to stratify new and existing drugs according to their proarrhythmic potential. We capitalize on recent developments in machine learning and integrate information across 10 orders of magnitude in space and time to provide a holistic picture of the effects of drugs, either individually or in combination with other drugs. We show, both experimentally and computationally, that drug-induced arrhythmias are dominated by the interplay between two currents with opposing effects: the rapid delayed rectifier potassium current and the L-type calcium current. Using Gaussian process classification, we create a classifier that stratifies drugs into safe and arrhythmic domains for any combinations of these two currents. We demonstrate that our classifier correctly identifies the risk categories of 22 common drugs exclusively on the basis of their concentrations at 50% current block. Our new risk assessment tool explains under which conditions blocking the L-type calcium current can delay or even entirely suppress arrhythmogenic events. Using machine learning in drug safety evaluation can provide a more accurate and comprehensive mechanistic assessment of the proarrhythmic potential of new drugs. Our study paves the way toward establishing science-based criteria to accelerate drug development, design safer drugs, and reduce heart rhythm disorders.


Assuntos
Arritmias Cardíacas , Preparações Farmacêuticas , Potenciais de Ação , Arritmias Cardíacas/induzido quimicamente , Simulação por Computador , Humanos , Aprendizado de Máquina , Medição de Risco
3.
Circulation ; 140(9): 765-778, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31315475

RESUMO

BACKGROUND: Restrictive cardiomyopathy is a rare heart disease associated with mutations in sarcomeric genes and with phenotypic overlap with hypertrophic cardiomyopathy. There is no approved therapy directed at the underlying cause. Here, we explore the potential of an interfering RNA (RNAi) therapeutic for a human sarcomeric mutation in MYL2 causative of restrictive cardiomyopathy in a mouse model. METHODS: A short hairpin RNA (M7.8L) was selected from a pool for specificity and efficacy. Two groups of myosin regulatory light chain N47K transgenic mice were injected with M7.8L packaged in adeno-associated virus 9 at 3 days of age and 60 days of age. Mice were subjected to treadmill exercise and echocardiography after treatment to determine maximal oxygen uptake and left ventricular mass. At the end of treatment, heart, lung, liver, and kidney tissue was harvested to determine viral tropism and for transcriptomic and proteomic analysis. Cardiomyocytes were isolated for single-cell studies. RESULTS: A one-time injection of AAV9-M7.8L RNAi in 3-day-old humanized regulatory light chain mutant transgenic mice silenced the mutated allele (RLC-47K) with minimal effects on the normal allele (RLC-47N) assayed at 16 weeks postinjection. AAV9-M7.8L RNAi suppressed the expression of hypertrophic biomarkers, reduced heart weight, and attenuated a pathological increase in left ventricular mass. Single adult cardiac myocytes from mice treated with AAV9-M7.8L showed partial restoration of contraction, relaxation, and calcium kinetics. In addition, cardiac stress protein biomarkers, such as calmodulin-dependent protein kinase II and the transcription activator Brg1 were reduced, suggesting recovery toward a healthy myocardium. Transcriptome analyses further revealed no significant changes of argonaute (AGO1, AGO2) and endoribonuclease dicer (DICER1) transcripts, and endogenous microRNAs were preserved, suggesting that the RNAi pathway was not saturated. CONCLUSIONS: Our results show the feasibility, efficacy, and safety of RNAi therapeutics directed towards human restrictive cardiomyopathy. This is a promising step toward targeted therapy for a prevalent human disease.


Assuntos
Cardiomiopatia Restritiva/patologia , Cadeias Leves de Miosina/metabolismo , Interferência de RNA , Alelos , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatia Restritiva/prevenção & controle , DNA Helicases/genética , DNA Helicases/metabolismo , Modelos Animais de Doenças , Redes Reguladoras de Genes , Vetores Genéticos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Contração Muscular , Mutagênese Sítio-Dirigida , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/antagonistas & inibidores , Cadeias Leves de Miosina/genética , RNA Interferente Pequeno/metabolismo
4.
EMBO J ; 33(23): 2798-813, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25349190

RESUMO

Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease-associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals.


Assuntos
Encéfalo/metabolismo , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Miócitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Dinaminas/genética , Tomografia com Microscopia Eletrônica , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Cadeias Pesadas de Miosina/genética , Ubiquitinação
5.
Am J Physiol Heart Circ Physiol ; 315(2): H348-H356, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29775410

RESUMO

The G protein-coupled receptor APJ is a promising therapeutic target for heart failure. Constitutive deletion of APJ in the mouse is protective against the hypertrophy-heart failure transition via elimination of ligand-independent, ß-arrestin-dependent stretch transduction. However, the cellular origin of this stretch transduction and the details of its interaction with apelin signaling remain unknown. We generated mice with conditional elimination of APJ in the endothelium (APJendo-/-) and myocardium (APJmyo-/-). No baseline difference was observed in left ventricular function in APJendo-/-, APJmyo-/-, or control (APJendo+/+, APJmyo+/+) mice. After exposure to transaortic constriction, APJendo-/- mice displayed decreased left ventricular systolic function and increased wall thickness, whereas APJmyo-/- mice were protected. At the cellular level, carbon fiber stretch of freshly isolated single cardiomyocytes demonstrated decreased contractile responses to stretch in APJ-/- cardiomyocytes compared with APJ+/+ cardiomyocytes. Ca2+ transients did not change with stretch in either APJ-/- or APJ+/+ cardiomyocytes. Application of apelin to APJ+/+ cardiomyocytes resulted in decreased Ca2+ transients. Furthermore, hearts of mice treated with apelin exhibited decreased phosphorylation in cardiac troponin I NH2-terminal residues (Ser22 and Ser23) consistent with increased Ca2+ sensitivity. These data establish that APJ stretch transduction is mediated specifically by myocardial APJ, that APJ is necessary for stretch-induced increases in contractility, and that apelin opposes APJ's stretch-mediated hypertrophy signaling by lowering Ca2+ transients while maintaining contractility through myofilament Ca2+ sensitization. These findings underscore apelin's unique potential as a therapeutic agent that can simultaneously support cardiac function and protect against the hypertrophy-heart failure transition. NEW & NOTEWORTHY These data address fundamental gaps in our understanding of apelin-APJ signaling in heart failure by localizing APJ's ligand-independent stretch sensing to the myocardium, identifying a novel mechanism of apelin-APJ inotropy via myofilament Ca2+ sensitization, and identifying potential mitigating effects of apelin in APJ stretch-induced hypertrophic signaling.


Assuntos
Receptores de Apelina/metabolismo , Apelina/farmacologia , Insuficiência Cardíaca/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Animais , Receptores de Apelina/genética , Sinalização do Cálcio , Células Cultivadas , Insuficiência Cardíaca/etiologia , Hipertrofia Ventricular Esquerda/complicações , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Troponina I/metabolismo
7.
Proc Natl Acad Sci U S A ; 111(4): 1551-6, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24453217

RESUMO

Chronic neurohormonal and mechanical stresses are central features of heart disease. Increasing evidence supports a role for the transient receptor potential canonical channels TRPC3 and TRPC6 in this pathophysiology. Channel expression for both is normally very low but is increased by cardiac disease, and genetic gain- or loss-of-function studies support contributions to hypertrophy and dysfunction. Selective small-molecule inhibitors remain scarce, and none target both channels, which may be useful given the high homology among them and evidence of redundant signaling. Here we tested selective TRPC3/6 antagonists (GSK2332255B and GSK2833503A; IC50, 3-21 nM against TRPC3 and TRPC6) and found dose-dependent blockade of cell hypertrophy signaling triggered by angiotensin II or endothelin-1 in HEK293T cells as well as in neonatal and adult cardiac myocytes. In vivo efficacy in mice and rats was greatly limited by rapid metabolism and high protein binding, although antifibrotic effects with pressure overload were observed. Intriguingly, although gene deletion of TRPC3 or TRPC6 alone did not protect against hypertrophy or dysfunction from pressure overload, combined deletion was protective, supporting the value of dual inhibition. Further development of this pharmaceutical class may yield a useful therapeutic agent for heart disease management.


Assuntos
Cardiomegalia/genética , Canais de Cátion TRPC/antagonistas & inibidores , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Ratos , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6
8.
Circ Res ; 114(5): 823-32, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24449818

RESUMO

RATIONALE: The heart is exquisitely sensitive to mechanical stimuli to adapt rapidly to physiological demands. In muscle lacking dystrophin, such as Duchenne muscular dystrophy, increased load during contraction triggers pathological responses thought to worsen the disease. The relevant mechanotransducers and therapies to target them remain unclear. OBJECTIVES: We tested the role of transient receptor potential canonical (TRPC) channels TRPC3 and TRPC6 and their modulation by protein kinase G (PKG) in controlling cardiac systolic mechanosensing and determined their pathophysiological relevance in an experimental model of Duchenne muscular dystrophy. METHODS AND RESULTS: Contracting isolated papillary muscles and cardiomyocytes from controls and mice genetically lacking either TRPC3 or TRPC6 were subjected to auxotonic load to induce stress-stimulated contractility (SSC, gradual rise in force and intracellular Ca(2+)). Incubation with cGMP (PKG activator) markedly blunted SSC in controls and Trpc3(-/-); whereas in Trpc6(-/-), the resting SSC response was diminished and cGMP had no effect. In Duchenne muscular dystrophy myocytes (mdx/utrophin deficient), the SSC was excessive and arrhythmogenic. Gene deletion or selective drug blockade of TRPC6 or cGMP/PKG activation reversed this phenotype. Chronic phosphodiesterase 5A inhibition also normalized abnormal mechanosensing while blunting progressive chamber hypertrophy in Duchenne muscular dystrophy mice. CONCLUSIONS: PKG is a potent negative modulator of cardiac systolic mechanosignaling that requires TRPC6 as the target effector. In dystrophic hearts, excess SSC and arrhythmia are coupled to TRPC6 and are ameliorated by its targeted suppression or PKG activation. These results highlight novel therapeutic targets for this disease.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Coração/fisiologia , Distrofia Muscular de Duchenne/fisiopatologia , Canais de Cátion TRPC/metabolismo , Animais , Proteínas Quinases Dependentes de GMP Cíclico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Distrofina/genética , Feminino , Coração/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Músculos Papilares/fisiologia , Inibidores da Fosfodiesterase 5/farmacologia , Estresse Mecânico , Sístole/efeitos dos fármacos , Sístole/fisiologia , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6
9.
Circ Res ; 109(12): 1410-4, 2011 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-22034490

RESUMO

RATIONALE: One of the physiological mechanisms by which the heart adapts to a rise in blood pressure is by augmenting myocyte stretch-mediated intracellular calcium, with a subsequent increase in contractility. This slow force response was first described over a century ago and has long been considered compensatory, but its underlying mechanisms and link to chronic adaptations remain uncertain. Because levels of the matricellular protein thrombospondin-4 (TSP4) rapidly rise in hypertension and are elevated in cardiac stress overload and heart failure, we hypothesized that TSP4 is involved in this adaptive mechanism. OBJECTIVE: To determine the mechano-transductive role that TSP4 plays in cardiac regulation to stress. METHODS AND RESULTS: In mice lacking TSP4 (Tsp4⁻/⁻), hearts failed to acutely augment contractility or activate stretch-response pathways (ERK1/2 and Akt) on exposure to acute pressure overload. Sustained pressure overload rapidly led to greater chamber dilation, reduced function, and increased heart mass. Unlike controls, Tsp4⁻/⁻ cardiac trabeculae failed to enhance contractility and cellular calcium after a stretch. However, the contractility response was restored in Tsp4⁻/⁻ muscle incubated with recombinant TSP4. Isolated Tsp4⁻/⁻ myocytes responded normally to stretch, identifying a key role of matrix-myocyte interaction for TSP4 contractile modulation. CONCLUSION: These results identify TSP4 as myocyte-interstitial mechano-signaling molecule central to adaptive cardiac contractile responses to acute stress, which appears to play a crucial role in the transition to chronic cardiac dilatation and failure.


Assuntos
Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Estresse Fisiológico/fisiologia , Trombospondinas/fisiologia , Animais , Insuficiência Cardíaca/fisiopatologia , Hipertensão/fisiopatologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Miócitos Cardíacos/citologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ratos , Trombospondinas/deficiência , Trombospondinas/genética
11.
Circ Res ; 106(1): 176-84, 2010 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-19893014

RESUMO

RATIONALE: Mechanical stress is known to alter the electrophysiological properties of the myocardium and may trigger fatal arrhythmias when an abnormal load is applied to the heart. OBJECTIVE: We tested the hypothesis that the structural heterogeneity of the ventricular wall modulates globally applied stretches to create heterogeneous strain distributions that lead to the initiation of arrhythmias. METHODS AND RESULTS: We applied global stretches to arterially perfused rabbit right ventricular tissue preparations. The distribution of strain (determined by marker tracking) and the transmembrane potential (measured by optical mapping) were simultaneously recorded while accounting for motion artifacts. The 3D structure of the preparations was also examined using a laser displacement meter. To examine whether such observations can be translated to the physiological condition, we performed similar measurements in whole heart preparations while applying volume pulses to the right ventricle. At the tissue level, larger stretches (> or = 20%) caused synchronous excitation of the entire preparation, whereas medium stretches (10% and 15%) induced focal excitation. We found a significant correlation between the local strain and the local thickness, and the probability for focal excitation was highest for medium stretches. In the whole heart preparations, we observed that such focal excitations developed into reentrant arrhythmias. CONCLUSIONS: Global stretches of intermediate strength, rather than intense stretches, created heterogeneous strain (excitation) distributions in the ventricular wall, which can trigger fatal arrhythmias.


Assuntos
Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Miocárdio/patologia , Estresse Fisiológico , Animais , Arritmias Cardíacas/etiologia , Perfusão , Coelhos
12.
Circ Genom Precis Med ; 15(4): e003563, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35671065

RESUMO

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.


Assuntos
Proteínas Correpressoras/metabolismo , Proteínas do Citoesqueleto/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Animais , Cálcio/metabolismo , Cardiomegalia/metabolismo , Cardiomiopatia Hipertrófica/metabolismo , Humanos , Ratos , Verapamil
13.
Nat Commun ; 13(1): 5107, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-36042219

RESUMO

The SARS-CoV-2 pandemic has differentially impacted populations across race and ethnicity. A multi-omic approach represents a powerful tool to examine risk across multi-ancestry genomes. We leverage a pandemic tracking strategy in which we sequence viral and host genomes and transcriptomes from nasopharyngeal swabs of 1049 individuals (736 SARS-CoV-2 positive and 313 SARS-CoV-2 negative) and integrate them with digital phenotypes from electronic health records from a diverse catchment area in Northern California. Genome-wide association disaggregated by admixture mapping reveals novel COVID-19-severity-associated regions containing previously reported markers of neurologic, pulmonary and viral disease susceptibility. Phylodynamic tracking of consensus viral genomes reveals no association with disease severity or inferred ancestry. Summary data from multiomic investigation reveals metagenomic and HLA associations with severe COVID-19. The wealth of data available from residual nasopharyngeal swabs in combination with clinical data abstracted automatically at scale highlights a powerful strategy for pandemic tracking, and reveals distinct epidemiologic, genetic, and biological associations for those at the highest risk.


Assuntos
COVID-19 , Pandemias , COVID-19/epidemiologia , Genoma Viral , Estudo de Associação Genômica Ampla , Humanos , SARS-CoV-2/genética
14.
J Clin Invest ; 118(2): 710-21, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18202748

RESUMO

To assess physiological and pathophysiological events that involve dynamic interplay between multiple cell types, real-time, in vivo analysis is necessary. We developed a technique based on confocal laser microscopy that enabled us to analyze and compare the 3-dimensional structures, cellular dynamics, and vascular function within mouse lean and obese adipose tissue in vivo with high spatiotemporal resolution. We found increased leukocyte-EC-platelet interaction in the microcirculation of obese visceral adipose tissue in ob/ob and high-fat diet-induced obese mice. These changes were indicative of activation of the leukocyte adhesion cascade, a hallmark of inflammation. Local platelet activation in obese adipose tissue was indicated by increased P-selectin expression and formation of monocyte-platelet conjugates. We observed upregulated expression of adhesion molecules on macrophages and ECs in obese visceral adipose tissue, suggesting that interactions between these cells contribute to local activation of inflammatory processes. Furthermore, administration of anti-ICAM-1 antibody normalized the cell dynamics seen in obese visceral fat. This imaging technique to analyze the complex cellular interplay within obese adipose tissue allowed us to show that visceral adipose tissue obesity is an inflammatory disease. In addition, this technique may prove to be a valuable tool to evaluate potential therapeutic interventions.


Assuntos
Gordura Intra-Abdominal/irrigação sanguínea , Gordura Intra-Abdominal/patologia , Microscopia Confocal/métodos , Obesidade/patologia , Paniculite/patologia , Animais , Anticorpos/farmacologia , Plaquetas/imunologia , Capilares/patologia , Permeabilidade Capilar , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/metabolismo , Endotélio Vascular/imunologia , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Gordura Intra-Abdominal/imunologia , Leucócitos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Obesos , Microcirculação , Obesidade/imunologia , Selectina-P/metabolismo , Paniculite/imunologia
15.
JCI Insight ; 6(13)2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34236044

RESUMO

Diagnosis of organ transplant rejection relies upon biopsy approaches to confirm alloreactive T cell infiltration in the graft. Immune molecular monitoring is under investigation to screen for rejection, though these techniques have suffered from low specificity and lack of spatial information. ImmunoPET utilizing antibodies conjugated to radioisotopes has the potential to improve early and accurate detection of graft rejection. ImmunoPET is capable of noninvasively visualizing the dynamic distribution of cells expressing specific immune markers in the entire body over time. In this work, we identify and characterize OX40 as a surrogate biomarker for alloreactive T cells in organ transplant rejection and monitor its expression by utilizing immunoPET. In a dual murine heart transplant model that has both syngeneic and allogeneic hearts engrafted in bilateral ear pinna on the recipients, OX40 immunoPET clearly depicted alloreactive T cells in the allograft and draining lymph node that were not observed in their respective isograft counterparts. OX40 immunoPET signals also reflected the subject's immunosuppression level with tacrolimus in this study. OX40 immunoPET is a promising approach that may bridge molecular monitoring and morphological assessment for improved transplant rejection diagnosis.


Assuntos
Rejeição de Enxerto , Transplante de Coração/efeitos adversos , Monitorização Imunológica/métodos , Ligante OX40 , Tomografia por Emissão de Pósitrons/métodos , Linfócitos T/imunologia , Animais , Antígenos de Diferenciação/análise , Biomarcadores/análise , Diagnóstico Precoce , Perfilação da Expressão Gênica/métodos , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/imunologia , Humanos , Programas de Rastreamento/métodos , Camundongos , Ligante OX40/análise , Ligante OX40/imunologia , Radioimunoensaio/métodos
16.
J Clin Invest ; 131(8)2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33855972

RESUMO

Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.


Assuntos
Interleucina-2/imunologia , Ativação Linfocitária , Receptores de Interleucina-2/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante , Animais , Interleucina-2/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Receptores de Interleucina-2/genética , Transdução de Sinais/genética , Linfócitos T Reguladores/citologia
17.
Circ Genom Precis Med ; 14(6): e003419, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34802252

RESUMO

BACKGROUND: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. METHODS: Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used co-immunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres. RESULTS: Patient-derived iPSC-cardiomyocytes were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca2+-signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc ultrastructure. In homozygous stop-gain cells, affinity-purification mass-spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 (alpha-actinin 1) and GJA1 (gap junction protein alpha 1), 2 sarcolemma-associated proteins, which may contribute to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene editing. CONCLUSIONS: Together, these data advance our understanding of the role of ACTN2 in the human heart and establish recessive inheritance of ACTN2 truncation as causative of disease.


Assuntos
Actinina , Cardiomiopatias , Actinina/genética , Actinina/metabolismo , Actinas/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Sarcômeros/genética
18.
J Clin Invest ; 131(16)2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34185705

RESUMO

The Western pattern diet is rich not only in fat and calories but also in phosphate. The negative effects of excessive fat and calorie intake on health are widely known, but the potential harms of excessive phosphate intake are poorly recognized. Here, we show the mechanism by which dietary phosphate damages the kidney. When phosphate intake was excessive relative to the number of functioning nephrons, circulating levels of FGF23, a hormone that increases the excretion of phosphate per nephron, were increased to maintain phosphate homeostasis. FGF23 suppressed phosphate reabsorption in renal tubules and thus raised the phosphate concentration in the tubule fluid. Once it exceeded a threshold, microscopic particles containing calcium phosphate crystals appeared in the tubule lumen, which damaged tubule cells through binding to the TLR4 expressed on them. Persistent tubule damage induced interstitial fibrosis, reduced the number of nephrons, and further boosted FGF23 to trigger a deterioration spiral leading to progressive nephron loss. In humans, the progression of chronic kidney disease (CKD) ensued when serum FGF23 levels exceeded 53 pg/mL. The present study identified calcium phosphate particles in the renal tubular fluid as an effective therapeutic target to decelerate nephron loss during the course of aging and CKD progression.


Assuntos
Fosfatos de Cálcio/metabolismo , Túbulos Renais/metabolismo , Insuficiência Renal Crônica/metabolismo , Animais , Líquidos Corporais/química , Fosfatos de Cálcio/química , Linhagem Celular , Cristalização , Dieta Ocidental/efeitos adversos , Progressão da Doença , Endocitose , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Homeostase , Humanos , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatos/administração & dosagem , Fosfatos/efeitos adversos , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/patologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
19.
Front Physiol ; 11: 181, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32231588

RESUMO

The myocardium has an intrinsic ability to sense and respond to mechanical load in order to adapt to physiological demands. Primary examples are the augmentation of myocardial contractility in response to increased ventricular filling caused by either increased venous return (Frank-Starling law) or aortic resistance to ejection (the Anrep effect). Sustained mechanical overload, however, can induce pathological hypertrophy and dysfunction, resulting in heart failure and arrhythmias. It has been proposed that angiotensin II type 1 receptor (AT1R) and apelin receptor (APJ) are primary upstream actors in this acute myocardial autoregulation as well as the chronic maladaptive signaling program. These receptors are thought to have mechanosensing capacity through activation of intracellular signaling via G proteins and/or the multifunctional transducer protein, ß-arrestin. Importantly, ligand and mechanical stimuli can selectively activate different downstream signaling pathways to promote inotropic, cardioprotective or cardiotoxic signaling. Studies to understand how AT1R and APJ integrate ligand and mechanical stimuli to bias downstream signaling are an important and novel area for the discovery of new therapeutics for heart failure. In this review, we provide an up-to-date understanding of AT1R and APJ signaling pathways activated by ligand versus mechanical stimuli, and their effects on inotropy and adaptive/maladaptive hypertrophy. We also discuss the possibility of targeting these signaling pathways for the development of novel heart failure therapeutics.

20.
medRxiv ; 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32766602

RESUMO

During COVID19 and other viral pandemics, rapid generation of host and pathogen genomic data is critical to tracking infection and informing therapies. There is an urgent need for efficient approaches to this data generation at scale. We have developed a scalable, high throughput approach to generate high fidelity low pass whole genome and HLA sequencing, viral genomes, and representation of human transcriptome from single nasopharyngeal swabs of COVID19 patients.

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