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Coronary heart disease (CHD) is a prevalent cardiac disease that causes over 370,000 deaths annually in the USA. In CHD, occlusion of a coronary artery causes ischemia of the cardiac muscle, which results in myocardial infarction (MI). Junctophilin-2 (JPH2) is a membrane protein that ensures efficient calcium handling and proper excitation-contraction coupling. Studies have identified loss of JPH2 due to calpain-mediated proteolysis as a key pathogenic event in ischemia-induced heart failure (HF). Our findings show that calpain-2-mediated JPH2 cleavage yields increased levels of a C-terminal cleaved peptide (JPH2-CTP) in patients with ischemic cardiomyopathy and mice with experimental MI. We created a novel knock-in mouse model by removing residues 479-SPAGTPPQ-486 to prevent calpain-2-mediated cleavage at this site. Functional and molecular assessment of cardiac function post-MI in cleavage site deletion (CSD) mice showed preserved cardiac contractility and reduced dilation, reduced JPH2-CTP levels, attenuated adverse remodeling, improved T-tubular structure, and normalized SR Ca2+-handling. Adenovirus mediated calpain-2 knockdown in mice exhibited similar findings. Pulldown of CTP followed by proteomic analysis revealed valosin-containing protein (VCP) and BAG family molecular chaperone regulator 3 (BAG3) as novel binding partners of JPH2. Together, our findings suggest that blocking calpain-2-mediated JPH2 cleavage may be a promising new strategy for delaying the development of HF following MI.
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Calpaína , Insuficiência Cardíaca , Proteínas de Membrana , Infarto do Miocárdio , Animais , Calpaína/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/etiologia , Humanos , Camundongos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Progressão da Doença , Masculino , Modelos Animais de Doenças , Proteólise , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Proteínas MuscularesRESUMO
BACKGROUND: Accessory pathways are a common cause of supraventricular tachycardia (SVT) and can lead to sudden cardiac death in otherwise healthy children and adults when associated with Wolff-Parkinson-White syndrome. The goal of this study was to identify genetic variants within a large family with structurally normal hearts affected by SVT and Wolff-Parkinson-White syndrome and determine causality of the gene deficit in a corresponding mouse model. METHODS: Whole exome sequencing performed on 2 distant members of a 3-generation family in which multiple members were affected by SVT or Wolff-Parkinson-White pattern (preexcitation) on ECG identified MRC2 as a candidate gene. Serial electrocardiograms, intracardiac electrophysiology studies, echocardiography, optical mapping studies, and histology were performed on both Mrc2 mutant and WT (wild-type) mice. RESULTS: A rare HET (heterozygous) missense variant c.2969A>G;p.Glu990Gly (E990G) in MRC2 was identified as the leading candidate gene variant segregating with the cardiac phenotype following an autosomal-dominant Mendelian trait segregation pattern with variable expressivity. In vivo electrophysiology studies revealed reentrant SVT in E990G mice. Optical mapping studies in E990G mice demonstrated abnormal retrograde conduction, suggesting the presence of an accessory pathway. Histological analysis of E990G mouse hearts showed a disordered ECM (extracellular matrix) in the annulus fibrosus. Finally, Mrc2 knockdown in human cardiac fibroblasts enhanced accelerated cell migration. CONCLUSIONS: This study identified a rare nonsynonymous variant in the MRC2 gene in individuals with familial reentrant SVT, Wolff-Parkinson-White ECG pattern, and structurally normal hearts. Furthermore, Mrc2 knock-in mice revealed an increased incidence of reentrant SVT and bypass tract formation in the setting of preserved cardiac structure and function.
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Linhagem , Taquicardia Supraventricular , Síndrome de Wolff-Parkinson-White , Síndrome de Wolff-Parkinson-White/genética , Síndrome de Wolff-Parkinson-White/fisiopatologia , Síndrome de Wolff-Parkinson-White/patologia , Humanos , Animais , Camundongos , Taquicardia Supraventricular/genética , Taquicardia Supraventricular/fisiopatologia , Taquicardia Supraventricular/patologia , Masculino , Feminino , Adulto , Mutação de Sentido Incorreto , Sequenciamento do Exoma , Eletrocardiografia , Pessoa de Meia-IdadeRESUMO
BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.
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3',5'-AMP Cíclico Fosfodiesterases , Cardiopatias , Receptores Adrenérgicos , Animais , Feminino , Humanos , Masculino , Camundongos , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cardiopatias/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Isoproterenol/farmacologia , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Overexpression of the CREM (cAMP response element-binding modulator) isoform CREM-IbΔC-X in transgenic mice (CREM-Tg) causes the age-dependent development of spontaneous AF. PURPOSE: To identify key proteome signatures and biological processes accompanying the development of persistent AF through integrated proteomics and bioinformatics analysis. METHODS: Atrial tissue samples from three CREM-Tg mice and three wild-type littermates were subjected to unbiased mass spectrometry-based quantitative proteomics, differential expression and pathway enrichment analysis, and protein-protein interaction (PPI) network analysis. RESULTS: A total of 98 differentially expressed proteins were identified. Gene ontology analysis revealed enrichment for biological processes regulating actin cytoskeleton organization and extracellular matrix (ECM) dynamics. Changes in ITGAV, FBLN5, and LCP1 were identified as being relevant to atrial fibrosis and structural based on expression changes, co-expression patterns, and PPI network analysis. Comparative analysis with previously published datasets revealed a shift in protein expression patterns from ion-channel and metabolic regulators in young CREM-Tg mice to profibrotic remodeling factors in older CREM-Tg mice. Furthermore, older CREM-Tg mice exhibited protein expression patterns reminiscent of those seen in humans with persistent AF. CONCLUSIONS: This study uncovered distinct temporal changes in atrial protein expression patterns with age in CREM-Tg mice consistent with the progressive evolution of AF. Future studies into the role of the key differentially abundant proteins identified in this study in AF progression may open new therapeutic avenues to control atrial fibrosis and substrate development in AF.
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Fibrilação Atrial , Modulador de Elemento de Resposta do AMP Cíclico , Fibrose , Átrios do Coração , Camundongos Transgênicos , Proteômica , Animais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/genética , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/genética , Proteômica/métodos , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Camundongos , Regulação da Expressão Gênica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Matriz Extracelular/metabolismo , MasculinoRESUMO
Introduction: Heterozygous autosomal-dominant single nucleotide variants in RYR2 account for 60% of cases of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia disorder associated with high mortality rates. CRISPR/Cas9-mediated genome editing is a promising therapeutic approach that can permanently cure the disease by removing the mutant RYR2 allele. However, the safety and long-term efficacy of this strategy have not been established in a relevant disease model. Aim: The purpose of this study was to assess whether adeno-associated virus type-9 (AAV9)-mediated somatic genome editing could prevent ventricular arrhythmias by removal of the mutant allele in mice that are heterozygous for Ryr2 variant p.Arg176Gln (R176Q/+). Methods and Results: Guide RNA and SaCas9 were delivered using AAV9 vectors injected subcutaneously in 10-day-old mice. At 6 weeks after injection, R176Q/+ mice had a 100% reduction in ventricular arrhythmias compared to controls. When aged to 12 months, injected R176Q/+ mice maintained a 100% reduction in arrhythmia induction. Deep RNA sequencing revealed the formation of insertions/deletions at the target site with minimal off-target editing on the wild-type allele. Consequently, CRISPR/SaCas9 editing resulted in a 45% reduction of total Ryr2 mRNA and a 38% reduction in RyR2 protein. Genome editing was well tolerated based on serial echocardiography, revealing unaltered cardiac function and structure up to 12 months after AAV9 injection. Conclusion: Taken together, AAV9-mediated CRISPR/Cas9 genome editing could efficiently disrupt the mutant Ryr2 allele, preventing lethal arrhythmias while preserving normal cardiac function in the R176Q/+ mouse model of CPVT.
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Myocardial lipid metabolism is critical to normal heart function, whereas altered lipid regulation has been linked to cardiac diseases including cardiomyopathies. Genetic variants in the JPH2 gene can cause hypertrophic cardiomyopathy (HCM) and, in some cases, dilated cardiomyopathy (DCM). In this study, we tested the hypothesis that JPH2 variants identified in patients with HCM and DCM, respectively, cause distinct alterations in myocardial lipid profiles. Echocardiography revealed clinically significant cardiac dysfunction in both knock-in mouse models of cardiomyopathy. Unbiased myocardial lipidomic analysis demonstrated significantly reduced levels of total unsaturated fatty acids, ceramides, and various phospholipids in both mice with HCM and DCM, suggesting a common metabolic alteration in both models. On the contrary, significantly increased di- and triglycerides, and decreased co-enzyme were only found in mice with HCM. Moreover, mice with DCM uniquely exhibited elevated levels of cholesterol ester. Further in-depth analysis revealed significantly altered metabolites from all the lipid classes with either similar or opposing trends in JPH2 mutant mice with HCM or DCM. Together, these studies revealed, for the first time, unique alterations in the cardiac lipid composition-including distinct increases in neutral lipids and decreases in polar membrane lipids-in mice with HCM and DCM were caused by distinct JPH2 variants. These studies may aid the development of novel biomarkers or therapeutics for these inherited disorders.
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Cardiomiopatias , Cardiomiopatia Dilatada , Cardiopatias , Animais , Humanos , Camundongos , Cardiomiopatias/genética , Cardiomiopatia Dilatada/genética , Ceramidas , Proteínas de Membrana/genética , MiocárdioRESUMO
Background: Overexpression of the CREM (cAMP response element-binding modulator) isoform CREM-IbΔC-X in transgenic mice (CREM-Tg) causes the age-dependent development of spontaneous AF. Purpose: To identify key proteome signatures and biological processes accompanying the development of persistent AF through integrated proteomics and bioinformatics analysis. Methods: Atrial tissue samples from three CREM-Tg mice and three wild-type littermates were subjected to unbiased mass spectrometry-based quantitative proteomics, differential expression and pathway enrichment analysis, and protein-protein interaction (PPI) network analysis. Results: A total of 98 differentially expressed proteins were identified. Gene ontology analysis revealed enrichment for biological processes regulating actin cytoskeleton organization and extracellular matrix (ECM) dynamics. Changes in ITGAV, FBLN5, and LCP1 were identified as being relevant to atrial fibrosis and remodeling based on expression changes, co-expression patterns, and PPI network analysis. Comparative analysis with previously published datasets revealed a shift in protein expression patterns from ion-channel and metabolic regulators in young CREM-Tg mice to profibrotic remodeling factors in older CREM-Tg mice. Furthermore, older CREM-Tg mice exhibited protein expression patterns that resembled those of humans with persistent AF. Conclusions: This study uncovered distinct temporal changes in atrial protein expression patterns with age in CREM-Tg mice consistent with the progressive evolution of AF. Future studies into the role of the key differentially abundant proteins identified in this study in AF progression may open new therapeutic avenues to control atrial fibrosis and substrate development in AF.
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Chronic kidney disease (CKD) is associated with a higher risk of atrial fibrillation (AF). The mechanistic link between CKD and AF remains elusive. IL-1ß, a main effector of NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, is a key modulator of conditions associated with inflammation, such as AF and CKD. Circulating IL-1ß levels were elevated in patients with CKD who had AF (versus patients with CKD in sinus rhythm). Moreover, NLRP3 activity was enhanced in atria of patients with CKD. To elucidate the role of NLRP3/IL-1ß signaling in the pathogenesis of CKD-induced AF, Nlrp3-/- and WT mice were subjected to a 2-stage subtotal nephrectomy protocol to induce CKD. Four weeks after surgery, IL-1ß levels in serum and atrial tissue were increased in WT CKD (WT-CKD) mice versus sham-operated WT (WT-sham) mice. The increased susceptibility to pacing-induced AF and the longer AF duration in WT-CKD mice were associated with an abbreviated atrial effective refractory period, enlarged atria, and atrial fibrosis. Genetic inhibition of NLRP3 in Nlrp3-/- mice or neutralizing anti-IL-1ß antibodies effectively reduced IL-1ß levels, normalized left atrial dimensions, and reduced fibrosis and the incidence of AF. These data suggest that CKD creates a substrate for AF development by activating the NLRP3 inflammasome in atria, which is associated with structural and electrical remodeling. Neutralizing IL-1ß antibodies may be beneficial in preventing CKD-induced AF.
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Fibrilação Atrial , Insuficiência Renal Crônica , Humanos , Camundongos , Animais , Inflamassomos/metabolismo , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Átrios do Coração/metabolismo , Interleucina-1beta/metabolismoRESUMO
Introduction: Heart failure (HF) is the leading cause of death worldwide. Most large and small animal disease models of HF are based on surgical procedures. A common surgical technique to induce HF is transverse aortic constriction (TAC), which induces pressure overload. The conventional TAC (cTAC) procedure is a highly invasive surgery that is associated with severe inflammation and excessive perioperative deaths. Aim: To establish an improved, minimally invasive TAC (mTAC) procedure that does not require thoracotomy. Methods and results: Following anesthesia, mice were intubated, and a small incision was made at the neck and chest. After cutting the sternum about 4 mm, the aortic arch was approached without opening the pleural cavity. A suture was placed between the brachiocephalic artery and the left common carotid artery. This model was associated with low perioperative mortality and a highly reproducible constriction evidenced by an increased right-to-left carotid blood flow velocity ratio in mTAC mice (5.9 ± 0.2) vs. sham controls (1.2 ± 0.1; P < 0.001). mTAC mice exhibited progressive cardiac remodeling during the 8 weeks post-TAC, resulting in reduced left ventricular (LV) contractility, increased LV end-systolic diameter, left atrial enlargement and diastolic dysfunction, and an increased heart weight to tibia length ratio (mTAC: 15.0 ± 0.8 vs. sham: 10.1 ± 0.6; P < 0.01). Conclusion: Our data show that the mTAC procedure yields a highly reproducible phenotype consisting of LV contractile dysfunction and enlargement, combined with left atrial enlargement and diastolic dysfunction. Potential impact of the findings: This model may be used to test the molecular mechanisms underlying atrial remodeling associated with HF development or to evaluate therapeutic strategies to treat these conditions.
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Right ventricular dysfunction (RVD) is a risk factor for mortality in multiple cardiovascular diseases, but approaches to combat RVD are lacking. Therapies used for left heart failure are largely ineffective in RVD, and thus the identification of molecules that augment RV function could improve outcomes in a wide-array of cardiac limitations. Junctophilin-2 (JPH2) is an essential protein that plays important roles in cardiomyocytes, including calcium handling/maintenance of t-tubule structure and gene transcription. Additionally, JPH2 may regulate mitochondrial function as Jph2 knockout mice exhibit cardiomyocyte mitochondrial swelling and cristae derangements. Moreover, JPH2 knockdown in embryonic stem cell-derived cardiomyocytes induces downregulation of the mitochondrial protein mitofusin-2 (MFN2), which disrupts mitochondrial cristae structure and transmembrane potential. Impaired mitochondrial metabolism drives RVD, and here we evaluated the mitochondrial role of JPH2. We showed JPH2 directly interacts with MFN2, ablation of JPH2 suppresses mitochondrial biogenesis, oxidative capacity, and impairs lipid handling in iPSC-CM. Gene therapy with AAV9-JPH2 corrects RV mitochondrial morphological defects, mitochondrial fatty acid metabolism enzyme regulation, and restores the RV lipidomic signature in the monocrotaline rat model of RVD. Finally, AAV-JPH2 improves RV function without altering PAH severity, showing JPH2 provides an inotropic effect to the dysfunction RV.
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INTRODUCTION: Abnormal calcium signaling between organelles such as the sarcoplasmic reticulum (SR), mitochondria and lysosomes is a key feature of heart diseases. Calcium serves as a secondary messenger mediating inter-organellar crosstalk, essential for maintaining the cardiomyocyte function. AREAS COVERED: This article examines the available literature related to calcium channels and transporters involved in inter-organellar calcium signaling. The SR calcium-release channels ryanodine receptor type-2 (RyR2) and inositol 1,4,5-trisphosphate receptor (IP3R), and calcium-transporter SR/ER-ATPase 2a (SERCA2a) are illuminated. The roles of mitochondrial voltage-dependent anion channels (VDAC), the mitochondria Ca2+ uniporter complex (MCUC), and the lysosomal H+/Ca2+ exchanger, two pore channels (TPC), and transient receptor potential mucolipin (TRPML) are discussed. Furthermore, recent studies showing calcium-mediated crosstalk between the SR, mitochondria, and lysosomes as well as how this crosstalk is dysregulated in cardiac diseases are placed under the spotlight. EXPERT OPINION: Enhanced SR calcium release via RyR2 and reduced SR reuptake via SERCA2a, increased VDAC and MCUC-mediated calcium uptake into mitochondria, and enhanced lysosomal calcium-release via lysosomal TPC and TRPML may all contribute to aberrant calcium homeostasis causing heart disease. While mechanisms of this crosstalk need to be studied further, interventions targeting these calcium channels or combinations thereof might represent a promising therapeutic strategy.
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Cardiopatias , Canal de Liberação de Cálcio do Receptor de Rianodina , Cálcio/metabolismo , Sinalização do Cálcio , Cardiopatias/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Post-therapeutic relapse remains the biggest challenge to breast cancer management. The re-initiation of proliferation of dormant tumor cells in either metastatic or primary tumor location marks the final rate-limiting step of malignancy and mortality. The underlying molecular mechanisms remain poorly understood. We have recently demonstrated that KLF8 promotes breast cancer metastasis via CXCR4 upregulation. Here we report a role and mechanisms for KLF8 in driving the recurrence-like tumor outgrowth in both secondary and primary sites in a CXCR4-dependent manner. Treatment of an MDA-MB-231 breast cancer cell variant with the CXCR4 ligand, CXCL12, induces formation of filopodia in monolayer culture and filopodium-like protrusions (FLPs) in 3D culture. The FLP+ cells proliferate significantly faster than FLP- cells in the 3D culture supplemented with CXCL12. Both the FLP formation and enhanced proliferation in the 3D culture can be prevented by silencing KLF8 expression in the cells. From this prevention, the cells can be rescued by overexpressing wild-type CXCR4 but not its inactive mutant form in the cells. Overexpression of KLF8 or CXCR4 in the cells dramatically enhances their invasive outgrowth and metastasis after being implanted into immunocompromised mice. Mechanistically, we found that the activated FAK was recruited to the nascent FLPs and that proliferation of the cells was completely prevented with a FAK-specific inhibitor. Taken together, these results shed new light on the role of KLF8 in promoting breast cancer recurrence, the fatal episode of the disease, by inducing CXCR4-dependent FLP formation.
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AIMS: Abnormal intracellular calcium (Ca2+) handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signalling pathway contributes to atrial remodelling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models. METHODS AND RESULTS: Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF (cAF). NFATc1 and NFATc2 mRNA levels were elevated in cAF patients compared with those in normal sinus rhythm (NSR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM:NFATc2-/- mice. Programmed electrical stimulation revealed that CREM:NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with wild-type controls, which was reversed in CREM:NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM:NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on ryanodine receptor type 2 (RyR2) in CREM:RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras and EF-hand domain-containing protein (Rasef) as a direct target of NFATc2-mediated transcription. CONCLUSION: Our findings reveal activation of the NFAT signalling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model.
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Fibrilação Atrial , Fatores de Transcrição NFATC , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Humanos , Camundongos , Fibrilação Atrial/genética , Fibrilação Atrial/prevenção & controle , Fibrilação Atrial/patologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/genética , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , RNA Mensageiro/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismoRESUMO
Loss of atrioventricular conduction system (AVCS) cells due to either inherited or acquired deficits leads to conduction diseases, which can deteriorate into fatal cardiac arrhythmias and sudden death. In this issue of the JCI, Wang et al. constructed a mouse model of atrioventricular block (AVB) by inducing AVCS cell-specific injury using the Cx30.2 enhancer to drive expression of diphtheria toxin fragment A. AVCS cell ablation in adult mice led to irreversible AVB. jkjkIn contrast, AVCS cell injury in neonatal mice was followed by spontaneous recovery in a subset of mice, revealing a limited postnatal time window during which the regeneration of AVCS cells can occur as a result of cellular plasticity. This exciting study paves the way for future research into biological or cellular treatment approaches for cardiac conduction diseases by exploiting the regenerative potential of AVCS cells.
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Regeneração , Animais , CamundongosAssuntos
Quinases Proteína-Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/genética , Fibrilação Atrial/genética , Cardiomiopatias/genética , Animais , Fibrilação Atrial/diagnóstico , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes/métodos , Átrios do Coração/metabolismo , Humanos , CamundongosRESUMO
Hyperphosphorylation of the calcium release channel/ryanodine receptor type 2 (RyR2) at serine 2814 (S2814) is associated with multiple cardiac diseases including atrial fibrillation and heart failure. Despite recent advances, the molecular mechanisms driving pathological changes associated with RyR2 S2814 phosphorylation are still not well understood. Methods: Using affinity-purification coupled to mass spectrometry (AP-MS), we investigated the RyR2 interactome in ventricles from wild-type (WT) mice and two S2814 knock-in mutants: the unphosphorylated alanine mutant (S2814A) and hyperphosphorylated mimic aspartic acid mutant (S2814D). Western blots were used for validation. Results: In WT mouse ventricular lysates, we identified 22 proteins which were enriched with RyR2 pull-down relative to both IgG control and no antibody (beads-only) pull-downs. Parallel AP-MS using WT, S2814A, and S2814D mouse ventricles identified 72 proteins, with 20 being high confidence RyR2 interactors. Of these, 14 had an increase in their binding to RyR2 S2814A but a decrease in their binding to RyR2 S2814D. We independently validated three protein hits, Idh3b, Aifm1, and Cpt1b, as RyR2 interactors by western blots and showed that Aifm1 and Idh3b had significantly decreased binding to RyR2 S2814D compared to WT and S2814A, consistent with MS findings. Conclusion: By applying state-of-the-art proteomic approaches, we discovered a number of novel RyR2 interactors in the mouse heart. In addition, we found and defined specific alterations in the RyR2 interactome that were dependent on the phosphorylation status of RyR2 at S2814. These findings yield mechanistic insights into RyR2 regulation which may guide future drug designs.
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Cardiomyocyte contraction relies on precisely regulated intracellular Ca2+ signaling through various Ca2+ channels and transporters. In this article, we will review the physiological regulation of Ca2+ handling and its role in maintaining normal cardiac rhythm and contractility. We discuss how inherited variants or acquired defects in Ca2+ channel subunits contribute to the development or progression of diseases of the heart. Moreover, we highlight recent insights into the role of protein phosphatase subunits and striated muscle preferentially expressed protein kinase (SPEG) in atrial fibrillation, heart failure, and cardiomyopathies. Finally, this review summarizes current drug therapies and new advances in genome editing as therapeutic strategies for the cardiac diseases caused by aberrant intracellular Ca2+ signaling.
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Sinalização do Cálcio/fisiologia , Cardiopatias/metabolismo , Coração/fisiologia , Animais , HumanosRESUMO
Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.