ABSTRACT
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.
Subject(s)
Pedigree , Tachycardia, Supraventricular , Wolff-Parkinson-White Syndrome , Wolff-Parkinson-White Syndrome/genetics , Wolff-Parkinson-White Syndrome/physiopathology , Wolff-Parkinson-White Syndrome/pathology , Humans , Animals , Mice , Tachycardia, Supraventricular/genetics , Tachycardia, Supraventricular/physiopathology , Tachycardia, Supraventricular/pathology , Male , Female , Adult , Mutation, Missense , Exome Sequencing , Electrocardiography , Middle AgedABSTRACT
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.
Subject(s)
Calpain , Heart Failure , Membrane Proteins , Myocardial Infarction , Animals , Calpain/metabolism , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Heart Failure/metabolism , Heart Failure/etiology , Humans , Mice , Membrane Proteins/metabolism , Membrane Proteins/genetics , Disease Progression , Male , Disease Models, Animal , Proteolysis , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Muscle ProteinsABSTRACT
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.
ABSTRACT
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.
Subject(s)
Atrial Fibrillation , Cyclic AMP Response Element Modulator , Fibrosis , Heart Atria , Mice, Transgenic , Proteomics , Animals , Atrial Fibrillation/metabolism , Atrial Fibrillation/genetics , Cyclic AMP Response Element Modulator/metabolism , Cyclic AMP Response Element Modulator/genetics , Proteomics/methods , Heart Atria/metabolism , Heart Atria/pathology , Mice , Gene Expression Regulation , Protein Interaction Maps , Proteome/metabolism , Disease Models, Animal , Gene Expression Profiling , Extracellular Matrix/metabolism , MaleABSTRACT
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.
ABSTRACT
BACKGROUND: Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility. METHODS: Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies. RESULTS: In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (P=2.0×10-2; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (P=1.4×10-6; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9×10-2 and 6.7×10-3, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (P=5.0×10-6; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (P=1.8×10-2 and 4.1×10-2, respectively; n=6, 6, and 5). CONCLUSIONS: Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.
Subject(s)
Atrial Fibrillation , Protein Phosphatase 1 , Stroke , Animals , Humans , Mice , Atrial Fibrillation/metabolism , Heart Atria/metabolism , Phosphorylation , Protein Phosphatase 1/genetics , Protein Phosphatase 1/metabolismABSTRACT
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.
Subject(s)
Atrial Fibrillation , Renal Insufficiency, Chronic , Humans , Mice , Animals , Inflammasomes/metabolism , Atrial Fibrillation/genetics , Atrial Fibrillation/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Renal Insufficiency, Chronic/genetics , Renal Insufficiency, Chronic/metabolism , Heart Atria/metabolism , Interleukin-1beta/metabolismABSTRACT
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.
ABSTRACT
Background: Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods: Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results: In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls ( P =2.0×10 -2 , n=12,12 in each group) with > 40% reduction in MLC2a phosphorylation ( P =1.4×10 -6 , n=12,12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF ( P =2.9×10 -2 and 6.7×10 -3 respectively, n=8,8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls ( P =5.0×10 -6 , n=12,8,12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls ( P =1.8×10 -2 and 4.1×10 -2 respectively, n= 6,6,5). Conclusions: AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.
ABSTRACT
Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are homologous cation channels that mediate release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR) and thereby are involved in many physiological processes. In previous studies, we determined that when the D2594 residue, located at or near the gate of the IP3R type 1, was replaced by lysine (D2594K), a gain of function was obtained. This mutant phenotype was characterized by increased IP3 sensitivity. We hypothesized the IP3R1-D2594 determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states. To test this possibility, the relationship between the D2594 site and IP3R1 regulation by IP3, cytosolic, and luminal Ca2+ was determined at the cellular, subcellular, and single-channel levels using fluorescence Ca2+ imaging and single-channel reconstitution. We found that in cells, D2594K mutation enhances the IP3 ligand sensitivity. Single-channel IP3R1 studies revealed that the conductance of IP3R1-WT and -D2594K channels is similar. However, IP3R1-D2594K channels exhibit higher IP3 sensitivity, with substantially greater efficacy. In addition, like its wild type (WT) counterpart, IP3R1-D2594K showed a bell-shape cytosolic Ca2+-dependency, but D2594K had greater activity at each tested cytosolic free Ca2+ concentration. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. Taken together, our functional studies indicate that the substitution of a negatively charged residue by a positive one at the channels' pore cytosolic exit affects the channel's gating behavior thereby explaining the enhanced ligand-channel's sensitivity.
Subject(s)
Calcium Signaling , Endoplasmic Reticulum , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Ligands , Mutation , Endoplasmic Reticulum/metabolism , Calcium/metabolismABSTRACT
BACKGROUND: Spontaneously depolarizing nodal cells comprise the pacemaker of the heart. Intracellular calcium (Ca2+) plays a critical role in mediating nodal cell automaticity and understanding this so-called Ca2+ clock is critical to understanding nodal arrhythmias. We previously demonstrated a role for Jph2 (junctophilin 2) in regulating Ca2+-signaling through inhibition of RyR2 (ryanodine receptor 2) Ca2+ leak in cardiac myocytes; however, its role in pacemaker function and nodal arrhythmias remains unknown. We sought to determine whether nodal Jph2 expression silencing causes increased sinoatrial and atrioventricular nodal cell automaticity due to aberrant RyR2 Ca2+ leak. METHODS: A tamoxifen-inducible, nodal tissue-specific, knockdown mouse of Jph2 was achieved using a Cre-recombinase-triggered short RNA hairpin directed against Jph2 (Hcn4:shJph2). In vivo cardiac rhythm was monitored by surface ECG, implantable cardiac telemetry, and intracardiac electrophysiology studies. Intracellular Ca2+ imaging was performed using confocal-based line scans of isolated nodal cells loaded with fluorescent Ca2+ reporter Cal-520. Whole cell patch clamp was conducted on isolated nodal cells to determine action potential kinetics and sodium-calcium exchanger function. RESULTS: Hcn4:shJph2 mice demonstrated a 40% reduction in nodal Jph2 expression, resting sinus tachycardia, and impaired heart rate response to pharmacologic stress. In vivo intracardiac electrophysiology studies and ex vivo optical mapping demonstrated accelerated junctional rhythm originating from the atrioventricular node. Hcn4:shJph2 nodal cells demonstrated increased and irregular Ca2+ transient generation with increased Ca2+ spark frequency and Ca2+ leak from the sarcoplasmic reticulum. This was associated with increased nodal cell AP firing rate, faster diastolic repolarization rate, and reduced sodium-calcium exchanger activity during repolarized states compared to control. Phenome-wide association studies of the JPH2 locus identified an association with sinoatrial nodal disease and atrioventricular nodal block. CONCLUSIONS: Nodal-specific Jph2 knockdown causes increased nodal automaticity through increased Ca2+ leak from intracellular stores. Dysregulated intracellular Ca2+ underlies nodal arrhythmogenesis in this mouse model.
Subject(s)
Calcium , Ryanodine Receptor Calcium Release Channel , Animals , Mice , Calcium/metabolism , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Myocytes, Cardiac/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/metabolism , Sinoatrial Node , Sodium-Calcium Exchanger/metabolismABSTRACT
Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that 'dispersion' of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged ß-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of ß-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA, which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.
Subject(s)
Heart Failure , Ryanodine Receptor Calcium Release Channel , Adrenergic Agents/metabolism , Adrenergic Agents/pharmacology , Animals , Calcium/metabolism , Calcium Signaling/physiology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Heart Failure/metabolism , Homeostasis , Mice , Myocytes, Cardiac/metabolism , Phosphorylation , Rats , Ryanodine Receptor Calcium Release Channel/metabolismABSTRACT
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is triggered by exercise or acute emotion in patients with normal resting electrocardiogram. The major disease-causing gene is RYR2, encoding the cardiac ryanodine receptor (RyR2). We report a novel RYR2 variant, p.Asp3291Val, outside the four CPVT mutation hotspots, in three CPVT families with numerous sudden deaths. This missense variant was first identified in a four-generation family, where eight sudden cardiac deaths occurred before the age of 30 in the context of adrenergic stress. All affected subjects harbored at least one copy of the RYR2 variant. Three affected sisters were homozygous for the variant. The same variant was found in two additional CPVT families. It is located in the helical domain 2 and changes a negatively charged amino acid widely conserved through evolution. Functional analysis of D3291V channels revealed a normal response to cytosolic Ca2+, a markedly reduced luminal Ca2+ sensitivity and, more importantly, an absence of normal response to 8-bromo-cAMP and forskolin stimulation in both transfected HEK293 and HL-1 cells. Our data support that the D3291V-RyR2 is a loss-of-function RyR2 variant responsible for an atypical form of CPVT inducing a mild dysfunction in basal conditions but leading potentially to fatal events through its unresponsiveness to adrenergic stimulation.
ABSTRACT
[Figure: see text].
Subject(s)
Calcium Signaling , Membrane Proteins/metabolism , Myocytes, Cardiac/metabolism , Ryanodine Receptor Calcium Release Channel/genetics , Tachycardia, Ventricular/genetics , Action Potentials , Animals , Binding Sites , Cells, Cultured , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mutation , Myocytes, Cardiac/physiology , Myocytes, Cardiac/ultrastructure , Protein Binding , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/metabolism , Tachycardia, Ventricular/metabolism , Tachycardia, Ventricular/pathologyABSTRACT
Ca2+ alternans (Ca-Alts) are alternating beat-to-beat changes in the amplitude of Ca2+ transients that frequently occur during tachycardia, ischemia, or hypothermia that can lead to sudden cardiac death. Ca-Alts appear to result from a variation in the amount of Ca2+ released from the sarcoplasmic reticulum (SR) between two consecutive heartbeats. This variable Ca2+ release has been attributed to the alternation of the action potential duration, delay in the recovery from inactivation of RYR Ca2+ release channel (RYR2), or an incomplete Ca2+ refilling of the SR. In all three cases, the RYR2 mobilizes less Ca2+ from the SR in an alternating manner, thereby generating an alternating profile of the Ca2+ transients. We used a new experimental approach, fluorescence local field optical mapping (FLOM), to record at the epicardial layer of an intact heart with subcellular resolution. In conjunction with a local cold finger, a series of images were recorded within an area where the local cooling induced a temperature gradient. Ca-Alts were larger in colder regions and occurred without changes in action potential duration. Analysis of the change in the enthalpy and Q10 of several kinetic processes defining intracellular Ca2+ dynamics indicated that the effects of temperature change on the relaxation of intracellular Ca2+ transients involved both passive and active mechanisms. The steep temperature dependency of Ca-Alts during tachycardia suggests Ca-Alts are generated by insufficient SERCA-mediated Ca2+ uptake into the SR. We found that Ca-Alts are heavily dependent on intra-SR Ca2+ and can be promoted through partial pharmacologic inhibition of SERCA2a. Finally, the FLOM experimental approach has the potential to help us understand how arrhythmogenesis correlates with the spatial distribution of metabolically impaired myocytes along the myocardium.
Subject(s)
Calcium , Myocytes, Cardiac , Calcium/metabolism , Calcium Signaling , Myocytes, Cardiac/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/metabolismABSTRACT
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.
Subject(s)
Calcium Signaling/physiology , Heart Diseases/metabolism , Heart/physiology , Animals , HumansABSTRACT
Proper cardiac Ca2+ homeostasis is essential for normal excitation-contraction coupling. Perturbations in cardiac Ca2+ handling through altered kinase activity has been implicated in altered cardiac contractility and arrhythmogenesis. Thus, a better understanding of cardiac Ca2+ handling regulation is vital for a better understanding of various human disease processes. 'Striated muscle preferentially expressed protein kinase' (SPEG) is a member of the myosin light chain kinase family that is key for normal cardiac function. Work within the last 5 years has revealed that SPEG has a crucial role in maintaining normal cardiac Ca2+ handling through maintenance of transverse tubule formation and phosphorylation of junctional membrane complex proteins. Additionally, SPEG has been causally impacted in human genetic diseases such as centronuclear myopathy and dilated cardiomyopathy as well as in common acquired cardiovascular disease such as heart failure and atrial fibrillation. Given the rapidly emerging role of SPEG as a key cardiac Ca2+ regulator, we here present this review in order to summarize recent findings regarding the mechanisms of SPEG regulation of cardiac excitation-contraction coupling in both physiology and human disease. A better understanding of the roles of SPEG will be important for a more complete comprehension of cardiac Ca2+ regulation in physiology and disease.
Subject(s)
Calcium/metabolism , Excitation Contraction Coupling , Heart Diseases/enzymology , Muscle Proteins/metabolism , Myocardial Contraction , Myocytes, Cardiac/enzymology , Protein Serine-Threonine Kinases/metabolism , Animals , Atrial Fibrillation/enzymology , Atrial Fibrillation/genetics , Atrial Fibrillation/pathology , Atrial Fibrillation/physiopathology , Genetic Predisposition to Disease , Heart Diseases/genetics , Heart Diseases/pathology , Heart Diseases/physiopathology , Heart Failure/enzymology , Heart Failure/genetics , Heart Failure/pathology , Heart Failure/physiopathology , Homeostasis , Humans , Muscle Proteins/genetics , Mutation , Myocytes, Cardiac/pathology , Protein Serine-Threonine Kinases/geneticsABSTRACT
The relationship between cardiac excitability and contractility depends on when Ca2+ influx occurs during the ventricular action potential (AP). In mammals, it is accepted that Ca2+ influx through the L-type Ca2+ channels occurs during AP phase 2. However, in murine models, experimental evidence shows Ca2+ influx takes place during phase 1. Interestingly, Ca2+ influx that activates contraction is highly regulated by the autonomic nervous system. Indeed, autonomic regulation exerts multiple effects on Ca2+ handling and cardiac electrophysiology. In this paper, we explore autonomic regulation in endocardial and epicardial layers of intact beating mice hearts to evaluate their role on cardiac excitability and contractility. We hypothesize that in mouse cardiac ventricles the influx of Ca2+ that triggers excitation-contraction coupling (ECC) does not occur during phase 2. Using pulsed local field fluorescence microscopy and loose patch photolysis, we show sympathetic stimulation by isoproterenol increased the amplitude of Ca2+ transients in both layers. This increase in contractility was driven by an increase in amplitude and duration of the L-type Ca2+ current during phase 1. Interestingly, the ß-adrenergic increase of Ca2+ influx slowed the repolarization of phase 1, suggesting a competition between Ca2+ and K+ currents during this phase. In addition, cAMP activated L-type Ca2+ currents before SR Ca2+ release activated the Na+-Ca2+ exchanger currents, indicating Cav1.2 channels are the initial target of PKA phosphorylation. In contrast, parasympathetic stimulation by carbachol did not have a substantial effect on amplitude and kinetics of endocardial and epicardial Ca2+ transients. However, carbachol transiently decreased the duration of the AP late phase 2 repolarization. The carbachol-induced shortening of phase 2 did not have a considerable effect on ventricular pressure and systolic Ca2+ dynamics. Interestingly, blockade of muscarinic receptors by atropine prolonged the duration of phase 2 indicating that, in isolated hearts, there is an intrinsic release of acetylcholine. In addition, the acceleration of repolarization induced by carbachol was blocked by the acetylcholine-mediated K+ current inhibition. Our results reveal the transmural ramifications of autonomic regulation in intact mice hearts and support our hypothesis that Ca2+ influx that triggers ECC occurs in AP phase 1 and not in phase 2.