Combined Neuro-Cardiogenic Epilepsy Syndromes and Novel Mechanistic Insights

Human-computer interactio

Deconstructing sarcomeric structure-function relations in titin-BioID knock-in mice

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin's Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context

Chapter Combined Neuro-Cardiogenic Epilepsy Syndromes and Novel Mechanistic Insights

Human-computer interactio

Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibition ameliorates arrhythmias elicited by junctin ablation under stress conditions

Background Aberrant calcium signaling is considered one of the key mechanisms contributing to arrhythmias, especially in the context of heart failure. In human heart failure, there is significant down-regulation of the sarcoplasmic reticulum (SR) protein junctin, and junctin deficiency in mice is associated with stress-induced arrhythmias. Objective The purpose of this study was to determine whether the increased SR Ca2+ leak and arrhythmias associated with junctin ablation may be associated with increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity and phosphorylation of the cardiac ryanodine receptor (RyR2) and whether pharmacologic inhibition of CaMKII activity may prevent these arrhythmias. Methods Using a combination of biochemical, cellular, and in vivo approaches, we tested the ability of KN-93 to reverse aberrant CaMKII phosphorylation of RyR2. Specifically, we performed protein phosphorylation analysis, in vitro cardiomyocyte contractility and Ca2+ kinetics, and in vivo ECG analysis in junctin-deficient mice. Results In the absence of junctin, RyR2 channels displayed CaMKII-dependent hyperphosphorylation. Notably, CaMKII inhibition by KN-93 reduced the in vivo incidence of stress-induced ventricular tachycardia by 65% in junctin null mice. At the cardiomyocyte level, KN-93 reduced the percentage of junctin null cells exhibiting spontaneous Ca2+ aftertransients and aftercontractions under stress conditions by 35% and 37%, respectively. At the molecular level, KN-93 blunted the CaMKII-mediated hyperphosphorylation of RyR2 and phospholamban under stress conditions. Conclusion Our data suggest that CaMKII inhibition is effective in preventing arrhythmogenesis in the setting of junctin ablation through modulation of both SR Ca2+ release and uptake. Thus, it merits further investigation as promising molecular therapy. © 2015 Published by Elsevier Inc. on behalf of Heart Rhythm Society

Resolving titin's lifecycle and the spatial organization of protein turnover in mouse cardiomyocytes

Cardiac protein homeostasis, sarcomere assembly, and integration of titin as the sarcomeric backbone are tightly regulated to facilitate adaptation and repair. Very little is known on how the >3-MDa titin protein is synthesized, moved, inserted into sarcomeres, detached, and degraded. Here, we generated a bifluorescently labeled knockin mouse to simultaneously visualize both ends of the molecule and follow titin's life cycle in vivo. We find titin mRNA, protein synthesis and degradation compartmentalized toward the Z-disk in adult, but not embryonic cardiomyocytes. Originating at the Z-disk, titin contributes to a soluble protein pool (>15% of total titin) before it is integrated into the sarcomere lattice. Titin integration, disintegration, and reintegration are stochastic and do not proceed sequentially from Z-disk to M-band, as suggested previously. Exchange between soluble and integrated titin depends on titin protein composition and differs between individual cardiomyocytes. Thus, titin dynamics facilitate embryonic vs. adult sarcomere remodeling with implications for cardiac development and disease

Impaired calciumhomeostasis is associated with sudden cardiac death and arrhythmias in a genetic equivalentmouse model of the human HRC-Ser96Ala variant

Aims The histidine-rich calcium-binding protein (HRC) Ser96Ala variant has previously been identified as a potential biomarker for ventricular arrhythmias and sudden cardiac death in patients with idiopathic dilated cardiomyopathy. Herein, the role of this variant in cardiac pathophysiology is delineated through a novel mouse model, carrying the human mutation in the homologous mouse position. Methods and results The mouse HRC serine 81, homologous to human HRC serine 96, was mutated to alanine, using knock-in gene targeting. The HRC-Ser81Ala mice presented increased mortality in the absence of structural or histological abnormalities, indicating that early death may be arrhythmia-related. Indeed, under stress-but not baseline-conditions, the HRC-Ser81Ala mice developed ventricular arrhythmias, whilst at the cardiomyocyte level they exhibited increased occurrence of triggered activity. Cardiac contraction was decreased in vivo, ex vivo, and in vitro. Additionally, Ca2 transients and SR Ca2 load were both reduced suggesting that cytosolic Ca2 overload is not the underlying proarrhythmic mechanism. Interestingly, total SR Ca2 leak was increased in HRC-Ser81Ala cardiomyocytes, without an increase in Ca2 spark and wave frequency. However, Ca2 wave propagation was significantly slower and the duration of the associated Na/Ca exchange current was increased. Moreover, action potential duration was also increased. Notably, Ca2/Calmodulin kinase II (CaMKII) phosphorylation of the ryanodine receptor was increased, whilst KN-93, an inhibitor of CaMKII, reduced the occurrence of arrhythmias. Conclusions The homologous mutation Ser81Ala in HRC in mice, corresponding to Ser96Ala in humans, is associated with sudden death and depressed cardiac function. Ventricular arrhythmias are related to abnormal Ca2 cycling across the SR. The data further support a role for CaMKII with the perspective to treat arrhythmias through CaMKII inhibition

Rationale, objectives, and design of the EUTrigTreat clinical study: A prospective observational study for arrhythmia risk stratification and assessment of interrelationships among repolarization markers and genotype

Aims The EUTrigTreat clinical study has been designed as a prospective multicentre observational study and aims to (i) risk stratify patients with an implantable cardioverter defibrillator (ICD) for mortality and shock risk using multiple novel and established risk markers, (ii) explore a link between repolarization biomarkers and genetics of ion (Ca 2, Na, K) metabolism, (iii) compare the results of invasive and non-invasive electrophysiological (EP) testing, (iv) assess changes of non-invasive risk stratification tests over time, and (v) associate arrythmogenomic risk through 19 candidate genes. Methods and resultsPatients with clinical ICD indication are eligible for the trial. Upon inclusion, patients will undergo non-invasive risk stratification, including beat-to-beat variability of repolarization (BVR), T-wave alternans, T-wave morphology variables, ambient arrhythmias from Holter, heart rate variability, and heart rate turbulence. Non-invasive or invasive programmed electrical stimulation will assess inducibility of ventricular arrhythmias, with the latter including recordings of monophasic action potentials and assessment of restitution properties. Established candidate genes are screened for variants. The primary endpoint is all-cause mortality, while one of the secondary endpoints is ICD shock risk. A mean follow-up of 3.3 years is anticipated. Non-invasive testing will be repeated annually during follow-up. It has been calculated that 700 patients are required to identify risk predictors of the primary endpoint, with a possible increase to 1000 patients based on interim risk analysis. ConclusionThe EUTrigTreat clinical study aims to overcome current shortcomings in sudden cardiac death risk stratification and to answer several related research questions. The initial patient recruitment is expected to be completed in July 2012, and follow-up is expected to end in September 2014. © The Author 2011

Caveolin3 stabilizes McT1-mediated lactate/proton transport in cardiomyocytes

RATIONALE: Caveolin3 variants associated with arrhythmogenic cardiomyopathy and muscular dystrophy can disrupt post-Golgi surface trafficking. As Caveolin1 was recently identified in cardiomyocytes, we hypothesize that conserved isoform-specific protein/protein interactions orchestrate unique cardiomyocyte microdomain functions. To analyze the Caveolin1 versus Caveolin3 interactome, we employed unbiased live-cell proximity proteomic, isoform-specific affinity, and complexome profiling mass spectrometry techniques. We demonstrate the physiological relevance and loss-of-function mechanism of a novel Caveolin3 interactor in gene-edited human iPSC-cardiomyocytes. OBJECTIVE: To identify differential Caveolin1 versus Caveolin3 protein interactions and to define the molecular basis of cardiac CAV3 loss-of-function. METHODS AND RESULTS: Combining stable isotope labeling with proximity proteomics, we applied mass spectrometry to screen for putative Caveolin3 interactors in living cardiomyocytes. Isoform-specific affinity proteomic and co-immunoprecipitation experiments confirmed the monocarboxylate transporter McT1 versus aquaporin1, respectively, as Caveolin3 or Caveolin1 specific interactors in cardiomyocytes. Superresolution STED microscopy showed distinct Caveolin1 versus Caveolin3 cluster distributions in cardiomyocyte transverse tubules. CRISPR/Cas9-mediated Caveolin3 knock-out uncovered a stabilizing role for McT1 surface expression, proton-coupled lactate shuttling, increased late Na+ currents, and early afterdepolarizations in human iPSC-derived cardiomyocytes. Complexome profiling confirmed that McT1 and the Na,K-ATPase form labile protein assemblies with the multimeric Caveolin3 complex. CONCLUSIONS: Combining the strengths of proximity and affinity proteomics, we identified isoform-specific Caveolin1 versus Caveolin3 binding partners in cardiomyocytes. McT1 represents a novel class of metabolically relevant Caveolin3-specific interactors close to mitochondria in cardiomyocyte transverse tubules. Caveolin3 knock-out uncovered a previously unknown role for functional stabilization of McT1 in the surface membrane of human cardiomyocytes. Strikingly, Caveolin3 deficient cardiomyocytes exhibit action potential prolongation and instability, reproducing human reentry arrhythmias in silico. Given that lactate is a major substrate for stress adaption both in the healthy and the diseased human heart, future studies of conserved McT1/Caveolin3 interactions may provide rationales to target this muscle-specific assembly function therapeutically