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In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca2+/calmodulin-dependent protein kinase II (CaMKII) can contribute to a further destabilization of RyR2 that results in triggered arrhythmias. Therefore, inhibition of CaMKII in such conditions has been suggested as a strategy to suppress RyR2 activity and arrhythmias. However, suppression of RyR2 activity can lead to the development of arrhythmogenic Ca2+ alternans. The aim of this study was to test whether the suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca2+ alternans. We studied spontaneous Ca2+ release events and Ca2+ alternans in isolated left ventricular cardiomyocytes from mice carrying the gain-of-function RyR2 mutation RyR2-R2474S and from wild-type mice. CaMKII inhibition by KN-93 effectively decreased the frequency of spontaneous Ca2+ release events in RyR2-R2474S cardiomyocytes exposed to the β-adrenoceptor agonist isoprenaline. However, KN-93-treated RyR2-R2474S cardiomyocytes also showed increased propensity for Ca2+ alternans and increased Ca2+ alternans ratio compared with both an inactive analog of KN-93 and with vehicle-treated controls. This increased propensity for Ca2+ alternans was explained by prolongation of Ca2+ release refractoriness. Importantly, the increased propensity for Ca2+ alternans in KN-93-treated RyR2-R2474S cardiomyocytes did not surpass that of wild type. In conclusion, inhibition of CaMKII efficiently reduces spontaneous Ca2+ release but promotes Ca2+ alternans in RyR2-R2474S cardiomyocytes with a gain-of-function RyR2 mutation. The dominant effect in RyR2-R2474S is to reduce spontaneous Ca2+ release, which supports this intervention as a therapeutic strategy in this specific condition. However, future studies on CaMKII inhibition in conditions with increased propensity for Ca2+ alternans should include investigation of both phenomena.NEW & NOTEWORTHY Genetically increased RyR2 activity promotes arrhythmogenic Ca2+ release. Inhibition of CaMKII suppresses RyR2 activity and arrhythmogenic Ca2+ release. Suppression of RyR2 activity prolongs refractoriness of Ca2+ release. Prolonged refractoriness of Ca2+ release leads to arrhythmogenic Ca2+ alternans. CaMKII inhibition promotes Ca2+ alternans by prolonging Ca2+ release refractoriness.

Rationale: 3′,5′-cAMP is a ubiquitous second messenger which, upon β-AR (β-adrenergic receptor) stimulation, acts in microdomains to regulate cardiac excitation-contraction coupling by activating phosphorylation of calcium handling proteins. One crucial microdomain is in vicinity of the cardiac RyR2 (ryanodine receptor type 2) which is associated with arrhythmogenic diastolic calcium leak from the sarcoplasmic reticulum often occurring in heart failure. Objective: We sought to establish a real-time live-cell imaging approach capable of directly visualizing cAMP in the vicinity of mouse and human RyR2 and to analyze its pathological changes in failing cardiomyocytes under β-AR stimulation. Methods and Results: We generated a novel targeted fluorescent biosensor Epac1 (exchange protein directly activated by cAMP 1)-JNC (junctin) for RyR2-associated cAMP and expressed it in transgenic mouse hearts as well in human ventricular myocytes using adenoviral gene transfer. In healthy cardiomyocytes, β 1 -AR but not β 2 -AR stimulation strongly increased local RyR2-associated cAMP levels. However, already in cardiac hypertrophy induced by aortic banding, there was a marked subcellular redistribution of PDEs (phosphodiesterases) 2, 3, and 4, which included a dramatic loss of the local pool of PDE4. This was also accompanied by measurable β 2 -AR/AMP signals in the vicinity of RyR2 in failing mouse and human myocytes, increased β 2 -AR–dependent RyR2 phosphorylation, sarcoplasmic reticulum calcium leak, and arrhythmia susceptibility. Conclusions: Our new imaging approach could visualize cAMP levels in the direct vicinity of cardiac RyR2. Unexpectedly, in mouse and human failing myocytes, it could uncover functionally relevant local arrhythmogenic β 2 -AR/cAMP signals which might be an interesting antiarrhythmic target for heart failure.

The incidence of aortic valve stenosis (AS), the most common reason for aortic valve replacement (AVR), increases with population ageing. While untreated AS is associated with high mortality, different hemodynamic subtypes range from normal left-ventricular function to severe heart failure. However, the molecular nature underlying four different AS subclasses, suggesting vastly different myocardial fates, is unknown. Here, we used direct proteomic analysis of small left-ventricular biopsies to identify unique protein expression profiles and subtype-specific AS mechanisms. Left-ventricular endomyocardial biopsies were harvested from patients during transcatheter AVR, and inclusion criteria were based on echocardiographic diagnosis of severe AS and guideline-defined AS-subtype classification: 1) normal ejection fraction (EF)/high-gradient; 2) low EF/high-gradient; 3) low EF/low-gradient; and 4) paradoxical low-flow/low-gradient AS. Samples from non-failing donor hearts served as control. We analyzed 25 individual left-ventricular biopsies by data-independent acquisition mass spectrometry (DIA-MS), and 26 biopsies by histomorphology and cardiomyocytes by STimulated Emission Depletion (STED) superresolution microscopy. Notably, DIA-MS reliably detected 2273 proteins throughout each individual left-ventricular biopsy, of which 160 proteins showed significant abundance changes between AS-subtype and non-failing samples including the cardiac ryanodine receptor (RyR2). Hierarchical clustering segregated unique proteotypes that identified three hemodynamic AS-subtypes. Additionally, distinct proteotypes were linked with AS-subtype specific differences in cardiomyocyte hypertrophy. Furthermore, superresolution microscopy of immunolabeled biopsy sections showed subcellular RyR2-cluster fragmentation and disruption of the functionally important association with transverse tubules, which occurred specifically in patients with systolic dysfunction and may hence contribute to depressed left-ventricular function in AS.

Junctophilins (JPHs) comprise a family of structural proteins that connect the plasma membrane to intracellular organelles such as the endo/sarcoplasmic reticulum (ER/SR). Tethering of these membrane structures results in the formation of highly organized subcellular junctions that play important signaling roles in all excitable cell types. There are four JPH isoforms, expressed primarily in muscle and neuronal cell types. Each JPH protein consists of six membrane occupation and recognition nexus (MORN) motifs, a joining region connecting these to another set of two MORN motifs, a putative alpha-helical region, a divergent region exhibiting low homology between JPH isoforms, and a carboxy-terminal transmembrane region anchoring into the ER/SR membrane. JPH isoforms play essential roles in developing and maintaining subcellular membrane junctions. Conversely, inherited mutations in JPH2 cause hypertrophic or dilated cardiomyopathy, while trinucleotide expansions in the JPH3 gene cause Huntington Disease-Like 2. Loss of JPH1 protein levels can cause skeletal myopathy, while loss of cardiac JPH2 levels causes heart failure and atrial fibrillation, among other disease. This review will provide a comprehensive overview of the JPH gene family, phylogeny, and evolutionary analysis of JPH genes and other MORN domain proteins. JPH biogenesis, membrane tethering, and binding partners will be discussed, as well as functional roles of JPH isoforms in excitable cells. Finally, potential roles of JPH isoform deficits in human disease pathogenesis will be reviewed.

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca

Ensuring scientific reproducibility and compliance with documentation guidelines of funding bodies and journals is a topic of greatly increasing importance in biomedical research. Failure to comply, or unawareness of documentation standards can have adverse effects on the translation of research into patient treatments, as well as economic implications. In the context of the German Research Foundation-funded collaborative research center (CRC) 1002, an IT-infrastructure sub-project was designed. Its goal has been to establish standardized metadata documentation and information exchange benefitting the participating research groups with minimal additional documentation efforts.Implementation of the self-developed menoci-based research data platform (RDP) was driven by close communication and collaboration with researchers as early adopters and experts. Requirements analysis and concept development involved in person observation of experimental procedures, interviews and collaboration with researchers and experts, as well as the investigation of available and applicable metadata standards and tools. The Drupal-based RDP features distinct modules for the different documented data and workflow types, and both the development and the types of collected metadata were continuously reviewed and evaluated with the early adopters.The menoci-based RDP allows for standardized documentation, sharing and cross-referencing of different data types, workflows, and scientific publications. Different modules have been implemented for specific data types and workflows, allowing for the enrichment of entries with specific metadata and linking to further relevant entries in different modules.Taking the workflows and datasets of the frequently involved experimental service projects as a starting point for (meta-)data types to overcome irreproducibility of research data, results in increased benefits for researchers with minimized efforts. While the menoci-based RDP with its data models and metadata schema was originally developed in a cardiological context, it has been implemented and extended to other consortia at GÃűttingen Campus and beyond in different life science research areas.

Introduction: Ensuring scientific reproducibility and compliance with documentation guidelines of funding bodies and journals is a topic of greatly increasing importance in biomedical research. Failure to comply, or unawareness of documentation standards can have adverse effects on the translation of research into patient treatments, as well as economic implications. In the context of the German Research Foundation-funded collaborative research center (CRC) 1002, an IT-infrastructure sub-project was designed. Its goal has been to establish standardized metadata documentation and information exchange benefitting the participating research groups with minimal additional documentation efforts. Methods: Implementation of the self-developed menoci-based research data platform (RDP) was driven by close communication and collaboration with researchers as early adopters and experts. Requirements analysis and concept development involved in person observation of experimental procedures, interviews and collaboration with researchers and experts, as well as the investigation of available and applicable metadata standards and tools. The Drupal-based RDP features distinct modules for the different documented data and workflow types, and both the development and the types of collected metadata were continuously reviewed and evaluated with the early adopters. Results: The menoci-based RDP allows for standardized documentation, sharing and cross-referencing of different data types, workflows, and scientific publications. Different modules have been implemented for specific data types and workflows, allowing for the enrichment of entries with specific metadata and linking to further relevant entries in different modules. Discussion: Taking the workflows and datasets of the frequently involved experimental service projects as a starting point for (meta-)data types to overcome irreproducibility of research data, results in increased benefits for researchers with minimized efforts. While the menoci-based RDP with its data models and metadata schema was originally developed in a cardiological context, it has been implemented and extended to other consortia at GÃűttingen Campus and beyond in different life science research areas.

Aims Cardiac arrhythmias are a major factor in the occurrence of morbidity and sudden death in patients with cardiovascular disease. Disturbances of Ca2+ homeostasis in the heart contribute to the initiation and maintenance of cardiac arrhythmias. Extrasystolic increases in intracellular Ca2+ lead to delayed afterdepolarizations and triggered activity, which can result in heart rhythm abnormalities. It is being suggested that the Ca2+-activated nonselective cation channel TRPM4 is involved in the aetiology of triggered activity, but the exact contribution and in vivo significance are still unclear. Methods and results In vitro electrophysiological and calcium imaging technique as well as in vivo intracardiac and telemetric electrocardiogram measurements in physiological and pathophysiological conditions were performed. In two distinct Ca2+-dependent proarrhythmic models, freely moving Trpm4−/− mice displayed a reduced burden of cardiac arrhythmias. Looking further into the specific contribution of TRPM4 to the cellular mechanism of arrhythmias, TRPM4 was found to contribute to a long-lasting Ca2+ overload-induced background current, thereby regulating cell excitability in Ca2+ overload conditions. To expand these results, a compound screening revealed meclofenamate as a potent antagonist of TRPM4. In line with the findings from Trpm4−/− mice, 10 µM meclofenamate inhibited the Ca2+ overload-induced background current in ventricular cardiomyocytes and 15 mg/kg meclofenamate suppressed catecholaminergic polymorphic ventricular tachycardia-associated arrhythmias in a TRPM4-dependent manner. Conclusion The presented data establish that TRPM4 represents a novel target in the prevention and treatment of Ca2+-dependent triggered arrhythmias.

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited arrhythmogenic disorder caused by missense mutations in the cardiac ryanodine receptors (RyR2), that result in increased β‐adrenoceptor stimulation‐induced diastolic Ca2+ leak. We have previously shown that exercise training prevents arrhythmias in CPVT1, potentially by reducing the oxidation of Ca2+/calmodulin‐dependent protein kinase type II (CaMKII). Therefore, we tested whether an oxidation‐resistant form of CaMKII protects mice carrying the CPVT1‐causative mutation RyR2‐R2474S (RyR2‐RS) against arrhythmias. Antioxidant treatment (N‐acetyl‐L‐cysteine) reduced the frequency of β‐adrenoceptor stimulation‐induced arrhythmogenic Ca2+ waves in isolated cardiomyocytes from RyR2‐RS mice. To test whether the prevention of CaMKII oxidation exerts an antiarrhythmic effect, mice expressing the oxidation‐resistant CaMKII‐MM281/282VV variant (MMVV) were crossed with RyR2‐RS mice to create a double transgenic model (RyR2‐RS/MMVV). Wild‐type mice served as controls. Telemetric ECG surveillance revealed an increased incidence of ventricular tachycardia and an increased arrhythmia score in both RyR2‐RS and RyR2‐RS/MMVV compared to wild‐type mice, both following a β‐adrenoceptor challenge (isoprenaline i.p.), and following treadmill exercise combined with a β‐adrenoceptor challenge. There were no differences in the incidence of arrhythmias between RyR2‐RS and RyR2‐RS/MMVV mice. Furthermore, no differences were observed in β‐adrenoceptor stimulation‐induced Ca2+ waves in RyR2‐RS/MMVV compared to RyR2‐RS. In conclusion, antioxidant treatment reduces β‐adrenoceptor stimulation‐induced Ca2+ waves in RyR2‐RS cardiomyocytes. However, oxidation‐resistant CaMKII‐MM281/282VV does not protect RyR2‐RS mice from β‐adrenoceptor stimulation‐induced Ca2+ waves or arrhythmias. Hence, alternative oxidation‐sensitive targets need to be considered to explain the beneficial effect of antioxidant treatment on Ca2+ waves in cardiomyocytes from RyR2‐RS mice., Inhibition of Ca2+/calmodulin‐dependent protein kinase type II (CaMKII) prevents arrhythmias in murine models of catecholaminergic polymorphic ventricular tachycardia (CPVT). CaMKII can be activated by the oxidation of methionines 281/282, and we show that antioxidant treatment may limit arrhythmogenic activity in CPVT. However, genetic inhibition of CaMKII oxidation at methionines 281/282 does not protect CPVT mice from arrhythmias. Other oxidation‐sensitive targets should be explored to explain the effect of antioxidants in CPVT.

In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a familial stress-induced arrhythmia syndrome, mostly caused by mutations in Ryanodine receptor 2 (RyR2), the sarcoplasmic reticulum (SR) Ca2+ release channel in cardiomyocytes. Pathogenetic mutations lead to gain of function in the channel, causing arrhythmias by promoting diastolic spontaneous Ca2+ release (SCR) from the SR and delayed afterdepolarizations. While the study of Ca2+ dynamics in single cells from murine CPVT models has increased our understanding of the disease pathogenesis, questions remain on the mechanisms triggering the lethal arrhythmias at tissue level. Here, we combined subcellular analysis of Ca2+ signals in isolated cardiomyocytes and in acute thick ventricular slices of RyR2R2474S knock-in mice, electrically paced at different rates (1–5 Hz), to identify arrhythmogenic Ca2+ dynamics, from the sub- to the multicellular perspective. In both models, RyR2R2474S cardiomyocytes had increased propensity to develop SCR upon adrenergic stimulation, which manifested, in the slices, with Ca2+ alternans and synchronous Ca2+ release events in neighboring cardiomyocytes. Analysis of Ca2+ dynamics in multiple cells in the tissue suggests that SCRs beget SCRs in contiguous cells, overcoming the protective electrotonic myocardial coupling, and potentially generating arrhythmia triggering foci. We suggest that intercellular interactions may underscore arrhythmic propensity in CPVT hearts with ‘leaky’ RyR2.

The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days. By trio whole-exome sequencing, we identified a novel homozygous missense mutation, c.662T > C, in YRDC affecting an evolutionary highly conserved amino acid (p.Ile221Thr). Functional characterization of patient-derived dermal fibroblasts revealed that this mutation impairs YRDC function and consequently results in reduced t6A modifications of tRNAs. Furthermore, we established and performed a novel and highly sensitive 3-D Q-FISH analysis based on single-telomere detection to investigate the impact of YRDC on telomere maintenance. This analysis revealed significant telomere shortening in YRDC-mutant cells. Moreover, single-cell RNA sequencing analysis of YRDC-mutant fibroblasts revealed significant transcriptome-wide changes in gene expression, specifically enriched for genes associated with processes involved in DNA repair. We next examined the DNA damage response of patient’s dermal fibroblasts and detected an increased susceptibility to genotoxic agents and a global DNA double-strand break repair defect. Thus, our data suggest that YRDC may affect the maintenance of genomic stability. Together, our findings indicate that biallelic variants in YRDC result in a developmental disorder with progeroid features and might be linked to increased genomic instability and telomere shortening.

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): South-Eastern Norway Regional Health Authority Background In catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) adrenergic activation of the cardiac ryanodine receptor (RyR2) causes spontaneous calcium release and triggers arrhythmias. Calcium/calmodulin-dependent protein kinase II (CaMKII) can contribute to such arrhythmogenic calcium release and has been proposed as a therapeutic target in CPVT1. To predict the efficacy and safety of this strategy, it is necessary to know whether the mechanism for CaMKII activation is important for its arrhythmogenic effects, and if inhibition has proarrhythmic effects. We tested (1) if oxidation of CaMKII contributes to spontaneous calcium release in CPVT1 and (2) if inhibition of CaMKII in this condition can induce calcium alternans. Methods Mice with the CPVT1-causative mutation RyR2-R2474S (RyR2-RS) were crossed with mice with the CaMKII M281/282V (MMVV) mutation that prevents CaMKII M281/282 oxidation, to create double mutants (RyR2-RSxMMVV). Telemetric ECG surveillance was used to study in vivo arrhythmias following an adrenergic challenge by i.p. administration of the beta-adrenoceptor agonist isoprenaline. Confocal line-scan imaging and whole-cell calcium imaging were used to study arrhythmogenic calcium release in isolated left ventricular cardiomyocytes during stimulation with isoprenaline. Results As expected, RyR2-RS mice exhibited more arrhythmic events and spontaneous calcium release (i.e. calcium sparks and calcium waves) compared to wild-type mice. Treatment of RyR2-RS cardiomyocytes with either the CaMKII inhibitor KN-93 or the antioxidant n-acetyl-cysteine reduced spontaneous calcium release (i.e. calcium sparks and calcium waves, for KN-93 and n-acetyl-cysteine, respectively). Interestingly, CaMKII inhibition by KN-93 also increased both incidence and degree of arrhythmogenic calcium alternans in RyR2-RS cardiomyocytes. This adverse effect was a result of prolonged refractoriness of calcium release. Furthermore, to test whether the protective effect of antioxidant treatment in RyR2-RS was mediated via CaMKII oxidation, we compared arrhythmias and spontaneous calcium release (i.e. calcium waves) in RyR2-RSxMMVV with RyR2-RS. However, these two genotypes did not differ in either incidence or severity of arrhythmias, and showed similar degree of spontaneous calcium release. Conclusions Inhibition of CaMKII protects against spontaneous calcium release in CPVT1, and is a promising therapeutic strategy. However, the fact that such inhibition also induces calcium alternans needs further exploration. Antioxidative agents also attenuate arrhythmogenic calcium release in CPVT1 cardiomyocytes, but this effect does not seem to involve the M281/282 CaMKII oxidation site. Future studies should explore other oxidation sites.

The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel required for skeletal muscle contraction. Here we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca2+, ATP and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca2+ alone induce conformational changes in the cytoplasmic assembly (‘priming’), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement and deformation of the S4-S5 linker, and conformational changes in the pseudo-voltage-sensor domain.

Rationale: CAV3 (caveolin3) variants associated with arrhythmogenic cardiomyopathy and muscular dystrophy can disrupt post-Golgi surface trafficking. As CAV1 (caveolin1) was recently identified in cardiomyocytes, we hypothesize that conserved isoform-specific protein/protein interactions orchestrate unique cardiomyocyte microdomain functions. To analyze the CAV1 versus CAV3 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 CAV3 interactor in gene-edited human induced pluripotent stem cell cardiomyocytes. Objective: To identify differential CAV1 versus CAV3 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 CAV3 interactors in living cardiomyocytes. Isoform-specific affinity proteomic and co-immunoprecipitation experiments confirmed the monocarboxylate transporter McT1 (monocarboxylate transporter type 1) versus aquaporin1, respectively, as CAV3 or CAV1 specific interactors in cardiomyocytes. Superresolution stimulated emission depletion microscopy showed distinct CAV1 versus CAV3 cluster distributions in cardiomyocyte transverse tubules. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9 nuclease)-mediated CAV3 knockout uncovered a stabilizing role for McT1 surface expression, proton-coupled lactate shuttling, increased late Na + currents, and early afterdepolarizations in human induced pluripotent stem cell-derived cardiomyocytes. Complexome profiling confirmed that McT1 and the Na,K-ATPase form labile protein assemblies with the multimeric CAV3 complex. Conclusions: Combining the strengths of proximity and affinity proteomics, we identified isoform-specific CAV1 versus CAV3 binding partners in cardiomyocytes. McT1 represents a novel class of metabolically relevant CAV3-specific interactors close to mitochondria in cardiomyocyte transverse tubules. CAV3 knockout uncovered a previously unknown role for functional stabilization of McT1 in the surface membrane of human cardiomyocytes. Strikingly, CAV3 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/CAV3 interactions may provide rationales to target this muscle-specific assembly function therapeutically.

Background: Enhanced diastolic calcium (Ca 2+ ) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca 2+ leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca 2+ /calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. Methods: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca 2+ leak in atrial cardiomyocytes was assessed using confocal Ca 2+ imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9. Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca 2+ spark frequency in atrial cardiomyocytes with Ca 2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca 2+ leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca 2+ release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.

The cardiac membrane protein phospholamban (PLN) is targeted by protein kinase A (PKA) at Ser16 and by Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr17 β-Adrenergic stimulation and PKA-dependent phosphorylation of Ser16 acutely stimulate the sarcoplasmic reticulum calcium pump (SERCA) by relieving its inhibition by PLN. CaMKII-dependent phosphorylation may lead to longer-lasting SERCA stimulation and may sustain maladaptive Ca2+ handling. Here, we demonstrated that phosphorylation at either Ser16 or Thr17 converted PLN into a target for the phosphoadaptor protein 14-3-3 with different affinities. 14-3-3 proteins were localized within nanometers of PLN and endogenous 14-3-3 coimmunoprecipitated with pentameric PLN from cardiac membranes. Molecular dynamics simulations predicted different molecular contacts for peptides phosphorylated at Ser16 or Thr17 with the binding groove of 14-3-3, resulting in varied binding affinities. 14-3-3 binding protected either PLN phosphosite from dephosphorylation. β-Adrenergic stimulation of isolated adult cardiomyocytes resulted in the membrane recruitment of endogenous 14-3-3. The exogenous addition of 14-3-3 to β-adrenergic-stimulated cardiomyocytes led to prolonged SERCA activation, presumably because 14-3-3 protected PLN pentamers from dephosphorylation. Phosphorylation of Ser16 was disrupted by the cardiomyopathy-associated ∆Arg14 mutation, implying that phosphorylation of Thr17 by CaMKII may become crucial for 14-3-3 recruitment to ∆Arg14 PLN. Consistent with PLN acting as a dynamic hub in the control of Ca2+ handling, our results identify 14-3-3 binding to PLN as a contractility-augmenting mechanism.

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.

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

Atrial dysfunction is highly prevalent and associated with increased severity of heart failure. While rapid excitation-contraction coupling depends on axial junctions in atrial myocytes, the molecular basis of atrial loss of function remains unclear. We identified approximately 5-fold lower junctophilin-2 levels in atrial compared with ventricular tissue in mouse and human hearts. In atrial myocytes, this resulted in subcellular expression of large junctophilin-2 clusters at axial junctions, together with highly phosphorylated ryanodine receptor (RyR2) channels. To investigate the contribution of junctophilin-2 to atrial pathology in adult hearts, we developed a cardiomyocyte-selective junctophilin-2–knockdown model with 0 mortality. Junctophilin-2 knockdown in mice disrupted atrial RyR2 clustering and contractility without hypertrophy or interstitial fibrosis. In contrast, aortic pressure overload resulted in left atrial hypertrophy with decreased junctophilin-2 and RyR2 expression, disrupted axial junctions, and atrial fibrosis. Whereas pressure overload accrued atrial dysfunction and heart failure with 40% mortality, additional junctophilin-2 knockdown greatly exacerbated atrial dysfunction with 100% mortality. Strikingly, transgenic junctophilin-2 overexpression restored atrial contractility and survival through de novo biogenesis of polyadic junctional membrane complexes maintained after pressure overload. Our data show a central role of junctophilin-2 cluster disruption in atrial hypertrophy and identify transgenic augmentation of junctophilin-2 as a disease-mitigating rationale to improve atrial dysfunction and prevent heart failure deterioration.

RationaleCyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which, upon β-adrenergic receptor (β-AR) stimulation, acts in microdomains to regulate cardiac excitation-contraction coupling by activating the cAMP-dependent protein kinase (PKA) phosphorylation of calcium handling proteins. One crucial microdomain is in vicinity of the cardiac ryanodine receptor type 2 (RyR2) which is associated with arrhythmogenic diastolic calcium leak from the sarcoplasmic reticulum (SR) often occurring upon RyR2 hyperphosphorylation by PKA and calcium/calmodulin-dependent kinase.ObjectiveWe sought to establish a real time approach capable of directly visualizing cAMP and its pathological changes in the vicinity of RyR2 by generating a proper targeted biosensor and transgenic mouse model to express it in adult cardiomyocytes.Methods and ResultsWe generated transgenic mice expressing a novel targeted fluorescent biosensor for RyR2-associated cAMP in adult mouse cardiomyocytes. In healthy cardiomyocytes, β1-AR but not β2-AR stimulation strongly increased local RyR2-associated cAMP levels. However, in cardiac hypertrophy induced by aortic banding, there was a marked subcellular redistribution of phosphodiesterases (PDEs) 2, 3 and 4, which included a dramatic loss of the local pool of PDE4. This was also accompanied by measurable β2-AR-induced cAMP signals, increased SR calcium leak and arrhythmia susceptibility.ConclusionsOur new targeted biosensor expressed in transgenic mice can visualize cAMP levels in the vicinity of cardiac RyR2 in healthy and diseased cardiomyocytes. In the future, this novel biosensor can be used to better understand alterations of RyR2-associated cAMP in cardiovascular diseases and local actions of new therapies.

Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (CaCirculating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on CaSN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased CaSN production is upregulated in conditions with cardiomyocyte Ca

This data article features supplementary figures and tables related to the article "Differential Multivariable risk prediction of appropriate shock vs. competing mortality - a prospective cohort study to estimate benefits from implantable cardioverter defibrillator therapy" (Bergau et al., 2018) [1]. The figures show the clinical study CONSORT graph (data that show the number of patients not-analyzable as well as a distribution of patients by outcomes) and the correlation scatter plot for risk scores of appropriate shock vs. mortality (data that show the calculated score values of the two scores plotted against each other). The tables show the results for the univariate Cox regressions for prediction of mortality and appropriate shock. For further information, please see Bergau et al. (2018) [1]. ispartof: DATA IN BRIEF vol:21 pages:2110-2116 ispartof: location:Netherlands status: published

Aims Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is caused by mutations in the cardiac ryanodine receptor (RyR2) that lead to disrupted Ca2+ handling in cardiomyocytes and ventricular tachycardia. The aim of this study was to test whether exercise training could reduce the propensity for arrhythmias in mice with the CPVT1-causative missense mutation Ryr2 -R2474S by restoring normal Ca2+ handling. Methods and results Ryr2 -R2474S mice (RyR-RS) performed a 2 week interval treadmill exercise training protocol. Each exercise session comprised five 8 min intervals at 80–90% of the running speed at maximal oxygen uptake (VO2max) and 2 min active rest periods at 60%. VO2max increased by 10 ± 2% in exercise trained RyR-RS (ET), while no changes were found in sedentary controls (SED). RyR-RS ET showed fewer episodes of ventricular tachycardia compared with RyR-RS SED, coinciding with fewer Ca2+ sparks and waves, less diastolic Ca2+ leak from the sarcoplasmic reticulum, and lower phosphorylation levels at RyR2 sites associated with Ca2+–calmodulin-dependent kinase type II (CaMKII) compared with RyR-RS SED. The CaMKII inhibitor autocamtide-2-related inhibitory peptide and also the antioxidant N -acetyl-l-cysteine reduced Ca2+ wave frequency in RyR-RS equally to exercise training. Protein analysis as well as functional data indicated a mechanism depending on reduced levels of oxidized CaMKII after exercise training. Two weeks of detraining reversed the beneficial effects of the interval treadmill exercise training protocol in RyR-RS ET. Conclusion Long-term effects of interval treadmill exercise training reduce ventricular tachycardia episodes in mice with a CPVT1-causative Ryr2 mutation through lower CaMKII-dependent phosphorylation of RyR2.

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BACKGROUND AND OBJECTIVE: We prospectively investigated combinations of risk stratifiers including multiple EP diagnostics in a cohort study of ICD patients. METHODS: For 672 enrolled patients, we collected history, LVEF, EP study and T-wave alternans testing, 24-h Holter, NT-proBNP, and the eGFR. All-cause mortality and first appropriate ICD shock were predefined endpoints. RESULTS: The 635 patients included in the final analyses were 63 ± 13 years old, 81% were male, LVEF averaged 40 ± 14%, 20% were inducible at EP study, 63% had a primary prophylactic ICD. During follow-up over 4.3 ± 1.5 years, 108 patients died (4.0% per year), and appropriate shock therapy occurred in n = 96 (3.9% per year). In multivariate regression, age (p

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA‐based protocols. Glyoxal acted faster than PFA, cross‐linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA‐based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

AIMS: Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) predisposes to ventricular tachyarrhythmias (VTs) during high heart rates due to physical or psychological stress. The essential role of catecholaminergic effects on ventricular cardiomyocytes in this situation is well documented, but the importance of heart rate per se for arrhythmia initiation in CPVT1 is largely unexplored. METHODS AND RESULTS: Sixteen CPVT1 patients performed a bicycle stress-test. Occurrence of VT triggers, i.e. premature ventricular complexes (PVC), depended on high heart rate, with individual thresholds. Atrial pacing above the individual PVC threshold in three patients did not induce PVCs. The underlying mechanism for the clinical observation was explored using cardiomyocytes from mice with the RyR2-R2474S (RyR2-RS) mutation, which exhibit exercise-induced VTs. While rapid pacing increased the number of Ca2+ waves in both RyR2-RS and wild-type (p

A hallmark of mature mammalian ventricular myocardium is a positive force-frequency relationship (FFR). Despite evidence of organotypic structural and molecular maturation, a positive FFR has not been observed in mammalian tissue engineered heart muscle. We hypothesized that concurrent mechanical and electrical stimulation at frequencies matching physiological heart rate will result in functional maturation. To this end, we investigated the role of such biomimetic mechanical and electrical stimulation in functional maturation in engineered heart muscle (EHM) comprising collagen type I and neonatal rat heart cells. Following tissue consolidation (8 days), EHM were subjected to electrical field stimulation at 0, 2, 4, or 6 Hz for 5 days, while strained on flexible poles to facilitate auxotonic contractions. EHM stimulated at 2 and 4 Hz displayed a similarly enhanced inotropic reserve, but a clearly diverging FFR. The positive FFR in 4 Hz stimulated EHM was associated with reduced calcium sensitivity, frequency-dependent acceleration of relaxation, and enhanced post-rest potentiation. This was paralleled on the cellular level with improved calcium storage and release capacity of the sarcoplasmic reticulum, increased amounts of SERCA2a and RyR2 protein, and enhanced T-tubulation. We demonstrate that electromechanical stimulation at a frequency matching closely the physiological heart rate supports functional maturation in mammalian EHM. The observed positive FFR in EHM has important implications for the applicability of EHM in cardiovascular research and drug testing.