Cardiac Isoform Research Articles

Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM.

Mutations in ryanodine receptors (RyRs), intracellular Ca2+ channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca2+ imaging, and cryo-electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca2+ binding site, which concur with the major "hyperactive" feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect.

Investigation of the safety and feasibility of AAV1/SERCA2a gene transfer in patients with chronic heart failure supported with a left ventricular assist device \u2013 the SERCA-LVAD TRIAL

The SERCA-LVAD trial was a phase 2a trial assessing the safety and feasibility of delivering an adeno-associated vector 1 carrying the cardiac isoform of the sarcoplasmic reticulum calcium ATPase (AAV1/SERCA2a) to adult chronic heart failure patients implanted with a left ventricular assist device. The SERCA-LVAD trial was one of a program of AAV1/SERCA2a cardiac gene therapy trials including CUPID1, CUPID 2 and AGENT trials. Enroled subjects were randomised to receive a single intracoronary infusion of 1 × 1013 DNase-resistant AAV1/SERCA2a particles or a placebo solution in a double-blinded design, stratified by presence of neutralising antibodies to AAV. Elective endomyocardial biopsy was performed at 6 months unless the subject had undergone cardiac transplantation, with myocardial samples assessed for the presence of exogenous viral DNA from the treatment vector. Safety assessments including ELISPOT were serially performed. Although designed as a 24 subject trial, recruitment was stopped after five subjects had been randomised and received infusion due to the neutral result from the CUPID 2 trial. Here we describe the results from the 5 patients at 3 years follow up, which confirmed that viral DNA was delivered to the failing human heart in 2 patients receiving gene therapy with vector detectable at follow up endomyocardial biopsy or cardiac transplantation. Absolute levels of detectable transgene DNA were low, and no functional benefit was observed. There were no safety concerns in this small cohort. This trial identified some of the challenges of performing gene therapy trials in this LVAD patient cohort which may help guide future trial design.

Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection.

Two subunits of the ternary troponin complex, I and C, have cardiac muscle specific isoforms, and hence could be applied as highly-selective markers of acute coronary syndrome. We aimed at paving the way for the development of a robust cardiac troponin I-detecting sandwich assay by replacing antibodies with nuclease resistant aptamer analogues, so-called spiegelmers. To complement the previously generated spiegelmers that were specific for the N-terminus of cTnI, spiegelmers were selected for an amino acid stretch in the proximity of the C-terminal part of the protein by using a D-amino acid composed peptide. Following the selection, the oligonucleotides were screened by filter binding assay, and surface plasmon resonance analysis of the most auspicious candidates demonstrated that this approach could provide spiegelmers with subnanomolar dissociation constant. To demonstrate if the selected spiegelmers are functional and suitable for cTnI detection in a sandwich type arrangement, AlphaLisa technology was leveraged and the obtained results demonstrated that spiegelmers with different epitope selectivity are suitable for specific detection of cTnI protein even in human plasma containing samples. These results suggest that spiegelmers could be considered in the development of the next generation cTnI monitoring assays.

Orthophosphate increases the efficiency of slow muscle-myosin isoform in the presence of omecamtiv mecarbil

Omecamtiv mecarbil (OM) is a putative positive inotropic tool for treatment of systolic heart dysfunction, based on the finding that in vivo it increases the ejection fraction and in vitro it prolongs the actin-bond life time of the cardiac and slow-skeletal muscle isoforms of myosin. OM action in situ, however, is still poorly understood as the enhanced Ca2+-sensitivity of the myofilaments is at odds with the reduction of force and rate of force development observed at saturating Ca2+. Here we show, by combining fast sarcomere-level mechanics and ATPase measurements in single slow demembranated fibres from rabbit soleus, that the depressant effect of OM on the force per attached motor is reversed, without effect on the ATPase rate, by physiological concentrations of inorganic phosphate (Pi) (1-10 mM). This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM-treated patients, whenever [Pi] increases with heart-beat frequency.

Double knockout of Akt2 and AMPK accentuates high fat diet-induced cardiac anomalies through a cGAS-STING-mediated mechanism

High fat diet intake contributes to undesired cardiac geometric and functional changes although the underlying mechanism remains elusive. Akt and AMPK govern to cardiac homeostasis. This study examined the impact of deletion of Akt2 (main cardiac isoform of Akt) and AMPKα2 on high fat diet intake-induced cardiac remodeling and contractile anomalies and mechanisms involved. Cardiac geometry, contractile, and intracellular Ca2+ properties were evaluated using echocardiography, IonOptix® edge-detection and fura-2 techniques in wild-type (WT) and Akt2-AMPK double knockout (DKO) mice receiving low fat (LF) or high fat (HF) diet for 4 months. Our results revealed that fat diet intake elicit obesity, cardiac remodeling (hypertrophy, LV mass, LVESD, and cross-sectional area), contractile dysfunction (fractional shortening, peak shortening, maximal velocity of shortening/relengthening, time-to-90% relengthening, and intracellular Ca2+ handling), ultrastructural disarray, apoptosis, O2−, inflammation, dampened autophagy and mitophagy. Although DKO did not affect these parameters, it accentuated high fat diet-induced cardiac remodeling and contractile anomalies. High fat intake upregulated levels of cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and STING phosphorylation while suppressing phosphorylation of ULK1 (Ser757 and Ser777), with a more pronounced effect in DKO mice. In vitro data revealed that inhibition of cGAS and STING using PF-06928215 and Astin C negated palmitic acid-induced cardiomyocyte contractile dysfunction. Biological function analysis for all differentially expressed genes (DEGs) depicted that gene ontology terms associated with Akt and AMPK signaling processes were notably changed in high fat-fed hearts. Our data indicate that Akt2-AMPK ablation accentuated high fat diet-induced cardiac anomalies possibly through a cGAS-STING-mechanism.

Transfer of a human gene variant associated with exceptional longevity improves cardiac function in obese type 2 diabetic mice through induction of the SDF-1/CXCR4 signalling pathway.

AimsHomozygosity for a four‐missense single‐nucleotide polymorphism haplotype of the human BPIFB4 gene is enriched in long‐living individuals. Delivery of this longevity‐associated variant (LAV) improved revascularisation and reduced endothelial dysfunction and atherosclerosis in mice through a mechanism involving the stromal cell‐derived factor‐1 (SDF‐1). Here, we investigated if delivery of the LAV‐BPIFB4 gene may attenuate the progression of diabetic cardiomyopathy.Methods and resultsCompared with age‐matched lean controls, diabetic db/db mice showed altered echocardiographic indices of diastolic and systolic function and histological evidence of microvascular rarefaction, lipid accumulation, and fibrosis in the myocardium. All these alterations, as well as endothelial dysfunction, were prevented by systemic LAV‐BPIFB4 gene therapy using an adeno‐associated viral vector serotype 9 (AAV9). In contrast, AAV9 wild‐type‐BPIFB4 exerted no benefit. Interestingly, LAV‐BPIFB4‐treated mice showed increased SDF‐1 levels in peripheral blood and myocardium and up‐regulation of the cardiac myosin heavy chain isoform alpha, a contractile protein that was reduced in diabetic hearts. SDF‐1 up‐regulation was instrumental to LAV‐BPIFB4‐induced benefit as both haemodynamic and structural improvements were inhibited by an orally active antagonist of the SDF‐1 CXCR4 receptor.ConclusionsIn mice with type‐2 diabetes, LAV‐BPIFB4 gene therapy promotes an advantageous remodelling of the heart, allowing it to better withstand diabetes‐induced stress. These results support the viability of transferring healthy characteristics of longevity to attenuate diabetic cardiac disease.

Do age-associated changes of voltage-gated sodium channel isoforms expressed in the mammalian heart predispose the elderly to atrial fibrillation?

Atrial fibrillation (AF) is the most common cardiac arrhythmia worldwide. The prevalence of the disease increases with age, strongly implying an age-related process underlying the pathology. At a time when people are living longer than ever before, an exponential increase in disease prevalence is predicted worldwide. Hence unraveling the underlying mechanics of the disease is paramount for the development of innovative treatment and prevention strategies. The role of voltage-gated sodium channels is fundamental in cardiac electrophysiology and may provide novel insights into the arrhythmogenesis of AF. Nav1.5 is the predominant cardiac isoform, responsible for the action potential upstroke. Recent studies have demonstrated that Nav1.8 (an isoform predominantly expressed within the peripheral nervous system) is responsible for cellular arrhythmogenesis through the enhancement of pro-arrhythmogenic currents. Animal studies have shown a decline in Nav1.5 leading to a diminished action potential upstroke during phase 0. Furthermore, the study of human tissue demonstrates an inverse expression of sodium channel isoforms; reduction of Nav1.5 and increase of Nav1.8 in both heart failure and ventricular hypertrophy. This strongly suggests that the expression of voltage-gated sodium channels play a crucial role in the development of arrhythmias in the diseased heart. Targeting aberrant sodium currents has led to novel therapeutic approaches in tackling AF and continues to be an area of emerging research. This review will explore how voltage-gated sodium channels may predispose the elderly heart to AF through the examination of laboratory and clinical based evidence.

Cannabidiol protects against high glucose-induced oxidative stress and cytotoxicity in cardiac voltage-gated sodium channels.

Cardiovascular complications are the major cause of mortality in diabetic patients. However, the molecular mechanisms underlying diabetes-associated arrhythmias are unclear. We hypothesized that high glucose could adversely affect Nav 1.5, the major cardiac sodium channel isoform of the heart, at least partially via oxidative stress. We further hypothesized that cannabidiol (CBD), one of the main constituents of Cannabis sativa, through its effects on Nav 1.5, could protect against high glucose-elicited oxidative stress and cytotoxicity. To test these ideas, we used CHO cells transiently co-transfected with cDNA encoding human Nav 1.5 α-subunit under control and high glucose conditions (50 or 100 mM for 24 hr). Several experimental and computational techniques were used, including voltage clamp of heterologous expression systems, cell viability assays, fluorescence assays and action potential modelling. High glucose evoked cell death associated with elevation in reactive oxygen species (ROS) right shifted the voltage dependence of conductance and steady-state fast inactivation, and increased persistent current leading to computational prolongation of action potential (hyperexcitability) which could result in long QT3 arrhythmia. CBD mitigated all the deleterious effects provoked by high glucose. Perfusion with lidocaine (a well-known sodium channel inhibitor with antioxidant effects) or co-incubation of Tempol (a well-known antioxidant) elicited protection, comparable to CBD, against the deleterious effects of high glucose. These findings suggest that, through its favourable antioxidant and sodium channel inhibitory effects, CBD may protect against high glucose-induced arrhythmia and cytotoxicity.

Luminal Calcium Control of Type-1 Inositol 1,4,5-Trisphosphate Receptor

Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are homologous endo/sarcoplasmic reticulum Ca2+ release channels. IP3R and RyR are tightly controlled by both cytosolic and luminal Ca2+. The negatively charged residue E4872 in RyR2 (cardiac isoform) electrostatically interacts with luminal Ca2+, explaining RyR2's luminal Ca2+ dependency (Chen et al., 2014). The corresponding residue in IP3R type 1 is D2594, but its role (if any) in IP3R function is unknown. We hypothesized the IP3R1-D2594 also electrostatically interacts with luminal Ca2+ to determine IP3R1 luminal Ca2+ sensitivity. To test this possibility, we created a mouse knock-in model harboring the D2594K mutation in which the negatively charged IP3R1-D2594 residue was replaced by lysine, a positively charged residue. IP3R1-WT and -D2594K cerebella microsomes were isolated and reconstituted into planar lipid bilayers. Single channel IP3R1 studies showed the conductance properties of IP3R1-WT and -D2594K channels are similar. The IP3R1-D2594K channels exhibited WT-like IP3 affinity, but had substantially greater IP3 efficacy. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. In IP3R1-WT channels, the decrease in activity at these low luminal Ca2+ levels (i.e. IP3R1 luminal Ca2+ sensitivity) depends on the membrane potential. Taken together, these results indicate the IP3R1-D2594 residue, as it does in RyR2, electrostatically interacts with luminal Ca2+, explaining at least in part, the IP3R's sensitivity to luminal Ca2+. This newly identified mechanism together with luminal Ca2+ feed-through may explain the luminal control of IP3R1.

Protocol Development for Expressing Functional Ryanodine Receptors in HEK293-6E Suspension Cells

We have developed a protocol for expressing ryanodine receptors (RyR), specifically the rabbit skeletal RyR isoform (rbRyR1) and human cardiac RyR isoform (hRyR2), that respond to known RyR modulators, with the goal of developing molecular tools for FRET-based structural readouts of RyRs. Both RyR1 and RyR2 are composed of four 565 kDa subunits and are the primary Ca2+ channels in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle, respectively. Triggered SR Ca2+ release is required for muscle contraction, but RyR dysregulation can result in SR Ca leak during rest, which is linked to several myopathies, diabetes, and neurological disorders.

A Cryo-EM Based Study of An Equivalent N Terminal Domain Mutation in Skeletal and Cardiac Ryanodine Receptor (RyR)

Mutations in ryanodine receptors (RyRs), large intracellular Ca2+ channels on the sarcoplasmic reticulum, are linked to disorders such as malignant hyperthermia and central core disease in the case of skeletal RyR1 isoform and catecholaminergic polymorphic ventricular tachycardia (CPVT) in the case of cardiac RyR2 isoform. RyRs are complex molecular machines whose activity is under precise regulation of interdomain interactions between the various subdomains as well as external binding partners. We examined equivalent single point mutations in the N-terminal A (NTD-A) domain of skeletal and cardiac RyR isoforms using cryo-EM to study their effect on the channel conformation and shed light on the possible underlying mechanism of leakiness. We resolved the 3D structures of RyR1 R164C and RyR2 R176Q at 3.5 Å and 3.9 Å, respectively. 3D analysis suggests that mutation R164C results in a tilting of the rhomboid structure (composed of NTDs, SPRY1-3, P1, HD1 and HD2 domains) indicative of a “primed” state in the absence of any activator ligands (such as Ca2+ or caffeine) and could be a result of disruption of the NTD-A interaction with the neighboring central domain. The effects of mutation R176Q in RyR2 were milder, suggesting isoform-specific effects on channel conformation despite the mutation being a loss of positively charged residue, arginine, in both isoforms. Analysis of these and other long-range conformational changes provides insight into the leaky phenotype of RyR1 and RyR2 produced by mutations situated far away (>80 Å) from the pore. Supported by grants AHA 19POST34430178 (K.A.I) and AHA 14GRNT19660003, MDA352845, NIH R01 AR068431, and NIH U24GM116789 (M.S.).

Structure of the Actin-Tropomyosin-TNT Complex

Thin filament-linked proteins actin, tropomyosin, and troponin are the major regulators of cardiac and skeletal muscle that activate contraction through calcium-mediated conformational changes. Single point mutations within the cardiac forms of these proteins cause hypertrophic (HCM) and dilated (DCM) cardiomyopathy, affecting more than 1 in every 500 people. In order to determine mechanisms of thin filament regulation and the initial mutation-associated insults that arise during disease development, it is necessary to define the molecular interactions of the thin filament components. The TnT tail (TnT1), and more specifically the N-terminal region (residues 1-156 in cardiac TnT isoform 6), tethers the troponin core domain (i.e. TnT2, TnC and TnI) to tropomyosin on the thin filament and thereby assumes the 40 nm tropomyosin periodicity. Additionally, TnT1 stabilizes the tropomyosin head-to-tail domain to form cables spanning the full filament. TnT1-156also contains more than one third of all thin filament mutations that cause HCM and DCM. Because no high-resolution structure of this region is available, residue-level interactions with tropomyosin as well as the positions of many HCM and DCM point mutants remain elusive. We are using cryo-electron microscopy to solve the high-resolution structure of the cardiac actin-tropomyosin-TnT1-156 complex. The complex has been fully optimized and 2-dimensional class averages display clear TnT density on actin-tropomyosin. 3-dimensional reconstructions reveal that TnT1-156 makes the tropomyosin appear broader and flatter, consistent with an α-helix binding alongside tropomyosin, rather than hovering over it. Given current cryo-EM optimization at nanometer resolution, we anticipate resolution to near-atomic levels, thus leading to the residue-level determination of the TnT-tropomyosin interaction and a more precise understanding of mutation-associated deficits.

Protective Effect of Cannabidiol Against Oxidative Stress and Cytotoxicity Evoked by High Glucose in Cardiac Voltage-Gated Sodium Channels

Cardiovascular complications are the major cause of mortality in diabetic patients. However, the molecular mechanisms underlying diabetes-associated arrhythmias are unclear. We hypothesized that high glucose could adversely affect Nav1.5, the major cardiac sodium channel isoform, at least partially via oxidative stress. We further hypothesized that cannabidiol (CBD), one of the main constituents of Cannabis sativa, could guard against diabetes induced cardiomyopathy. We postulated that CBD, through its effects on Nav1.5, could protect against high glucose elicited oxidative stress and cytotoxicity. To test these ideas, we used Chinese hamster ovarian (CHO) cells transiently transfected with cDNA encoding Nav1.5 α-subunit and incubated the cells in control (10 mM) and high glucose (50 or 100 mM) conditions for 24 hours. High glucose evoked cell death associated with elevation in reactive oxygen species (ROS), right shifted the voltage dependence of conductance and steady state fast inactivation, and increased persistent current leading to prolongation of action potential (hyperexcitability) which could result in long QT-3 arrhythmia. CBD mitigated the deleterious effects provoked by high glucose. Perfusion with lidocaine (a sodium channels inhibitor with anti-oxidant effects), or co-incubation of Tempol (an anti-oxidant) elicited protection, comparable to CBD, against the deleterious effects of high glucose. These findings suggest that, through its favorable anti-oxidant and sodium channel inhibitory effects, CBD may protect against high-glucose induced arrhythmia and cytotoxicity.

Non-coronarogenic causes of increased cardiac troponins in the practice of physicians (literature review)

Cardiac isoforms of troponins are the most sensitive and specific biomarkers of myocardial damage and new high-sensitivity methods can reveal very minor damages on the heart muscle. However, elevated troponin levels indicate cardiac damage, but do not define the cause of the damage. Therefore, cardiac troponin elevations are common in many disease states and do not necessarily indicate the presence of acute myocardial infarction. In clinical practice, interpretation of positive troponins may be challenging. In our article, we consider the main non-coronarogenic causes of increased cardiac troponins. The first part of the review discusses the mechanisms of increase and the diagnostic value of cardiac troponins during physical exertion, inflammatory and toxic myocardial injuries (endocarditis, myocarditis, sepsis), and renal failure. The second part of the literature review describes the mechanisms of increase and the diagnostic value of troponins in pulmonary thromboembolism, dissecting aortic aneurysms, neurogenic pathologies (stroke, subarachnoid hemorrhages), and treatment with cardiotoxic drugs (chemotherapy). Special attention is paid to the false positive causes of elevation of troponins.

Metabolism of cardiac troponins (literature review)

The review summarizes all recent data on the metabolism of cardiac troponin isoforms. The main mechanisms of troponin release from intact myocardium are described. These mechanisms ensure its baseline levels (less than the 99th percentile) in all healthy individuals. There are various fragments of troponin that circulate in the blood flow as a heterogeneous pool. Their circulation is related to various intracellular and extracellular proteases. In-depth understanding ofthese mechanisms is required to improve the diagnostic process. The article provides new insights into the evaluation of cardiac troponins in other human biological fluids: pericardial, cerebrospinal, amniotic, urine, and oral fluid. The measurements of saliva and urine levels of troponins seem to be promising alternative for non-invasive diagnosis. Recent circadian patterns of high-sensitive cardiac troponin T alterations are reported. These patterns should be taken into account while practicing fast diagnostic algorithms.

A multicenter, randomized, double-blind, controlled study to evaluate the efficacy and safety of dantrolene on ventricular arrhythmia as well as mortality and morbidity in patients with chronic heart failure (SHO-IN trial): rationale and design

BackgroundLeakage of Ca2+ from the sarcoplasmic reticulum (SR) is a critical contributing factor to heart failure pathophysiology. Therefore, reducing SR Ca2+ leaks may provide significant additive benefits when used in combination with conventional therapies. Dantrolene, a drug routinely used to treat malignant hyperthermia, also stabilizes the cardiac isoform of the release channel (RyR2), thus decreasing SR Ca2+ leaks. The purpose of this study is to evaluate the effect of chronic administration of dantrolene on heart failure and lethal arrhythmia in patients with chronic heart failure and reduced ejection fraction in a multicenter, randomized, double-blind, controlled study. MethodsPatients with chronic heart failure who had functional status of New York Heart Association class II and III and a left ventricular ejection fraction <40% were treated according to the Japanese Circulation Society, the European Society of Cardiology, and the American Heart Association/the American College of Cardiology guidelines for diagnosis and treatment of acute and chronic heart failure. Patients were randomized and divided into two groups in a double-blind fashion: dantrolene group and placebo group (target sample size: 300 cases). These drugs were administered for 96 weeks. The primary endpoint is cardiovascular death, first hospitalization for exacerbation of heart failure, or lethal arrhythmia [ventricular tachycardia (VT) storm, sustained VT, ventricular fibrillation] for 2 years after starting administration of dantrolene 1 cap (25mg) three times daily (if not tolerable, two times daily) or matching placebo. ResultsThis paper presents the rationale and trial design of the study. Recruitment for the study started on 8 December 2017. ConclusionsThe results of this trial will clarify the efficacy and safety of dantrolene for ventricular arrhythmia, as well as mortality and morbidity in patients with chronic heart failure and reduced ejection fraction during guideline-directed medical treatment.

Overexpression of Na+-HCO3– cotransporter contributes to the exacerbation of cardiac remodeling in mice with myocardial infarction by increasing intracellular calcium overload

The role of the cardiac isoform of the electrogenic sodium-bicarbonate ion cotransporter (NBCe1) in cardiac remodeling is not fully understood. The aim of this study was to assess the effects of NBCe1 overexpression on cardiac remodeling induced by myocardial infarction (MI) in mice. We generated NBCe1 transgenic (Tg) mice and NBCe1 overexpressing adult mouse ventricular myocytes (AMVMs) to investigate the role of NBCe1 on post-MI remodeling and calcium kinetics. Tg mice showed a markedly higher mortality rate and larger infarct size after MI. At 6 weeks after MI, the maximum rising rates of left ventricular pressure (dp/dt), contractility index, and the exponential time constant of relaxation (τ) were markedly lower, and there was higher cardiomyocyte apoptosis, in Tg mice compared with WT mice. In cultured AMVMs, overexpression of NBCe1 decreased sarcomere shortening and calcium amplitude. In WT AMVMs, the rates of the rise and decay phase of calcium transients, indicated by the rising time (Tpeak, time to peak) and decay time constant (τd), and the number of apoptotic cells, were increased following hypoxia, while overexpression of NBCe1 further increased Tpeak and cellular apoptosis, but not τd. Intracellular resting calcium and sodium concentrations were significantly increased following both hypoxia and NBCe1 overexpression. Co-treatment with S0859, an NBCe1 antagonist, blocked the hypoxia-induced increase in Tpeak, τd, intracellular resting calcium and sodium concentrations, and apoptosis in cardiomyocytes. These findings indicate that NBCe1 overexpression promotes cardiac remodeling by increasing intracellular calcium overload. Therefore, NBCe1 should be a potential target for treatment of cardiac remodeling.

Low mutation rate in the TTN gene in paediatric patients with dilated cardiomyopathy \u2013 a pilot study

Idiopathic dilated cardiomyopathy (DCM) is a common cardiomyopathy with the prevalence of 1:250, and at least one-third of all the cases are inherited. Mutations in the TTN gene are considered as the most frequent cause of inherited DCM and cover 10–30% of the cases. The studies were mainly focused on the adult or mixed age group of patients with DCM. The mutation rate in the TTN gene, the characteristics of manifestations and their prognostic significance in childhood have not been studied. To determine TTN mutation rate in children with DCM and the relevance of including this gene in the DNA diagnostic protocol for paediatric DCM, complete clinical and instrumental examination of 36 DCM patients (up to 18 years) with the manifestation of the disease was conducted in specialised cardiology centres. Molecular genetic testing included sequencing of coding and adjacent regulatory regions of the major cardiac TTN isoform N2BA using IonTorrent ™ semiconductor sequencing (for 25 isolated cases) and trio whole exome sequencing (trio WES)on the Illumina platform (for 11 family cases). Our pilot group included 36 probands with DCM diagnosis first established on the basis of the generally accepted criteria at the age of 5 days to 18 years(average age: 6.5 years). The sex ratio (M:F) was 23: 8. There were 25 sporadic DCM cases and 11 cases of familial DCM (at least one of the parents and/or siblings were also diagnosed with DCM). The only likely pathogenic truncating variant p.Arg33703*in the TTN gene (TTNtv) was found in a 16-year-oldmale proband out of 36 (3%). Apparently, TTN-dependent forms of DCMs manifest later at a young (but older than 18 years) or more mature age, and TTN gene cannot be considered as the first-line genetic testing for DCM in the paediatric group, despite several studies have reported a generally high mutation rate in this gene with DCM. Further research is needed to compare the representation of mutations in the TTN gene in different age groups of DCM patients.

The N-termini of GRK2 and GRK3 simulate the stimulating effects of RKIP on β-adrenoceptors

The Raf kinase inhibitor protein (RKIP) activates β-adrenoceptors (β-AR) and thereby induces a well-tolerated cardiac contractility and prevents heart failure in mice. Different to RKIP-mediated β-AR activation, chronic activation of β-AR by catecholamines was shown to be detrimental for the heart. RKIP is an endogenous inhibitor of G protein coupled receptor kinase 2 (GRK2); it binds GRK2 and thereby inhibits GRK2 mediated β-AR phosphorylation and desensitization.Here, we evaluate RKIP-mediated effects on β-AR to explore new strategies for β-AR modulation. Co-immunoprecipitation assays and pull-down assays revealed subtype specificity of RKIP for the cardiac GRK isoforms GRK2 and GRK3 – not GRK5 – as well as several RKIP binding sites within their N-termini (GRK21−185 and GRK31−185). Overexpression of these N-termini prevented β2-AR phosphorylation and internalization, subsequently increased receptor signaling in HEK293 cells and cardiomyocyte contractility. Co-immunoprecipitation assays of β2-AR with these N-terminal GRK fragments revealed a direct interaction suggesting a steric interference of the fragments with the functional GRK-receptor interaction.Altogether, N-termini of GRK2 and GRK3 efficiently simulate RKIP effects on β-AR signaling in HEK293 cells and in cardiomyocytes by their binding to β2-AR and, thus, provide important insights for the development of new strategies to modulate β2-AR signaling.

P6344Volume overload-triggered pathological myocardial remodeling occurs in the absence of calcium/calmodulin-dependent protein kinase type II delta

Abstract Background Calcium/calmodulin-dependent protein kinase type II delta (CamKIIδ), the predominant cardiac CaMKII isoform, has been implicated in the progression of myocardial infarction- and pressure overload-induced pathological remodeling and heart failure, but its role in volume overload (VO) has not been defined. We have previously reported an activation of CamKII during transition to HF in VO. Purpose Here, we analyzed the impact of CamKIIδ deletion in VO-triggered myocardial remodeling and heart failure development. Methods CaMKIIδ knockout (CaMKIIδ-KO) and wild-type (WT) littermates were exposed to aorto-caval shunt-induced VO, and the progression of myocardial remodeling was assessed by serial echocardiography, histological and molecular analyses. Results CaMKIIδ-KO and WT littermates exhibited similar mortality pattern in response to VO. Serial echocardiographic measurements showed a comparable eccentric myocardial remodeling, altered left ventricle geometry and perturbed ventricular function after shunt. At 12 weeks post-shunt both CaMKIIδ-KO and WT mice experienced comparable increases in relative heart weight, cardiomyocyte diameter, cardiac apoptosis, and hypertrophic genes expression. Conclusion We therefore conclude that CaMKIIδ signaling is dispensable for the progression of pathological cardiac remodeling induced by VO. This should be considered before CaMKII inhibition is approved therapeutically for HF treatment.