The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool

Missense mutations in the cardiac ryanodine receptor type 2 (RyR2) characteristically cause catecholaminergic arrhythmias. Reminiscent of the phenotype in patients, RyR2-R2474S knockin mice develop exercise-induced ventricular tachyarrhythmias. In cardiomyocytes, increased mitochondrial matrix Ca2+ uptake was recently linked to non-linearly enhanced ATP synthesis with important implications for cardiac redox metabolism. We hypothesize that catecholaminergic stimulation and contractile activity amplify mitochondrial oxidation pathologically in RyR2-R2474S cardiomyocytes. To investigate this question, we generated double transgenic RyR2-R2474S mice expressing a mitochondria-restricted fluorescent biosensor to monitor the glutathione redox potential (EGSH). Electrical field pacing-evoked RyR2-WT and RyR2-R2474S cardiomyocyte contractions resulted in a small but significant baseline EGSH increase. Importantly, β-adrenergic stimulation resulted in excessive EGSH oxidization of the mitochondrial matrix in RyR2-R2474S cardiomyocytes compared to baseline and RyR2-WT control. Physiologically β-adrenergic stimulation significantly increased mitochondrial EGSH further in intact beating RyR2-R2474S but not in RyR2-WT control Langendorff perfused hearts. Finally, this catecholaminergic EGSH increase was significantly attenuated following treatment with the RyR2 channel blocker dantrolene. Together, catecholaminergic stimulation and increased diastolic Ca2+ leak induce a strong, but dantrolene-inhibited mitochondrial EGSH oxidization in RyR2-R2474S cardiomyocytes.

Activation of RAGE- dependent endoplasmic reticulum stress associates with exacerbated post- myocardial infarction ventricular arrhythmias in diabetes

Association between receptor for advanced glycation end products (RAGE) and post- myocardial infarction (MI) ventricular arrhythmias (VAs) in diabetes was investigated. Correlation between premature ventricular contractions (PVCs) and serum advanced glycation end products (AGEs) content was analyzed in a cohort consisted of 101 ST segment elevated MI (STEMI) patients. MI diabetic rats were treated with anti- receptor for AGE (RAGE) antibody. Electrocardiography was used to record VAs. Myocytes were isolated from adjacent area around infracted region. Immunofluorescent stains were used to evaluate association between FKBP12.6 (FK506-bindingprotein 12.6) and ryanodine receptor 2 (RyR2). Calcium sparks were evaluated by confocal microscope. Protein expression and phosphorylation were assessed by Western blotting. Calcineurin (CaN) enzymatic activity and RyR2 channel activity were also determined. In the cohort study, significantly increased amount of (PVCs) were found in STEMI patients with diabetes (P<0.05). Serum AGEs concentration was significantly positively correlated with PVCs amount in STEMI patients (r=0.416, P<0.001). Multivariate analysis showed serum AGEs concentration was independently and positively related to frequent PVCs (adjusted hazard ratio, 1.86; 95% CI, 1.09-3.18, P=0.022). In the animal study, increased glucose regulated protein 78 (GRP78) expression, protein kinase RNA-like ER kinase (PERK) phosphorylation, CaN enzymatic activity, FKBP12.6-RyR2 disassociation, RyR2 channel opening and endoplasmic reticulum (ER) calcium releasing were found in diabetic MI animals, which were attenuated by anti-RAGE antibody treatment. This RAGE blocking also significantly lowered the VAs amount in diabetic MI animals. Activation of RAGE- dependent ER stress- mediated PERK/CaN/RyR2 signaling participated in post-MI VAs in diabetes.

RYR2 Channel Inhibition Is the Principal Mechanism 0f Flecainide Action in CPVT

Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive cardiac ryanodine receptor (RyR2) calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum (SR) Ca release and for preventing ventricular tachycardia in vivo. Methods and Results: We synthesized N-methylated flecainide analogues (QX-FL and NM-FL) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-Methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a calsequestrin knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes — lacking intact sarcolemma and devoid of sodium channel contribution — flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin (TTX) to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous SR Ca release, while QX-FL and NM-FL did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas NM-FL had no significant effect on arrhythmia burden, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

Hyper-Activation of Endoplasmic Reticulum Stress PERK/Calcineurin/RyR2 Signaling Pathway is Involved in AGEs-Exacerbated Post- Myocardial Infarction Ventricular Arrhythmias

Background: The current study was aimed to investigate the mechanisms of advanced glycation end products (AGEs) in exacerbating post- myocardial infarction (MI) ventricular arrhythmias (VAs). Methods: Correlation between premature ventricular contractions (PVCs) and serum AGEs concentrations was analyzed in a cohort consisted of 101 STEMI patients with culprit vessel of left anterior descending artery (LAD). Established MI rat model were treated with AGEs and/or anti- receptor for AGE (RAGE) antibody. Electrocardiography was used to record VAs. Myocytes were isolated from adjacent area around infracted region. Immunofluorescent stains were used to evaluate association between FKBP12.6 (FK506-bindingprotein 12.6) and ryanodine receptor 2 (RyR2). Calcium sparks were evaluated by confocal microscope. Protein expression and phosphorylation were assessed by Western blotting. A colorimetric method was used to determine the enzymatic activity of calcineurin (CaN). [3H]-ryanodine binding assay was carried out to detect the RyR2 channel activity. Results: In the cohort study, significantly increased amount of (PVCs) were found in STEMI patients with diabetes (P<0.05). Serum AGEs concentration was significantly positively correlated with PVCs amount in STEMI patients (r=0.416, P<0.001). Multivariate analysis showed serum AGEs concentration was independently and positively related to frequent PVCs (adjusted hazard ratio, 1.86; 95% CI, 1.09-3.18, P=0.022). In the animal study, increased glucose regulated protein 78 (GRP78) expression, protein kinase RNA-like ER kinase (PERK) phosphorylation, CaN enzymatic activity, FKBP12.6-RyR2 disassociation, RyR2 channel opening and endoplasmic reticulum (ER) calcium releasing were found in MI animals exposed to AGEs, which were attenuated by anti-RAGE antibody treatment. This RAGE blocking also significantly lowered the VAs amount in MI animals exposed to AGEs. Conclusions: Hyper-activation of ER stress- mediated PERK/CaN/RyR2 signaling participated in AGEs- exacerbated post-MI VAs.

P5025Structural insights into catecholaminergic polymorphic ventricular tachycardia-associated RyR2 mutant channels using a three-dimensional in silico model

Background The cardiac ryanodine receptors (RyR2) are large tetrameric calcium-permeant ion channels found in cardiac muscle sarcoplasmic reticulum, which play an important role in the control of intracellular Ca2+ release and cardiac contraction. Mutations in the RYR2 gene are associated with lethal arrhythmia diseases including catecholaminergic polymorphic ventricular tachycardia (CPVT) resulting from increased diastolic Ca2+ leak from mutant channels. RyR2 is a huge protein that each subunit of tetramer is comprised of 4967 amino acids, which hampers the detailed in vitro analysis of RyR2 mutant channels. Purpose We aimed to analyze the structural features of RyR2 mutant channels identified in our cohort with inherited arrhythmias using RyR2 three-dimensional (3D) in silico model to reveal the arrhythmogenic mechanisms. Methods A targeted next-generation sequencing panel for inherited arrhythmias was employed for genetic diagnosis of the patients. Then, we mapped the identified mutations on RyR2 3D structural model developed by cryo-EM images (PDB: 5go9, 5goa, Peng Science 2016) and investigated the relationship between the location of the mutations and specific functional sites. Results As a result of genetic analysis, we identified 93 RYR2 mutations from 112 probands with CPVT (n=93) or long-QT syndrome (LQTS) (n=19).64 of 93 (69%) RYR2 mutations are located in three “hot-spot” area (N-terminal (residues 77–466), central (2246–2534), and channel (3778–4959) hotspot. RyR2 3D in silico modeling revealed that the mutations are regionally distributed mainly in three parts: N-terminal, periphery, and channel part (Figure A). In N-terminal part (1–642 amino acid), 9 of 13 mutations alter the charges of the amino acids (Figure B). Especially, R169L, R169Q, and G172E are close to the interface between two neighboring subunits (∼20Å). These mutations which change the amino acid charge may cause a complete disruption of the ionic pair network and result in largest structural changes, which facilitates RyR2 channel opening. In periphery part (643–3528aa), 22 of 33 mutations are close to the two predicted binding sites of FKBP12.6, a stabilizer of RyR2 (∼5–40Å, Figure C). The mutations are supposed to disturb the binding affinity to the FKBP12.6 resulting in RyR2 channel instability. In channel part (3613–4968aa), 16 of 40 mutations are located near two interface. (FigureD) 12 mutations are close to the Ca2+ sensor and the other 4 mutations are adjacent to the pore-forming segment. Especially, V4821I is just located on this segment and strongly expected to impair the channel function. Above all, RyR2 3D in silico modeling revealed that 63 of all 93 (68%) identified mutations are supposed to be pathogenic. Location of RYR2 mutations in 3D model Conclusion 3D structural model of RyR2 is useful for the investigation of the pathogenic mechanisms of CPVT-related mutations. Further studies are needed to elucidate the relationship between the location of the mutations and clinical phenotypes.

Phosphorylation of the ryanodine receptor 2 at serine 2030 is required for a complete β-adrenergic response

During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in protein kinase A (PKA)–mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent “hyperphosphorylation” of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation–contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch–clamp recordings and confocal Ca2+ imaging during β-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (ICa) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during β-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.

Increased mitochondrial nanotunneling activity, induced by calcium imbalance, affects intermitochondrial matrix exchanges

Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca2+imbalance by depressing RyR2 channel activity during excitation–contraction coupling, resulting in random bursts of Ca2+release probably due to Ca2+overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2A4860G+/−cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower.