Correction: “Ryanopathies” and RyR2 dysfunctions: can we further decipher them using in vitro human disease models?

The regulation of intracellular calcium (Ca2+) homeostasis is fundamental to maintain normal functions in many cell types. The ryanodine receptor (RyR), the largest intracellular calcium release channel located on the sarco/endoplasmic reticulum (SR/ER), plays a key role in the intracellular Ca2+ handling. Abnormal type 2 ryanodine receptor (RyR2) function, associated to mutations (ryanopathies) or pathological remodeling, has been reported, not only in cardiac diseases, but also in neuronal and pancreatic disorders. While animal models and in vitro studies provided valuable contributions to our knowledge on RyR2 dysfunctions, the human cell models derived from patients’ cells offer new hope for improving our understanding of human clinical diseases and enrich the development of great medical advances. We here discuss the current knowledge on RyR2 dysfunctions associated with mutations and post-translational remodeling. We then reviewed the novel human cellular technologies allowing the correlation of patient’s genome with their cellular environment and providing approaches for personalized RyR-targeted therapeutics.

The RyR2-E189D mutation increased the propensity for SOICR, without altering the FKBP12.6 affinity to bind to the channel. [1] G230C This novel CPVT mutation enhances RyR2 cytosolic Ca 2+ sensitivity, which leads to diastolic SR Ca 2+ leak under stress conditions. RyR2 leak was associated with a depletion of the stabilizing FKBP12.6 protein, which eventually provoked arrhythmias. [2] ΔExon 3 The RYR2 exon 3 deletion causes an NTD alteration and results in a Ca 2+ release properties adjustment. Although this deletion is rescued by the β strand switching, it affects interfaces with other RYR2 domains. This suggests some N-terminal domain and channel pore coupling. [3]

G357S
The RyR2-G357S mutation reduced the expression of the RyR2 protein and increased the arrhythmogenic SOICR in HEK293 cells, which might be responsible for the CPVT syndrome. [4]

A165D
The RyR2-A165D mutation was first identified in a CPVT patient. When using a knock-in mice model, the A165D mutation induced SR Ca 2+ release triggering DADs. The A165D mutation was located in the conformational stability loop, which explained the occurrence of some diastolic leak that is responsible for arrhythmias. [5] Helical domain 1 S2246L Increase of Ca 2+ release in HL-1 cardiomyocytes expressing mutant hRyR2, after caffeine and β-adrenergic activation. [6]

P2328S
This mutation decreases FKBP12.6 binding to the RyR2. Sensitivity increases with cytosolic Ca 2+ allowing a higher open probability of RyR2 channels at low diastolic levels, causing SR Ca 2+ leaks in the CPVT1 syndrome. The JTV519 Rycal molecule rescued a normal RyR2 function. [7] R2401H RyR2-R2401H mutation is located in the FKBP12.6 RyR2 binding region, which could affect the CICR and the ECC resulting in a CPVT. [8] S2246L, R2474S RyR2 mutations increased both store-overload-induced Ca 2+ release (SOICR) activity and sensitivity towards luminal calcium, without affecting the channel affinity for the FKBP12.6 in CPVT. [9]

R2267H
A novel mutation was identified in sudden infant death syndrome cases. When using some heterologous system expression, this mutation was leaky under beta-adrenergic stimulation, leading to a PKA-phosphorylation that triggers cardiac arrhythmias. Interestingly, another study demonstrated a lack of pathogenicity of this variant. Thus, the in vitro functional findings were not translated to human phenotype. [11,12]

R2474S
The RyR2-R2474S mutation perturbed the interdomain conformational changes, which destabilized the closed state of the RyR2 and lead to a leaky channel. [13,14] Central domain N4104K See findings of the S2246L mutation. [6] Q4201R See findings of the P2328S mutation. [7] Q4201R See findings of the S2246L and R2474S mutations. [9] S4153R This novel RyR2 heterozygous mutation was first described in a 25-year-old CPVT syndrome female patient. This mutation is characterized by some RyR2 gain-of-function that is induced by the SOICR threshold reduction and some propensity increase for spontaneous calcium release. [15,16] Channel domain R4497C See findings of the S2246L mutation. [6] V4653F See findings of the P2328S mutation. [7] I4867M, See findings of the S2246L and R2474S mutations. [9] A4860G When using mice models and HEK293 cells, the RyR2-A4860G mutation reduced the channel activity by inhibiting Ca 2+ release during the diastole and by overloading the SR with Ca 2+. Consequently, it prolonged Ca 2+ release and corresponding AP, leading to the activation of the NCX exchanger. The I Ti current triggers the early afterdepolarizations (EADs) that are responsible for CPVT pathogenesis. [17,18] S4565R Two novel mutations were identified in sudden infant death syndrome cases. When using some heterologous system expression, these 2 mutations were leaky under beta-adrenergic stimulation, leading to a PKA-phosphorylation that triggers cardiac arrhythmias. [11] Y. Sleiman et al.  [19,20] K4750Q The RyR2-K4750Q mutation mediated-CPVT induced diastolic SR Ca 2+ leak was caused by an enhancement of propensity to activation of cytosolic and luminal Ca 2+ and by the loss of cytosolic Ca 2+ /Mg 2+ -mediated inactivation. [21]

I4855M
The RyR2-I4855M mutation was present in 2 members of a CPVT-affected family. The RyR2-I4855M shows some loss of function and is characterized by some CICR inhibition of the HEK293 cells. The I4855A may interfere with Ca 2+ permeation and may affect interactions between the RyR2 pore subunits. [22] Case reports and genotyping studies of patient cohorts N-terminal domain R414L, I419F, P164S Novel RyR2 mutations were associated with the CPVT1 syndrome in a swimming-triggered arrhythmia syndrome using direct DNA sequencing and denaturing high-performance liquid chromatography. The 388 unrelated patients were chosen according to family or personal history of drowning or swimming-related cardiac events. However, considering the large number of the cohort, they did not specify the cardiac phenotype of each patient. [23] ΔExon 3, A77V In a 17-year-old boy postmortem study, the RyR2-A77V mutation was associated with both an arrhythmogenic right ventricular cardiomyopathy and a CPVT syndrome, in the same family. This 17-year-old boy presented right ventricular fibrofatty and fatty myocardium replacement and calcium phosphate deposits in right ventricular cardiomyocytes that were mostly restrained into mitochondria. His mother and his sister presented normal right and left ventricles volume and no kinetic alterations. The exercise treadmill stress test revealed polymorphic ventricular tachycardia that was successfully abolished with β-blocker (Acebutolol) treatment. The same RyR2-A77V mutation led to distinct diseases in the same family members. This reflects the complexity of clinical diagnosis and the variable phenotype that can be present even among family members of the same family. De novo RYR2 exon 3 deletions were reported in a severe CPVT case. This patient also developed some left ventricular noncompaction (LVNC), which exacerbates the arrhythmia. This patient showed no sign of endomyocardial inflammation and displayed normal heart structure. Multiform premature ventricular contractions, ectopic atrial rhythm, and ventricular triplet was observed during exercise. She experienced ventricular fibrillation and underwent ICD implantation together with the administration of Metoprolol and then Satolol treatment. Due to the severity of her phenotype, she started Flecainide and Nadolol treatment and underwent bilateral sympathectomy. The interaction between RyR2-ΔExon 3 and LVNC that may represent a predictive clinical marker for a more severe CPVT phenotype remains unclear.

R414C
The molecular autopsy revealed novel mediated CPVT syndrome RyR2 mutations in 2 unexplained drowning cases. This patient carrying the RyR2-R414C variant experienced syncope and seizure-like symptoms. Unexceptional and unremarkable EEG and physical examination were found. She was first diagnosed with acute seizure activity secondary to trauma. Due to the nature of the sudden death, direct DNA sequencing, and polymerase chain reaction, denaturing high-performance liquid chromatography was performed, which revealed this missense novel RyR2 mutation. As this patient presented a normal structural heart and absence of fatty infiltration, she was considered as a CPVT patient. [26] V186M, P164S Four patients (3 males) out of 8 patients, were presented with RyR2 mutations associated with some CPVT syndrome. Each patient presented specific symptoms which reflect the heterogeneity of CPVT phenotypes. Some patients had palpitations and seizure-like activity others had a cardiac arrest with ventricular fibrillation. Unfortunately, they did not match each RyR2-variant with its specific phenotype. [27]

R169Q
One RyR2 novel heterozygous mutation in exon 8 was screened in an 18-year-old female patient presenting a CPVT syndrome. This patient presented sudden collapse due to exercise and had bidirectional ventricular tachycardia during the exercise stress test. She had a good response to the β-blocker treatment. This same mutation was found recently in three unrelated females. Interestingly, all of these patients presented left ventricular non-compaction cardiomyopathy, and two of them survived sudden cardiac arrest. In vitro, functional analysis of this mutation revealed an increase of the Ca 2+ fractional release [28][29][30] Y. Sleiman et al. It was suggested that this RyR2-R169Q mutation leads to local structural abnormalities within or near the hot-spot regions, which in turn leads to functional perturbations. It leads to allosteric dysregulation by reducing the side chain size and diminishing the positive charge and stacking interaction of the RyR2 protein.
L62F, M81L, P164S, E243K, F329L, R332W, V377M, G357S, T415R, R420Q, V507I, A549V, S616L, H240R A cohort of CPVT patients was screened to investigate RYR2 gene mutations. 34 novel mutations were identified. They did not specify the clinical phenotype of the 155 unrelated patients examined in this study. Interestingly, they proposed a novel targeted genetic testing for CPVT syndrome. They emphasized also the genotype/phenotype relationship as the majority of these mutations were localized in the so-called hot-spot regions. [31] D242V, E243K The long-term follow-up of 101 CPVT patients showed high cardiac events, despite some β-blockers treatment in 21% of patients with 13% of fatal or near-fatal events. Some of these patients survived cardiac arrest and presented palpitations and syncope accompanied or not with seizures. 80% of these patients were treated with β-blockers (mostly with Nadolol but also with Propranolol, Bisoprolol, Acebutolol, and Pindolol). ICD implantation and Verapamil were added to some patients after the 1 st cardiac event. Even though β-blockers lower the cardiac events rate, they are not sufficient alone to prevent arrhythmias. [32]

R169L
This mutation was identified in an 8 years-old boy with CPVT and Left Ventricular Hypertrophy. This boy presented with two episodes of emotion-triggered syncope and could not survive the third one, which led to sudden cardiac death. This patient carried two other mutations, the G1339 variant in ATP-binding cassette, sub-family C member 9 (ABCC9), and the R52H variant in Potassium Inwardly Rectifying Channel Subfamily J Member 5 (KCNJ5). These 2 variants have unknown significance. The combination of CPVT and Left Ventricular Hypertrophy might lead to a more severe fatal phenotype. However, more studies are needed to elucidate the pathophysiological mechanism underlying the structural alterations of this RyR2 mutation. This same mutation was also reported in another 9 years-old girls who experienced a syncopal episode. The ECG findings were not reported.

R2311D, E2311D
The arrhythmogenic events occurred in young RyR2 mutationsaffected patients when compared to ungenotyped CPVT patients, with a higher risk of syncope for males. [40] V2306I, P2328S Novel mutations were found to be associated with the CPVT syndrome in 12 Finnish probands. [41] A2387P Novel RyR2 mutation was screened and identified using the DHPLC approach. [42] A2403T See findings of the R414L mutation. [23] L2487I RyR2 mutation was detected in 6% of unrelated genotypenegative and atypical LQTS, that were considered CPVT patients. [43] A2254V, A2394G Independently of the localization of the RyR2 mutations, all CPVT patients presented some bradycardia and responded to the β-blockers treatment. 9-year-old was the median age of symptoms onset.

G2337V
The β-blockers treatment suppressed severe arrhythmias in stress-induced CPVT-related RyR2 mutations, though it did not prevent the less severe ones. [48]

L2527W
Determination of a novel RyR2 heterozygous mutation in a 9year-old Chinese boy, misdiagnosed with epilepsy and CPVT syndrome. The β-blocker (Metoprolol) treatment proved unfavorable. [49] E2296K This RyR2-E2296K mutation was identified in a 5-year-old Chinese boy with CPVT using whole exome sequencing. This mutation might reduce protein stability. However, further investigations are needed to prove its causality. [50]

V2193L
The RyR2-V2193L mutation was identified in a 9-year-old Chinese boy who presented with both epilepsy and CPVT syndrome. The exercise stress test revealed frequent PPVB and PMVT with the presence of R on T. His electroencephalogram (EEG) showed frequent epileptiform discharges during stage II, stage III, and REM sleep. He was successfully treated with Metoprolol and Levetiracetam. [51]

C2277R
The RyR2-C2277R variant, located in the calstabin-binding domain, was identified in 8 members of the same family. The proband and her other family members presented ventricular extrasystoles (VE), bigeminy and/or trigeminy, doublets, and non-sustained VT upon exercise stress test and adrenaline test. These patients showed similar responses but different ventricular arrhythmias complexity degrees. The proband was treated with a combination of ICD implantation, Flecainide, and Nadolol. The other family members were treated either with Atenolol, Nadolol, or with the combination of Nadolol and Flecainide or Atenolol and Flecainide, which proved effective. [52]

G3037D
Identification of a novel RyR2 heterozygous mutation in a 2 years old patient exhibiting some CPVT syndrome. [53] Y. Sleiman et al. See findings of the mutation S2246L. The RyR2-N4104K variant was identified in a 14-year-old boy who presented nonsustained bidirectional VT upon exercise stress test. This proband was efficiently treated with Atenolol. [37,38] Central domain Q4201R Missense RyR2 gene mutation was identified in CPVT patients, which could affect myocardial calcium signaling. [39] L3778F, G3946S See findings of the R2311D mutation. [40,54] N4097S, E4146K, T4158P In a postmortem genetic testing model, 3 novel mutations were identified in 7 cases of sudden unexplained death, that might potentially cause CPVT. [55] F4020L, E4076K, N4104I, H4108N, H4108Q Independently of the localization of the RyR2 mutations, all CPVT patients presented some bradycardia and responded to the β-blockers treatment. 9-year-old was the median age of symptoms onset. Unspecified domain K4392R Case report of an athlete woman harboring some gain-offunction RyR2-K4392R mutation associated CPVT syndrome. [58]

R4497C
See findings of the mutation S2246L. The RyR2-R4497C variant was identified in a 30-year-old female who presented nonsustained bidirectional polymorphic VT upon exercise stress test. Two of her sisters died suddenly at the age of 14 and 16, respectively. Variable age-related manifestation of the disease has been thus suggested. This proband was treated with ICD implantation. [37,38] Channel domain V4653F Missense RyR2 gene mutation was identified in CPVT patients, which could affect myocardial calcium signaling. [39] V4771I, A4860G, I4867M, N4895D, E4950K See findings of the R2311D mutation. [40] P4902L, R4959Q Three novel mutations were found to be associated with the CPVT syndrome in 12 Finnish probands. [41] N4504I, A4608P, V4880A, M4504I, A4607P Four novel RyR2 mutations were screened and identified using the DHPLC approach. [42] F4499C, A4510T, G4671R, I4848V See findings of the R414L mutation. [23] A4556T, 4657-4658EYinsertion, G4671R RyR2 mutations were detected in 6% of unrelated genotypenegative and atypical LQTS, that were considered CPVT patients. [43] G4662S, H4762P, P4902S Independently of the localization of the RyR2 mutations, all CPVT patients presented some bradycardia and responded to the β-blockers treatment. 9-year-old was the median age of symptoms onset. The probands carrying the RyR2-G4662S and [35] Y. Sleiman et al.  [60] N-terminal domain S406L The β-adrenergic stimulation by isoproterenol induced DADs and diastolic Ca 2+ leak, which were reduced with the Dantrolene treatment. [61] E2311D/ Q231D Increased spontaneous calcium sparks and DADs, that were normalized by a CaMKII inhibition. [62]

R420Q
Non-ionotropic and lusitropic effects increased arrhythmias and intracellular Ca 2+ associated with immature ultrastructural features. [63] ΔExon 3 Dantrolene treatment reduced the premature ventricular complexes and the abnormal Ca 2+ release in 4 CPVT patients and CPVT hiPSC-CMs. However, Dantrolene was not effective to treat patients carrying mutations in or near the transmembrane domain of the RyR2. [64] Helical domain 1 F2483I The reduction of Ca 2+ stores induced by a higher CICR mechanism led to abnormal Ca 2+ homeostasis. These abnormalities were verified in 2018 in gene-edited CPVT hiPSC-CMs generated by the CRISPR/Cas9 technology. [65-67]

P2328S
The abnormal calcium homeostasis and the reduction of the SR Ca 2+ load led to EADs and DADs at baseline and under isoproterenol stimulation. Another study found that the CPVT hiPSC-CMs exhibit increased non-alternating variability of Ca 2+ transients and slow depolarization under isoproterenol stimulation. [68,69] P2328S, T2538R See findings of the ΔExon 3 mutation. [64] Y2476D Arrhythmic events and impairment of the calcium handling and beating properties of CPVT hiPSC-CMs. These abnormalities were more pronounced under β-adrenergic stress. [70] Central domain M4109R The β-adrenergic stimulation induces DADs and irregular Ca 2+ transients that were abolished with the Flecainide and Thapsigargin treatments. [71] L4115F, Q4201R See findings of the ΔExon 3 mutation. [64]

L3741P
The Flecainide treatment abolished the DADs and the spontaneous calcium sparks. [72] D3638A The RyR2 macromolecular complex remodeling, including FKBP12.6 depletion, SR Ca 2+ leak, and impaired contractile properties, were observed in RyR2-D3638A hiPSC-CMs under stress conditions. Abnormal release of Ca 2+ was prevented with the Flecainide and S107 treatments but not with the Metoprolol. [73]

R4651I
CPVT tissues display re-entrant rhythms under stress that are prevented by CaMKII inhibition. [60] Channel domain V4653F See findings of the P2328S mutation. [64] I4587V DADs and abnormal diastolic Ca 2+ release were observed under β-adrenergic stress. The S107 treatment reduced the occurrence of DADs. [74]