Abstract Background Ryanodine receptor (RyR2) mutations have largely been associated with catecholaminergic polymorphic ventricular tachycardia (PMVT). The role of RyR2 mutations in the pathogenesis ...of arrhythmias and syncope at rest is unknown. We sought to characterize the clinical and functional characteristics associated with a novel RyR2 mutation found in a mother and daughter with PMVT at rest. Methods and results A 31-year-old female with syncope at rest and recurrent short-coupled premature ventricular contractions (PVCs) initiating PMVT was found to be heterozygous for a novel RyR2-H29D mutation. Her mother, who also had syncope at rest and short-coupled PMVT, was found to harbor the same mutation. Human RyR2-H29D mutant channels were generated using site-directed mutagenesis and heterologously expressed in HEK293 cells together with the stabilizing protein calstabin2 (FKPB12.6). Single channel measurements of RyR2-H29D mutant channels and wild type (WT) RyR2 channels were compared at varying concentrations of cytosolic Ca 2 + . Binding affinities of the RyR2-H29D channels and RyR2-WT channels to calstabin2 were compared. Functional characterization of the RyR2-H29D mutant channel revealed significantly higher open probability and opening frequency at diastolic levels of cytosolic Ca 2 + under non-stress conditions without protein kinase A treatment. This was associated with a modest depletion of calstabin2 binding under resting conditions. Conclusions The RyR2-H29D mutation is associated with a clinical phenotype of short-coupled PMVT at rest. In contrast to catecholaminergic PMVT-associated RyR2 mutations, RyR2-H29D causes a leaky channel at diastolic levels of Ca 2 + under non-stress conditions. Leaky RyR2 may be an under-recognized mechanism for idiopathic PMVT at rest.
RATIONALE:Mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT-associated RyR2 mutations cause fatal ...ventricular arrhythmias in young individuals during β-adrenergic stimulation.
OBJECTIVE:This study sought to determine the effects of a novel RyR2-G230C mutation and whether this mutation and RyR2-P2328S alter the sensitivity of the channel to luminal calcium (Ca).
METHODS AND RESULTS:Functional characterizations of recombinant human RyR2-G230C channels were performed under conditions mimicking stress. Human RyR2 mutant channels were generated by site-directed mutagenesis and heterologously expressed in HEK293 cells together with calstabin2. RyR2 channels were measured to examine the regulation of the channels by cytosolic versus luminal sarcoplasmic reticulum Ca. A 50-year-old white man with repeated syncopal episodes after exercise had a cardiac arrest and harbored the mutation RyR2-G230C. cAMP-dependent protein kinase–phosphorylated RyR2-G230C channels exhibited a significantly higher open probability at diastolic Ca concentrations, associated with a depletion of calstabin2. The luminal Ca sensitivities of RyR2-G230C and RyR2-P2328S channels were WT-like.
CONCLUSIONS:The RyR2-G230C mutant exhibits similar biophysical defects compared with previously characterized CPVT mutationsdecreased binding of the stabilizing subunit calstabin2 and a leftward shift in the Ca dependence for activation under conditions that simulate exercise, consistent with a “leaky” channel. Both RyR2-G230C and RyR2-P2328S channels exhibit normal luminal Ca activation. Thus, diastolic sarcoplasmic reticulum Ca leak caused by reduced calstabin2 binding and a leftward shift in the Ca dependence for activation by diastolic levels of cytosolic Ca is a common mechanism underlying CPVT.
Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a genetic disorder characterized by ventricular tachycardia, that can cause the heart to stop beating leading to death. The prevalence ...is 1/10.000 and in approximately 60% of cases, the syndrome can be due to a mutation of the cardiac ryanodine receptor gene (RyR2). We derived an induced pluripotent stem cell (iPSC) line from an 11-year-old patient blood-cells, carrying a heterozygous missense mutation on the 8th exon of the RyR2 N-terminal part. This reprogramed CPVT line displayed normal karyotype, expressed pluripotent markers and had a capacity to differentiate in trilineage embryonic layers.
Background
Duchenne muscular dystrophy (DMD) is an X‐linked disorder characterized by progressive muscle weakness due to the absence of functional dystrophin. DMD patients also develop dilated ...cardiomyopathy (DCM). We have previously shown that DMD (mdx) mice and a canine DMD model (GRMD) exhibit abnormal intracellular calcium (Ca2+) cycling related to early‐stage pathological remodelling of the ryanodine receptor intracellular calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) contributing to age‐dependent DCM.
Methods
Here, we used hiPSC‐CMs from DMD patients selected by Speckle‐tracking echocardiography and canine DMD cardiac biopsies to assess key early‐stage Duchenne DCM features.
Results
Dystrophin deficiency was associated with RyR2 remodelling and SR Ca2+ leak (RyR2 Po of 0.03 ± 0.01 for HC vs. 0.16 ± 0.01 for DMD, P < 0.01), which led to early‐stage defects including senescence. We observed higher levels of senescence markers including p15 (2.03 ± 0.75 for HC vs. 13.67 ± 5.49 for DMD, P < 0.05) and p16 (1.86 ± 0.83 for HC vs. 10.71 ± 3.00 for DMD, P < 0.01) in DMD hiPSC‐CMs and in the canine DMD model. The fibrosis was increased in DMD hiPSC‐CMs. We observed cardiac hypocontractility in DMD hiPSC‐CMs. Stabilizing RyR2 pharmacologically by S107 prevented most of these pathological features, including the rescue of the contraction amplitude (1.65 ± 0.06 μm for DMD vs. 2.26 ± 0.08 μm for DMD + S107, P < 0.01). These data were confirmed by proteomic analyses, in particular ECM remodelling and fibrosis.
Conclusions
We identified key cellular damages that are established earlier than cardiac clinical pathology in DMD patients, with major perturbation of the cardiac ECC. Our results demonstrated that cardiac fibrosis and premature senescence are induced by RyR2 mediated SR Ca2+ leak in DMD cardiomyocytes. We revealed that RyR2 is an early biomarker of DMD‐associated cardiac damages in DMD patients. The progressive and later DCM onset could be linked with the RyR2‐mediated increased fibrosis and premature senescence, eventually causing cell death and further cardiac fibrosis in a vicious cycle leading to further hypocontractility as a major feature of DCM. The present study provides a novel understanding of the pathophysiological mechanisms of the DMD‐induced DCM. By targeting RyR2 channels, it provides a potential pharmacological treatment.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of heart samples revealed a higher PPARγ gene expression level in the ACM-heart bearing the DSC2 mutation than in the ...control heart (Figure S1). ...when we cultured DSC2-hiPSC-CMs in the presence of T007 between D20 and D60 and then removed the drug at D100, the cells exhibited the typical disease phenotype (Figure S5). ...early and continuous exposure of DSC2-hiPSC-CMs to T007 is required to maintain a wild-type-like cellular electrophysiological signature (Figure S6). ...the repressing effect of T007 on PPARγ transcriptional activity maintained regular electrical activity and Ca2+ handling in hiPSC-CMs bearing the DSC2 (c.394C>T) mutation.
Duchenne Muscular Dystrophy (DMD) is a X-linked degenerative pathology with a prevalence of 1/3600–6000 boys due to the absence of functional dystrophin in muscles. This muscular disease leads to ...skeletal muscle damages, respiratory failure and in the later stages dilated cardiomyopathy (DCM) leading to heart failure. We generated iPSC lines from three different DMD patients carrying respectively deletions of exons 1, 52 and 55 in the dystrophin gene. The reprogrammed iPSC lines showed expression of pluripotent markers, capacity to differentiate in trilineage embryonic layers and a normal karyotype.
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