Abstract
Current mechanisms of arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia (CPVT) require spontaneous Ca(2+) release via cardiac ryanodine receptor (RyR2) channels affected by gain-of-function mutations. Hence, hyperactive RyR2 channels eager to release Ca(2+) on their own appear as essential components of this arrhythmogenic scheme. This mechanism, therefore, appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations. We aimed to elucidate arrhythmia mechanisms in a RyR2-linked CPVT mutation (RyR2-A4860G) that depresses channel activity. Recombinant RyR2-A4860G protein was expressed equally as wild type (WT) RyR2, but channel activity was dramatically inhibited, as inferred by [(3)H]ryanodine binding and single channel recordings. Mice heterozygous for the RyR2-A4860G mutation (RyR2-A4860G(+/-)) exhibited basal bradycardia but no cardiac structural alterations; in contrast, no homozygotes were detected at birth, suggesting a lethal phenotype. Sympathetic stimulation elicited malignant arrhythmias in RyR2-A4860G(+/-) hearts, recapitulating the phenotype originally described in a human patient with the same mutation. In isoproterenol-stimulated ventricular myocytes, the RyR2-A4860G mutation decreased the peak of Ca(2+) release during systole, gradually overloading the sarcoplasmic reticulum with Ca(2+). The resultant Ca(2+) overload then randomly caused bursts of prolonged Ca(2+) release, activating electrogenic Na(+)-Ca(2+) exchanger activity and triggering early afterdepolarizations. The RyR2-A4860G mutation reveals novel pathways by which RyR2 channels engage sarcolemmal currents to produce life-threatening arrhythmias.
Highlights
In the heart, ryanodine receptor (RyR2) channels release massive amounts of Ca2+ from the sarcoplasmic reticulum (SR) in response to membrane depolarization, in turn modulating cardiac excitability and triggering ventricular contractions [1, 2]
Point mutations in RYR2, the gene encoding for the cardiac RyR channel, are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) [5], a highly arrhythmogenic syndrome triggered by sympathetic stimulation that may lead to sudden cardiac death, especially in children and young adults [6]
The diary of channel openings (Fig. S2D) for wild type (WT) channels displayed periods of steady activity interspaced by bursts of high Po and almost no sweeps of inactivity, but the latter were the characteristic event of RyR2-A4860G channels
Summary
Ryanodine receptor (RyR2) channels release massive amounts of Ca2+ from the sarcoplasmic reticulum (SR) in response to membrane depolarization, in turn modulating cardiac excitability and triggering ventricular contractions [1, 2]. To date, delayed afterdepolarizations (DADs) triggered by spontaneous Ca2+ release stand as the most accepted cellular mechanism to explain cardiac arrhythmias in CPVT In this scheme, RyR2 channels destabilized by gain-of-function mutations release Ca2+ during diastole, generating a depolarizing transient inward current (Iti) as the sarcolemmal Na+-Ca2+ exchanger (NCX) extrudes the released Ca2+. Most RyR2linked CPVT mutations characterized to date produce hyperactive RyR2 channels [9,10,11,12] This scheme appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations [13]. When expressed in HEK293 cells, recombinant RyR2-A4860G channels displayed a dramatic depression of activity, manifested mainly as a loss of luminal Ca2+ sensitivity [13] This in vitro characterization was insufficient to elucidate the mechanisms by which these hypoactive channels generate cellular substrates favorable for cardiac arrhythmias. We generated an animal model of CPVT that harbors a loss-of-function mutation that was originally described in humans and elucidated novel mechanisms by which hypoactive RyR2 channels trigger malignant arrhythmias
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