Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a difficult-to-diagnose cause of sudden cardiac death (SCD). We identified a family of 1400 individuals with multiple cases of CPVT, ...including 36 SCDs during youth.
We sought to identify the genetic cause of CPVT in this family, to preventively treat and clinically characterize the mutation-positive individuals, and to functionally characterize the pathogenic mechanisms of the mutation.
Genetic testing was performed for 1404 relatives. Mutation-positive individuals were preventively treated with β-blockers and clinically characterized with a serial exercise treadmill test (ETT) and Holter monitoring. In vitro functional studies included caffeine sensitivity and store overload-induced calcium release activity of the mutant channel in HEK293 cells.
We identified the p.G357S_RyR2 mutation, in the cardiac ryanodine receptor, in 179 family members and in 6 SCD cases. No SCD was observed among treated mutation-positive individuals over a median follow-up of 37 months; however, 3 relatives who had refused genetic testing (confirmed mutation-positive individuals) experienced SCD. Holter monitoring did not provide relevant information for CPVT diagnosis. One single ETT was unable to detect complex cardiac arrhythmias in 72% of mutation-positive individuals, though the serial ETT improved the accuracy. Functional studies showed that the G357S mutation increased caffeine sensitivity and store overload-induced calcium release activity under conditions that mimic catecholaminergic stress.
Our study supports the use of genetic testing to identify individuals at risk of SCD to undertake prophylactic interventions. We also show that the pathogenic mechanisms of p.G357S_RyR2 appear to depend on β-adrenergic stimulation.
Highlights • Catecholaminergic polymorphic ventricular tachycardia is a cause of sudden death. • Molecular autopsy should be performed in sudden death cases showing normal autopsy. • Next Generation ...Sequencing technology allows a comprehensive genetic analysis. • Familial assessment is crucial to identify relatives at risk of sudden death
The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol DiBAC...(3) has been reported as a novel large-conductance Ca...-activated K+ (BK) channel activator with selectivity for ...its β...- or β...-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory β...-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the β...-subunit, we explored the effects of DiBAC...(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC...(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic β...-knockout mice in excised patches. BK channels from brain arteries, with or without the β...-subunit, were similarly activated by DiBAC...(3). In addition, we found that saturating concentrations of DiBAC...(3) (~30 ...M) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as -300 mV. This "sweet spot" for persistent activation is independent of Ca... and/or the β...subunits and is fully achieved when DiBAC...(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC...(3) varies across species and/or vascular beds. DiBAC...(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators. (ProQuest: ... denotes formulae/symbols omitted.)
The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol DiBAC
4
(3) has been reported as a novel large-conductance Ca
2+
-activated K
+
(BK) channel activator with selectivity for ...its β
1
- or β
4
-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory β
1
-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the β
1
-subunit, we explored the effects of DiBAC
4
(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC
4
(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic β
1
-knockout mice in excised patches. BK channels from brain arteries, with or without the β
1
-subunit, were similarly activated by DiBAC
4
(3). In addition, we found that saturating concentrations of DiBAC
4
(3) (∼30 μM) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as −300 mV. This “sweet spot” for persistent activation is independent of Ca
2+
and/or the β
1–4
-subunits and is fully achieved when DiBAC
4
(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC
4
(3) varies across species and/or vascular beds. DiBAC
4
(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators.
The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol DiBAC₄(3) has been reported as a novel large-conductance Ca²⁺-activated K⁺ (BK) channel activator with selectivity for its ...β₁- or β₄-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory β₁-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the β₁-subunit, we explored the effects of DiBAC₄(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC₄(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic β₁-knockout mice in excised patches. BK channels from brain arteries, with or without the β₁-subunit, were similarly activated by DiBAC₄(3). In addition, we found that saturating concentrations of DiBAC₄(3) (~30 μM) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as -300 mV. This "sweet spot" for persistent activation is independent of Ca²⁺ and/or the β₁₋₄-subunits and is fully achieved when DiBAC₄(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC₄(3) varies across species and/or vascular beds. DiBAC₄(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators.
Large-conductance Ca
2+
-activated (BK) channels, expressed in a variety of tissues, play a fundamental role in regulating and maintaining arterial tone. We recently demonstrated that the slow ...voltage indicator DiBAC
4
(3) does not depend, as initially proposed, on the β
1
or β
4
subunits to activate native arterial smooth muscle BK channels. Using recombinant mslo BK channels, we now show that the β
1
subunit is not essential to this activation but exerts a large potentiating effect. DiBAC
4
(3) promotes concentration-dependent activation of BK channels and slows deactivation kinetics, changes that are independent of Ca
2+
. K
d
values for BK channel activation by DiBAC
4
(3) in 0 mM Ca
2+
are approximately 20 μM (α) and 5 μM (α+β
1
), and G-V curves shift up to −40mV and −110 mV, respectively. β
1
to β
2
mutations R11A and C18E do not interfere with the potentiating effect of the subunit. Our findings should help refine the role of the β
1
subunit in cardiovascular pharmacology.
PDF
