RyR mutations - CPVT Flashcards
What is malignant hyperthermia?
Symptoms?
Treatment?
Malignant Hyperthermia
Anesthetics are weak activators of RyR1 which can be fatal.
Symptoms include muscle rigidity, fever and tachycardia - anaesthetists cautious
Dantrolene is the treatment of choice and selectively inhibit RyR1.
What is CPVT, what mutation causes, incidence, what are the symptoms and what are the treatments?
Catecholaminergic Polymorphic Ventricular Tachycardia
Mutations in the cardiac ryanodine receptor gene (RyR2) underlie catecholaminergic polymorphic ventricular tachycardia (CPVT).
Rare condition with 1/60000 incidence.
Symptoms are recurrent syncope and ventricular arrhythmias triggered by stress or exercise. High incidence of sudden cardiac death.
Treatment includes:
- Beta blockers
suitable in 60-70% patients but wear off in between doses
- Implanted defibrillators
can cause psychological problems if it happens often – feels like being hit in the chest which can be a cause of anxiety, worrying if it will go off.
What mechanism causes CPVT?
3
Causes
- Pathological SR Ca2+ waves cause delayed after-depolarisations’ (DADs).
- DADs arise when removal of wave Ca2+ via the Na/Ca exchanger (NCX) induces a transient inward current (Iti)
- If the resulting depolarisation exceeds threshold, then an action potential will arise, with the potential to cause an arrhythmia. SR needs to be very full of calcium or channels have to behave differently to facilitate propagated activity.
When do DADs occur?
What are the properties of the propagated waves?
When cells become Ca2+ overloaded, or when specific RyR2 mutations are present:
- Local control of SR Ca2+ release fails due to high luminal feedback on RyR2, caused by mutations cause RyR2 channels to behave abnormally
- Ca2+ sparks propagate to form waves, which are slower, spontaneous, non-uniform and do not require an action potential
Describe the locations of RyR mutations
What is reduced in CPVT?
- They tend to cluster with mutations associated in the N terminus, Central domain and Transmembrane region
- Inactive channels are stabilised by inter-domain interactions which is reduced in CPVT mutations thus leading to channel instability - increased RyR Ca release
What evidence is there for the pathology of the mutations?
1
Yang et al., 2006
- Permeabilized rat ventricular myocytes
- Introduction of DPc10, a instability peptide to a region of the central domain, induced a transient increase in spark frequency and a sustained rise in resting [Ca2+] - visualised by confocal microscopy
- Equilibration with DPc10, the cytosolic [Ca2+] threshold for spontaneous, propagated Ca release was markedly reduced and the proportion of spontaneously active cells increased
- Results suggest that DPc10 (and CPVT mutations) increases the sensitivity to CICR as caffeine does, but also to potentiate Ca2+leakage from the SR which can trigger DADs.
What results further backed up the argument for RyR2 mutations?
Which has more weight?
2
Thomas et al., 2004
- Similar results
- Expressed recombinant human wild-type (WT) and SCD-linked RyR2 mutations in human embryonic kidney (HEK) cells and confocal laser scanning microscopy analysed Ca release
- RyR2 mutations exhibited a marked reduction in sensitivity to channel activation.
- HEK cells which have their limitations
- Yang et al. (2006) in vivo so more weight but in rats which may exhibit species differences
Name a novel therapeutic target for CPVT?
Therapeutic Target – Flecainide
- Inhibits Nav1.5 (Class 1C antiarrhythmic agent)
- Cannot be used in patients with structural heart disease
Give the initial evidence for the use of flecainide in CPVT - mouse model? 1
Watanabe et al., 2009
Mouse Model
- Used mice ventricular myocytes isolated with gene-targeted deletion of Casq2 (Casq2−/− mice) induces CPVT
1) Flecainide significantly suppressed the rate of spontaneous SR Ca2+ releases - for a prolonged period of time
2) No autoregulation
3) Reduced DAD beats by 69% - could be due to the role of blocking the Na channels and preventing action potential that way.
4) The heart rate remained regular and catecholamine/exercise challenge did not induce ventricular arrhythmias
5) Lidocaine, a Na+ channel blocker that does not inhibit RyR2 channels did not prevent exercise-induced ventricular tachycardia further indicating that RyR2 inhibition is crucial for flecainide’s antiarrhythmic efficacy in CPVT.
What evidence for the use of flecainide in CPVT - human model? 2
Watanabe et al., 2009
- 12-week flecainide treatment in two human subjects with CPVT, who had remained highly symptomatic on conventional drug therapy
1) ICD records showed ‘markedly less’ VT however data was not provided in the paper which questions how much of a decrease was seen
2) Exercise tests showed complete suppression of VT
However routine flecainide use cannot be recommended until further clinical studies define its risks and benefits in humans with CPVT.
What further evidence supported Watanabe’s views? 3
Hilliard et al., 2010
Supported theory and provided insight into mechanism of action
1) Ventricular myocytes isolated from a Casq2-/- mice model for CPVT, used permeabilized patch clamp recordings
Flecainide significantly reduced spark amplitude and spark width, resulting in a 40% reduction in spark mass but significantly increased spark frequency.
As a result, no significant effect on spark-mediated SR Ca2+ leak or SR Ca2+ content so no autoregulation (IMPORTANT)
Advantage of permeabilised cells is that Nav1.5 channels inactivated so must be acting on RyR.
2) Sheep myocyte SR vesicles containing RyR2 channels were obtained and reconstituted into lipid bilayers, flecainide inhibited RyR2 channels by open state block.
Suggested that the smaller spark mass contributes to flecainide’s antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca2+ release units – a possible therapeutic target.
Study used two species to demonstrate same effect which gives it more weight
What evidence contradicted the use of flecainide as a therapeutic target?
Explain the limitations to this study 1
Sikkel et al., 2013
Disagreed and suggested the mechanism involves INa reduction rather than direct ryanodine receptor (RyR2) inhibition.
Used healthy adult rat myocytes, perforated patch clamp, confocal imaging observed spark frequency, wave frequency, and wave velocity were reduced when INa is active
Sole inhibition of INa via tetrodotoxin produced similar changes.
Reduced INa results in increased Ca2+ efflux via NCX across the sarcolemma, reducing Ca2+ concentration near the RyR2.
Limitation
Flecainide is 99% positively charged (i.e. membrane impermeant) at physiological pH. It takes time for the 1% uncharged form of flecainide to diffuse into cells and accumulate to a level sufficient to affect RyR2 which may explain the result.
Voltage clamp technique used designed to eliminate INa, could also alter NCX function during the contraction.
Watanabe already injected lidocaine which inhibits Nav1.5 but has no effect on RyR2.
What evidence using bilayers was found to disprove use of flecainide? Is it powerful? 2
Bannister et al., 2015
Single WT human RyR2 channels were incorporated into bilayers and permeabilized cardiac myocytes showed that flecainide did not inhibit the channel.
Limitations
The experiments were performed on WT human RyR2 so irrelevent in the treatment of CPVT.
Lipid bilayer studies is not necessarily operating as it would in the intact cell, because the ionic conditions used are unphysiological and partly because accessory proteins that modify channel function are absent (e.g. calmodulin).
Summarise use of flecainide as a therapeutic target
- Although controversial, there is compelling evidence that flecainide acts on RyR2 to inhibit spontaneous SR Ca2+ waves, e.g. flecainide affects sparks and waves in permeabilized cells where effects on Nav1.5 are completely absent
- However, the simultaneous inhibition of Nav1.5 will also provide strong secondary antiarrhythmic protection IF SR Ca2+ waves occur in the presence of flecainide, i.e. inhibition of Nav1.5 increases the threshold for triggering of an AP. Hence DADs will be less likely to trigger Aps
- Also, inhibition of Nav1.5 will tend to lower cellular [Na+]i. When [Na+] decreases, [Ca2+]i tends to decrease due to effects on NCX. This is a third potential antiarrhythmic mechanism
In vivo, it is reasonable to assume that flecainide can act via all 3 mechanisms.