C2. Sudden cardiac death- experiments Flashcards
Summary
LQT
Brugada Syndrome
CPVT
HCM
Ventricular Fibrillation
Treatment
- Bagnall 2016
Prospective study 1-35 ages Aus NZ
SCD data autopsies included
CAD and Cardiomyopathies in 40%
27% had clinically relevant mutation
Likely to be an underestimate
- Sanguinetti 1996
Transfected CHO cells with channel cDNA showed unique IKs-like properties
Depolarising stimuli were followed by a brief delay then a repolarising outward potassium current
Could have transfected the mutant protein to see effects of this
- Curran 1995
Linkage and physical mapping on LQT2 Kindreds
Identified potential hERG gene
Single strand DNA seq confirmed hERG mutations
Northern blotting of protein in cardiac tissue
Should have isolated protein and expressed it
- Suessbrich 1996
hERG channels expressed in xenopus and voltage clamped to see current in response to depolarising
Haloperidol decreased hERG current in a concentration dependent manner
Should study in situe, maybe iPSCs
- Bennett 1995
LQT3 mutant and WT channels in xenopus oocyte
200ms depolarisation similar activation
Current through mutant channel did not decay within 200ms
Could have done in cardiomyocytes
- Moretti 2010
iPSCs from dermal fibroblasts of LQT R190 mutations in KCNQ1
Reverted to pluripotency then cardiac differentiation
Characteristic prolonged action potential and slower repolarisation preserved in iPSCs.
- Jiang 2004
Transfected HEK293 Cells with WT or CPVT RyR2
Line-Scan More Calcium Sparks in CPVT Transfected
Question Validity of Kidney Cell Line
Validated in Other Human and Mouse Models
- Brunello 2013
Casq2 mutant mice and measured frequency of calcium events when paced with Fluo-3
Casq2 mutant mice had consistent diastolic calcium release after systole
In intact muscle preparations there was temporal alignment of DCRs
Membrane potential imaging demonstrated this could result in extra-systolic activity.
- Hilliard 2010
Flecainide Thought to Block VGSCs
Wistar Rats with Casq2-/- Model of CPVT
Flecainide Reduced Amplitude + Intensity of Calcium Sparks in CPVT
Prevented Arrhythmogenic Wave Formation
- Kryshtal 2021
Similar Experimental Design to Hilliard 2010
Showed Effects of Flecainide Persisted in Absence of TTX
NM-FL – Non-Membrane Permeant Unable to Improve Symptoms in CPVT Mice
- Kyndt 2001
Identified Brugada patients, exome sequencing of SCN5A gene identifying mutation
Cloned WT and mutant genes into COS-7 cells and whole cell patch clamping
Reduced current through SCN5a channels and loss of function
Could use iPSCs
- Veldkamp 2000
Effects of SCN5A mutations on repolarisations
Introduced C-terminal mutation in SCN5A and expressed in HEK293 cells
Recorded whole cell currents and applied a sustained depolarising stimulus
In mutant cells, stimulus led to a current plateau indicative of failure of fast inactivation
Between stimuli a two-pulse protocol assessed slow inactivation
Demonstrated the slow-inactivation was augmented in this protocol, which was shown to have an effect on channel availability in the paired protocol
HEK293 cells are a non-cardiac heterologous expression system, potentially limiting the physiological relevance and clinical translation of findings related to SCN5A channel function
- Paavloa 2007
15 CPVT patients recorded endocardial action potentials with silver catheter in response to epinephrine
DADs present in 4 of these patients
HEK cells transfected with mutant and not-wild-type RyR2s showed the incidence of calcium waves
- Allessie 1973
Atrial tissue paced for 20 beats
Ectopic stimuli and surface electrograms at different points on the heart
Propagation of impulse in circular pathway
No anatomical impulse
Extensive preparation artefacts
- Gibson 2008
Retrospective analysis of coronary angiograms of STEMI patients and compared those with VF and VT
Patients with more impaired perfusion had higher incidence of ventricular arrhythmias and mortality
Hard to replicate in animal models
- Li et al in 1997
Used multiplex RT-PCR to identify gene expression of a variety of growth factors in patients with HCM
Used ventricular biopsies obtained from patients with HCM, aortic stenosis and stable angina
IGF-1 and TGF-beta mRNA were upregulated in HCM biopsies compared with other disease states
Confirmed by immunohistochemical staining for the two growth factors
Weren’t able to gather data from a healthy, control group due to ethical difficulties associated with performing operations on healthy patients.
- Sepp 1996
Role of Distribution of Cx43 in HCM
Obtained Six HCM Necropsy hearts compared with age matched controls
Immunostaining and confocal showed promiscuous expression and formation of abnormal gap junctions
Interesting to correlate these findings with ECGs or ex vivo myocyte studies to compare conduction.
- Corrado 1998
Italian Law requiring athletes for Clinical evaluation
12-Lead ECG + Echo when abnormalities
22 athletes disqualified for HCM and of SCDs only 1 in the 17-year period was due to HCM, 48 others for various regions
Would need to compare to a similar region with no screening
- Jabbari 2015
Characterised the risk of ventricular fibrillation in patients with STEMI before coronary interventions
Performed a case-control study of patients presenting within 12 hours of STEMI
Compared the incidence of VF within 12 hours of admission to hospital in patients, and demonstrated an increased incidence of VF in STEMI patients relative to control
2.8-fold increase in risk for atrial fibrillation
12-hour limit for onset of ventricular fibrillation does not accurately represent the sudden cardiac death
- Hill 1981
LAD Ligation in pig
Extracellular electrodes to measure K+
Rising levels of K+
Reference electrode and Vfib
Could deplete the extracellular space of potassium
- Lu 1999
Rats subject to IR and measured VFIB in presence of lidocaine and verapamil to inhibit sodium and calcium loading
Attenuate onset of arrhythmias
Anaesthesia may influence coronary action potential
- De Bakker 1993
Demonstrated that scar tissue contributes to creation of slow conduction that is necessary for re-entry
Measured the spread of activation in infarcted papillary muscles from patients who had undergone heart transplant because of infarction
High-resolution mapping was performed revealing slow-conduction velocity in fibrotic areas
Concluded that the slow conduction perpendicular to the fibre direction in infarcted myocardial tissue is caused by a “zigzag” course of activation
Conclusions rely exclusively on ex vivo mapping of isolated infarcted papillary muscle tissue, which may not fully represent the complexity of conduction patterns occurring in vivo in intact hearts
- Kemp 2021
hERG activators in xenopus oocytes with hERG and LQT mutant proteins
Whole cell voltage clamping following large depolarisations
RPR restored the repolarising current
However, only at a high dose of 30 uM
