C1. Calcium sparks- experiments Flashcards
Pneumonic
ITWFCDPAAAC
Summary
Initiation and termination
Transcients
Waves
Function
Calcium clocks
Disputing calcium clocks
Phosphorylation and luminal calcium sensing sites
Atrial vs ventricular myocytes and IP3Rs
Arrhythmogenesis
Atrial fibrillation
CPVT and heart failure
- Cheng 1993
Fluo-3 linescan microscopy of resting rat cardiomyocytes
Spontaneous short-lived calcium rises
Calcium-free baths and cadmium – not extracellular entry
Ryanodine increased the frequency of sparks
3pa calcium flux but this is debated
Discovery of calcium sparks
Remains debate over number of RyR2 in calcium sparks
- Cheng 1996
Isolated ventricular myocytes
Fluo-3am line scan microscopy
Increasing extracellular concentration (1-10mm) of calcium increased number of wave events
80% of waves preceded by calcium sparks
Other calcium waves may have been initiated outside of imaging plane
- Venkataraman 2012
Imaging whole rat hearts after 15 min ischaemia by LAD ligation
Fura2 2000frame per second camera
Calcium waves increased above and below point of ligation, but mainly below
Reversed by reperfusion
Good model of reperfusion? – ex vivo
- Barcenas-Ruiz 1987
Voltage clamp depolarisations of guinea pig ventricular myocytes
Fura2 + digital microscope
Step depolarisations elicited calcium transients
Blocked by either verapamil or ryanodine in high concentration
Not like physiological spread along gap junctions
- Rigg & Terrar 1996
Extracellular electrodes measure heart rate guinea pig atrium
Ryanodine and cyclopiazonic acid impair heart rate
Verified at single cell with microelectrodes
Gradient of diastolic depolarisation gradient decreased
No hyperpolarising pulses to investigate funny current
- Capel & Terrar 2015
Isolated guineae pig san cells
Bapta to pipette in voltage glamp
Rapid cessation of pacemaker activation
Concurrent loading with fluo5f
Showed oscillatory calcium transients until bapta addition
- Van Oort 2010
Knock-in mice of constituively phosphorylated variant of RyR2
Mice killed, myocytes isolated, fluorescent calcium indicator
Increased prevalence of calcium sparks
However, debated by other studies
Probably differences in the amino acid residues used
- Gyorke 1998
Lipid bilayer with canine sr microsomes
Varied calcium concentration of luminal and cytosolic side of membrane
Single channel recordings
Open probability increased by higher luminal calcium
Membrane potential across bilayer not physiological
- Gyorke 2004
Lipid bilayer with purified RyR2
Imprevious to luminal calcium
Added casq, triadin 1 and junctin to purified receptors
Able to recapitulate the results in sr microsomes
Sr calcium overload in heart failure, thus important to understand calcium sensing
- Zima 2010
Calcium sparks are insufficient to account for all the calcium leak seen from the sr at physiological concentrations of sr calcium
Isolated ventricular myocytes loaded with Fluo-5N
Observed the changes in sr [ca2+] relative to the spark frequency, using confocal microscopy
Inactivation of SERCA by thapsigagrin caused the sr [ca2+] to fall, along with spark frequency
Total attenuation of spark frequency at 279um sr [ca2+] the calcium leak remained, albeit at a slower rate
- Fowler 2018
Confocal microscopy with improved spatiotemporal resolution
Late calcium release events during rabbit VM action potential
Surprising as channels thought to be in refractory period
Hypothesised this was responsible for action potential prolongation
- Fowler 2020
Myocytes from rabbit models of HFrEF after LAD ligation
Increased frequency of late calcium events
Current clamp showed AP prolongation
Appears to show late calcium sparks contributing to EADs
Lad not representative of broad clinical spectrum
- Bogeholz 2016
Ncx over-expressing mice
Subjected isolated atrial mycoytes to pacing
Patch clamp showed no increase in DADs
But number of action potentials triggered by DADs increased nearly 20fold
- Hove-Madsen 2004
Excised atrial appendage from AF patients
Fluo-3am and confocal
Frequency of calcium sparks higher in AF
Maybe phosphorylation of RyR2
- Vest 2005
Phosphorylation specific antibodies for RyR2 immunoblots
From canine models of AF
Hyperphosphorylatiojn of RyR2 in canine model
Doesn’t replicate cardiac remodelling in human AF
- Voigt 2012
Atrial samples from 70 AF and matched controls
Calcium leak during diastole increased
More calcium sparks in fluo4am
Dads visible on current clamps
Right atrium may not be site of ectopic activity
- 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
- Hilliard 2010
Flecainide thought to block vgscs
Wistar rats with casq2-/- model of CPVT
Flecainide reduced amplitude + intensity of calcium sparks in CPVT
Prevented arrythmogenic 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
- Marx 2000
Ryr2 phosphorylation with antibodies specific to phosphorylation
Radio-actively labelled ATP and anti-phosphoserine immunoblots
Failing heats from human cardiac transplants and canine failing hearts by rapid pacing
Upregulation of RyR2 phosphorylation
Canine samples single channel recordings by incoporating purified RyR2 into lipid bilayer
Open probability significantly increased
Was the RyR2 whole apparatus transferred to bilayer
- Shan 2010
Ryr2 knockin in hyperphosphorylated
Mimicked chronic PKA with isoporterenol caused reduded ejection fraction
Channels isolated and single channel lipid bilayers showed greater open probability
After LAD ligation mice more disposed to vt and death
Could have studied calcium transients in myocytes isolated from canine models
- Oahara et al in 2002
LAD in dogs is capable of pre-disposing to atrial fibrillation
Compared the responses of control and MI dogs to rapid atrial pacing
Observed an increased vulnerability to prolonged atrial fibrillation in the MI dog
Limited by difficulty in establishing consistent ischaemia in dogs due to coronary collateralisation
- Schwarzl 2016
Porcine model of early AF
Radio controlled pacemaker in right atrial wall
600bpm pacing for 6 weeks
Animals in paced group succumbed to sustained AF for longer than a 60-minute period
100% success rate
- Brunner 2008
Rabbit model of lqt1 and lqt2
Insertion of human mutant KCNQ1 and KCNH2 respectively into rabbit heart expression under a rabbit myosin heavy chain promoter prior to injection into embryos
Survival of the lqt2 model was significantly reduced by greater than 50% at one year
Correlated with the development of spontaneous polymorphic ventricular tachycardia
Current clamp conditions showed that the average action potential was significantly longer in knockout rabbits
Current densities in voltage clamp conditions of Iks and Ikr were both significantly reduced in both models.
Non-physiological overexpression or ectopic expression patterns
