C2. Pacemaking- experiments Flashcards
Summary
Pacemaker Activity in a Clinical Context
Membrane Clock
Calcium Clock
Coupled Clock
Dysregulation of Coupled Clock
Localisation to the SAN
- Keith & Flack 1907
Reported on the presence of a region in the sino-auricular junction responsible for the heart’s automaticity
Described the passage of electricity from this fibrous tissue to the interauricular septum
Validated early physiological studies that showed the heart’s rhythm began in the neighbourhood of the great veins
- Protze 2017
iPSCs exploited understanding of development
Observed a population of cells with pacemaker like gene expression
Increased culture efficiency with BMP4
Cells had pacemaker activity in vitro and in vivo with transplantation and assessment of ventricular ectopic beats
Survival of the grafts was limited
- Qu 2003
HCN2 as a target for gene therapy using adenoviral vectors with GFP as well
Electrodes were implanted to stimulate vagal tone and inhibit natural pacemaker
ECG recorded electrical activity in left atrium where gene therapy was directed
Isolated myocytes verified this
Only 3-4 days after cardiac injection, no longevity
- Brown, DiFrancesco & Noble 1979
Advances in whole-cell patch clamping
Administration of hyperpolarising pulses activated an inward, funny current in rabbit SAN myocytes
Replicated in Human myocytes by Verkerk 2007 confirming current sensitivity to Cs+
This patient had Paroxysmal tachycardias
- DiFrancesco & Tortora 1991
Excised patches from rabbit SAN cells, measured funny currents in response to hyperpolarising pulses
cAMP addition to internal side of patch increased funny current
Perfusion with PKA was not sufficient
cAMP shifted activation curve of If to more membrane positive values
Not studying the channels in situ
- Santoro 1998
Isolated mBCNG-1 channel and expressed in xenopus oocytes
Rapid hyperpolarising pulses elicited inward current
Showed sensitivity to Cs+ and cAMP
Similar properties to HCN
- Shi 1999
Verified HCN1 & HCN4 (later named) were selectively expressed in the SAN
Used to argue that If is fundamental to pace-making
Expression of the channels facilitating If is localised to the SAN and other neurons that exhibit pacemaker-like activity
- 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
- Rigg 2000
Isolated hearts ryanodine shifted dose-response of isoprenaline in terms of heart rate to the right
Isoprenaline increased frequency of calcium transients in Indo-1 loaded isolated SAN cells
Ryanodine attenuated these transients
Conduct again with nifedipine to show intracellular origin of calcium
- Baruscotti 2011
Floxed-HCN4 with inducible Cre under aMHC
Tamoxifen administration caused severe bradycardia shown in vivo ECG
Isolated cells with decreased DD slope in current clamp
Off-target effects of knockout
- Hoesl 2008
HCN4 promoter expressing Cre with floxed HCN4
No change in heart rate or reduction in funny current in isolated cells exposed to hyperpolarising pulses
Not inducible knockout
- Moore 1986
Spontaneously-firing bullfrog sinus venosus cells were exposed to hyperpolarising command signals
Did not generate funny current
May be explained by the developmental stage of the sinus venosus
- Himeno 2011
BAPTA-AM chelation of current clamped SAN myocytes did not reduce pacemaker activity
Failure to measure intracellular calcium to show BAPTA did inhibit calcium signals.
- Capel & Terrar 2015
Isolated Guineae Pig SAN Cells
BAPTA to Pipette in Voltage Glamp
Rapid Cessation of Pacemaker Activation
Concurrent Loading with Fluo5F
Sowed Oscillatory Calcium Transients Until BAPTA Addition
- Rigg 2003
Voltage clamped SAN myocytes loaded with rapid BAPTA chelator
Hyperpolarising stimuli less If with BAPTA chelation
W-7 a calmodulin antagonist mimics effects of calcium chelation
Multiple off target effects of W-7
- Mattick 2007
RT-PCR and Immunoblotting to show calcium-sensitive AC1 and AC8 specificially in SAN
Forskolin rescued negative chronotropy in BAPTA-loaded cells under voltage clamp conditions
Could develop small molecule isoform specific inhibitors
- Wu 2009
Alternative mechanism inhibiting CaMKII in mice with genetically encodable cardiospecific promoter
Isolated SAN cells and mice had reduced DD gradients
CaMKII inhibition did not reduce funny current suggesting alternate effects on L-or T-tpe VGCCs
- Yaniv 2014
Importance of membrane and calcium clocks in maintaining beating intervals in isolated rabbit sinoatrial node cells
Treated these cells with ivabradine to inhibit funny current, or inhibited SERCA2 with cyclopiazonic acid to disrupt the membrane clock
Voltage clamped these isolated SAN cells, and were able to show that the frequency of action potential firing was decreased, as was the variability of the action potential intervals
Voltage clamp conditions artificially disrupts the physiological interplay between membrane and calcium clocks, potentially limiting the physiological relevance of their findings
- Wirth (Unpublished)
Adenosine to isolated SAN myocytes reduced temporal correlation between LCRs and global calcium transients
Distinguished by computer learning
Superimposition of LCRs onto action potentials again showed asynchrony between intracellular calcium events and membrane depolarisation
Could examine whether adenosine induces arrhythmia in guinea pigs
- Yeh 2009
Atrial tachypacing of dogs to mimic sick sinus syndrome
After a week of tachypacing was 50% reduced expression of HCN2/4 in SAN and resulting decrease in funny current when isolated
Could compare frequency of LCRs as well to assess clock uncoupling
- Liu 2014
Reduced firings of aged rat SAN cells
Blunted response to PKA by IBMX induced PDE inhibition
Correlated with reduced LCRs in response to PDE inhibition
Measure correlation between LCR ensembles and action potentials
- D’Souza 2014
Exercise in rats by training with uphill running was sufficient to generate bradycardia
Attributable to electrophysiological remodelling of SAN cells as persisted in the presence of autonomic nervous blockade with propranolol and atropine in vivo
Patch-clamped isolated rat cells from trained and untrained animals in the whole cell configuration
Rapid hyperpolarising pulses showed reduction in whole-cell If conductance in trained mice
Examined expression of the HCN4 mRNA in sinus node cells using qPCR
Reduction in HCN4 expression in trained rats compared with sedentary correlated with reduced levels of Tbx3 and increased expression of miR1
Membrane clock dominates in causing bradycardia after training
Did not examine calcium release in trained SAN cells, which may have also been important in the response to training
- Chandler 2009
Human SAN from right atrial sample
qPCR showed expression of HCN4 but less in the right atrium and paranodal area
Also reduced SERCA2A expression in the SA node
No comparison to ventricular tissue
- Mattick 2007
Showed expression of AC1 in the SAN, but not ventricles
At both protein and mRNA levels using immunohistochemistry and RT-PCR respectively
- Coppen 1999
Immuno for Cx45, Cx40 and Cx43
Expression of Cx45 and 40 in SAN but not crista terminalis
Cx43 not in SA node
See if inducible knockout of Cx45 in SA node could attenuate electrical activity.
- Dobrzynksi 2003
Excitation contraction uncoupled easier to study AVn
Voltage sensitive dye to determine area of spontaneity
Applied caesium and observed the cessation of electrical activity
Selective expression of Cx45 again similar to SAN
Also expressed HCN4
Combination of dissection trauma and electrical may have shifted electrical activity
