Theme 2: Cardiac Electricity and Arrhythmia Flashcards
Define voltage.
- Potential difference between two points.
- Work done in moving a unit of positive charge from a lower potential to a higher potential. It takes 1 joule to move 1 coulomb of charge across a potential difference of 1 volt.
What is the typical resting membrane potential of an electrically excitable cell?
-70mV
Define current.
- Rate of flow of charge.
- The convention is that current flows from positive to negative.
- Basic unit is an amp (symbol A). 1 amp = 1 coulomb/second.
What magnitude currents are seen through cell membranes and through single channels?
- Cell membranes can pass currents of nA-uA.
- Single channels pass currents in the pA range.
What is Ohm’s law and how else can it be written?
V = IR
Can also be written as:
I = Vg (where g = 1/R)
What are the units for conductance?
Siemens (S) but ohm-1 and mho also used.
What is a voltage clamp?
A device that controls the potential difference (voltage) between two points by passing a current between those two points.
What does a steeper line on an I-V plot indicate?
A steeper line indicates greater conductance.
What is the equilibrium potential in a cell?
- Diffusion of X+ through a channel is driven by both the concentration gradient of X+ and the electrical potential difference across the membrane.
- The potential at which the electrical driving force is equal in magnitude but opposite in direction to the chemical driving force is known as the equilibrium potential (EX ) (as there is no net flow of X+).
How is a cell membrane represented in an electric diagram?
- The ion channel is drawn as a resistor
- The ion gradient is drawn as a battery with an emf equivalent to the equilibrium potential
Draw an electric circuit for a voltage clamp of a membrane channel.
The ion channel is the resistor and the ion gradient is the battery with emf E.
Describe how a voltage clamp holds voltage at a constant level.
- When no current is applied by the voltage clamp, the voltage is constant at E (the potential difference created by the ion gradient) and there is no current through the channel
- Positive current is required to clamp voltage positive to E and negative current to clamp voltage negative to E.
- Thus the direction of current flow reverses when V=E, this is called the reversal potential.
- The steepness of the slope of the I-V relation reflects the conductance of the ion channel. The magnitude of current flow through this circuit depends upon the difference between V (actual voltage) and E (the reversal potential), such that: I = g(V-E).
For a channel permeable to only one ion, what is the equation for the reversal potential?
The Nernst potential:
Ex = (RT/zF) x ln([X]o/[X]i)
What is the problem with this voltage clamp model?
- It assumes that the ion channel acts as a fixed value resistor
- In reality, real ion channels often show changes in conductance with voltage. This property is called “rectification”.
What effect does rectification have on I/V plots for an ion channel?
- The current is the product of the driving force and the conductance
- From this, the I/V plot can be deduced
Draw the design of a microelectrode used in electrophysiology studies.
What are the two types of microelectrode used in electrophysiology studies?
- Sharp electrodes = <0.5um tip
- Patch electrodes = 1-2um tip
How are sharp electrodes used in electrophysiology? What are the issues?
- Sharp electrodes are impaled into cells.
- Impaling can damage the membrane leading to current leak and membrane depolarisation. The smaller the cell the sharper the electrode needs to be.
- Sharp electrodes have high resistance and can develop unstable voltage offsets (tip potentials).
- High resistance electrodes are also unsuitable for voltage clamp.
How are patch electrodes used in electrophysiology? What are the issues?
- Large tip diameter >= 1um. Tip often smoothed by fire polishing.
- Electrode is placed against cell membrane and gentle suction applied. This pulls a patch of membrane into the electrode which, with luck, will adhere to the glass generating a seal with very high resistance (1-40 Gohm).
- Further suction is then used to break the patch of membrane allowing access to the inside of the cell.
- High seal resistances allow this technique to be used with the smallest of cells.
- The inside of the pipette and the inside of the cell can mix easily so the electrode must be filled with the right solution.
The large tip diameter of patch electrodes used in whole cell recording means that there is rapid exchange between the contents of the cell and the contents of the electrode. How is this problem overcome?
Patch pipette filling solutions are used that must be compatible with the intracellular environment:
- They must be isotonic otherwise the cell will either shrink (hypotonic) or swell & burst (hypertonic).
- Ionic concentrations should be the same, or similar to those inside the cell. e.g. high (140 mm) K+ , low Na+ (10 mM), low Ca2+ (e.g. 100 nM buffered with EGTA or similar), pH 7.2 (buffered with HEPES). Cl- should also be similar to that found in cells but is often much higher.
- ATP is also often included.
What is perforated patch whole cell recording?
- In perforated patch recording, the electrode filling solution contains a pore forming molecule, usually either nystatin or amphotericin.
- The electrode is sealed to the membrane to form a cell attached patch.
- The pore forming molecule then inserts itself into the patch of membrane under the electrode tip.
- The resulting pores are permeable to both K+ & Na+ (and some other small monovalent cations) but nothing else.
- These pores thus allow current flow between the electrode and the cell interior whilst keeping all other cellular constituents inside the cell. This means that voltage clamping can be done without disturbing the cell’s function.
How does a voltage-clamp work? Draw out the theory.
Define inwards and outwards current.
Inwards (- current):
- Positively-charged ions flowing in to cell OR
- Negatively-charged ions flowing out
- Depolarises cell
Outwards (+ current):
- Negatively-charged ions flowing in to cell OR
- Positively-charged ions flowing out
- Repolarises cell
Define inward and outward rectification.
- Inward rectification is when a channel becomes permeable so as to facilitate inward currents
- Outward rectification is when a channel becomes permeable so as to facilitate outward currents
Describe how data from a voltage clamp experiment is presented.
- A baseline voltage is decided, at which the cell is held
- The voltage is then changed to a series of values in turn
- At each voltage, the current resulting from this change is recorded
- Because these are very fast events, they are superimposed on a graph with the same time base
In voltage clamp experiments, the currents produced may be the result of multiple ion currents. How can the overall current be separated into the individual ionic currents?
- Each ion current can be eliminated using a substitution:
- To eliminate Na+-currents replace Na+ with N-methyl-d-glucamine (NMDG) or choline.
- To eliminate Ca2+ currents remove Ca2+ and add a chelator e.g. EGTA
- To eliminate K+ currents replace K+ with Cs+
- If you substitute the substituted line from the overall line, you have a graph for the current of only that one ion
- Alternatively, you can also use pharmacology to block certain types of channels (e.g. TTX)
How can the relationship between voltage and conductance (i.e. channel activation) be calculated in a voltage clamp experiment?
- The equation I = (Vclamp-Ereversal) x g can be rearranged to give:
- g = I / (Vclamp-Erev)
- The conductance can then be plotted against voltage
How can the time-dependent inactivation of a channel be studied in a voltage clamp experiment?
- A voltage step can be applied to a channel
- At each voltage, the current (or conductance) is measured over time
- The rate at which inactivation occurs can be determined by fitting an exponential function to the declining phase of the current (or conductance).
- The exponential constant derived from this fit is called a time constant and is a measure of the speed of inactivation (long time constant = slow; short time constant = fast).
How can the voltage-dependent inactivation of a channel be studied in a voltage clamp experiment?
- The voltage is clamped at a holding potential
- It is then increased to a pre-pulse potential (this is the independent variable) -> This may inactivate some of the channels
- Then a test pulse occurs, which is a more depolarised potential which is of sufficient amplitude to fully activate the current
- The process is then repeated several times using increasingly more positive pre-pulse potentials and the peak current recorded during the test pulse plotted as a function of the pre-pulse voltage.
- In the diagram below, you can see that the peak current is higher after the pre-pulse at more negative voltages. The graph shows how the relative peak height changes with voltage.
Describe how voltage clamp data about channel activation and inactivation can be combined.
- Graphs of channel activation and inactivation agaisnt voltage can be combined on the same axes
- This shows the proportion of channels that are activated and inactivated at any given voltage
- In some cases, there may be an area of overlap, known as a window current -> This is a situation where there may be a sustained current because channels can go through a cycle of being activated, inactivated and then activated again
How can the time dependence of recovery from inactivation of a channel be studied in a voltage clamp experiment?
- A double pulse protocol is used.
- The first pulse must be of sufficient amplitude and duration to fully inactivate the channel.
- A second pulse then follows after a variable time delay and the amplitude of the current evoked by the second pulse is then plotted against the time delay between the first & second pulse.
Summarise what a patch clamp is.
It’s a type of voltage clamp where you use a patch electrode to isolate a small patch of membrane with only 1 or a few ion channels.
What are the 4 configurations of a patch clamp?
- Cell attached
- Inside out
- Outside out
- Perforated vesicle
Write an equation for the total membrane current generated by any single type of ion channel.
Write the basics of single channel open/close kinetics.
Describe how single channel open/close kinetics are studied.
- Studying the length of time a channel spends in the open or closed state (dwell times) can yield important information about channel kinetics.
- Measuring the duration of open, or closed, events requires the use of two thresholds set at sufficiently different current amplitudes to prevent noise in the recording from being mistaken for a real transition between open & closed states.
- Where there is only one kinetic state corresponding to the open (or closed) state the frequency distribution of dwell times can be described by a single exponential function.
- Where there are multiple kinetic states, as is often the case with closed states, the frequency distribution of closed times has several exponential components. These are best revealed by plotting the data using a logarithmic time base (and logarithmic time bins). Distinct kinetic states are then revealed as multiple peaks in the frequency distribution.
What are the 3 reasons why we have action potentials in the heart?
- Generating rhythm (i.e. pacemaker)
- Transmission across electrical synapses
- Coupling onto second messenger cascades (i.e. EC coupling)
Draw the 3 main types of action potential in the heart and describe what makes them different.
What factors can be changed in order to trigger membrane currents?
- A current can be triggered by changing either the driving force or the conductance
- The conductance is the easier factor to change
- It can be changed by changing the channel expression and targeting, pore permeability and selectivity, time, ligands and Vm
Draw the currents underpinning a ventricular cardiac action potential.
By convention, inward current has a _____ sign.
Negative
Describe the currents that make up the ventricular action potential.
- The action potential is a combination of sodium, calcium and potassium currents.
- However, these all have different temporal patterns.
Compare the human and mouse ventricular action potentials.
- The mouse action potential is similar but has different currents.
- It is also much faster since the mouse heart beats much faster than the human heart.
- These differences are important to consider in animal studies.
For each of the currents that make up the ventricular action potential, name the clone (i.e. the gene that encodes the channel for that current).
Describe how a heterologous expression system works for study of membrane channels.
- DNA from a cell is extracted and inserted into the model cell (e.g. a xenopus oocyte)
- This DNA is eventually expressed and the channel is expressed on the surface of the cell
- This means it can now be studied using patch clamp techniques
What are some limitations of heterologous expression system for study of membrane channels?
- Are the modulatory influences present? (e.g. does phosphorylation happen in the model cell?)
- Are the channels expressed in the right environment?
- Are the channels anchored correctly?
- Are all relevant accessory sub-units also included? (e.g. beta subunits)
What channel is involved in holding the membrane potential constant between ventricular action potentials?
Inward rectifier potassium channels (3 types)
What is the role of inward rectifier potassium channels in ventricular myocytes?
- The inward rectifier potassium channels have a very large conductance around the resting membrane potential
- This means that, at more negative voltages they produce a large inward current, while at more positive voltages they produce a large outward current -> This serves to very rapidly return the membrane to the resting membrane potential when there are small unwanted disturbances (i.e. noise)
- However, at much more positive voltages, the conductance falls and the channels are blocked, which is important in allowing action potentials to be fired
What channels are involved in the rapid depolarisation during a ventricular action potential? What can this be blocked by?
- Voltage-gated sodium channels
- Can be blocked by TTX and local anaesthetics
How can the location of the voltage sensor in voltage-sensitive sodium channels be studied?
Voltage-clamp fluorimetry:
- A single leucine is replaced with a cysteine
- This cysteine is reactive and can be tagged with rhodamine, which is fluorescent
- If the cysteine is in the voltage sensor, depolarisation drags the rhodamine into the membrane, so that the fluorescence is reduced
- The location of the cysteine can be changed to see where the voltage sensor is
Give some experimental evidence for how inactivation of voltage-gated sodium channels works. How is this clinically relevant?
(Armstrong, 1973):
- Administered pronase
- This cuts the inactivation loop in the ball and chain model of inactivation and prevents inactivation of the voltage-gated sodium channels
In long QT syndrome (LQT3), there is a mutation in the linker between the 3rd and 4th domain, which means that sodium channel inactivation does not fully happen and there is a persistent sodium current that elongates the QT interval.
Describe the different currents that make up the plateau and repolarisation phase of the ventricular action potential. What is the role of each?
- L-type calcium current -> Enables calcium entry for contraction
- Transient outwards potassium current (ITO) -> Allows a very small repolarisation right before the plateau phase, which ensures a sufficient driving force for calcium entry.
- Ultra-rapid potassium current (IKur) -> Enables repolarisation
- hERG current (potassium) -> Accelerates repolarisation right at the end of the action potential
What can the L-type calcium current be blocked by?
Verapamil
Draw when the L-type calcium current occurs in the ventricular action potential.
Draw when the transient outward potassium current (ITO) occurs in the ventricular action potential. What is its function?
- It occurs right after full depolarisation
- It allows some slight repolarisation (after which the channels close and inactivate)
- This repolarisation increases the driving force for the calcium current, so more calcium can enter
Give some experimental evidence for the importance of the transient outward potassium current (ITO) in the ventricular action potential.
(Cooper, 2010):
- Studied the importance of ITO by removing the notch in the action potential that it creates
- Did this by voltage clamping rat ventricular myocytes with human action potentials and recording intracellular Ca2+ with fluorescent dyes.
- Loss of the notch resulted in about a 50% reduction in the initial phase of the Ca2+ transient due to reduced ability of the L-type Ca2+ channel to trigger release.
- There was also desynchronisation of myocyte contraction.
- Loss of the notch is observed in heart failure, so this is a possible mechanism.
Draw when the ultra-rapid potassium current (IKur) occurs in the ventricular action potential. What is its function?
- It is activated during the plateau and continues until repolarisation
- It enables slow repolarisation, which is important for calcium entry
What can the ultra-rapid potassium current (IKur) be blocked by?
Quinidine
Draw when the hERG potassium current occurs in the ventricular action potential. What is its function?
- The hERG current is small throughout the plateau and then becomes very large right at the end of the action potential
- This enables very rapid termination of the action potential, protecting against delayed after depolarisations
- The hERG channel allows this by having a very large conductance at intermediate voltages
What is the clinical relevance of hERG channels?
- They are sensitive to extracellular [K+] -> This means that hypokalemia can lead to prolonged action potentials
- They are targets for many drugs (if this is unwanted, the drug may not be viable)
- Mutations can lead to channelopathies: e.g. LQT2
Draw how ventricular tachycardia and ventricular fibrillation appear on an ECG.
- Top = Normal
- Middle = Ventricular tachycardia
- Bottom = Ventricular fibrillation
Give some experimental evidence for what ventricular tachycardia and ventricular fibrillation are.
(Nash, 2006):
- Used an electrode sock to study the electrical activity of the heart during heart surgery (when the heart is often placed in ventricular fibrillation to allow operation)
- Recordings often showed that normal activity became ventricular tachycardia (resulting from a single reentrant circuit) and then ventricular fibrillation (when there were several reentrant circuits)
What causes ventricular tachycardia/fibrillation? How can this be studied?
Rare monogenetic disorders:
- Structural cardiomyopathies:
- Hypertrophic cardiomyopathies
- Dilated cardiomyopathies
- Arrhythmogenic right ventricular cardiomyopathies (due to desmosome proteins)
- Primary electrical disease (due to mutations in ion channels)
Genome wide association studies (GWAS):
- SNPs
Describe a model for how ventricular arrhythmias may occur. Give a reference.
(Herring, 2019):
- Suggest a probabilistic model for arrhythmogenesis, where each event must occur in sequence in order for the arrhythmia to be established
- First, a trigger is required, which can be a delayed/early afterdepolarization or abnormal automaticity
- This then requires a substrate for propagation, which could be an electrical/structural heterogeneity that is static or dynamic
- Next, a re-entry mechanism is required (or else it is just a single irregular heartbeat) -> This leads to ventricular tachycardia
- If wavebreak occurs, this is ventricular fibrillation
- The reason this model is difficult to study is because each event may be influenced by a huge number of factors
Give some experimental evidence for how delayed after depolarisations occur.
(Marban, 1986):
- Found that ouabain, isoprenaline and increased [Ca2+]o increased the likelihood of DADs, which can subsequently lead to the ectopic firing of another action potential or the formation of an arrhythmia after stimulation is stopped
- These DADs can be prevented by ryanodine or BAPTA (calcium buffer), which suggests that DADs may be generated by SR calcium overload, leading to increased release and subsequent increased activity of the NCX (which is electrogenic)
Give some experimental evidence for how early after depolarisations occur.
(Marban, 1986):
- Used drugs to induce a prolonged action potential
- These prolonged action potentials frequently had an EAD, which can lead to arrhythmias
(January, 1989):
- Used voltage-clamping to mimick an action potential -> Depolarised the cell, held it at the plateau voltage, and then repolarised around the time an EAD would occur
- At the time of the EAD, an inward current was measured
- This current was increased by a calcium channel activator, stopped by a calcium channel blocker, and unaffected by TTX
- This suggests that EADs occur when the calcium channels have time to re-activate and produce a calcium current
Describe how a substrate may allow arrhythmias to form.
- The right substrate may allow re-entry of an action potential into an area of the heart that would usually be refractory
- This can occur due to abnormal conduction velocity, refractory period or size of the circuit
- For example, a model of how this could happen in the heart is where there is an area of anatomical or functional conduction block. This causes the action potential from limb A to loop back round limb B through a zone of slow conduction or unidirectional block. This circuit can then continue.
Give experimental evidence for how a substrate may allow arrhythmias to form.
STRUCTURAL
(Arenal, 2004):
- Obtained left ventricular electroanatomic voltage maps using a catheter in patients who had had a myocardial infarction that left scar tissue
- Studied the scar tissue in more depth and found that there were often re-entrant circuits within the scar tissue that produced arrythmias such as ventricular tachycardia
- Clinically, the exit points of these circuits could be sealed off to prevent these circuits, but this is not usually successful
FUNCTIONAL
(Nash, 2006):
- The closer in time an ectopic action potential is to the normal action potential before it, the shorter its duration
- However, this effect has heterogeneity in different cardiac myocytes -> This can be illustrated an a polar map that shows that different parts of the heart are heterogenous
- This heterogeneity can result in wavefronts breaking and causing circuits around an area where there is a large different in action potential duration
(Tomek, 2019):
- Studied calcium handling at the edge of a myocardial infarct in rats by using Langendorff perfused hearts
- When the time between action potentials is shortened, alternans (alternating short and long action potential duration) begins to occur at the edge of the infarct -> This is due to differences in calcium handling which alter action potential duration
- This effect could be reversed by norepinephrine, which increased the amplitude/duration of each action potential so that the alternans is reduced
(Gardner, 2015):
- Studied the idea that sympathetic innervation of a myocardial infarct affects the susceptibility to arrhythmias
- Compared control mice heterozygous for protein tyrosine phosphatase receptor σ (PTPσ) with PTPσ knockout mice -> The KO increases innervation
- These mice were then implanted with ECG telemetry transmitters and then subjected to sham or MI surgery.
- Ten days after surgery, the mice were injected with 10 μg of the beta agonist isoproterenol (ISO), which is used to induce arrythmias.
- The KO mice (innervated) had less heterogeneity in action potential duration and were less susceptible to arrhythmias
The take home message is that any heterogeneity in the myocardium can result in arrythmias.
State the classification of anti-arrhythmic drugs.
Give some experimental evidence for the success of anti-arrhythmic drugs.
(Echt, 1991):
- Compared the effectiveness of encainide/flecainide (the first anti-arrhythmic drugs) against placebo in preventing cardiac events
- The drugs showed much higher rates of cardiac events and the trail was ended early
How can we determine which patients should receive an implantable cardioverter defibrillator (ICD)?
- In general, the patients at highest risk of sudden cardiac death are:
- Those with a prior myocardial infarction
- Survivors of cardiac arrest and VT/VF
- Those with an ejection fraction below 35%
- These therefore benefit most from an ICD
- Patients who should not be given an ICD include:
- When there is also bypass surgery
- Immediately//shortly after a heart attack
- Older patients without CHD
The high-risk groups form a small part of the population so ultimately they only make up a small percentage of total sudden cardiac deaths, but the frequency of events in the high-risk population make it worth getting an ICD.
Give experimental evidence for NPY in cardiac arrhythmias.
(Kalla, 2020):
- Studied an isolated Langendorff perfused rat heart
- Prolonged high frequency stimulation of the right stellate ganglia or left stellate ganglia caused the release of neuropeptide Y into the perfusate.
- This shows that sympathetic stimulation releases not only noradrenaline but also NPY onto the heart.
- Set up optical mapping of voltage and calcium transients.
- Even in the presence of full beta blocking, sympathetic stimulation led to larger calcium transients. This effect was blocked by Y1 receptor antagonism.
- This suggests that NPY is involved as a product of sympathetic stimulation and it leads to changes in intracellular calcium handling
- Also used a ventricular fibrillation induction protocol to induce arrhythmias.
- This required less energy during sympathetic stimulation and beta blocker compared to a just beta blocker. However, there is no difference between sympathetic stimulation and no sympathetic stimulation when there is a beta blocker and NPY blocker present.
- This suggests the importance of NPY in arrhythmias.
- Studied 78 patients shortly after myocardial infarction and found that those with high NPY had significantly higher incidence of VT and VF.
- In rats after myocardial infarction, VT and VF occur significantly more with NPY administration compared to control. This increase is blocked by a Y1R blocker.
Overall, this shows the importance of NPY as a marker of arrhythmia risk and also as a target for prevention.
Draw a flowchart to show what causes sudden cardiac death.
Provide a summary of the biophysical events triggering cardiac arrhythmias.
- Channelopathies can lead to alterations in currents that lead to abnormal action potentials:
- Long QT syndrome -> Commonly caused by minK mutations that affect IKs or by hERG mutations that affect IKr -> These prolong the action potential
- Catecholaminergic polymorphic ventricular tachycardia (CPVT) -> Caused by mutations in the RyR2 receptor on the SR, leading to a leak of calcium
- These abnormalities may be aggravated by emotional or physical stress, due to sympathetic activation -> This leads to cardiac arrhythmias
Describe what causes long QT syndrome and how it can lead to arrhythmias.
- Long QT syndrome features a prolonged action potential due to altered delayed rectifier potassium current
- This current is the sum of two currents: IKr (HERG channel) and IKs (minK). A mutation in either of these channels can lead to long QT.
- Long QT features increased calcium entry and sequestration, which means that more calcium must be removed by the NCX between action potentials.
- The NCX is electrogenic so it can lead to early after depolarisations (EADs), which can lead to arrhythmias.
- A series of the EADs can present as Torsade de Pointes.
Give some experimental evidence for how long QT syndrome can lead to arrhythmias.
(Winbo, 2021):
- Co-cultured stem cell-derived sympathetic neurons along with myocytes from long QT patients.
- Current-clamped the myocytes and stimulated the neurons with nicotine (to release noradrenaline)
- This led to increased action potential duration and signs of early after-depolarisations.
Give an example of a molecular signal that could be disrupted, leading to sudden cardiac death. Give experimental evidence.
(Chang, 2008):
- CAPON is a NOS adaptor protein -> Its role is to translocate neuronal NO around the cell
- It has previously been identified as containing one of the only SNPs shown to increase the risk of sudden cardiac death -> This is because it can lead to long QT syndrome
- Found that overexpression of CAPON in ventricular myocytes leads to shortening of the action potential, which shortens the QT interval
- This is thought to be because CAPON-mediated translocation of NO leads to decreased calcium channel activity and increased IKr
CAPON is also present in sympathetic neurons, where overexpression leads to reduced exocytosis of neurotransmitter.
Describe how changes in innervation of the heart can lead to sudden cardiac death.
- Developmental disorders can lead to uneven innervation of the heart, which causes arrhythmias.
- Scar formation after myocardial infarction may involved hyperinnervation around the scar, leading to heterogeneity in innervation and therefore arrhythmias.
- Diabetes mellitus may involved denervation of the heart, so there is no feedback from the heart. This can lead to silent ischaemia (where no angina is felt), which can lead to arrhythmias.
Why can mental stress trigger sudden cardiac death? Give experimental evidence.
- Stress leads to the release of catecholamines
- This leads to stimulation of beta adrenergic receptors on cardiac myocytes
- (Lubbe, 1992):
- Plotted VFT threshold and tissue cAMP against perfusate adrenaline
- VFT threshold decreased and tissue cAMP increased as adrenaline increased
- This effect was exacerbated by theophylline (phosphodiesterase inhibitor) and blocked by atenolol
- Beta adrenergic receptors are also found on sympathetic neurons, meaning that catecholamines can cross the blood brain barrier and stimulate the neurons to release noradrenaline onto the cardiac myocytes.
Name an alternative to ICDs as a preventative measure against sudden cardiac death. Give experimental evidence.
(Herring, 2019):
- Stellectomy (removal of the stellate ganglia) is a preventative measure
- Compared unilateral stellectomy with bilateral stellectomy
- Bilateral stellectomy led to improved survival at 12 month follow-up
- The problem with this operation is that Horner’s syndrome may occur if the surgeon is not skilled
How does the ECG appear in atrial fibrillation?
- P wave is absent and f waves are present instead
- RR intervals vary in length (irregular ventricular rhythm)
- QRS complex is normal
Draw a general model of how atrial fibrillation arises.
Atrial fibrillation arises due to a combination of triggers that induce the arrhythmia and the substrate that sustains it:
- Triggers -> Triggers include sympathetic or parasympathetic stimulation, bradycardia, atrial premature beats or tachycardia, accessory AV pathways, and acute atrial stretch. Recently identified as triggers are ectopic foci occurring in “sleeves” of atrial tissue within the pulmonary veins or vena caval junctions.
- Substrate -> These are either (1) structural or (2) electrical changes in the atria (termed remodelling).
What sort of electrical remodelling can lead to atrial fibrillation?
- A short action potential is a hallmark of AF
- This can be due to reduced influx of calcium and increased efflux of potassium during the action potential
Is atrial fibrillation progressive?
- Yes, because electrical remodelling and structural remodelling are self-reinforcing, as well as driven by AF itself.
- This is the concept that “AF begets AF”.
Give some experimental evidence for the idea that atrial fibrillation begets atrial fibrillation (i.e. that atrial fibrillation is self-reinforcing and progressive).
(Wijfells, 1995):
- Studied awake chronically instrumented goats
- Subjected the goats to a period of burst pacing (high energy stimulation) to induce atrial fibrillation
- 24 hours of burst pacing produced atrial fibrillation, but 2 weeks of burst pacing produced sustained atrial fibrillation
- This is evidence for the idea that AF begets AF
How does atrial remodelling sustain atrial fibrillation?
- AF can be sustained by either re-entry mechanisms or rapid focal ectopic firing (where firing spreads from a site, such as the pulmonary vein).
- Remodelling creates a substrate that is prone to re-entry due to:
- Shortened refractory period
- Slowed conduction
- Increased atrial size
- Paroxysmal (short-lasting) AF is usually caused by ectopic firing from the pulmonary vein, while progression to permanent AF is usually caused by remodelling of the atria to allow re-entry mechanisms
What mechanisms can lead to the ectopic firing that triggers atrial fibrillation?
- Enhanced automaticity -> When there is some slight depolarisation during diastole due to decreased IK3 and increased If
- EADs -> When there is prolonged action potential duration due to slowed repolarisation (IK), allowing channels to reactivate and depolarisation to occur
- DADs -> When there is calcium overload or RyR dysfunction, so that there is excess calcium release from the SR and the electrogenic NCX has to deal with it. This leads to depolarisation.
These situations leave the cell prone to early action potential firing and thus arrhythmias.
Give a summary of the mechanisms causing atrial fibrillation.
Give some experimental evidence for the role of oxidative stress in atrial fibrillation.
(Reilly, 2011):
- Studied how superoxide (a measure of oxidative stress) varies with duration of atrial fibrillation
- Used a goat model of pacing-induced atrial fibrillation
- Found that superoxide was increased in the left atria after 2 weeks and in both atria after 6 months
- After 2 weeks, the major source of superoxide in the left atria was NADPH oxidase
- After 6 months, the major sources of superoxide in the right atria were mitochondrial oxidase and uncoupled nitric oxide synthase, and mitochondrial oxidase in the left atria
- The superoxide can affect electrical remodelling and structural remodelling
(Zheng, 2016):
- Carried out a meta-anlysis of the use of peri-operative rosuvastatin in patients undergoing elective cardiac surgery
- Statins reduce the concentration of superoxide radicals
- Perioperative statin therapy did not prevent postoperative atrial fibrillation or perioperative myocardial damage.
- This suggests that free radicals may not contribute to atrial fibrillation as much as the previous study would suggest.
Give some experimental evidence for the role of microRNA in atrial fibrillation.
(Reilly, 2016):
- Studied the idea that miR-31 (microRNA-31) may be involved atrial fibrillation by depleting dystrophin and neuronal nitric oxide synthase.
- miR-31 leads to decay of the mRNA that encodes nNOS. It also represses dystrophin translation, which means that nNOS subcellular localization is altered.
- In nNOS knockout mice, the probability of AF induction by atrial burst pacing and duration of the induced AF episodes was greater than in wild type mice.
- Immunostaining for nNOS and dystrophin in atrial myocytes showed that the dystrophin-nNOS complex is disrupted in atrial myocytes in AF.
Is there an interaction between atrial and ventricular function in atrial fibrillation?
Yes, atrial dysfunction can lead to ventricular dysfunction and vice versa.
Describe the main principles of treatment of atrial fibrillation.
Treatment falls into 3 categories:
- Rate control
- Rhythm control
- Stroke prevention
Describe an alternative schema for how stroke occurs in atrial fibrillation.
- Classically, strokes in AF are thought to occur after turbulent blood flow in the atria leads to thrombus formation
- However, there is now some evidence that vascular risk factors (such as endothelial dysfunction) may underlie the AF and also lead to non-AF related stroke mechanisms
Does ablation improve prognosis of atrial fibrillation? Give experimental evidence.
(Marrouche, 2018):
- Compared ablation and medical therapy in terms of probability of survival free of hospital admission, with 60 month of follow up
- Ablation was shown to be worse in terms of survival
- However, criticism of the study included:
- Underpowered and highly selected population
- Exclusion of patients and events post-randomisation
- High rate of loss to follow-up
- Small number of events
(Packer, 2019) - CABANA trial:
- Compared ablation and medical therapy in 2204 patients in terms of cardiovascular events and death, with 60 month of follow up
- Among patients with atrial fibrillation, catheter ablation, compared with medical therapy, did not significantly reduce the primary composite outcome.
So overall there is contradictory evidence about whether ablation is superior to anti-arrhythmic therapy in preventing cardiovascular events and death.
What are SAN and AVN disease that can be caused by problems such as right coronary artery ischaemia?
- SAN disease -> When the SAN is unable to consistently produce a regular sinus rhythm
- AVN disease
- Second-degree heart block, type 2 mobitz -> Where there is usually a fixed number of non-conducted P waves for every successfully conducted QRS complex (e.g. 2:1 block)
- Third-degree heart block -> Where there is complete heart block, so no SAN impulses reach the ventricles and therefore the AVN generates the rhythm for the ventricles (much slower)
Describe how a pacemaker works.
- It is implanted into the chest
- It has two leads:
- One lead runs to the SAN, in case its function needs to be corrected
- One lead runs to the ventricles, to assist with their function
Draw the currents that make up the SAN action potential.
Note that the sustained inward current is not shown here.
Give some experimental evidence for the importance of the lack of the inward rectifier potassium channels in the SAN for pacemaking.
- (Kodama, 1997) carried out block of ICaL (L-type calcium channels) by nifedipine
- (Lei, 2011) carried out block of IKr (delayed recitifer potassium channels) by E-4031
- Blocking each of these channels results in cessation of pacemaker activity
- However, the membrane potential does not stay at the most negative potential reached between beats -> This is evidence for the lack of inward rectifier potassium channels present in the SAN (since these are what typically make the resting membrane potential so negative)
- (Maike, 2002):
- Used an adeno-associated viral vector to suppress IK1 (inward rectifier potassium channels) in VENTRICULAR myocytes
- This caused them to start spontaneously contracting, similar to SAN cells
- This suggests that the lack of inward rectifier potassium channels in the SAN is important for function
What is the sustained inward current and what is its importance in the SAN for pacemaking? Give experimental evidence.
- Sustained inward current is a sodium-based current that is passed through Cav1.3 channels (along with the L-type calcium current)
- (Mangoni, 2003):
- Knockout of the Cav1.3 channel in the SAN results in a much slower cycle length, so the heart rate is reduced compared to wild-type
- Voltage clamping of the wild-type and knockout mice shows that the Cav1.3 channel enables a small inward current at the range of diastolic depolarisation
- (Toyoda, 2017):
- Compared wild-type and Cav1.3 knockout mice SAN cells
- In both the wild-type and knockout mice, lowering extracellular calcium close to 0 reduced the total current at the range of diastolic depolarisation
- However, nifedipine had no further effect in the knockout mice but it did in the wilf-type mice
- This is evidence for the idea that the sustained inward current is via Cav1.3 channels (and is therefore sensitive to nifedipine) but is not calcium-mediated
Summarise the different contributions of the NCX to the coupled clock mechanism of pacemaking.
Explanation of final point:
- When there is more than 100nm calcium in the cell, there is a baseline of NCX activity that maintains the cell slightly more depolarised than it would otherwise be
- This is important because pacemaking would not work otherwise
Summarise the different potential roles of calcium in SAN pacemaking.
Draw a diagram to summarise the coupled clock model.
The diagram shows a membrane clock and a calcium clock that are highly interlinked. Both of these clocks may also be modulated by the autonomic nervous system.
How does resting and maximum heart rate change with age?
- Intrinsic heart rate gradually falls with age -> A constant resting heart rate is maintained by a degree of parasympathetic tone that decreases with age and then eventually a degree of sympathetic tone takes over
- By switching off the parasympathetic tone and maximising sympathetic tone, a maximum heart rate can be achieved -> This decreases with age due to the falling intrinsic heart rate
What is mechano-electric coupling?
It is the idea that mechanical changes in the heart (e.g. physical stimuli) can feed back onto the electrical activity of the heart.
What is the main type of mechano-sensitive ion channel in the heart? Describe the properties.
- Cation-Nonselective Stretch-Activated Channels (SACNS)
- Since these are non-selective, they tend to show a linear I-V relationship
- This makes it easy to model their effects on membrane potential
However, there are also other types of SACs, such as SACK which carry a potassium current and have a much more negative reversal potential.
What factors determine the effects that SACs have on a cardiac action potential?
There are both SACNS and SACK present. Each will try and drag the membrane potential towards its reversal potential (SACK is much more negative), so the effect on the action potential depends on which channels are open at any given time. This is affected by:
- Stretch target (affected by environment) -> For example, ischaemia leads to favoured opening of SACK.
- Stretch timing -> The timing at which stretch happens determines what the membrane potential is at that given time and therefore how the membrane potential will change.
- Amplitude of stretch -> A high enough amplitude may lead to action potential firing.
Describe how commotio cordis may occur.
- There is a very narrow time window just before the peak of the T wave where the heart is vulnerable to stretch-induced VF
- This is because this is when the repolarising myocytes are susceptible to EADs and DADs -> This is the trigger for an arrhythmia
- The heterogeneity in the repolarisation of myocytes acts as the sustaining mechanism
- The combination of the trigger and sustaining mechanism means that an arrhythmia can arise
Describe in words the timing and space that a mechanical stimulus must occur in order to produce an arrhythmia. Give experimental evidence.
- Stretch-induced arrhythmogenesis requires supra-threshold stimulus overlapping with trailing edge of preceding excitation.
- In other words, the stretch must produce a depolarisation that catches up with the wave of repolarisation after the last action potential.
- This produces a region of functional block between the areas of depolarisation and repolarisation.
- In turn, this creates the possibility for re-entrant circuits (i.e. arrhythmias).
- Timing is very important in order for this areas of functional block to be produced and a re-entrant circuit to form.
(Quinn, 2017):
- Applied mechanical stimuli to isolated rabbit hearts during optical voltage mapping and combined this with pharmacological block of SACNS and SACK channels.
- Showed that local mechanical stimulation reliably triggers premature ventricular excitation at the contact site. This premature ventricular excitation was diminished by pharmacological block of SACNS.
- In hearts where electrocardiogram T waves involve a well-defined repolarization edge traversing the epicardium, PVEM can reliably provoke ventricular fibrillation if, and only if, the mechanical stimulation site overlaps the repolarization wave edge.
- SACK block hadno effect on PVEM inducibility, but shifts it to later time points by delaying repolarization and prolonging refractoriness.
Give a summary of mechano-electric coupling in the heart.
Draw the waves of depolarisation in sinus rhythm, ventricular tachycardia and ventricular fibrillation.
