Electrical Activity in the SAN

Question 1

What role does electrical activity have in the heart?

Answer 1

• Electrical activity is the trigger and co-ordinator of contractile activity in the heart.

Question 2

What happens in the absence of rhythmical activity?

Answer 2

• In the absence of rhythmic electrical activity, the heart cannot pump blood effectively and death results.

Question 3

How can electrical activity be measured?

Answer 3

• The electrical activity can be measured from the whole heart using an electrocardiogram, which measures the movement of a depolarising wave front across the heart (vector) relative to detection sites on the chest on the body surface
• Single cells produce action potentials which can be measured

Question 4

What is the heterogeneity of electrical activity?

Answer 4

• The electrical activity in the heart is heterogenous, meaning each area of the heart has slightly different action potential shapes and durations

Question 5

How are neighbouring cells connected?

Answer 5

• The neighbouring cardiac myocytes contact each other by intercalated discs and gap junctions, primarily connexin, ensure rapid conduction and for the heart to behave as a functional syncytium

Question 6

Which cells have an ability to pace?

Answer 6

• The cells with this ability are:
   Sinoatrial Node
   Atrioventricular Node
   Purkinje Fibres

Question 7

What properties to pacemaker cells have?

Answer 7

an intrinsic, spontaneous time dependent depolarisation of the cell membrane leading to an action potential

Question 8

What is the primary pacemaker and what stimulation is it constantly under?
What is the frequency bpm?

Answer 8

• The SAN is the primary pacemaker and is under constant vagal stimulation which supress its intrinsic frequency to approx. 60 bpm
• Failure of the SAN leads to sick sinus syndrome

Question 9

Properties of the SAN electrical activity
MDP
DD
Regulation

Answer 9

• The maximum diastolic potential of -60mV in SAN cells is less negative than the ventricle
• Pacemaker cells have an intrinsic diastolic depolarisation (DD)
  Regulation
• The autonomic nervous system (NA and ACh)
• Endocrine Function (Adrenaline)
• Mechanical Stimulation (Stretch)

Question 10

Give the currents responsible for phases of SAN AP 0-4

Answer 10

• Phase 0
  o Upstroke slow from -40mV to +15mV
  o INa is absent and phase 0 is due to ICaL and ICaT
• Phase 1
  o Absent in SAN
• Phase 2
  o Short Plateau maintained by ICaL and INCX (inward currents)
• Phase 3
  o Repolarisation due to activation of IK (outward)
• Phase 4
  o Diastolic Depolarisation, rate determined by intrinsic frequency
• MDP of -60mV due to lack of IK1

Question 11

What two theories are there about the pacemaker current?

Answer 11

1) Hyperpolarisation-activated cyclic nucleotide gated (HCN) channels - funny channel (DiFrancesco)
2) Calcium clock channel (Lakatta)

Question 12

Who found the properties of the funny current?

And what did they find? - 1

Answer 12

• Yanighara & Irisawa, 1980
  o Little specificity to any particular ion, and its reversal potential was −25 mV
  o began to activate at −50 mV and fully saturated at about −100 mV
  o This current is more inward at greater negative potentials, and not voltage dependent within pacemaker potential range of the SAN

Question 13

What ions does the funny current carry?

Answer 13

• Using the Nernst Equation, the funny current corresponds to an inward flux of Na and K ions – non-specific cation current

Question 14

Is If able to be regulated?

Who found this out and what did they do? -2

Answer 14

• Regulation of funny current was found that the autonomic products were able to shift the activation curves, providing evidence that the funny current may be the pacemaker current
  o DiFrancesco et al., 1986
   Single cell voltage clamp on rabbit SAN cells showed that adrenaline and noradrenaline increased the funny current and accelerated its activation at more negative voltages
  o Similarly, ACh was found to inhibit the current and prolong the rate of current flowing which reduced pacing – supporting the idea of the funny current controlling heart rate in pacemaker cells

Question 15

What was the further support of the regulation of If in pacemaker currents? - 3

Answer 15

o Bucchi et al., 2007
 Used rabbit SAN again and found the selective If blocked ivabradine at 3uM was able to slow rate of depolarisation and also the steepness of early DD
 They also examined the effects of isopranaline and ACh to modify the current, this affected the EDD only and not the take off potential associated with SR Ca2+ transients and the calcium clock.

Question 16

What mutations were found to provide further evidence about the HCN channels? - 4

Answer 16

o Milanesi et al., 2006
 A family with a hereditary form of asymptomatic bradycardia associated with a mutation in the pacemaker-channel α-subunit HCN4 - S672R
 Wild-type and mutant human HCN4 channel cDNA was transfected into HEK293 cells
 Found that S672R mutation shifts the current activation range to hyperpolarized voltages and slowing current deactivation; these changes mimic those caused by a low concentration (10 to 30 nM) of acetylcholine

Question 17

What effect did training have on If? -5

Answer 17

o D’Souza et al., 2014
 Found that exercise training reduces resting heart rate via downregulation of the funny channel HCN4
 The cycle length was significantly longer and the rate was significantly slower in the trained mice and also in the rat
 There was no evidence of an increase in vagal tone following training in mice.
 Tissue samples (1 mm) were taken from the sinus node (and neighbouring right atrial muscle) of sedentary and trained rats. qPCR measuring the amount of mRNA of many channels showed a downregulation of mRNA for HCN and protein levels of HCN4 assessed by immunohistochemistry where there was a significant decrease

Question 18

What channel is involved in the process of the calcium clock hypothesis?
Role?
Net movement?
What causes depolarisation of channel?

Answer 18

• The channel responsible for this theory is the sodium calcium exchanger (NCX)
• Role is to remove Ca2+ from the cytoplasm.
• Exchanges 3Na+ for 1 Ca2+ , there is a net movement of ions/charge in the direction that Na+ moves
• When Ca2+ efflux is favoured (1Ca2+ out 3Na+ in) = inward (depolarising) current
• Regular, local, release of Ca2+ from the SR into the sub-membrane space cause depolarising INCX - creating DD.

Question 19

Describe this process of calcium clock hypothesis

Answer 19

1) Release of Ca2+ from small clusters of RyR receptors in SR increases Ca2+ locally and causes neighbouring release
2) Ca2+ efflux favoured by NCX
3) DD
4) Em reaches threshold for Ica and phase 0 depolarisation occurs

Question 20

What key point backs up this hypothesis?

Answer 20

• A key point of the Ca2+-clock theory is that during DD, INCX is increasing while If is decreasing suggesting a greater role for INCX

Question 21

What did Lakatta say about the theory and its regulation? - 1

Answer 21

• Lakatta et al., 2010
  o Localized subsarcolemmal Ca2releases are generated by the sarcoplasmic reticulum via ryanodine receptors during late diastolic depolarization and are referred to as an intracellular “Ca2+clock,” because their spontaneous occurrence is periodic during voltage clamp
  o GPCR signaling ensures pacemaker flexibility, effecting rate regulation by impacting on these very same factors that regulate coupled-clock Ca2cycling to guarantee basal state pacemaker stability and robustness

Question 22

What evidence shows that calcium clock may be more stronger in DD? - 2

Answer 22

• Lakatta & Maltsev, 2012
  o The INCX is larger in amplitude and activates more rapidly than If
  o When the coupled clock system is impaired and If intact, sympathetic stimulation does not change rate as much as in controls indicating If is not sufficient for rate acceleration

Question 23

There was a paper by Gao et al. (2013) which suggested NCX was not needed. Who counter-argued this statement and what was concluded? -3

Answer 23

• Maltsev et al., 2013
  o A recent study published by Gao et al. (2013) used NCX1-KO to explore the role of the exchanger in cardiac pacemaker cells. They deemed that NCX is not needed for resting heart rate.
  o The authors did not simulate the NCX current under physiological conditions in the incomplete KO SAN cells
  o We found that minimum diastolic Ca2+ levels and INCX amplitudes generated by remaining NCX molecules (only 20% of control) remained almost unchanged.
  o They concluded that the remaining NCX molecules in the incomplete KO model likely produce almost the same diastolic INCX as in wild-type cells