Autonomic regulation of cardiac function

Question 1

Describe the properties of the parasympathetic innervation to the heart

Answer 1

Parasympathetic (acetylcholine)

• Ganglia near the SA node and AV node
• Ventricle not really innervated
• Vagal effects are brief with short latency – ACh is quickly broken down by acetylcholinesterases and there is no 2nd messenger so short latency
• Mostly mediated through rate (chronotropy)

Question 2

Describe the properties of the sympathetic innervation to the heart

Answer 2

Sympathetic (Noradrenaline)

• Extensive innervation throughout all chambers of heart – pass over epicardial surface and follow circulation
• Time-course of effects slower (not beat-to-beat) – NA taken back up in nerve terminals which takes longer and relies on 2nd messenger
• Circulating adrenaline contributes to sympathetic effects – A for beta-adrenoreceptors and NA for alpha-adrenoreceptors

Question 3

What does acute sympathetic stimulation occur through and what are the effects?

Answer 3

Mediated through B1-adrenoreceptor (AR) with B2 and A involved in disease.
Stimulation of B1 AR leads to increases in:
- Rate (chronotropy)
- Force (inotropy)
- Relaxation (Lusitropy)

Question 4

Describe the mechanism of B1 AR stimulation

Both the direct and indirect pathways

Answer 4

B1 AR stimulation increases cAMP which has direct and indirect effects

• Noradrenaline released from nerve terminals, or circulating adrenaline from the adrenal gland, binds to B1 AR –dissociation of alpha subunit of stimulatory G protein (Gs)
• Gs activates adenyl cyclase which produces cAMP from ATP
• cAMP directly binds to HCN channels
• cAMP indirectly stimulates uncoupling of catalytic sub-units of PKA and goes on to phosphorylate targets

Question 5

What are the chronotropic effects of B1 AR on HCN channel

Answer 5

• β 1 AR stimulation shifts activation curve: increased inward current at any Em
• Increases rate of phase 4 depolarisation in SA node
• Via direct cAMP binding to the channel

Question 6

Give the evidence for the modulation of HCN channels in B1 AR stim

Answer 6

DiFrancesco et al., 1986
Isolated SAN cells from rabbit hearts were studied in the whole cell configuration and voltage clamp technique used to study the Em dependence of the If activation.
B-adrenergic agonist, e.g. isoproterenol, shifts the activation curve to more depolarised potentials resulting in a more rapid diastolic depolarisation and thereby heart rate.
This was found to be directly due to cAMP activating with the channels at their cytoplasmic side.
This provides rapid and efficient mode of channel control that doesn’t require metabolic input.

Question 7

Give the evidence for b1 AR stim modulating the Ca clock hypothesis

Answer 7

Lakatta et al., 2008
Each spontaneous cycle of the SANC LCR clock can be envisioned to initiate the occurrence of an AP via INCX activation. Ca2+ influx via ICaL during AP triggers CICR.

Vinogradova et al., 2004
Confocal linescan images revealed persistent LCRs in cells acutely voltage-clamped at the maximum diastolic potential.
In spontaneously beating cells, in both transient and steady states, LCR period was highly correlated with the spontaneous cycle length.
Diastolic LCRs reflect rhythmic intracellular Ca2+ cycling that does not require the concomitant membrane depolarization.

Question 8

Give the evidence for inotropic effect on L type calcium channel - what amino acid?
Describe the alternative phosphorylation target found

Answer 8

Yuan & Bers, 1995
shows the increase in Ca current seen following stimulation with forskolin, a direct activator of AC, which is absent when PKA is inhibited using the drug H-89.
PKA phosphorylation will increase Ca influx (trigger for CICR) and SR Ca load which will increase the size of the Ca transient.
For many years it was accepted that PKA regulation of the L_type Ca channel was mediated through phosphorylation at Ser1928 in the pore-forming alpha1C subunit of the channel.

Minobe et al. (2014) identified a new phosphorylation site in baby hamster kidney (BHK) cells expressing ʟ-type calcium channel.
Ser(1574) channel modulation.

Although future studies are necessary to confirm this idea in situ as BHK cells are not an honest depiction of in vivo studies. It is possible that there may be multiple phosphorylation sites or species dependent changes.

Question 9

Give the evidence for ionotropic effects of B1 AR in PLB

Answer 9

Wegener et al., 1989
Localized the sites of PʟB phosphorylation in intact guinea pig ventricles
B1 agonist isoproterenol, found to be cAMP-dependent PKA phosphorylation at Serine 16. Two major tryptic phosphopeptides containing greater than 90% of the radioactivity were obtained from PʟB- serine 16 and threonine 17.

Question 10

Give the evidence for the B1AR stimulation of RyR - inotropic

Answer 10

Marks et al. (2000)
PKA-mediated phosphorylation of RyR increase RyR Ca2+-mediated open probability. At a single serine, Ser2808.
Not universally accepted due to inability to confirm components and the contradiction between a sustained increase in contractility despite autoregulation having an effect.
MacDonnell et al., 2008
Used a genetically modified mouse in which S2808A to prevent phosphorylation at this site.
Isoproterenol caused significant increases in cardiac function, both in vivo and in isolated hearts, and there were no differences in these contractile effects in wild-type and S2808A hearts.
These results show Ser2808 does not have a major role in sympathetic nervous system regulation and that PKA mediated increases in the ICaL and PʟB

Question 11

Describe the lusitropic effects of B1 AR stimulation on PLB and Troponin I

Answer 11

Increased SERCA2 activity increases rate of Ca removal

Phosphorylation of TnI decreases TnC affinity for Ca. Increased rate of Ca dissociation from myofilaments

Question 12

Give the evidence for the lusitropic effects of PLB and TnI

Answer 12

Li et al., 2000

Measured relaxation rates before and after maximal Iso treatment for amplitudes in ventricular myocytes and muscle from wild-type (WT) mice and mice which the PLB gene was knocked out (PLB-KO).
In KO mice, isoproterenol had no effect on myocyte relaxation despite increase in TnI phosphorylation.
This suggests that most of the lusitropic effect is due to phospholamban phosphorylation

Question 13

What effect does the parasympathetic system have on the heart?

Answer 13

Parasympathetic Stimulation
Effects mediated through M2 receptor which decreases:
- Rate
- Force
M2 receptor stimulation has effects via Gβγ and cAMP (direct and PKA)

Question 14

Describe the mechanism of indirect effects of parasympathetci stimulation

Answer 14

Indirect
Gi protein is activated – alpha subunit can go on to inhibit adenyl cyclase activity – reduces cAMP and activation of channels gated by cAMP like HCN channels and inhibits PKA by causing reassociation of catalytic subunits.

Question 15

Describe the mechanism of direct effects of parasympathetic stimulation

Answer 15

Direct

Beta & gamma subunit – which activates the KIR channel – mediated without the need of a 2nd messenger so faster

Question 16

Give the evidence for the chronotropic effects of M2 stimulation on If

Answer 16

DiFrancesco et al., 1986
Showed that M2 receptor stimulation by adding acetylcholine into SAN rabbit cells which shifts activation curve left: decreased inward current at any Em
Decreases rate of phase 4 depolarisation via direct unbinding of cAMP

Question 17

Describe the chronotropic effects of M2 stimulation of Ikach

Answer 17

• ¬ACh-activated K channel is activated by beta-gamma subunit of the GPCR
• Strong inwardly rectifying current which stabilises Em near Ek by opposing inward current during phase 4 – prominent in SAN and AVN
• Centrally important in slowing the heart rate to increase the threshold of inward current needed for AP activation

Question 18

Give the evidence for the chronotropic effects of M2 stimulation of Ikach

Answer 18

Wickman et al., 1998
Assessed the role of IKACh in heart rate regulation in vivo
Generated a mouse line deficient in IKACh by targeted disruption of the gene coding for GIRK4, one of the channel subunits
A diminished bradycardic response in the KO mice found that IKACh mediated approximately half of the negative chronotropic effects of vagal stimulation and is necessary for the fast fluctuations in heart rate responsible for beat-to-beat control of heart activity, both at rest and after vagal stimulation.
Surprisingly, resting mean heart rate did not differ between knockout and wild-type mice which indicates that other signalling pathways involved in heart rate regulation can balance the missing IKACh current

Question 19

Give the ionotropic effects of M2 stimulation on Ical

and give the evidence for it

Answer 19

Wang & Lipsius, 1995
Perforated patch whole-cell recording method in cat atrial myocytes.
ACh decreased basal ICa,L. Within approximately 30 s of returning to ACh-free solution, basal ICa,L exhibited a rebound increase above the control level.
There is an inhibition of AC immediately which reduces the current initially but the conclusion is that atrial rebound responses involve a delayed facilitation of a cAMP-dependent mechanism is supported by the fact that ACh-induced rebound stimulation of Ca2+ channel activity is blocked by inhibitors of PKA.

Question 20

What change in autonomic innervation in ʜFʔ

At myocyte level what does this meanʔ

Answer 20

Heart failure (HF) is associated with imbalance between sympathetic () and parasympathetic () tone

At the myocyte level, chronotropic, inotropic and lusitropic responses to β1 stimulation (e.g. exercise) are reduced