Transcript Oral presentation

Do stretch activated ion channels
have a physiological role in the
heart?
Douglas Kelly
Cellular Biophysics Laboratory
Department of Physiology
University of Adelaide
Investigation of MEF and the
Frank-Starling relationship in the
heart
Mechano-electric Feedback
• The electrical activity of the heart drives its
mechanical activity
MEF
– Excitation Contraction Coupling
– Calcium-induced calcium release (CICR)
• Changes in electrical activity can be brought about
by changes in mechanical activity
– Stretch activated ion channels
Mechano-electric Feedback
• Observations of cardiac rhythm disturbances and
sudden death caused by non-penetrating
mechanical impacts to the chest.
– Post mortem sectioning shows no signs of internal or
structural damage.
– This condition is referred to as Commotio cordis
( disturbance of the heart).
Experimental work on Commotio cordis
• Wooden hammers of varying weights and diameters
used to apply impacts to the precordial region of
anaesthetised animals. (Georg Schlomka, 1930s)
 Impacts provoked cardiac arrhythmia.
 almost always induced by extra-systoles.
 Ventricular fibrillation
 Caused sudden death occurred in 20% of animals
• These arrhythmias might be due to myocardial
stretch which alter cellular electrophysiological
properties
Mechano-electric Feedback
Escape beats
Balloon Volume
Monophasic action potential (MAP) recording from
the epicardial surface of a rabbit ventricle in
response to volume pulses applied to a balloon
inserted into the left ventricle.
Effects of Stretch
• Depolarises cardiomyocyte during diastole
– Stretch activated non-selective cation channels (Na+, Ca2+)
• Repolarises cardiomyocyte during systole.
– Stretch activated K+ channels
– Stretch activated non-selective cation channels (K+)
• Shortens the action potential duration
– Stretch activated K channels
• Decreases action potential amplitude ? (K+)
Membrane Potential (mV)
Cardiac Action Potential - Stretch
  K+
Decreased AP
  K+
Amplitude
Decreased AP
Duration
Ca2+
 K+
Cross-over  Na+ & Ca2+
100
50
Na+
0
+
2+
K+   Na & Ca
AP Prolongation
-50
Control
Stretch
  Na+ & Ca2+
Diastolic
Depolarisation
-100
0
750
Time (ms)
1500
Frank Starling Mechanism
• Greater diastolic volume results in an increase in
cardiac performance
 Heart responds to increases in mechanical stress by
increasing the force of contraction and heart rate.
• Thought to be due to mechanical alterations in
myocyte.
– Could MEF contribute ???
• We speculate that changes in the Action Potential
could be altering contractility on a beat-to-beat
basis.
Contractility
Frank-Starling Relationship
0
Diastolic Pressure (mmHg)
25
Aims
• To observe the effects of modulating SAC activity
on Frank-Starling curve
- Streptomycin (20 - 100 M, to block all SACs)
- Gadolinium (1 - 20 M to block all SACs)
- GsTx-4 (10 - 500 nM, to selectively block all SACs)
- Chlorpromazine (0.1 - 1 M, to block SAPCs)
- Chloroform (0.1 - 0.8 mM, to stimulate SAPCs)
- Halothane ? (also stimulates SAPCs)
• Relate changes in Frank-Starling curve to
suggested activity of SACs.
Hypotheses
• SACs are involved in the Frank-Starling curve, & thus in
the beat-to-beat regulation of the heart.
• SACs contribute to the shape or plateau of the FrankStarling curve.
• The Frank-Starling response is partly the result of changes
in the cardiac action potential
Methods
• Heart quickly excised from anaethesised male
Sprague-Dawley Rat.
• A latex balloon on a catheter, attached to a
pressure transducer, is inserted via the left atrium
into the left ventricle.
• Volume of balloon adjusted in steps to produce 2.5
mmHg pressure changes
Methods
• Varying concentrations of SAC blocking &
stimulating agents will be circulated in the
solution perfusing the heart.
• Effect on agents on Frank-Starling mechanism
analysed.
• The pressure transducer monitors left ventricular
pressure and the timing of balloon inflation.
• Contact electrodes will record monophasic action
potentials from the left left ventricle
2.5 mmHg
Steps
Diastolic
-dV/dt
Systolic
dV/dt
Phase I Protocol
Stabilisation
period
Soln Change
(10 min)
50uM
Streptomycin
100uM
Streptomycin
> 20 min
Control
Pulse
Curves
Pressure
n Change
Sol
Re-Control
20uM
Washout
Streptomycin
Rate of
Contraction
Time (minutes)
Final Curves
(following washout)
Contractility
+20
stretch
0
(mV)
Membrane Potential
Expected Results
Increasing SAC
Blocker
Concentration
-90
Action Potential Duration (ms)
L-Type Ca2+
channel block
- Gd 3+
- Streptomycin ?
0
Diastolic Pressure (mmHg)
25
Preliminary Results
dP/dt (mmHg/second)
4000
Legend:
Control
20M Strept
Re-Control
50M Strept
3500
3000
2500
2000
100M Strept
1500
1000
500
0
-1
4
9
14
19
Diastolic Pressure (mmHg)
24
29
Phase II Experiments
Cardiac cycle-dependent contribution of
SACs
•
Diagram of ventricular action potential showing the development of a secondary depolarisation
during the terminal part of phase 3 (early afterdepolarisation, EAD) (A) and following repolarisation
(delayed afterdepolarisation, DAD) (B). In each case when the amplitude of the EAD or DAD
reaches threshold (fourth action potentials in each panel), it triggers a premature action potential.
Diagram from Taggart and Sutton (1999).
Membrane Potential (mV)
Cardiac Action Potential - Stretch
  K+
Decreased AP
  K+
Amplitude
Decreased AP
Duration
Ca2+
 K+
Cross-over  Na+ & Ca2+
100
50
Na+
0
+
2+
K+   Na & Ca
AP Prolongation
-50
Control
Stretch
  Na+ & Ca2+
Delayed After
Depolarisation
-100
0
750
Time (ms)
1500
Membrane Potential (mV)
Cardiac Action Potential - Stretch
Decreased AP
Amplitude
Decreased AP
Duration
100
50
Cross-over
0
Control
Stretch
  Na+ & Ca2+
Early After
Depolarisation
-50
-100
0
750
Time (ms)
1500
Unphysiological Stretch
• If the myocardium is subjected to unphysiological
amounts of stretch during the repolarisation phase
of the action potential, depolarisations can occur.
• Triggered activities arise when the magnitude of
these depolarisations are sufficient to induce
premature action potentials.
• Triggered activities may result in runs of
tachycardia or arrhythmia.
Aims (Part II)
• To characterise cardiac cycle-dependent effects of
stretch in the heart.
• To determine the contribution of various SACs
throughout the cardiac cycle.
Hypotheses
• Non-selective SACs are most important during
diastole
• K+-selective SACs are more specifically involved
in the early termination of the cardiac AP.
• Both populations of SACs are important and their
individual effects are cardiac phase-dependent (ie
dependent on timing and the cardiac cycle).
Expected Results (Pt II)
Membrane 0
Potential
mV
-90
EAD
300ms
• Due to non-selective SACs
 Blocked by:
– Streptomycin
– Gadolinium
– GsTx-4
Expected Results (Pt II)
Membrane 0
Potential
mV
-90
DAD
300ms
• Due to non-selective SACs
 Blocked by:
– Streptomycin
– Gadolinium
– GsTx-4
Expected Results (Pt II)
Early
Termination
Membrane 0
Potential
mV
-90
300ms
• Due to both non-selective and K+ selective SACs
 Partially Blocked By
–
–
–
–
Streptomycin
Gadolinium
GsTx-4
Chlorpromazine
Expected Results (Pt II)
Early
Termination
Membrane 0
Potential
mV
-90
300ms
• Due to both non-selective and K+ selective SACs
 Partially Stimulated By
– Chloroform
– Halothane
Phase III
Stretch-Activated Ion Channels in Human
Atria
Effect of Stretch on Atrium
• In humans, a change in the venous return had been
found to affect heart rate. Donald and Shepherd
(1978) reported that healthy volunteers who were
required to remain in a supine position had an
increase in heart rate when their legs were
elevated.
• SACs located within the sino-atrial node (SAN),
are proposed to be involved in the positive
chronotropic response of the heart to mechanical
stress (Kohl, et. al., 1999)
• The shorter the action potential duration, the
earlier the subsequent action potential can be
elicited.
• As SACs activation can be involved in the
shortening of APD, a patient with chronic atrial
dilation would be more susceptible to re-entry
arryhthmias.
• Stretch-induced arrhythmia inhibited by
Streptomycin & Gadolinium
• Stretch-induced arrhythmia not inhibited by Ltype / Na channel blockers
– Verapamil, nifedipine, TTX
• Non-selective SACs induce a depolarising current when
activated during diastole while the TREK-1 current is
hyperpolarising. Half-maximal activation of these cationic
non-selective channels occur at 1.5mm Hg whereas for
TREK-1, it occurs at a higher level of stretch, ie. around 12
mmHg (Terrenoire, et. al. 2001; Kim, 1992), suggesting
that TREK-1 activation will occur secondary to the
activation of these cationic non-selective SACs
(Terrenoire, et al., 2001). Hence, TREK-1 channels could
function as a negative feedback to the stretch-activated
cationic non-selective channels.