Dissertation

Mechanisms of subcellular alternans in cardiac myocytes
Cardiac Electrodynamics Laboratory September 2nd, 2009
Stephen Gaeta
Cardiac alternans
1 ECG
T-wave alternans is a beat-to-beat alternation in the amplitude of the Twave that occurs with rapid pacing of the heart. Often seen to precede the onset of potentially lethal reentrant arrhythmias.
1Narayan, J Am Coll Cardiol., 47(2): 269-281. 2006.
Cardiac alternans
Cellular alternans Given su ciently fast pacing, the action potential duration (APD) and Ca2+-transient amplitude of individual cardiac myocytes alternate on consecutive beats.
Slow pacing (T=300ms) T=300ms T=300ms Rapid pacing (T=180ms) T=180ms T=180ms
Normally, the AP and the Ca2+transient are at a period-1 rhythm, in which every beat is the same.
With rapid pacing, the APD and Ca2+transient amplitude alternate (Isolated rabbit ventricular myocyte).
1Chudin et al. Biophys J, 77(6): 2930-41. 1999.
Cardiac alternans
Cellular alternans Given su ciently fast pacing, the action potential duration (APD) and Ca2+-transient amplitude of individual cardiac myocytes alternate on consecutive beats.
T=300ms whole-cell concordant alternans

Peak Ca2+ of consecutive beats.
Rapid pacing (T=180ms) T=180ms 2
Normally, the Ca2+-transient is approximately spatially uniform within an individual myocyte. subcellular alternans (Isolated guinea pig myocyte)
beat n
beat n+1
beat n
beat n+1
2Gaeta et al. Circ Res. 105(4): 335-42. 2009.
Subcellular alternans
beat n
beat n+1
subcellular alternans 1
Peak consecutive beats. Ca2+ of
Ca2+-transients in adjacent subcellular regions (within the same cell) alternate with opposite phase.
beat n
beat n+1
Average [Ca2+]i of top and bottom half of the cell above from six beats.
(Isolated guinea pig ventricular myocyte)
1Gaeta et al. Circ Res. 105(4): 335-42. 2009.
Subcellular alternans has been reported during rapid pacing of multiple species and cell-types, in both isolated cells and within individual cells of the intact heart. ! ! ! ! Canine ventricular myocytes (Cordeiro et al., 2007) Cat atrial myocytes (Kockskamper and Blatter, 2002) Intact rat heart (Aistrup et al., 2006; Aistrup et al., 2009, Kapur et al., 2009) Intact guinea pig heart (Aistrup et al., 2006)
Why is subcellular alternans important?
Previously uncharacterized cardiac myocyte dynamics. Predisposes to intracellular waves of calcium release, which are known to cause arrhythmogenic afterdepolarizations.
255
[Ca2+] (a.u.)
0
Imaging line
Linescan imaging of [Ca2+]i from ve consecutive beats during subcellular alternans. (Isolated rat ventricular myocyte)1
1Diaz et al. Circ Res. 91(7): 585-93. 2002.
Ca2+ wave (mini)
255
[Ca2+] (a.u.)
0
Imaging line
Linescan imaging of [Ca2+]i from ve consecutive beats during subcellular alternans. (Isolated rat ventricular myocyte)1
1Diaz et al. Circ Res. 91(7): 585-93. 2002.
Two mechanisms have been proposed for pacing-induced subcellular alternans, neither has been veri ed in vitro. 1. Anatomical mechanism Di erences in the machinery of Ca2+
release/reuptake in adjacent subcellular regions can cause the di erences in Ca2+-transients seen.1
o! Proposed gradients in Ca2+-cycling properties are not experimentally observed.
1Aistrup et al. Circ Res. 104 (5): 639-49. 2009.
Two mechanisms have been proposed for pacing-induced subcellular alternans, neither has been veri ed in vitro. 1. Anatomical mechanism Di erences in the machinery of Ca2+
release/reuptake in adjacent subcellular regions can cause the di erences in Ca2+-transients seen.1
o! Proposed gradients in Ca2+-cycling properties are not experimentally observed.
2. Dynamical mechanism given speci c coupling between the

AP and the Ca2+ transient, subcellular alternans is predicted to arise spontaneously by a well-known mechanism of dynamical pattern formation.2
o! Only predicted subcellular alternans in an unusual subset of myocytes.
1Aistrup et al. Circ Res. 104 (5): 639-49. 2009.
2Shiferaw and Karma. PNAS. 103 (15): 5670-5. 2006.
Results: Subcellular alternans is induced by this same dynamical mechanism in the remaining, more common subset of myocytes during pacing with a simple feedback control pacing algorithm.
static pacing
alternans control pacing
Background
Outline (Background)
o! Mechanisms of APD-alternans and Ca2+-alternans. o! Coupling between the AP and the Ca2+-transient. o! Previously proposed dynamical mechanism of subcellular alternans.
Background
Alternans mechanisms
What causes the APD-alternans and Ca2+-alternans seen during rapid pacing?
T=300ms
T=180ms
1
T=300ms
! APD-alternans can occur independently of Ca2+-transient alternans.
T=180ms
1
T=300ms
! APD-alternans can occur independently of Ca2+-transient alternans.
T=180ms
1
Vm-driven alternans the dependence of the APD on the preceding diastolic interval (DI) causes APD-alternans during rapid pacing.
T=300ms
! APD-alternans can occur independently of Ca2+-transient alternans. ! Ca2+-alternans can occur independently of APD-alternans.
T=180ms
1
T=300ms
! APD-alternans can occur independently of Ca2+-transient alternans. ! Ca2+-alternans can occur independently of APD-alternans.
T=180ms
1
Ca2+-driven alternans the load dependence of sarcoplasmic reticulum (SR) Ca2+ release can cause Ca2+-transient alternans during rapid pacing.
T=300ms
! APD-alternans can occur independently of Ca2+-transient alternans. ! Ca2+-alternans can occur independently of APD-alternans. ! Bidirectional coupling between the APD and the Ca2+ transient means alternans in one will cause secondary alternans in the other.
T=180ms
1
T=300ms
! APD-alternans can occur independently of Ca2+-transient alternans. ! Ca2+-alternans can occur independently of APD-alternans. ! Bidirectional coupling between the APD and the Ca2+ transient means alternans in one will cause secondary alternans in the other.
T=180ms
1
Alternans is most likely primarily caused by Ca2+-cycling dynamics (Ca2+-driven); APDs alternate secondarily.
Background
Bidirectional coupling of Vm and Ca2+
Ca2+-alternans causes secondary APD-alternans (Ca2+ to Vm coupling). APD-alternans causes secondary Ca2+-alternans (Vm to Ca2+ coupling).
Bidirectional coupling of Vm and Ca2+
Ca2+-alternans causes secondary APD-alternans (Ca2+ to Vm coupling). APD-alternans causes secondary Ca2+-alternans (Vm to Ca2+ coupling).
positive coupling
secondary = alternans are in-phase
secondary negative = alternans are coupling out-of-phase
Ca2+ to Vm coupling
The Ca2+-transient in uences the AP through the e ects of several Ca2+-sensitive membrane currents.
o! Na+/Ca2+-exchanger (3:1 stoichiometry makes Ca2+-extrusion depolarizing). o! Ca2+-induced inactivation of L-type Ca2+ channels (net hyperpolarizing).
Dominant e ect is a species, temperature dependent characteristic.
Ca2+ to Vm coupling
C
Vm
dn-1
An exchange current dominates) Cn

[Ca ]
2+
Positive Ca2+ to Vm coupling (Na+/Ca2+

o! Larger Ca2+-transient causes longer APD. o! Ca2+-alternans causes in-phase APD-alternans. 2+
Vm
[Ca2+]
[Ca ]i
region 1
Vm
region 2
Ca2+ to Vm coupling
B
dn-1
An inactivation dominates) channel C [Ca ] [Ca ] o! Less commonly seen.
Negative Ca2+ to Vm coupling (L-type Ca2+

o! Larger Ca2+-transient causes shorter APD. o! Ca2+-alternans causes out-of-phase APDn 2+alternans. 2+ V
m
Vm
Vm
[Ca2+]
[Ca2+]
Vm
[Ca2+]i
Vm
region 1
regi
Vm to Ca2+ coupling
The duration of each AP in uences the subsequent Ca2+-transient by changing the duration of the diastolic interval (DI).
o! A shortened DI causes a smaller subsequent Ca2+-transient because less L-type Ca2+ channel current is available to trigger SR Ca2+ release.
Vm to Ca2+ coupling
coupling
Vm
2+ [Ca2+]i
region 1
Vm
region 2
Vm to Ca2+ coupling
Positive Vm to
Ca2+
coupling
Vm Vm
o! APD-alternans causes in-phase Ca2+-alternans. o! Assumed to be true during static pacing.
[Ca2+ [Ca2+ ]ii
region 1
Vm
region 2
Vm to Ca2+ coupling
Negative Vm to Ca2+ coupling has never been observed experimentally
Background
Subcellular alternans mechanism
A previous theoretical study1 showed analytically that given proper Ca2+ to Vm and Vm to Ca2+ coupling, subcellular alternans will arise due to a well known, generic mechanism of pattern formation (Turing instability). However, ! Conditions are only satis ed in an uncommon subset of myocytes (those with negative Ca2+ to Vm coupling). ! Never experimentally validated.
1Shiferaw and Karma, PNAS, 2006.

Subcellular alternans is modeled as the formation of a gradient in Ca2+alternans amplitude (!c) along the length of a single myocyte.
A A
x
x
1 1
Vm
1
region 1
region 1 region 2
B B
c
c
x
[Ca ]i
2+
region 1
2 region 2
x!
c
region 2
node
node
!"#$%&'()*+#

Turing instability The interaction of only two di using substances
region 1 region 2
(morphogens) can cause a concentration gradient to form from a nearly homogeneous substrate.
APDalternans
x
1
B
c
Vm
x
c
Ca2+[Ca2+ alternans]i
region 1
region 2
node
!"#$%&'()*+#

region 1 region 2
Ca2+ to Vm 1 coupling
Vm
Vm to Ca2+ coupling
B
c
x
c
[Ca ]i
2+
region 1
region 2
node
!"#$%&'()*+#

region 1 region 2
Vm
Vm to Ca2+ coupling
B
c
x
c
[Ca ]i
2+
region 1
region 2
node
!"#$%&'()*+#
A gradient in Ca2+-alternans amplitude (subcellular alternans) will form if local Ca2+-alternans grows and generates secondary APD-alternans that then produces out-of-phase Ca2+ alternans more distantly.

region 1 region 2
Vm
Vm to Ca2+ coupling
B
c
x
c
[Ca ]i
2+
region 1
region 2
node
!"#$%&'()*+#
A gradient in Ca2+-alternans amplitude (subcellular alternans) will form if local Ca2+-alternans grows and generates secondary APD-alternans that then produces out-of-phase Ca2+ alternans more distantly. Ca2+-driven alternans

Given Ca2+-driven alternans, the conditions for a Turing instability are satis ed in myocytes with negative Ca2+ to Vm coupling during static pacing (positive Vm to Ca2+ coupling).
x
1
Vm
region 1 region 2
B
c
x
c
[Ca ]i
2+
region 1
region 2
node
!"#$%&'()*+#

Given Ca2+-driven alternans, the conditions for a Turing instability are satis ed in myocytes with negative Ca2+ to Vm coupling during static pacing (positive Vm to Ca2+ coupling).
x
1
Vm
region 1 region 2
B
c
x
c
[Ca ]i
2+
1.! Local Ca2+-alternans produces out-of-phase APD-alternans. 2.! During static pacing, this APD-alternans causes Ca2+-alternans more distantly without switching the phase back.
region 1
region 2
node
!"#$%&'()*+#

Given Ca2+-driven alternans, the conditions for a Turing instability are also satis ed in myocytes with positive Ca2+ to Vm coupling if Vm to Ca2+ coupling is negative.
x
1
Vm
region 1 region 2
B
c
x
c
ion 2
[Ca ]i
2+
region 1
region 2
node
!"#$%&'()*+#

x
1
Vm
region 1 region 2
B
c
x
c
ion 2
[Ca ]i
2+
region 1
region 2
node
!"#$%&'()*+#
1.! Local Ca2+-alternans produces in-phase APD-alternans. 2.! This APD-alternans causes out-of-phase Ca2+-alternans more distantly.

x
1
Vm
region 1 region 2
B
c
x
c
ion 2
[Ca ]i
2+
region 1
region 2
node
!"#$%&'()*+#
This possibility was not previously studied because negative Vm to Ca2+ coupling has not been experimentally observed
Hypothesis
We hypothesize that during pacing with a simple feedback control algorithm (alternans control pacing), Vm to Ca2+ coupling is e ectively negative. This makes the novel prediction that subcellular alternans is predicted to occur during alternans control pacing in myocytes with positive Ca2+ to Vm coupling (and Ca2+-driven alternans).
Thesis work
Alternans control pacing induces subcellular alternans by a Turing instability in cardiac myocytes with positive Ca2+ to Vm coupling and Ca2+-driven alternans.
Thesis work
Alternans control pacing induces subcellular alternans by a Turing instability in cardiac myocytes with positive Ca2+ to Vm coupling and Ca2+-driven alternans.
o! Extended previous mathematical modeling work to analytically predict conditions for pattern formation during alternans control pacing. o! Con rmed and characterized this mechanism in a detailed computational model of an isolated myocyte. o! Experimentally veri ed this mechanism in isolated guinea pig myocytes.
(Gaeta et al. Circ Res. 2009: 105(4): 335-42)
o! Identi ed a possible role for this mechanism in the formation of subcellular alternans in the intact heart using computational modeling of intact tissue.
Alternans control pacing

Feedback control pacing algorithm intended to eliminate APD-alternans
o! extends the cycle length following long APs and shortens the cycle length following short APs.
T*
Vm!
Tn
Cycle length of each beat is calculated by
Tn = T* + g(An-An-1)
An-1
An
where T* is the target cycle length and g is the control gain.

Feedback control pacing algorithm intended to eliminate alternans
o! extends the cycle length following long APs and shortens the cycle length following short APs.
alternans control
CL
Alternans control pacing forces alternating APDs of a guinea pig ventricular myocyte towards an unstable 1:1 rhythm.
duration (ms)
Vm to Ca2+ coupling during alternans control pacing

During alternans control pacing, Vm to Ca2+ coupling is e ectively negative (above a critical gain).
Static pacing
coupling
Vm
region 2
2+ [Ca2+]i
Following an extended APD, the subsequent Ca2+-transient is smaller (positive Vm to Ca2+ coupling).
[Ca2+]i
region 1
Vm
Vm to Ca2+ coupling during alternans control pacing

During alternans control pacing, Vm to Ca2+ coupling is e ectively negative (above a critical gain).
Static pacing
coupling
Vm
region 2
Vm
Vm
2+ [Ca2+]i
Following an extended APD, the subsequent Ca2+-transient is smaller (positive Vm to Ca2+ coupling).
region 1 Following an extended APD, the cycle length is prolonged, and the subsequent Ca2+-transient is larger (negative Vm to Ca2+ coupling).
[Ca2+]i 2+] [Ca
region 2
[Ca2+]i
region 1
Vm
Conditions for subcellular alternans
Alternans control pacing is predicted to dynamically induce subcellular alternans in isolated myocytes with positive Ca2+ to Vm coupling when both of the following are true: ! Alternans control gain is above a critical threshold (causing negative Vm to Ca2+ coupling). ! Alternans is Ca2+-driven.
Computational model
Alternans control pacing tested in the spatially extended SSK model of an isolated ventricular myocyte.
o! 1-dimensional chain of 75 identical sarcomeres, each with its own Ca2+ concentrations and dynamics. o! Adjacent sarcomeres coupled by Ca2+di usion. o! Vm determined by the average currents of all sarcomeres (spatially uniform). o! Well characterized parameter adjustments control the sign of Ca2+ to Vm coupling and whether alternans is Ca2+ or Vm-driven.
k
k k
k-1
k+1
Ca2+-cycling components of 3 adjacent sarcomeres (of 75 total).
Computational modeling results

Subcellular alternans is induced during alternans control pacing of the cell model with positive Ca2+ to Vm coupling and Ca2+-driven alternans.
alternans control
Vm
Whole cell 2+ [Ca2+ ]i average [Ca ]i
Alternans control pacing successfully eliminates alternans in APD and whole cell average Ca2+-transient.
After stable alternans was induced by rapid, static pacing, alternans control pacing was initiated at the same cycle length (T*=300ms).

Subcellular alternans is induced during alternans control pacing of the cell model with positive Ca2+ to Vm coupling and Ca2+-driven alternans.
alternans control
Vm
Whole cell 2+ [Ca2+ ]i average [Ca ]i
600ms
Alternans control pacing causes the Ca2+-transients in the two halves of the cell to alternate out-of-phase.
After stable alternans was induced by rapid, static pacing, alternans control pacing was initiated at the same cycle length (T*=300ms).
1.5 0 45 60 30 75
c (M)
-1.5
600ms
100 1 15 beat sarcomere (k)" number (n)"

Ca2+-alternans amplitude (!c) quanti ed as the di erence in peak [Ca2+]i (c) between each beat and the last:
20
60

During static pacing, Ca2+-alternans in every sarcomere has the same phase (!c>0).
1.5 0 45 60 30 75
c (M)
Alternans control induces a gradient in Ca2+-alternans amplitude (!c with opposite sign).
600ms
-1.5
600ms
100 1 15 beat sarcomere (k)" number (n)"

Ca2+-alternans amplitude (!c) quanti ed as the di erence in peak [Ca2+]i (c) between each beat and the last:
20
60

The same model cell can exhibit di erent patterns of subcellular alternans in di erent trials (with identical protocol), dependent on the distribution of noise in initial Ca2+ concentrations.
Alternans control initiated from steady-state alternans (plus noise); T*=300ms.
Alternans control pacing tested at cycle lengths from 200 to 400ms. Subcellular alternans was induced during alternans control pacing of the cell model when Ca2+ to Vm coupling was positive when both of the following were true: ! Alternans control gain was above a low threshold (g0.2-0.4 for most cycle lengths). ! Alternans was Ca2+-driven.
!! !!
Subcellular alternans only induced during static pacing when Ca2+ to Vm coupling was negative. The same model cell can exhibit di erent patterns of subcellular alternans in di erent trials.
Experimental studies
Does this work in real cardiac myocytes?
Does this work in real cardiac myocytes?
! Enzymatically isolated adult guinea pig ventricular myocytes (which have positive Ca2+ to Vm coupling) by Langendor retrograde perfusion method. ! Cells loaded with Ca2+-sensitive uorescent dye (Fluo-4 AM). ! Perforated patch clamped cells, paced by small depolarizing current injections (room temperature). ! After stable alternans was induced by rapid pacing, alternans control pacing was initiated (at the same cycle length) and Ca2+ imaging begun.
Alternans control pacing induces a 2-region subcellular alternans pattern. Resumption of static pacing causes resynchronization of Ca2+-transient. (T=340ms)
Alternans control pacing of an isolated guinea pig ventricular myocyte induces a 3-region subcellular alternans.
Alternans control pacing at T*=300ms.
(n)!
(k)!
Ca2+ uorescence binned into 12 regions (perpendicular to the long axis of the cell).
The same cell exhibited a di erent pattern of subcellular alternans (2 regions) in a di erent trial at the same cycle length.
After subcellular alternans is induced (by alternans control pacing), resuming static pacing causes resynchronization of the Ca2+-transient.
Alternans control pacing used to induce 3-region subcellular alternans (prior to this video), after which static pacing was resumed (T=300ms).
After subcellular alternans is induced (by alternans control pacing), resuming static pacing causes resynchronization of the Ca2+-transient.
Alternans control pacing used to induce 2-region subcellular alternans (not shown). Static pacing resumed following the rst beat (T=250ms).
Experimental results
Alternans control pacing robustly induces subcellular alternans in isolated guinea pig ventricular myocytes.
o! 51 total trials in 16 di erent cells. o! Alternans control successfully suppressed APD-alternans in 45 of 51 trials. o! Subcellular alternans was seen in 42 of these 45 trials. o! None of the trials in which control failed showed subcellular alternans. o! 5 of 16 cells experienced di erent patterns (numbers of regions) of subcellular alternans in di erent trials.
Subcellular alternans in intact tissue

Is alternans control pacing physiologically relevant?
During alternans in intact tissue, beat-to-beat changes in conduction velocity (CV) cause alternans control-like (non-static) pacing of cells distant from the pacing site. Hypothesis: the same dynamical mechanism may contribute to subcellular alternans seen during static pacing of the intact heart.
Conduction velocity (CV) restitution1
APs following short DIs propagate more slowly (due to incomplete recovery from inactivation of Na+ channels).
1Watanabe et al. J Cardiovasc Electrophysiol. 12(2): 196-206. 2001.
Stimulus following long AP is delayed.

cell 1 cell k
1-dimensional fiber of cardiac myocytes
Even during static pacing, CV restitution will cause alternans control-like pacing of cells distant from the pacing site.

APD (ms)
Spatially discordant alternans (SDA)1

300 200 100 0
distance from stimulus site (mm)
80
APDs of two consecutive beats in a 1dimensional ber of simulated cardiac myocytes.
Tissue simulations
1-dimensional cable of 400 cell models, coupled by voltage di usion. Each cell is itself a 1-dimensional cable of 75 sarcomeres.
k k k
Parameters chosen for positive Ca2+ to Vm coupling and Ca2+-driven alternans.

k
k-1
k+1
Tissue simulations
Pacing Protocol
1.! Statically paced (from the left side of the cable) for 300 beats at T1=400ms (at which there is no alternans). 2.! Pacing period decreased to either T2=330ms or T2=320ms for the next 300 beats. (Random noise added to all Ca2+-concentrations before each stimulus, =0.5% of steady-state concentrations)
cc(M) APD (ms) APD (ms) (M) c (M) S
240 220 200 3.0 260 2.0 240 1.0 220 0 200 1.5 3.0 1.0 2.0 0.5 1.0
0 1.5 1.0 0.5 5 15 0 25 35 45 55 5 65 15 75 25
35 1 45 55 15 65 30 75
T2=330ms, beat 419 and 420
cc(M) (M) S
c (M) APD (ms) APD (ms)
Tissue simulations A
260
260 240 220 200 3.0 260 2.0 240
APD and whole-cell average Ca2+1.0 220 0 transient amplitude ( c ) of every cell 200 1.5 3.0 alternates, but spatially discordant 1.0 2.0 alternans (SDA) is not seen. 0.5 1.0
0 1.5 1.0 0.5 0 50 100 150 200 250 cell number (k) 300 350 400
cS (M)
C
C
sarcomere
50
100
150 200 250 cell number (k)
300
350
400
sarcomere sarcomere
25
30
35
cS (M)
50
100
300
350
400
50
100
300
350
400
time (s)
40
45
50
0.6 1.2 1.8 2+ [Ca ]i (M)
55
2.4
sarcomere
45 1 60 15 75 3037
45 60 75 37
25
30
35
time (s)
40
45
50
0.6
55
2.4
38 time (s)
39
1.2 1.8 [Ca2+]i (M)
38 time (s)
39
Tissue simulations
APD (ms) APD (ms)
T2=330ms, beat 419 and 420

260 240 220 200 3.0 2.0
260 240
c (M)
cS (M)
1.0 0.5 0 50 100 150 200 250 cell number (k) 300 350 400
cS (M)
1.0 0 1.5
c (M)
APD and whole-cell average Ca2+220 transient amplitude ( c ) of every cell 200 3.0 alternates, but spatially discordant 2.0 alternans (SDA) is not seen. 1.0
0 1.5 1.0 No signi cant subcellular alternans seen 0.5 in any cell. 0 50 100 150 200 250 cell number (k) 300 350 400
C
sarcomere
5 15 25 35 45 55 65 75
cs = di erence in amplitude between the largest and smallest

Ca2+-transients within each cell, during a given beat.
30 35 time (s) 40 25 45 50
0.6 1.2 1.8 [Ca2+]i (M)
sarcomere
15 30 45 60 75 37 38 time (s) 39
55
2.4
Tissue simulations
APD (ms)
T2=320ms, beat 419 and 420

260 240 220 200 3.0 2.0
c (M)
Spatially discordant alternans seen in both APD and whole-cell average Ca2+transient amplitude ( c ).
cS (M)
1.0 0 1.5 1.0 0.5 0 50 100 150 200 250 cell number (k) 300 350 400
50
400
Transient subcellular alternans seen in cells adjacent to the node of tissue-level SDA.
35
time (s)
40
45
50
0.6 1.2 1.8 2+ [Ca ]i (M)
55
2.4
APD (ms)
APD (ms)
240 220 200 3.0 2.0
240 220 200 3.0 2.0
Tissue simulations
APD (ms)
c (M)
c (M)
cS (M)
1.0 0.5 0 50
260 240
150 200 250 cell number (k) 300 350 400
cS (M)
1.0 0 1.5
T2=320ms, beat 419 and 420

220 100 200 3.0 2.0
1.0 0 1.5 1.0 0.5 0
sarcomere
260
260
cell 213
15 30 45 60 75 37 1
5 15 25 35 45 55 65 75
25
30
50
5 15 25 35 45 55 65 75
sarcomere
cS (M)
1.0 0 1.5 1.0 0.5 0 50

30 100
sarcomere
50
100
300
350
400
c (M)
sarcomere
15 30 45 60 75 37 38 time (s)
time (s)
400
25
150 200 35250 cell number (k)
300 350 40 400 time (s)
45
38 time (s)
50
0.6
39
55
2.4
1.2 1.8 [Ca2+]i (M)
39
40 45 50
0.6 1.2 1.8 2+ [Ca ]i (M)
35
55
2.4
cS (
Tissue simulations 0
50
APD (ms)
cSS(M) c (M) sarcomere APD (ms)
220 200 3.0 2.0
APD (ms)
50
5 220 15 200 3.0 25 2.0 35 1.0 0 1.5 45 1.0 55 400 0.5 0 65 75 C 5

260 240
sarcomere sarcomere
sarcomere
100 150 200 250 300 350 400 cell number (k) T2=320ms, beat 419 and 420
B
50
sarcomere
0.5
cS (
1.0
0.5
cell 213
260 240 220 200 3.0 2.0
5 15 25 35 45 55 100 65 75
150 200 250 300 350 400 cell number (k)

25
260 240
15 30 45 60 75 37 38 time (s)
30
c (M)
c (M)
cS (M)
1.0 0 1.5 1.0 0.5 0 50 100 150 200 250 cell number (k) 300 350
400
cSS(M)
1.0 0 1.5 1.0 0.5 0 50
39
300 350 400
50
100
cell 213
35
15 25 35 45 55 40 65 time (s) 75
sarcomere sarcomere sarcomere
15 30 45 60
25
300
350
400
100
30
35
50
time (s)
55
2.4 time (s)
40
45
50
55
45
15 30 45 60 75 37
25
30
0.6
35 1.2 1.8 2+ [Ca ]i (M)
40
45
Node of subcellular alternans (dashed line) migrates across the cell, until the entire cell has reversed phase of Ca2+-alternans.
38 time (s) 39
0.6 1.2 1.8 2.4 50 55 2+ [Ca ]i (M) 2.4 0.6 1.2 1.8
2+ [Ca2+]ii (M)
Tissue simulations
A B
APD (ms)
220 200 3.0 2.0
APD (ms)
260 240
260 240 220 200 3.0 2.0
c (M)
c (M)
cSS(M)
1.0 0.5 0 50 100 150 200 250 cell number (k) 300
cSS(M)
1.0 0 1.5
Line scan imaging of Ca2+ within a single myocyte in 1.0 intact rat ventricle during rapid, static pacing 0.5
350 400 0
1.0 0 1.5
Aistrup et al. Circ Res. 104(5): 639-49. 2009.

50 100 150 200 250 cell number (k) 300
350
400
C
sarcomere sarcomere
5 15 25 35 45 55 65 75
cell 213
sarcomere sarcomere
15 30 45 60 75 37
25
30
35
time (s)
40
45
50
0.6 1.2 1.8 2+ [Ca2+]ii (M)
55
2.4
Node of subcellular alternans (dashed line) migrates across the cell, until the entire cell has reversed phase of Ca2+-alternans.
38 time (s) 39
Conclusions
o! Subcellular alternans can be dynamically induced in isolated cardiac myocytes by a pattern forming Turing instability.
Conclusions
!! This may be the rst experimental demonstration of a biological Turing instability in which the morphogens are clearly identi able.
Conclusions
!! This may be the rst experimental demonstration of a biological Turing instability in which the morphogens are clearly identi able.
o! Subcellular alternans may also be dynamically induced in the intact heart static pacing by this same mechanism.
Future Directions
Alternans control pacing is a useful tool for future studies of subcellular alternans and related phenomena.
o! o! Alternans mechanisms is alternans really always Ca2+-driven? Ca2+-wave studies.
High resolution (confocal) imaging studies. 2 dimensional tissue-level computational modeling.
Acknowledgements
Christini Lab
Rebecca Ahrens-Nicklas Jonathan Bettencourt Peter Jordan Uche Kanu Trine Krogh-Madsen Anat Maoz Jonathan Moreno Byron Roberts
David Christini
Thesis Committee Geo Abbott Lab (WCMC) Sources of Funding Geo Abbott Jason Banfelder, PBTech (WCMC) NIH R01RR020115 (D.C.) Olaf Andersen Gil Bub (Oxford) NIH MSTP grant GM07739 (S.G.) Marcelo Magnasco Robert Gilmour Lab (Cornell) NIH 1 F30 HL095324-01 (S.G.) Alan Weinstein Yohannes Shiferaw (CSUN) Kenny Gordon Foundation Lai-Hua Xie (UMDNJ) Tri-Institutional MD-PhD Program Andrew Zygmunt (MMRL)

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Mechanisms of subcellular alternans in cardiac myocytes

Cardiac Electrodynamics Laboratory September 2nd, 2009

1Narayan, J Am Coll Cardiol., 47(2): 269-281. 2006.

1Chudin et al. Biophys J, 77(6): 2930-41. 1999.

T=300ms whole-cell concordant alternans

Rapid pacing (T=180ms) T=180ms 2

1Chudin et al. Biophys J, 77(6): 2930-41. 1999.

2Gaeta et al. Circ Res. 105(4): 335-42. 2009.

1Gaeta et al. Circ Res. 105(4): 335-42. 2009.

1Diaz et al. Circ Res. 91(7): 585-93. 2002.

Ca2+ wave (mini)

1Diaz et al. Circ Res. 91(7): 585-93. 2002.

1Aistrup et al. Circ Res. 104 (5): 639-49. 2009.

2. Dynamical mechanism given speci c coupling between the

2Shiferaw and Karma. PNAS. 103 (15): 5670-5. 2006.

alternans control pacing

1Chudin et al. Biophys J, 77(6): 2930-41. 1999.

1Chudin et al. Biophys J, 77(6): 2930-41. 1999.

1Chudin et al. Biophys J, 77(6): 2930-41. 1999.

1Chudin et al. Biophys J, 77(6): 2930-41. 1999.

Bidirectional coupling of Vm and Ca2+

Bidirectional coupling of Vm and Ca2+

secondary = alternans are in-phase

secondary negative = alternans are coupling out-of-phase

Dominant e ect is a species, temperature dependent characteristic.

An exchange current dominates) Cn

Positive Ca2+ to Vm coupling (Na+/Ca2+

Dominant e ect is a species, temperature dependent characteristic.

An inactivation dominates) channel C [Ca ] [Ca ] o! Less commonly seen.

Negative Ca2+ to Vm coupling (L-type Ca2+

o! APD-alternans causes in-phase Ca2+-alternans. o! Assumed to be true during static pacing.

[Ca2+ [Ca2+ ]ii

Negative Vm to Ca2+ coupling has never been observed experimentally

Subcellular alternans mechanism

1Shiferaw and Karma, PNAS, 2006.

Subcellular alternans mechanism

Subcellular alternans mechanism

Subcellular alternans mechanism

Subcellular alternans mechanism

Subcellular alternans mechanism

Subcellular alternans mechanism

Subcellular alternans mechanism

Subcellular alternans mechanism

Subcellular alternans mechanism

Subcellular alternans mechanism

Alternans control pacing

Cycle length of each beat is calculated by

where T* is the target cycle length and g is the control gain.

Alternans control pacing

Vm to Ca2+ coupling during alternans control pacing

Vm to Ca2+ coupling during alternans control pacing

Alternans control pacing

[Ca2+]i 2+] [Ca

Conditions for subcellular alternans

Ca2+-cycling components of 3 adjacent sarcomeres (of 75 total).

Computational modeling results

Computational modeling results

Computational modeling results

100 1 15 beat sarcomere (k)" number (n)"

Computational modeling results

100 1 15 beat sarcomere (k)" number (n)"

Computational modeling results