Effects of Mechanical uncouplers, Diacetyl Monoxime and Cytochalasin-D on the

Electrophysiology of Perfused Mouse Hearts

Linda C. Baker, Robert Wolk, Bum-Rak Choi, Simon Watkins, Patricia Plan, Anisha Shah2, Guy

Salama1

University of Pittsburgh Department of Cell Biology and Physiology and the Department of Medicine, Division of

Cardiology2

Pittsburgh, PA 15261

Running Title: Anti-Arrhythmic Actions of DAM and Cyto-D in Mouse Hearts

Discipline: Experimental; Object of Study: Heart; Level: Organ; Field of Study: Electrophysiology.

Key words and Abbreviations: optical action potentials, APs; intracellular calcium, [Ca2+]i;cytochalasin-D, cyto-D; diacetyl monoxime, DAM; restitution kinetics, RK;

1Corresponding Author:

Guy Salama, PhD University of Pittsburgh, School of Medicine Dept. of Cell Biology and Physiology and Physiology S314 Biomedical Science Tower 3500 Terrace St.Pittsburgh, PA 15261 Phone: 412-648-9354 Fax: 412-648-8330 E-mail: gsalama@pitt.edu

Articles in PresS. Am J Physiol Heart Circ Physiol (June 10, 2004). 10.1152/ajpheart.00234.2004

Copyright © 2004 by the American Physiological Society.

Abstract

Chemical uncouplers, diacetyl monoxime (DAM) and cytochalasin-D (cyto-D) are used

to abolish cardiac contractions in optical studies, yet alter intracellular Ca2+ ([Ca2+]i)

handling and vulnerability to arrhythmias in a species dependent manner. The effects of

uncouplers were investigated in perfused mouse hearts labeled with Rhod-2/AM or di-4-

ANEPPS to map [Cai2+]i transients ( em=585 20 nm) and action potentials (APs), ( em >

610 nm; ex= 530 20 nm). Confocal images showed that Rhod-2 is primarily in the

cytosol. DAM (15 mM) and cyto-D (5 M) increased AP durations (APD75=20.0 3 to 46.6

5 and 39.9 8 ms, respectively, n=4) and refractory periods (RPs=45.14 12.1 to 82.5 3.5

and 78 4.24 ms, respectively). Cyto-D reduced conduction velocity by 20% within 5 min

and DAM by 10% gradually in 1 hour (n=5 each). Uncouplers did not alter the direction and

gradient of repolarization which progressed from apex to base, in 15 3 ms. Peak systolic

[Ca2+]i increased with cyto-D from 743 47 (n=8) to 944 17 nM (n=3, p=0.01) but

decreased with DAM to 398 44 nM (n=3, p < 0.01). Diastolic [Ca2+]i was higher with

cyto-D (544 80 nM, n = 3) and lower with DAM (224 31, n = 3) compared to controls

(257 30 nM, n=3). DAM prolonged [Ca2+]i transients at 75% recovery (54.3 5 to 83.6 1.9

ms), while cyto-D had no effect (58.6 1.2 ms; n=3). Burst pacing routinely elicited long

lasting ventricular tachycardia VT but not fibrillation. Uncouplers flattened the slope of AP

restitution kinetic curves and blocked VT induced by burst pacing.

2

Introduction

Molecularly engineered mice have been extensively used to genetically alter a specific

component of a complex signaling process and to develop models of human diseases. Transgenic

mice are used as models for various cardiac diseases and offer an effective strategy to elucidate

the mechanisms underlying long QT-related arrhythmias, metabolic diseases and the pathology

of heart failure (29). A limitation of mouse models is the small size of the heart making it

difficult to study changes in contractility, electrophysiology and vulnerability to arrhythmias in

intact hearts. The challenge of measuring changes in cardiac phenotype has been partly

overcome by applying optical technique to map electrical activity but a major technical difficulty

in the application of optical techniques to measure APs and [Ca2+]i transients has been the

distortion of the signals by movement due to muscle contractions.

Several approaches have been used to reduce movement artifacts: a) perfusion in Ca2+

free Tyrode’s solution to abolish contractions, an approach applicable to amphibian hearts (38);

b) design perfusion chambers to mechanically stabilize the heart (18, 37); c) perfusion with an

inhibitor of L-type voltage-gated Ca2+ channels, ICa,L to reduce [Ca2+]i and force generations

(16); d) perfusion with a chemical uncoupler of excitation-contraction like diacetyl monoxime

(DAM) and cytochalasin D (cyto-D) to block force by a direct inhibition of the contractile

filaments (5, 14).

Chemical uncouplers can potentially provide a practical approach to block movement

artifacts and have been used to inhibit contractions during optical recordings, particularly for

measurements of the recovery phase of the AP and [Ca2+]i transients which tend to be distorted

by movement artifacts.

DAM acts as a chemical phosphatase, exerts its biological effects by altering protein

phosphorylation and blocks contractions through the inhibition of myosin ATPase activity such

3

that the rise of intracellular [Ca2+]i elicited by an AP fails to generate force (2, 6). However,

DAM also alters repolarization and reduces AP durations (APD) in several species of cardiac

tissues, such as cat ventricular muscles (43), dog Purkinje fibres (4) and guinea pig papillary

muscle (27). In contrast, DAM prolonged APDs in rat Purkinje fibres (12, 13) and mouse

ventricles (3). In sheep and guinea pig ventricular muscles, DAM reduced calcium and

potassium conductance (27).

Cytochalasin-D (Cyto-D) was shown to disrupt F-actin filaments in the cytoskeleton and

unexpectedly blocked contractions by disrupting F-actin in myofibrils with little effect on APDs

of ventricular rat (40) and canine myocytes (5). Cyto-D was considered to be a better uncoupler

than DAM because of its negligible effects on APDs, transmural propagation velocity and

repolarization gradients (5, 44). However, cyto-D was shown to abolish Ca2+ mediated inward

rectification of K+ channels (31) and to modulate the kinetics of voltage-gated Na+ current (40)

by disrupting the cytoskeleton of cardiac myocytes.

The interpretation of optical data obtained using chemical uncouplers must be carefully

re-examined in light of their multiple effects on ionic channels, gap junctions and intracellular

Ca2+ handling in myocytes (12, 13, 21, 27, 28, 30, 39, 42-44). There is also little doubt that the

electrophysiological effects of cyto-D and DAM are species dependent (39) yet they have not

been extensively studied in murine hearts. Here, we examine the effects of cyto-D and DAM

(using the lowest concentrations that block force reliably) on APs and [Ca2+]i transient and on the

vulnerability to arrhythmias in mouse hearts.

4

Methods

Preparations

All animal procedures complied with NIH guidelines and were approved by the IACUC of

the University of Pittsburgh. FVB mice were anaesthetized with pentobarbital (50 mg/kg) and

heparinized (35 mg/kg) with an intra-peritoneal injection. The heart was rapidly excised,

cannulated, placed in a chamber specially designed to immobilize the ventricles, paced, and a

chosen region was imaged on a photodiode array. The perfusate contained (in mM): 112 NaCl,

1.0 KH2PO4, 25.0 NaHCO3, 1.2 MgSO4, 5.0 KCl, 50.0 Dextrose, 1.8 CaCl2, at pH 7.4 and was

gassed with 95% O2 and 5% CO2. Perfusion pressure was adjusted to 60-80 mm Hg by

controlling the flow rate of a peristaltic pump. The temperature of the bath surrounding the heart

was kept at 37o C by continuously monitoring the temperature with a thermistor, which

controlled a heating coil located in the back of the chamber, via a feedback amplifier. In pilot

studies, left ventricular pressure was measured using an intra-ventricular balloon (23) to ensure

that the concentrations of uncoupler effectively blocked contractions. Left ventricular diastolic

and systolic pressures were measured, the heart was allowed to reach a stable steady state then

various concentrations of DAM or cyto-D were added to the perfusate to arrest contractions. The

minimum concentrations of DAM and cyto-D that reliably reduced developed pressure by > 90%

were 15 mM and 5 M, respectively, after 2-5 min of continuous perfusion. Left peak systolic,

end-diastolic pressures, and maximum and minimum dP/dt were measured with a Digi-Med

heart performance analyzer.

Hearts were stained with the voltage-sensitive dye di-4-ANEPPS (10.0 l of 1 mg/ml

dissolved in DMSO) or the calcium indicator dye, Rhod-2 (0.2 mg in 0.2 ml DMSO). The dyes

were delivered as a single one-time bolus through a port in the bubble trap, which served as a

5

compliance chamber and was located proximal to the aortic cannula. [Ca2+]i was calibrated as

previously described (11). Briefly, Rhod-2 exhibits >100-fold increase in fluorescence emission

(F) at 585 nm upon binding to Ca2+ with ex = 530 20 nm. Fmax determined by adding 100 M

2,2’ dithiodipyridine and 10 M A23187 to the port in the bubble trap in perfusate containing 5

mM Ca2+, resulting in a peak F in 2 min. A rapid saturation of [Ca2+]i was achieved because 2,2’

dithiodipyridine elicited rapid release of Ca2+ from the sarcoplasmic reticulum by oxidizing

critical sulfhydryl activating ryanodine receptors while A23187 facilitated Ca2+ entry in heart

cells. (32) Fmin was then determined by perfusing the hearts with perfusate containing 5 mM

EGTA for 20-30 min. The absolute fluorescence intensity recorded during a cardiac AP or a Ca2+

transient is dependent on the optical apparatus, the depth of staining and the physiological

condition of the heart; under the current experimental conditions, the mouse ventricular AP

upstroke and Ca2+ transient produced fractional fluorescence change of 10-12 % and 30-35%,

respectively.

Confocal Images

The subcellular distribution of Rhod-2 was examined by confocal microscopy in perfused mouse

hearts loaded with Rhod-2 to discriminate between mitochondrial vs. cytosolic loading. The

perfused heart was mounted horizontally in a chamber with a Sylgard bottom carved in the shape

of the heart and a 3 mm diameter glass window (0.2 mm thick) on the bottom. The chamber was

placed on the stage of the inverted microscope such that the objective viewed the left ventricular

epicardium. To acquire confocal images, KCl (20 mM) was added to the perfusate to arrest the

heart. An argon laser excited the epicardial surface and fluorescence was collected through a

confocal aperture by a photomultiplier under manual gain and black-level control. Confocal

images were recorded from hearts loaded with Rhod-2 then after perfusion with 20 M digitonin

6

and 2 M free Ca2+ to permeabilize cell membranes and release Rhod-2 trapped in the cytosol

while retaining Rhod-2 trapped in mitochondria and other subcellular organelles. (15)

Optical Apparatus, Computer Interface, Analysis

The optical apparatus, computer interface, and analysis of APs have been previously

described (3). Briefly, light from a 100-W tungsten-halogen lamp was collimated, passed

through a 530 20 nm interference filter, and focused on the epicardial surface of the left

ventricle of the mouse heart. The fluorescence from dye bound to the heart was passed through a

cut-off filter (>610nm) for di-4-ANEPPs or an interference filter (585 20 nm) for Rhod-2. An

image of the heart was focused on a 12X12-element photodiode array of which 124 diodes were

simultaneously monitored. Each diode recorded the summed electrical activity from a 312X312

m2 region of the ventricle with a depth of 70 m. Image magnification was X 4.5 and a 4 X 4

mm2 tissue area was viewed by the array. The photocurrent from each diode was passed through

a current-to-voltage converter (50 M feedback resistor), AC or DC coupled, amplified (1, 50,

200 or 100X), digitized at 2000 frames/sec at a 12-bit resolution (DAP 3200e/214 Microstar

Laboratories) and stored in computer memory.

APDs were determined from measurements of the time point of maximum upstroke velocity,

(dF/dt)max minus the time-point at which the downstroke recovered to 75% back to baseline, that

is APD75. APs with signal to noise ratios of <10 or excessive movement artifact were not

included in the analysis. Conduction velocities were calculated as previously described. (36) The

duration of [Ca2+]i transients was determined from the maximum first derivative of the [Ca2+]i

upstroke to the time point of 75% recovery of [Ca2+]i to its original baseline (DCaT75). The rise

time of the Ca2+ transient was taken as the time-to-peak from the minimum to the maximum of

the [Ca2+]i upstroke (3). [Ca2+]i was calibrated from hearts loaded with Rhod-2 AM, using the

equation: [Ca2+] = Kd * [(F-Fmin)/ (Fmax-F)] where Kd is 710 nM, Fmin is the Rhod-2 fluorescence

7

when all the dye is in the free form or in zero Ca2+ and Fmax is the Rhod-2 fluorescence when all

the dye is bound to Ca2+ or in a Ca2+ saturated solution, as previously described. (11)

Programmed Stimulation

APD was characterized as a function of basic cycle length (CL) using basic pacing at S1-S1

intervals of 40 -200ms, in increments of 20 ms. Based on the measurements of spatial dispersion

of repolarization, single premature stimuli were delivered at decreasing coupling intervals at the

base or apex of the heart. The heart was paced at a basic interval or CL (e.g., S1-S1= 200 ms) for

10 beats to obtain a stable APD. Every 10th beat, an extra impulse S2 was applied to interrupt the

basic CL. The S1-S2 interval was gradually decreased in 1 or 2 ms steps (particularly in the steep

zone of the restitution curve) until S2 failed to capture an AP. The refractory period at that site

was defined as the shortest S1-S2 interval that elicited a propagating AP. Ventricular arrhythmias

in the murine heart were induced by applying a train of stimuli, burst pacing. Burst pacing

consisted of 10 electrical impulses, 1 millisecond in duration, with 15 ms inter-pulse interval at 3

times threshold voltage.

Statistics

Data are presented as mean standard deviation and changes in APDs recorded under

different conditions were compared by paired or unpaired Student's t test, as appropriate. The

results were considered significant for p < 0.05.

Results

Effects of DAM and Cyto-D on mouse AP

A digital picture of a perfused mouse heart is shown mounted in a chamber to abate

movement artifacts without chemical uncouplers, with examples of 4 simultaneously recorded

APs from different sites on the heart (Fig. 1A). The left epicardium faces the optical apparatus

8

and a silhouette of the array is superimposed on the heart to identify the region of optical

recordings. In Figure 1 (panels b-d), the silhouette of the array is illustrated with the AP recorded

by each diode drawn in its respective location from hearts perfused with control solutions (panel

b), 15 mM DAM (panel c) or 5 M cyto-D (panel d). Below each panel, a trace of AP recordings

from a diode at the center of the field of view is shown at a fast sweep speed. Upon the addition

of the chemical uncouplers, there was an immediate (within a minute) and marked prolongation

of APDs with APD75 increasing from 20.0 3 ms in controls (n = 8) to 46.6 5 ms in DAM (n =

4) and 39.9 ms in cyto-D (n = 4) (Table 1). Both uncouplers increased the refractory periods of

the epicardial APs, which would in principle increase the wavelength of reentrant circuits (Table

1). The chemical uncouplers caused marked changes in the shape and time course of APs. In

particular, note the spike and dome appearance of APs in hearts treated with cyto-D.

In control mouse hearts, APDs are shorter at the apex than the base of the left ventricle

producing a gradient of repolarization of 10.4 4.1 ms. (3) Gradients of APDs were within

experimental error similar in controls and hearts treated with chemical uncouplers. In DAM and

cyto-D, APDs increased progressively in going from apex to base with APD75 increasing from

63 4 ms to 69 5 ms with DAM and from 45 5 to 50 8ms with cyto-D.

Effects of Uncouplers on Restitution Kinetics and Conduction Velocity

Abrupt changes in heart rate or the firing of a premature impulse can produce dynamic

heterogeneities of AP amplitudes (APA) and durations. In mouse hearts, APDs are considerably

shorter than in other mammalian hearts, show only a slight variation as a function of rate (Fig.

2A) and tend to have flat APD restitution curves. In the presence of DAM and cyto-D, APDs

varied steeply as a function of cycle length (CL) for long CLs; however, for short CL that are 10-

15 ms above the refractory period, the curve remained flat and close to zero (Fig. 2A). In Figure

2B, the restitution kinetics curve of the AP amplitude is compared before and after the addition

9

of DAM or cyto-D by plotting the AP amplitude (APA) as a function of S1-S2 intervals. With

cyto-D and DAM, the shortest possible S1-S2 intervals were considerably longer than in controls

because the uncouplers increased APDs and refractory periods. As a result, the steep phases of

the restitution kinetics curves at S1-S2 < 75 ms were abolished. From the analysis of AP

recordings from 8 sites per heart, DAM and cyto-D increased the refractory periods from 45.14

2.1 (n = 8 hearts) to 82.5 3.5 (n = 4) and 78 4.24 ms (n = 4), respectively. At the longer S1-S2

intervals (> 75 ms), the chemical uncouplers had a slightly steeper restitution kinetics curves

compared to controls. Cyto-D reduced conduction velocity from 0.55 0.03 m/s in controls to

0.47 0.08 m/s (n=4) which was statistically significant (p <0.01, ANOVA) and there was a

tendency by DAM to reduce velocity from 0.58 0.06 m/s to 0.54 0.04 m/s DAM (n=5) that did

not reach statistical significance (Fig. 2C).

Effects of Uncouplers on Intracellular Ca2+ transients

Hearts were loaded with Rhod-2/AM and the left ventricles were imaged on the array to

record [Ca2+]i transients from multiple sites. A symbolic map of the array and the [Ca2+]i

transients recorded by each diode are shown in their respective locations (Fig. 3A). The shape

and time course of [Ca2+]i transients are shown for 4 diodes at faster sweep speeds (Fig. 3B) from

a heart paced at 200 ms cycle length. Figure 3C compares Ca2+ transients from a control heart

and in the presence of DAM or cyto-D where all 3 signals were calibrated in terms of free

cytosolic Ca2+, as described in methods (Fig. 3C). DAM decreased diastolic and systolic [Ca2+]i

whereas cyto-D increased both (Table 2). DAM produced a statistically significant prolongation

of the duration of [Ca2+]i transients whereas cyto-D had negligible effects (Table 2).

10

Distribution of Rhod-2 in Mouse Ventricular Myocytes

The intracellular distribution of Rhod-2 in perfused murine hearts was examined by

confocal microscopy from hearts loaded with Rhod-2/AM using identical conditions that were

used to load the hearts with Rhod-2 to map [Ca2+]i transients. Confocal images of the Rhod-2

distribution in a cell on the epicardium of the perfused heart revealed a pattern of cytosolic

milieu without the punctate appearance of mitochondrial loading (Fig. 4A, n=4). The loading

procedure resulted in a similar distribution of dye throughout the epicardium with no apparent

hot spots of high Rhod-2 fluorescence which would be expected if the dye was accumulated in

subcellular organelles with higher concentrations of hydrolyzed dye and/or high [Ca2+]i (ie. in the

sarcoplasmic reticulum network or the mitochondria). To verify that Rhod-2 was not trapped in a

subcellular compartment, the heart was perfused with digitonin for 10 min to increase the

permeability of the cellular plasma membrane cells without compromising the integrity of

subcellular organelles. Low Ca2+ (2 M) was used in the perfusate containing digitonin to

maintain a high level of Rhod-2 fluorescence and a normal mitochondrial potential (15). As

shown in Figure 4B, perfusion with digitonin resulted in an extensive loss of Rhod-2 from all

regions of the cells with similar observations made throughout the epicardium, indicating that the

dye was not trapped in subcellular compartments (n=4 hearts).

Anti-Arrhythmic Actions of Uncouplers

The chemical uncouplers prolonged APDs and refractory periods and eliminated the

steep phase of restitution kinetic curves without significantly changing gradients of refractoriness

but decreasing conduction velocity. Because these changes in the myocardial substrate can

potentially reduce the vulnerability to arrhythmias, we tested for the propensity to arrhythmias by

applying burst stimulation in attempts to elicit ventricular tachycardia (VT). The incidence of

arrhythmias in control hearts was compared to that in hearts perfused with a chemical uncoupler.

11

In most control hearts (n = 8/10), one or two bursts (10 pulses per burst) were sufficient to elicit

an immediate monomorphic VT. In a few hearts, several bursts were required to elicit VT

(n=2/10). Most VTs were long lasting, 30 s to an hour (n = 6/10 hearts) (Fig. 5a) or

spontaneously returned to sinus rhythm in 15-20 min (n=4/10). In contrast, burst pacing of

isolated hearts perfused with DAM (Fig. 5b) or cyto-D (Fig. 5c) triggered the firing of APs but

the hearts became quiescent immediately after the end of the burst (n = 5 for cyto-D and n = 5

for DAM). In control hearts, patterns of activation during VTs were highly reproducible

exhibiting stable frequencies ranging from 10-19 Hz, (n = 6) for the duration of the arrhythmia

(Fig. 6).

Discussion

Optical mapping has become an established technique to investigate mechanisms

underlying cardiac arrhythmias, metabolic diseases, and the pathology of heart failure by

providing accurate recordings of cardiac APs and [Ca2+]i transients at high spatial and temporal

resolution. However, muscle contractions produce movement artifacts, which can distort optical

signals raising concerns regarding their validity, particularly of the recovery phase of APs and

[Ca2+]i transients. Mechanical immobilization of the heart has been extensively used to abate

movement artifacts relative to the voltage-dependent component of the optical signal (18, 37).

Another approach has been to block force and movement artifacts by reducing [Ca2+]i with Ca2+

free Tyrode’s solution (38) or blocking L-type Ca2+ channels (16) which alters the substrate

being investigated. Alternatively, pharmacological agents were used as chemical uncouplers of

contractions which ideally eliminate force generation without affecting the shape, time course or

propagation of the cardiac AP or of [Ca2+]i transients. DAM and cyto-D have been extensively

used as chemical uncouplers in cardiomyocytes because they interfere with the contractile

12

apparatus at the level of the myofibrils presumably without significantly changing [Ca2+]i (5, 14)

Unfortunately, these uncouplers are far from ideal and are found to alter the myocardial substrate

through changes intracellular Ca2+ handling, ion channels and AP characteristics in a species

dependent manner.

Multiple Effects of DAM

DAM was first introduced as a nucleophilic agent with phophatase-like activity, that acts

by removing the phosphate groups required for protein activation. Its phosphatase activity

appears to be nonspecific having numerous effects at the cellular level by altering the properties

of ion channels, gap junctions and other intracellular processes regulated by phosphorylation. In

cat ventricular muscle, DAM was found to inhibit contractions and depress the plateau phase of

the action potential (43). DAM has been shown to alter delayed rectifier K+ current and L-type

Ca2+ current in certain species. DAM prolongs APD in mouse and rat but shortens APD in rabbit

and guinea pig hearts yet depresses contraction in all these species. Since DAM has opposite

effects on APDs in different species, the depression of contractions cannot be explained simply

by an inhibition of L-type Ca2+ currents. It has been suggested that the lengthening effect of

DAM on APD results mainly from the simultaneous reduction of both the slow inward calcium

current and the transient outward current, two antagonistic currents with unequal influences on

AP plateau development.(13) In guinea pig myocytes, DAM reduced voltage gated Ca2+ currents

and the inward and delayed rectifying K+ currents. (28)

In rat ventricular myocytes, DAM produced a rapid, dose-dependent, and reversible

blockade of gap junctional conductance (4), that potentially reduced conduction velocity in heart

muscle. Interestingly, DAM reduced gap junction conductance in neonatal rat cardiomyocytes

without changing the phosphorylation state of connexin 43, the main gap junctional protein in rat

hearts.(17) The reduced conductance of connexin 43 without a change in protein phosphorylation

13

state suggested that DAM acted at associated regulatory proteins that determine the functional

state of gap junctions (17) or acted by an entirely different mechanism. Our current results on

mouse hearts are consistent with these previous observations since DAM tended to decrease

conduction velocity in mouse ventricles and prolonged APDs, consistent with observations in rat

heart APs.

DAM was also shown to reduce L-type Ca2+ current in rabbit, rat, and guinea pig

ventricular myocytes. In rat heart trabeculae, DAM at 10-20 mM decreased peak systolic [Ca2+]i

with no significant changes in the time course of [Ca2+]i transients.(1) However, in murine

hearts, we found a substantial prolongation in the duration of [Ca2+]i transients induced by DAM.

Others have shown an increased sensitivity of mouse hearts to changes in [Ca2+]o within the

physiologic range compared to the rat (7) which could account for the different effects of DAM

on [Ca2+]i transient in mice and rats. In the current experiments, the rise time of [Ca2+]i

transients measured at 200 ms cycle length were increased by DAM from 14.1 1.2 to

25.0 3.31 ms (n=5). It is interesting to note that the rise times of [Ca2+]i in mouse hearts (14.1

1.2 ms) were considerably shorter than in guinea pig hearts (25.65 5 ms). (11)

Actions of Cyto-D

The exact molecular basis for cardiac contraction failure induced by cyto-D

remains unknown. Cytochalasins selectively bind to rapidly polymerizing and depolymerizing

actin filaments and disrupt F-actin of the cytoskeleton by binding to the net polymerizing end of

actin filament. Cyto-D has been the agent of choice to disrupt cytoskeletal actin but in skinned

rat myocytes, cyto-D interacts with sarcomeric actin and shifts the force vs. pCa curve to lower

pCa values. (8) The direct interaction of cyto-D with sarcomeric actin can account for the

inhibition of cardiac contractions and the interaction with cytoskeletal actin may account for the

various effects on ion channels. Other experiments suggest that cyto-D (10 M) does not directly

14

bind to cytoskeletal actin but reduces the activation of an actin-depolymerizing factor (cofilin)

that binds to actin, leading to the depolymerization of F-actin and the subsequent reduction of the

Ca2+ current, ICa-L. (35) Similarly, cyto-D was found to reduce the Na+ current through a decrease

in open probability and perhaps by slowing the inactivation rate. (40) The interaction of cyto-D

(10 M) with the cytoskeleton of guinea pig ventricular myocytes has been implicated as the

mechanism for reducing the inward and delayed rectifying K+ currents (31) and for the

acceleration of the run-down of ATP-sensitive K+ (KATP) channels.(19) Jalife et al recorded

optical APs from murine hearts and reported a prolongation of APDs by cyto-D and at higher

concentrations of cyto-D (80 M) reported a ‘hump’ on the plateau phase.(22) In conclusion, the

interplay of all these effects of cyto-D on ion channels must be considered to explain the changes

in AP and [Ca2+]i transients that are elicited by cyto-D.

Restitution Kinetics and Ventricular Arrhythmias

The restitution kinetics of APs was measured by pacing the heart at a basic cycle length

(S1-S1=200 ms) for 10 beats then applying a premature stimulus at varying S1-S2 intervals. The

short duration of the mouse heart AP made it difficult to detect small decreases in APDs of the

premature beats, particularly during the steep slope of the restitution curve. We therefore plotted

the restitution of APA that depends on the restitution of inward currents (INa and ICa) and

indirectly on K+ repolarizing currents. We have previously shown that APA decreases with

decreasing S1-S2 interval in mice. (3)

DAM and cyto-D prolonged APDs in mouse ventricles as well as the APD vs. cycle

length and AP amplitude restitution curve. The changes in APD as a function of cycle length are

most likely mediated by rate dependent changes in Ca2+ and K+ conductance. (10) DAM and

cyto-D caused marked changes in the restitution curve of AP amplitude; both increased

refractoriness, made the curve steeper at long cycle lengths and flatter at short cycle lengths.

15

Their effects on refractoriness may be due to their actions on both depolarizing and repolarizing

currents which in the mouse are dominated by voltage gated sodium and calcium channels, INa,

ICa-L and by the transient outward currents I t.o.,f and I t.o.,s. more related to the delayed rectifier

potassium current as previously described in other species.

Electrical restitution has been suggested to play an important role in the initiation and

maintenance of arrhythmias.(24) The rationale behind the restitution kinetics hypothesis is that a

dynamic change in APD (i.e. long to short APD) causes the subsequent wave front to encounter

refractory myocardium resulting in unidirectional conduction block and wave breakup, which

promotes the initiation and maintenance of VF. (24) Theoretical and experimental studies have

proposed that the slope of the restitution curve can serve as an index of vulnerability to

arrhythmia. (9, 20, 25, 33, 34) If the slope is > 1, a small perturbation in diastolic interval (DI)

produces a larger change in APD, which becomes amplified upon iteration. Eventually, DI

reaches a value shorter than the refractory period resulting in local conduction block, wave break

and turbulence.

Hearts treated with cyto-D or DAM had flatter restitution curves and had a reduced

vulnerability to arrhythmias, which is consistent with the restitution kinetics hypothesis. Indeed,

burst pacing consistently elicited ventricular arrhythmias in control mice but in hearts treated

with an uncoupler, all attempts to induce an arrhythmia failed (n = 5/5 for DAM and n = 5/5 for

DAM). The anti-arrhythmic actions of chemical uncouplers in murine hearts could also be due to

the lengthening of reentrant wavelengths as a result of prolonged APDs and slower conduction

velocity. In swine hearts, Lee et al showed that DAM converts ventricular fibrillation (VF) to

tachycardia whereas cyto-D did not alter the organization or dynamics of fibrillation. (26)

16

Can Mouse Ventricles Fibrillate?

Vaidya et al challenged the critical mass hypothesis for fibrillation by showing that VT

(n=4/5) and VF (n=4/7) could be induced by burst pacing the small heart of a mouse. (41) In

contrast, the present experiments failed to elicit VF even in mice that were not treated with a

chemical uncoupler. Here, VTs were readily obtained by burst pacing, were long lasting and did

not progress to VF. It should be noted; however, that the stimulation protocol used by Vaidya et

al was significantly different from the current protocol, as multiple bursts (20 stimuli each) of

different cycle length (shorter than that needed for 1:1 capture), of various stimulation strengths

and at various location were needed to elicit VT or VF.(41) In addition, VF was only observed in

hearts that were not perfused with DAM and the numbers of bursts needed to elicit VF and the

duration of VF episodes was not reported. (41)

Distribution of Rhod-2 in mouse Hearts

This study validates the measurement of cytosolic [Ca2+]i in perfused mouse hearts with

Rhod-2. A criticism against the use of Rhod-2 to measure cytosolic [Ca2+]i is that the dye has a

positive charge, which results in dye accumulation in the mitochondria (due to their negative

potential inside-120 to –180 mV). The mitochondrial content could raise the background

fluorescence and give errors in diastolic and systolic [Ca2+]i. We have previously shown in

perfused rabbit hearts that Rhod-2 can be used to selectively measure intracellular cytosolic

calcium with negligible contributions from dye loaded in mitochondria, sarcoplasmic reticulum

or nuclei. In addition, Rhod-2 loaded in endothelial cells was minimal making it the best dye to

measure cytosolic [Ca2+]i transients in intact hearts. (15) We further examined the distribution of

Rhod-2 in isolated guinea pig hearts and showed that the dye was remarkably selective for the

cytosol and did not load in mitochondria compared to Fura-2 and Fluo-3. (15) Here, we

examined the distribution of Rhod-2 in mouse hearts by loading perfused hearts with Rhod-2,

17

immobilizing the heart with 20 mM KCl and placing the immobilized heart on a confocal

microscope. As shown in figure 4A, epicardial cells from the perfused heart were effectively and

rapidly loaded with Rhod-2 within 5-10 min after passing a bolus of dye with the coronary

perfusate. Rhod-2 fluorescence did not exhibit a punctate appearance of mitochondrial loading

that is typically seen with tetramethylrhodamine ethyl ester (TMRE, a voltage sensitive dye used

to measure mitochondrial potential). (15) The subsequent perfusion of the same hearts with

digitonin revealed that Rhod-2 became freely permeable across the plasma membrane and within

5-10 min diffused out of the cells and was washed out by the perfusion. No significant dye was

found trapped in subcellular organelles (Fig. 4B). Similar results were obtained with confocal

images of mouse, guinea pig and rabbit myocytes. Thus, reports of mitochondrial dye

accumulation appear to be highly dependent on staining conditions (temperature and time).

Reversibility of Chemical Uncouplers

Most studies observed an effective recovery of contractions after washing out DAM but

not after wash out cyto-D. In mouse hearts perfused with DAM or cyto-D (at concentrations that

decreased left ventricular pressure by 90%), the subsequent wash out of the uncouplers

partially reversed the suppression of developed pressure by 50% of the original pressure with

cyto-D and 70% with DAM. Other studies in mouse myocytes and canine trabeculae showed that

the block of force by cyto-D could not be reversed by extensive washing. (5, 22) Our partial

recovery of force after treatment with cyto-D in the present experiments was most likely due to

the lower concentrations used to block contractions.

In summary, DAM and cyto-D inhibit contraction but are far from being ideal uncouplers

and should be used with caution in studies of arrhythmia mechanisms because of marked effects

on APs, [Ca2+]i transients, APDs, conduction velocity and refractoriness.

18

Acknowledgements

This study was supported by National Institutes of Health (NIH) grants HL

59614, HL 57929 and HL 69097 to Dr. G. Salama, HL-70250 to Dr. Barry London and

Postdoctoral Fellowships from the Western Pennsylvania Affiliate of the American Heart

Association to Drs. L Baker, R. Wolk, A. Shah and B-R Choi. The authors thank William

Hughes of our Departmental machine shop for the construction of optical components

and the mouse chamber and Greg J. Szekeres of our Departmental electronic shop for

building the computer interface.

19

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16. Dillon SM. Optical recordings in the rabbit heart show that defibrillation strength shocks prolong the duration of depolarization and the refractory period. Circ Res 69: 842-856, 1991. 17. Duthe F, Dupont E, Verrecchia F, Plaisance I, Severs NJ, Sarrouilhe D, and Herve JC. Dephosphorylation agents depress gap junctional communication between rat cardiac cells without modifying the Connexin43 phosphorylation degree. Gen Physiol Biophys 19: 441-449, 2000.18. Efimov IR, Huang DT, Rendt JM, and Salama G. Optical mapping of repolarization and refractoriness from intact hearts. Circulation 90: 1469-1480, 1994. 19. Furukawa T, Yamane T, Terai T, Katayama Y, and Hiraoka M. Functional linkage of the cardiac ATP-sensitive K+ channel to the actin cytoskeleton. Pflugers Arch 431: 504-512, 1996.20. Gilmour RF and Chialvo DR. Electrical restitution, critical mass, and the riddle of fibrillation. J Cardiovasc Electrophysiol 10: 1087-1089., 1999. 21. Gwathmey JK, Hajjar RJ, and Solaro RJ. Contractile deactivation and uncoupling of crossbridges. Effects of 2,3-butanedione monoxime on mammalian myocardium. Circ Res 69: 1280-1292, 1991. 22. Jalife J, Morley GE, Tallini NY, and Vaidya D. A fungal metabolite that eliminatesmotion artifacts. J Cardiovasc Electrophysiol 9: 1358-1362, 1998. 23. Kameyama T, Chen Z, Bell SP, Fabian J, and LeWinter MM. Mechanoenergetic studies in isolated mouse hearts. Am J Physiol 274: H366-374, 1998. 24. Karma A. Electrical alternans and spiral wave breakup in cardiac tissue. Chaos 4: 461-472, 1994. 25. Karma A. Spiral breakup in model equations of action potential propagation in cardiac tissue. Physical Review Letters 71: 1103-1106, 1993. 26. Lee MH, Lin SF, Ohara T, Omichi C, Okuyama Y, Chudin E, Garfinkel A, Weiss JN, Karagueuzian HS, and Chen PS. Effects of diacetyl monoxime and cytochalasin D on ventricular fibrillation in swine right ventricles. Am J Physiol Heart Circ Physiol 280: H2689-2696, 2001. 27. Li T, Sperelakis N, Teneick RE, and Solaro RJ. Effects of diacetyl monoxime on cardiac excitation-contraction coupling. J Pharmacol Exp Ther 232: 688-695, 1985. 28. Liu Y, Cabo C, Salomonsz R, Delmar M, Davidenko J, and Jalife J. Effects of diacetyl monoxime on the electrical properties of sheep and guinea pig ventricular muscle.Cardiovasc Res 27: 1991-1997, 1993. 29. London B. Cardiac arrhythmias: from (transgenic) mice to men. J CardiovascElectrophysiol 12: 1089-1091, 2001. 30. Lopatin AN and Nichols CG. 2,3-Butanedione monoxime (BDM) inhibition of delayed rectifier DRK1 (Kv2.1) potassium channels expressed in Xenopus oocytes. J Pharmacol Exp Ther 265: 1011-1016, 1993. 31. Mazzanti M, Assandri R, Ferroni A, and DiFrancesco D. Cytoskeletal control of rectification and expression of four substates in cardiac inward rectifier K+ channels. Faseb J 10: 357-361, 1996. 32. Prabhu SD and Salama G. Reactive disulfide compounds induce Ca2+ release fromcardiac sarcoplasmic reticulum. Arch Biochem Biophys 282: 275-283, 1990. 33. Qu Z, Weiss JN, and Garfinkel A. Cardiac electrical restitution properties and stability of reentrant spiral waves: a simulation study. Am J Physiol 276: H269-283., 1999. 34. Riccio ML, Koller ML, and Gilmour RF. Electrical restitution and spatiotemporalorganization during ventricular fibrillation. Circ Res 84: 955-963., 1999.

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35. Rueckschloss U and Isenberg G. Cytochalasin D reduces Ca2+ currents via cofilin-activated depolymerization of F-actin in guinea-pig cardiomyocytes. J Physiol 537: 363-370, 2001.36. Salama G, Kanai A, and Efimov IR. Subthreshold stimulation of Purkinje fibers interrupts ventricular tachycardia in intact hearts. Experimental study with voltage-sensitive dyes and imaging techniques. Circ Res 74: 604-619, 1994. 37. Salama G, Lombardi R, and Elson J. Maps of optical action potentials and NADH fluorescence in intact working hearts. Am J Physiol 252: H384-394, 1987. 38. Salama G and Morad M. Merocyanine 540 as an optical probe of transmembraneelectrical activity in the heart. Science 191: 485-487, 1976. 39. Sellin LC and McArdle JJ. Multiple effects of 2,3-butanedione monoxime. PharmacolToxicol 74: 305-313, 1994. 40. Undrovinas AI, Shander GS, and Makielski JC. Cytoskeleton modulates gating of voltage-dependent sodium channel in heart. Am J Physiol 269: H203-214, 1995. 41. Vaidya D, Morley GE, Samie FH, and Jalife J. Reentry and fibrillation in the mouseheart. A challenge to the critical mass hypothesis. Circ Res 85: 174-181, 1999. 42. Verrecchia F and Herve JC. Reversible blockade of gap junctional communication by 2,3-butanedione monoxime in rat cardiac myocytes. Am J Physiol 272: C875-885, 1997. 43. Wiggins JR, Reiser J, Fitzpatrick DF, and Bergey JL. Inotropic actions of diacetyl monoxime in cat ventricular muscle. J Pharmacol Exp Ther 212: 217-224, 1980. 44. Wu J, Biermann M, Rubart M, and Zipes DP. Cytochalasin D as excitation-contraction uncoupler for optically mapping action potentials in wedges of ventricular myocardium. J Cardiovasc Electrophysiol 9: 1336-1347, 1998.

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Table 1. Effects of chemical uncouplers on APDs and Refractory Periods

Control DAM Cyto-DAPD75 (ms) 20.0 3 46.6 5 39.9 8

RefractoryPeriods (ms)

45.1 12 82.5 4 78 4

All values are means SD, p values compared APs in controls vs. the presence of the chemical uncoupler.

Table 2. Calibration of [Ca2+]i transients with and without Chemical Uncouplers

Diastolic [Ca2+]i Systolic [Ca2+]i Duration of[Ca2+]i

transientsControl (n = 4) 257 30 nM 743 47 nM 54.3 5 ms

DAM (n = 4) 224 31 nM, p = 0.25 398 44 nM, p = 0.0007 83.6 1.9 ms

Cyto-D (n = 4) 544 180 nM, p = 0.06 944 17 nM, p = 0.01 58.6 1.2 ms

All values are means SD, p values compared [Ca2+]i in controls vs. the presence of the chemical uncoupler.

23

Figure Legends

Figure 1. Mapping Electrical Activity of Mouse Hearts

a: Digital picture of a mouse heart placed in a chamber designed to reduce movement artifacts. A

silhouette of the array is superimposed on the left ventricle of the mouse heart to identify the

region of tissue viewed by the photodiode array. APs recorded simultaneously from 4 regions of

the ventricle are illustrated to demonstrate the quality of signals sampled at 12-bit resolution,

1,000 frames per s, from a 330x330 m2 area of tissue.

b, c and d: Effects of Chemical Uncouplers on mouse APs

Optical APs were recorded from control (b), DAM (c) and Cyto-D (d) treated hearts then

displayed on a symbolic map of the array, where each box represents a diode in which the AP

recorded by that diode is shown. Optical APs from 1 diode are shown at a fast sweep speed to

illustrate the marked changes in the time course and shape of AP elicited by DAM and cyto-D.

Note the prolongation of APDs with DAM and cyto-D and the spike and dome appearance of AP

in cyto-D.

Figure 2. Effects of DAM and cyto-D on Restitution Kinetics and Conduction velocity

a) Plot of APD75 vs. CL for control hearts compared to perfusion with DAM and cyto-D.

Each data point represents the mean of APD75 measured from 8 diodes at the center of the

array times 4 hearts, before and after perfusion with DAM or cyto-D. Data obtained before

perfusion with a chemical uncoupler are grouped in one trace. DAM and cyto-D enhanced

the rate dependence of APDs in mouse hearts. The SD of each data point was 5% of the

mean.

b) Restitution kinetics of the AP amplitude. Hearts were paced at a basic CL (S1-S1=200

ms) and every 10th beat, a premature impulse was applied at varying S1-S2 intervals.

24

S1-S2 varied in steps of 5 ms and then steps of 1-ms as S1-S2 approached the refractory

period. Note that DAM and cyto-D increased refractory periods. The SD of each data

point was 5% of the mean.

c) Conduction velocities in control, DAM or cyto-D. At time, t = 0 DAM or cyto-D were

added to the perfusate and conduction velocity was measured every 5 min for an hour.

The SD of each data point was 5% of the mean.

Figure 3. Effects of Chemical Uncouplers on [Ca2+]i transients

a) Map of simultaneously recorded [Ca2+]i transients from a mouse heart loaded with Rhod-

2. The [Ca2+]i transients recorded by each diode are drawn in their location in the

symbolic map of the array.

b) [Ca2+]i transients recorded in control conditions from four diodes are shown at fast

sweep speed.

c) Calibration of [Ca2+]i transients for controls and hearts perfused with DAM or cyto-D.

Diastolic and systolic [Ca2+]i levels increased with Cyto-D but decreased with DAM.

Figure 4. Distribution of Rhod-2 in Murine Heart

a) Confocal fluorescence image of epicardial cells from a mouse heart loaded with Rhod-

2/AM and perfused with dye-free Ringer’s solution. Rhod-2 images did not produce a

punctuate appearance typical of mitochondrial dye loading but exhibited a rather

homogeneous distribution of fluorescence with the expected exclusion of dye from

regions dense with contractile proteins.

b) The possibility that Rhod-2/AM diffuses, accumulates and becomes trapped in the

mitochondria was further tested by permeabilizing the cells with digitonin, which

25

allowed for the washout of cytosolic Rhod-2 with no detectable levels of Rhod-2 trapped

in mitochondria.

Figure 5. Anti-Arrhythmic Effects of Uncouplers

Burst stimulation was applied after a basic drive rate (S1-S1=200 ms) of 10 beat and was

followed by an interruption of pacing. Burst stimulation applied near the apex of the left

ventricle failed to produce arrhythmias in hearts perfused with DAM (n = 5) (A) or cyto-D (n =

5) (B). In contrast, burst pacing of control mouse hearts induced VT (n = 8/10). Dark Bar

denotes the interval during which burst pacing was applied.

Figure 6. A) Action Potentials from a mouse heart one minute after the arrhythmia began.

This an example of optical action potentials from one diode recorded from a control mouse

with no addition of DAM or Cyto-D.

B) FFT Spectra of a mouse heart one minute after the arrhythmia began. FFT spectra were

analyzed over a 1.25 second time interval. Per spectra of voltage oscillations displayed a

monomorphic VT with one single dominant frequency of 13 Hz.

C) Activation Map from a mouse heart one minute after the arrhythmia began. In this and

subsequent activation maps, the first site to depolarize is depicted in “light gray” (i.e., time

t=0.0ms) and subsequent depolarizations are depicted in increasingly darker shades, with

isochronal lines 1 ms apart.

D) Action Potentials from a mouse heart three minutes after the arrhythmia began.

26

E) FFT Spectra of a mouse heart three minutes after the arrhythmia began. The FFT

spectrum analyzed over a 1.25 second interval, displayed a power spectrum with one single

dominant frequency of 12.5 Hz indicative of a monomorphic VT.

F) Activation map from the mouse heart three minutes after the onset of VT.

27

Figure 1

Time (sec)

Time (sec) Time (sec)

0.2 sec

a) Ventricular Action Potentials b) Control

c) DAM d) Cyto-D

Figure 2

0

20

40

60

80

100

120

0 20 40 60 80 100 120 140 160 180 200

S1-S2 Interval (ms)

% A

PA

ControlDAMCyto-D

b)

a)

c)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40 50 60Minutes

Velo

city

(m/s

)

ControlDAMCyto-D

0

10

20

30

40

50

60

70

40 60 80 100 120 140 160 180 200

Cycle Length (ms)

APD

75(m

s)

ControlDAMCyto-D

Figure 3

a)

b)

0.5 1.0 1..5 2.0

0

200

400

600

800

1000

Control Cyto-D

DAM

Time (sec)

Cai

Time (sec)

c)

(nM)

Figu

re 4

a)

b)

Figure 5

a) DAM

b) Cyto-D

c) Control

Time (sec)

Time (sec)

Time (sec)

Burst Pacing

Burst Pacing

Pacing

Figure 6

Time (sec)

Hertz

a) 1 min of arrhythmia b) FFT spectrum c) Activation Map

d) 3 min of arrhythmia e) FFT spectrum f) Activation Map

Hertz

Time (sec)