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Am J Physiol Heart Circ Physiol 293: H2472-H2478, 2007. First published July 27, 2007; doi:10.1152/ajpheart.00359.2007
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Effects of metabolic inhibition on conduction, Ca transients, and arrhythmia vulnerability in embryonic mouse hearts

Fuhua Chen,1,2 Carlos De Diego,3,4 Lai-Hua Xie,3,4 Jun-Hai Yang,3,4 Thomas S Klitzner,1 and James N Weiss1,3,4

1Cardiovascular Research Laboratory and Departments of 2Pediatrics (Cardiology), 3Medicine (Cardiology), and 4Physiology, David Geffen School of Medicine at University of California, Los Angeles, California

Submitted 21 March 2007 ; accepted in final form 25 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Developing myocardium is more dependent on glycolysis than adult myocardium, yet the effects of selectively inhibiting glycolysis versus oxidative phosphorylation on embryonic heart function have not been well characterized. Accordingly, we investigated how selective metabolic inhibition affects membrane voltage and intracellular Ca (Cai) transients in embryonic mouse hearts, including their susceptibility to arrhythmias. A total of 136 isolated embryonic mouse hearts were exposed to either 1) 2-deoxyglucose (2DG; 10 mM) or iodoacetate (IAA; 0.1 mM) with 10 mM pyruvate in place of glucose to selectively inhibit glycolysis or 2) the mitochondrial uncoupler protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 500 nM) with 10 mM glucose present to selectively inhibit oxidative phosphorylation. Using confocal imaging, we found that mitochondrial membrane potential monitored with tetramethylrhodamine methyl ester (200 nM) remained stable with 2DG or IAA but depolarized within 5 min after exposure to FCCP. IAA and FCCP decreased heart rate, inhibited Cai transient amplitude, shortened action potential duration at 80% repolarization (APD80), and prolonged atrioventricular conduction time to similar extents. Although 2DG decreased heart rate and Cai transient amplitude, it did not significantly affect APD80 and AV conduction time. In addition, spontaneous arrhythmias occurred in 77 of 136 embryonic hearts (57%) after exposure to IAA (28/53) or FCCP (49/83). There were no significant differences in the types or incidence of arrhythmias induced by IAA and FCCP. These data support the idea that both glycolysis and oxidative phosphorylation play critical metabolic roles in regulating cardiac function in the embryonic mouse heart.

embryonic heart development; calcium transient; arrhythmias

THE EMBRYONIC HEART operates and develops normally in a relatively hypoxic microenvironment. During embryogenesis, cardiac mitochondria convert fatty acids to energy at a very low rate compared with adult heart (25, 26) and glycolysis contributes more substantially to overall ATP production (17). This confers a benefit to the developing heart, since glucose requires less O2 than fatty acids to produce a given amount of ATP. Previous studies have shown that hypoxia causes less severe suppression of mechanical function in neonatal rabbit and canine hearts than in adult hearts from these species (14). It has been proposed, therefore, that embryonic and newborn hearts are better adapted to support mechanical function during hypoxia via enhanced metabolic flux through the glycolytic pathway.

On the other hand, mitochondria are abundant at early stages in embryonic hearts. The percent volume of mitochondria in rat myocytes was 17% at embryonic day 14 (E14) and increased to 35% at birth (12). In chicken embryos at Hamburg-Hamilton stages 18 to 29 (equivalent to mouse E11.5 to E14), mitochondrial-to-cell volume averaged 11% (7). As early as Hamburg-Hamilton stage 12 (approximately equivalent to E10 in the mouse embryo), electron micrographs showed that mitochondria were visible throughout the cytoplasm, although they were not specifically aligned with the myofilaments as in adult myocytes (20).

In the present study, we investigated the functionality of mitochondria in embryonic mouse heart during early cardiogenesis by comparing the effects of selective inhibition of oxidative phosphorylation versus glycolysis on action potential duration (APD), intracellular Ca (Cai) transients, and heart rhythm. Using optical mapping techniques to record mitochondrial membrane potential ({Delta}{Psi}m), sarcolemmal membrane potential, and Cai in embryonic mouse hearts, we found that selective inhibition of oxidative phosphorylation or glycolysis has roughly equivalent effects of decreasing heart rate, shortening APD, suppressing the amplitude of Cai transients, prolonging atrioventricular (AV) conduction time, and causing arrhythmias. These findings support the hypothesis that oxidative phosphorylation and glycolysis are both critically important for embryonic cardiac function.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Preparation and solutions. CD-1 mice were time-mated, and the day on which copulation plugs were observed was designated day 0.5 of pregnancy. On the desired gestational day, pregnant female mice were first sedated by inhalation of isoflurane and then killed by cervical dislocation (5, 30). The uterus was dissected, and whole embryos were exposed. Entire embryonic mouse hearts were then carefully excised from explanted embryos using a dissecting microscope. During the dissection procedure, the embryonic mice were bathed in modified oxygenated Tyrode's solution containing (in mM) 136 NaCl, 5.4 KCl, 0.1 CaCl2, 0.33 NaH2PO4, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.3). The same solution with 1.8 mM CaCl2 was used as the standard bath solution to superfuse embryonic hearts at 37°C in the experimental chamber. To selectively inhibit glycolysis, glucose was replaced with 10 mM pyruvate, and 10 mM 2-deoxyglucose (2DG) or 0.1 mM iodoacetate (IAA) was added (32). To selectively inhibit oxidative phosphorylation, the protonophore mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 500 nM) was added to the standard bath solution (32). All fluorophores were obtained from Molecular Probes (Eugene, OR). Other chemicals were obtained from Sigma Chemical (St. Louis, MO). This study conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996), and protocols were approved by the Chancellor's Animal Research Committee of the University of California at Los Angeles.

Confocal imaging of mitochondrial membrane potential. To monitor the change in mitochondrial membrane potential ({Delta}{Psi}m), the hearts were superfused with Tyrode's solution containing tetramethylrhodamine methyl ester (TMRM; 200 nM). TMRM is a lipophilic, membrane-permeant, monovalent cationic fluorescent dye that distributes itself in the mitochondrial matrix following the Nernst equation (16, 24). Time series and z stacks of hearts were acquired with a Zeiss LSM 5 Pascal confocal laser scanning microscope (Carl Zeiss, Jena, Germany) equipped with an inverted Axiovert 200 microscope and x10 objective. The combination of a 488-nm argon laser and 505-nm long-pass emission filters was used to visualize TMRM fluorescence. Subsequent image restoration and analysis (such as the intensity measurements) were done with LSM510 VisArt (Carl Zeiss) and Image J (NIH, Bethesda, MD).

Optical mapping. The experimental setup and methods for simultaneous optical imaging of membrane potential and Cai have been described in detail previously (30). In brief, for dual voltage and Cai mapping, isolated E10.5 to E11.5 embryonic mouse hearts were incubated with 5 µM rhod-2 acetoxymethyl ester (rhod-2 AM) dissolved in dimethyl sulfoxide and Pluronic F-127 (0.1%) for 40 min to image Cai, followed by a 5-min incubation with 5 µM RH-237 to image membrane voltage. Embryonic mouse hearts were then washed in Tyrode's solution before transfer to an experimental chamber (37°C) on a modified inverted microscope. Cai and voltage signals were simultaneously recorded using two electron-multiplier charge-coupled device (CCD) cameras operating at 200–500 frames/s (Cascade 128+; Photometrics, Tucson, AZ) with a spatial resolution of 128 x 128 pixels. In control experiments, we documented that with the filter settings used (30), RH-237 and rhod-2 AM fluorescence did not exhibit significant cross talk, confirming previous reports by other investigators using this dye combination (6, 9, 22). In some experiments Cai imaging alone was performed, substituting fluo-4 AM for rhod-2 AM using the same dye loading protocol, with a different CCD camera (model LCL 811K; Watec America, Las Vegas, NV) using an ATI video capture card (30 frames/s).

Data were analyzed using custom software, and detailed methods for measuring propagation, Cai transient, and APD are the same as described previously (30). Cai transient amplitude was measured as the fluorescence intensity difference ({Delta}F) immediately before systole to the peak during systole, averaged for four to six successive beats. Although activation sequences could be reliably measured from the timing of upstrokes of action potentials or Cai transients from all regions of the hearts, atrial and AV ring voltage recordings typically contained too much motion artifact to measure APD accurately. Therefore, APD at 80% repolarization (APD80) was measured only from ventricular tissue by selecting regions with the least amount of motion. Because the images were not confocal, the fluorescence traces represented an average of cells from both the anterior and posterior regions of the chamber. Care was taken to avoid overlap with the outflow tract, whose activation is delayed relative to the ventricles. AV conduction time was measured as the average time delay between the action potential upstroke (or Cai transient upstroke) from atrial and ventricular recording sites on either side of the AV ring. Student's t-test was used to compare means, with P < 0.05 considered statistically significant.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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Basal conduction properties of mouse embryo hearts. Figure 1 A shows simultaneous dual voltage (Vm) and Cai fluorescence images of a representative isolated E10.5 embryonic mouse heart during sinus rhythm, indicating the anatomical locations of the atria, AV ring, and ventricles. Figure 1B shows traces of Vm and Cai recorded from representative sites in the atria, AV ring, and ventricles, respectively, during control perfusion with 1.8 mM Ca-Tyrode's solution at 37°C. Figure 1C illustrates the conduction sequence in the form of a space-time plot, in which the y-axis shows the fluorescence intensity along the red line shown in Fig. 1A, plotted as a function of time along the x-axis. The action potential or Cai transient duration is thus indicated by the thickness of the bright (red-white) region, whereas conduction velocity through the atria, AV ring, and ventricles is proportional to the slope of the left edge of fluorescence trace through that region. The plot demonstrates that the heart beat originated in the atria and propagated slowly through the AV ring (shallow slope) and then rapidly through the ventricles (steep, almost vertical slope). Figure 1D shows pseudocolorized snapshots of Cai fluorescence at the various times indicated from another E10.5 embryonic mouse heart. Figure 1E shows an isochronal map constructed from the activation times, illustrating rapid conduction through atria, slow conduction through the AV ring (indicated by the bunching together of isochrome lines), and rapid conduction through the ventricles (see also Supplemental Video 1). Supplemental data for this article is available online at the American Journal of Physiology-Heart and Circulatory Physiology website.


Figure 1
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  		Fig. 1. Dual optical mapping of voltage and intracellular Ca (Cai) transient in an embryonic day 10.5 (E10.5) embryo mouse heart. A: representative example of simultaneously recorded voltage (top) and Cai fluorescence images (bottom) from an E10.5 heart dually loaded with RH-237 and rhod-2 AM. Locations of the atria (A), AV ring (AVR), and ventricles (V) are indicated. B: representative simultaneously recorded traces of voltage (black) and Cai fluorescence ({Delta}F/F; red) from sites in the atria, AVR, and ventricles, respectively, illustrating the conduction pattern in sinus rhythm. C: space-time plot of Cai fluorescence along the red line shown in A, demonstrating rapid activation of the atria (top), slow conduction through the AVR (upper middle), and rapid activation of the ventricles (bottom half). D: pseudocolored snapshots of Cai fluorescence at the various times indicated from another E10.5 mouse heart, demonstrating the activation sequence in sinus rhythm. E: isochronal map of propagation during normal sinus rhythm, illustrating bunching of isochromes due to slow conduction in the AVR. See Supplemental Video 1 for details.

 
Effects of selective metabolic inhibition on mitochondrial membrane potential. To image mitochondria, embryonic mouse hearts were superfused at room temperature with control Tyrode's solution containing 200 nM TMRM for 20 min in a chamber positioned on an inverted laser confocal microscope. Figure 2 A (see also Supplemental Video 2) illustrates a representative experiment from an E10.5 heart showing the z stack of TMRE fluorescence images. Bright TMRM fluorescence in the atria, AV ring, and ventricle indicates well-polarized mitochondria in the E10.5 embryonic mouse heart, although individual mitochondria cannot be visualized at this magnification. Similar findings were obtained in 10 additional hearts.


Figure 2
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  		Fig. 2. Effects of selective metabolic inhibition on mitochondrial membrane potential ({Delta}{Psi}m). A–C: by using tetramethylrhodamine methyl ester (TMRM) to image {Delta}{Psi}m, confocal images at 1-min intervals reveal that {Delta}{Psi}m remains well-polarized under control conditions (A) and during selective inhibition with glycolysis by iodoacetate (IAA; B) but is rapidly depolarized by the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) during selective inhibition of oxidative phosphorylation (C). D: summary of changes in TMRM fluorescence intensity in 6 control hearts, 5 exposed to IAA and 5 exposed to FCCP. TMRM fluorescence of the whole heart is expressed as a percentage of basal fluorescence vs. time, with images obtained every 15 s. Error bars indicate SD. [See Supplemental Video 2 for laser scanning of stacked (10-µm sections) TMRM images in an E10.5 embryonic mouse heart.]

 
Figure 2, B and C, shows the effects of selective inhibition of glycolysis (0.1 mM IAA and 10 mM pyruvate replacing glucose) versus oxidative phosphorylation (500 nM FCCP with 10 mM glucose present) on TMRM fluorescence. Mean fluorescence intensity was measured from confocal images every 15 s and expressed as the percentage of initial intensity after background subtraction. Neither control superfusion (Fig. 2A) nor selective glycolytic inhibition with IAA (Fig. 2B) caused any significant decrease in TMRM intensity, documenting that IAA did not cause mitochondrial depolarization. In contrast, selective inhibition of oxidative phosphorylation with the mitochondrial uncoupler FCCP significantly deceased TMRM fluorescence in all regions of the heart within 5 min (Fig. 2C), consistent with the loss of mitochondrial membrane potential ({Delta}{Psi}m). The average time course of changes in TMRM fluorescence from five hearts in each group is summarized in Fig. 2D. Similar to IAA, selective inhibition of glycolysis with 10 mM 2DG and 10 mM pyruvate replacing glucose had no significant effect on TMRM fluorescence (data not shown).

Effects of selective metabolic inhibition on electrophysiological function and Cai. Using dual voltage and Cai optical mapping, we next characterized the effects of selective inhibition of glycolysis or oxidative phosphorylation on heart rate, conduction time through the AV ring, and ventricular APD80. The top panels of Fig. 3, A–C, illustrate isochronal activation maps during sinus rhythm in three different embryonic mouse hearts under control conditions (A) and after superfusion with 0.1 mM IAA (B) or 500 nM FCCP (C). The middle panels show corresponding Vm and Cai traces (superimposed) from ventricles, and the bottom panels show space-time plots along the lines indicated in the middle panels. Figure 3D summarizes the average changes in heart rate, AV conduction time, and ventricular APD80 after 2DG, IAA, or FCCP. 2DG, IAA, and FCCP all slowed heart rate to a comparable degree, from 83 ± 2 to 71 ± 3 beats/min (n = 22, P < 0.05) after 2DG, from 89 ± 2 to 64 ± 2 beats/min (n = 34, P < 0.001) after IAA, and from 88 ± 3 to 49 ± 3 beats/min (n = 34, P < 0.001) after FCCP. 2DG had no significant effect on AV conduction time or ventricular APD80, whereas both IAA and FCCP significantly prolonged AV conduction time, from 201 ± 15 ms (n = 24) to 243 ± 28 ms with IAA (n = 21, P < 0.001) and 268 ± 22 ms with FCCP (n = 24, P < 0.001), and shortened ventricular APD80, from 203 ± 14 ms (n = 18) to 135 ± 11 ms with IAA (n = 18, P < 0.001) and 123 ± 10 ms with FCCP (n = 19, P < 0.001). The slower heart rate during metabolic inhibition cannot account for the APD shortening, since slowing heart rate typically prolongs APD.


Figure 3
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  		Fig. 3. Effects of selective inhibition of glycolysis (IAA) vs. oxidative phosphorylation (FCCP) on heart rate, AV conduction time, and ventricular action potential at 80% repolarization (APD80). A–C: isochronal maps (top), superimposed Vm (black) and Cai fluorescence traces (red), and space-time plots during sinus rhythm under control conditions (A) or 10 min after IAA (B) or FCCP (C) (bottom). Also see Fig. 1 legend. D: averaged data showing changes of heart rate (top), AV conduction time (middle), and ventricular APD80 (bottom). The number of hearts in each group is indicated in each column; bpm, beats/min. Error bars are SD. ***P < 0.001.

 
Figure 4, A–C, shows the effects of 2DG, IAA, and FCCP on the ventricular Cai transient amplitude in three representative embryonic hearts, using each heart as its own control. After 10 min of exposure to selective metabolic inhibition, Cai transient amplitude declined by 37 ± 7% after 2DG (n = 18, P < 0.05), 47 ± 6% after IAA (n = 18, P < 0.05), and 42 ± 5% after FCCP (n = 20, P < 0.05). Figure 4D summarizes the data from all preparations. In contrast, under control conditions with maintained superfusion with Tyrode's solution, the Cai transient amplitude decrease due to dye bleaching, and other factors averaged 12 ± 5% over 25–30 min (n = 35), that is, <5% over the comparable 10-min time course over which the effects of metabolic inhibitors were studied. The modest decrease in heart rate during metabolic inhibition also is unlikely to account for the decrease in Cai transient amplitude, since under control conditions, increasing heart rate by pacing from 175 to 430 beats/min led to a progressive decrease (averaging 55% in 8 hearts), rather than an increase in Cai transient amplitude (the typical negative staircase in rodent heart).


Figure 4
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  		Fig. 4. Effects of selective inhibition of glycolysis [2-deoxyglucose (2DG), IAA] vs. oxidative phosphorylation (FCCP) on Cai transient amplitude in embryonic mouse hearts. A–C: Cai transients ({Delta}F) from a representative site in the ventricles before and after perfusion for 10 min with 2DG (A), IAA (B), and FCCP (C) in 3 E10.5 mouse hearts. The vertical positioning of the 2 traces is arbitrary, and the effects of bleaching on the signals (<5% in control experiments) have been disregarded. D: summary of average Cai transient amplitudes before and 10 min after selective metabolic inhibition. The number of myocytes in each group is indicated. *P < 0.05.

 
Arrhythmias induced by selective metabolic inhibition. In 136 mouse embryo hearts exposed to selective inhibition of glycolysis or oxidative phosphorylation, 77 developed arrhythmias of various types. Arrhythmias occurred with a similar incidence (50–60%) in both forms of metabolic inhibition (Fig. 5). The most common types of arrhythmias observed included sinus bradycardia/sinus arrest, AV block, AV ring reentry, and various ventricular arrhythmias, such as premature ventricular contractions, ectopic ventricular rhythms with intact ventricular-atrial conduction, and ventricular tachycardia (VT). Ventricular fibrillation (VF) was not observed in any hearts. Figure 6 A (see also Supplemental Video 3) shows a typical example of sinus rhythm with 2:1 AV block during exposure to FCCP in an E10.5 heart loaded with fluo-4 AM. In this case, every other atrial Cai transient failed to propagate through the upper portion of the AV ring area.


Figure 5
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  		Fig. 5. Arrhythmias during selective inhibition of glycolysis (IAA) or oxidative phosphorylation (FCCP). A: incidence of arrhythmias in IAA and FCCP groups. B: frequency of different types of arrhythmias observed: sinus bradycardia (SB), sinus arrest (SA), AV block (AVB), premature ventricular contraction (PVC), ventricular tachycardia (VT), ventricular atrial conduction (VAC), and AVR reentry (AVRR). Other arrhythmias (miscellaneous, Misc) include 1 junctional rhythm and 1 ectopic wandering atrial rhythm. The number of arrhythmias of type is indicated in B.

 

Figure 6
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  		Fig. 6. Examples of arrhythmias during metabolic inhibition. A: AV conduction block (2:1) during selective metabolic inhibition with FCCP. Left, Cai fluorescence images illustrating the locations of the right (RA) and left atria (LA), AVR, and right (RV) and left ventricles (LV) in an E10.5 embryonic mouse heart loaded with fluo-4 AM. Right, space-time plot along the line shown in A, demonstrating 2:1 conduction between the atria and ventricles. The site of block is located at the entrance to the AVR. (See also Supplemental Video 3). B: AVR reentry during selective metabolic inhibition with FCCP. Top, representative Cai fluorescence traces from selected sites in the atria (A), AVR, and ventricles (V) during AVR reentry in an E10.5 embryonic mouse heart loaded with fluo-4 AM. Bottom, corresponding space-time plot of Cai fluorescence along a line extending from the atria through the AVR to the ventricles, illustrating anterograde and retrograde conduction through the AVR. Note that the first atrial beat conducts anterogradely through the AVR to the ventricles and then retrogradely back to the atria. The second atrial beat then conducts anterogradely through the AV ring to the ventricles but is unable to conduct retrogradely because of a premature atrial beat, with which it collides in the AVR, terminating reentry. The pattern then repeats (see also Supplemental Video 4). C: nonsustained ventricular tachycardia during selective metabolic inhibition with FCCP. Top, Cai fluorescence traces. Bottom, corresponding space-time plot along a line crossing from the atria through the AVR to the ventricles in an E10.5 embryonic myocyte loaded with fluo-4 AM. Sinus beats are followed by 2–5 beat bursts of VT that conduct retrogradely to the atria (see also Supplemental Video 5).

 
Figure 6B (see also Supplemental Video 4) shows an example of AV ring reentry in a fluo-4 AM-loaded E10.5 heart exposed to FCCP. Spatially dissociated anterograde and retrograde conduction pathways through the AV ring led to reciprocating tachycardia, analogous to dual AV nodal pathway conduction in adult hearts. Anterograde conduction from the atria proceeded through the AV ring, activated the ventricle, and then immediately turned back through the AV ring to activate the atria retrogradely. The reentrant atrial excitation then failed to propagate through the AVR, terminating reentry. This sequence repeated itself multiple times. In other hearts, reentry was self-sustained for 3–25 min, similar to our group's previous description (30) of AV ring reentry in embryonic mouse hearts induced by combined beta-adrenergic and muscarinic stimulation. Among the nine hearts exhibiting AV ring reentry, five occurred with FCCP and two with IAA.

Figure 6C (see also Supplemental Video 5) shows an example of nonsustained VT in a fluo-4 AM-loaded E10.5 heart during exposure to FCCP. Cai transient recordings (top) and the corresponding space-time plot (bottom) demonstrate that following each sinus beat, there was a fast salvo of two to five ventricular beats that conducted retrogradely through the AV ring to the atria. To our knowledge, this is the first documentation of rapid VT at such an early embryonic stage. VT was observed in three hearts, two after FCCP and one after IAA.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding of this study is that the isolated embryonic mouse heart responds similarly to selective inhibition of either glycolysis or oxidative phosphorylation. Specifically, both forms of metabolic inhibition caused comparable slowing of heart rate, prolongation of AV conduction time, shortening of the ventricular APD, suppression of the ventricular Cai transient, and a similar incidence and spectrum of arrhythmias. A limitation is that, for technical reasons, we were unable to accurately characterize changes in diastolic Cai, which may be particularly important for arrhythmogenesis. We also did not pace the heart to exclude the possibility that changes in heart rate contributed to changes in APD or Cai transient amplitude. However, in the adult rodent heart, APD and Cai transient amplitude typically increase as heart rate slows (2, 1), opposite to the changes caused by metabolic inhibition. In control experiments, we also directly confirmed that Cai transient amplitude in embryonic mouse hearts also decreased at faster heart rates.

We documented that {Delta}{Psi}m remained normal during selective glycolytic inhibition with either 2DG or IAA, suggesting that with pyruvate present, oxidative phosphorylation was unaffected, whereas selective inhibition of oxidative phosphorylation with the protonophore FCCP caused severe mitochondrial depolarization. Although we did not measure cellular high-energy phosphate levels in the present study, Murphy et al. (21) reported a significant decrease in ATP during exposure to the oxidative phosphorylation inhibitor rotenone (0.1 mM), which was accelerated when combined with glycolytic inhibition by IAA (1 mM). These data suggest that both oxidative and glycolytic pathways are important in maintaining ATP in embryonic hearts. On the other hand, glycolytic ATP accounts for only 5% of total ATP generation in adult mammalian heart (29), and selective inhibition of glycolysis does not acutely depress cellular high-energy phosphate levels as long as a substrate for oxidative phosphorylation such as pyruvate is available (32). Whether the same is true in embryonic hearts, however, remains to be demonstrated. Since the developing cardiovascular system normally operates in a hypoxic environment, vertebrate embryonic hearts display a relatively low rate of oxidative phosphorylation (17, 26). Consequently, it is generally believed that embryonic hearts are more heavily dependent on glycolysis. In the adult heart, selective inhibition of glycolysis has qualitatively different functional consequences than selective inhibition of oxidative phosphorylation (27, 32). These differences may be related to preferential energy channeling as the heart matures (28, 34). Specifically, it has been proposed that glycolytically derived ATP may be preferentially available to membrane-bound pumps and ion channels (3, 4, 11, 13, 15, 18, 23, 27, 32, 33), whereas mitochondrial ATP may be preferentially utilized by the contractile apparatus, with creatine kinase and adenylate kinase acting to facilitate high-energy phosphate delivery to these compartments in the heart. Since our study in intact embryonic heart revealed no obvious differences between selective inhibition of glycolysis and oxidative phosphorylation, this may imply that the development of cardiac energy channeling occurs postnatally, in response to the greater diffusion limitations of high-energy phosphates as the size of cardiac myocytes increases, as well as with the switch of excitation-contraction coupling from primarily transsarcolemmal Ca fluxes to predominantly intracellular Ca cycling as the sarcoplasmic reticulum matures. However, it is also possible that our optical mapping techniques are not sufficiently sensitive to detect subtle differences in metabolic responses. Different functional effects of selective metabolic inhibition have been noted previously in cultured embryonic chick heart cells (8, 10), although cultured cells may rapidly differentiate (6). Finally, since we studied isolated embryonic hearts, we cannot exclude the possibility that embryonic hearts in vivo behave differently.

The mechanism of arrhythmias during selective metabolic inhibition is unknown. However, the similar incidence and types of arrhythmias observed after selective inhibition of either glycolysis or oxidative phosphorylation suggests that they share a common metabolic etiology. Sinus bradycardia may be related to suppression of the Cai transient, which has recently been shown to play a key role in pacemaking by sinus node (19) and embryonic heart cells (31). Our findings establish that reentry during metabolic inhibition is possible despite the small size of the embryonic heart at this stage (<2 mm in diameter), since we observed AV ring reentry (Fig. 6) in 9 of 136 hearts. Whether the ventricular arrhythmias were due to automaticity, triggered activity, or reentry, however, remains a matter of speculation. In none of 136 hearts exposed to selective metabolic inhibition was VF detected. The heart may be too small at this stage to support multiple wavelets, since even in adult murine hearts, VF is uncommon and usually nonsustained.

In summary, using optical mapping techniques to record mitochondrial membrane potential, membrane voltage, and Cai in embryonic mouse hearts, we found that selective inhibition of oxidative phosphorylation or glycolysis has roughly equivalent effects of decreasing heart rate, shortening APD, suppressing the amplitude of the Cai transients, prolonging AV conduction time, and causing arrhythmias. These findings support the hypothesis that oxidative phosphorylation and glycolysis are both critically important for embryonic cardiac function.


   GRANTS
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 GRANTS
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The study was partially funded by American Heart Association Western States Affiliate and the Laubisch and Kawata Endowments.


   ACKNOWLEDGMENTS
 
We thank L. Pan, A. Dave, and M. Valderrábano for experimental assistance and J. Parker, S. Lamp, and T. Duong for technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. N. Weiss, David Geffen School of Medicine at UCLA, 675 Charles Young Drive So. 3645 MRL, Los Angeles, CA 90095-1760 (e-mail: jweiss{at}mednet.ucla.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 DISCUSSION
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  1. Anumonwo JM, Tallini YN, Vetter FJ, Jalife J. Action potential characteristics and arrhythmogenic properties of the cardiac conduction system of the murine heart. Circ Res 89: 329–335, 2001.[Abstract/Free Full Text]
  2. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. Dordrecht, The Netherlands: Kluwer, 1991, p. 167–170.
  3. Bessman SP, Geiger PJ. Transport of energy in muscle: the phosphorylcreatine shuttle. Science 211: 448–452, 1981.[Abstract/Free Full Text]
  4. Bricknell OL, Opie LH. Effects of substrates on tissue metabolic changes in the isolated rat heart during underperfusion and on release of lactate dehydrogenase and arrhythmias during reperfusion. Circ Res 43: 102–115, 1978.[Abstract/Free Full Text]
  5. Chen F, Klitzner TS, Weiss JN. Autonomic regulation of calcium cycling in developing embryonic mouse hearts. Cell Calcium 39: 375–385, 2006.[CrossRef][ISI][Medline]
  6. Choi BR, Salama G. Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans. J Physiol 529: 171–188, 2000.[Abstract/Free Full Text]
  7. Clark EB, Hu N, Dummett JL, Vandekieft GK, Olson C, Tomanek R. Ventricular function and morphology in chick embryo from stages 18 to 29. Am J Physiol Heart Circ Physiol 250: H407–H413, 1986.[Abstract/Free Full Text]
  8. Doorey AJ, Barry WH. The effects of inhibition of oxidative phosphorylation and glycolysis on contractility and high-energy phosphate content in cultured chick heart cells. Circ Res 53: 192–201, 1983.[Free Full Text]
  9. Fast VG. Simultaneous optical imaging of membrane potential and intracellular calcium. J Electrocardiol 38: 107–112, 2005.[CrossRef][ISI][Medline]
  10. Hasin Y, Doorey A, Barry WH. Electrophysiologic and mechanical effects of metabolic inhibition of high-energy phosphate production in cultured chick embryo ventricular cells. J Mol Cell Cardiol 16: 1009–1021, 1984.[CrossRef][ISI][Medline]
  11. Higgins TJC, Bailey PJ. The effects of cyanide and iodoacetate intoxication and ischaemia on enzyme release from the perfused rat heart. Biochim Biophys Acta 762: 67–75, 1983.[Medline]
  12. Hirakow R, Gotoh TA. A quantitative ultrastructural study on the developing rat heart. In: Developmental and Physiological Correlates of Cardiac Muscle, edited by Lieberman M, and Sano T. New York: Raven, 1975, p. 37–49.
  13. Hoerter JA, Ventura-Clapier R, Kuznetsov A. Compartmentation of creatine kinases during perinatal development of mammalian heart. Mol Cell Biochem 133–134: 277–286, 1994.[CrossRef]
  14. Jarmakani JM, Nakazawa M, Nagatomo T, Langer GA. Effect of hypoxia on mechanical function in the neonatal mammalian heart. Am J Physiol Heart Circ Physiol 235: H469–H474, 1978.[Abstract/Free Full Text]
  15. Jeremy RW, Koretsune Y, Marban E, Becker LC. Relation between glycolysis and calcium homeostasis in postischemic myocardium. Circ Res 70: 1180–1190, 1992.[Abstract/Free Full Text]
  16. Korge P, Weiss JN. Thapsigargin directly induces the mitochondrial permeability transition. Eur J Biochem 265: 273–280, 1999.[ISI][Medline]
  17. Lopaschuk GD, Collins-Nakai RL, Itoi T. Developmental changes in energy substrate use by the heart. Cardiovasc Res 26: 1172–1180, 1992.[Abstract/Free Full Text]
  18. Lynch RM, Paul RJ. Compartmentation of glycolytic and glycogenolytic metabolism in vascular smooth muscle. Science 222: 1344–1346, 1983.[Abstract/Free Full Text]
  19. Maltsev VA, Vinogradova TM, Lakatta EG. The emergence of a general theory of the initiation and strength of the heartbeat. J Pharmacol Sci 100: 338–369, 2006.[CrossRef][ISI][Medline]
  20. Manasek FJ. Histogenesis of the embryonic myocardium. Am J Cardiol 25: 149–168, 1970.[CrossRef][Medline]
  21. Murphy E, LeFurgey A, Lieberman M. Biochemical and structural changes in cultured heart cells induced by metabolic inhibition. Am J Physiol Cell Physiol 253: C700–C706, 1987.[Abstract/Free Full Text]
  22. Omichi C, Lamp ST, Lin SF, Yang J, Baher A, Zhou S, Attin M, Lee MH, Karagueuzian HS, Kogan B, Qu Z, Garfinkel A, Chen PS, Weiss JN. Intracellular Ca dynamics in ventricular fibrillation. Am J Physiol Heart Circ Physiol 286: H1836–H1844, 2004.[Abstract/Free Full Text]
  23. Parker JC, Hoffman JF. The role of membrane phosphoglycerate kinase in the control of glycolytic rate by active cation transport in human red blood cells. J Gen Physiol 50: 893–916, 1967.[Abstract/Free Full Text]
  24. Petronilli V, Penzo D, Scorrano L, Bernardi P, Di Lisa F. The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ. J Biol Chem 276: 12030–12034, 2001.[Abstract/Free Full Text]
  25. Pugh E, Sidbury JB Jr. Fatty acid oxidation in embryonic chick tissues. Biochim Biophys Acta 239: 376–383, 1971.[Medline]
  26. Romano R, Rochat AC, Kucera P, De Ribaupierre Y, Raddatz E. Oxidative and glycogenolytic capacities within the developing chick heart. Pediatr Res 49: 363–372, 2001.[ISI][Medline]
  27. Runnman EM, Lamp ST, Weiss JN. Enhanced utilization of exogenous glucose improves cardiac function in hypoxic rabbit ventricle without increasing total glycolytic flux. J Clin Invest 86: 1222–1233, 1990.[ISI][Medline]
  28. Saks V, Dzeja P, Schlattner U, Vendelin M, Terzic A, Wallimann T. Cardiac system bioenergetics: metabolic basis of the Frank-Starling law. J Physiol 571: 253–273, 2006.[Abstract/Free Full Text]
  29. Stanley WC, Recchia FA, Lopaschuk GD. Myocardial substrate metabolism in the normal and failing heart. Physiol Rev 85: 1093–1129, 2005.[Abstract/Free Full Text]
  30. Valderrabano M, Chen F, Dave AS, Lamp ST, Klitzner TS, Weiss JN. Atrioventricular ring reentry in embryonic mouse hearts. Circulation 114: 543–549, 2006.[Abstract/Free Full Text]
  31. Viatchenko-Karpinski S, Fleischmann BK, Liu Q, Sauer H, Gryshchenko O, Ji GJ, Hescheler J. Intracellular Ca2+ oscillations drive spontaneous contractions in cardiomyocytes during early development. Proc Natl Acad Sci USA 96: 8259–8264, 1999.[Abstract/Free Full Text]
  32. Weiss J, Hiltbrand B. Functional compartmentation of glycolytic versus oxidative metabolism in isolated rabbit heart. J Clin Invest 75: 436–447, 1985.[ISI][Medline]
  33. Weiss JN, Lamp ST. Glycolysis preferentially inhibits ATP-sensitive K+ channels in isolated guinea pig cardiac myocytes. Science 238: 67–69, 1987.[Abstract/Free Full Text]
  34. Weiss JN, Yang L, Qu Z. Systems biology approaches to metabolic and cardiovascular disorders: network perspectives of cardiovascular metabolism. J Lipid Res 47: 2355–2366, 2006.[Abstract/Free Full Text]




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