AJP - Heart Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 287: H1029-H1038, 2004. First published May 20, 2004; doi:10.1152/ajpheart.00814.2003
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/3/H1029    most recent
00814.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Grandy, S. A.
Right arrow Articles by Howlett, S. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grandy, S. A.
Right arrow Articles by Howlett, S. E.

Overexpression of human {beta}2-adrenergic receptors increases gain of excitation-contraction coupling in mouse ventricular myocytes

Scott A. Grandy, Eileen M. Denovan-Wright, Gregory R. Ferrier, and Susan E. Howlett

Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

Submitted 22 August 2003 ; accepted in final form 11 May 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study investigated cardiac excitation-contraction coupling at 37°C in transgenic mice with cardiac-specific overexpression of human {beta}2-adrenergic receptors (TG4 mice). In field-stimulated myocytes, contraction was significantly greater in TG4 compared with wild-type (WT) ventricular myocytes. In contrast, when duration of depolarization was controlled with rectangular voltage clamp steps, contraction amplitudes initiated by test steps were the same in WT and TG4 myocytes. When cells were voltage clamped with action potentials simulating TG4 and WT action potential configurations, contractions were greater with long TG4 action potentials and smaller with shorter WT action potentials, which suggests an important role for action potential configuration. Interestingly, peak amplitude of L-type Ca2+ current (ICa-L) initiated by rectangular test steps was reduced, although the voltage dependencies of contractions and currents were not altered. To explore the basis for the altered relation between contraction and ICa-L, Ca2+ concentrations were measured in myocytes loaded with fura 2. Diastolic concentrations of free Ca2+ and amplitudes of Ca2+ transients were similar in voltage-clamped myocytes from WT and TG4 mice. However, sarcoplasmic reticulum (SR) Ca2+ content assessed with the rapid application of caffeine was elevated in TG4 cells. Increased SR Ca2+ was accompanied by increased frequency and amplitudes of spontaneous Ca2+ sparks measured at 37°C with fluo 3. These observations suggest that the gain of Ca2+-induced Ca2+ release is increased in TG4 myocytes. Increased gain counteracts the effects of decreased amplitude of ICa-L in voltage-clamped myocytes and likely contributes to increased contraction amplitudes in field-stimulated TG4 myocytes.

calcium current; calcium transient; sarcoplasmic reticulum; calcium stores; calcium sparks

IT IS WIDELY ACCEPTED that sympathetic stimulation plays a major role in modulating the strength of cardiac contraction. The effects of sympathetic activity are mediated primarily through activation of {beta}-adrenergic receptors ({beta}-ARs) in cardiac muscle. Both {beta}1- and {beta}2-ARs are expressed in heart (5, 30). These two types of ARs can alter cardiac contraction by actions on one or more steps involved in excitation-contraction (EC) coupling.

Disruption of the cardiac {beta}-AR system is known to occur in chronic congestive heart failure (27). To determine whether augmentation of {beta}-AR signaling has therapeutic potential in heart failure, a number of studies have examined effects of {beta}-AR overexpression on EC coupling in myocytes from transgenic animals (19, 22). One approach utilizes cardiac-specific overexpression of human {beta}2-AR in a line of transgenic mice (TG4) (22). TG4 mice exhibit significant increases in heart rate, left ventricular developed pressure, and rate of pressure development (4, 19, 22, 24). In addition, left ventricular relaxation is enhanced in TG4 hearts (22, 24). In vitro studies indicate that both adenylyl cyclase activity and cAMP levels are increased in TG4 hearts, even in the absence of exogenous agonist (4, 19, 22, 24, 31, 32). This and other observations suggest that these nonnative {beta}2-receptors are constitutively active (12, 25, 30, 32, 34).

Several studies have reported that contraction and Ca2+ transient amplitudes are significantly increased in field-stimulated TG4 myocytes examined at 22°C (29, 31–33). However, other studies reported no change in contraction amplitudes in field-stimulated TG4 myocytes examined at 32°C (11, 14). In cardiac muscle, it is generally accepted that L-type Ca2+ current (ICa-L) can trigger Ca2+ release from the sarcoplasmic reticulum (SR) by a process called Ca2+-induced Ca2+ release (CICR) (3). However, ICa-L measured in voltage-clamped myocytes from TG4 mice was found to be unchanged (32, 33) or even decreased (14, 19) compared with myocytes from wild-type (WT) mice. Thus measurements of ICa-L in TG4 mice suggest that the relationship between ICa-L and initiation of contraction may be altered in TG4 myocytes. However, measurements of contraction were conducted in field-stimulated myocytes, whereas measurements of ICa-L were conducted separately in voltage-clamped myocytes. Because these studies were not conducted in the same cells or under the same experimental conditions, the relationship between ICa-L and contraction in TG4 mice is unclear.

Overexpression of {beta}2-AR results in an increase in basal cAMP levels (32). Increased cAMP could be expected to increase SR Ca2+ stores by phosphorylation of phospholamban and removal of phospholamban inhibition of SR Ca2+-ATPase (10, 18, 21). Thus increased SR Ca2+ stores could possibly explain the positive inotropic effect of {beta}2-AR overexpression observed in several studies (29, 31, 33). Indeed, the frequency and amplitudes of Ca2+ sparks are increased in TG4 myocytes at room temperature (33), which is consistent with an increase in SR Ca2+ stores. Nonetheless, amplitudes of caffeine-induced Ca2+ transients detected with fluo 3 were not increased in TG4 myocytes in the same study (33). However, these experiments utilized fluo 3, which does not directly measure Ca2+ concentration, and were conducted at room temperature, which has marked effects on SR Ca2+ load, Ca2+ sparks, EC coupling, and phosphorylation levels (2, 8, 9). Thus it is unclear whether SR Ca2+ content is altered in TG4 myocytes at physiological temperature.

To evaluate the effects of {beta}2-AR overexpression on cardiac EC coupling it is essential to measure differences in contraction, Ca2+ transients, ICa-L, and SR Ca2+ content either simultaneously or under comparable experimental conditions. The objectives of this study were 1) to compare amplitudes of contraction in field-stimulated TG4 and WT ventricular myocytes at physiological temperature, 2) to measure contraction and ICa-L simultaneously in voltage-clamped ventricular myocytes where effects of action potential configuration are eliminated, 3) to assess SR Ca2+ content and Ca2+ transients with fura 2 under conditions identical to those used to evaluate contraction and ICa-L, and 4) to compare the frequency and properties of Ca2+ sparks at physiological temperature in TG4 and WT mouse myocytes.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation. Experiments were conducted on cardiac ventricular myocytes from 24- to 32-wk-old male and female TG4 mice and WT littermates. Initial breeding pairs were made up of female WT (B6SJLF1/J) and male TG4 [B6SJL-TgN(Wtbeta2)4Wjk] mice obtained from Jackson Laboratories. The colony was then maintained by breeding TG4 animals with WT littermates. Because TG4 mice are hemizygous for the transgene, mice carrying the transgene were identified by genotyping with a protocol based on that provided by Jackson Laboratories (www.jax.org/jaxmice/micetech). All experiments were performed in accordance with guidelines published by the Canadian Council on Animal Care and were approved by the Dalhousie University Committee on Animal Care. Myocytes were isolated as described previously (8). Briefly, animals were anesthetized with pentobarbital sodium (200–300 mg/kg ip) coinjected with heparin (100 units) to inhibit blood coagulation. Hearts were cannulated in situ and perfused retrogradely through the aorta (2 ml/min) with oxygenated (100% O2, 37°C) Ca2+-free solution of the following composition (mM): 130 NaCl, 5 KCl, 25 HEPES, 0.33 NaH2PO4, 1.0 MgCl2, 20 glucose, 3.0 Na-pyruvate, and 1.0 lactic acid (pH 7.4 with NaOH). Hearts were then perfused for 10 min with Ca2+-free solution supplemented with 50 µM Ca2+, collagenase (24 mg/30 ml; Worthington type I), protease (0.33 mg/ml; Boehringer-Mannheim), and trypsin (1 mg/ml; Sigma). Ventricles were then minced in a high-K+ buffer containing (mM) 80 KOH, 50 glutamic acid, 30 KCl, 30 KH2PO4, 20 taurine, 10 HEPES, 10 glucose, 3 MgSO4, and 0.5 EGTA (pH 7.4 with KOH).

General methods. Cells were placed in a 0.75-ml chamber on the stage of an inverted microscope. After settling, cells were superfused at 3 ml/min with a standard buffer solution with the following composition (mM): 145 NaCl, 10 glucose, 10 HEPES, 4 KCl, 1 CaCl2, and 1 MgCl2 (pH 7.4 with NaOH). All experiments were conducted at 37°C.

In some experiments, cells were field stimulated at 2 Hz through platinum electrodes. Unloaded cell shortening was recorded with a video edge detector (Crescent Electronics, Sandy, UT) and a video camera (model TM-640, Pulnix America) operating at 120 Hz. In experiments in which cells were voltage clamped, the standard buffer solution was supplemented with 4 mM 4-aminopyridine to block transient outward current and 0.3 mM lidocaine to block inward Na+ current. Discontinuous single electrode voltage clamp (5–8 kHz) was conducted with high-resistance microelectrodes (18–26 M{Omega}, 2.7 M KCl) to minimize cell dialysis and distortion of differences in cytosolic composition between WT and TG4 myocytes. Voltage clamp was conducted with pCLAMP software (version 8.0, Axon Instruments) and an Axoclamp 2B amplifier (Axon Instruments). Test steps were preceded by five 200-ms conditioning pulses to 0 mV from the holding potential (–80 mV) to provide a consistent history of activation. Details of specific voltage-clamp protocols are provided in the appropriate sections of RESULTS.

Ca2+ concentrations. Myocytes were loaded with fura 2 by incubation with cell-permeant fura 2-AM (5 µM) for 10 min at room temperature. Fluorescence was measured with a DeltaRam photometer (Photon Technology International) and Felix software (version 1.4, Photon Technology International) as described previously (8). Fura 2 was excited at 340 and 380 nm, and emission was measured at 510 nm. The 340- to 380-nm emission ratio was converted to intracellular Ca2+ concentration ([Ca2+]i) with an in vitro calibration curve determined with the same light path used experimentally. Ca2+ transients were elicited by voltage clamp steps from –80 mV to 0 mV.

Fura 2 Ca2+ transients elicited by rapid application of caffeine were used as an index of SR Ca2+ load in voltage-clamped cells (2, 24). After five 200-ms conditioning pulses to 0 mV, cells were repolarized to –60 mV and caffeine was applied with a rapid solution switcher for 1 s. The rapid switcher is a computer-controlled device that allows complete change of the solution bathing the myocyte in <0.5 s while maintaining the temperature at 37°C (15). Caffeine was applied in a solution of the following composition (in mM): 10 caffeine, 140 LiCl, 4 KCl, 10 glucose, 5 HEPES, 4 MgCl2, 2.5 4-aminopyridine, and 2.5 lidocaine. Na+ and Ca2+ were omitted from this solution to minimize loss of Ca2+ through Na+/Ca2+ exchange (17). However, because Na+- and Ca2+-free solution was not applied in advance of caffeine it is possible that some Na+/Ca2+ exchange could occur during the initial period of caffeine application.

Ca2+ sparks. Ca2+ sparks were recorded with a Zeiss LSM 510 laser scanning confocal microscope with techniques described previously (8). Myocytes were incubated with 20 µM fluo 3-AM for 25 min. Cells were transferred to an experimental chamber on the microscope stage and superfused with the standard buffer solution at 37°C. Ca2+ sparks were measured in quiescent WT and TG4 myocytes. Changes in free Ca2+ were detected in line scan mode with excitation at 488 nm and emission measured at 525 nm (Zeiss oil-immersion objective, x40, 1.3 numerical aperture). Cells were repetitively scanned along the length of the cell at 1.5-ms intervals, for a maximum of 6 s. Each line was composed of 512 pixels. The confocal pinhole was adjusted to maximize x-y-z resolution to 0.26 x 0.26 x 0.75 µm. Laser intensity was reduced to ≤5% maximum to minimize cytotoxicity and dye bleaching. Line scan diagrams were constructed by stacking emission lines in temporal order.

Data analysis. Contraction amplitude was measured as peak shortening with respect to cell length immediately before cell shortening. Contraction measures were normalized as percent resting cell length, to allow comparison of cell shortening between the TG4 and WT groups. Time to peak contraction was measured as time between the initiation of contraction and maximal cell shortening, and time to half-relaxation was the time required for contraction to relax by 50%. Depolarization- and caffeine-induced Ca2+ transients were measured with reference to diastolic [Ca2+]i. We used the Ca2+ channel blocker Cd2+ to validate our measurements of ICa-L. In these experiments, we used the rapid solution switcher to apply Cd2+ for 3 s after the conditioning pulse train and during the test step (Fig. 1). We subtracted the current in the presence of Cd2+ (Fig. 1B) from the current in the absence of Cd2+ (Fig. 1A) to obtain the Cd2+-sensitive current (Fig. 1C). We then compared the amplitudes of peak Cd2+-sensitive current with amplitudes of peak ICa-L measured as the difference between peak inward current and a reference point at the end of the voltage step. There was no significant difference in amplitudes of ICa-L measured with the two techniques. Therefore, we measured the amplitude of ICa-L as the difference between peak inward current and a reference point at the end of the voltage step. Cell membrane area was determined by integrating capacitive transients with pCLAMP software; ICa-L was normalized by cell capacitance and expressed as current density.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. Measurements of L-type Ca2+ current (ICa-L) reflect the Cd2+-sensitive current in mouse ventricular myocytes. Cells were voltage clamped with the protocol shown at top. A: a voltage clamp step from –65 to –40 mV was used to discharge and inactivate Na+ current. A second step from –40 to 0 mV activated inward current. B: inward current was abolished by rapid application of 100 µM Cd2+. C: Cd2+-sensitive difference current was obtained by subtraction of the current in the presence of Cd2+ from the current in the absence of Cd2+. D: amplitude of ICa-L measured as the peak Cd2+-sensitive current is similar to the amplitude of peak ICa-L measured as the difference between peak inward current and a reference point at the end of the voltage step. n = 12 Wild-type (WT) myocytes.

 
Ca2+ sparks were visualized and analyzed with Image J (National Institutes of Health). Ca2+ sparks were identified as local peak elevations of fluorescent intensity (F) that were ≥1.5 times the surrounding background levels (F0). Both the percentage of cells exhibiting Ca2+ sparks (incidence) and frequency of Ca2+ sparks (sparks·100 µm–1·s–1) were calculated for WT and TG4 myocytes. Amplitude (F/F0), full spatial width at half-maximum amplitude (FWHM), time to peak amplitude, and time to half-decay were calculated for individual sparks to characterize and compare sparks in WT and TG4 myocytes.

Statistical analyses. The differences between means for WT and TG4 groups for the field stimulation data, voltage clamp data, fluorescence data and spark data were assessed with a t-test. Differences in contraction-voltage and current-voltage relationships between the two groups were evaluated with a two-way repeated-measures ANOVA. Differences in incidence of Ca2+ sparks between WT and TG4 cells were assessed with a {chi}2-test. t-Test and ANOVA calculations were performed with SigmaStat 2.03 (Jandel) or SAS (SAS Canada, Toronto, ON, Canada). Data are presented as means ± SE. No more than two myocytes from one heart were included in any one data set.

Chemicals. Lidocaine, HEPES buffer, EGTA, MgCl2, anhydrous DMSO, 4-aminopyridine, and caffeine were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Fura 2-AM, fluo 3-AM, and Pluronic F-127 were purchased from Molecular Probes (Hornby, ON, Canada). All other chemicals were purchased from BDH (Toronto, ON, Canada). Stock solutions of fura 2-AM were prepared by dissolving 50 µg of fura 2-AM in 20 µl of anhydrous DMSO. Fluo 3-AM (1 mg) was dissolved in 0.86 ml of anhydrous DMSO. To this was added 9 ml of a solution prepared by sonicating 300 µl of Pluronic F-127 in 12 ml of fetal calf serum (Invitrogen, Burlington, ON, Canada). All other chemicals were dissolved in deionized water.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The first series of experiments compared the amplitudes of contraction of WT and TG4 myocytes at 37°C in cells that were field stimulated at 2 Hz. Representative recordings of unloaded cell shortening from WT and TG4 myocytes are shown in Fig. 2, A and B, respectively. The amplitude of contraction was much greater in the TG4 myocyte than in the WT cell, although the time courses of contractions were similar. Mean data for these parameters are shown in Fig. 2, C–F. Figure 2C shows that the amplitudes of contraction were significantly greater in TG4 myocytes compared with WT controls. However, diastolic cell length (Fig. 2D) was similar in the two groups, as were time to peak shortening (Fig. 2E) and time to half-relaxation (Fig. 2F). Thus, although the amplitude of contraction was significantly greater in field-stimulated TG4 cells at 37°C, neither time course of contraction nor diastolic cell length was different between the two groups.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 2. Amplitudes of contraction were significantly greater in field-stimulated TG4 myocytes compared with WT cells. WT and TG4 myocytes were field stimulated at 2 Hz in experiments at 37°C. A and B: representative contractions for WT and TG4 myocytes, respectively. C: mean WT and TG4 contraction data show that amplitudes of contraction were significantly larger in TG4 myocytes compared with WT cells. Cell shortening is expressed as % of maximum cell length. D: diastolic cell length was similar in WT and TG4 cells. There was no significant difference in time to peak contraction (E) or time to half-relaxation (F) between the two groups. *Significantly different from WT (P < 0.05) (n = 5–9 WT myocytes and 5 TG4 myocytes).

 
We next determined whether the difference in amplitudes of contraction between WT and TG4 cells persisted when the duration of depolarization was controlled by voltage clamp. Test steps were preceded by trains of five 200-ms-long conditioning pulses (2 Hz, –80 to 0 mV) to provide comparable activation histories in each cell (Fig. 3, top). Trains of conditioning pulses were followed by a postconditioning potential of either –40 or –65 mV. Test steps to activate transmembrane currents and contractions were made from the postconditioning potential. Figure 3, A and B, shows representative recordings of contractions and currents from WT and TG4 cells depolarized with a 250-ms test step from –65 to 0 mV. Interestingly, when cells were voltage clamped, there was no difference in the amplitudes of contraction between WT and TG4 myocytes. Furthermore, ICa-L was smaller in the TG4 myocyte compared with the WT cell (Fig. 3, A and B). Mean data for these experiments are shown in Fig. 3C. Figure 3C, top, shows that the amplitudes of contraction were not significantly different between WT and TG4 myocytes. However, the magnitude of peak ICa-L was significantly smaller in TG4 myocytes than in WT cells (Fig. 3C, bottom).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3. The difference in amplitudes of contraction between WT and TG4 myocytes was abolished when cells were voltage clamped. After a train of conditioning pulses, cells were depolarized from either –65 or –40 mV to 0 mV as illustrated in the schematic at top. A and B: representative contractions (top) and currents (bottom) recorded from –65 mV in WT and TG4 cells, respectively. C: when cells were depolarized from –40 mV, mean contraction amplitudes (top) were similar in WT and TG4 myocytes. In contrast, mean peak ICa-L (bottom) was significantly smaller in TG4 myocytes than WT. D: similar results were obtained when cells were depolarized from –65 mV. E: when cells were depolarized from –40 mV, mean times to peak contraction (top) and half-relaxation times (bottom) were similar in WT and TG4 myocytes. F: mean time courses of contraction also were similar in WT and TG4 myocytes when cells were depolarized from –65 mV. *Significantly different from WT (P < 0.05); {dagger}significantly different from a postconditioning potential of –40 mV (P < 0.05) (n = 12–13 WT myocytes and 6–8 TG4 myocytes).

 
We showed previously (9, 16) that components of both contraction and inward current may be inactivated when a postconditioning potential of –40 mV is used. Therefore, in an additional series of experiments cells were depolarized from –65 mV. Mean data for these experiments are shown in Fig. 3D. Comparison of Fig. 3, C and D, shows that contractions were larger in magnitude when cells were activated from –65 mV, and this increase was significant for the WT cells. However, there still was no significant difference between the amplitudes of contractions recorded from WT and TG4 myocytes. In addition, peak inward current remained significantly smaller in TG4 myocytes despite the change in postconditioning potential. We also determined whether the time course of contraction activated by voltage clamp steps differed between WT and TG4 myocytes. Mean data presented in Fig. 3E show that both times to peak contraction and half-relaxation times were similar in WT and TG4 myocytes when cells were activated by steps from –40 mV. Similar results were obtained when cells were activated by steps from –65 mV (Fig. 3F).

Our results demonstrate that contractions are larger in field-stimulated TG4 cells than in WT myocytes, although these differences are abolished when the cells are depolarized with a rectangular voltage-clamp test step. Because action potential duration is prolonged in TG4 myocytes compared with WT cells (33), it is possible that differences in contraction amplitudes between TG4 and WT myocytes may reflect differences in action potential duration. To test this possibility we used simulated action potential waveforms and conducted experiments under voltage-clamp conditions. We used previously published values for resting potentials, action potential amplitudes, and action potential durations at 50% and 90% repolarization to simulate action potentials from WT and TG4 mouse myocytes (33). All values were similar in WT and TG4 cells except that action potential duration at 90% repolarization was 48 ms in WT cells and 104 ms in TG4 cells (33). Cells were held at the resting potential of –70 mV and stimulated with trains of either WT or TG4 action potentials, and contractions were recorded. Figure 4A shows a representative simulated WT action potential (top) and contraction (bottom) recorded from a WT cell. Figure 4B shows that contraction increased markedly when the same cell was activated with a simulated TG4 action potential. Mean data demonstrate that the increase in action potential duration caused a significant increase in magnitudes of contractions (Fig. 4C). Thus prolongation of the action potential could contribute to the increase in magnitude of contraction in field-stimulated TG4 myocytes.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 4. Contraction amplitudes are greater when cells are voltage clamped with action potentials designed to mimic TG4 action potentials than when simulated WT action potentials are used. A: a representative WT mouse ventricular myocyte was voltage clamped with a simulated action potential waveform designed to mimic a WT action potential (top). The simulated WT action potential triggered a contraction (bottom). B: duration (time to 90% repolarization) of the simulated action potential in A was increased from 48 ms to 104 ms to mimic a TG4 action potential (top). The amplitude of contraction of the same myocyte shown in A increased when action potential duration was prolonged (bottom). C: mean amplitudes of contraction increased when myocytes were voltage clamped with simulated TG4 action potentials over those when cells were voltage clamped with simulated WT action potentials. *Significantly different from WT (P < 0.05) (n = 13 myocytes).

 
The smaller amplitude of peak ICa-L in TG4 myocytes could be caused either by a reduction in maximum current or by a shift in the current-voltage relationship. To differentiate between these possibilities the voltage dependencies of contraction and current were determined. The voltage-clamp protocol is illustrated in Fig. 5, top. In these experiments, test steps were initiated from a postconditioning potential of –40 mV after trains of conditioning pulses. Test steps to different potentials were made with each repetition of the protocol. Contraction and current amplitudes were plotted as a function of test step voltage to construct contraction-voltage and current-voltage relationships for WT and TG4 myocytes (Fig. 5, A and B). Figure 5A shows that the contraction-voltage relationships were bell-shaped with a peak near 0 mV. Similarly, current-voltage relationships were bell-shaped with peaks near 0 mV (Fig. 5B). Clearly, there were no shifts in the voltage dependence of either contraction or current in TG4 compared with WT myocytes. Interestingly, although contraction amplitudes were virtually identical in the two cell types, the amplitudes of ICa-L were significantly smaller near the peak of the current-voltage relation in TG4 myocytes (Fig. 5B). Thus the amplitudes of contraction were maintained despite a significant reduction in the amplitude of ICa-L in TG4 myocytes. These findings indicate that the relationship between ICa-L and contraction is altered in TG4 myocytes and suggest that the gain of EC coupling is increased in TG4 myocytes.



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 5. Contraction amplitude is maintained in TG4 myocytes despite a significant reduction in the magnitude of ICa-L. Cells were depolarized to different potentials in 10-mV increments from –40 to +60 mV (top). A: mean contraction-voltage relationships were very similar in WT and TG4 cells. B: mean current-voltage relationships recorded from WT and TG4 cells showed that the maximum amplitude of ICa-L was smaller in TG4 cells than in WT myocytes. This difference was statistically significant at membrane potentials from –10 to +20 mV. *Significantly different from WT (P < 0.05) (n = 12 WT myocytes and 6 TG4 myocytes).

 
It is not clear how full-sized contractions are maintained when peak inward ICa-L is reduced ~50% in TG4 myocytes. An increase in EC coupling gain in TG4 myocytes could reflect a change in myofilament Ca2+ sensitivity and/or a change in the sensitivity of CICR. To explore the mechanism of altered EC coupling gain, we conducted a separate series of experiments to compare and characterize diastolic Ca2+ concentrations and Ca2+ transient amplitudes in TG4 and WT myocytes. In these experiments [Ca2+]i was measured with fura 2. Cells were held at a potential of –80 mV and depolarized with trains of voltage-clamp steps to 0 mV at a frequency of 2 Hz. Figure 6, A and B, shows representative Ca2+ transients for WT and TG4 myocytes. The representative traces show that resting and peak Ca2+ concentrations were similar in the two types of myocytes. Figure 6C shows that mean diastolic Ca2+ concentrations were not significantly different between WT and TG4 myocytes. In addition, the mean amplitudes of Ca2+ transients elicited by depolarizing steps also were not significantly different between cell types (Fig. 6D). These observations indicate that Ca2+ transients, like contractions, have similar amplitudes in voltage-clamped TG4 and WT myocytes. Thus the difference in gain between cell types is not likely explained by a difference in myofilament Ca2+ sensitivity. Rather, these data indicate that the higher gain is largely caused by release of a similar amount of SR Ca2+ despite a smaller trigger influx of Ca2+.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 6. Diastolic intracellular Ca2+ concentrations ([Ca2+]i) and amplitudes of Ca2+ transients are similar in WT and TG4 myocytes. Ca2+ transients were elicited by voltage-clamp steps from –80 to 0 mV. A and B: representative recordings of electrically induced Ca2+ transients in WT and TG4 myocytes. Amplitudes of Ca2+ transients were similar in WT and TG4 cells. C: mean data showed there was no significant difference between diastolic [Ca2+]i in the 2 groups. D: mean amplitudes of Ca2+ transients were not significantly different in WT and TG4 cells. n = 10 WT myocytes and 6 TG4 myocytes per group.

 
Several studies have shown that the sensitivity of CICR is increased when SR Ca2+ load is elevated (1, 20, 28). Therefore, we explored the possibility that increased gain in TG4 myocytes is accompanied by increased SR Ca2+ stores relative to WT myocytes. These experiments were conducted in voltage-clamped cells loaded with fura 2. SR Ca2+ content was assessed by rapid application of 10 mM caffeine for 1 s. The voltage-clamp protocol is shown in Fig. 7 (inset). Cells were activated with five conditioning pulses delivered at a frequency of 2 Hz. Cells were then repolarized to –60 mV, and caffeine was applied with a rapid solution switcher. Caffeine was applied in a 0 mM Na+-0 mM Ca2+ solution to minimize loss of Ca2+ through the Na+/Ca2+ exchanger (17). Figure 7, A and B, shows representative caffeine-induced Ca2+ transients recorded from WT and TG4 myocytes. The caffeine-induced Ca2+ transient was larger in the TG4 myocyte than in the WT myocyte. Mean data for these experiments are presented in Fig. 7C. Mean amplitudes of caffeine-induced Ca2+ transients were significantly greater in TG4 myocytes than in WT cells. These results show that SR Ca2+ stores are significantly greater in TG4 myocytes than in WT myocytes and therefore may be responsible for the increased gain of EC coupling observed in TG4 cells.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 7. SR Ca2+ load was significantly greater in TG4 myocytes compared with WT cells. SR stores were assessed with rapid (1 s) applications of 10 mM caffeine. The voltage-clamp protocol used in these experiments is shown in inset, bottom right. A and B: representative examples of Ca2+ transients induced by rapid application of caffeine in WT and TG4 cells. C: mean amplitudes of caffeine-induced transients were significantly greater in TG4 myocytes than in WT controls. Caffeine-induced Ca2+ transients were measured in the same cells in which depolarization-induced Ca2+ transients shown in Fig. 6 were measured. *Significantly different from WT (P < 0.05) (n = 10 WT myocytes and 7 TG4 myocytes per group).

 
To determine whether Ca2+ release was sensitized at the level of the fundamental SR Ca2+ release units, we measured and compared the occurrence and characteristics of spontaneous Ca2+ sparks in WT and TG4 cells. These experiments were conducted in non-voltage-clamped myocytes. Sparks were detected at physiological temperature because temperature is known to alter the frequency and characteristics of Ca2+ sparks (8). Figure 8, A and B, shows representative line scan diagrams, which illustrate spontaneous Ca2+ sparks recorded from WT and TG4 myocytes. More Ca2+ sparks are apparent in the TG4 line scan. Furthermore, the sparks recorded from the TG4 cell appeared brighter than sparks recorded from the WT cell. The fraction of cells exhibiting spontaneous sparks within line scans with durations of 6 s also was markedly different between cell types. This is illustrated in Fig. 8C, which shows that >90% of TG4 cells exhibited spontaneous Ca2+ sparks whereas only 20% of WT cells exhibited spontaneous sparks. The difference in incidence of cells exhibiting sparks was significant. The frequency of Ca2+ sparks also was significantly greater in TG4 myocytes compared with WT controls, as shown in Fig. 8D. Thus Ca2+ release events were increased in both incidence and frequency in TG4 myocytes compared with WT cells.



View larger version (60K):
[in this window]
[in a new window]
  		Fig. 8. Incidence and frequency of spontaneous Ca2+ sparks are greater in myocytes from TG4 mice than WT mice. A and B: line scan diagrams showing representative Ca2+ sparks recorded from WT and TG4 myocytes. Diastolic Ca2+ levels are indicated in orange, and localized increases in Ca2+ corresponding to Ca2+ sparks are indicated in yellow. Ca2+ sparks were more frequent in the TG4 myocyte. C: % of cells exhibiting spontaneous Ca2+ sparks (incidence) was significantly higher in TG4 cells compared with WT cells, as determined by {chi}2-test. D: frequency of Ca2+ sparks was increased significantly in TG4 myocytes compared with WT cells. *Significantly different from WT (P < 0.05) (n = 25 WT and 12 TG4 cells).

 
We also measured the amplitudes, spatial widths, and time courses of individual spontaneous Ca2+ sparks in WT and TG4 cells at physiological temperature. Figure 9, A–D, compares mean values for these parameters in WT and TG4 myocytes. Figure 9A shows that the amplitude of Ca2+ sparks, expressed as F/F0, was significantly greater in TG4 myocytes compared with WT cells. However, the spatial width, measured as FWHM, was not significantly different between the two groups (Fig. 9B). Similarly, neither the time to peak amplitude nor the time to half-decay of Ca2+ sparks differed significantly in WT and TG4 myocytes (Fig. 9, C and D). Thus Ca2+ sparks had greater amplitudes and occurred with a greater frequency in TG4 myocytes.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 9. Amplitudes of Ca2+ sparks recorded from TG4 myocytes were greater than in WT cells, but widths and times courses were unchanged. A: amplitudes of Ca2+ sparks expressed as the ratio of peak to preceding diastolic fluorescence (F/F0). Peak amplitudes of Ca2+ sparks were greater in TG4 myocytes compared with WT cells. B: spatial width [full width at half-maximum amplitude (FWHM)] was not significantly different between TG4 and WT myocytes. C and D: times to peak and times to half-decay (T0.5) were not significantly different between WT and TG4 myocytes. *Significantly different from WT (P < 0.05) (n = 19 WT sparks and 22 TG4 sparks).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The goal of this study was to evaluate the effects of {beta}2-AR overexpression on cardiac EC coupling. In particular, we wished to explore the relationship between amplitudes of ICa-L, SR Ca2+ release, and amplitudes of contraction in myocytes from TG4 mice. To do this, we determined the effects of {beta}2-AR overexpression on contraction, ICa-L, Ca2+ sparks, and SR Ca2+ load under comparable experimental conditions at 37°C. Our results showed that contraction amplitudes were significantly greater in TG4 myocytes than in WT cells in field-stimulated myocytes. However, when contractions were initiated with rectangular voltage-clamp steps, the amplitudes of contraction no longer were significantly different between WT and TG4 myocytes. This difference between contractions in field-stimulated and voltage-clamped TG4 cells likely reflects prolongation of the action potential, which is characteristic of TG4 cells. When cells were voltage clamped with action potentials designed to mimic TG4 action potentials, contraction amplitudes were greater than when simulated WT action potentials were used. In experiments with rectangular voltage clamp steps, simultaneous measurement of Ca2+ current in the same cells showed that peak ICa-L was markedly smaller in the TG4 myocytes. In addition, the amplitudes of Ca2+ transients did not differ between the two groups of cells. Because the amplitudes of contractions and Ca2+ transients were maintained despite a reduction in the amplitude of peak ICa-L, these results suggest that the gain of CICR is increased in TG4 myocytes. This increase in gain might reflect SR Ca2+ load, which was significantly greater in TG4 myocytes. Increased SR Ca2+ load was accompanied by sensitization of Ca2+ release as indicated by increased incidence, frequency, and amplitude of spontaneous Ca2+ sparks compared with WT myocytes.

In the present study, we examined EC coupling at 37°C in myocytes from 24- to 32-wk-old mice stimulated at 2 Hz in the presence of 1 mM Ca2+. We found that the amplitude of contraction was larger in field-stimulated TG4 cells compared with WT myocytes, although there was little difference in time to peak contraction and time to half-relaxation. Earlier studies reported that contractions are either larger (29, 31–33) or unchanged (11, 14) in field-stimulated TG4 myocytes. These studies were conducted at 22°C and 32°C, respectively, and utilized cells from animals of different ages, different stimulation rates, and different concentrations of extracellular Ca2+. Therefore, differences in results may reflect experimental conditions used in these studies. It also is possible that differences in action potential configuration may have contributed to the conflicting results obtained with different experimental conditions in earlier studies. This is suggested by our observation that the difference in amplitudes of contraction between cell types disappeared when cells were voltage clamped to control the time course of depolarization.

We also found that the magnitude of ICa-L was significantly smaller in TG4 myocytes than in WT myocytes at 37°C. This result is in agreement with several studies conducted at room temperature with myocytes from TG4 mice (13, 14, 19). However, several other reports have concluded that there was no difference in the magnitudes of ICa-L between WT and TG4 myocytes (32, 33). These latter studies were conducted in myocytes from relatively young mice (2–3 mo), and it has been suggested that reduction in ICa-L may develop with age in TG4 mice (13). Indeed, all of the studies reporting a decrease in ICa-L (including the present study) were conducted in mice aged between 3 and 8 mo (13, 14). There are several possible explanations for a reduction in the magnitude of ICa-L in cells from TG4 mice: 1) increased Ca2+-induced inactivation of ICa-L related to elevated SR stores of Ca2+, 2) decreased open probability of L-type Ca2+ channels related to {beta}2-AR overexpression, and 3) reduced expression of L-type Ca2+ channels in TG4 myocytes. The present study presents evidence that it is unlikely that ICa-L is reduced in amplitude in response to increased Ca2+ release from the SR, as the amplitudes of Ca2+ transients were not different in WT and TG4 myocytes. On the other hand, L-type Ca2+ channels have been shown to have a reduced open probability in cell-attached patch-clamp studies in WT and TG4 myocytes (13). The decreased open time resulted in a decreased maximum ensemble current in TG4 myocytes. The same study also demonstrated that the maximum peak ICa-L in TG4 myocytes could be restored when myocytes were pretreated with pertussis toxin to disrupt inhibitory G protein function (13). Because {beta}2-ARs are known to couple to inhibitory G proteins, this suggests that ICa-L is decreased because of constitutive {beta}2-AR activity in TG4 myocytes (13). Furthermore, restoration of ICa-L by pertussis toxin suggests that expression of L-type channels was not reduced in TG4 myocytes.

It is not clear which of the changes observed in TG4 myocytes is primarily responsible for the altered EC coupling observed in these cells. It is possible that increased SR Ca2+ stores result, at least in part, from constitutive {beta}2-AR activity, which could promote SR Ca2+ uptake in TG4 myocytes (23, 24). However, Ca2+ dynamics in cardiac cells involve the interplay between influx, efflux, storage, and release of Ca2+. A balance between these factors is achieved at steady state, and perturbation of one or more of these factors may alter the others (6, 7).

Interestingly, an increase in SR Ca2+ stores may compensate for the decreased magnitude of trigger Ca2+ entering the cell as ICa-L. It has been demonstrated that increased SR Ca2+ content sensitizes Ca2+-release channels and can increase the gain of CICR (1, 20, 28). Eventually the increase in gain of CICR results in Ca2+ transients that are the same amplitude as those seen in WT myocytes, even though the amplitude of ICa-L is smaller. Sensitization of Ca2+-release channels also can be detected as an increase in frequency of Ca2+ sparks (28) and agrees with the observations in the present study. This balance between SR stores and Ca2+ release may therefore explain the maintenance of normal contractions and Ca2+ transients despite a reduction in ICa-L in voltage-clamped TG4 myocytes.

The above considerations by themselves cannot explain the increase in contraction amplitudes observed in field-stimulated myocytes. One possibility is that there is an increase in myofilament Ca2+ sensitivity in TG4 myocytes compared with WT cells. However, this does not seem likely because Ca2+ transients and contractions measured in voltage-clamp experiments were similar in WT and TG4 myocytes. It also is possible that the increase in contraction in field-stimulated TG4 cells is related to differences in action potential duration in WT and TG4 cells, as reported previously (33). Although action potential amplitudes and early repolarization were similar, the time to 90% repolarization was doubled to ~104 ms in TG4 myocytes (33). Indeed, our studies demonstrated that when cells were voltage clamped with action potentials designed to mimic TG4 action potentials, contractions were significantly larger than when simulated WT action potentials were used. It is possible that slowed repolarization in TG4 myocytes could increase Ca2+ influx by slowing deactivation of ICa-L even though peak ICa-L is reduced in TG4 myocytes. Slowed repolarization also may reduce efflux of released Ca2+ through the sarcolemma by reducing the electrochemical gradient for Ca2+ efflux through Na+/Ca2+ exchange. Both of these effects would be expected to increase peak cytosolic Ca2+ levels and amplitudes of contraction in TG4 myocytes. Thus it is possible that the increased amplitude of contraction in field-stimulated myocytes with {beta}2-AR overexpression results from a further shift in the balance between Ca2+ influx and efflux related to prolongation of action potential duration.

The results of this study demonstrate that overexpression of nonnative {beta}2-ARs in murine cardiac myocytes can dramatically alter cardiac EC coupling at the cellular level. These findings are potentially important, because overexpression of {beta}2-ARs may have therapeutic potential in augmentation of contraction in diseases such as heart failure where {beta}-AR signaling is disrupted (27). Therefore, it is important to understand the consequences of {beta}2-AR overexpression on cardiac function and EC coupling. However, the findings reported here apply only to overexpression of nonnative {beta}2-ARs in murine ventricular myocytes. Whether overexpression of human {beta}2-ARs receptors would produce similar effects on EC coupling in human ventricular myocytes is not clear and will require further investigation.

The present study supports earlier studies that reported that overexpression of {beta}2-ARs is accompanied by an increase in magnitude of contraction in field-stimulated myocytes and whole animals (19, 22, 31–33). However, the present study, conducted at physiological temperature, further investigates this observation by exploring the relationship between ICa-L and contraction measured simultaneously in the same cells. This study shows that the positive inotropic effect disappears in voltage-clamped myocytes and that the magnitude of ICa-L is actually smaller in TG4 myocytes than in WT myocytes. Nonetheless, the gain of CICR is clearly increased and likely is related to increased SR Ca2+ stores. Thus our results show that cardiac EC coupling is modified in myocytes overexpressing {beta}2-AR by changes that include reduced magnitude of ICa-L coupled to increased SR Ca2+ stores and increased gain of CICR. Increased gain likely counteracts the effects of decreased amplitude of ICa-L in voltage-clamped myocytes and may contribute to increased contraction amplitudes in field-stimulated TG4 myocytes.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Nova Scotia. S. A. Grandy was supported by a studentship from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada.


   ACKNOWLEDGMENTS
 
The authors thank C. Mapplebeck, S. Foster, and P. Nicholl for excellent technical assistance. The authors also thank Stephen Whitefield for excellent technical assistance with the confocal microscope experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. E. Howlett or G. R. Ferrier, Dept. of Pharmacology, Sir Charles Tupper Medical Bldg., Dalhousie Univ., Halifax, NS, Canada B3H 4H7 (E-mail: Susan.Howlett{at}dal.ca or Gregory.Ferrier{at}dal.ca).

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
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bassani J, Yuan W, and Bers D. Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes. Am J Physiol Cell Physiol 268: C1313–C1319, 1995.[Abstract/Free Full Text]
  2. Bers D. Excitation-Contraction Coupling and Cardiac Contractile Force. Boston, MA: Kluwer Academic, 2001.
  3. Bers DM. Cardiac excitation-contraction coupling. Nature 415: 198–205, 2002.[CrossRef][Medline]
  4. Bond R, Johnson TD, Milano CA, Rockman HA, McMinn TR, Apparsundaram S, Hyek MF, Kenakin TP, Allen LF, and Lefkowitz RJ. Physiological effects of inverse agonists in transgenic mice with myocardial overexpression of the {beta}2-adrenoceptor. Nature 16: 214, 1995.
  5. Dzimiri N. Regulation of {beta}-adrenoceptor signaling in cardiac function and disease. Pharmacol Rev 51: 465–502, 1999.[Abstract/Free Full Text]
  6. Eisner D, Choi HS, Diaz ME, O'Neill SC, and Trafford AW. Integrative analysis of calcium cycling in cardiac muscle. Circ Res 87: 1087–1094, 2000.[Abstract/Free Full Text]
  7. Eisner D, Trafford AW, Diaz ME, Overend CL, and O'Neill SC. The control of Ca release from the cardiac sarcoplasmic reticulum: regulation versus autoregulation. Cardiovasc Res 38: 589–604, 1998.[Abstract/Free Full Text]
  8. Ferrier G, Smith RH, and Howlett SE. Calcium sparks in mouse ventricular myocytes at physiological temperature. Am J Physiol Heart Circ Physiol 285: H1495–H1505, 2003.[Abstract/Free Full Text]
  9. Ferrier GR and Howlett SE. Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction. Am J Physiol Heart Circ Physiol 280: H1928–H1944, 2001.[Abstract/Free Full Text]
  10. Frank K and Kranias E. Phospholamban and cardiac contractility. Ann Med 32: 572–578, 2000.[Web of Science][Medline]
  11. Gong H, Adamson DL, Ranu HK, Koch WJ, Heubach JF, Ravens U, Zolk O, and Harding SE. The effect of Gi-protein inactivation on basal, and {beta}1- and {beta}2AR-stimulated contraction of myocytes from transgenic mice overexpressing the {beta}2-adrenoceptor. Br J Pharmacol 131: 594–600, 2000.[CrossRef][Web of Science][Medline]
  12. Gurdal H, Bond RA, Johnson MD, Friedman E, and Onaran HO. An efficacy-dependent effect of cardiac overexpression of {beta}2-adrenoceptor on ligand affinity in transgenic mice. Mol Pharmacol 52: 187–194, 1997.[Abstract/Free Full Text]
  13. Heubach J, Graf E, Molenaar P, Jager A, Schroder F, Herzig S, Harding S, and Ravens U. Murine ventricular L-type Ca2+ current is enhanced by zinterol via {beta}1-adrenoceptors, and is reduced in TG4 mice overexpressing the human {beta}2-adrenoceptor. Br J Pharmacol 133: 73–82, 2001.[CrossRef][Web of Science][Medline]
  14. Heubach J, Trebess I, Wettwer E, Himmel H, Michel M, Kaumann A, Koch W, Harding S, and Ravens U. L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac {beta}2-adrenoceptor. Cardiovasc Res 42: 173–182, 1999.[Abstract/Free Full Text]
  15. Hobai I, Howarth FC, Pabbathi VK, Dalton GR, Hancox JC, Zhu JQ, Howlett SE, Ferrier GR, and Levi AJ. "Voltage-activated Ca release" in rabbit, rat and guinea-pig cardiac myocytes, and modulation by internal cAMP. Pflügers Arch 435: 164–173, 1997.[CrossRef][Web of Science][Medline]
  16. Howlett S, Zhu JQ, and Ferrier GR. Contribution of the voltage-sensitive calcium release mechanism to contraction in cardiac ventricular myocytes. Am J Physiol Heart Circ Physiol 274: H155–H170, 1998.[Abstract/Free Full Text]
  17. Katoh H, Schlotthauer K, and Bers DM. Transmission of information from cardiac dihydropyridine receptor to ryanodine receptor: evidence from BayK 8644 effects on resting Ca2+ sparks. Circ Res 87: 106–111, 2000.[Abstract/Free Full Text]
  18. Koss K and Kranias EG. Phospholamban: a prominent regulator of myocardial contractility. Circ Res 79: 1059–1063, 1996.[Free Full Text]
  19. Liggett S, Tepe NM, Lorenz JN, Canning AM, Jantz TD, Mitarai S, Yatani A, and Dorn GW. Early and delayed consequences of {beta}2-adrenergic receptor overexpression in mouse hearts: critical role for expression level. Circulation 101: 1707–1714, 2000.[Abstract/Free Full Text]
  20. Lukyanenko V, Gyorke I, and Gyorke E. Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes. Pflügers Arch 432: 1047–1054, 1996.[CrossRef][Web of Science][Medline]
  21. Luo W, Grupp IL, Harrer J, Ponniah S, Grupp G, Duffy JJ, Doetschman T, and Kranias EG. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation. Circ Res 75: 401–409, 1994.[Abstract/Free Full Text]
  22. Milano C, Allen LF, Rockman HA, Dobler PC, McMinn TR, Chien KR, Johnson TD, Bond RA, and Lefkowitz RJ. Enhanced myocardial function in transgenic mice overexpressing the {beta}2-adrenergic receptor. Science 264: 582–586, 1994.[Abstract/Free Full Text]
  23. Reiken S, Gaburjakova M, Guatimosim S, Gomez AM, D'Armiento J, Burkhoff D, Wang J, Vassort G, Lederer WJ, and Marks AR. Protein kinase A phosphorylation of the cardiac calcium release channel (ryanodine receptor) in normal and failing hearts. Role of phosphatases and response to isoproterenol. J Biol Chem 278: 444–453, 2003.[Abstract/Free Full Text]
  24. Rockman H, Hamilton R, Jones LR, Milano CA, Mao L, and Lefkowitz RJ. Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the {beta}2-adrenergic receptor is associated with reduced phospholamban protein. J Clin Invest 97: 1618–1623, 1996.[Web of Science][Medline]
  25. Samama P, Bond RA, Rockman HA, Milano CA, and Lefkowitz RJ. Ligand-induced overexpression of a constitutively active {beta}2-adrenergic receptor: pharmacological creation of a phenotype in transgenic mice. Proc Natl Acad Sci USA 94: 137–141, 1997.[Abstract/Free Full Text]
  26. Trafford A, Diaz ME, and Eisner DA. Coordinated control of cell Ca2+ loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca2+ current. Circ Res 88: 195–201, 2001.[Abstract/Free Full Text]
  27. Vatner SF, Vatner DE, and Homcy CJ. {beta}-Adrenergic receptor signaling: an acute compensatory adjustment-inappropriate for the chronic stress of heart failure? Insights from Gs{alpha} overexpression and other genetically engineered animal models. Circ Res 86: 502–506, 2000.[Free Full Text]
  28. Viatchenko-Karpinski S and Gyorke S. Modulation of the Ca2+-induced Ca2+ release cascade by {beta}-adrenergic stimulation in rat ventricular myocytes. J Physiol 533: 837–848, 2001.[Abstract/Free Full Text]
  29. Xiao R, Avdonin P, Zhou Y, Cheng H, Akhter SA, Eschenhagen T, Lefkowitz RJ, Koch WJ, and Lakatta EG. Coupling of {beta}2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes. Circ Res 84: 43–52, 1999.[Abstract/Free Full Text]
  30. Xiao RP. {beta}-Adrenergic signaling in the heart: dual coupling of the {beta}2-adrenergic receptor to Gs and Gi proteins. SciSTKE 104: RE15, 2001.
  31. Zhang S, Cheng H, Zhou Y, Wang D, Zhu W, Ziman B, Spurgoen H, Lefkowitz R, Lakatta E, Koch W, and Xiao R. Inhibition of spontaneous {beta}2-adrenergic activation rescues {beta}1-adrenergic contractile response in cardiomyocytes overexpressing {beta}2-adrenoceptor. J Biol Chem 275: 21773–21779, 2000.[Abstract/Free Full Text]
  32. Zhou Y, Cheng H, Song L, Wang D, Lakatta E, and Xiao R. Spontaneous {beta}2-adrenergic signaling fails to modulate L-type Ca2+ current in mouse ventricular myocytes. Mol Pharmacol 56: 485–493, 1999.[Abstract/Free Full Text]
  33. Zhou Y, Song L, Lakatta E, Xiao R, and Cheng H. Constitutive {beta}2-adrenergic signalling enhances sarcoplasmic reticulum Ca2+ cycling to augment contraction in mouse heart. J Physiol 521: 351–361, 1999.[Abstract/Free Full Text]
  34. Zhou Y, Yang D, Zhu W, Zhang S, Wang D, Rohrer D, Devic E, Kobilka B, Lakatta E, Cheng H, and Xiao R. Spontaneous activation of {beta}2- but not {beta}1-adrenoceptors expressed in cardiac myocytes from {beta}1{beta}2 double knockout mice. Mol Pharmacol 58: 887–894, 2000.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Physiol.Home page
E. M. Snyder, S. T. Turner, M. J. Joyner, J. H. Eisenach, and B. D. Johnson
The Arg16Gly polymorphism of the {beta}2-adrenergic receptor and the natriuretic response to rapid saline infusion in humans
J. Physiol., August 1, 2006; 574(3): 947 - 954.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. E. Howlett, S. A. Grandy, and G. R. Ferrier
Calcium spark properties in ventricular myocytes are altered in aged mice
Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1566 - H1574.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
E. M. Snyder, K. C. Beck, N. M. Dietz, J. H. Eisenach, M. J. Joyner, S. T. Turner, and B. D. Johnson
Arg16Gly polymorphism of the {beta}2-adrenergic receptor is associated with differences in cardiovascular function at rest and during exercise in humans
J. Physiol., February 15, 2006; 571(1): 121 - 130.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/3/H1029    most recent
00814.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Grandy, S. A.
Right arrow Articles by Howlett, S. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Grandy, S. A.
Right arrow Articles by Howlett, S. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.