(Circulation. 1999;99:1618-1622.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Adenylylcyclase Increases Responsiveness to Catecholamine Stimulation in Transgenic Mice
From the Departments of Medicine, VAMC-San Diego and University of California San Diego, and Department of Anesthesiology (D.M.R.), University of California San Diego, La Jolla, Calif.
Correspondence to H. Kirk Hammond, MD (111-A), VAMC-San Diego, 3350 La Jolla Village Dr, San Diego, CA 92161. E-mail khammond{at}ucsd.edu
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background—The cellular content of cAMP generated by activation of adenylylcyclase (AC) through the ß-adrenergic receptor (ßAR) is a key determinant of a cell's response to catecholamine stimulation. We tested the hypothesis that increased AC content, independently of ßAR number, increases responsiveness to catecholamine stimulation in vivo.
Methods and Results—Transgenic mice with cardiac-directed expression of ACVI showed increased transgene AC expression but no change in myocardial ßAR number or G-protein content. When stimulated through the ßAR, cardiac function was increased, and cardiac myocytes showed increased cAMP production. In contrast, basal cAMP and cardiac function were normal, and long-term transgene expression was not associated with abnormal histological findings or deleterious changes in cardiac function.
Conclusions—The amount of AC sets a limit on cardiac ß-adrenergic signaling in vivo, and increased AC, independent of ßAR number and G-protein content, provides a means to regulate cardiac responsiveness to ßAR stimulation. Overexpressing an effector (AC) does not alter transmembrane signaling except when receptors are activated, in contrast to receptor/G-protein overexpression, which yields continuous activation and has detrimental consequences. Our findings establish the importance of AC content in modulating ß-adrenergic signaling in the heart, suggesting a new target for safely increasing cardiac responsiveness to ßAR stimulation.
Key Words: receptors, adrenergic, beta • proteins • adenylylcyclase
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Adenosine 3',5'-cyclic monophosphate (cAMP) is a pivotal second messenger that regulates cellular function. A large number of agonists act via membrane receptors and GTP-binding proteins (such as the stimulatory GTP-binding protein, Gs) to regulate cAMP generation via adenylylcyclase (AC). ß-Adrenergic receptors (ßARs) are key targets of agonists that promote cAMP generation in a wide variety of cells and are particularly important in cardiac myocytes, in which they regulate cardiac contraction and relaxation as well as heart rate.
The estimated molar proportions of the elements of the
ßAR/Gs/AC complex in cardiac myocytes are
1:200:3,1 suggesting that ßAR number or the amount of AC
may limit ßAR-mediated transmembrane signaling. Increasing ßAR
number 20- to 200-fold in cultured cardiac myocytes2 and
transgenic mice3 achieved only 2-fold increases in cAMP
production. Similarly, cardiac-directed overexpression of
Gs
in transgenic mice only minimally increased
cardiac adrenergic responsiveness.4 A fixed amount of AC
may have limited the degree to which increased ßAR or
Gs could provoke a substantial increase in second
messenger generation and physiological response.
Indeed, in 1969 it was suggested that AC may limit cellular
responsiveness to agonist stimulation,5 and recent studies
showed that increased expression of AC in cultured cardiac myocytes
yields a proportional increase in cAMP
production.6 These studies underscore the
importance of AC in transmembrane signaling but do not establish that
AC content governs adrenergic responsiveness in vivo. Furthermore,
previous studies that overexpressed the ßAR in the heart found that
heart rate, function, and cAMP generation were increased, even in the
unstimulated state.3 It was concluded that sustained
ß-adrenergic stimulation resulted in
cardiomyopathy and substantial cardiac
histological abnormalities in hearts of older
transgenic mice overexpressing cardiac
Gs.7 Similar deleterious
consequences of cardiac-directed overexpression of ßARs have been
reported.8 Here we test the hypothesis that increasing the
expression of the effector (AC) will increase the responsiveness of the
heart to ßAR stimulation and that increasing the amount of AC,
without changing ßAR number or Gs, provides a
means for the same number of receptors to transduce signals more
effectively in vivo. We thought that increasing AC without altering
cell-surface ßAR number or G-protein content might elude the
potentially deleterious effects of sustained adrenergic
stimulation.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Generation of Transgenic Mice
The use of animals was in accordance with institutional guidelines. A 10.8-kb fragment containing the expression cassette of the construct (Figure 1a
) was used for
microinjection. Sixteen of 70 offspring were positive for the
transgene, as shown by polymerase chain reaction (PCR) of the tail tip
(confirmed by Southern blotting; data not shown). Founder animals were
crossbred with wild-type mice of the same strain, and selected animals
were killed for analysis of cardiac transgene expression
reverse-transcription (RT)-PCR (for quantitative assessment of the
level of expression; data not shown), Northern blotting, and Western
blotting (Figure 1b and 1c). ACVI mRNA
expression levels were markedly increased but variable (Figure 1b). Even so, ACVI protein expression
(Figure 1c and 1d) was constant, and lines with similar
ACVI protein expression were used for the study,
with transgene-negative siblings serving as controls. Litter size was
normal, and mortality by 19 months was rare and similar between
transgene-positive and control animals.
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Documentation of Transgene Expression
Total RNA was extracted by
homogenization of tissue in 2 mL of RNA STAT-60
(Tel-Test Inc). Denatured total RNA (20 µg) was
electrophoresed in 1x MOPS/EDTA buffer on a 1.0% agarose gel. RNA was
transferred onto a nylon membrane in 20x SSC solution,
immobilized (80°C, 2 hours), and hybridized with a
[32P]dCTP-labeled murine
ACVI cDNA probe. RT-PCR was used to estimate the
quantities of transgene ACVI mRNA (Figure 1
). Total RNA (1 µg) was mixed with 1 µg of
transgene-specific primer. The RT reaction was carried out by use of
the Superscript II kit and instruction (Gibco-BRL Life Technologies).
RT product (5 µL) was used as template for PCR with
transgene-specific primers (indicated as T1 and T2 in Figure 1).
To document transgene protein expression, a polyclonal antibody recognizing ACV and ACVI protein (Santa Cruz Biosciences) was used in Western blots conducted on cardiac homogenates.6 Left ventricular (LV) samples were homogenized in 1 mL cold Tris buffer (25 mmol/L Tris HCl, 25 mmol/L Tris Base, 8 mmol/L MgCl2, and 0.5 mmol/L EGTA, pH 7.5). Protease inhibitors were added (final concentrations, in µg/mL: leupeptin 10, aprotinin 10, pepstatin 10, and 4-[2-aminoethyl]benzenesulfonyl fluoride [Pefabloc] 100), followed by centrifugation (40 000g, 20 minutes, 4°C). The pellet was resuspended in Tris buffer and sonicated. For Western blot analysis, 100 µg protein was separated on 7.0% PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with anti-ACV/ACVI antibody diluted 1:100 in blocking buffer (5% nonfat dry milk in Tris buffer: 0.9% NaCl, 10 mmol/L Tris HCl, pH 7.5). Primary antibodies were detected with goat anti-rabbit IgG horseradish peroxidase conjugate (Gibco-BRL Life Technology) in blocking buffer. The antigen was visualized with chemiluminescent substrates A and B (Kirkegaard and Perry Laboratories) and exposed to x-ray film.
Echocardiography
Echocardiography was performed in
anesthetized mice as previously reported.9 Animals
were anesthetized with ketamine 100 mg/kg IP and
xylazine 5 mg/kg IP. An echocardiograph (Interspec Apogee
CX) with dynamically focused symmetrical annular array technology for
2-dimensional and M-mode imaging was used (9-MHz transducer). With mice
in the left lateral decubitus position, a parasternal short-axis view
was obtained as a guide for LV M-mode imaging at the papillary muscle
level. M-mode tracings were recorded on videotape. Pulsed
Doppler images of estimated peak LV outflow velocity and mitral
inflow velocities were obtained in a modified parasternal long-axis
view. The M-mode images were digitized and visualized with a scanner to
facilitate accurate measurement. The images were measured with NIH
Image (version 1.52) on a computer screen with leading-edge
techniques.9 Chamber dimensions and the velocity of
circumferential fiber shortening were obtained. Data were acquired and
analyzed without knowledge of whether animals were
transgene-positive or controls.
In Vivo Physiology
Mice were anesthetized with ketamine 50
mg/kg IP and thiobutabarbital 100 mg/kg IP (Inactin, Research
Biochemical International). A cervical incision was made and the
trachea intubated and connected to a volume-cycled ventilator. A 1.4F
micromanometer catheter (Millar Instruments) was
inserted via the right carotid artery and advanced into the LV. The
left carotid artery was cannulated with flame-stretched PE 50 tubing
and connected to a low-compliance pressure transducer (Abbott
Laboratories). Bilateral vagotomy was performed to minimize confounding
effects of reflex activation during agonist infusion. The left external
jugular vein was cannulated with flame-stretched PE 50 tubing for
venous access and delivery of agonists. LV and arterial
pressures were recorded at baseline and 45 seconds after bolus
injection (0.1-mL volume) of isoproterenol that was given in increasing
doses at 5-minute intervals. NKH477 (Nippon Kayaku Co, Ltd), a
water-soluble forskolin derivative10 (1.0 ng/g IV), was
delivered in separate experiments. Data were digitized, recorded on
disk, and analyzed (Dataq DI-400, Windaq Software, Dataq
Instruments). Ten sequential beats were averaged for each measurement.
Data were obtained and analyzed without knowledge of whether
animals were transgene-positive or controls.
Ex Vivo Physiology
Cardiac function was determined in response to adrenergic
stimulation in isolated perfused hearts with an
intraventricular balloon catheter to determine
isovolumic LV pressure as recently described.11 Mice were
anesthetized with ketamine 100 mg/kg IP and xylazine 5
mg/kg IP, and heparin was given (5000 IU/kg IP). Hearts were excised
after midline sternotomy. The aortic root was cannulated with a
22-gauge cannula and suspended in an isolated perfused heart
apparatus. Retrograde perfusion of the heart was
established with warmed Krebs-Henseleit solution oxygenated
with 95% O2 and 5% CO2.
The hearts were perfused at a constant pressure (80 mm Hg) while
a polyethylene balloon was placed in the LV through a small incision in
the left atrium. The fluid-filled balloon was connected via 2 lumens to
a 100-µL syringe for filling the balloon and a 1.8F high-fidelity
pressure transducer for the measurement of LV pressure. After
cannulation and balloon placement, the hearts were allowed to
equilibrate for 10 minutes. Baseline heart rate and LV pressure
development were recorded. The hearts were then perfused with
isoproterenol in bolus doses equal to final concentrations of 0.1 to
1000 nmol/L at 5-minute intervals. LV developed pressure was
recorded for 10 sequential beats at the peak of the response. Data
were recorded on disk and analyzed as above. Peak rates of
LV pressure development (LV dP/dt) and relaxation (LV –dP/dt) were
calculated after acquisition at a sampling rate of 3000 per second
(Dataq DI-400, Windaq Software).
Isolation of Cardiac Myocytes and cAMP Generation
LV myocytes were isolated by use of modifications of
methods previously described.12 Mice were heparinized
(5000 IU/kg) and anesthetized with ketamine 100 mg/kg
and xylazine 5 mg/kg IP, hearts were removed, and aortas were
cannulated and connected to a perfusion apparatus. Hearts
were perfused for 3 minutes (2 mL/min) with calcium-free medium
containing Joklik-modified minimal essential medium (Gibco-BRL) with
final concentrations (mmol/L) of NaCl 113, KCl 4.7,
MgCl2 1,
NaH2PO4 ·
2H2O 8.4, glucose 20,
NaHCO3 12, KHCO3 10,
Na-HEPES 10, taurine 30, carnitine 2, and creatine 2 (pH 7.36). The
perfusion medium then was switched to the above solution with
collagenase (Worthington Type II, 33 IU/mg, 1 mg/mL) for 20
minutes, and hearts were removed from the apparatus. Atria
were removed and ventricles cut into small pieces and triturated with a
wide-bore pipette in collagenase solution (10 mL for 10
minutes). Free myocytes were centrifuged twice (400 rpm for 2
minutes) and washed in 1% BSA. Before treatment of cells, growth
medium was removed and cells were equilibrated for 30 minutes (25°C)
in serum-free and NaHCO3-free DMEM supplemented
with 20 mmol/L HEPES (pH 7.2).
Equal numbers of viable cardiac myocytes were incubated (10 minutes, 25°C) in fresh DMEM containing no addition (basal), isoproterenol 10 µmol/L, or forskolin 10 µmol/L. Aspiration of medium and addition of 7.5% ice-cold trichloroacetic acid (TCA) terminated the reaction. TCA extracts were frozen (-20°C) until assayed. Intracellular cAMP levels were determined by radioimmunoassay (Amersham Life Science).
ßAR Number and G-Protein and G-Protein Receptor Kinase
Content
ßARs were estimated in radioligand-binding
experiments using [125I]iodocyanopindolol 212
pmol/L; binding in the presence of 10-4 mol/L
isoproterenol defined nonspecific binding.6 Experiments
were performed on triplicate samples. Polyclonal antibodies recognizing
Gs, Gi2, and
G-protein receptor kinase types 2 (GRK2) and 5 (GRK5) were used in
Western blots conducted on cardiac homogenates as
previously described.13
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Transgenic Mice
Using standard procedures for the generation of transgenic mice, we obtained substantial and selective cardiac expression of ACVI, documented by PCR, RT-PCR, Northern blotting, and immunoblotting (Figure 1
).
ACVI mRNA was increased 10- to 80-fold in hearts
from transgene-positive animals, and ACVI protein
was consistently increased 20-fold in hearts of animals from
these lines. No mRNA was detected in other tissues by Northern blot
analysis (data not shown). Body weight (control: 31±2 g, n=23;
transgene: 30±1, n=27), tibial length (control: 18.4±0.2 mm,
n=11; transgene: 18.2±0.2, n=18), and right ventricular
and LV wet weight (control: 105±6 mg, n=23; transgene: 102±4 mg,
n=27) and dry weight (control: 28±2 mg, n=9; transgene: 28±1 mg,
n=12) were unchanged by transgene expression.
Histological assessment showed no fibrosis or other
abnormalities in the heart or other organs, even in 15- and
19-month-old animals (n=3, data not shown).
Echocardiography
Despite increased cardiac AC expression, basal heart rate and
contractile function were unchanged when examined by
echocardiography
(Table). Heart rates were similar in
transgene-positive and control animals, and basal cardiac contractile
function, as estimated by the velocity of circumferential fiber
shortening, also was unaltered. Even in older animals, there was no
evidence for cardiac enlargement or declining LV function
(Table).
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In Vivo Physiology
Stimulation of the heart in vivo with the ßAR agonist
isoproterenol showed that these phenotypically normal hearts responded
to adrenergic stimulation in a unique manner (Figure 2). LV +dP/dt and -dP/dt as well as
heart rate and systolic blood pressure (Figure 2) were
increased in hearts of transgene-positive animals, indicating that
increased AC content augments cardiac responsiveness to
catecholamine stimulation. We also assessed the effects of
a water-soluble forskolin derivative (NKH447; see Reference 1010 ), which
directly stimulates AC without interacting with the ßAR, and again
found that transgene-positive animals exhibited increased rates of LV
dP/dt (control: 5790±450 mm Hg/s, n=4; transgene:
12 982±2720 mm Hg/s, n=5; P=0.028) and LV -dP/dt
(control: -5722±135 mm Hg/s, n=4; transgene:
-12 362±1263 mm Hg/s, n=5; P<0.001) as well as
heart rate (control: 440±13 bpm, n=4; transgene: 557±36 bpm, n=5;
P=0.028).
|
, Mean values from 5
control animals; •, from 5 transgene-positive animals. In graphs in
all figures, error bars denote 1 SEM.
Ex Vivo Physiology
When hearts of transgene-positive and control mice were isolated
from neural input and the circulation, they showed similar basal
intrinsic heart rates (control: 364±25 bpm, n=6; transgene: 408±19
bpm, n=11; P=0.12) and basal LV dP/dt (control:
2860±325 mm Hg/s, n=6; transgene: 3189±263 mm Hg/s, n=11;
P=NS) (Figure 3). However, LV
dP/dt in response to isoproterenol stimulation was increased in
transgene-positive animals through a wide range of isoproterenol
concentrations (P<0.0001, Figure 3), even though
heart rate was unchanged (Figure 3). LV
end-diastolic pressures were stable throughout the studies
and were not different between groups (control: 13±1 mm Hg;
range, 12 to 13 mm Hg; transgene: 12±1 mm Hg; range,
11 to 13 mm Hg).
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Transmembrane ßAR Signaling
Cardiac myocytes from transgene-positive animals showed
increased cAMP production when stimulated by forskolin and
isoproterenol (Figure 4a). These data
document that cardiac myocytes expressing transgene
ACVI have increased adrenergic responsiveness not
only to direct stimulation of AC by forskolin, reflecting increased
amounts of AC, but also to isoproterenol, indicating that increased AC
is functionally coupled and recruitable through ßAR stimulation.
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To determine whether increased AC expression, with attendant
alterations in cAMP production, might have affected the
expression of other elements in the ßAR transmembrane signaling
cascade,14 we assessed myocardial ßAR number, cardiac
G-protein content, and GRK2 and GRK5 (predominant GRK isoforms in
mammalian heart13 ). GRK uncouples the ßAR from
Gs, attenuating signal transduction.
Radioligand binding assay and
immunoblotting indicated similar ßAR number and
Gs and Gi2 content in
hearts from transgene-positive and control animals (Figure 4b and 4c). In contrast, GRK2 content (but not GRK5) was increased (Figure 4d).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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To explore the influence of increased AC content on heart function and transmembrane signaling, we overexpressed ACVI, a major AC isoform in cardiac myocytes,13 in transgenic mice. This allowed us to assess the effects of variations in myocardial ACVI content on cardiac structure and function in vivo and facilitated detailed studies of adrenergic signaling in isolated cardiac myocytes. The relationships between myocardial AC content, signal transduction, and heart function were our focus.
Despite increased cardiac AC expression, basal heart rate and
contractile function were unchanged when examined by
echocardiography (Table). Basal cAMP
generation in cardiac myocytes from transgene-positive mice also was
unchanged (Figure 4). These data indicate that basal cardiac
ßAR responsiveness is unaltered in transgene-positive animals. Why
then were the LV dP/dt and heart rate increased in the unstimulated
state (0 isoproterenol, Figure 2)? Measurement of LV dP/dt
required surgical intervention and mechanical ventilation, which
increase catecholamine release.15 In this
setting, higher LV dP/dt and heart rates in transgene-positive animals
most likely reflect increased responsiveness to elevated
endogenous catecholamines. To determine whether
this was in fact the case, we isolated hearts of transgene-positive and
control mice from neural input and the circulation, and they showed
similar intrinsic heart rates but increased LV dP/dt in response to
adrenergic stimulation at matched heart rates (Figure 3). The
noninvasive echocardiographic studies did not require
surgery or mechanical ventilation and are in closer accord with the
unstimulated state.
A 2.1-fold increase in GRK2 content makes the increase in ßAR-stimulated cAMP generation even more impressive and may help to explain why basal cAMP generation and cardiac function are unchanged despite enhanced responsiveness of both to ßAR stimulation. Thus, increased adrenergic signaling resulted from increased ACVI content, not from alterations in other elements in the ßAR signaling pathway, and additional ACVI was coupled to endogenous ßARs. These results indicate that increasing the expression of the effector (AC) can influence the responsiveness of a cell to ßAR stimulation without changing the amount of the receptor or G protein.
Augmentation of transmembrane adrenergic signaling by increasing cardiac ßAR or Gs expression2 3 4 or inhibiting GRK function16 achieves no more than a 2-fold increase in cAMP production. In contrast, the present study indicates that overexpression of ACVI in cardiac myocytes is associated with a robust amplification of ßAR-mediated signaling (2.7-fold), despite unchanged ßAR and G-protein expression and increased GRK2. It appears that AC holds a pivotal position in transmembrane signaling and is the limiting factor governing intracellular cAMP generation in response to neurohumoral adrenergic stimulation.
Cardiac-directed expression of AC results in anatomically normal
hearts with normal basal function, and there is no decline in function
in older mice (Table); myocardial fibrosis is not present
even in 19-month-old animals. This is in contrast to cardiac-directed
overexpression of Gs or ßARs, which results in
dilated cardiomyopathy and cardiac fibrosis as
animals age.7 8 The disparities between previous models
and the present study with regard to cardiac structure and function
suggest an intrinsic difference between receptor/transducer versus
effector amplification. This is likely to be that overexpression of
ßAR and Gs (but not AC) results in sustained
ßAR activation,3 7 which, ultimately, has detrimental
consequences.7 8
In conclusion, we have shown that transgenic mice with cardiac-directed expression of ACVI have structurally normal hearts with normal basal function. Cardiac responsiveness to adrenergic stimulation is increased, with amplified transmembrane signaling and increased physiological function. Cardiac myocytes isolated from transgene-positive hearts respond to adrenergic stimulation with increased cAMP production. These data indicate that the amount of AC sets a limit on cardiac ß-adrenergic signaling in vivo and that increased AC, independent of ßAR number and G-protein content, provides a means to regulate cardiac responsiveness to adrenergic stimulation. Our findings establish the importance of AC content in modulating ß-adrenergic signaling in the heart and potentially for other AC-linked receptors in other cells. Our data suggest a potential target for increasing cardiac responsiveness to adrenergic stimulation.
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Acknowledgments |
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This research was supported by a Merit Award from the Department of Veteran's Affairs (Dr Hammond) and Specialized Center of Research on Congestive Heart Failure 1 P50 HL-53773-01 (Dr Hammond). Additional support was provided by Institutional National Research Service Award HL-07444 (Dr Gao) and a grant from the Foundation for Anesthesia Education Research/Society of Cardiovascular Anesthesiologists (Dr Roth). Portions of this research were supported by an unrestricted grant from Collateral Therapeutics, Inc, San Diego, Calif, for which Dr Hammond consults. The authors thank Dr Jeffrey Robbins for providing the
-myosin heavy
chain promoter; Nippon Kayaku Co, Ltd, for providing NKH477; Dr Robert
Ross for helpful comments; and Drs Tamsin Lisa Kelly and Paul A. Insel
for reviewing the manuscript. |
Footnotes |
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Drs Lai and Roth and Mr Zhou contributed equally to this study.
Received August 17, 1998; revision received October 10, 1998; accepted November 23, 1998.
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T. Takahashi, T. Tang, N. C. Lai, D. M. Roth, B. Rebolledo, M. Saito, W. Y.W. Lew, P. Clopton, and H. K. Hammond Increased Cardiac Adenylyl Cyclase Expression Is Associated With Increased Survival After Myocardial Infarction Circulation, August 1, 2006; 114(5): 388 - 396. [Abstract] [Full Text] [PDF]
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 H. K. Hammond Why FRET? Focus on "{beta}-Adrenergic and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes using a FRET-based biosensor" Am J Physiol Cell Physiol, August 1, 2005; 289(2): C246 - C247. [Full Text] [PDF]
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 T. Tang, M. H. Gao, D. M. Roth, T. Guo, and H. K. Hammond Adenylyl cyclase type VI corrects cardiac sarcoplasmic reticulum calcium uptake defects in cardiomyopathy Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H1906 - H1912. [Abstract] [Full Text] [PDF]
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K. Iwatsubo, S. Minamisawa, T. Tsunematsu, M. Nakagome, Y. Toya, J. E. Tomlinson, S. Umemura, R. M. Scarborough, D. E. Levy, and Y. Ishikawa Direct Inhibition of Type 5 Adenylyl Cyclase Prevents Myocardial Apoptosis without Functional Deterioration J. Biol. Chem., September 24, 2004; 279(39): 40938 - 40945. [Abstract] [Full Text] [PDF]
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M. H. Gao, T. Tang, T. Guo, S. Q. Sun, J. R. Feramisco, and H. K. Hammond Adenylyl Cyclase Type VI Gene Transfer Reduces Phospholamban Expression in Cardiac Myocytes via Activating Transcription Factor 3 J. Biol. Chem., September 10, 2004; 279(37): 38797 - 38802. [Abstract] [Full Text] [PDF]
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 S. Rosenkranz TGF-{beta}1 and angiotensin networking in cardiac remodeling Cardiovasc Res, August 15, 2004; 63(3): 423 - 432. [Abstract] [Full Text] [PDF]
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C. Sesti and R. A. Kloner Gene Therapy in Congestive Heart Failure Circulation, July 20, 2004; 110(3): 242 - 243. [Full Text] [PDF]
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N. C. Lai, D. M. Roth, M. H. Gao, T. Tang, N. Dalton, Y. Y. Lai, M. Spellman, P. Clopton, and H. K. Hammond Intracoronary Adenovirus Encoding Adenylyl Cyclase VI Increases Left Ventricular Function in Heart Failure Circulation, July 20, 2004; 110(3): 330 - 336. [Abstract] [Full Text] [PDF]
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 S. D. Carvalho-Bianco, B. W. Kim, J. X. Zhang, J. W. Harney, R. S. Ribeiro, B. Gereben, A. C. Bianco, U. Mende, and P. R. Larsen Chronic Cardiac-Specific Thyrotoxicosis Increases Myocardial {beta}-Adrenergic Responsiveness Mol. Endocrinol., July 1, 2004; 18(7): 1840 - 1849. [Abstract] [Full Text] [PDF]
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D. M. Roth, N. C. Lai, M. H. Gao, J. D. Drumm, J. Jimenez, J. R. Feramisco, and H. K. Hammond Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H172 - H177. [Abstract] [Full Text] [PDF]
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X. Liu, R. S. Ostrom, and P. A. Insel cAMP-elevating agents and adenylyl cyclase overexpression promote an antifibrotic phenotype in pulmonary fibroblasts Am J Physiol Cell Physiol, May 1, 2004; 286(5): C1089 - C1099. [Abstract] [Full Text] [PDF]
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M. L. Williams, J. A. Hata, J. Schroder, E. Rampersaud, J. Petrofski, A. Jakoi, C. A. Milano, and W. J. Koch Targeted {beta}-Adrenergic Receptor Kinase ({beta}ARK1) Inhibition by Gene Transfer in Failing Human Hearts Circulation, April 6, 2004; 109(13): 1590 - 1593. [Abstract] [Full Text] [PDF]
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 X.-M. Gao, A. Agrotis, D. J. Autelitano, E. Percy, E. A. Woodcock, G. L. Jennings, A. M. Dart, and X.-J. Du Sex Hormones and Cardiomyopathic Phenotype Induced by Cardiac {beta}2-Adrenergic Receptor Overexpression Endocrinology, September 1, 2003; 144(9): 4097 - 4105. [Abstract] [Full Text] [PDF]
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S. Okumura, J.-i. Kawabe, A. Yatani, G. Takagi, M.-C. Lee, C. Hong, J. Liu, I. Takagi, J. Sadoshima, D. E. Vatner, et al. Type 5 Adenylyl Cyclase Disruption Alters Not Only Sympathetic But Also Parasympathetic and Calcium-Mediated Cardiac Regulation Circ. Res., August 22, 2003; 93(4): 364 - 371. [Abstract] [Full Text] [PDF]
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 S. Okumura, G. Takagi, J.-i. Kawabe, G. Yang, M.-C. Lee, C. Hong, J. Liu, D. E. Vatner, J. Sadoshima, S. F. Vatner, et al. Disruption of type 5 adenylyl cyclase gene preserves cardiac function against pressure overload PNAS, August 19, 2003; 100(17): 9986 - 9990. [Abstract] [Full Text] [PDF]
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 Genetically Modified Animals in Endocrinology Endocr. Rev., August 1, 2003; 24(4): 554 - 555. [Full Text] [PDF]
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P. Most, A. Remppis, and H. A Katus Conditional AC type VI expression in the heart: relevant insights into function of inducible target gene expression Cardiovasc Res, November 1, 2002; 56(2): 181 - 183. [Full Text] [PDF]
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M. H. Gao, H. Bayat, D. M Roth, J. Yao Zhou, J. Drumm, J. Burhan, and H Kirk Hammond Controlled expression of cardiac-directed adenylylcyclase type VI provides increased contractile function Cardiovasc Res, November 1, 2002; 56(2): 197 - 204. [Abstract] [Full Text] [PDF]
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S. Rosenkranz, M. Flesch, K. Amann, C. Haeuseler, H. Kilter, U. Seeland, K.-D. Schluter, and M. Bohm Alterations of beta -adrenergic signaling and cardiac hypertrophy in transgenic mice overexpressing TGF-beta 1 Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H1253 - H1262. [Abstract] [Full Text] [PDF]
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 J. T. Auman, F. J. Seidler, C. A. Tate, and T. A. Slotkin Are developing beta -adrenoceptors able to desensitize? Acute and chronic effects of beta -agonists in neonatal heart and liver Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2002; 283(1): R205 - R217. [Abstract] [Full Text] [PDF]
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 P. Jourdain, F. Funck, Y. Fulla, A. Hagege, M. Bellorini, N. Guillard, J. Loiret, B. Thebault, and M. Desnos Myocardial contractile reserve under low doses of dobutamine and improvement of left ventricular ejection fraction with treatment by carvedilol Eur J Heart Fail, June 1, 2002; 4(3): 269 - 276. [Abstract] [Full Text] [PDF]
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B. J. A. Janssen and J. F. M. Smits Autonomic control of blood pressure in mice: basic physiology and effects of genetic modification Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2002; 282(6): R1545 - R1564. [Abstract] [Full Text] [PDF]
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A. M. Feldman Adenylyl Cyclase: A New Target for Heart Failure Therapeutics Circulation, April 23, 2002; 105(16): 1876 - 1878. [Full Text] [PDF]
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N. N Petrashevskaya, I. Bodi, M. Rubio, J. D Molkentin, and A. Schwartz Cardiac function and electrical remodeling of the calcineurin-overexpressed transgenic mouse Cardiovasc Res, April 1, 2002; 54(1): 117 - 132. [Abstract] [Full Text] [PDF]
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M. J. Lohse and S. Engelhardt Protein Kinase A Transgenes: The Many Faces of cAMP Circ. Res., November 23, 2001; 89(11): 938 - 940. [Full Text] [PDF]
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R. S. Ostrom, C. Gregorian, R. M. Drenan, Y. Xiang, J. W. Regan, and P. A. Insel Receptor Number and Caveolar Co-localization Determine Receptor Coupling Efficiency to Adenylyl Cyclase J. Biol. Chem., November 2, 2001; 276(45): 42063 - 42069. [Abstract] [Full Text] [PDF]
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X.-J. Du Sympathoadrenergic mechanisms in functional regulation and development of cardiac hypertrophy and failure: findings from genetically engineered mice Cardiovasc Res, June 1, 2001; 50(3): 443 - 453. [Full Text] [PDF]
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 R. J. Lefkowitz and J. T. Willerson Prospects for Cardiovascular Research JAMA, February 7, 2001; 285(5): 581 - 587. [Abstract] [Full Text] [PDF]
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X.-J. Du, X.-M. Gao, B. Wang, G. L Jennings, E. A Woodcock, and A. M Dart Age-dependent cardiomyopathy and heart failure phenotype in mice overexpressing {beta}2-adrenergic receptors in the heart Cardiovasc Res, December 1, 2000; 48(3): 448 - 454. [Abstract] [Full Text] [PDF]
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N. C. Lai, D. M. Roth, M. H. Gao, S. Fine, B. P. Head, J. Zhu, M. D. McKirnan, C. Kwong, N. Dalton, K. Urasawa, et al. Intracoronary Delivery of Adenovirus Encoding Adenylyl Cyclase VI Increases Left Ventricular Function and cAMP-Generating Capacity Circulation, November 7, 2000; 102(19): 2396 - 2401. [Abstract] [Full Text] [PDF]
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X.-J. Du, X.-M. Gao, G. L. Jennings, A. M. Dart, and E. A. Woodcock Preserved ventricular contractility in infarcted mouse heart overexpressing beta 2-adrenergic receptors Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2456 - H2463. [Abstract] [Full Text] [PDF]
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R. S. Ostrom, S. R. Post, and P. A. Insel Stoichiometry and Compartmentation in G Protein-Coupled Receptor Signaling: Implications for Therapeutic Interventions Involving Gs J. Pharmacol. Exp. Ther., August 1, 2000; 294(2): 407 - 412. [Abstract] [Full Text]
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 R. S. Ostrom, J. D. Violin, S. Coleman, and P. A. Insel Selective Enhancement of beta -Adrenergic Receptor Signaling by Overexpression of Adenylyl Cyclase Type 6: Colocalization of Receptor and Adenylyl Cyclase in Caveolae of Cardiac Myocytes Mol. Pharmacol., May 1, 2000; 57(5): 1075 - 1079. [Abstract] [Full Text]
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L. Lipskaia, N. Defer, G. Esposito, I. Hajar, M.-C. Garel, H. A. Rockman, and J. Hanoune Enhanced Cardiac Function in Transgenic Mice Expressing a Ca2+-Stimulated Adenylyl Cyclase Circ. Res., April 14, 2000; 86(7): 795 - 801. [Abstract] [Full Text] [PDF]
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S. F. Vatner, D. E. Vatner, and C. J. Homcy {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., March 17, 2000; 86(5): 502 - 506. [Full Text] [PDF]
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 O.-E. Brodde and M. C. Michel Adrenergic and Muscarinic Receptors in the Human Heart Pharmacol. Rev., December 1, 1999; 51(4): 651 - 690. [Abstract] [Full Text] [PDF]
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S. F. Steinberg The Molecular Basis for Distinct {beta}-Adrenergic Receptor Subtype Actions in Cardiomyocytes Circ. Res., November 26, 1999; 85(11): 1101 - 1111. [Full Text] [PDF]
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J. L. Zeiders, F. J. Seidler, and T. A. Slotkin Agonist-Induced Sensitization of beta -Adrenoceptor Signaling in Neonatal Rat Heart: Expression and Catalytic Activity of Adenylyl Cyclase J. Pharmacol. Exp. Ther., November 1, 1999; 291(2): 503 - 510. [Abstract] [Full Text]
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D. M. Roth, M. H. Gao, N. C. Lai, J. Drumm, N. Dalton, J. Y. Zhou, J. Zhu, D. Entrikin, and H. K. Hammond Cardiac-Directed Adenylyl Cyclase Expression Improves Heart Function in Murine Cardiomyopathy Circulation, June 22, 1999; 99(24): 3099 - 3102. [Abstract] [Full Text] [PDF]
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C. L. Antos, N. Frey, S. O. Marx, S. Reiken, M. Gaburjakova, J. A. Richardson, A. R. Marks, and E. N. Olson Dilated Cardiomyopathy and Sudden Death Resulting From Constitutive Activation of Protein Kinase A Circ. Res., November 23, 2001; 89(11): 997 - 1004. [Abstract] [Full Text] [PDF]
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D. M. Roth, H. Bayat, J. D. Drumm, M. H. Gao, J. S. Swaney, A. Ander, and H. K. Hammond Adenylyl Cyclase Increases Survival in Cardiomyopathy Circulation, April 23, 2002; 105(16): 1989 - 1994. [Abstract] [Full Text] [PDF]
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