(Circulation. 2000;102:2396.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Intracoronary Delivery of Adenovirus Encoding Adenylyl Cyclase VI Increases Left Ventricular Function and cAMP-Generating Capacity
From San Diego VA Healthcare System (D.M.R., B.P.H., J.Z., C.K., H.K.H.); the Departments of Medicine (N.D., H.K.H.) and Anesthesia (D.M.R.), University of California at San Diego; the Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Sapporo, Japan (K.U.); and Collateral Therapeutics, Inc, San Diego, Calif (N.C.L., M.H.G., S.F., M.D.M., D.A.R.).
Correspondence to H. Kirk Hammond, MD, VAMC-San Diego (111-A), 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail khammond{at}ucsd.edu
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Abstract |
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Background—We tested the hypothesis that intracoronary injection of a recombinant adenovirus encoding adenylyl cyclase type VI (ACVI) would increase cardiac function in pigs.
Methods and Results—Left ventricular (LV) dP/dt and cardiac output in response to isoproterenol and NKH477 stimulation were assessed in normal pigs before and 12 days after intracoronary delivery of histamine followed by intracoronary delivery of an adenovirus encoding lacZ (control) or ACVI (1.4x1012 vp). Animals that had received ACVI gene transfer showed increases in peak LV dP/dt (average increase of 1267±807 mm Hg/s; P=0.0002) and cardiac output (average increase of 39±20 mL · kg-1 · min-1; P<0.0001); control animals showed no changes. Increased LV dP/dt was evident 6 days after gene transfer and persisted for at least 57 days. Basal heart rate, blood pressure, and LV dP/dt were unchanged, despite changes in cardiac responsiveness to catecholamine stimulation. Twenty-three hour ECG recordings showed no change in mean heart rate or ectopic beats and no arrhythmias. LV homogenates from animals receiving ACVI gene transfer showed increased ACVI protein content (P=0.0007) and stimulated cAMP production (P=0.0006), confirming transgene expression and function; basal LV AC activity was unchanged. Increased cAMP-generating capacity persisted for at least 18 weeks (P<0.0002).
Conclusions—Intracoronary injection of a recombinant adenovirus encoding AC provides enduring increases in cardiac function.
Key Words: receptors, adrenergic, beta • gene transfer • heart failure • histamine • myocardial contraction
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Introduction |
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We recently demonstrated that the amount of adenylyl cyclase (AC) is the primary limiting factor of a cell’s ability to generate cAMP in response to ß-adrenergic receptor (ßAR) stimulation.1 We subsequently showed that transgenic mice with cardiac-directed expression of AC type VI (ACVI) have increased responses to catecholamine2 and that when cardiac ACVI is overexpressed in a cardiomyopathic background, left ventricular (LV) function is improved3 and life is prolonged (unpublished data).
However, there are enormous differences between the transgenic mouse paradigm and exogenous gene transfer to adult animals. For example, the use of a cardiac-directed promoter in transgenic mice can provide high-level transgene expression exclusively and ubiquitously in cardiac myocytes. These features are in stark contrast to exogenous gene transfer to adult animals, where the efficiency of gene transfer is limited, transgene expression not as robust, and cell specificity more difficult to obtain with commonly employed vectors.
This is an important issue scientifically, but it also has potential clinical implications. For example, if these impediments to successful gene transfer could be overcome, it is possible that clinical dilated heart failure could be treated more effectively. Clinical application would require a method of gene transfer that is safe, effective, and preferably nonsurgical and that would have enduring effects on LV function. The goal of the present study was to determine if global LV function could be increased by intracoronary delivery of an adenovirus expressing ACVI.
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Methods |
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A total of 63 pigs were used in accordance with the National Institutes of Health and institutional guidelines. Fifty-four animals (mean weight, 38±1 kg) underwent intracoronary delivery of a recombinant adenovirus encoding either ACVI or lacZ; 9 additional animals (mean weight, 59±8 kg) did not receive gene transfer and were used as controls for LV cAMP studies and for histological examination. In initial studies (n=5 pigs), we performed a qualitative assessment of the cardiac gene transfer obtained with an intracoronary infusion of histamine (25 µg/min for 3 minutes) followed by an intracoronary injection of 1012 vp of a recombinant adenovirus encoding nuclear-tagged lacZ. Myocardial samples were prepared as previously reported.4 These studies indicated substantial LV gene transfer 5 days after injection (Figure 1
).
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We next instrumented 18 pigs for studies of LV contractile
function, which were performed 12±1 days after gene transfer
(ACVI, n=10; lacZ, n=8). To determine the onset
and duration of the physiological effects, studies
were conducted in a subset of these animals at earlier (6 days) and
later (
30 and 60 days) time points. In addition, 8 pigs were used to
determine whether intracoronary delivery of adenovirus in doses
of 5x1012 vp (n=3) and
1.4x1012 vp (n=5), without precedent histamine,
would also result in an increased LV cAMP-generating capacity.
Finally, 23 uninstrumented animals (ACVI, n=16; lacZ, n=7) were used to determine the effects of ACVI gene transfer on cAMP production in LV membranes at 4, 8, 12, and 18 weeks after gene transfer. Twenty-three hour ECG recordings (Holter Recorder Model 461A, Del Mar Medical Systems) were obtained in 17 of these pigs (ACVI, n=11, lacZ, n=6) before and 10±1 days after gene transfer.
Instrumentation
LV pressure transducers (Konigsberg Instruments, Inc),
pulmonary arterial or aortic flow probes (Transonic
Inc), ECG wires, and left atrial, aortic, and pulmonary
arterial catheters were placed in 18 pigs. Then, 19±2 days
later, hemodynamic measurements and assessment of LV
global function were performed before and during agonist
infusions.
Gene Transfer
E1-deleted, replication-incompetent adenovirus vectors encoding
murine ACVI or nuclear-tagged bacterial
ß-galactosidase (lacZ), driven by a cytomegalovirus promoter, were
generated by homologous recombination, as previously
described.1 For instrumented animals, gene transfer was
performed 3±1 days after completion of pharmacological tests. All
animals except those enrolled in the no-histamine studies received a
3-minute intracoronary infusion of histamine diphosphate (25
µg/min; Sigma) into each of the 3 major coronary branches,
followed by a total dose of 1.4x1012 vp
adenovirus (left anterior descending, 0.7x1012
vp; left circumflex, 0.4x1012 vp; right
coronary artery, 0.3x1012 vp). Animals
that were not given precedent intracoronary histamine received
1.4x1012 vp (lacZ, n=5;
ACVI, n=5), 5x1012 vp
(ACVI, n=3), or no gene transfer (n=9). In
instrumented animals, hemodynamics and LV global
function before and during agonist infusions were repeated 12±1 days
after gene transfer. Pigs were killed and tissues were collected 14±4
days after gene delivery for histopathological and biochemical studies.
Eighteen animals that had received ACVI gene
transfer were killed 4 (n=4), 8 (n=6), 12 (n=4), and 18 (n=4) weeks
after gene transfer. Five additional animals that had received lacZ
gene transfer were used as controls for these studies.
LV Contractile Function
Echocardiographic assessments (Hewlett-Packard
Sonos 1000) were obtained in conscious animals4 to
determine the effects of gene transfer on LV size and basal function.
Two or 3 tests conducted on separate days were averaged before and
after gene transfer. Basal LV pressure, cardiac output, and heart rate
were recorded before and after administration of the glycopyrrolate
(0.07 mg/kg) used to remove vagal tone. The response after graded
intravenous bolus injections of (-)-isoproterenol (0.01,
0.03, 0.1, 0.3, 1.0, and 3.0 µg/kg) was assessed. Subsequently, LV
pressure was recorded during the intravenous
administration of 3, 10, and 30 µg/kg NKH477, a water-soluble
forskolin derivative (Nippon Kayaku, Tokyo, Japan). Data
analyses were blinded.
AC Assays
Methods were modified from Salomon, as previously
reported.1 NKH477 (0.1 to 100 µmol/L) was used to
stimulate cAMP production to assess AC function.
ACVI Protein Content
LV homogenates were centrifuged through a
5% to 35% discontinuous sucrose gradient. Fractions containing
caveolin-3 were isolated5 and concentrated with a
size-exclusion centrifugal filter (Amicom Centriprep). Then, an
anti-ACV/VI primary antibody (Santa Cruz
Biotechnology) and a secondary antibody coupled to horseradish
peroxidase were used to detect ACVI from these
fractions (1.5 µg of protein). A glutathione
S-transferase-human-ACVI fusion protein
was loaded with each sample to ensure that equal amounts of protein
were added in each lane. The intensity of the
ACVI bands was measured by densitometry (Storm
840, Molecular Dynamic).
LV ßAR, G Protein, and G Protein Receptor-Coupled Kinase-2
Content
Crude LV membrane homogenates were used in
immunoblotting studies to determine the protein
contents of ß1 and
ß2-adrenergic receptors and of G
s,
Gi2, and G protein receptor-coupled kinase-2
(GRK2), using methods previously described.2
Assessment of Inflammation and Fibrosis
Hematoxylin and eosin and Masson’s trichrome stains were used
to detect inflammatory cell infiltrates, cell necrosis, and fibrosis in
samples from the lung, liver, and LV in uninstrumented animals that had
received gene transfer of ACVI at
1.4x1012 vp (with histamine) killed 4 weeks
after gene transfer (n=4) and 5.0x1012 vp (no
histamine) killed 2 weeks after gene transfer (n=3). Five animals that
had not received adenovirus were used as controls.
Histological analysis was blinded.
Data Analysis
Differences in hemodynamic measurements and in
cAMP production were assessed by ANOVA. Protein contents were
compared using Student’s t test for paired or unpaired data
(2-tailed).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Gene Transfer and Histamine Pretreatment
Figure 1
indicates that there is a relationship between the
amount of adenovirus encoding ACVI delivered and
the effect on LV cAMP generation measured 12 days later and that the
effect is increased by precedent intracoronary histamine
infusion. In the absence of precedent intracoronary histamine,
5x1012 vp (but not
1.4x1012 vp) of adenovirus encoding
ACVI increased LV cAMP above control values. In
contrast, when intracoronary delivery of
1.4x1012 vp of an adenovirus encoding
ACVI was preceded by histamine infusion, LV cAMP
production increased 3-fold (versus lacZ); production
increased 2.8-fold over what was seen when the same dose had been
administered without histamine.
LV Contractile Function
LV dimensions were unaltered by gene transfer (Table 1), but basal fractional
shortening was mildly increased after ACVI gene
transfer. Basal hemodynamics, LV dP/dt, and cardiac
output were unaffected by gene transfer (Table 1). During
adrenergic stimulation, pigs that received lacZ gene transfer showed no
differences in LV dP/dt or cardiac output response compared with their
responses before gene transfer (Figure 2). In contrast,
ACVI gene transfer was associated with increased
LV dP/dt and cardiac output during isoproterenol stimulation, with
maximum gains of 1267±807 mm Hg/s in peak LV dP/dt
(P=0.0002) and of 39±20 mL ·
kg-1 · min-1 in
cardiac output (P<0.0001) versus the pretransfer values.
NKH477 stimulation was associated with a maximum gain of 2515±890
mm Hg/s in peak LV dP/dt. Changes in LV dP/dt were evident 6 days
after gene transfer and persisted for at least 57 days (Figure 3D), which was the time of final testing.
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), whereas a positive y value indicates
that gene transfer was associated with increased responsiveness
(ACVI, •). A, ACVI gene transfer was
associated with increased LV dP/dt (versus pretransfer value) through a
wide range of concentrations of infused isoproterenol
(ACVI, n=10; lacZ, n=8). B, ACVI gene transfer
was associated with increased LV peak negative dP/dt, indicating
increased relaxation and diastolic function
(ACVI, n=10; lacZ, n=8). C, Cardiac output (CO) stimulated
by isoproterenol was increased after ACVI gene transfer
through a wide range of concentrations of infused isoproterenol
(ACVI, n=7; lacZ, n=7). D, Direct stimulation of
ACVI (NKH477) also showed increased contractile
responsiveness in animals after ACVI gene transfer
(ACVI, n=7; lacZ, n=5). Error bars denote 1 SEM.
P value is from 2-way ANOVA.
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ECG Recordings
Gene transfer (Table 1) did not alter mean heart rate over
a 23.0±0.5-hour period. A normal diurnal variation in heart rate was
evident, with no difference between groups.
Supraventricular and ventricular
tachycardias were not present. Isolated rare unifocal
premature ventricular contractions (PVCs) were not
increased by gene transfer (ACVI: pretransfer,
2±1 PVCs; post-transfer, 2±1 PVCs; lacZ: pretransfer, 8±8 PVCs,
post-transfer, 1±1 PVCs; P=0.26, 2-way ANOVA).
ACVI Protein Content and cAMP Production
ACVI protein content was increased
(P=0.0007) in purified LV membranes from animals that had
received ACVI gene transfer (Figures 3A and 3B). Increased LV ACVI protein content
was associated with a 2-fold increase in cAMP-generating capacity 2
weeks after gene transfer (P=0.0006). This effect persisted
for at least 18 weeks (Figure 3C). LV membranes obtained 14 days
after ACVI gene transfer showed no increase in
basal AC activity (lacZ: 36±7 pmol ·
mg-1 · min-1, n=5;
ACVI: 28±5 pmol ·
mg-1 · min-1, n=5;
P=0.37).
LV ßAR, G Protein, and GRK2 Content
No differences in ß1- or
ß2-adrenergic receptors, Gs, or
Gi2 were detected in LV membranes from animals
that had received ACVI gene transfer 12 days
before testing (Table 2). However,
ACVI gene transfer was associated with a 13%
increase in immunodetectable GRK2 in LV samples.
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Histopathology
No differences existed in inflammatory cell infiltrates, cell
necrosis, or fibrosis in samples from the lung, liver, and LV of
control animals (n=5) versus samples from animals that had received
gene transfer of ACVI at
1.4x1012 vp (n=4) or
5.0x1012 vp (n=3). We did not examine the liver
and lung for the presence of ACVI transgene
expression.
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Discussion |
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Our data indicated that 12 days after ACVI gene transfer, the heart’s contractile response to ßAR stimulation increased in parallel with increased LV ACVI protein content and cAMP-generating capacity. These changes did not occur after intracoronary delivery of an adenovirus encoding lacZ. Changes in LV contractility were observed 6 days after gene transfer and persisted for at least57 days. Increased LV cAMP-generating capacity persisted for at least 18 weeks after a single intracoronary injection of adenovirus encoding ACVI.
There was no alteration in basal LV function or basal AC activity. The
LV content of ß1- and
ß2-adrenergic receptors and of the stimulatory
and inhibitory GTP-binding proteins (Gs and
Gi2) was also unchanged. The small increase in
GRK2 would not be expected to contribute to increased LV cAMP
responsiveness, but it might mildly attenuate signal transduction.
Therefore, the favorable effects on LV contractile function and cAMP
generation were the result of increased transgene expression of
ACVI in LV membranes. Gene transfer was not
associated with histological abnormalities in the
heart, liver, or lung, even at doses of 5x1012
vp. This confirms the results of a previous study.4 We
previously showed, using transgenic mice, the favorable effects of
prolonged cardiac-directed AC expression2 and its
effectiveness in treating experimental
cardiomyopathy.3 The favorable
alteration in global LV function after intracoronary delivery
of an adenovirus encoding ACVI suggests that this
strategy can be used as an alternative to transgenic mice, thereby
circumventing problems associated with the transgenic mice paradigm. It
also opens the door for possible future clinical applications.
We recently showed that intracoronary delivery of an adenovirus
encoding an angiogenic gene improves regional myocardial contractile
function in myocardial ischemia4 and in heart
failure.6 Angiogenic gene therapy does not require
ubiquitous gene transfer because the secreted transgene has a second
effect, namely, the elaboration of new blood vessels. In contrast, the
present objective was to upregulate an integral membrane protein
(ACVI), and we presumed that the degree of
increase in LV function would directly reflect the extent of gene
transfer. The ability of intracoronary gene transfer to achieve
global alterations in LV function was somewhat surprising, because the
extent of gene transfer obtained after intracoronary delivery
of an adenovirus is estimated to be <30%.7 Agents that
increase vascular permeability reportedly increase the extent of gene
transfer conferred by intracoronary delivery of an adenovirus
in ex vivo–perfused hearts.8 In the current study, we
used intracoronary infusion of histamine followed by delivery
of the adenovirus, because our initial pilot studies demonstrated
substantial gene transfer in the heart using this technique (Figure 1). We then determined that intracoronary histamine
before intracoronary adenovirus infusion increased the effect
on LV cAMP generation by nearly 3-fold (Figure 1).
Intracoronary histamine infusion may increase
intracoronary gene transfer through its effects on vascular
permeability or by increased transvesicular transport across
endothelial cells.9
Measurement of LV contractile function required the maintenance
of a pressure transducer in the LV chamber. This device rarely gives
suitable data for longer than 2 months; therefore, we were unable to
obtain data regarding LV contractile function beyond 57 days after gene
transfer (Figure 3D). It is noteworthy that peak LV dP/dt in
response to isoproterenol is as high 57 days after gene transfer as it
was 12 days after gene transfer, suggesting that the
physiological effect does not decay over this
interval. The persistent elevation in LV cAMP-generating capacity
throughout the 18-week study (Figure 3C) may indicate that LV
contractile function may be increased this long as well.
These persistent changes in transgene expression and function are longer than expected after adenovirus-mediated gene transfer.7 Perhaps this reflects a long biological half-life of the ACVI protein, although we can find nothing in the literature regarding the turnover rate of newly synthesized ACVI. Other considerations are that the cardiac myocyte is postmitotic, so a dilutional effect of cell division on the proportion of cells expressing the transgene is not a factor. Furthermore, the absence of myocardial inflammation, which we confirmed by the absence of CD4-and CD8-expressing T lymphocytes in previous studies,4 might result in more prolonged gene expression than observed in other settings that are associated with an inflammatory response.
Sustained stimulation of the heart and provocation of arrhythmias are reasonable concerns with amplification of cardiac adrenergic responsiveness. However, there was no increase in basal cardiac output, LV dP/dt, or blood pressure associated with ACVI gene transfer. Likewise, basal cAMP was unchanged, which is consistent with previous findings in transgenic mice with cardiac-directed overexpression of ACVI2 and in adenovirus-mediated ACVI gene transfer in cultured cardiac myocytes.1 Finally, 23-hour ECG recordings showed unchanged mean heart rates and no arrhythmias after ACVI gene transfer. Provocation of arrhythmia may be more likely in the setting of heart failure, but cardiac overexpression of ACVI is associated with improved heart function3 and reduced mortality (unpublished data), suggesting that ACVI treatment does not provoke life-threatening arrhythmias in experimental heart failure.
Three studies have used direct or indirect intracoronary gene transfer to alter global LV function.10 11 12 These studies substantiate the effectiveness of an intracoronary injection of recombinant adenovirus to change cardiac function, confirming in our initial report in 1996.4 Weig et al10 used a coronary infusion of low calcium and serotonin followed by an adenovirus encoding V2 vasopressin receptors (1010 pfu). This was associated with a 20% increase in LV dP/dt response at 3 doses of desmopressin acetate in anesthetized animals in studies performed an unspecified time after gene transfer. However, this was an unblinded study in anesthetized rabbits, and LV dP/dt was only 40% of normal because of the negative inotropic effects of the anesthetic agents that were used. Maurice et al11 used adenosine pretreatment in rabbits followed by indirect intracoronary injection of an adenovirus encoding ß2-adrenergic receptors (1012 vp). They observed increased LV dP/dt in anesthetized animals in response to 4 doses of isoproterenol (the mean increase was 13% versus lacZ-treated animals at 6 days and 20% at 21 days). However, cardiac overexpression of ßAR13 14 or Gs15 provides sustained adrenergic stimulation with undesirable consequences, including increased mortality, in experimental cardiomyopathy,14 cardiac fibrosis,13 15 and cardiomyopathy.13 15 Hajjar et al12 used thoracotomy followed by indirect intracoronary delivery in rats of an adenovirus encoding phospholamban and reported a 39% reduction in LV dP/dt when anesthetized animals were studied 2 days later. However, the effect was gone by 5 days. In these studies,12 the amount of adenovirus delivered to the rat heart was 30- to 90-fold higher per gram of rat heart than what we delivered to the pig heart. This may explain the cardiac inflammation seen in the rat heart, which could cause the short-lived effects.
A number of clinical trials of inotropic agents that increase cAMP levels in the failing heart indicate that such a strategy is contraindicated. However, the agents studied in these trials resulted in sustained elevations of cAMP via stimulation of the ßAR with dobutamine infusion16 or by blocking cAMP breakdown with the phosphodiesterase inhibitor milrinone.17 It may be the unrelenting stimulation of the heart from sustained cAMP elevations that led to deleterious consequences in these clinical trials. In contrast, with ACVI gene transfer, there is no change in basal levels of cAMP in cardiac myocytes.1 2 3 The beneficial effects of ACVI overexpression in the setting of experimental heart failure3 support the possibility that this mode of treatment could improve LV contractile responsiveness in clinical heart failure without detrimental effects. Clearly, tests in animal models are not conclusive, but the data justify further evaluation of this strategy in clinical heart failure.
In conclusion, intracoronary delivery of an adenovirus expressing ACVI increases global LV contractile function in normal pigs. AC overexpression provided increased recruitable adrenergic responsiveness, resulting in increased LV contractile function. This effect was present 6 days after gene transfer and persisted for at least 57 days. Increased function was associated with increased LV ACVI protein content and an increased ability of cardiac myocytes to generate cAMP, an effect that persisted for at least 18 weeks. There was no evidence of sustained adrenergic activation or arrhythmias. These data, together with previous reports,2 3 suggest that this approach may be suitable for the treatment of clinical heart failure.
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Acknowledgments |
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Supported by the Department of Veteran’s Affairs (Merit award to H.K.H. and Research Career Development Award to D.M.R.), grants from the National Institutes of Health (NIH1P50HL-53773-01 and 2P50HL-53773-06 to H.K.H.), and Collateral Therapeutics, Inc. We thank Matthew Spellman, Muriel Spooner, Xin Guo, Gary Anderson, Mona Logan, and Bob Worth for technical assistance; Dr Joe Voland for histological review; Nippon Kayaku for providing NKH477; and Dr Tamsin Lisa Kelly for reviewing the manuscript.
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Footnotes |
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Dr Hammond is the scientific founder of Collateral Therapeutics, Inc (CTI); he also owns CTI stock, but is not an employee. Dr David M. Roth serves as a consultant to Collateral Therapeutics, Inc; he, too, owns CTI stock. CTI was one of the supporters of the study.
Received September 5, 2000; revision received September 22, 2000; accepted September 28, 2000.
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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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 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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 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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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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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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