Published online before print July 8, 2004, doi: 10.1161/01.RES.0000138449.85324.c5
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2004 American Heart Association, Inc.
Cellular Biology |
Creation of a Genetic Calcium Channel Blocker by Targeted Gem Gene Transfer in the Heart
From the Institute of Molecular Cardiobiology and Division of Cardiology, Department of Medicine, The Johns Hopkins University, Baltimore, Md.
Correspondence to Eduardo Marbán, MD, PhD, Institute of Molecular Cardiobiology, The Johns Hopkins University, 720 Rutland Ave, Ross 844, Baltimore, MD 21205. E-mail marban{at}jhmi.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Calcium channel blockers are among the most commonly used therapeutic drugs. Nevertheless, the utility of calcium channel blockers for heart disease is limited because of the potent vasodilatory effect that causes hypotension, and other side effects attributable to blockade of noncardiac channels. Therefore, focal calcium channel blockade by gene transfer is highly desirable. With a view to creating a focally applicable genetic calcium channel blocker, we overexpressed the ras-related small G-protein Gem in the heart by somatic gene transfer. Adenovirus-mediated delivery of Gem markedly decreased L-type calcium current density in ventricular myocytes, resulting in the abbreviation of action potential duration. Furthermore, transduction of Gem resulted in a significant shortening of the electrocardiographic QTc interval and reduction of left ventricular systolic function. Focal delivery of Gem to the atrioventricular (AV) node significantly slowed AV nodal conduction (prolongation of PR and AH intervals), which was effective in the reduction of heart rate during atrial fibrillation. Thus, these results indicate that gene transfer of Gem functions as a genetic calcium channel blocker, the local application of which can effectively modulate cardiac electrical and contractile function.
Key Words: calcium channel blocker • gene therapy
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Intracellular Ca2+ plays a pivotal role in diverse biological processes, including gene regulation, memory, and cell death.1,2 In the heart, Ca2+ is essential as the activator of muscle contraction.3 Although Ca2+ is vital for normal function, excessive increases in intracellular Ca2+ foster arrhythmias, hypertrophy, apoptosis, and cardiac remodeling. Modulation of Ca2+ homeostasis could be useful for the therapeutic manipulation of such pathophysiological processes.4–6 Among the cell’s many Ca2+ handling proteins, the L-type calcium channel, initiating excitation-contraction coupling, represents a logical target for intervention. Indeed, in animal experiments, calcium channel blockers reduce cardiac hypertrophy in spontaneously hypertensive rats,7 and inhibit the electrical remodeling induced by rapid atrial pacing.8 Unfortunately, the use of calcium channel blockers is often accompanied by side effects (eg, hypotension, heart block, constipation),6 which limits their utility for the treatment of heart disease. It would be highly desirable to be able to achieve calcium channel blockade in the heart (or part of the heart) without affecting other tissues.
Such a motivation prompted us to establish a novel method to modulate calcium channel activity focally. This was accomplished by viral gene transfer of Gem into the heart in vivo. Gem is a member of a small GTP-binding family of proteins within the Ras superfamily,9,10 and was recently found to suppress L-type calcium currents in PC12 cells by inhibiting the trafficking of calcium channel 
subunits to the plasma membrane.11 Reasoning that a similar effect might be recruitable in native heart cells, we transduced an adenovirus encoding Gem into the heart, resulting in the suppression of L-type calcium currents and a concomitant reduction of cardiac contractility, as expected with cardiac-specific calcium channel blockade. As proof of principle for the value of focal Ca2+ channel blockade in vivo, we demonstrated that Gem was effective in slowing atrioventricular (AV) conduction when delivered into the porcine AV node, resulting in the reduction of heart rate during atrial fibrillation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Plasmid Construction and Adenovirus Preparation
The full-length coding sequences of Gem (kindly supplied by Dr Katherine Kelly, NIH, Bethesda, Md, and independently by Dr Susumu Seino, Chiba, Japan) was cloned into the multiple cloning site of the adenovirus shuttle vector pAdCIG to generate pAdCIG-Gem. This construct is bicistronic (through an internal ribosome entry site) driven by a cytomegalovirus promoter and carrying green fluorescent protein (GFP) as a reporter. The point mutation W269G was introduced into Gem by site-directed mutagenesis, creating the vector pAdCIG-Gem W269G. Detailed methods of adenovirus vector construction have been described.12–14 In vivo adenoviral transduction into guinea-pig hearts was performed as described.15 Adenoviruses (160 µL, equivalent to
3x109 plaque-forming units, pfu) were injected in the left ventricular (LV) cavity of guinea pigs (280 to 340 g), whereas the aorta and pulmonary artery were clamped for 50 to 60 seconds.
Myocyte Isolation and Electrophysiology
Seventy-two hours after gene delivery, myocytes were isolated from the left ventricles of guinea pigs, using enzymatic digestions as previously described.13 Membrane currents and action potentials were recorded using whole-cell patch clamp with an Axopatch 200B amplifier (Axon Instruments, Foster City, Calif). All myocyte recordings were performed at 37°C. Cells were superfused in solution containing (in mmol/L) 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, 2 CaCl2, and 10 glucose (pH 7.4 adjusted with NaOH). For ICa,L recordings, the external solution was replaced with solution containing (in mmol/L) 140 cholineCl, 5 CsCl, 1 MgCl2, 10 HEPES, 2 CaCl2, and 10 glucose (pH 7.4 adjusted with CsOH), after establishing the whole-cell clamp mode. The micropipette electrode solution for ICa,L was composed of (in mmol/L) 120 CsCl, 10 TEACl, 1 MgCl2, 10 HEPES, 10 EGTA, and 5 MgATP. Action potentials and IK1 were recorded with an internal solution composed of (in mmol/L) 130 K-glutamate, 9 KCl, 1 MgCl2, 10 Na-HEPES, 2 EGTA, and 5 Mg-ATP (pH 7.2 adjusted with KOH). L-type calcium currents were elicited by 300 ms-depolarizing steps from –40 to 60 mV in 10 mV increments. For action potential recordings followed by the measurement of nitrendipine-sensitive Ca2+ currents, the external solution contained (in mmol/L) NaCl 140, KCl 5, CaCl2 2, MgCl2 1.0, glucose 10, and HEPES 10 (pH 7.4 with NaOH), and the pipette solution contained (in mmol/L) K-glutamate 130, KCl 9.0, MgCl2 1.0, EGTA 2.0, Mg-ATP 5.0, and Na-HEPES 10 (pH 7.3 with KOH). After recording the action potential, currents were elicited by the standard pulse protocol for calcium currents as shown before and after application of 10 µmol/L nitrendipine, and subtracted currents were considered as nitrendipine-sensitive calcium currents. For IK1 recordings, CaCl2 was reduced to 100 µmol/L, CdCl2 (200 µmol/L) was added to block ICa,L, and INa was steady-state inactivated by using a holding potential of –40 mV. To obtain IK1 as a Ba2+-sensitive current, currents recorded before and after the addition of Ba2+ (500 µmol/L) were subtracted.
Borosilicate glass pipettes were pulled and fire-polished to final tip resistances of 1 to 3 M
when filled with internal recording solution. Uncompensated capacitance currents in response to small hyperpolarizing voltage steps were recorded for off-line integration to measure cell capacitance. Cells were allowed >5 minutes to equilibrate after whole-cell access was obtained. Action potentials were initiated by short depolarizing current pulses (2 ms, 100 to 300 pA, 10% to 15% over the threshold) at every 3 seconds. APD was measured as the time from the overshoot to the indicated percentage of repolarization. A xenon arc lamp was used to view green fluorescent protein (GFP) fluorescence at 488/530 nm (excitation/emission). Transduced cells were recognized by their obvious green fluorescence.
To measure gating currents, ionic currents were blocked by adding 2 mmol/L CdCl2 and 0.1 mmol/L LaCl3 to the bath solution. Leaks and capacitative transients were subtracted by a P/–4 protocol from a –100 mV holding potential. Charge movement was quantified by calculating the area under the curve for each trace, using the steady-state level of the current as a baseline.
Electrocardiograms
Body surface electrocardiograms (ECGs) were recorded within 2 hours after operation (baseline) and 72 hours after adenovirus injection, as previously described.13 Guinea pigs were anesthetized with isoflurane, and needle electrodes were placed under skin. Needle electrode positions were marked postoperatively on the skin to ensure exactly the same electrode position for 72-hour recordings. Measured QT intervals were corrected (QTc) for heart rate as previously described.16
Cardiac Hemodynamic Studies
Seventy-two hours after LV injection, guinea-pigs were anesthetized with isoflurane. The right carotid artery was isolated and a micromanometer catheter (Millar Instruments, Houston, Texas) was inserted via the right carotid artery and passed retrogradely into the left ventricle. Position was confirmed by the characteristic decrease in diastolic pressure that occurred with passage of the catheter across the aortic valve into the LV cavity, after which LV pressure and the first derivative of LV pressure (dP/dt) were recorded.
Focal Gene Transfer Into AV Node
Adenoviral gene transfer into AV node was performed as described.17 Briefly, immediately before catheterization, domestic swine (25 to 30 kg) received 25 mg sildenafil orally. The right carotid artery, right internal jugular vein, and right femoral vein were accessed by sterile surgical technique, and introducer sheaths were inserted into each vessel. After baseline EP study, the right coronary artery was catheterized via the right femoral artery. The AV nodal branch was selected with a 0.014-inch guide wire, over which a 2.7F infusion catheter was inserted into the AV nodal artery. The following solutions were infused through the catheter: 10 mL normal saline (NS) containing 5 µg VEGF165 and 200 µg nitroglycerin >3 minutes, 1 mL NS containing 1.0x1010 pfu adenovirus and 20 µg nitroglycerin >30 seconds, and 2.0 mL NS >30 seconds.
Western Blot Analysis
Expression of Gem protein in the heart was determined by the immunoblot technique. For this purpose, crude homogenates of the left ventricle were prepared, normalized for protein content, and SDS-PAGE was performed on 4% to 12% gradient gels.
Monoclonal anti-Gem antibody (gift from Dr Katherine Kelly, NIH, Bethesda, Md) was used as a primary antibody. A peroxidase-conjugated goat anti-mouse IgG (Amersham Biosciences, Uppsala, Sweden) was used as a secondary antibody. Signals were visualized with enhanced chemiluminescence.
Statistical Analysis
All the data shown are mean±SEM. Statistical differences were determined using repeated measures ANOVA and Student paired t test, where appropriate, and P<0.05 was considered to indicate statistical significance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Overexpression of Gem in Guinea Pig Ventricular Cardiomyocytes
We first investigated whether Gem overexpression could inhibit L-type calcium current (ICa,L) in guinea-pig ventricular cardiomyocytes. Adenoviruses were injected into the left ventricular (LV) cavity of guinea-pig hearts, and 3 days later, LV cells were isolated. Overexpression of AdCIG-wild-type (WT) Gem resulted in a dramatic decrease of ICa,L from a peak density of 4.7±0.5 pA/pF at 10 mV (n=11) in AdCIG-transduced (control) cells to 0.5±0.2 pA/pF at 10 mV (n=8) in AdCIG-WT Gem-transduced cells (Figure 1a and 1b). The inhibitory effect of Gem on ICa,L attributable to the prevention of interaction between
and ß subunits of L-type calcium channels by scavenging ß subunits.11 Based on this, we examined the effect of the less-effective AdCIG-W269G mutant.11 Overexpression of the AdCIG-W269G mutant reduced ICa,L modestly, but significantly (30% inhibition versus control, 3.3±0.2 pA/pF at 10 mV, n=10).
|
, n=8), compared with control cells (
, n=11), whereas W269G mutant had a slight effect (
, n=10). #P<0.05 vs control, *P<0.01 vs control. c, Representative Ik1 currents in an AdCIG-transduced (control) and an AdCIG wild-type (WT) Gem-transduced cell. Vertical scale bar=10 pA/pF; horizontal scale bar=100 ms; dash marks designate zero current. d, Current-voltage relationships of Ik1 currents in control (Because Gem is a GTP binding protein possibly involved in many signal transduction pathways, it is possible that Gem might have effects such on other ion currents. We therefore investigated the effects on other ion currents such as IK1, INa, and ICa,T before and after Gem gene transfer. No changes in IK1 were observed (at –50 mV, 3.5±0.2 pA/pF, n=5 versus 3.4±0.9 pA/pF, n=5, in AdCIG-WT Gem-transduced, and AdCIG-transduced cells, respectively; Figure 1c and 1d). Additionally, neither INa (at –40 mV, 25.8±2.5 pA/pF, n=5 versus 26.5±3.2 pA/pF, n=6, in AdCIG-WT Gem-transduced, and AdCIG-transduced cells, respectively), nor ICa,T (at –20 mV, 0.43±0.13 pA/pF, n=4 versus 0.42±0.17 pA/pF, n=5, in AdCIG-WT Gem-transduced, and AdCIG-transduced cells, respectively) were affected; thus, transduction of Gem appears to specifically affect L-type Ca2+ channels.
If, as previously proposed, Gem binds to ß-subunits of calcium channels and thereby inhibits the trafficking of -subunits to the plasma membrane,11 a decrease in the number of functional channels would be predicted. To assess channel number electrophysiologically, we measured the gating charge attributable to L-type calcium channels and isolated the calcium channel-specific component using 10 µmol/L nitrendipine, a pharmacological calcium channel blocker.18,19 Overexpression of AdCIG-WT Gem resulted in a marked reduction of nitrendipine-sensitive gating currents compared with control (Figure 2a and 2b). The gating currents during depolarization were integrated to calculate charge movements during depolarization (Qon). Control Qon was significantly greater than that in AdCIG-WT Gem-transduced myocytes (2.2±0.2 fC/pF at +30 mV, n=6, versus 0.67±0.1 fC/pF at +30 mV, n=6; P=0.001). The voltage dependence of Qon was comparable in AdCIG-transduced (control) and AdCIG-WT Gem-transduced cells, as demonstrated by simultaneously fit Boltzmann distributions to the mean data sets, with V1/2=0.5 mV, k=13.3 mV in control, and V1/2=–0.3 mV, k=9.8 mV in AdCIG-WT Gem-transduced cells. AdCIG-W269G mutant only modestly affected these parameters (1.8±0.1 fC/pF at +30 mV, V1/2=5.5 mV, k=15.5 mV, n=6). These results indicate that the number of functional calcium channels in the cell membrane is indeed decreased in Gem-transduced cells, compared with AdCIG-transduced (control) cells. Those channels that do make it to the surface, however, appear to have normal gating properties.
|
Next, we investigated the effects of Gem overexpression on action potentials in ventricular cardiomyocytes. Overexpression of AdCIG-WT Gem resulted in the abbreviation of action potential duration (APD) without any change in resting membrane potential (–84.6±0.4 mV versus –84.9±0.4 mV) or phase 0 depolarization (dV/dtmax) (–86.1±3.5 V/s versus –82.2±3.9 V/s) (Figure 3a). Both APD50 and APD90 were significantly shortened in AdCIG-WT Gem-transduced cells compared with control cells, whereas overexpression of AdCIG-W269G mutant had little effect (Figure 3a and 3b). Notably, the robust plateau phase was blunted in WT Gem-transduced myocytes.
|
The modification of action potentials became more pronounced as ICa,L decreased. There was a clear correlation between ICa,L density, calculated as the nitrendipine-sensitive ionic current, and APD90; both were reduced in AdCIG-WT Gem-transduced myocytes compared with AdCIG-transduced (control) cells (n=21, r=0.87, P<0.0001) (Figure 3c).
In Vivo Phenotype of Cardiac Calcium Channel Blockade
We previously reported that the LV cavity injection method resulted in a transduction efficiency of 15% to 25% in the heart.20 Western blot analysis (Figure 4a) confirmed Gem overexpression in all cardiac chambers, amounting to a 300±60% increase in the AdCIG-WT Gem-transduced animals relative to control levels (P=0.002). The presence of endogenous Gem in heart was unexpected, and gives reason to wonder whether it may play a physiological role in myocardial Ca2+ homeostasis; however, this possibility was not explored in the present study.
|
To assess the electrophysiological phenotype in intact animals, electrocardiograms were performed 3 to 4 days after injection of adenoviruses into the LV cavity. The QT interval19 was shortened in AdCIG-WT Gem-transduced animals compared with AdCIG-transduced (control) animals (eg, Figure 4b). Consistent with action potential recordings in isolated AdCIG-WT Gem-transduced myocytes, the QTc intervals of the ECG measured 3 days after transduction were abbreviated in AdCIG-WT Gem-transduced animals compared with the same animals immediately after surgery (165±3.5 ms versus 148±2.3 ms, n=9; P<0.05). In contrast, no change in the QTc interval was observed in the animals transduced with AdCIG (165±3.3 ms versus 166±1.8 ms, n=6) or AdCIG-W269G mutant (166±1.8 ms versus 163±1.1 ms, n=7). Interestingly, we observed PQ interval prolongation in one of the AdCIG-WT Gem-transduced animals (central panel, Figure 4b), which was presumably induced by fortuitously intense expression of Gem in the AV node.
Effects of Gem on Cardiac Hemodynamics
Next, we examined the effect of WT Gem transduction on cardiac hemodynamics. Heart rate did not change (Figure 5a), but transduction of AdCIG-WT Gem resulted in the reduction of contractile activity in guinea pig hearts. The peak LV systolic pressure (LVSP) was reduced 3 days after gene delivery in AdCIG-WT Gem-transduced animals compared with AdCIG-transduced (control) animals (65.8±5.2 mm Hg, n=5, versus 85.8±2.7 mm Hg, n=4; P<0.05) (Figure 5b). Furthermore, the maximum first derivative of LV pressure (dP/dtmax) was reduced as well (4028±150 mm Hg/s, n=5, versus 4842±158 mm Hg/s, n=4; P=0.01) (Figure 5c). These data indicate that transduction of Gem produced a significant negative inotropic effect, as expected with myocardial calcium channel blockade.
|
Focal Modification of AV Nodal Conduction by Gem Gene Transfer
Atrial fibrillation is a disturbance of cardiac rhythm in which a rapid heart rate produces breathlessness and decreased exercise tolerance. Inhibition of AV nodal conduction, by calcium channel blockade, is the mainstay of drug therapy, but such therapy is fraught with side effects that are attributable to calcium channel blockade outside the AV node. We previously developed an intracoronary perfusion model for adenoviral gene delivery in pigs, and succeeded in the modification of AV nodal conduction by overexpression of the inhibitory G protein, Gi2.17 We reasoned that overexpression of Gem in the AV node would likewise slow AV nodal conduction, with possible benefit for rate control in atrial fibrillation. Seven days after gene transfer in the same swine model, AdCIG-WT Gem-transduced animals revealed prolongation of the PR interval on the surface ECG and the AH interval (but not the HV interval) on the intracardiac electrogram, confirming slowed conduction in the AV node (Figure 6b through 6e). During acute episodes of atrial fibrillation (Figure 6a), overexpression of AdCIG-WT Gem in the AV node caused a 20% reduction in the ventricular rate during atrial fibrillation (Figure 6f). This effect persisted in the setting of ß-adrenergic stimulation as well as cholinergic inhibition (Figure 6g and the Table). Given the previous demonstration17 that adenoviral transduction per se does not affect AV nodal conduction, we conclude that focal calcium channel blockade induced by Gem gene transfer into the AV node effectively reduces the heart rate in atrial fibrillation.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The utility of calcium channel blockers for heart disease has been limited by potent vasodilatation and hypotension attributable to blockade of noncardiac channels. In this regard, gene therapy would be a feasible strategy for organ-specific or regionally specific treatment within an organ. In this study, we reported the novel finding that gene transfer of a ras-related small G-protein, Gem could be useful as a genetic calcium channel blocker in the heart, expressed globally for the depression of contractility, or in part of the heart (the AV node), delivered focally to alter conduction.
Overexpression of AdCIG-WT Gem prominently inhibited ICa,L in guinea-pig ventricular cardiomyocytes, resulting in marked abbreviation of APD, whereas W269G mutant had directionally similar but weaker effects. Furthermore, in vivo delivery of AdCIG-WT Gem shortened the QT interval, consistent with the abbreviation of APD seen in isolated cardiomyocytes. The prominent inhibitory effect of Gem WT on ICa,L was effective in genetic modification of AV nodal conduction to treat atrial fibrillation, a common arrhythmia that afflicts >2 million Americans. Once AF becomes chronic, therapy is directed at achieving rate control with the use of AV nodal blocking agents.21 Because ICa,L underlies impulse conduction in the AV node, calcium channel blockers are preferred agents for rate control during AF, but often are not tolerated because of contractile depression from block of non-AV nodal calcium channels in the heart, or hypotension from block of noncardiac channels. In this study, we have achieved focal modification of AV nodal conduction by gene transfer of Gem WT via the AV nodal artery; the resultant regionally selective ICa,L blockade is effective at rate control during AF, without undermining calcium channel function in the pumping chambers of the heart.
Based on the marked inhibitory effect of WT Gem on ICa,L, complete AV block might have been a potential consequence of WT Gem gene transduction. Anticipating this possibility, we introduced electronic pacemakers into all animals for backup purposes before gene transfer. Nevertheless, AV conduction was significantly slowed without high-grade AV block. This effect is not inconsistent with the observed efficacy of gene transfer to the AV node (40% of AV nodal cells are transduced by our delivery method,17 which might not suffice for complete AV block to develop.
A second application is for modulation of cardiac contractility. Hemodynamic studies showed that there was a significant negative inotropy in AdCIG-WT Gem-transduced hearts compared with AdCIG-transduced (control) hearts in guinea pigs, indicating that gene transfer of Gem could be useful to reduce cardiac contractility. Negative inotropic drugs are first-line treatment for patients with hypertrophic obstructive cardiomyopathy (HOCM),22 to reduce contractile activity in the hypertrophic heart as a means of improving overall pumping efficacy. In accordance with this idea, iatrogenic myocardial infarction has been developed as another means of treating severe HOCM.23–25 However, the utility of this radically destructive therapy is limited by its side effects, including inflammation, fibrosis, and arrhythmogenesis. Focal gene therapy may represent an attractive alternative approach to HOCM, in that part of the myocardium would be rendered regionally passive whereas remaining alive and excitable. More work will be necessary to reduce this idea to practice.
Although Gem is weakly expressed in the heart, its function has never been elucidated. In other cell types such as fibroblast and neurons, Gem was reported to interact with KIF9 and Rho kinase, resulting in changes in cell morphology and cytoskeletal organization.26,27 We are now investigating Gem-mediated signal transduction pathways in cardiomyocytes, with a view to determining its potential utility in the suppression of hypertrophy. However, such data are beyond the scope of the present study.
The present study was designed for proof of concept. For such a purpose, adenoviruses are well-suited; they can be readily made and grown to high titers, they are highly effective at transducing cardiac myocytes, and they lead to intense expression of the transgene for days to weeks. Longer-term expression is limited with adenoviruses, and adverse effects have occurred clinically with their systemic use. Although our adenoviral transduction strategy should lead to higher expression in the heart, there could inevitably be some contaminated infection in other organs such as liver and kidney. For these reasons, other vectors such as adeno-associated virus or usage of a cardiac-specific promoter would be better-suited for chronic experiments, including those which will be required before the present approach can be translated to patients.
With the appropriate vector and delivery method, the present strategy is generalizable. If we can selectively block cardiac calcium channels, why not target arterioles to control hypertension as a durable surrogate for lifetime antihypertensive therapy? Might specific regions of the brain involved in memory benefit from local suppression of L-type channel activity? These and many other intriguing possibilities are now amenable to study by straightforward modifications of the novel approach described here.
|
Acknowledgments |
|---|
This study was supported by the NIH (R37 HL36957 and P50 HL52307 to E.M.) and the Donald W. Reynolds Cardiovascular Clinical Research Center at Johns Hopkins. E.M. holds the Michel Mirowski, MD, Professorship of Cardiology. We thank B. O’Rourke, M.K. Leppo, and Peihong Dong for technical advice and helpful discussions.
|
Footnotes |
|---|
Under a licensing agreement between Excigen, Inc. and the Johns Hopkins University, Drs Marbán, Donahue, and Murata are entitled to a share of royalty and milestone payments received by the University on sales of products described in this article. Drs Marbán and Donahue own Excigen, Inc. stock, which is subject to certain restrictions under University policy. Drs Marbán and Donahue also are paid consultants to Excigen, Inc. and paid members of the company’s scientific advisory board. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.
This manuscript was sent to Michael Rosen, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received April 1, 2004; revision received June 24, 2004; accepted June 28, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Muth JN, Varadi G, Schwartz A. Use of transgenic mice to study voltage-dependent Ca2+ channels. Trends Pharmacol Sci. 2001; 22: 526–532.[CrossRef][Medline] [Order article via Infotrieve]
2. Carafoli E, Santella L, Branca D, Brini M. Generation, control, and processing of cellular calcium signals. Crit Rev Biochem Mol Biol. 2001; 36: 107–260.[CrossRef][Medline] [Order article via Infotrieve]
3. Bers DM. Cardiac excitation-contraction coupling. Nature. 2002; 415: 198–205.[CrossRef][Medline] [Order article via Infotrieve]
4. Isner JM. Myocardial gene therapy. Nature. 2002; 415: 234–239.[CrossRef][Medline] [Order article via Infotrieve]
5. Kaprielian R, del Monte F, Hajjar RJ. Targeting Ca2+ cycling proteins and the action potential in heart failure by gene transfer. Basic Res Cardiol. 2002; 97: I136–45.[Medline] [Order article via Infotrieve]
6. Missiaen L, Robberecht W, van den Bosch L, Callewaert G, Parys JB, Wuytack F, Raeymaekers L, Nilius B, Eggermont J, De Smedt H. Abnormal intracellular Ca2+ homeostasis and disease. Cell Calcium. 2000; 28: 1–21.[CrossRef][Medline] [Order article via Infotrieve]
7. Feron O, Salomone S, Godfraind T. Action of the calcium channel blocker lacidipine on cardiac hypertrophy and endothelin-1 gene expression in stroke-prone hypertensive rats. Br J Pharmacol. 1996; 118: 659–664.[Medline] [Order article via Infotrieve]
8. Kurita Y, Mitamura H, Shiroshita-Takeshita A, Yamane A, Ieda M, Kinebuchi O, Sato T, Miyoshi S, Hara M, Takatsuki S, Ogawa S. Daily oral verapamil before but not after rapid atrial excitation prevents electrical remodeling. Cardiovasc Res. 2002; 54: 447–455.
9. Cohen L, Mohr R, Chen YY, Huang M, Kato R, Dorin D, Tamanoi F, Goga A, Afar D, Rosenberg N, et al. Transcriptional activation of a ras-like gene (kir) by oncogenic tyrosine kinases. Proc Natl Acad Sci U S A. 1994; 91: 12448–12452.
10. Maguire J, Santoro T, Jensen P, Siebenlist U, Yewdell J, Kelly K. Gem: an induced, immediate early protein belonging to the Ras family. Science. 1994; 265: 241–244.
11. Beguin P, Nagashima K, Gonoi T, Shibasaki T, Takahashi K, Kashima Y, Ozaki N, Geering K, Iwanaga T, Seino S. Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem. Nature. 2001; 411: 701–706.[CrossRef][Medline] [Order article via Infotrieve]
12. Hardy S, Kitamura M, Harris-Stansil T, Dai Y, Phipps ML. Construction of adenovirus vectors through Cre-lox recombination. J Virol. 1997; 71: 1842–1849.[Abstract]
13. Hoppe UC, Marban E, Johns DC. Distinct gene-specific mechanisms of arrhythmia revealed by cardiac gene transfer of two long QT disease genes, HERG and KCNE1. Proc Natl Acad Sci U S A. 2001; 98: 5335–5340.
14. Johns DC, Marx R, Mains RE, O’Rourke B, Marban E. Inducible genetic suppression of neuronal excitability. J Neurosci. 1999; 19: 1691–1697.
15. Mazhari R, Nuss HB, Armoundas AA, Winslow RL, Marban E. Ectopic expression of KCNE3 accelerates cardiac repolarization and abbreviates the QT interval. J Clin Invest. 2002; 109: 1083–1090.[CrossRef][Medline] [Order article via Infotrieve]
16. Hayes E, Pugsley MK, Penz WP, Adaikan G, Walker MJ. Relationship between QaT and RR intervals in rats, guinea pigs, rabbits, and primates. J Pharmacol Toxicol Methods. 1994; 32: 201–207.[CrossRef][Medline] [Order article via Infotrieve]
17. Donahue JK, Heldman AW, Fraser H, McDonald AD, Miller JM, Rade JJ, Eschenhagen T, Marban E. Focal modification of electrical conduction in the heart by viral gene transfer. Nat Med. 2000; 6: 1395–1398.[CrossRef][Medline] [Order article via Infotrieve]
18. Bolger GT, Gengo PJ, Luchowski EM, Siegel H, Triggle DJ, Janis RA. High affinity binding of a calcium channel antagonist to smooth and cardiac muscle. Biochem Biophys Res Commun. 1982; 104: 1604–1609.[CrossRef][Medline] [Order article via Infotrieve]
19. Bean BP. Nitrendipine block of cardiac calcium channels: high-affinity binding to the inactivated state. Proc Natl Acad Sci U S A. 1984; 81: 6388–6392.
20. Miake J, Marban E, Nuss HB. Functional role of inward rectifier current in heart probed by Kir2.1 overexpression and dominant-negative suppression. J Clin Invest. 2003; 111: 1529–1536.[CrossRef][Medline] [Order article via Infotrieve]
21. Wyse DG, Waldo AL, DiMarco JP, Domanski MJ, Rosenberg Y, Schron EB, Kellen JC, Greene HL, Mickel MC, Dalquist JE, Corley SD. A comparison of rate control and rhythm control in patients with atrial fibrillation. N Engl J Med. 2002; 347: 1825–1833.
22. Spirito P, Seidman CE, McKenna WJ, Maron BJ. The management of hypertrophic cardiomyopathy. N Engl J Med. 1997; 336: 775–785.
23. Nielsen CD, Spencer WH, 3rd. Role of controlled septal infarct in hypertrophic obstructive cardiomyopathy. Cardiol Rev. 2002; 10: 108–118.[CrossRef][Medline] [Order article via Infotrieve]
24. Sigwart U. Non-surgical myocardial reduction for hypertrophic obstructive cardiomyopathy. Lancet. 1995; 346: 211–214.[CrossRef][Medline] [Order article via Infotrieve]
25. Chang SM, Nagueh SF, Spencer WH III, Lakkis NM. Complete heart block: determinants and clinical impact in patients with hypertrophic obstructive cardiomyopathy undergoing nonsurgical septal reduction therapy. J Am Coll Cardiol. 2003; 42: 296–300.
26. Piddini E, Schmid JA, de Martin R, Dotti CG. The Ras-like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin-like protein KIF9. EMBO J. 2001; 20: 4076–4087.[CrossRef][Medline] [Order article via Infotrieve]
27. Ward Y, Yap SF, Ravichandran V, Matsumura F, Ito M, Spinelli B, Kelly K. The GTP binding proteins Gem and Rad are negative regulators of the Rho- Rho kinase pathway. J Cell Biol. 2002; 157: 291–302.
This article has been cited by other articles:
 |
|
 |
|
  N. Beetz, L. Hein, J. Meszaros, R. Gilsbach, F. Barreto, M. Meissner, U. C. Hoppe, A. Schwartz, S. Herzig, and J. Matthes Transgenic simulation of human heart failure-like L-type Ca2+-channels: implications for fibrosis and heart rate in mice Cardiovasc Res, December 1, 2009; 84(3): 396 - 406. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Levy, O. Beharier, Y. Etzion, M. Mor, L. Buzaglo, L. Shaltiel, L. A. Gheber, J. Kahn, A. J. Muslin, A. Katz, et al. Molecular Basis for Zinc Transporter 1 Action as an Endogenous Inhibitor of L-type Calcium Channels J. Biol. Chem., November 20, 2009; 284(47): 32434 - 32443. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Xu and H. M. Colecraft Engineering Proteins for Custom Inhibition of CaV Channels Physiology, August 1, 2009; 24(4): 210 - 218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-P. Leyris, C. Gondeau, A. Charnet, C. Delattre, M. Rousset, T. Cens, and P. Charnet RGK GTPase-dependent CaV2.1 Ca2+ channel inhibition is independent of CaV{beta}-subunit-induced current potentiation FASEB J, August 1, 2009; 23(8): 2627 - 2638. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Davis, M. V. Westfall, D. Townsend, M. Blankinship, T. J. Herron, G. Guerrero-Serna, W. Wang, E. Devaney, and J. M. Metzger Designing Heart Performance by Gene Transfer Physiol Rev, October 1, 2008; 88(4): 1567 - 1651. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Savelieva and J. Camm Anti-arrhythmic drug therapy for atrial fibrillation: current anti-arrhythmic drugs, investigational agents, and innovative approaches Europace, June 1, 2008; 10(6): 647 - 665. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Cingolani, G. A. Ramirez Correa, E. Kizana, M. Murata, H. C. Cho, and E. Marban Gene Therapy to Inhibit the Calcium Channel {beta} Subunit: Physiological Consequences and Pathophysiological Effects in Models of Cardiac Hypertrophy Circ. Res., July 20, 2007; 101(2): 166 - 175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yada, M. Murata, K. Shimoda, S. Yuasa, H. Kawaguchi, M. Ieda, T. Adachi, M. Murata, S. Ogawa, and K. Fukuda Dominant Negative Suppression of Rad Leads to QT Prolongation and Causes Ventricular Arrhythmias via Modulation of L-type Ca2+ Channels in the Heart Circ. Res., July 6, 2007; 101(1): 69 - 77. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Beguin, Y. J. A. Ng, C. Krause, R. N. Mahalakshmi, M. Y. Ng, and W. Hunziker RGK Small GTP-binding Proteins Interact with the Nucleotide Kinase Domain of Ca2+-channel beta-Subunits via an Uncommon Effector Binding Domain J. Biol. Chem., April 13, 2007; 282(15): 11509 - 11520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Seu and G. S. Pitt Dose-dependent and Isoform-specific Modulation of Ca2+ Channels by RGK GTPases J. Gen. Physiol., November 1, 2006; 128(5): 605 - 613. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Crump, R. N. Correll, E. A. Schroder, W. C. Lester, B. S. Finlin, D. A. Andres, and J. Satin L-type calcium channel {alpha}-subunit and protein kinase inhibitors modulate Rem-mediated regulation of current Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1959 - H1971. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. S. Finlin, R. N. Correll, C. Pang, S. M. Crump, J. Satin, and D. A. Andres Analysis of the Complex between Ca2+ Channel beta-Subunit and the Rem GTPase J. Biol. Chem., August 18, 2006; 281(33): 23557 - 23566. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. S. Finlin, A. L. Mosley, S. M. Crump, R. N. Correll, S. Ozcan, J. Satin, and D. A. Andres Regulation of L-type Ca2+ Channel Activity and Insulin Secretion by the Rem2 GTPase J. Biol. Chem., December 23, 2005; 280(51): 41864 - 41871. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Chen, H. L. Puhl III, S.-L. Niu, D. C. Mitchell, and S. R. Ikeda Expression of Rem2, an RGK Family Small GTPase, Reduces N-Type Calcium Current without Affecting Channel Surface Density J. Neurosci., October 19, 2005; 25(42): 9762 - 9772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sasaki, T. Shibasaki, P. Beguin, K. Nagashima, M. Miyazaki, and S. Seino Direct Inhibition of the Interaction between {alpha}-Interaction Domain and {beta}-Interaction Domain of Voltage-dependent Ca2+ Channels by Gem J. Biol. Chem., March 11, 2005; 280(10): 9308 - 9312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. Balijepalli, J. D. Foell, and T. J. Kamp Blocking the L-type Ca2+ Channel With a Gem: A Paradigm for a More Specific Ca2+ Channel Blocker Circ. Res., August 20, 2004; 95(4): 337 - 339. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||