© 2001 American Heart Association, Inc.
Editorial |
Adenoviral-Mediated SERCA Gene Transfer Into Cardiac Myocytes
How Much Is Too Much?
From the College of Medicine, University of Cincinnati, Cincinnati, Ohio.
Correspondence to Muthu Periasamy, PhD, College of Medicine, University of Cincinnati, 231 Albert-Sabin Way, Cincinnati, OH 45267-0542. E-mail muthu.periasamy{at}uc.edu
Key Words: excitation-contraction coupling • heart failure • SERCA-SR Ca2+ ATPase • gene transfer
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Introduction |
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 Top Introduction Ca2+ Handling and
Heart...Adenoviral-Mediated Gene...Collateral Effects of Adenoviral...Effects of Long-Term SERCA...ConclusionsReferences |
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In the last decade, a great deal of attention has been focused on adenoviral-mediated gene transfer into somatic cells as a possible therapeutic approach. Somatic gene transfer into postmitotic cells (such as cardiomyocytes) provides a very powerful means to deliver the protein of interest, which is either functionally defective or missing because of loss of gene expression.
In recent years, gene therapy for heart failure has gained considerable interest, mainly because of improvements in vector technology, cardiac gene delivery, and a better understanding of the molecular basis of heart failure.1 2
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Ca2+ Handling and Heart Failure |
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TopIntroductionCa2+ Handling and
Heart...Adenoviral-Mediated Gene...Collateral Effects of Adenoviral...Effects of Long-Term SERCA...ConclusionsReferences |
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Heart failure provides an attractive candidate for gene therapy, because several targets have been identified as either functionally impaired or defective. Studies using animal models and failing human hearts have identified several abnormalities that affect excitation-contraction coupling. In particular, changes at the level of sarcolemmal/sarcoplasmic reticulum Ca2+ transport and contractile proteins are thought to contribute to depressed contractile function. Cardiomyocytes from failing animal and human hearts reveal abnormal Ca2+ homeostasis, such as reduced sarcoplasmic reticulum (SR) Ca2+ release, elevated diastolic Ca2+, and reduced rate of Ca2+ removal.3 4 5 There is strong evidence that reduced expression or activity of the SR Ca2+ ATPase (SERCA) and increased expression of Na+-Ca2+ exchanger are key changes contributing to alterations in calcium homeostasis in the heart.6 7 8 9 10 It is also believed that abnormalities in calcium cycling are responsible for blunting of the frequency potentiation of contractile force in the failing human heart.11 Thus, SR Ca2+ ATPase plays a dominant role in removing cytosolic Ca2+ and is the main mechanism for restoring SR Ca2+ load. The Na+-Ca2+ exchanger, on the other hand, transports calcium outside the cell and is a competitor of SR Ca2+ ATPase with respect to SR calcium accumulation and availability for Ca2+ release. Therefore, stimulation of SR Ca2+ ATPase activity either by increasing pump expression or inhibiting phospholamban interaction, a modulator of SR pump function, should increase SR Ca2+ transport and SR Ca2+ load.
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Adenoviral-Mediated Gene Transfer Into Myocytes |
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TopIntroductionCa2+ Handling and
Heart...Adenoviral-Mediated Gene...Collateral Effects of Adenoviral...Effects of Long-Term SERCA...ConclusionsReferences |
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Recombinant adenoviruses have been the vector of choice to transfer genes of interest into cardiac myocytes because of their ease to produce in high titers as well as their ability to transduce nonreplicating myocytes.12 13 Recombinant adenovirus has proven to be a very effective vector to deliver high levels of exogenous protein expression with 100% efficiency of transduction. Therefore, several investigators have used adenoviral-mediated gene transfer of SERCA gene expression into myocytes.14 15 16 17 18 19 20
The studies of Hajjar and colleagues14 15 were the first to demonstrate that expression of SERCA2a in cardiac myocytes by adenoviral gene transfer results in increased contractility and a faster relaxation rate of the Ca2+ transient. Inesi and colleagues17 18 19 went on to additionally document that SERCA2a and ectopically SERCA1a can be expressed at high levels in embryonic chicken and neonatal rat cardiomyocytes by adenoviral vectors. Although both isoforms were expressed at equal levels, SERCA1a activity was 2-fold greater than SERCA2a activity because of intrinsic differences in turnover rates. Furthermore, the rate of decay of cytosolic Ca2+ transients in cells expressing SERCA1 was reduced by 30% to 40% but did not alter resting Ca2+ level or peak amplitudes. These studies provided important information toward the use of SERCA gene as a therapeutic reagent.
More recently, Hajjar and colleagues21 22 used a catheter-based technique of adenoviral gene transfer to achieve global myocardial transduction of SERCA2a in vivo in live animals. These authors chose to restore SERCA2a activity in a rat model of pressure-overload hypertrophy in transition to failure, in which SERCA2a levels and activity were decreased and severe contractile dysfunction was evident.21 Overexpression of SERCA2a by gene transfer in vivo restored both systolic and diastolic dysfunction to normal levels. SERCA overexpression decreased left ventricular size and restored the slope of the end-diastolic pressure-dimension relationship to control levels. Similarly, infection of senescent rat hearts with adenovirus carrying SERCA2a increased Ca2+ ATPase activity and improved rate-dependent contractility and diastolic function.22 These studies provide strong evidence and support the idea that increased SERCA expression can be used to restore Ca2+ transport and contractility. Furthermore, these data suggest the feasibility of cardiac gene transfer into failing hearts as a therapeutic intervention.
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Collateral Effects of Adenoviral Gene Therapy |
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TopIntroductionCa2+ Handling and
Heart...Adenoviral-Mediated Gene...Collateral Effects of Adenoviral...Effects of Long-Term SERCA...ConclusionsReferences |
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In general, adenoviral-mediated SERCA gene transfer was shown to improve Ca2+ homeostasis and contractile function in myocytes. However, the collateral effects of adenoviral infection and exogenous protein expression on myocytes have received little attention. In this issue of Circulation Research, O’Donnell et al23 identify several important collateral effects of adenoviral gene transfer of SERCA into neonatal rat and chicken embryonic cardiac myocytes. This study shows that viral titers >5 to 6 pfu/cell produce cytotoxic effects resulting in apoptotic cell death, more prominently in rat than in chicken myocytes. The differences in cytotoxicity between the 2 cell types may be a function of viral receptor density and cell proliferation capacity. Interestingly, this phenomenon was observed both with a wild-type and mutant/inactive SERCA protein, suggesting that too much active or inactive SERCA protein can lead to cytotoxic effects and cell death.
Such cytotoxic effects were also observed with an adenovirus encoding for enhanced green fluorescent protein and an empty adenovirus but at higher titers than SERCA. Therefore, these collateral cytotoxic effects reported may result from a combination of factors, including viral side effects and high levels of SERCA protein levels. The observation that mutant SERCA can also induce apoptotic cell death may suggest that increased SERCA pump activity is not the cause of this phenotype. It is also possible that because of a paucity of SR membrane in the fetal myocytes, high levels of exogenous SERCA delivered by adenoviral gene transfer could result in overcrowding and alteration in membrane structure. Disruption of SR/endoplasmic reticulum structure might have profound effects on intracellular Ca2+ regulation, protein synthesis, protein folding, and trafficking. These events may also trigger a stress response, leading to apoptotic cell death. In addition, overexpressing the fast twitch isoform, SERCA1, in cardiac myocytes may result in abnormal intracellular trafficking and increased toxicity. Furthermore, either by transgenic approaches or adenoviral gene transfer, the overexpression level of the cardiac form of SERCA has not exceeded 1.5- to 2-fold.14 15 21 22 24 25 26 This suggests that different SERCA isoforms may be trafficked differently in cardiac myocytes.
These authors additionally document that the positive effect of SERCA overexpression on Ca2+ transport can be obtained by maintaining SERCA-virus titer between 1 to 4 pfu/cell. This would suggest that viral titer is a critical factor in preventing the side effects. In addition, the infected myocytes showed hypertrophic growth response with an increase in cell size and protein synthesis. This increase in growth response occurred only in the presence of serum; thus, it is most likely that viral infection alters membrane permeability and allows better delivery of growth stimulants from the media. In summary, the studies by O’Donnell et al23 are highly valuable and should serve as a warning to investigators working with adenoviral gene transfer. These studies emphasize the need for careful evaluation of viral titer and protein expression before attempting to rescue cardiac function in human myocytes.
It is important to note that the studies of O’Donnell et al23 were carried out in fetal/neonatal myocytes for gene transfer. A potential shortcoming using fetal/neonatal myocytes is that they have limited SR volume compared with adult myocytes, and a several-fold increase in SR Ca2+ ATPase within a 48- to 72-hour period can lead to cytotoxic effects, including membrane disruption. Therefore, it would be important to examine whether adult myocytes transduced with adenoviral vectors undergo similar phenomena. In particular, Hajjar and colleagues21 22 24 have shown that adenoviral gene transfer is very effective in rescuing contractile function in failing adult myocytes and in intact myocardium. It is also important to point out that SERCA expression in noncontracting myocytes may have different outcome over the beating heart, as described below.
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Effects of Long-Term SERCA Overexpression in Animal Models |
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TopIntroductionCa2+ Handling and
Heart...Adenoviral-Mediated Gene...Collateral Effects of Adenoviral...Effects of Long-Term SERCA...ConclusionsReferences |
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The strategy to improve cardiac contractility by SERCA overexpression has also been tested in vivo in transgenic animal models. He et al25 and Baker et al26 have reported that overexpression of SERCA2a was associated with increased rates of contraction and relaxation and faster cytosolic Ca2+ transients. Studies by Loukianov et al27 showed that ectopic expression of SERCA 1 cDNA, the fast skeletal isoform, in the mouse heart under the
-myosin heavy chain promoter enhanced cardiac
contractility. SERCA1a expression increased total SERCA protein levels
2.5-fold and resulted in increased SR Ca2+
transport and enhanced rates of contraction and relaxation. Chronic
overexpression of SERCA1a in vivo did not result in cardiac pathology
or cardiac hypertrophy. Thus, high levels of SERCA protein expression
itself are not detrimental to the myocardium. Interestingly, SERCA1a
overexpression in mouse hearts results in the downregulation of
endogenous SERCA2a to 
50% of its level, suggesting that an increase
in exogenous SERCA is compensated by a decrease in the endogenous pump.
These data suggest that SERCA1a pump can functionally substitute for
SERC2a and can be used effectively to enhance cardiac
function. |
Conclusions |
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TopIntroductionCa2+ Handling and
Heart...Adenoviral-Mediated Gene...Collateral Effects of Adenoviral...Effects of Long-Term SERCA...ConclusionsReferences |
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In summary, the studies of SERCA gene transfer both in vivo and in vitro have provided strong support that SERCA gene transfer can be effectively used to enhance cardiac contractility and rescue contractile function in the failing myocardium. The studies by O’Donnell et al23 additionally emphasize the importance of careful evaluation of adenoviral gene transfer, including viral titer and protein expression. Of course, these studies also raise many additional questions. For instance, how much SERCA expression is too much? What is the physiological level of SERCA needed to maintain optimal function? Are there differences between fetal and adult myocytes with regard to viral infection and protein expression? What are the long-term effects of SERCA overexpression (in vivo) compared with short-term? Future experiments will address many of these important questions. Although SERCA gene therapy for heart failure awaits additional experimentation, there is reason for optimism in the future.
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Acknowledgments |
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This work was supported by the National Institutes of Health (grant RO1-HL64140-01). The author is grateful to Roger J. Hajjar, MD, and Sabine Huke, PhD, for critical reading of the manuscript.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionCa2+ Handling and
Heart...Adenoviral-Mediated Gene...Collateral Effects of Adenoviral...Effects of Long-Term SERCA...ConclusionsReferences |
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