Cardiovascular Research

Cardiovascular Research 1998 37(2):360-366; doi:10.1016/S0008-6363(97)00270-8
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Sarcoplasmic reticulum Ca²⁺-ATPase overexpression by adenovirus mediated gene transfer and in transgenic mice

Markus Meyer and Wolfgang H Dillmann^*

Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0618, USA

^* Corresponding author. Tel. (+1-619) 534 9934; Fax (+1-619) 534 9932; E-mail: wdillman@ucsd.edu

Received 28 July 1997; accepted 23 October 1997

	Abstract

Top Abstract 1 Introduction 2 Adenoviral mediated SERCA2a... 3 Transgenic mice overexpressing... 4 Conclusions and perspectives References

 
The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) is a major determinant of cardiac relaxation. It has been demonstrated that the steady state levels of the mRNA coding for this pump are reduced in human heart failure due to dilated cardiomyopathy. Although results regarding the protein level are controversial, most functional studies indicate decreased SERCA2a activity in heart failure. The extent to which a potential decrease in the calcium sequestering function of this protein could contribute to the contractile dysfunction in heart failure, and whether a reconstitution of SERCA2a could alleviate heart failure, are yet unknown. To further investigate these questions two methodological approaches were chosen. Adenovirus mediated gene transfer provides an approach to study functional consequences of SERCA2a overexpression in cardiac myocytes in vitro [1], and a transgenic mouse model allows the effects of cardiac overexpression of SERCA2a to be examined in vivo [2].

KEYWORDS Sarcoplasmic reticulum Ca²⁺-ATPase; Adenovirus; Transgenic mice; Cardiac contractility; Relaxation; Calcium transients

	1 Introduction

Top Abstract 1 Introduction 2 Adenoviral mediated SERCA2a... 3 Transgenic mice overexpressing... 4 Conclusions and perspectives References

 
Dynamic regulation of the calcium concentration within cardiomyocytes is critical to the cardiac contraction. Studies performed on tissue from failing human hearts have indicated that abnormal calcium handling may contribute to the contractile dysfunction in heart failure [1]. Since it has been described that gene expression of the sarcoplasmic reticulum calcium pump (SERCA2a) is reduced in rat models of heart failure due to pressure overload [2, 3], it was conjectured that alterations in expression levels of SERCA2a might contribute as well to the contractile alterations observed in human heart failure. In accordance with the results from the animal models of heart failure, SERCA2a mRNA steady state levels were found to be decreased in samples from human hearts with dilated cardiomyopathy of either ischemic or idiopathic origin [4–9]. An assay which employs the SERCA mediated uptake of a calcium isotope into isolated sarcoplasmic reticulum vesicles or crude myocardial homogenates demonstrates the functional relevance of this finding. Significant reductions in uptake activity were found in several studies using ventricular homogenates from failing hearts when compared to homogenates from normal hearts [8, 10–12]; whereas in one study using purified sarcoplasmic reticulum vesicles no difference was detected [13]. As antibodies against SERCA2a became available, the semiquantitative method of immunoblotting was used to further determine whether SERCA2a protein levels are reduced in human heart failure. Again, observations were subject to controversy; some indicating depressed SERCA2a protein levels [7, 14]and others showing no changes [8, 9, 15]. Additionally, some alternative functional approaches to investigate SERCA2a involvement in altered contractile function have shown that the calcium sequestering function of the sarcoplasmic reticulum is affected in human heart failure [8, 11, 12, 16–21]. A chronological summary of the findings related to alterations of SERCA2a in human heart failure is given in Table 1. Although most studies of human tissue support an association between decreased sarcoplasmic reticulum calcium sequestration in heart failure and impaired cardiac contractility, their data do not offer proof of a functional relationship. In addition, numerous other molecular changes have been described which could promote alterations in contractile behavior. Phospholamban, a potent modulator of SERCA2a activity at submicromolar calcium concentrations, has been shown to be altered in heart failure [14]. It has been suggested that beta-receptor downregulation and altered expression of other elements of signaling cascades are reputedly potential causes of a disturbed cardiac relaxation [22]. Expression levels of two other proteins involved in calcium transport, the sodium-calcium exchanger and the sarcoplasmic ryanodine channel, are reportedly altered in human heart failure [23, 7]. These findings indicate the complexity of molecular changes in human heart failure and the subsequent difficulty of determining the functional relevance of most of these findings.

View this table: [in this window] [in a new window]   Table 1 Summary of studies measuring SERCA2a expression and/or function in human dilated cardiomyopathy. Values reflect averaged relative changes between failing and normal hearts

 
In order to enhance our understanding of the role of SERCA2a in cardiac performance we chose experimental models in which we could achieve and study isolated changes in SERCA2a protein levels. Since most studies of human heart failure as well as several animal models of heart failure displayed a reduction of SERCA2a expression, it was desirable to design experimental systems in which the reduced SERCA2a expression could be rescued by transgenic overexpression of SERCA2a. Two independent approaches were chosen to demonstrate the feasibility of SERCA2a transgene expression in cardiomyocytes and to study its functional consequences. An adenoviral vector was constructed to examine the effects of increased SERCA2a levels on a cellular level. This gene vector enables us to investigate the effects of SERCA2a overexpression in isolated cardiomyocytes. A transgenic mouse line was generated and described in order to further study the effects of a SERCA2a transgene in vivo. We would like to focus this review on our recent results and some of the difficulties which we encountered with regard to overexpression of SERCA2a in cardiomyocytes.

	2 Adenoviral mediated SERCA2a overexpression

Top Abstract 1 Introduction 2 Adenoviral mediated SERCA2a... 3 Transgenic mice overexpressing... 4 Conclusions and perspectives References

 
The aim of this project was to create a strong gene vector which would enable transfer of the SERCA2a gene into myocytes in order to enhance SERCA2a gene expression. Since standard transient transfection techniques lead to very low yields of transfected cardiomyocytes, it was necessary to create a viral vector. The human adenoviruses 2 and 5 have proven to be highly effective in both infection yields and amounts of transgene expression in neonatal and adult cardiomyocytes. The human adenovirus 5 vector which we used has deleted early genes (E1A and E1B), and is therefore replication deficient in cells other than their packaging cell line. The packaging cells (293 cells) represent a stable cell line carrying the viral early genes in their genome. They can therefore be used for the production and propagation of the virus. The E1 region of the adenovirus is replaced by the rat SERCA2a cDNA driven by a thymidin kinase promotor with tandem SV40 enhancers.

When neonatal rat cardiomyocytes were infected with the SERCA2a adenovirus, immunoblot analysis confirmed SERCA2a transgene expression [24]. The same finding was also reported by Hajjar et al. with a different adenoviral construct [25]. We performed calcium uptake studies in order to affirm that the transgene expression leads to a functional protein. In neonatal myocytes infected with an adenovirus carrying no transgene (empty virus) oxalate-facilated calcium uptake was unchanged when compared to the uninfected control myocytes 48 h after infection (21.9±2.6 nmol·min^–1mg^–1 and 20.6±0.7 nmol·min^–1·mg^–1), whereas SERCA2a infected myocytes showed a significant 26% increase in uptake activity when compared to empty virus infected control myocytes (27.7±0.5 nmol·min^–1·mg^–1). These experiments were performed at a calculated free calcium concentration of 7 µM which excludes phospholamban mediated inhibition of SERCA2a activity (Fig. 1). Overexpression of functional SERCA2a transgene was further confirmed by the measurement of Indo-1 transients of SERCA2a adenovirus infected neonatal myocytes (These measurements were performed by PM McDonough, San Diego State University). When Indo-1 Ca²⁺ transients were normalized to peak calcium, the SERCA2a adenovirus infected myocytes showed a marked acceleration of diastolic calcium removal when compared to the uninfected control myocytes. The time from peak transient to 50% of the transient (t_1/2 [Ca²⁺]i) as an index for the speed of cytoplasmatic calcium removal was abbreviated by 32.6% (p<0.001) in SERCA2a infected cardiomyocytes. This data indicated the feasibility of a functional SERCA2a transgene expression in neonatal cardiomyocytes.

View larger version (35K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Effects of adenoviral SERCA2a overexpression in rat neonatal cardiomyocytes on uptake activity 48 h after infection. Cells were infected at a 5-fold excess of infectious adenoviral particles over cells. To control for the effects of adenoviral infection on the uptake, one group of myocytes was infected with an adenovirus carrying no transgene (empty virus). No difference between uninfected control myocytes (n=8) and empty virus infected myocytes (n=8) was found. SERCA2a adenoviral infection (n=9) increased the uptake by 26% when compared to the empty virus infected control myocytes.

 
To further investigate the potential of adenovirus mediated SERCA2a overexpression in a model system where SERCA2a levels have been described as considerably depressed, neonatal myocytes were treated with phorbol myristate acetate (PMA) for 72 h. This substance, commonly used to study protein kinase C activity, has been shown to lead to reduced SERCA2a levels in cardiomyocytes [26, 27]. The mechanism by which PMA reduces SERCA2a levels is not completely understood. After three days of treatment, SERCA2a mRNA and protein levels were found to be reduced by 75% and 40% respectively when compared to untreated control myocytes (both p<0.05). When PMA treated myocytes were infected with the SERCA2a adenovirus, mRNA levels and protein levels were found to be increased by 150% and 10% respectively when compared with untreated control myocytes (both p<0.05). This indicated that infection with SERCA2a adenovirus could reconstitute SERCA2a at a molecular level. Measurements of calcium transients were performed, in order to study the functional consequences of this rescue in SERCA2a levels. When compared with PMA treated myocytes which were infected with an empty adenovirus, the SERCA2a adenovirus infected myocytes showed a 32.4% reduction in t_1/2 [Ca²⁺]i (p<0.05), indicating a marked acceleration of diastolic calcium removal (Fig. 2). This data showed for the first time that SERCA2a overexpression can compensate for prolonged intracellular calcium transients due to decreased SERCA2a levels. Hajjar et al. confirmed this finding in a recent study where prolongation of calcium transients due to adenoviral phospholamaban overexpression was counteracted by adenoviral SERCA2a coinfections [28].

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Differences in Indo-1 Ca2+ transients (normalized to peak transient) in PMA treated neonatal myocytes infected with SERCA2a adenovirus or an adenovirus without transgene (empty virus). After 24 h of PMA treatment myocytes were infected with adenovirus (5-fold excess of infectious adenoviral particles over cells). After 72 h Indo-1 Ca2+ transient measurements were performed (15–20 cells in each group were analyzed in three independent experiments). Infection with SERCA2a adenovirus results in a 32.6% reduction in the time from peak transient to 50% of the transient (t1/2 [Ca2+]i) as an index for the speed of cytoplasmatic calcium removal (p<0.001).

 
Although the results from the neonatal rat myocytes are promising, it needs to mentioned that overexpression of SERCA2a in cardiomyocytes is a difficult task. This problem is reflected in the rather moderate 26% increase in the calcium uptake activity (see Fig. 1) in rat neonatal myocytes after infection with the SERCA2a adenovirus. The abundance of endogenous SERCA2a, which encounters the transgenic protein, is a potential explanation for this finding. Compared with other species, rodent cardiomyocytes contain very high amounts of SERCA2a [29]. When the calcium uptake experiments were performed in L6AM cells, a rat skeletal cell line, under identical conditions, the activity was found to be increased by 75% (p<0.05). Furthermore in non-myogenic cells calcium uptake activities are reported to increase routinely up to 10–20 fold after transfection with SERCA constructs [30]. From these findings it was evident that the SERCA2a cDNA has to be driven a by strong promotor-enhancer construct to increase SERCA2a protein levels significantly in cardiac myocytes. Despite these difficulties and in view of the apparent value of a transgenic in vivo approach in further studies of the effects of SERCA2a overexpression and reconstitution in heart failure, a transgenic animal model was established. Since genetically modified mice are an established and cost effective model to study the consequences on cardiac contractility, transgenic mice were generated.

	3 Transgenic mice overexpressing SERCA2a

Top Abstract 1 Introduction 2 Adenoviral mediated SERCA2a... 3 Transgenic mice overexpressing... 4 Conclusions and perspectives References

 
To overexpress SERCA2a, a strong promotor consisting of a human cytomegalovirus enhancer chicken β-actin promotor was chosen to drive a SERCA2a mini gene in transgenic animals. This mini gene includes the first two introns of the SERCA gene followed by the rat SERCA2a cDNA ligated to the third exon. Several lines of transgene positive mice were obtained as confirmed by Southern Blot analysis [31]. Transgene transcription was confirmed at the mRNA level. In the mouse line used for the physiological measurements, mRNA levels were found to be increased by 160% (p<0.05). In comparison, protein levels were increased by only 20% (p<0.05). This discrepancy between the increase in SERCA2a transcription and protein expression is a further indication that a significant addition of SERCA2a protein in cardiomyocytes is difficult to achieve. Although the increase in SERCA2a protein levels in the hearts of these mice was rather small, physiological measurements revealed a substantial acceleration of cardiac relaxation. Left ventricular pressure measurements in the mice were performed in collaboration with Howard A Rockman. In the anesthetized mice the chest was opened and a 1.8 or 2 F micromanometer catheter was inserted into the left atrium and advanced into the cavity of the left ventricle. When baseline recordings in SERCA2a transgenic mice were compared to their transgene negative littermates, the maximum rate of relaxation as expressed by –dP/dt was found to be increased by 16% in the SERCA2a transgenic mice (–4032±256 mmHg.s^–1 in transgene negative animals (Co) versus –4674±323 mmHg.s^–1 in SERCA2a transgenic animals (Serca), p=0.01). This finding was accompanied by an 29% increase in the maximum rate of pressure rise during systole dP/dt (Co: 5428±387 mmHg.s^–1, Serca: 7006±331 mmHg.s^–1, p<0.001), suggesting an increase in sarcoplasmic reticulum calcium loading. The in vivo findings were confirmed in isolated myocytes obtained from these animals. The maximum rate of relengthening (+dL/dt) as an indicator for myocyte relaxation was increased by 39% (Co: 190±8 µm.s^–1, Serca: 265±6 µm.s^–1, p<0.05), and the maximum rate of shortening (–dL/dt) was increased by 36% (Co: –277±10 µm.s^–1, Serca: –377±16 µ·ms^–1, p<0.05). Along with the enhanced contractile behavior, intracellular calcium transients as measured by Indo-1 revealed a 23% abbreviated t_1/2 [Ca²⁺]i (p<0.01). See Table 2. These results which indicated an accelerated cardiac relaxation was further supported by the findings in isolated left ventricular papillary muscles. In papillary muscles from SERCA2a mice (isometric contractions, stimulation rate 120 s^–1) time from peak tension to half maximal relaxation (RT50) was accelerated by 18% when compared with transgene negative papillary muscles (Co: 39.8±1.3 ms, Serca: 33.8±1.3 ms, p=0.002). Postrest potentiation experiments were performed as an indirect method to study calcium loading of the sarcoplasmic reticulum. When the stimulation of the papillary muscles was interrupted for increasing time periods (0.5–15 s) the resumption of the stimulation resulted in a larger contraction when compared to the steady state contraction. When the time course of the post rest potentiation was analyzed, time to half maximal potentiation was found to be abbreviated by 33% in SERCA2a transgenic papillary muscles (Co: 3.19±0.33 s, Serca: 2.13±0.27 s, p=0.024). This finding indicates that loading of the sarcoplasmic reticulum in papillary muscles of SERCA2a positive animals occurred indeed at a significantly faster rate.

View this table: [in this window] [in a new window]   Table 2 Summary of molecular and physiological data obtained in SERCA2a transgenic mice and their transgene negative littermates

 
Since the hemodynamic data together with the findings in isolated cardiomyoyctes and in isolated papillary muscles clearly showed a SERCA2a transgene dependent acceleration of cardiac relaxation, it was important to look closely at the parameters which would reflect a difference in sarcoplasmic reticulum calcium load. The significant acceleration in the upstroke of the contraction, expressed as the maximum rate of myocyte shortening in isolated myocytes (–dL/dt) and the maximum rate of pressure rise (dP/dt) in the hemodynamic study, was already indicative. An increase in calcium load of the sarcoplasmic reticulum should be reflected in an enhanced cardiac contractility. Neither mean systolic left ventricular pressure in the hemodynamic study (Co: 77±2 mmHg, Serca: 78±2 mmHg) nor mean tension in the isolated papillary muscles (Co: 1.8±0.5 mN/mm², Serca: 3.3±1.0 mN/mm², p=0.19) were found to be significantly increased. Fractional shortening which is the indicator of contractility in isolated myocytes was found to be significantly increased by 17% in myocytes obtained from SERCA2a transgenic animals when compared to myocytes from control animals (Co:18.3±0.5%, Serca: 21.5±0.2% p<0.05). The latter data showed that SERCA2a overexpression increases cardiac contractility in addition the effects observed on time parameters of contraction (Fig. 3). The inability to detect a difference in mean systolic pressures is presumably due to the multifactorial regulation of blood pressure, whereas in isolated papillary muscles developed tension varied considerably between preparations leading to large standard deviations in both groups.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Difference in fractional shortening as measured by video edge detection in adult cardiomyocytes obtained from SERCA2a transgenic animals (n=38) and transgene negative control littermates (n=33). The myocytes were stimulated to contract at 0.3 Hz at a calcium concentration of 2 mM. This plot reflects the averaged data of all myocytes in both groups.

 
In summary, the data obtained in the transgenic SERCA2a mouse model demonstrate, that in vivo overexpression of SERCA2a is feasible. Transgenic SERCA2a leads to abbreviated cardiac contraction and increases in myocyte shortening as an index of cardiac contractility. This model will be very helpful in investigating the effects of SERCA2a transgene expression under pathophysiological conditions which have shown to alter SERCA2a levels.

	4 Conclusions and perspectives

Top Abstract 1 Introduction 2 Adenoviral mediated SERCA2a... 3 Transgenic mice overexpressing... 4 Conclusions and perspectives References

 
Our studies provide evidence that overexpression of SERCA2a in cardiomyocytes is a feasible approach. Overexpression of SERCA2a in transgenic mice leads to abbreviated cardiac contractions and increased cardiac contractility. Adenoviral SERCA2a delivery into neonatal rat cardiomyocytes with decreased SERCA2a levels due to PMA treatment can reconstitute SERCA2a levels as shown by biochemical studies and analysis of calcium transients. The transgenic SERCA2a mice as well as the SERCA2a adenovirus will be helpful in further exploring the contribution of SERCA2a to the contractile changes in heart failure. It should emphasize that our results do not easily translate into other species. Since there are significant species differences in the contribution of SERCA2a to cardiac relaxation, the outcome of SERCA2a overexpression will be different between species. For example in rabbits, the contribution of the Na-Ca exchanger has been found to contribute about one third of the cardiac relaxation [32], and in the failing human heart the expression of the Na-Ca exchanger is increased, indicating a compensatory effect on cytoplasmic calcium removal [7]. The SERCA2a adenovirus will be a valuable tool to elucidate the functional consequences of SERCA2a transgene expression in human cardiomyocytes from failing hearts. Although the result of SERCA2a overexpression in human cardiomyocytes from failing hearts is difficult to predict, the reconstitution of SERCA2a levels presents an attractive approach towards the improvement of cardiac performance in the diseased human heart. How such an increase in SERCA2a expression could be achieved in patients is presently not clear. Thyroid hormone is known to increase SERCA2a expression at a transcriptional level [33]and has recently been shown to compensate for the delayed relaxation in a rat model heart failure [34]. It is doubtful that this approach is easily applicable to human heart failure since excess thyroid hormone levels in humans can be detrimental to the cardiac function due to tachycardia and atrial fibrillation. In terms of gene transfer it must be concluded from the studies available and our own experiences that adenovirus mediated gene transfer into mammal hearts by means of a coronary infusion is impeded by major problems such as insufficient infection efficacy, immune responses directed against the adenoviral vector, loss of transgene expression and the distribution of the virus to other organs. The development of new viral vectors and alternative methods of gene transfer into the heart will bring us closer to gene transfer into human hearts. These ongoing new developments need to be paralleled by the search for potential molecular targets. The calcium pump of the sarcoplasmic reticulum represents such a molecular target, however future research needs to prove that this protein is a major contributor of the contractile dysfunction in human heart failure.

Time for primary review 34 days.

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Top Abstract 1 Introduction 2 Adenoviral mediated SERCA2a... 3 Transgenic mice overexpressing... 4 Conclusions and perspectives References

 

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