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European Journal of Heart Failure 2008 10(1):47-54; doi:10.1016/j.ejheart.2007.10.013
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© 2008 European Society of Cardiology

rAAV-asPLB transfer attenuates abnormal sarcoplasmic reticulum Ca2+-ATPase activity and cardiac dysfunction in rats with myocardial infarction

Xiao-Yan Zhaoa, Shen-Jiang Hua,c,*, Jiang Lia, Yun Moua, Ka Bianb,c, Jian Suna and Zhao-Hui Zhua

a Institute of Cardiology, The First Affiliated Hospital, College of Medicine, Zhejiang University 79 Qingchun Road, Hangzhou 310003, China
b Department of Integrative Biology and Pharmacology, The University of Texas-Houston Medical School 6431 Fannin, Houston, TX, 77030, USA
c E-Institute of Shanghai Universities, Division of Nitric Oxide and Inflammatory Medicine China

* Corresponding author. Institute of Cardiology, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, China. Tel.: +86 571 87236569; fax: 86 571 87236628. E-mail address: s0hu0001{at}hotmail.com


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 
Background: Diminished myocardial sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity and upregulated phospholamban (PLB) level during cardiac dysfunction, had been reported in many studies.

Aims: The current study was designed to examine the effects of rAAV-antisense phospholamban (asPLB) gene transfer on cardiac function, SERCA expression and activity, as well as PLB expression and phosphorylation (Pser16-PLB), in a rat myocardial infarction (MI) model.

Methods and results: Rat MI model was generated by ligating the left anterior descending coronary artery. Four weeks later, left ventricular ejection fraction (LVEF), left ventricular systolic pressure (LVSP), the maximal rates of increase and decrease in intraventricular pressure (±dp/dtmax) were significantly depressed, and left ventricular end diastolic pressure (LVEDP) was increased. Myocardial PLB was markedly increased while both SERCA activity and Pser16-PLB level were decreased. In rAAV-asPLB transfected rats, rAAV-asPLB, which was injected into the myocardium around the infarction area immediately after the coronary artery ligation, effectively attenuated the depression of cardiac function, significantly inhibited the expression of PLB, restored Pser16-PLB level and enhanced myocardium SERCA activity.

Conclusion: rAAV-asPLB transfer in rats with MI effectively prevented the progression of heart failure.

Key Words: Heart failure • Gene therapy • RNA, antisense

Received February 15, 2007; Revised August 22, 2007; Accepted October 19, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 
Medical intervention and surgical treatment have substantially reduced the mortality from coronary heart disease, but chronic heart failure after myocardial infarction (MI) remains a leading cause of morbidity and mortality. Heart failure is closely associated with abnormal handling of intracellular Ca2+ during myocardial contraction, such as elevated resting Ca2+, and decreased amplitude and prolonged duration of the Ca2+ transient [1]. These changes are the direct result of reduced sarcoplasmic reticulum (SR) Ca2+-ATPase pump (SERCA2) activity [2,3]. SERCA2 is regulated mainly by phospholamban (PLB). The PLB/SERCA2 interaction controls the Ca2+ content of the SR and ultimately controls cardiac contractility. To restore the failing heart, it is important to recover the PLB/SERCA2 ratio by either increasing the expression of SERCA2 or decreasing the level of PLB. Over-expression of SERCA2 by gene transfer in failing human cardiomyocytes significantly restores calcium handling and contractility [4]. On the other hand, inhibition of PLB expression has shown positive results in heart failure therapy [5,6]. Decreasing PLB expression by antisense PLB (asPLB) increases the velocity of both contraction and relaxation in cardiomyocytes isolated from failing human hearts [7]. The PLB-null mutation was reported to prevent systolic dysfunction and exercise intolerance in a murine model of hypertrophic cardiomyopathy [8]. When a pseudophosphorylated mutant PLB gene was delivered into hamsters with cardiomyopathy, the usually rapid progression of cardiac dysfunction over the 7-month study period was substantially alleviated [9]. In rats with established heart failure resulting from MI, mutant PLB gene transfer prevents progressive cardiac dysfunction and pathological remodelling [10].

Decreased SERCA2 activity may also be caused by an alteration of the phosphorylation state of PLB with no change in the expression levels of PLB. Unphosphorylated PLB inhibits SERCA2 activity, whereas phosphorylated PLB dissociates from SERCA2 and leads to enhanced pump activity. PLB can be phosphorylated at the serine16 residue by β-adrenergic signalling and at the threonine17 residue via calcium/calmodulin kinase II [11]. It has been shown that reduced serine16 phosphorylation of PLB (Pser16-PLB) in the failing rat myocardium is a major contributor to decreased SERCA2 activity [12]. However, to the best of our knowledge, it is still not known whether the inhibition of PLB expression can impact on Pser16-PLB levels.

In the present study, we constructed a recombinant adeno-associated viral vector expressing antisense PLB (rAAV-asPLB) and transferred it to a rat model of acute MI. The possible effects of asPLB treatment on the Pser16-PLB level and progression of heart failure in MI rats were explored.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Limitations
 References
 
2.1. Reagents
The AAV Helper-Free System, which provides pAAV-MCS vector, pAAV-LacZ vector, pAAV-RC plasmid, pHelper plasmid and AAV-293 cells, was obtained from Stratagene (Netherlands). M-MLV reverse transcriptase was obtained from Promega (China), Pyrobest DNA polymerase from Takara (Japan), mouse monoclonal antibodies to PLB and SERCA from ABR (USA), rabbit monoclonal antibody to Pser16-PLB from Upstate Biotechnology Inc. (USA), HRP-conjugated antibody to GAPDH from Kangchen Biotech (China), HRP-conjugated goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG from Amersham Pharmacia Biotech (UK), and the enhanced chemiluminescence (ECL) kit was obtained from Amersham Corp (USA).

2.2. Construction of recombinant adeno-associated virus vector
rAAV-asPLB was constructed as described previously [13]. In brief, pAAV-asPLB was generated by cloning the PLB cDNA in reversed orientation relative to the promoter of pAAV-MCS (multiple cloning sites). pAAV-asPLB, pAAV-RC (plasmid encoding AAV rep-cap gene) and pHelper were co-transfected into 293 cells to obtain rAAV-asPLB. In addition, pAAV-lacZ, pAAV-RC and pHelper were co-transfected into 293 cells to obtain rAAV-lacZ. The rAAV vectors were propagated and purified. The titers of rAAV-asPLB and rAAV-lacZ were adjusted to 1x1010 infectious units/mL (IU) for the following experiments.

2.3. Generation of MI rats and gene transfection
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). The experimental procedures were approved by the Committee of Animal Care and Use at Zhejiang University. Experiments were carried out on adult male Wistar rats (clean grade, provided by Shanghai Laboratory Animal Center, Chinese Academy of Sciences) weighing 250-300 g. All rats were housed under similar conditions on a 12 h light/dark cycle with the temperature maintained at 21±1 °C, humidity at 55±5%, with free access to standard rat chow and water.

MI was induced by ligation of the left coronary artery as described elsewhere [10]. In brief, rats were anesthetized with chloral hydrate and mechanically ventilated with room air. A left thoracotomy was performed, and the left anterior descending coronary artery was ligated between the right ventricular outflow tract and the left atrium. Special care was taken to ligate the same anatomical site of the artery to produce similar degrees of MI. Then administration of rAAV-asPLB or rAAV-lacZ was performed as described below. Sham-operated rats underwent the same procedure but without coronary artery ligation.

MI rats were randomly divided into 3 groups: MI-saline, MI-rAAV-lacZ and MI-rAAV-asPLB groups. In the MI-rAAV-asPLB group, immediately after the coronary artery ligation, 120 µL (1.2x109 IU) of rAAV-asPLB was injected through a 30-gauge needle into 4 sites of the myocardium around the infarction area. In the MI-rAAV-lacZ group, 120 µL (1.2x109 IU) of rAAV-lacZ was injected in the same way. In the MI-saline group, 120 µL of saline was injected.

In the normal-rAAV-asPLB group, normal rats were anesthetized, the thorax opened, and an intramyocardial injection of the same dose of rAAV-asPLB was administered to the same region using the same procedure.

2.4. Echocardiography
One week after the operation, echocardiography was performed to estimate left ventricular infarct size using the centerline method, as described elsewhere [10,14]. Briefly, light anaesthesia was induced. The infarct region was then determined by the definition of systolic akinesis or dyskinesis in two-dimensional short-axis images. Infarction size was expressed as the percentage of the infarct region to the left ventricular circumference. The size and the percentage were estimated at three levels (mid-papillary muscle, chordae tendineae, and near the apex) and averaged.

Four weeks after induction of MI, another echocardiogram was performed to assess left ventricular size and cardiac function [15,16]. Briefly, after induction of light anaesthesia, two-dimensional parasternal long-axis images were obtained perpendicular to the short-axis, these were deemed appropriate at the site where left ventricular length was maximal and both mitral and aortic valves were contained in the image. Left ventricular end diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD) were measured, and left ventricular ejection fraction (LVEF) was calculated.

2.5. Haemodynamic measurement
Rats were anaesthetized with chloral hydrate (400 mg/kg body weight, injected intraperitoneally). A 20-gauge catheter-tip pressure transducer was then introduced into the left ventricle through the right carotid artery, for measurement of left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and maximal rates of increase and decrease in intraventricular pressure (±dp/dtmax). All data were stored in a computer and analyzed using MedLab biosoftware. After completion of the haemodynamic measurements, hearts were arrested with 10% potassium chloride injected into the left ventricle. Left ventricles were immediately excised and frozen in liquid nitrogen.

2.6. Evaluation of SERCA activity
SERCA activity was determined following the method of Larsen [17], with some modifications. The myocardium of the left ventricle was homogenized in tissue buffer (20 mmol/L Hepes, 2 mmol/L EDTA and 250 mmol/L sucrose, pH 7.4) at 4 °C. The homogenate (1 g wet weight/L) was added to the reaction medium (20 mmol/L Hepes at pH 7.4, 1 mmol/L MgCl2, 1 mmol/L EGTA, 0.01{per thousand} TritonX-100, 100 mmol/L KCl and 0.8 mmol/L CaCl2) and pre-incubated at 37 °C for 10 min. The reaction was initiated by an addition of 10 mmol/L p-Npp, incubated at 37 °C for 30 min, stopped by using double the volume of cold buffer (500 mmol/L Tris, 55 mmol/L EDTA), and quantified by absorption spectrophotometry at a wavelength of 410 nm.

2.7. Western blot analysis
Left ventricles were homogenized in an extraction buffer (10 mmol/L Tris-HCl, pH 7.4, 0.32 mmol/L sucrose) at 4 °C. For inhibition of endogenous phosphatases, one protease inhibitor cocktail tablet (Roche Diagnostics, Germany) was added. The concentration of the protein in each lysate was determined with Coomassie brilliant blue G-250. Equal amounts of protein (20 µg/sample) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinyl difluoride membranes. Non-fat milk (5%) in Tris-buffered saline (25 mmol/L Tris and 150 mmol/L NaCl, containing 0.05% Tween 20) was used to block non-specific sites on the membrane for 1 h at room temperature. Then the membranes were incubated with rabbit monoclonal antibody to Pser16-PLB or mouse monoclonal antibodies to PLB or SERCA2 (1:500) at 4 °C overnight, and incubated with a dilution of peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:2000) for 1 h. The immune complexes were visualized by the ECL chemiluminescence method. Quantification of the bands was carried out using densitometric analysis software Quantity One (Bio-Rad). To ensure equal loading, GAPDH was used as an endogenous control.

2.8. Statistical analysis
All values were expressed as mean±SD and statistically analyzed by one-way ANOVA with post hoc comparisons by the LSD test. P values less than 0.05 were defined as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Limitations
 References
 
3.1. Survival rate and infarct size
Overall 92 rats were included in the study. Two rats in the sham-operation group died 24 h after the operation. In addition, 25 rats died from MI, of these 20 died within the first 24 h after the operation, and five died 1 week after the operation. There were no significant differences in the survival rates between the three MI groups 24 h after the operation or during the 4 week observation period. Five rats with small (<30%) or very large (>40%) infarctions were excluded from the study. There were five treatment groups in the study: normal (n=10), normal-rAAV-asPLB (n=10), sham-operated (n=10), MI-saline (n=11), MI-rAAV-lacZ (n=10) and MI-rAAV-asPLB (n=9). There was no significant difference in infarct size (%) between the three MI groups (34±3.4, 36±3.3 and 35±3.5, respectively).

3.2. Transfection efficiency of rAAV vector
We previously reported that the transfection efficiency of the rAAV vector in isolated rat cardiomyocytes is about 80% by using rAAV-lacZ as a reporter gene [13]. In the current study, we also used this reporter gene, but did not calculate the precise transfection efficiency because the intramyocardial injection method limited the definition of the total number of cardiomyocytes. However, X-gal staining showed that the cytoplasm of a large number of cardiomyocytes around the infarction area was stained blue, indicating an effective transfection (Fig. 1). To evaluate the safety of rAAV vectors, we performed the X-gal staining on brain, kidney, liver and lung tissue slices from the MI-rAAV-lacZ group, and found no positive staining (data not shown).


Figure 01
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  		Fig. 1 Representative images of X-gal staining followed by H&E staining of the myocardium. A. MI-rAAV-lacZ group. B. MI-saline group. Positive cells are those with cytoplasm stained blue.

 
3.3. Cardiac function
Echocardiography was performed 4 weeks after the induction of MI and gene transfection, to evaluate left ventricular size and LVEF (Table 1). Left ventricular function was also determined by haemodynamic measurements (Table 2). In normal rats, rAAV-asPLB transfer had no effect on left ventricular dimensions or cardiac function. Compared with normal control and sham-operated rats, the left ventricles were dilated and LVEF decreased markedly in the MI-saline and MI-rAAV-lacZ groups. Haemodynamic parameters also indicated left ventricular dysfunction in MI rats, such as decreased LVSP, reduced ±dp/dtmax and increased LVEDP. rAAV-asPLB treatment significantly attenuated cardiac dysfunction in MI rats. Both systolic and diastolic function were significantly enhanced, and LVEF, LVSP, ±dp/dtmax and LVEDP were all markedly improved (P<0.05) in the MI-rAAV-asPLB group compared with the MI-saline and MI-rAAV-lacZ groups. However, LVEDD was not restored by rAAV-asPLB treatment despite the normalized LVESD.


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  		Table 1 Echocardiographic parameters 4 weeks after the induction of MI and gene transfection

 


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  		Table 2 Cardiac function evaluated by haemodynamic measurements

 
3.4. Myocardial SERCA activity and SERCA protein expression
Compared with the normal control group, myocardial SERCA activity was significantly decreased in the MI-saline group (2.21±0.74 versus 3.21±0.27, P<0.05) and the MI-rAAV-lacZ group (2.11±0.61 versus 3.21±0.27, P<0.05) (Fig. 2A). In non-MI rats (normal-rAAV-asPLB group), rAAV-asPLB treatment did not affect myocardial SERCA activity, while in MI rats (MI-rAAV-asPLB group), rAAV-asPLB treatment improved myocardial SERCA activity significantly (MI-rAAV-asPLB versus MI-saline, 3.14±0.51 versus 2.21±0.74, P<0.05) at 4 weeks (Fig. 2A). There were no significant differences in myocardial SERCA protein expression level between the experimental groups (Fig. 2B,C).


Figure 02
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  		Fig. 2 Myocardial SERCA activity (µmol/min g) (A) and SERCA protein expression level (B, C). Data are expressed as mean±SD. A. Myocardial SERCA activity. B. Representative Western blot image of SERCA protein expression in each group. Lane 1: normal control; lane 2: normal-rAAV-asPLB group; 3: sham-operated group; lane 4: MI-saline group; lane 5: MI-rAAV-lacZ group; lane 6: MI-rAAV-asPLB group. C. SERCA protein level expressed as mean±SD. To ensure equal loading, GAPDH was included as an endogenous control. *P<0.05 versus sham-operated groups. {blacktriangleup}P<0.05 versus MI-saline group.

 
3.5. Myocardial PLB and Pser16-PLB expression levels
As assessed by Western blot (Fig. 3), PLB protein expression was upregulated in all MI rats compared with the sham-operated group. rAAV-asPLB treatment markedly inhibited the PLB protein over-expression (Fig. 3A,C). Phosphorylation of PLB at the ser16 site was decreased in MI rats, and this was significantly restored by rAAV-asPLB transfection. In normal rats, rAAV-asPLB treatment slightly inhibited PLB expression, but this did not reach statistical significance (normal-rAAV-asPLB versus normal control, 0.69±0.19 versus 0.96±0.18, P=0.11) and had no influence on ser16 PLB phosphorylation (Fig. 3B,D).


Figure 03
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  		Fig. 3 Myocardial PLB and Pser16-PLB protein expression levels detected by Western blot. A. Representative image of PLB expression in each group. B. Representative image of Pser16-PLB expression in each group. Lanes from 1 to 6 represent the same groups as Fig. 2. C. PLB protein level expressed as mean±SD. D. Pser16-PLB protein level expressed as mean±SD. To ensure equal loading, GAPDH was included as an endogenous control. *P<0.05 versus sham-operated groups. pP<0.05 versus MI-saline group.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Limitations
 References
 
The current study demonstrates that in vivo delivery of the rAAV-asPLB gene was highly effective in preventing left ventricular dysfunction caused by acute MI. rAAV-asPLB gene treatment significantly improved left ventricular function, as evaluated by echocardiography and haemodynamic parameters.

In order to validate the efficiency and safety of the rAAV-asPLB vector before applying it to the pathological MI model, we tested the effects of rAAV-asPLB transfection in normal rats. Intramyocardial injection of rAAV-asPLB in normal control rats did not influence short-term mortality. In myocardial tissues transfected with rAAV-asPLB, a slight down-regulation of PLB protein level was detected, but this was not statistically significant. There was also no effect of rAAV-asPLB transfection on PLB phosphorylation, SERCA protein expression and activity, or cardiac function. Thus, we concluded that rAAV-asPLB is relatively safe, and its effects in rats with MI could be evaluated.

Failing heart muscle generally exhibits distinct changes in intracellular Ca2+ handling, including impaired removal of cytosolic Ca2+, reduced Ca2+ loading of the cardiac SR, and defective SR Ca2+ release, which contribute to the impairment of cardiac function [18]. Increasing evidence indicates that decreased SERCA activity is the key to altered cardiac function [2,3,18]. PLB is the most important regulator of cardiac SERCA activity. Phosphorylation of PLB enhances SERCA activity and dephosphorylation of PLB inhibits it. Furthermore, in human myocardium, SERCA activity is regulated by cAMP-dependent phosphorylation of PLB at position ser16 [18]. Both in vitro and in vivo studies confirm that ablation or inhibition of PLB expression is beneficial to restoring Ca2+ homeostasis and to improving the contractile function of the failing myocardium [5,6,7,10]. The function of PLB is mainly regulated via ser16 phosphorylation by cAMP-dependent protein kinase, and/or Thr17 phosphorylation by CaMKII [19]. It has been proposed that the level of ser16 phosphorylation can be used as an index for determining SERCA2 activity in the failing heart after MI [12]. Despite several attempts to alter protein expression levels of PLB and SERCA or the phosphorylation level of PLB in heart failure after MI, the therapeutic effects have not been consistent [4,6,9,10]. It is possible that differences in study design have contributed to these discrepant results. In this in vivo study, we transferred the target gene by intramyocardial injection to ensure transfection efficiency, and thus get more precise results.

In previous studies, some groups [20,21] have reported that the levels of mRNAs and proteins of myocardial SERCA and PLB were not altered in the post-infarction rat model. Sjaastad et al. found that myocardial SERCA protein expression was significantly decreased while the PLB protein level was unchanged in rats 6 weeks after coronary artery ligation [22]. Other groups have demonstrated that both SERCA and PLB proteins decrease in animal models of post-MI heart failure [23,24]. However, in this study we found that in rats with intermediate size MI, with moderate LV remodelling and mild heart failure, following left anterior descending coronary artery ligation for 4 weeks, myocardial SERCA protein was unchanged, PLB protein was upregulated, and ser16 phosphorylation of PLB was significantly down-regulated. In the present study, total protein was extracted from the myocardial tissue but not the infarction scar, which may partly explain the discrepant results, since SERCA and PLB protein levels have been shown to change differently in different regions of the ischaemic heart [25]. Prunier F et al. generated a rat model with large-size MI and severe heart failure and found markedly down-regulated SERCA gene expression [26], while the present study included rats with middle-sized MI and moderate heart failure and found that expression of SERCA was unchanged. Thus we deduce that the extent of the MI and the severity of cardiac dysfunction may influence myocardial SERCA expression significantly. Therefore, the results and conclusions of this study only apply to MIs of intermediate size, and not to severe MIs. In addition, many other factors such as MI extent, MI persistence period, feeding and behavioural activity may affect the expression levels of SERCA and PLB proteins.

Although targeting PLB for the treatment of experimental heart failure has shown promising results, whether PLB inhibition at the acute phase of MI can prevent the occurrence of heart failure is unclear to date. In this study, heart failure developed in the MI-saline and MI-rAAV-lacZ treated rats, and was accompanied by diminished SERCA activity and increased PLB protein level and decreased phosphorylation of PLB at ser16. Transfection of the asPLB gene at the acute phase of MI normalized PLB phosphorylation at ser16, partially restored the diminished SERCA activity and prevented the progression of heart failure. However, the reasons why asPLB gene treatment restored the phosphorylation state of PLB requires further investigation.

In this study we applied rAAV-asPLB and rAAV-lacZ by using an AAV Helper-Free System. The single-stranded DNA virus vector rAAV has been widely used in recent years because of its advantages of non-pathogenicity and long-term expression in a variety of cell types [27,28], including cardiomyocytes [29,30]. AAV is a naturally defective virus, requiring provision of several factors in trans for productive infection, and has not been associated with any human disease. In this study, we found no marked pathological alterations in the myocardium in response to rAAV-asPLB and rAAV-lacZ, and no increased mortality. The rAAV vector can infect both dividing and non-dividing cells with high transfection efficiency, as confirmed by our previous study [13], in which we found that the efficiency of rAAV-asPLB in isolated rat cardiomyocytes was about 80%. In this in vivo study, although we did not evaluate the precise transfection efficiency, X-gal staining showed a large number of cardiomyocytes positively stained blue in and around the injection region in rats of the MI-rAAV-lacZ group. We regard gene transfection by intramyocardial injection a promising method, because it has great biosafety considering the absence of target genes in other organs such as brain, liver and kidney.

This study established that, in a rat MI model, intramyocardial injection of rAAV-asPLB in the acute phase, inhibited over-expression of myocardial PLB and significantly restored its phosphorylation state, so increasing SERCA activity and attenuating cardiac dysfunction. To our knowledge, this is the first study to report the effects of rAAV-asPLB in an animal model of MI in the acute phase using the transfection method of intramyocardial injection.


   5. Limitations
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
References
 
Although this study showed that rAAV-asPLB transfer in rats with intermediate size MI effectively prevented the progression of heart failure, the long-term survival rate and/or the effect of the infarct area on survival should be explored in further study. Also, to understand more precisely the effects of asPLB on cardiac function, further studies are needed to measure the effects on intracellular calcium transients/concentrations. In addition, protein expression in both ischaemic and remote LV myocardium was not investigated and further study will be necessary to clarify the relevance of these findings. In this study, we used a single dose of the target gene, and further studies are also needed to test the dose dependency.


   Acknowledgements
 
We express our sincere gratitude to Professor Kun-Chen (College of Medicine, Zhejiang University) for handling the statistical analysis and to Professor Iain C Bruce (University of Hong Kong) for revising this manuscript. This study was supported by the Scientific and Technological Agency of Zhejiang Province (No. 021107817 and 2006C23035) and the Health Agency of Zhejiang Province (No. 2003A041 and 2006A032), and supported in part by the E-Institutes of the Shanghai Municipal Education Commission (No. E-04010).


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 

  1. Frank K.F., Bolck B., Brixius K., Kranias E.G., Schiwinger R.H. Modulation of SERCA: implications for the failing human heart. Basic Res Cardiol (2002) 97:I72–I78.[Medline]
  2. Jiang M.T., Lokuta A.J., Farrell E.F., Wolff M.R., Haworth R.A., Valdivia H.H. Abnormal Ca2+ release, but normal ryanodine receptors, in canine and human failure. Circ Res (2002) 91:1015–1022.[Abstract/Free Full Text]
  3. Koss K.L., Kranias E.G. Phospholamban: a prominent regulator of myocardial contractility. Circ Res (1996) 79:1059–1063.[Free Full Text]
  4. del Monte F., Harding S.E., Schmidt U., et al. Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer of SERCA2a. Circulation (1999) 100:2308–2311.[Abstract/Free Full Text]
  5. Eizema K., Fechner H., Bezstarosti K., et al. Adenovirus-based phospholamban antisense expression as a novel approach to improve cardiac contractile dysfunction. Circulation (2000) 101:2193–2199.[Abstract/Free Full Text]
  6. He H., Meyer M., Martin J.L., et al. Effects of mutant and antisense RNA of phospholamban on SR Ca2+-ATPase activity and cardiac myocyte contractility. Circulation (1999) 100:974–980.[Abstract/Free Full Text]
  7. del Monte F., Harding S.E., Dec G.W., Gwathmey J.K., Hajjar R.J. Targeting phospholamban by gene transfer in human heart failure. Circulation (2002) 105:904–907.[Abstract/Free Full Text]
  8. Freeman K., Lerman I., Kranias E.G., et al. Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy. J Clin Invest (2001) 107:967–974.[Web of Science][Medline]
  9. Hoshijima M., Ikeda Y., Iwanaga Y., et al. Chronic suppression of heart failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery. Nat Med (2002) 8:864–871.[Web of Science][Medline]
  10. Iwanaga Y., Hoshijima M., Gu Y., et al. Chronic phospholamban inhibition prevents progressive cardiac dysfunction and pathological remodeling after infarction in rats. J Clin Invest (2004) 113:727–736.[CrossRef][Web of Science][Medline]
  11. Drago G.A., Colyer J. Discrimination between two sites of phosphorylation on adjacent amino acids by phosphorylation site-specific antibodies to phospholamban. J Biol Chem (1994) 269:25073–25077.[Abstract/Free Full Text]
  12. Sande J.B., Sjaastad I., Hoen I.B., et al. Reduced level of serine16 phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity. Cardiovasc Res (2002) 53:382–391.[Abstract/Free Full Text]
  13. Li J., Hu S.J., Sun J., et al. Construction of phospholamban antisense RNA recombinant adeno-associated virus vector and its effects in rat cardiomyocytes. Acta Pharmacol Sin (2005) 26:51–55.[CrossRef][Web of Science][Medline]
  14. Sheehan F.H., Bolson E.L., Dodge H.T., Mathey D.G., Schofer J., Woo H.W. Advantages and applications of the centerline method for characterizing regional ventricular function. Circulation (1986) 74:293–305.[Abstract/Free Full Text]
  15. Tanaka N., Dalton N., Rockman H.A., et al. Transthoracic echocardiography in the normal and abnormal mouse heart. Circulation (1996) 94:1109–1117.[Abstract/Free Full Text]
  16. Prunier F., Gaertner R., Louedec L., Michel J.B., Mercadier J.J., Escoubet B. Doppler echocardiographic estimation of left ventricular end-diastolic pressure after MI in rats. Am J Physiol Heart Circ Physiol (2002) 283:H346–H352.[Abstract/Free Full Text]
  17. Larsen J.S., Kjeldsen K. Quantification in crude homogenates of rat myocardial Na+, K+- and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes. Basic Res Cardiol (1995) 90:323–331.[CrossRef][Web of Science][Medline]
  18. Schwinger R.H., Munch G., Bolck B., Karczewski P., Krause E.G., Erdmann E. Reduced Ca2+-sensitivity of SERCA 2a in failing human myocardium due to reduced serine-16 phospholamban phosphorylation. J Mol Cell Cardiol (1999) 31:479–491.[CrossRef][Web of Science][Medline]
  19. Sjaastad I., Wasserstrom J.A., Sejersted O.M. Heart failure—a challenge to our current concepts of excitation-contraction coupling. J Physiol (2003) 546:33–47.[Abstract/Free Full Text]
  20. Yue P., Long C.S., Austin R., Chang K.C., Simpson P.C., Massie B.M. Post-infarction heart failure in the rat is associated with distinct alterations in cardiac myocyte molecular phenotype. J Mol Cell Cardiol (1998) 30:1615–1630.[CrossRef][Web of Science][Medline]
  21. Huang B., Wang S., Qin D., Boutjdir M., El-Sherif N. Diminished basal phosphorylation level of phospholamban in the postinfarction remodeled rat ventricle: role of beta-adrenergic pathway, G(i) protein, phosphodiesterase, and phosphatases. Circ Res (1999) 85:848–855.[Abstract/Free Full Text]
  22. Sjaastad I., Bokenes J., Swift F., Wasserstrom J.A., Sejersted O.M. Normal contractions triggered by I (Ca, L) in ventricular myocytes from rats with postinfarction CHF. Am J Physiol Heart Circ Physiol (2002) 283:H1225–H1236.[Abstract/Free Full Text]
  23. Guo x., Chapman D., Dhalla N.S. Partial prevention of changes in SR gene expression in congestive heart failure due to myocardial infarction by enalapril or losartan. Mol Cell Biochem (2003) 254:163–172.[CrossRef][Web of Science][Medline]
  24. Shao Q., Ren B., Saini H.K., Netticadan T., Takeda N., Dhalla N.S. Sarcoplasmic reticulum Ca2+ transport and gene expression in congestive heart failure are modified by imidapril treatment. Am J Physiol Heart Circ Physiol (2005) 288:H1674–H1682.[Abstract/Free Full Text]
  25. Thomas S.A., Fallavollita J.A., Suzuki G., Borgers M., Canty J.M. Jr. Dissociation of regional adaptations to ischemia and global myolysis in an accelerated swine model of chronic hibernating myocardium. Circ Res (2002) 91:970–977.[Abstract/Free Full Text]
  26. Prunier F., Chen Y., Gellen B., et al. Left ventricular SERCA2a gene down-regulation does not parallel ANP gene up-regulation during post-MI remodelling in rats. Eur J Heart Fail (2005) 7:739–747.[Abstract/Free Full Text]
  27. Ohashi K., Nakai H., Couto L.B., Kay M.A. Modified infusion procedures affect recombinant adeno-associated virus vector type 2 transduction in the liver. Hum Gene Ther (2005) 16:299–306.[CrossRef][Web of Science][Medline]
  28. Lai L., Lin K., Foulks G., Ma L., Xiao X., Chen K. Highly efficient ex vivo gene delivery into human corneal endothelial cells by recombinant adeno-associated virus. Curr Eye Res (2002) 30:213–219.[CrossRef]
  29. Maeda Y., Ikeda U., Oya K., et al. Adeno-associated virus-mediated transfer of endothelial nitric oxide synthase gene inhibits protein synthesis of rat ventricular cardiomyocytes. Cardiovasc Drugs Ther (2001) 15:19–24.[CrossRef][Web of Science][Medline]
  30. Su H., Joho S., Huang Y., et al. Adeno-associated viral vector delivers cardiac-specific and hypoxia-inducible VEGF expression in ischemic mouse hearts. Proc Natl Acad Sci USA (2004) 101:16280–16285.[Abstract/Free Full Text]

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