Transcriptional changes following restoration of ... - The FASEB Journal

The FASEB Journal express article 10.1096/fj.04-1714fje. Published online July 9, 2004.

Transcriptional changes following restoration of SERCA2a levels in failing rat hearts Federica del Monte,* Rishikesh Dalal,* Adel Tabchy,† Jennifer Couget,‡ Kenneth D. Bloch,* Randall Peterson,* and Roger J. Hajjar* *Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129; †Massachusetts Institute of Technology, Department of Mechanical Engineering and Center for Cancer Research, Cambridge, Massachusetts 02139; ‡Harvard University Bauer Center for Genomics Research, Boston, Massachusetts Corresponding author: Federica del Monte, M.D., Ph.D., Cardiovascular Research Center, Massachusetts General Hospital, East 149 13th Street, Charlestown, MA 02129; E-mail: [email protected] ABSTRACT Heart failure is characterized at the cellular level by impaired contractility and abnormal Ca2+ homeostasis. We have previously shown that restoration of a key enzyme that controls intracellular Ca2+ handling, the sarcoplasmic reticulum Ca2+ ATPase (SERCA2a), induces functional improvement in heart failure. We used high-density oligonucleotide arrays to explore the effects of gene transfer of SERCA2a on genetic reprogramming in a model of heart failure. A total of 1,300 transcripts were identified to be unmodified by the effect of virus alone. Of those, 251 transcripts were found to be up- or down-regulated upon failure. A total of 51 transcripts which were either up- (27) or down- (24) regulated in heart failure were normalized to the nonfailing levels by the restoration of SERCA2a by gene transfer. The microarray analysis identified new genes following SERCA2a restoration in heart failure, which will give us insights into their role in the normalization of multiple pathways within the failing cell. Key Words: heart failure • calcium ATPase • transcript profile

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eart failure represents the end stage of familial or acquired cardiac diseases where compensatory mechanisms are no longer able to restore cardiac function and can further accelerate the disease process. A wide array of genes have been shown to be altered in that process and a number of them have been explored in human and animal models of heart failure (1–5). The analysis of transcriptional reprogramming in heart failure enables us to explore the events that trigger the cascade of changes that progress to heart failure as well as the compensatory mechanisms that take place to preserve cardiac function. Beyond the many structural changes, failing hearts show a permanent decline in contractile function. At the cellular level, the functional defect is detected as a reduction in the amplitude of cell shortening, velocities of shortening and relaxation, as well as abnormal intracellular Ca2+ homeostasis (6). At the molecular level, despite differences in the triggering event, a defect in protein level and function of SERCA2a is a common finding (7). We have shown that the restoration of the defective protein levels of SERCA2a, that regulates the cyclic

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compartmentalization of Ca2+ upon contraction and relaxation, leads to an improvement in the contractile function in vitro and in vivo (8–10). The restoration of Ca2+ homeostasis leading to improved myocardial function is accompanied by improved survival, normalization of metabolism and intracellular signaling pathways, and abrogation of ventricular arrhythmias (11, 12). Those positive effects may correlate with transcriptional reprogramming underlying the compensatory changes produced by the triggered or by primary genetic alterations. We have explored the pattern of gene expression changes that occur in heart failure after restoration of the intracellular Ca2+ homeostasis and contractile function using in vivo adenoviral gene transfer of SERCA2a. Gene transfer is likely to induce a further perturbation that is not accounted for by the genetic changes linked to the overexpression of the targeted gene. Such “viral” effect was explored by looking at gene expression profiles after adenoviral gene transfer of a reporter gene. After accounting for the effects of viral infection and reporter gene expression, 251 genes were shown to be altered in heart failure. METHODS Construction of recombinant adenoviruses To construct the adenovirus containing SERCA2a cDNA, we used the method described by He et al. (13), whereby the backbone vector, containing most of the adenoviral genome (pAd. EASY1) is used and the recombination is performed in Escherichia coli. SERCA2a cDNA was subcloned into the adenoviral shuttle vector (pAd.TRACK), which uses the cytomegalovirus (CMV) long terminal repeat as a promoter. The shuttle vector used also has a concomitant green fluorescent protein (GFP) under the control of a separate CMV promoter. An adenovirus containing both β-galactosidase (β-gal) and GFP controlled by separate CMV promoters (Ad.GFP) was used as a control. The adenoviruses were propagated in 293 cells. The titers of stocks used for these studies measured by plaque assays were 3×1011 pfu/mL for Ad.ßgal-GFP and 1.8×1011 pfu/mL for Ad.SERCA2a, with particle/pfu ratios of 8:1 and 18:1, respectively. These recombinant adenoviruses were tested for the absence of wild-type virus by polymerase chain reaction (PCR) of the early transcriptional unit E1. Experimental protocol Four-week-old Sprague-Dawley rats (Charles River, Mass; 70 to 80 g) were anesthetized with pentobarbital (60 mg/kg IP) and placed on a ventilator. As described previously (14), a small suprasternal incision was made, exposing the aortic root, and a tantalum clip with an internal diameter of 0.58 mm (Weck, Inc.) was placed on the ascending aorta. Animals in the sham group underwent a similar procedure without insertion of a clip. The supraclavicular incision was then closed, and the rats were transferred back to their cages. The animals were initially divided into 2 groups: 1 group of 45 animals with aortic banding and a second group of 42 animals that were sham-operated. Three animals in the aortic banding group did not survive the initial operation, and 2 animals in the sham-operated group did not survive. In the aortic-banded animals, we waited 26 to 28 weeks for the animals to develop left ventricular


(LV) dilatation before cardiac gene transfer. In the banded group as well as in the sham-operated group, 14 animals did not undergo gene transfer and were followed longitudinally. The rest of the animals (28 and 26) underwent adenoviral gene transfer with either Ad.SERCA2a or Ad.GFP. Serial echocardiographic assessment After 18 weeks of banding, serial echocardiograms were performed weekly in lightly anesthetized animals (pentobarbital 40 mg/kg IP). Transthoracic M-mode and 2D echocardiography was performed with a Hewlett-Packard Sonos 5500 imaging system with a 12MHz broadband transducer. A midpapillary level LV short-axis view was used, and measurements of posterior wall thickness, LV diastolic dimension, and fractional shortening were collected. Gene transfer was performed within 3 days of detection of a drop in fractional shortening of >25% compared with the fractional shortening at 18 weeks after banding. In the sham-operated rats, gene delivery was performed at 27 weeks. Adenoviral delivery protocol The group of animals subjected to aortic banding was further subdivided into 3 groups of 14, 14, and 14 receiving Ad.SERCA2a, Ad.GFP, or no adenovirus, respectively. The group of shamoperated animals was also subdivided into 3 groups of 13, 13, and 14 receiving Ad.SERCA2a, Ad.GFP, or no adenovirus. The adenoviral delivery system has been described previously by our group in detail (14, 15). Preparation of cRNA for microarray analysis Total RNA was extracted from rat hearts in each experimental group using TRIzol (Invitrogen) according to the manufacturer’s recommendations. RNA was resuspended in diethyl pyrocarbonate-treated H2O and further purified using the Qiagen (Chatsworth, CA) RNeasy total RNA isolation kit. RNA was quantified and was used to generate cDNA using the Superscript Choice system (Invitrogen, Carlsbad, CA) according to the Affymetrix protocol (Affymetrix, Santa Clara, CA). Resulting cDNA was used to generate biotin-labeled cRNA using the ENZO Bioarray High Yield transcript labeling kit (Affymetrix). cRNA (20 µg) was fragmented in fragmentation buffer [40 mM Tris (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate] for 35 min at 94°C. The quality of the cRNA was checked by hybridization to Test3 arrays (Affymetrix). Subsequently, samples were hybridized to Affymetrix mU34A microarrays containing 8800 genes, and staining and scanning according to Affymetrix protocols identified bound sequences. Analysis of microarray data Data acquisition The acquisition and initial processing of the array data was done using the Affymetrix® Microarray Suite. The software generated an estimate of the relative abundance of each transcript on the array. Transcripts were first rejected based on the quality of measurements. To enable comparison between experiments, expression data are globally scaled to an average intensity of 1,500. A minimum value of 150 was assigned to all average differences (AvDiffs) with an


intensity measurement below 150. Two parameters, the AvDiffs and the absolute call (present or absent), extracted from the Affymetrix data files were used by the analysis software dChip (Harvard University, Cambridge, MA) to generate 4,237 genes on the basis of quality of measurements and normalized data. 4,237 out of the 8,800 transcripts on the microarrays were identified as “present” in at least 2 out of 10 samples based on the abundance of each transcript and were used for further analysis. Samples were duplicated and averaged. The 4,237 transcripts extracted from the Affymetrix software output as filtered intensities were fed into a Perl algorithm specifically tailored to the goal of our study. The algorithm was designed to remove first the viral effect, and subsequently evaluate the genes potentially affected by SERCA2a overexpression in the hearts in transition from failing (F) to failing + SERCA2a overexpression (FS). We calculated intensity ratios between different experimental samples and a reference sample. The intensity ratio calculated for each gene reflects the relative abundance of mRNA in the experimental sample vs. a reference sample. By using a common reference microarray, we get a relative expression level of each gene across the experimental samples (arrays). We used a 1.2 cutoff value for the ratios. Thus, the upper cutoff value is 1.2, and the lower cutoff value is 1/1.2 = 0.83. Subtraction of viral effects Viral effects were noted as effects brought about by vector elements other than SERCA2a. To remove viral effects, we considered as unaffected by virus (at 1.2 cutoff) any gene that did not change their level of expression significantly when virus lacking SERCA2a infected nonfailing hearts and failing hearts (as compared with levels in nonfailing and failing hearts, respectively). By using a 1.2 fold-change cutoff, any change potentially introduced by the virus, even if very small, would be rejected. This filtering virtually eliminates all virus effects, thus decreasing false positives, at the cost of increasing false negatives. Extraction of SERCA2a affected genes We then compared the microarray data among 3 groups: nonfailing heart (NF), failing heart (F), and failing heart transfected with SERCA2a (FS). Starting with the 1,300 genes, we extracted the genes that were changed upon failure. We also extracted genes changed from failure level following SERCA2a infection. We further refined our selection criteria by extracting those genes, which in the Failing heart (F) were changed from their expression level in the nonfailing heart (NF), but then were restored back to normal (NF) when SERCA2a was added (FS). Consistent with our analysis above, we also used a 1.2 fold-change cutoff. When a 1.2 cutoff is used as a measure of change (as opposed to being used as a measure of nochange like in the subtraction of virus effect above), we expect false positive results, which are tolerated here, as they will not be due to virus effect. Quantitative reverse transcriptase PCR (QRT-PCR) analysis Total RNA was isolated and purified from the rat hearts as described above. Following purification, RNA was quantified using the Agilent Bioanalyzer according to the manufacturer’s instructions. RNA (15 µg) was treated (10 min at 20°C) with amplification grade DNase I (Promega; Madison, WI) following which the DNase I was heat-inactivated (15 min at 65°C).


QRT-PCR was performed in duplicate using the Qiagen Quantitect RT-PCR kit containing SYBR Green I (1:30,000, Sigma), forward and reverse primers (50 nM each), and sample RNA (