Douglas Sawyer has received research support from Acorda Therapeutics, Inc. that helped to fund parts of this study. Several authors as noted on the title page are employees of Acorda Therapeutics, Inc., a company that is developing GGF2 for the treatment of cardiovascular and neurodegenerative disease. The authors note that these competing interests do not alter their adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Conceived and designed the experiments: MFH AVP AM HMS CLG LP XP CGL OO DBF MWK SZ ZZ YS FEH MS AG JI TJP AOC DBS. Performed the experiments: MFH AVP AM HMS CLG LP XP CGL OO DBF MWK SZ ZZ DBS. Analyzed the data: MFH AVP AM HMS CLG LP XP CGL OO DBF MWK SZ ZZ YS FEH MS AG JI TJP AOC DBS. Contributed reagents/materials/analysis tools: DBF SZ ZZ YS FEH MS AG JI TJP AOC. Wrote the paper: MFH AVP AM HMS CLG LP XP CGL OO DBF MWK SZ ZZ YS FEH MS AG JI TJP AOC DBS.
Current address: Division of Cardiovascular Medicine, University of Louisville School of Medicine, Louisville. Kentucky, United States of America
Recombinant Neuregulin (NRG)-1β has multiple beneficial effects on cardiac myocytes in culture, and has potential as a clinical therapy for heart failure (HF). A number of factors may influence the effect of NRG-1β on cardiac function via ErbB receptor coupling and expression. We examined the effect of the NRG-1β isoform, glial growth factor 2 (GGF2), in rats with myocardial infarction (MI) and determined the impact of high-fat diet as well as chronicity of disease on GGF2 induced improvement in left ventricular systolic function. Potential mechanisms for GGF2 effects on the remote myocardium were explored using microarray and proteomic analysis.
Rats with MI were randomized to receive vehicle, 0.625 mg/kg, or 3.25 mg/kg GGF2 in the presence and absence of high-fat feeding beginning at day 7 post-MI and continuing for 4 weeks. Residual left ventricular (LV) function was improved in both of the GGF2 treatment groups compared with the vehicle treated MI group at 4 weeks of treatment as assessed by echocardiography. High-fat diet did not prevent the effects of high dose GGF2. In experiments where treatment was delayed until 8 weeks after MI, high but not low dose GGF2 treatment was associated with improved systolic function. mRNA and protein expression analysis of remote left ventricular tissue revealed a number of changes in myocardial gene and protein expression altered by MI that were normalized by GGF2 treatment, many of which are involved in energy production.
This study demonstrates that in rats with MI induced systolic dysfunction, GGF2 treatment improves cardiac function. There are differences in sensitivity of the myocardium to GGF2 effects when administered early vs. late post-MI that may be important to consider in the development of GGF2 in humans.
Congestive heart failure (CHF) subsequent to myocardial infarction (MI) and other forms of cardiac injury are common clinical problems with a poor prognosis
The biologic effects of NRG-1β are mediated through ErbB2, 3 and 4 receptor tyrosine kinases, where ErbB3 and 4 bind NRGs directly, and ErbB2 acts as a heterodimerization partner. The activity of NRG-1β will therefore vary with conditions that alter ErbB receptor expression or activity. In cultured myocytes, for example, antibodies to ErbB2 receptors alter cellular response to NRG-1β
One of the multiple isoforms of NRG-1β is the kringle-containing form originally named for its mitogenic effects on glial cells as glial growth factor 2 (GGF2)
Sprague Dawley rats were obtained from Charles River Laboratories (Wilmington, MA). Experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee at Vanderbilt University Medical Center (Protocol ID#: M/09/269). The investigation conforms to the
Male Sprague Dawley rats (175–200 g body weight) were anaesthetized intraperitoneally with 30 mg/kg of pentobarbital. Absence of a response to pinching the toe was used as an indicator of the appropriate level of anesthesia. Rats were then rapidly intubated and mechanically ventilated (tidal volume, 1 mL/100 g body weight; ventilation rate, 65 strokes/min) by a constant volume small animal ventilator (Model 683, Harvard Apparatus). A left thoracotomy was performed at the fourth intercostal space, and the left coronary artery was then ligated by irreversible tightening of a 6–0 suture loop. MI was confirmed by regional cyanosis of the myocardial surface distal to the suture, accompanied by S-T segment elevation on the electrocardiogram. Following successful induction of MI, the chest cavity was compressed to evacuate any air before being tightly sealed. Sham-operated animals underwent the same surgical procedure with the exception that the left coronary artery was not ligated. The rats were given buprenorphine 0.05 mg/kg post-operatively, then Q 8–12 hours post-operatively by subcutaneous injection. The rats that survived through day 7 post-MI were randomly assigned to various groups as outlined in the experimental design.
All rats were maintained on a normal chow diet of 12% calories from fat, 28% calories from protein, and 60% calories from carbohydrate (LabDiet) prior to coronary ligation surgery. Following surgery, rats in the study examining effects of GGF2 early after MI were also randomly assigned to either a normal diet or a high saturated fat diet of 43.5% calories from fat, 22.2% calories from protein, and 34.3% calories from carbohydrate (LabDiet) beginning 7 days post-MI and continuing until completion of the study. All rats in the study examining effects of GGF2 late after MI were continuously maintained on a normal chow diet.
Recombinant GGF2 (96.0% purity by SEC-HPLC) was provided by Acorda Therapeutics, Inc. and stored at 4°C. GGF2 was used at low dose (0.625 mg/kg) or high dose (3.25 mg/kg) based upon rat weights taken at each day of treatment. GGF2 was administered via the right lateral tail vein every second day to surviving MI rats starting one or 8 weeks after MI. Vehicle-treated rats received equal volume injections of sterile GGF2 diluent (20 mM histidine, 100 mM arginine, 100 mM sodium sulfate, 1% mannitol, pH 6.5).
Rats in the study examining effects of GGF2 late after MI underwent microPET imaging to evaluate myocardial metabolic changes and myocardial viability post-infarction. Anesthesia was induced using 2–3% isoflurane/97–98% oxygen supplied via nosecone. Fasting rats were administered an intraperitoneal injection of 18F-fluorodeoxyglucose (18FDG) and anesthesia was discontinued. After a sixty minute rest period to allow for uptake of radioisotope tracer into tissues, anesthesia was re-induced for the imaging procedure. A small-animal tomograph (Siemens MicroPET Focus 220) acquired full body PET images for approximately 30–45 minutes. Images were later analyzed with Amide software. Mean cardiac standard uptake values (SUVs) were calculated from transverse slices (0.47 mm thickness) by drawing 3-dimensional, free hand regions of interest that completely encircled the heart. Regions of interest approximately 0.5 cm in diameter were also drawn on each animal's right shoulder skeletal muscle. Mean cardiac SUVs were then normalized to shoulder muscle SUVs. After completing the PET procedure, animals were placed in a recovery cage and monitored until fully ambulatory. Rats were scanned at 8, 10, and 12 weeks post-MI. All scanning and interpretation was performed blinded to the treatment group.
Transthoracic echocardiographic images of hearts from all groups of rats were obtained using a 12-MHz ultrasound probe (model s12, Agilent Technologies) and an echograph (SONOS 5500, Hewlett Packard) while rats were immobilized under anesthesia (1.5–2.0% isoflurane). For M-mode recordings, the parasternal short-axis view was used to image the heart in two dimensions at the level of the papillary muscles. End-diastolic and end-systolic LV cavity dimensions were measured using software resident on the ultrasonograph. LV fractional shortening (FS) was calculated from M-mode-derived left ventricular inner diameters in diastole (LVIDd) and systole (LVIDs) using the formula (LVIDd – LVIDs)/LVIDd x100%. All imaging and interpretation of results was done blinded to treatment group. In all studies rats underwent serial echocardiograms at baseline prior to initiation of treatment, and subsequently at two week intervals until sacrifice.
All histological evaluation was performed in a blinded fashion. Paraffin-embedded sections (4 µm) of hearts from all of the groups were stained with Masson's trichome. Cardiac fibrosis in the LV remote from the site of infarction was measured in using NIH ImageJ software and was expressed in arbitrary units as a percentage of the total area of the tissue section stained.
We examined myocardial oxidative stress by measuring myocardial protein carbonylation as previously described
Remote, non-infarct left ventricular tissue from all groups was homogenized using a liquid nitrogen chilled mortar and pestle, in a liquid nitrogen chilled CryoGrinder. Homogenization was performed using RIPA buffer (Millipore), supplemented with protease inhibitor cocktail (Roche). Proteins were quantified with Bradford assay (Bio-Rad). 50 micrograms of protein in SDS sample buffer was heated using a Dry Bath incubator (Fisher scientific) for 10 minutes and after centrifugation, loaded onto 4–12% NuPage gels (Invitrogen). After electrophoresis, proteins were transferred to an immunoblot PVDF membrane, using the Semi-Dry transfer apparatus at 20 V for 30 mins. Membranes were blocked using PBST-5% BSA and then incubated overnight at 4°C with antibodies directed against ErbB2 and ErbB4 (clone F-11 and clone C-18 respectively, Santa Cruz Biotechnology), each diluted 1∶500. Secondary horseradish peroxidase-conjugated antibody (Cell Signaling) was applied for 1 hour at room temperature. Blots were visualized using the SuperSignal West Pico chemiluminiscent substrate (Pierce) and analyzed with NIH ImageJ densitometry software.
Total RNA was extracted from the non-infarcted LV using Trizol (Invitrogen Co.) and RNeasy columns (Qiagen, Valencia, CA), according to the manufacturers' instructions, followed by DNAse treatment (Qiagen). RNA quality measurements using a 2100 Bioanalyzer (Agilent, Santa Clara, CA) and further sample processing were performed in the Genome Sciences Resource (GSR) Core at Vanderbilt. Processed and labeled samples were subsequently hybridized to the rat Gene 1.0 ST whole-transcriptome array (Affymetrix, Santa Clara, CA). Three biological replicates were used for each sample type as follows: Sham-operated, early MI rats (vehicle and low dose [0.625 mg/kg] GGF2-treated), and late MI rats (vehicle and high dose [3.25 mg/kg] GGF2-treated). Therefore, there were a total of 15 samples (5 sample types, n = 3). Only rats on the standard diet were chosen for transcriptome analysis, due to minimal differences in GGF2-mediated responses in rats fed a high versus low-fat diet.
Subsequent to import of raw data (.CEL) files, Robust Multi-chip Average (RMA) normalization
Gene functions, as presented in significant gene list tables, were determined using publicly available records using NCBI Entrez Gene, Stanford SOURCE, Aceview, and Pubmed databases. Statistical analyses (including correction for multiple hypothesis testing) for identification of overrepresented ontologies and functions were performed using Ingenuity Pathway Analysis (IPA) software (ingenuity Systems, Redwood City, CA).
For comparison of our gene expression results to previous studies, raw microarray data (CEL or text files) from four rat MI studies were downloaded from Gene Expression Omnibus (GEO) (
To link our gene expression results (i.e., gene expression profiles of GGF2-treated early and late MI rats) to results obtained from the various other animal and human studies, a perl script was written to match genes across the various lists by accession number (for same species results) or gene symbol (for inter-species results comparisons). If neither the accession number nor gene symbol matched perfectly, then the gene descriptions were compared and manually checked if the difference did not exceed 25%. If a data set contained more than one match for a single data point in a published study, only the top three matches were recorded, with the best match ordered first. This perl script was later modified to compute averages for the multiple matches. This method would be expected to fail to identify some homologous genes in which there are name designation differences between species (e.g., the mouse version of human IL-8 is referred to as KC or GRO). However, our intention was to greatly minimize false positives and thus compare only those genes that were truly the same between the various data sets. To ensure that this was indeed the case, all final gene lists representing overlap between study results were also manually examined and any genes that were computationally misidentified were subsequently removed.
Real-time RT-PCR was performed to quantify transcript levels for a subset of genes using the CFX96 C-1000 (Bio-Rad, Hercules, CA) using SYBR Green I dye (QIAGEN), per manufacturer's instructions. Briefly, each 25-μl reaction contained 100 ng of RNA, 2.5 μl of primers (Quantitect Primer Assays; QIAGEN), 12.5 μl of SYBR Green PCR master mix and 0.25 μl of reverse transcriptase. Negative controls containing water instead of RNA were concomitantly run to confirm that the samples were not cross-contaminated. Targets were normalized to reactions performed using Quantitect TPT1 primers (QIAGEN), which amplify a translationally-controlled transcript. Fold change was determined using the comparative threshold method
Myocardial tissue originating from the remote, viable LV in all groups of rats was immediately frozen in liquid nitrogen and stored at –80°C until use. Frozen viable LV tissues were homogenized in lysis buffer consisting of 7 M urea, 2 M thiourea, 30 mM Tris, 5 mM magnesium acetate, 4% CHAPS, and 58 mM DTT using a ratio of 1 g tissue:10 ml lysis buffer. The lysate was centrifuged at 12,000×g for 1 h at 10°C. The pellet was discarded and the protein concentration of the supernatant was determined using a 2-D Quant protein assay kit (Amersham Biosciences/GE Healthcare, Piscataway, New Jersey).
250 µg from each of the samples were precipitated with methanol/chloroform
All equipment was manufactured by GE Healthcare/Amersham Biosciences (Piscataway, New Jersey) unless otherwise noted. The samples were separated by standard 2D gel electrophoresis using a manifold-equipped IPGphor first-dimension isoelectric focusing unit and 24 cm 4–7 immobilized pH gradient (IPG) strips, followed by second-dimension 12% SDS-PAGE homogenous on hand-cast gels that had one plate pre-silanized to ensure accurate robot picking in subsequent steps, using an Ettan DALT 12 unit according to the manufacturer's protocols. The Cy2 (mixed standard), Cy3 (sample X) and Cy5 (sample Y) components of each gel were individually imaged at 100 µm resolution with mutually-exclusive excitation/emission wavelengths using a Typhoon 9400 Variable Mode Fluorescence Imager. A Sypro Ruby total protein post-stain (Invitrogen/Molecular Probes) was used to ensure accurate protein excision, as the low stoichiometry of Cy-dyes label only 1–3% of the total protein.
DeCyder software v6.5 was used for simultaneous comparison of abundance changes across all sample pairs with statistical confidence and without interference from gel-to-gel variation
Proteins of interest were robotically excised, digested into peptides in-gel with modified porcine trypsin protease (Trypsin Gold, Promega) and peptides extracted using the automated Ettan Spot Handling Workstation (GE Healthcare) using 20 μL 20 mM NH4HCO3 containing 0.01 μg/μL Trypsin Gold (Promega) for 3 h at 37°C for protein digestion. Peptides were extracted by two rounds of incubation with 60% acetonitrile, 0.1% trifluoroacetic acid, dried and reconstituted in 15 μL 0.1% formic acid and placed into autosampler vials. 5 μL of each peptide hydrosylate was separately analyzed by C18 reverse-phase LC-MS/MS using a Thermo LTQ ion trap mass spectrometer equipped with a Thermo MicroAS autosampler and Eksigent HPLC nanoLC pump system, nanospray source, and Xcalibur 2.0 instrument control using standard data-dependent methods. Tandem MS data were analyzed with the Sequest algorithm, searching the IPI_rat-v342 database (Apr 2008) that contained a concatenated reverse decoy database to estimate false-discovery rates. Search results were filtered by cross-correlation scores (<1.0 for singly-charged peptides, <1.8 for doubly-charged, and <2.5 for triply-charged) with an overall 2.4% false-discovery rate. Protein identifications were based on a minimum of 2 peptides passing these criteria for each protein. The number of unique peptides and the total number of peptide identifications (spectral counts) for each protein are listed, and the predicted MW and pI were correlated with the relative gel position from which the protein was excised.
Gene expression and real-time RT-PCR statistical analyses were performed as described above. All other statistical analyses were completed using statistical software R version 2.13.0 (2011–04–13). Echocardiography data were analyzed by using the generalized least squares model. This linear regression model accounts for correlation across time within animals and was fit to investigate whether GGF2 treatment and diet affected the 35-day outcome (FS %). Estimated mean values were fit using restricted maximum likelihood or REML and are presented with its 95% confidence interval. Non- linearity was addressed by applying a restricted cubic spline term, and transformation was applied when the normality assumption did not hold. Time by treatment and time by diet interactions were added to the model, and Wald statistics were used to assess the individual and joint significance of regression coefficients. Other data were analyzed by ordinary least squares and one-way ANOVA as indicated.
In total, 103 rats underwent surgery, with 98 rats receiving coronary artery ligation (MI) and 5 rats receiving sham operation. Overall survival rate among the MI rats was 87.8%. All of the sham-operated rats survived. This resulted in 86 MI and 5 sham-operated animals that were included in the present study. No deleterious side effects were observed in any of the GGF2-treated rats throughout the treatment periods.
In the first group of rats, the effects of two doses of intravenous GGF2 with treatment started 7 days after MI were compared. Rats were randomly assigned to treatment groups. Echocardiographic assessment of left ventricular systolic function one week after MI or sham operation showed similar cardiac dysfunction in all groups compared to sham-operated rats. FS% was improved in GGF2 treated groups compared to those in the vehicle treated groups by the end of 4 weeks treatment (
Serial measurements of fractional shortening (FS) were acquired from post-MI rats at baseline (1 week post-MI), 2 weeks after GGF2 treatment, and 4 weeks after GGF2 treatment. Echocardiographic assessment revealed that residual left ventricular (LV) FS% values were significantly higher (p = 0.0001) in GGF2 treated animals compared to those in the vehicle groups at the end of the study. The estimated mean FS% and range at 35 days post MI was 43.6 (41.0, 46.3) for the high dose treated group (n = 9), 42.0 (39.1, 45.0) for the low dose treated group (n = 9), and 36.6 (34.2, 39.1) for the vehicle group (n = 10. Individual rat FS % values trended downwards in the vehicle animals with time, whereas a progressive increase in FS % was observed in both the low dose and high dose GGF2 treatment groups. Moreover, the results indicate that high-fat feeding did not impair the effects of high dose GGF2 treatment on cardiac function.
Group | Week | FS %† | P Value† | LVIDd‡ | Heart/Body§ |
Normal Diet, Vehicle | 1 | 36.5 | 0.75 | ||
3 | 34.7 (32.2, 37.1) | NS | 0.77 (0.74, 0.81) | ||
5 | 36.6 (34.2, 39.1) | NS | 0.79 (0.75, 0.82) | 1.42 (1.29,1.57) | |
Low Dose GGF2 | 1 | 36.5 | 0.75 | ||
3 | 40.1 (37.1, 43.1) | NS | 0.74 (0.71, 0.78) | ||
5 | 42.0 (39.1, 45.0) | 0.005* | 0.76 (0.72, 0.80) | 1.27 (1.13, 1.43) | |
High Dose GGF2 | 1 | 36.5 | 0.75 | ||
3 | 40.3 (37.7, 42.9) | NS | 0.76 (0.73, 0.80) | ||
5 | 43.6 (41.0, 46.3) | 0.0001* | 0.77 (0.73, 0.80) | 1.49 (1.34, 1.65) | |
Fatty Diet, Vehicle | 1 | 36.5 | 0.75 | ||
3 | 34.6 (32.0, 37.2) | NS | 0.74 (0.71, 0.78) | ||
5 | 35.6 (33.0, 38.2) | NS | 0.75 (0.71, 0.78) | 1.38 (1.25,1.53) | |
High Dose GGF2 | 1 | 36.5 | 0.75 | ||
3 | 40.2 (37.3, 43.1) | NS | 0.73 (0.70, 0.77) | ||
5 | 42.6 (39.7, 45.4) | 0.0001* | 0.73 (0.69, 0.77) | 1.44 (1.30,1.60) |
To evaluate the potential effects of diet on GGF2 cardiac function, some animals were randomized to high fat diet vehicle and high fat diet GGF2-treated groups beginning 7 days post-MI and continuing until the end of the study. Results indicated that fatty diet had no significant influence on FS% values (p = 0.77) compared to normal diet, similar to previous reports
We examined the effects of GGF2 treatment on LV function and remodeling late after MI in rats. The late-infarct model underwent the same MI procedure; however, the rats were left untreated for 8 weeks prior to starting GGF2 or vehicle treatment. The first delayed treatment rats received intravenous low dose GGF2 (0.625 mg/kg) or vehicle three times per week. Transthoracic echocardiography was used to evaluate LV function at 8 weeks post-MI (pre-treatment) and after 2 and 4 weeks of treatment. FS% values were not significantly higher (p = 0.27) in low dose GGF2 treated compared to the vehicle group by the end of study (
A) Low dose GGF2: Rat FS % values were progressively reduced in both vehicle (n = 14) and low dose GGF-2 treated (n = 14) animals with time. The estimated mean FS% and range at 12 weeks post-MI was 32.9 (30.5, 35.3) for the low dose treated group and 31.7 (29.4, 33.9) for the vehicle group. Overall, the low dose treatment regimen did not restore late-post infarct LV structure and function because the average treatment effect was statistically insignificant (p = 0.51). B) High dose GGF2: FS % values decreased in vehicle (n = 4) animals and rose in high dose GGF2 treated (n = 8) rats with time. FS % was significantly increased in the high dose treated animals at 10,12,14, and 16 weeks post-MI (p = 0.013, p = 0.002, p = 0.002, and p = 0.017 respectively.) The estimated mean FS% and range at 16 weeks post-MI was 27.8 (24.1, 32.0) for the high dose treated group and 21.7 (18.1, 26.0) for the vehicle group. The average treatment effect of high dose GGF2 was found to be statistically significant (p = 0.005).
We therefore repeated the experiment using a higher dose of GGF2 (3.25 mg/kg) in a new cohort of rats with initiation of therapy at 8 weeks post-MI. We further extended the treatment period by another 4 weeks (8 weeks total). In contrast to low dose GGF2 treatment late after MI, we found that FS % was significantly improved in high dose GGF2 treated animals compared to the vehicle group at 10,12,14, and 16 weeks post-MI (p = 0.013, p = 0.0016, p = 0.0018, and p = 0.017 respectively) (
Data from
Positron emission tomography (PET) was used to assess metabolic trends in post-MI rat myocardium treated with GGF2 both early and late after MI. Standard Uptake Values (SUVs) acquired by positron emission tomography were compared between all groups at the time points indicated above. In both infarct models, 18FDG uptake was similarly low in vehicle and GGF2-treated animal's infarct and remote regions (
Short axis (transverse sections) images representative of sham, MI, and MI + GGF2 treated rats are displayed above. The sham rats showed prominent 18F-FDG uptake in all regions of the myocardium, whereas MI and MI + GGF2 treated rats show very little uptake in the infarct zone. Statistical analysis of cardiac standard uptake values was conducted by ANOVA, and results indicated that GGF2 treatment did not increase PET uptake in either remote or infarct regions (MI+LD, p = 0.79; MI+HD, p = 0.50).
We examined whether changes in ErbB receptor expression, as observed in end-stage heart failure, could explain the differences in myocardial responsiveness to early versus late post-MI GGF2 treatment. Myocardial ErbB2 and ErbB4 receptor expression was analyzed from tissue lysates by immunoblot in rats in the groups treated with vehicle both early and late after MI (
) Representative Western blot for ErbB2 and ErbB4 in early post-MI (5 weeks) vs late post-MI (12 weeks).
Cardiac fibrosis was examined in formalin fixed sections of remote, non-infarcted myocardium from rats in the early post-MI experiment. Compared to the hearts of sham MI rats, the hearts from the MI rats treated with the vehicle demonstrated significantly greater fibrosis in LV myocardium remote from the site of infarction (p = 0.025) (data not shown). Treatment with low or high dose GGF2 did not alter the extent of myocardial fibrosis compared to vehicle-treated MI rats.
To determine whether GGF2 altered myocardial oxidative protein modification, post-MI heart homogenates were assayed for reactive carbonyl derivatives. While carbonylated protein content was more consistently low in the high dose GGF2-treated early-post MI group, there was wide variability in the sham operated and Vehicle-treated groups, and no significant difference was detectable by ANOVA (p = 0.62) (data not shown).
In an effort to gain insight into the molecular mechanisms of GGF2-mediated restoration in cardiac function, we examined the effects of GGF2 treatment on the post-MI cardiac transcriptome. We chose post-MI rats given low dose GGF2 early after MI and included three biological replicates for the Sham-operated rats. This resulted in three biological replicates (Sham, VEH, and GGF2). Normalization followed by statistical analysis of the normalized hybridization signals resulted in 1,616 probe sets significantly altered (p value <0.05, fold-change at least 1.5) in untreated MI rats (VEH), compared to Sham-operated control animals (
A) Hierarchical clustering of RNA normalized fluorescent signal values was performed for 1,720 genes determined to be differentially expressed (p value <0.05, FC >1.5) in LV samples collected 4 weeks post-MI. Color indicates relative levels of expression replicates, with bright red indicating the highest fluorescent signal values and bright blue representing the lowest in the various samples shown. Each row represents one probe set, found as up- or down-regulated in GGF2-treated rats at 4 weeks post-MI. Columns represent the three replicates for each group, color-coded by sample type (Sham: pink; vehicle: yellow; GGF2: green). Two prominently differential clusters were generated that separated Sham-operated rats from those that received vehicle treatment. Two of the GGF2-treated samples were more similar to Sham-operated controls, indicating a normalization of MI-induced gene expression for these two biological replicates. This pattern was not detected for the third GGF2-treated sample, which clustered with vehicle-treated rats. B) GGF2 treatment in the two biological replicate ‘responders’ showed reversal of 853 genes altered by MI, compared to Sham-operated rats.
For GGF2-treated compared to vehicle-treated MI rats, there were 959 significantly altered genes. The majority of these shared common functions, most notably genes encoding mitochondrial proteins essential for energy production. Of these 959 genes, 855 were altered in vehicle- treated MI rats compared to sham-operated controls as well as altered by GGF2 treatment compared to vehicle- treated MI. All but two of these 855 genes were normalized by GGF2 treatment (
The vast majority of the 853 normalized genes (787 genes) were up-regulated in response to induction of MI and down-regulated in GGF2-treated rats, compared to vehicle-treated controls. The remaining 66 genes were down-regulated in MI rats, compared to Sham-operated control animals, and induced in GGF2-treated rats. The inverse correlation between the entire set of 853 normalized genes was −0.87, based on Pearson's correlation coefficient, and the R-squared value was 0.92 when plotted and fit with a polynomial trend line. Functional analysis using Ingenuity Pathway Analysis software indicated that the most enriched functions for GGF2 normalized genes were energy production processes, including genes involved in the electron transport chain and metabolism related to production of energy precursors (Benjamini and Hochberg (B&H) corrected p values 5.8×10−4–1.1×10−30) (
Eight genes were chosen for quantitative RT-PCR (qRT-PCR). Each bar represents one of four fold-differences obtained for vehicle-treated rats, compared to Sham-operated controls obtained using microarrays (black bars) or qRT-PCR (small, vertical striped bars) or GGF2-treated rats, compared to vehicle controls obtained using microarrays (large, vertical striped bars) or qRT-PCR (stippled white bars), as shown in the legend. Actual fold-changes are shown as a table, beneath each set of bars. Statistical analyses showed significant changes (n = 3 for each group, p<0.05) except where indicated (nc = no change).
Over-represented Gene Ontology Category | MI-Veh vs. Sham | MI-GGF2 vs. MI-Veh |
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cellular respiration | 35 | 26 |
electron transport chain | 37 | 24 |
acetyl-CoA catabolic process | 18 | 15 |
oxidation reduction | 124 | 83 |
aerobic respiration | 21 | 18 |
tricarboxylic acid cycle | 18 | 15 |
fatty acid catabolic process | 17 | 13 |
respiratory electron transport chain | 15 | 9 |
ATP metabolic process | 29 | 12 |
lipid oxidation | 17 | 12 |
glycolysis | 16 | 13 |
oxidative phosphorylation | 26 | 11 |
generation of precursor metabolites and energy | 94 | 62 |
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cellular macromolecule catabolic process | 94 | 54 |
cofactor metabolic process | 60 | 44 |
coenzyme metabolic process | 52 | 40 |
cellular carbohydrate catabolic process | 23 | 17 |
glucose catabolic process | 20 | 14 |
cellular lipid catabolic process | 25 | 18 |
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response to oxidative stress | 42 | 19 |
protein catabolic process | 82 | 47 |
ubiquitin-dependent protein catabolic process | 53 | 33 |
negative regulation of protein modification process | 31 | 22 |
protein complex assembly and biogenesis | 73 | 35 |
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mitochondrion organization | 40 | 29 |
ribonucleotide metabolic process | 40 | 18 |
purine ribonucleotide metabolic process | 39 | 18 |
RNA processing | 71 | 31 |
nucleoside triphosphate metabolic process | 36 | 15 |
macromolecular complex subunit organization | 97 | 43 |
purine nucleotide metabolic process | 45 | 23 |
mitotic cell cycle | 48 | 26 |
macromolecular complex assembly | 90 | 41 |
intracellular transport | 79 | 37 |
In addition to whole transcriptome analysis, we examined the effects of low-dose GGF2 treatment early after MI on the post-MI cardiac proteome. A total protein stain of a representative gel is shown in
Representative 2D gel image of total protein stain from the 5-gel coordinated DIGE experiment. A total of 300 µg of protein was loaded onto each DIGE gel. Altered proteins are indicated by numbers which correspond to line entries for identified proteins listed in
GGF2 treatment resulted in the down-regulation of several MI-induced proteins involved in metabolism and oxidative phosphorylation (
Protein | Gene | Function | MS Measurements | MI/Sham | HF | GGF2/VEH | |||||||||
This Study | Humans | This Study | |||||||||||||
Spot | pI | MW | Unique peptides | Spectral counts | MS | Array | Array | MS | Array | ||||||
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NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial |
Ndufs1 | Electron transport | 17 | 5.6 | 79 | 6 | 6 | 1.7 | 2.9 | 2.5 | −1.6 | −1.4 | |||
Electron-transfer- flavoprotein, beta polypeptide |
Etfb | Electron transport | 37 | var | 28 | 4 | 4 | 1.4 | 1.7 | 2.2 | −1.4 | −1.5 | |||
Isocitrate dehydrogenase 3 (NAD+) beta |
Idh3b | TCA cycle | 33 | 7.8 | 39 | 4 | 4 | nd | 2.9 | 1.7 | −1.4 | −1.6 | |||
Enolase 3, beta, muscle | Eno3 | Glycolysis, may play a role in muscle development and regeneration | 35 | 7.1 | 47 | 7 | 8 | nd | 2.3 | +/− | −1.3 | −1.4 | |||
Glycogen phosphorylase |
Pygm | Muscle enzyme involved in glycogenolysis | 14 | 6.9 | 97 | 13 | 16 | nd | 1.8 | −1.7 | −1.3 | −1.4 | |||
Propionyl coenzyme A carboxylase, beta polypeptide | Ppcb | Lipid metabolism | 24 | 7.2 | 58.6 | 3 | 4 | 1.4 | 1.5 | nd | −1.3 | −1.5 | |||
Succinate dehydrogenase complex, subunit A, flavoprotein |
Sdha | Major catalytic component of mitochondrial respiratory chain | 18 | 6.1 | 68 | 7 | 8 | 1.4 | 1.9 | 3.6 | −1.2 | −1.5 | |||
Phosphoglycerate mutase 2 (muscle) | Pgam2 | Glycolysis, striated muscle contraction, gluconeogenesis | 37 | var | 28 | 4 | 5 | 1.4 | 2.4 | nd | −1.4 | −1.9 | |||
3-hydroxybutyrate dehydrogenase, type 2 |
Bdh2 | Plays a key role in iron homeostasis and transport | 37 | var | 28 | 3 | 3 | 1.4 | 1.8 | 1.8 | −1.4 | −1.5 | |||
Polymerase I and transcript release factor§ | Ptrf | Suggested to play an essential role in the formation of caveolae and the stabilization of caveolins, may also be involved in control of lipolysis | 27 | 5.4 | 44 | 7 | 11 | −1.3 | −1.2 | 2 | 1.4 | 1.3 | |||
Glutathione peroxidase 3§ | Gpx3 | Functions in the detoxification of hydrogen peroxide | 40 | 6.4 | 23 | 3 | 3 | nd | −1.3 | −1.6 | 1.4 | nd | |||
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Collagen, type I, alpha 1 | Col1a1 | Structural constituent of the extracellular matrix, focal adhesion | 7 | 5.7 | 138 | 3 | 3 | −1.6 | −1.2 | +/− | 1.9 | 1.2 | |||
Procollagen, type VI, alpha 3 |
Col6a3 | Muscle organ development, cell adhesion | 1 | 8.4 | 288 | 18 | 18 | −1.3 | −1.5 | −1.6 | 1.3 | 1.5 | |||
Troponin I type 3 (cardiac) ‡ | Tnni3 | Heart development, regulation of smooth muscle contraction and vasculogenesis | 39 | 9.6 | 24 | 2 | 2 | −1.4 | nd | nd | 1.5 | nd | |||
Myosin binding protein C, cardiac | Mybpc3 | Cardiac muscle contraction, cell adhesion | 3 | 6.1 | 141 | 29 | 39 | nd | nd | 2.2 | −1.5 | nd | |||
Vimentin | Vim | Structural constituent of the cytoskeleton | 26 | 5 | 54 | 11 | 13 | nd | −1.3 | nd | 1.3 | nd | |||
Guanine deaminase‡ | Gda | Potentially involved in neuronal development | 30 | 5.5 | 51 | 8 | 8 | −1.3 | 1.2 | nd | 1.5 | 1.5/nd | |||
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Inner membrane protein, mitochondria |
Immt | Unknown function, associates with Opa1 and Chchd3 | 17 | 5.4 | 82 | 3 | 3 | 1.7 | 1.7 | 1.5 | −1.6 | −1.4 | |||
Coiled-coil-helix- coiled-coil-helix domain containing 3 |
Chchd3 | Mitochondrial protein, unknown function | 37 | var | 28 | 3 | 3 | 1.4 | 2.7 | 2.2 | −1.4 | −1.6 |
Supported by protein, gene expression, and human repository data; ‡supported by protein data only; conflicting results for protein and gene expression data §; nd = no measurable difference;
We compared the proteomics results to our rat gene expression data and also to genes transcriptionally altered in failing human hearts. As shown in
The present study is one of a series that demonstrates that recombinant NRG-1β can improve cardiac function after myocardial infarction or other forms of cardiac injury in animals. The NRG-1β treatment effects were preserved in the setting of high fat diet, as well as later post-MI. GGF2 treated myocardium showed reversal of gene and protein expression changes induced by MI. The effects of GGF2 on myocardial gene and protein expression offer some possible explanations for the benefit of recombinant NRG-1β for CHF.
While the myocyte protective effects of recombinant NRG-1β are lost in the presence of increased levels of saturated fat
It appears that the duration of heart failure after MI does alter sensitivity to GGF2. Increased myocardial ErbB2 expression early after MI may explain this result. Other investigators have shown that chronic heart failure is associated with reduced ErbB mRNA expression in both rodents and humans
Bersell et al. have reported that injection of NRG-1β into post-MI mice for 12 weeks resulted in a decreased infarct size which coincided with improved myocardial function
The molecular pathways that mediate NRG-1β's effects on the heart remain unclear despite several studies demonstrating improved cardiac function. There are multiple cells in the heart that express ErbB receptors, respond to recombinant NRG, and could contribute to the observed improvement in cardiac function
Other genes of note that were regulated by NRG-1β treatment in this and prior studies include
To assess the potential relevancy of this study's microarray results to human heart failure, the data were compared to raw data from eight studies representing 4 rat heart failure model studies and 4 human heart failure data sets obtained from online data repositories. The data were analyzed using the same methods employed to examine transcriptional changes in our rat model of MI, and genes altered at least 1.5-fold (p value <0.05) from all studies were compared (
Study | Group | n | # Genes altered | Overlap To Current Dataset | |
MI-Veh vs. Sham | MI-GGF2 vs. MI-Veh | ||||
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This Study | Sham | 3 | |||
MI – Vehicle treated (v. Sham) | 3 | 1,616 | - | ||
MI – GGF2 treated (v. MI + V) | 2 | 959 | - | ||
GEO data (4 sets) | Sham | 15 | |||
MI or IR | 37 | 2,399 | 294 | 130 | |
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C. Kirchoff Lab, Univ. Hospital Hamburg, E-TABM-480 | Controls | 4 | |||
Idiopathic | 5 | 1,875 | 273 | 118 | |
Cardiogenomics Lab, Harvard GSE1145 | Normal | 15 | |||
Cardiomyopathy | 92 | 4,628 | 585 | 194 | |
JM Hare Lab, Johns Hopkins, Internal Medicine, GSE1869 | Unused Donor | 6 | |||
Pre-LVAD Heart Failure | 31 | 3,469 | 448 | 136 | |
TP Cappola Lab, U Penn School of Medicine, GSE5406 | Non-Failing | 16 | |||
Cardiomyopathy | 194 | 309 | 42 | 15 | |
Total Across Human Studies | 363 | 7,551 | 894 | 308 |
We also compared our data to those of a previous study that assessed the effects of a NRG-1β fragment at 8 weeks post-MI in rats
Gene Symbol | Name | GGF2 | rhNRG | Function |
Aldh6a1 | Aldehyde dehydrogenase 6 family, member A1 | −1.9 | −1.4 | Amino acid and lipid metabolism |
Atp5g1 | ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C1 | −1.9 | −1.3 | Energy production |
Dcxr | Dicarbonyl L-xylulose reductase | −1.6 | −1.4 | Glucose metabolism |
Dhrs7c | Dehydrogenase/reductase (SDR family) member 7C | −1.7 | −1.3 | Putative oxidoreductase |
Eln | Elastin | 1.6 | 1.2 | Structural protein |
Gdi1 | GDP dissociation inhibitor 1 | 1.5 | 1.6 | Small GTPase mediated signal transduction |
Gstm7 | Glutathione S-transferase, mu 7 | −1.6 | −1.4 | Glutathione and xenobiotic metabolism |
Hmgn1 | High-mobility group nucleosome binding domain 1 | 1.6 | 2.1 | Chromatin organization |
Hopx | Homeodomain only protein x | −1.6 | −1.3 | Heart development, regulation of contraction |
Macrod1 | MACRO domain containing 1 | −2.1 | −1.3 | Estrogen signaling, cell proliferation |
Mut | Methylmalonyl-CoA mutase | −1.5 | −1.3 | Amino acid metabolism |
Pgam2 | Phosphoglycerate mutase 2 (muscle) | −1.9 | −1.4 | Gluconeogenesis, muscle contraction |
Pnn | Pinin, desmosome associated protein | 1.7 | 2.7 | RNA processing and transport |
Prpf39 | PRP39 pre-mRNA processing factor 39 homolog | 1.5 | 2.1 | RNA processing and transport |
Ptma | Prothymosin alpha | 1.7 | 1.6 | Anti-apoptosis |
Rfk | Riboflavin kinase | −1.9 | −1.3 | Riboflavin metabolism |
Snrp70 | U1 small nuclear ribonucleoprotein polypeptide A | 1.6 | 1.3 | RNA splicing |
Srrm2 | similar to serine/arginine repetitive matrix 2 | 1.6 | 1.9 | RNA splicing |
Tada2b | Transcriptional adaptor 2 (ADA2 homolog, yeast)-beta | 1.7 | 2.2 | Transcriptional adaptor protein |
Tst | Thiosulfate sulfurtransferase, mitochondrial | −1.5 | −1.3 | Mitochondrial rRNA import |
Fold-changes in gene expression in treated compared to untreated animals are reported; negative value indicates downregulation in treated compared to controls.
In summary, we report that treatment with the GGF2 isoform of NRG-1β can improve LV function after MI in rats. The myocardial sensitivity to GGF2 is increased early compared to late after MI, and this correlates with increased myocardial ErbB2 receptor expression at the early time point. Furthermore, GGF2 treatment was associated with ‘normalization’ of the expression of many genes in the myocardium, providing directions to pursue in defining possible mechanisms of these effects. These results support further exploration of GGF2 as a therapeutic for systolic heart failure following MI.
Complete list of genes altered by MI vs. Sham and/or GGF2 treatment.
(XLSX)
We thank Lian Li and Zhizhang Wang for technical assistance with animal surgery and echocardiography. We thank Todd Peterson and the Vanderbilt University Insititute for Imaging Sciences for conducting Positron Emission Tomography imaging studies. Cardiac surgery and echocardiography were conducted in the Mouse Metabolic Phenotyping Center which is supported by NIH Grant DK59637.