G Protein-coupled Receptor Kinase-2 Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts*

Cardiac fibroblasts (CF) make up 60–70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction and the transition to heart failure. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. We investigated the potential role of G protein-coupled receptor kinase-2 (GRK2) in modulating collagen synthesis by adult human CF isolated from normal and failing left ventricles. Baseline collagen synthesis was elevated in failing CF and was not inhibited by β-agonist stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in the failing CF, and expression and activity of GRK2 were increased 3-fold. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following β-agonist stimulation. In contrast, knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity in failing CF by expression of the GRK2 inhibitor, GRK2ct, or siRNA-mediated knockdown restored β-agonist-stimulated inhibition of collagen synthesis and decreased collagen synthesis in response to TGFβ stimulation. GRK2 appears to play a significant role in regulating collagen synthesis in adult human CF, and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.

alloproteinase activity dominates the early, adaptive wound healing response after MI, enhanced collagen synthesis is a feature of the later stages of healing and results in increased ECM deposition (3). Early after an injury such as MI, a series of cellular responses are activated to promote tissue repair and scar formation in the infarct zone. However, in some cases the repair process involves myocardial tissue remote from the infarct, resulting in superfluous fibrous tissue being deposited in noninfarcted myocardium, leading to pathological fibrosis (4). Fibrosis appears to underlie most cardiac pathologies where overproduction of ECM can alter the structure and architecture of the heart with deleterious effects on cardiac function (5). The formation of myocardial scar tissue is believed to progress to congestive HF and/or cardiac arrhythmias that account for significant morbidity and mortality (6).
Myofibroblast formation is controlled by a variety of growth factors, cytokines, and mechanical stimuli. Recent work has demonstrated that angiotensin II and TGF␤-stimulated cardiac myofibroblast formation and collagen synthesis are significantly decreased by increasing intracellular cAMP levels in adult rat CF (7). These data clearly define the critical downstream role of cAMP in regulating ECM synthesis in the heart. cAMP, a ubiquitous second messenger produced in response to activation of ␤ 2 -ARs and adenylyl cyclase, influences growth, death, and differentiated functions of many cell types, primarily by promoting protein phosphorylation via cAMP-dependent protein kinase (PKA). G protein-coupled receptor agonists that signal through the stimulatory G protein, G s to activate adenylyl cyclase and stimulate cAMP production can inhibit collagen synthesis (8). Although previous work suggests that increased cAMP production inhibits collagen synthesis via an inhibition of rat fibroblast to myofibroblast transformation, no studies have examined the effects of increased cAMP on myofibroblast formation in the human heart, and, importantly, there have been no studies investigating how CF ␤-AR signaling and adenylyl cyclase activity may be regulated, particularly in response to profibrotic and hypertrophic stimuli such as myocardial infarction, which can transition to a HF phenotype. In addition, the ability of catecholamines and ␤-AR stimulation to influence ECM protein synthesis by CF remains unclear.
G protein-coupled receptor kinase-2 (GRK2) is the primary GRK expressed in the heart and has been shown to be a critical regulator of cardiac function in vivo when overexpressed or inhibited in cardiac myocytes (9). Despite the significance of GRK2 in regulating cardiac myocyte function and ventricular contractility, the biological and physiological roles of GRKs have not been investigated in CF. We hypothesized that GRK2 plays an important role in regulating CF ␤-AR signaling, myofibroblast formation, and collagen synthesis.

EXPERIMENTAL PROCEDURES
All cell culture reagents were purchased from Invitrogen except FBS, which was obtained from Atlanta Biologicals (Lawrenceville, GA). Unless stated otherwise, all additional chemicals were obtained from Sigma-Aldrich (St. Louis, MO). All antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) except ␣-SMA and vimentin, which were obtained from Sigma; collagen types I and III, which were obtained from Calbiochem; and collagen type VI, which was obtained from Fitzgerald Industries International (Acton, MA).
Isolation and Culture of Adult Human Cardiac Fibroblasts-All procedures for tissue procurement in this study were performed in compliance with institutional guidelines for human research and an approved Institutional Review Board protocol at the University of Chicago Medical Center. Left ventricular tissue was taken from patients with severe left ventricular dysfunction undergoing heart transplantation. The indication for transplant in all patients (n ϭ 10) was end-stage ischemic heart failure. Tissue was taken from a region remote from the territory of infarction, most commonly the left circumflex/posterobasal segment. Failing cardiac fibroblasts were isolated by a modified method of Turner et al. (10). Biopsy specimens were minced and digested in DMEM containing 0.05% BSA, 1000 units/ml collagenase 2 (Worthington) and 0.003% trypsin at 37°C with continuous shaking for 90 min. The dissociated cells were plated for 1 h to allow fibroblasts to adhere. Following removal of non-adherent cells, CF were cultured to confluence in fresh growth medium. Experiments were performed on early passage cells (Յ2) from 10 different patients. Non-failing adult human left ventricular cardiac fibroblasts (control) were purchased from Cell Applications, Inc. (San Diego, CA). Four different control fibroblast cultures were obtained. To prevent spontaneous differentiation to myofibroblasts, all studies were carried out in low-serum (2.5% FBS) medium using early passage cells (Յ2) plated at a density of ϳ200 cells/mm 2 (7). HF fibroblasts were used within 2 weeks of culturing to ensure preservation of the failing phenotype.
GRK2 Activity by Rhodopsin Phosphorylation-CF cells were lysed in buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM EGTA, 10 mM MgCl 2 , 10 g/ml leupeptin, 20 g/ml aprotinin, and 1 mM phenyl-methylsulfonyl fluoride. Tissue lysates (60 g of total protein) were incubated with rhodopsin-enriched rod outer segments in 60 l of lysis buffer with 10 mM MgCl 2 and 0.1 mM ATP containing [␥-32 P]ATP. After an incubation period of 30 min in white light at room temperature, reactions were quenched with ice-cold lysis buffer and centrifuged for 15 min at 13,000 ϫ g. Sedimented proteins were resuspended in 25 l of protein gel-loading dye and subjected to 12% SDS-PAGE. Phosphorylated rhodopsin was visualized by autoradiography of dried polyacrylamide gels. To confirm that the total activity in these assays was exclusively that of GRK2 and not GRK5, GRK2 or GRK5 antibodies (1 g/l) were added to the reaction mix to inhibit the respective activity of each GRK. Rod outer segments were obtained from Invision BioResources (Seattle, WA). Each kinase activity reaction mixture contained 50 g of purified rhodopsin. Collagen Synthesis [ 3 H]proline Incorporation-Cardiac fibroblasts (both control and HF) were treated with ␤-agonist ISO (10 Ϫ7 to 10 Ϫ5 M for dose response experiments or 10 Ϫ5 M for others), TGF␤ (10 ng/ml), or both as indicated in legends for Figs. 2-6. Cells were stimulated with ISO or TGF␤ for 48 h. In cases where both agonists were used, cells were stimulated with ISO for 20 min prior to 48-hour stimulation with TGF␤. [ 3 H]proline incorporation was measured according to the method of Swaney et al. (7). Both total and collagenase-sensitive proline incorporation was measured. Cells were grown to 80% confluence on 12-well plates, serum-starved for 24 h, and incubated with [ 3 H]proline (1 Ci/well, PerkinElmer Life Sciences) for 48 h. Cellular protein was precipitated overnight with 20% trichloroacetic acid, washed three times with 1 ml of 5% trichloroacetic acid plus 0.01% proline, and then dissolved in 0.2 M NaOH. For collagenase-sensitive proline incorporation, collagenase type 2 (100 l, 2 mg/ml, Worthington) in Tris/ CaCl 2 /N-ethylmaleimide buffer was added to each tube prior to trichloroacetic acid precipitation. The activity of [ 3 H]proline in the supernatant of both fractions was determined by liquid scintillation counting.
Intracellular cAMP Quantitation-80% confluent CF cultured on 12-well plates were equilibrated for 24 h in low-serum (2.5% FBS) DMEM and assayed for intracellular cAMP accumulation by a 15-min incubation with 0.2 mM isobutylmethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor, followed by addition of ISO (10 Ϫ5 M) for an additional 15 min. Reactions were terminated by aspiration of culture medium and addition of 150 l of 0.1 M HCl to each well. HCl extracts were assayed for cAMP content by direct ELISA kit (Assay Designs, Ann Arbor, MI).
Protein Immunoblotting-Cells were lysed in buffer containing 25 mM HEPES, 1 mM EDTA, 125 mM NaCl, 0.5 mM NaF, 0.25% Nonidet P-40, 5% glycerol (pH 6.8), and protease inhibitor mixture (Calbiochem). Equal amounts of protein for each sample were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted. Bands were visualized with ECL Western blotting substrate (Thermo Scientific, Rockford, IL). The band intensity was quantitated using Bio-Rad Quantity One software. GAPDH was used as a loading control.
Real time PCR was performed in a 20-l reaction, 96-well format (0.2 l cDNA, 250 nM forward and reverse primers, 1ϫ DyNAmo HS SYBR Green Master Mix-Finnzymes) using an Opticon 2 real-time PCR machine (Bio-Rad). Three samples were measured in triplicate for each experimental group. Realtime PCR data analysis was carried out using the ⌬⌬ ct (threshold cycle) method. GAPDH was used as an internal control.
Confocal Microscopy-CF were grown to 60% confluence on 12-mm coverslips, washed with PBS, fixed with 3.7% formaldehyde for 15 min, and permeabilized with 0.1% Triton X-100 for 10 min. Following blocking in 3% BSA in PBS, the cells were treated with primary antibody (␣-SMA and Vimentin 1:400 dilution; GRK2, collagen types I, III, and VI; and fibronectin 1:50 dilution) overnight at 4°C and thereafter with either Alexa Fluor 594 goat anti-mouse IgG (1:1000; Invitrogen) or goat anti-rabbit IgG-FITC (1:100) secondary antibody for 1 h. After extensive washing in PBS, cells were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen). Cells were visualized using a Leica SP2 laser scanning microscope.
siRNA Transfection of Cardiac Fibroblasts-Target-specific siRNA duplexes were designed using the sequence from the open reading frame of human GRK2 mRNA to knock down mRNA and protein expression of GRK2. The target sequence from 16 to 36 bases downstream of the start codon of human GRK2 mRNA (5Ј-GCUCGCAUCCCUUCUCGAA-3Ј) was utilized, and the selected sequence was then submitted to a BLAST search to ensure that GRK2 mRNA was exclusively targeted. A scrambled oligo-ribonucleotide complex that was not homologous to any mammalian genes was utilized as a control. Both siRNAs were composed of 19 nucleotides and were obtained from Dharmacon Research (Lafayette, CO). Transfection of siRNA to human GRK2 in cardiac fibroblasts was accomplished with DharmaFECT Duo transfection reagent. Briefly, 60 -70% confluent cells in 12-well plates were transfected with 15 nmol of siRNA-GRK2 and siRNA-scrambled (scr) control in RNase-free medium. Protein expression of GRK2 was examined after 48 -72 h of transfection.
Statistical Analysis-All data are expressed as mean Ϯ S.E. and were analyzed using Student's t test. cAMP quantitation was done using GraphPad Prism 5 (GraphPad Software, San Diego, CA). Values of p Ͻ 0.05 were considered significant.

Fibroblasts Isolated from Failing Human Left Ventricles Display a Myofibroblast-predominant
Phenotype-CF isolated from failing human left ventricles had a 2.2-fold increase in ␣-SMA expression by protein immunoblotting, indicative of transformation to a myofibroblast phenotype compared with the normal control CF (Fig. 1A). In addition, they displayed a robust (7-fold) increase in the extracellular matrix glycoprotein fibronectin typical of the failing phenotype. Confocal imaging again demonstrated the up-regulation of ␣-SMA expression in the failing CF as well as increased expression of fibronectin. Vimentin is a fibroblast marker protein that was studied to verify the purity of the CF cultures (Fig. 1B). Collagen type I is the most abundant isoform in the myocardial ECM. Collagen type 1␣ mRNA expression was up-regulated 3.5-fold in the failing CF relative to the control (Fig. 1C). Protein expression of collagen types I␣ and III were also significantly increased by 2-fold, whereas collagen type VI was increased by 10-fold in CF isolated from the failing left ventricles (Fig. 1D). Increased collagen types I, III, and VI expression in failing CF is also clearly illustrated in the confocal imaging studies relative to normal control ventricular fibroblasts (Fig. 1E).
␤-Adrenergic Receptor Signaling Is Uncoupled, and GRK2 Activity Is Up-regulated in Failing Cardiac Fibroblasts-Increasing intracellular levels of cAMP have been shown previously to inhibit collagen synthesis in adult rat CF (7). We first examined the effects of ␤-agonist stimulation on collagen synthesis in normal and failing CF. Control adult human CF demonstrated a dose-dependent decline in ␤-agonist (isoproterenol)-stimulated collagen synthesis as measured by [ 3 H]proline incorporation ( Fig. 2A). In contrast, failing CF had a significantly higher base-line level of collagen synthesis, which remained unchanged following ␤-agonist stimulation ( Fig. 2A). To investigate the potential mechanism for this finding, we studied CF ␤-AR signaling in a comprehensive manner. Basal cAMP production was significantly lower in failing CF compared with the control (1.9 Ϯ 0.3 versus 4.8 Ϯ 0.75 pmol cAMP/ ml; p Ͻ 0.03) (Fig. 2B). Isoproterenol-stimulated cAMP production was also severely blunted in the HF group versus control (2.9 Ϯ 0.2 versus 12.1 Ϯ 0.3 pmol cAMP/ml; p Ͻ 0.02). However, cAMP production in response to forskolin stimulation was robust and not different between groups (Fig. 2B). These data suggest that the adenylyl cyclase moiety is intact in failing CF and that the mechanism of impaired ␤-agonist-stimulated cAMP production occurs upstream in this signaling pathway. There was no difference in ␤ 2 -AR mRNA or total protein expression between failing and normal CF (Fig. 2C). ␤ 2 -ARs are the predominant adrenergic receptors expressed in CF that couple to adenylyl cyclase. To determine whether ␤-AR desensitization may be the mechanism of impaired cAMP production in the failing CF, we studied expression of GRK2, the highest expressed GRK in the heart (11). There was an ϳ4-fold increase in GRK2 expression in the failing CF compared with normal controls (Fig. 2D). Confocal imaging also demonstrated increased GRK2 expression in the failing CF and robust membrane localization (Fig. 2E), which is consistent with the greater than 2-fold increase in GRK2 activity relative to the control as assessed by rhodopsin phosphorylation (Fig. 2F). As both GRK2 and GRK5 are expressed in the myocardium (12), monoclonal antibodies for GRK2 and GRK5 were added to the rhodopsin phosphorylation reactions and revealed that all GRK activity in the failing CF is attributable to GRK2 (Fig. 2F).
GRK2 Regulates Collagen Synthesis in Normal Human Cardiac Fibroblasts-To directly investigate the role of GRK2 in regulating collagen synthesis in adult human CF, adenoviralmediated overexpression of GRK2 was performed in normal CF. The level of increased GRK2 expression was similar to the failing CF, ϳ3.5-fold, which was also demonstrated by confocal imaging (Fig. 3A). This led to severely blunted isoproterenolstimulated cAMP production (Fig. 3B) and the relative loss of ␤-agonist-mediated inhibition of collagen synthesis compared with normal CF infected with an identical titer of a null adeno-viral construct (Fig. 3C). Increased intracellular cAMP has been shown previously to inhibit myofibroblast formation and collagen synthesis in adult rat CF by TGF␤, a potent profibrotic growth factor (13). GRK2 overexpression did not alter collagen synthesis in response to TGF␤ stimulation; however, there was loss of inhibition of TGF␤-stimulated collagen synthesis following ␤-agonist stimulation in Ad-GRK2 infected CF compared with those infected with Ad-Null (Fig. 3D). To more specifically investigate regulation of CF transformation and collagen synthesis by GRK2, GRK2 expression was knocked down using a siRNA approach in normal adult human ventricular CF. Expression of the GRK2 siRNA construct led to an ϳ50% decrease in CF GRK2 expression compared with the scrambled siRNA control (Fig. 4A). Knockdown of GRK2 significantly increased ␤-agonist-stimulated cAMP production compared with CF treated with the scrambled siRNA (88.9 Ϯ 1.5 versus 53.9 Ϯ 6.2 pmol cAMP/ml, p Ͻ 0.04) (Fig. 4B). There was no difference between groups in baseline collagen synthesis (2417 Ϯ 78 versus 2528 Ϯ 230 cpm/mg protein, p Ͼ 0.05), but there was much greater isoproterenol-stimulated inhibition of collagen synthesis following GRK2 knockdown versus the control (802 Ϯ 32 versus 1434 Ϯ 113 cpm/mg protein, p Ͻ 0.04) (Fig. 4C). Decreased GRK2 expression in normal CF did not alter collagen synthesis following TGF␤ stimulation alone (Fig.  4D). However, there was even greater ␤-agonist-mediated inhibition of TGF␤-stimulated collagen synthesis with GRK2 knockdown (748 Ϯ 30 versus 1247 Ϯ 164 cpm/mg protein, p Ͻ 0.04) (Fig. 4D). These overexpression and knockdown studies in normal adult human CF support the hypothesis that GRK2 plays an important role in the regulation of collagen synthesis in response to ␤-agonist and TGF␤ stimulation, both of which are increased in the setting of HF.

Restoration of ␤-Adrenergic Signaling in Failing Cardiac Fibroblasts via Inhibition of GRK2 Inhibits Collagen Synthesis
Stimulated by TGF␤-We then evaluated the potential therapeutic strategy of inhibiting GRK2 activity or expression in adult human CF isolated from failing left ventricles at the time of heart transplantation. The etiology of heart failure was ischemic cardiomyopathy in all patients. The previously described GRK2 inhibitor, GRK2ct, corresponding to the carboxyl-termi- nal 194 amino acids of GRK2 (14) was expressed in failing CF using an adenoviral-mediated approach driven by the CMV promoter. Expression of GRK2ct was confirmed by protein immunoblotting (Fig. 5A, upper panel), and confocal imaging demonstrates less GRK2 membrane localization consistent with decreased activity (Fig. 5A, lower panel). Inhibition of GRK2 activity by GRK2ct led to decreased ␣-SMA expression following stimulation by isoproterenol (Fig. 5B). Inhibition of GRK2 activity by GRK2ct also restored normal ␤-AR signaling in these failing CF, as measured by increased cAMP production following stimulation with isoproterenol as compared with the uncoupled ␤-AR signaling present in failing CF infected with an empty (null) adenoviral vector (28.3 Ϯ 1.4 versus 6.4 Ϯ 0.3 pmol cAMP/ml, p Ͻ 0.01) (Fig. 5C). Base-line collagen types I, III, and VI expression in failing CF was not altered by expression of GRK2ct, but there was a significant decline following isoproterenol stimulation as measured by protein immunoblotting (10.1 Ϯ 1.2 versus 20.2 Ϯ 1.7, 9 Ϯ 0.8 versus 18.7 Ϯ 1.2, and   6.11 Ϯ 0.8 versus 13.91 Ϯ 1.7 arbitrary units, respectively, for collagens type I, III, and VI; p Ͻ 0.03 for each analysis) (Fig. 5D) and seen by confocal imaging (Fig. 5E). GRK2ct expression also significantly inhibited collagen synthesis in response to ␤-agonist stimulation in a dose-dependent fashion similar to that seen in normal control CF (Fig. 5F). TGF␤-stimulated collagen synthesis in failing CF was not altered by expression of GRK2ct, but there was restoration of the ability of isoproterenol to inhibit the stimulation of collagen synthesis by TGF␤ as shown in normal CF (Fig. 5G). We also utilized a siRNA approach to inhibit GRK2 activity in failing human CF. GRK2 protein expression was decreased by ϳ75% using this strategy (Fig. 6A). Similar to our results with GRK2ct, siRNA-mediated knockdown of GRK2 resulted in a significant increase in isoproterenol-stimulated cAMP production in the failing CF (9.8 Ϯ 1.3 versus 5.5 Ϯ 1.1 pmol cAMP/ml, p Ͻ 0.04) (Fig. 6B). Knockdown of GRK2 expression led to ␤-agonist-mediated inhibition of collagen synthesis as present in the normal phenotype (1735 Ϯ 148 versus 4318 Ϯ 440 pmol/mg protein, p Ͻ 0.02) (Fig.  6C). The ability of ␤-agonist stimulation to inhibit TGF␤-mediated collagen synthesis in the failing CF was also restored by knockdown of GRK2 (1951 Ϯ 154 versus 3992 Ϯ 71 cpm/mg protein, p Ͻ 0.02) (Fig. 6D).

DISCUSSION
HF is a frequent complication of myocardial infarction (MI) that is associated with adverse ventricular remodeling (15). Well healed infarcts contain large amounts of ECM proteins, which can occupy up to 80% of the infarct area (16). However, collagen deposition also occurs in the non-infarcted remote myocardial region, predominantly in the interstitium, where it contributes to ventricular dysfunction. CF make up 60 -70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling. Cardiac fibrosis is characterized by overproduction of ECM, predominantly collagen types I and III, into the interstitial and perivascular space (17). Excessive collagen deposition leads to myocardial stiffening, impaired cardiac relaxation and filling (diastolic dysfunction), and overload of the heart, perhaps as a consequence of transformation of quiescent fibroblasts to activated myofibroblasts (18). Mechanisms of CF to myofibroblast transformation are just beginning to be understood; however, no targeted therapies currently exist to prevent myofibroblast formation and excessive collagen synthesis in the remote myocardium.
Our data show for the first time that GRK2 is robustly expressed in adult human CF and that expression and activity of this kinase are up-regulated in CF in the setting of chronic HF. This appears to be the primary mechanism of ␤ 2 -AR desensitization in CF, which results in impaired cAMP production, increased myofibroblast formation, and enhanced collagen synthesis, which are important mechanisms of adverse ventricular remodeling. Local myocardial and circulating levels of the catecholamines norepinephrine and epinephrine are elevated in advanced HF to stimulate myocyte contractility and increase cardiac output. This ␤-agonist stimulation would be expected to inhibit CF transformation and collagen synthesis through increased intracellular cAMP production; however, this effect is completely abolished as a result of GRK2-mediated ␤-AR desensitization. Overexpression of GRK2 in normal CF recapitulates this HF phenotype with loss of ␤-agonist-mediated inhibition of TGF␤-stimulated collagen synthesis. In contrast, knockdown of GRK2 expression by 50% in normal CF leads to decreased TGF␤-mediated collagen synthesis following ␤-agonist stimulation. To investigate a potential novel therapeutic strategy to inhibit collagen synthesis in adult human CF isolated from patients with end-stage ischemic cardiomyopathy, we inhibited GRK2 activity through expression of the well  described GRK2 inhibitor GRK2ct or inhibited expression using a siRNA approach. Both strategies led to restoration of the normal phenotype with ␤-agonist-stimulated inhibition of collagen synthesis in a dose-dependent manner. Inhibition of GRK2 in the failing CF also significantly diminished the profibrotic response to TGF␤ following pretreatment with isoproterenol.
␤ 2 -ARs are the predominant adrenergic receptors expressed in CF that couple to adenylyl cyclase. ␤-AR signaling is regulated by a member of the family of serine-threonine kinases known as GRKs, specifically GRK2, which phosphorylates and uncouples agonist-occupied ␤-ARs. GRK2 is up-regulated in cardiac myocytes in the development of hypertrophy and in HF and is a critical negative regulator of cardiac myocyte contractility (19). In contrast, the potential role of GRK2 in CF has not been investigated previously, including the regulation of myofibroblast formation and collagen synthesis following injury. Cyclic AMP appears to exert its antifibrotic action via multiple intracellular signaling pathways. One is the inhibition of Smad-mediated transcription via competition between cAMP response element binding protein (CREB) and Smad for key transcriptional co-activators (13). Another is cAMP-mediated inhibition of certain non-Smad signaling pathways activated by TGF␤, including ERK1/2 and JNK MAP kinases. These and likely other antifibrotic actions of cAMP appear to occur through activation of both PKA and exchange protein activated by cAMP-1 (Epac-1) (20).
The fibrosis that develops in regions remote to the infarction represents the majority of connective tissue found in ischemic cardiomyopathy and contributes to adverse structural remodeling in the failing human heart (21). Our data suggest that GRK2 plays a prominent role in the regulation of CF biology in the normal and failing heart and that CF-specific inhibition of GRK2 may provide a mechanism to prevent the maladaptive ventricular remodeling and remote fibrosis that commonly occurs following myocardial infarction and eventually progresses to HF despite revascularization. Previous studies confirm that CF biology in response to various stimuli can differ significantly on the basis of species and level of development (1). This study involves only adult human CF isolated from normal and failing left ventricles, providing greater clinical relevance. A recent study demonstrated that long-term myocardial expression of GRK2ct in the rat heart post-MI prevented adverse remodeling with decreased collagen I and TGF␤ mRNA expression and resulted in improved left ventricular function (22). The adeno-associated viral vector was directly injected into the myocardium, and there was likely significant expression of GRK2ct in the fibroblasts in addition to cardiac myocytes. Thus, it appears that inhibition of GRK2 has a beneficial effect on post-infarction remodeling in both of these predominant cell types in the heart. This strategy may also be beneficial in preventing fibrosis of other organs, such as the lung, liver, and kidney, which also leads to significant morbidity and mortality.