Author + information
- Received March 3, 2017
- Revision received May 26, 2017
- Accepted June 15, 2017
- Published online August 14, 2017.
- Joshua G. Travers, BSa,
- Fadia A. Kamal, PharmD, PhDa,b,
- Iñigo Valiente-Alandi, PhDa,
- Michelle L. Nieman, BSc,
- Michelle A. Sargent, BSa,
- John N. Lorenz, PhDc,
- Jeffery D. Molkentin, PhDa,d and
- Burns C. Blaxall, PhDa,∗ ()
- aDepartment of Pediatrics, Division of Molecular Cardiovascular Biology, The Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
- bThe Center for Musculoskeletal Research, Department of Orthopedics, University of Rochester Medical Center, Rochester, New York
- cDepartment of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio
- dHoward Hughes Medical Institute, Chevy Chase, Maryland
- ↵∗Address for correspondence:
Dr. Burns C. Blaxall, The Heart Institute, Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Medical Center, 240 Albert Sabin Way, MLC 7020, Cincinnati, Ohio 45229-3039.
Background Cardiac fibroblasts are a critical cell population responsible for myocardial extracellular matrix homeostasis. Upon injury or pathological stimulation, these cells transform to an activated myofibroblast state and play a fundamental role in myocardial fibrosis and remodeling. Chronic sympathetic overstimulation, a hallmark of heart failure (HF), induces pathological signaling through G protein βγ (Gβγ) subunits and their interaction with G protein−coupled receptor kinase 2 (GRK2).
Objectives This study investigated the hypothesis that Gβγ-GRK2 inhibition and/or ablation after myocardial injury would attenuate pathological myofibroblast activation and cardiac remodeling.
Methods The therapeutic potential of small molecule Gβγ-GRK2 inhibition, alone or in combination with activated fibroblast- or myocyte-specific GRK2 ablation—each initiated after myocardial ischemia−reperfusion (I/R) injury—was investigated to evaluate the possible salutary effects on post-I/R fibroblast activation, pathological remodeling, and cardiac dysfunction.
Results Small molecule Gβγ-GRK2 inhibition initiated 1 week post-injury was cardioprotective in the I/R model of chronic HF, including preservation of cardiac contractility and a reduction in cardiac fibrotic remodeling. Systemic small molecule Gβγ-GRK2 inhibition initiated 1 week post-I/R in cardiomyocyte-restricted GRK2 ablated mice (also post-I/R) still demonstrated significant cardioprotection, which suggested a potential protective role beyond the cardiomyocyte. Inducible ablation of GRK2 in activated fibroblasts (i.e., myofibroblasts) post-I/R injury demonstrated significant functional cardioprotection with reduced myofibroblast transformation and fibrosis. Systemic small molecule Gβγ-GRK2 inhibition initiated 1 week post-I/R provided little to no further protection in mice with ablation of GRK2 in activated fibroblasts alone. Finally, Gβγ-GRK2 inhibition significantly attenuated activation characteristics of failing human cardiac fibroblasts isolated from end-stage HF patients.
Conclusions These findings suggested consideration of a paradigm shift in the understanding of the therapeutic role of Gβγ-GRK2 inhibition in treating HF and the potential therapeutic role for Gβγ-GRK2 inhibition in limiting pathological myofibroblast activation, interstitial fibrosis, and HF progression.
Heart failure (HF) is a devastating disease characterized by interstitial fibrosis, chamber remodeling, and reduced ventricular compliance. Cardiovascular disease remains the predominant cause of mortality in the United States, presenting a considerable economic burden, with estimated annual direct and indirect costs totaling approximately $320 billion (1). Regardless of etiology, HF generally involves adverse myocardial remodeling characterized by excessive deposition of extracellular matrix proteins by pathologically activated cardiac fibroblasts; this reduces tissue compliance, promotes arrhythmogenesis, and accelerates HF progression (2). Despite the critical importance of fibrosis in HF, there are essentially no clinical interventions that effectively target the cardiac fibroblast nor its pathological contributions to disease progression.
The adrenergic system plays a fundamental role in the physiological regulation of the myocardium; however, chronic stimulation can induce cardiac hypertrophy and fibrosis, which are important components of HF pathophysiology (3). Stimulation of the β-adrenergic receptor (β-AR) induces conformational changes in G protein βγ (Gβγ) subunits, ultimately resulting in activation and membrane recruitment of G protein−coupled receptor kinase 2 (GRK2) (4). Expression of GRK2 is known to be elevated in patients with HF (4), and recent studies have suggested that this is associated with β-AR uncoupling and downregulation in fibroblasts, which can promote a pro-fibrotic phenotype (5).
We and others have explored several approaches to specifically target GRK2 and its interaction with Gβγ subunits. These efforts have demonstrated the therapeutic potential of Gβγ-GRK2 inhibitory peptides (6) or compounds (7–9). The beneficial effects of the small molecule gallein, which selectively inhibits the interaction between Gβγ and GRK2 (7,10), were recently demonstrated in several animal models of HF (7,8). Although the Gβγ-GRK2 interface represents an important target of therapeutic interventions for HF, the mechanisms and therapeutic potential of Gβγ-GRK2 inhibition specifically within cardiac fibroblasts and the progression of fibrosis have yet to be elucidated.
In the present study, small molecule inhibition of Gβγ-GRK2 initiated 1 week after myocardial ischemia−reperfusion (I/R) ameliorated the progression of cardiac dysfunction and pathological cardiac remodeling, particularly regarding infarct expansion. Furthermore, inducible ablation of GRK2 in the pathologically activated cardiac fibroblasts 1 week post-I/R was found to be equally cardioprotective, whereas gallein provided significant cardioprotection in animals with post-I/R ablation of GRK2 in cardiomyocytes. This cardioprotection in vivo correlated with a reduction in the activation state of primary mouse and human HF-derived cardiac fibroblasts when treated with gallein. These data support a paradigm shift in proposed mechanisms behind the protective effects of Gβγ-GRK2 inhibition in the treatment of HF.
We recently reported a possible therapeutic role for interdicting pathological Gβγ-GRK2 binding interactions with the small molecule gallein (8). In the present study, gallein was evaluated for its therapeutic efficacy in a more clinically relevant I/R model of HF; mice were subjected to I/R through coronary artery occlusion, followed by 4 weeks of reperfusion. Gallein administration was initiated 1 week post-I/R at 2.5 mg/kg/day and titrated to a maximum dose of 10 mg/kg/day over 3 weeks, followed by assessment of cardiac function by echocardiography and histological analysis of fibrotic remodeling 4 weeks after injury. To biochemically assess injury severity, transcript expression of fibrotic and HF markers was assessed by quantitative polymerase chain reaction (qPCR).
Conditional cardiomyocyte-targeted GRK2 knockout mice were achieved by crossing GRK2fl/fl animals with mice possessing tamoxifen-inducible Cre recombinase under the control of the endogenous promotor for α-myosin heavy chain (α-MHCMCM) (11). GRK2fl/fl animals were also crossed with mice that expressed inducible Cre recombinase under the control of the endogenous promoter for periostin (PostnMCM) (12). Tamoxifen administration via the chow was initiated after surgery and continued for 2 weeks to achieve inducible GRK2 ablation in a cell-specific manner.
Detailed materials and methods are included in the Online Appendix.
Optimum dosing and administration, along with initial therapeutic efficacy of gallein, were first evaluated and established in wild-type C57Bl/6 mice subjected to I/R injury. Gallein treatment conferred substantial protection against myocardial dysfunction and dilation (Online Figures 1A to 1C). Furthermore, examination of collagen deposition by Masson’s trichrome staining showed a reduction in fibrotic expansion in animals treated with gallein (Online Figure 1D).
Corroboration of the cardioprotective attributes of small molecule Gβγ-GRK2 inhibition was assessed in animals possessing the GRK2fl/fl allele, because these mice served as controls for subsequent genetic ablation studies when crossed with various Cre lines. Gallein treatment was initiated 1 week post-injury as described to investigate its therapeutic efficacy in treating extant HF (Figure 1A). Animals that received vehicle demonstrated significant deterioration in overall cardiac function 4 weeks post-I/R. Remarkably, mice treated with gallein initiated 1 week post-I/R exhibited significant preservation of contractile performance and ventricular volumes (Figure 1B) as measured by percent fractional shortening (Figure 1C), ejection fraction (Figure 1D), and left ventricular (LV) volume (Figure 1E). Echocardiographic strain analysis supported this finding, revealing significant restoration in peak wall contraction in animals that received gallein, which indicated attenuation of progressive LV dysfunction (Figure 1F). Subsequent qPCR analysis of injured mice that received gallein post-I/R demonstrated a significant reduction in the mRNA levels of natriuretic peptide B (Nppb) (Figure 1G), β-myosin heavy chain (Myh7) (Figure 1H), and natriuretic peptide A (Nppa) (Online Figure 2A) in comparison to vehicle-treated control mice.
The observed preservation of cardiac function by post-I/R gallein treatment occurred commensurate with an overall reduction in post-I/R fibrosis, particularly with regard to infarct expansion. Serial cardiac longitudinal sections were stained by picrosirius red to determine the extent of collagen deposition following injury (Figure 2A). Fibrosis was quantified in relation to total LV area, which revealed a significant reduction in pathological fibrotic expansion from the initial infarct region in gallein-treated mice (Figure 2B). Immunofluorescence revealed a parallel reduction in the presence of periostin, which is produced specifically by activated fibroblasts following tissue injury (12), in gallein-treated mice compared with vehicle controls (Figure 2C). Furthermore, gene expression analysis revealed a concomitant reduction in the transcript expression of collagen type I α1 (col1a1) (Figure 2D), periostin (Postn) (Figure 2E), and fibronectin 1 (Fn1) (Online Figure 2C), together with trends toward a decrease in the expression of collagen type III α1 (Col3a1) (Online Figure 2B) in mice treated with gallein. Overall, pharmacological Gβγ-GRK2 inhibition initiated post-I/R provided functional protection and reduced post-I/R infarct expansion following ischemic myocardial injury.
Effect of cardiomyocyte-specific GRK2 inhibition
The therapeutic efficacy of cardiomyocyte-specific GRK2 inhibition and ablation has been investigated extensively for its role in slowing HF progression in numerous animal models (13,14). Cardiomyocyte-restricted GRK2 ablation was achieved by placing GRK2fl/fl × α-MHCMCM mice on tamoxifen chow following I/R, and functional assessment was performed by echocardiography 4 weeks post-injury (Figure 3A, Online Figure 3A). Successful knockdown was confirmed in isolated cardiomyocytes by a reduction in both transcript expression of GRK2 (Adrbk1) (Figure 3B) and protein expression (Online Figure 3B). GRK2fl/fl control mice subjected to I/R injury showed significant signs of impaired cardiac function compared with sham control mice, as measured by echocardiography 4 weeks post-injury (Figures 3C and 3D). Cardiac functional assessment of αMHCMCM mice by investigator-blinded echocardiography revealed a nearly identical level of cardiac dysfunction following I/R, as was observed in the GRK2fl/fl mice, which was consistent with previous reports (Online Table 1, Online Figure 4) (13). Cardiomyocyte-specific GRK2 ablation offered modest protection from myocardial dysfunction compared with GRK2fl/fl control mice as measured by percent ejection fraction (Figure 3C) (p = 0.277) and fractional shortening (Figure 3D) (p = 0.276). Histologically, cardiomyocyte-specific GRK2 ablation only modestly reduced LV fibrosis, as observed by picrosirius red staining (Figures 3E and 3F) or periostin immunofluorescence (Figure 3G). However, there did appear to be some preservation in cardiomyocyte viability, as previously described (Figure 3E) (13).
To investigate potential cardiomyocyte-independent properties of pharmacological Gβγ-GRK2 inhibition, gallein was initiated 1 week post-I/R by intraperitoneal injection to GRK2fl/fl × αMHCMCM mice (Online Figure 3A). Significant improvement in GRK2fl/fl × αMHCMCM mice treated with gallein post-I/R compared with GRK2fl/fl control mice was observed by cardiac ejection fraction and fractional shortening (Figures 3C and 3D). Furthermore, there was a trend toward improvement in the peak strain percentage (Online Figure 3C), together with a reduction in overall ventricular dyssynchrony (Online Figure 3D) when mice were assessed by strain analysis. Post-I/R gallein treatment offered numerical functional improvement in GRK2fl/fl × αMHCMCM mice after injury. Assessment of cardiomyocyte-specific GRK2 knockout mice treated with gallein post-I/R revealed a significant decrease in pathological fibrotic expansion and periostin secretion (Figures 3E to 3G). Collectively, these data validated Gβγ-GRK2 as a therapeutic target and suggested potential functional significance of Gβγ-GRK2 inhibition in cell types beyond the cardiomyocyte.
Activated fibroblast-specific GRK2 ablation post-i/r
Based on the beneficial effects of post-I/R gallein treatment observed in cardiomyocyte-specific GRK2 knockout mice, the therapeutic potential of activated fibroblast restricted GRK2 ablation was investigated post-I/R with or without gallein treatment. Knockdown of GRK2 was achieved in GRK2fl/fl × PostnMCM mice through tamoxifen administration via the chow. These mice provided a powerful method by which to inducibly ablate GRK2 only in the pathologically activated fibroblasts (Online Figure 5A) (12). Previous reports demonstrated that protein knockdown might not occur until 4 to 6 days after tamoxifen administration via the chow (12), which coincided with initiation of gallein treatment 1 week post-injury. To demonstrate successful gene knockdown, flow cytometry was used to select for CD31/CD45 negative and platelet-derived growth factor receptor-α positive cells (Online Figure 5B); qPCR analysis revealed a significant reduction in the transcript expression of GRK2 (Adrbk1) in this cell population following myocardial injury and tamoxifen administration (Online Figure 5C).
Following injury, GRK2fl/fl control mice presented with significant cardiac dysfunction 4 weeks after injury, demonstrating reductions in functional parameters by echocardiography compared with mice that had sham surgery (Figures 4A to 4C). Mice in which GRK2 was ablated post-I/R, solely in activated cardiac fibroblasts, maintained nearly normal contractile performance, including significant improvements in percent ejection fraction (Figure 4A) and fractional shortening (Figure 4B) versus GRK2fl/fl control mice. Post-I/R GRK2 ablation in periostin-expressing cells also significantly normalized cardiac dimensions following injury. Surprisingly, addition of the small molecule Gβγ-GRK2 inhibitor gallein 1 week post-I/R offered no observable further cardioprotection over fibroblast-specific GRK2 ablation alone as assessed by M-mode echocardiography (Figures 4A and 4B). However, although GRK2fl/fl × PostnMCM mice trended toward improvement in the peak percentage by echocardiographic strain analysis, the addition of gallein induced a significant improvement over control animals (Figure 4D). Overall, these findings demonstrated that inducible post-I/R GRK2 ablation in activated cardiac fibroblasts was cardioprotective in an I/R model of HF and that addition of a small molecule Gβγ-GRK2 inhibitor provided no further functional protection as assessed by standard echocardiographic measures (Online Table 2). Interestingly, gallein improved advanced strain-based imaging measures, suggesting a possible role in attenuating diastolic dysfunction. These data suggested that pathological Gβγ-GRK2 signaling plays a significant role in the activated cardiac fibroblasts and contributes to cardiac remodeling and dysfunction.
Despite specifically targeting activated cardiac fibroblasts, the effect of this fibroblast-specific GRK2 ablation on cardiomyocyte contractility was explored. Cardiomyocytes were isolated from the hearts of GRK2fl/fl × PostnMCM mice and GRK2fl/fl control mice 4 weeks post-I/R, and sarcomeric shortening was evaluated. Interestingly, cardiomyocytes isolated from mice in which GRK2 was deleted solely in activated cardiac fibroblasts possessed significantly enhanced contractility compared with control mice (Figure 4E), with representative sarcomeric length traces shown in Figure 4F. These data suggested that GRK2 signaling in the activated fibroblast could affect resident cardiomyocyte contractility.
The hearts of GRK2fl/fl × PostnMCM mice treated with or without gallein were evaluated histologically to determine the effects of post-I/R Gβγ-GRK2 inhibition and targeted GRK2 ablation in activated fibroblasts on pathological cardiac remodeling following establishment of the initial infarct immediately post-I/R. Picrosirius red staining showed a significant reduction in collagen deposition and infarct expansion in mice in which GRK2 was ablated in activated cardiac fibroblasts to a similar degree regardless of gallein treatment (Figures 5A and 5B). Immunofluorescence staining for periostin revealed an expression pattern similar to that of collagen, which was reduced in mice in which GRK2 was ablated post-I/R in activated cardiac fibroblasts compared with GRK2fl/fl control mice following injury (Figure 5C). To confirm these results, the expression of several fibrotic markers was assessed in the LV tissue; qPCR analysis revealed significant reductions in the mRNA levels of collagen type I α1 (Col1a1), Postn, and fibronectin (Fn1) (Figures 5D to 5F). Taken together, these findings suggested that improvements observed in overall cardiac function were potentially due to restored ventricular compliance as the result of a lessened post-I/R fibrotic burden caused by attenuation of pathological Gβγ-GRK2 signaling in activated fibroblasts.
Pharmacological Gβγ-GRK2 inhibition in vitro
To determine the functional effects of attenuating Gβγ-GRK2 signaling ex vivo, primary cardiac fibroblasts were isolated from the ventricles of adult mice to assess the efficacy of Gβγ-GRK2 inhibition in preventing pathological fibroblast activation. These cells were cultured in collagen gels, stimulated with transforming growth factor (TGF)-β to induce activation, and treated with either gallein or vehicle. Time-dependent contraction of the collagen gels was observed in fibroblasts treated with TGF-β, which was significantly attenuated in cells treated with gallein (Figure 6A). Furthermore, expression of the contractile protein smooth muscle α-actin (α-SMA), which confers contractile properties to activated fibroblasts, was evaluated in vitro; gallein treatment significantly reduced the expression of α-SMA both by immunofluorescence and Western blotting in response to TGF-β stimulation (Figures 6B to 6D). To explore a possible mechanism for this reduction in fibroblast activation, the effect of pharmacological Gβγ-GRK2 inhibition on modulating cyclic adenosine monophosphate (cAMP) production in response to β-AR stimulation was investigated, because cAMP was previously described as antifibrotic (15). Interestingly, gallein treatment in mouse cardiac fibroblasts significantly increased cAMP production in response to stimulation by isoproterenol (Figure 6E).
Failing human cardiac fibroblasts were isolated from the hearts of end-stage HF patients who underwent LV assist device implantation. Approximately 98% of these cells stained positive for vimentin, which offered some reassurance that these failing human cells consisted of a relatively pure population of cardiac fibroblasts (Online Figures 6A and 6B). As with murine cardiac fibroblasts, small molecule Gβγ-GRK2 inhibition significantly attenuated the ability of these human HF fibroblasts to contract collagen gels (Figure 7A). Gallein treatment also significantly reduced the proliferative rate of the failing human cardiac fibroblasts (Figure 7B), together with their ability to migrate across a cell-permeable membrane (Figure 7C). Importantly, gallein was shown to have no negative impact on fibroblast integrity (Online Figure 6C). These cells also possessed a blunted response to adrenergic stimulation compared with nonfailing control subjects, as seen by a significant attenuation in cAMP production following stimulation with isoproterenol (Figure 7D). Gallein treatment significantly restored cAMP production induced by adrenergic stimulation in cardiac fibroblasts isolated from patients with end-stage HF (Figure 7E). Finally, although no significant alternations in the overall expression of α-SMA were observed (data not shown), gallein treatment appeared to reduce the formation of prominent α-SMA stress fibers (Figure 7F).
The transformation of cardiac fibroblasts to activated myofibroblasts plays a key role in myocardial fibrosis and remodeling, which contribute significantly to HF progression (2). Currently, no clinical interventions effectively target this important cell population and its pathological contributions to disease progression; development of such a therapy would significantly enhance current approaches in HF treatment.
The adrenergic system plays a fundamental role in the physiological regulation of the myocardium; however, long-term overstimulation can induce both cardiac hypertrophy and fibrosis (3). This is also associated with upregulation in the expression of GRK2 in human HF, in which this protein is known to play a central role in the uncoupling and desensitization of β-ARs (4). The therapeutic potential of pathological Gβγ-GRK2 signaling inhibition has been extensively explored in various animal models of HF. We recently described a small molecule known as gallein that targets specific Gβγ protein interactions, including its association with GRK2 (7,10). Disruption of pathological Gβγ-GRK2 signaling, using various inhibitory peptides (6) and small molecules (7–9), was proven highly successful. We recently demonstrated the therapeutic efficacy of gallein-mediated Gβγ-GRK2 inhibition in cardiorenal syndrome, where it directly attenuated both myocardial and renal dysfunction, as well as fibrosis (16). However, the cellular specificity of this systemic inhibition was not previously thoroughly investigated, particularly regarding the cardiac fibroblast. In this study, we examined the therapeutic potential for small molecule Gβγ-GRK2 inhibition administered following myocardial I/R, including whether it would offer further protection over the previously established beneficial effects of GRK2 deletion in cardiomyocytes. GRK2 ablation post-I/R in activated cardiac fibroblasts, in the presence or absence of systemic post-I/R small molecule Gβγ-GRK2 inhibition, was also investigated for its therapeutic potential in treating extant HF (Central Illustration).
The present study used murine cardiac I/R injury, a more clinically relevant mouse model of ischemic HF that is initially characterized by a robust inflammatory response and extensive cardiomyocyte necrosis within the immediate infarction region (3). To prevent cardiac rupture, this area of ischemic injury is restored by fibrotic tissue deposition (replacement fibrosis) through elevated secretion of extracellular matrix components by activated cardiac fibroblasts. Fibroblast persistence then allows for the development of pathological infarct expansion beyond the original infarct region, which leads to ventricular noncompliance and HF (17). Disruption of pathological Gβγ-GRK2 signaling by the small molecule inhibitor gallein was initiated 1 week post-injury with dose titration to assess its therapeutic efficacy in the setting of existing cardiac damage (8).
Gallein treatment initiated 1 week post-I/R injury offered significant protection against myocardial dysfunction and attenuated cardiac dilation compared with vehicle-treated animals. These mice were also assessed by echocardiographic strain analysis, which provided a global assessment of ventricular wall function and a valuation of dyssynchrony between various wall segments (18). Significant rescue of peak strain percentage confirmed the preservation of ventricular wall integrity with gallein treatment. In addition, staining for collagen deposition revealed a significant amelioration of fibrotic infarct expansion and a concomitant reduction in fibrotic marker expression in gallein-treated mice, which suggested interference in the persistent activation of cardiac fibroblasts. These data were consistent with our previous report of pharmacological Gβγ-GRK2 inhibition with gallein in pressure-overload HF (8), and in a study investigating high-dose paroxetine in myocardial infarction (9). Because gallein is delivered systemically, it was imperative to investigate the cellular specificity of pharmacological Gβγ-GRK2 inhibition by exploring the effects of GRK2 ablation in various resident myocardial cells.
Targeting of the cardiomyocyte for the treatment of cardiovascular disease has been at the forefront of cardiac research for several decades. Abundant evidence has suggested salutary properties of cardiomyocyte-specific GRK2 inhibition both in improving contractility in isolated cardiomyocytes and in rescuing cardiac function following cardiac injury (13,14,19). The aim of this study was to evaluate potential cardiomyocyte-independent properties of gallein when given to inducible, cardiomyocyte-specific GRK2 knockout mice subjected to I/R injury. In the present study, GRK2 ablation in cardiac myocytes was induced following I/R to assess the potential of treating extant HF as described previously (13). Tamoxifen administration via the chow was limited to the duration required to induce gene ablation, while attempting to avoid potential off-target effects. Of note, high doses of tamoxifen, in combination with various inducible mouse lines, can induce focal cardiac fibrosis. In addition, several reports indicated that high levels of Cre expression itself can be cytotoxic (20). Successful knockdown was confirmed in isolated cardiomyocytes by reduced GRK2 transcript and protein expression following 2 weeks of tamoxifen chow feeding.
Following injury, a modest level of cardioprotection was observed in cardiomyocyte-specific GRK2 knockout mice, in both functional parameters and fibrotic remodeling. These mice possessed numerically improved cardiac performance with what appeared to be reduced cardiomyocyte apoptosis and subsequent replacement fibrosis. Interestingly, gallein administration initiated 1 week post-I/R in GRK2fl/fl × α-MHCMCM mice provided significant cardioprotection, which suggested that cardiomyocyte-independent properties of pharmacological Gβγ-GRK2 inhibition were likely important for its salutary effects. To that end, an inducible system was used to ablate GRK2 under the control of the promoter for periostin (12), a secreted extracellular matrix protein expressed by activated fibroblasts within areas of cardiac injury (21), which allowed us to assess the effects of inducible, post-I/R GRK2 ablation in activated cardiac fibroblasts.
Remarkably, mice in which GRK2 was inducibly ablated post-I/R in activated cardiac fibroblasts demonstrated nearly complete preservation of cardiac function. This included a significant decrease in pathological fibrotic expansion together with concomitant reductions in the transcript expression levels of several pro-fibrotic markers. Altogether, these findings suggested that improvements observed in overall cardiac function were potentially due to restored ventricular compliance as the result of a decreased fibrotic burden. As an unexpected consequence, the addition of pharmacological Gβγ-GRK2 inhibition 1 week post-I/R to the GRK2fl/fl × PostnMCM mice offered no further improvements to cardiac function as seen by M-mode echocardiography, nor further reductions in fibrotic expansion or fibrotic marker gene expression. However, a significant improvement was detected when fibroblast-specific GRK2 knockout mice treated with gallein were assessed by strain analysis, which was not achieved by GRK2 knockdown alone, which suggested a potential reduction in diastolic dysfunction through the addition of small molecule Gβγ-GRK2 inhibition. These findings suggested that the cardioprotection proffered by small molecule Gβγ-GRK2 inhibition might be substantially mediated through its effects in the pathologically activated cardiac fibroblasts. However, considering the nearly complete level of protection observed with post-I/R GRK2 ablation in the myofibroblast, we could not rule out the possibility that gallein might elicit salutary effects in other cells beyond the fibroblast.
To assess potential extra-fibroblast effects that might have contributed to the observed functional protection in activated fibroblast specific GRK2 knockout mice following injury, the contractility of isolated cardiomyocytes from these animals was quantified; cardiomyocytes from these mice demonstrated enhanced levels of sarcomeric contractility. This might be the result of enhanced crosstalk between cardiac fibroblasts and myocytes or simply that cardiomyocytes were isolated from healthier hearts. Although the exact mechanism behind this preserved cardiomyocyte function remains unclear, the enhanced contractility of cardiomyocytes in mice with post-I/R activated fibroblast-specific GRK2 ablation likely contributed to the improvement observed in overall cardiac function following injury.
Cardiac fibroblasts respond to pathological stress and environmental stimuli by transforming into activated fibroblasts that express elevated levels of various pro-fibrotic factors, possess enhanced migratory and proliferative capacities, and acquire increased contractile properties (2). These features, which can be evaluated in vitro, lead to the development of adverse changes in ventricular structure and compliance. Primary cardiac fibroblasts isolated from the ventricles of adult mice were stimulated with TGF-β to recapitulate the activation state of fibroblasts following a cardiac event in vivo. Because the contraction of the collagen network following myocardial injury is a key function of the activated fibroblast, measuring this phenomenon in culture can provide important information regarding cellular activation status. The decrease in the extent of collagen gel contraction mediated by cells treated with gallein suggested a reduction in their activation state. The protein α-SMA, which has been used extensively to identify myofibroblasts in vitro (in addition to its other functions) (2), grants contractile properties to activated fibroblasts, which allow fibrotic scar contraction. In association with the reduction in collagen gel contraction, a reduction in α-SMA protein expression was also observed by immunofluorescence and Western blotting, potentially accounting for this phenomenon. Collectively, these findings suggested that pharmacological Gβγ-GRK2 inhibition could attenuate fibroblast activation characteristics in vitro. The efficacy of small molecule Gβγ-GRK2 inhibition in reducing fibroblast activation characteristics was also assessed in cardiac fibroblasts isolated from patients with end-stage HF. In a similar fashion, gallein considerably reduced the contractile properties of these cells, evidenced by a reduction in their ability to contract collagen gels in which they were cultured. Furthermore, both the proliferative and migratory capacities of these cells were significantly attenuated, which suggested a reduction in their pathological activation state.
A recent study explored the effects of GRK2 ablation in collagen1-α−expressing cells initiated 3 weeks before ischemic myocardial injury; animals were then evaluated up to 72 h post-I/R (22). The authors described preventative maintenance of cardiac function and a relative reduction in replacement fibrosis. Interestingly, no alterations in α-SMA expression were described as a result of pre-I/R GRK2 ablation upon gross histological observation; however, no further characterization of the myofibroblast transition with established methods was reported in vivo or ex vivo (12,20).
As mentioned previously, the adrenergic system plays an important role in regulating the myocardium. Although several subtypes of the β-AR exist, the β2-AR appears to be the form that is predominantly expressed by cardiac fibroblasts (23). Chronic stimulation of this receptor, which would occur in HF, leads to Gβγ-GRK2−mediated β2-AR desensitization and has been shown to induce fibroblast proliferation, collagen secretion, and other characteristics of the activated fibroblast phenotype (23). It is well-established that acute (but not chronic) stimulation of the β2-AR directly increases the levels of cAMP, which was shown to attenuate the proliferation of fibroblasts (23) and to inhibit the synthesis and secretion of collagen (24). Furthermore, elevated levels of cAMP could inhibit activation of fibroblasts induced by stimulation with TGF-β (24,25). We demonstrated that failing human cardiac fibroblasts lost the ability to respond to adrenergic stimulation, as seen by an attenuation of isoproterenol-induced cAMP production compared with nonfailing cardiac fibroblasts. However, treatment of either primary adult mouse fibroblasts or failing human ventricular fibroblasts with the small molecule Gβγ-GRK2 inhibitor gallein increased the production of cAMP following stimulation by isoproterenol. This restoration of adrenergic responsiveness potentially accounted for the reduction in fibroblast activation observed in isolated cells treated with gallein and for the reduction in fibroblast activation following cardiac injury in vivo in which there was chronically elevated catecholamine exposure (Central Illustration).
Although this study provided evidence of the therapeutic efficacy of Gβγ-GRK2 inhibition in cardiac myofibroblasts after ischemic myocardial injury, it did not preclude the possibility that gallein might exert beneficial effects in myocardial cell types beyond the fibroblast. Further investigation will help determine the specific molecular mechanisms responsible for enhancing isolated cardiomyocyte contractility in the setting of fibroblast-specific GRK2 ablation. In addition, further exploration of the off-target effects of gallein will be important to fully characterize its salutary properties.
These findings demonstrated the therapeutic efficacy of the selective small molecule Gβγ-GRK2 inhibitor gallein initiated 1 week post-I/R in preserving cardiac function and attenuating pathological cardiac remodeling in a more clinically relevant ischemic model of HF. Furthermore, we reported the cardioprotective properties of inducible post-I/R GRK2 ablation in activated cardiac fibroblasts, which suggested a paradigm shift in our understanding of the therapeutic role of Gβγ-GRK2 inhibition in the treatment of HF. This work added further evidence for the beneficial effects of Gβγ-GRK2 inhibition in treating cardiovascular disease. This small molecule and fibroblast-targeted approach might lead to refinement of existing targets and compounds, and possibly development of novel therapeutics for HF treatment.
COMPETENCY IN MEDICAL KNOWLEDGE: Fibrosis is a key process in the progression of heart failure, and chronic sympathetic overstimulation causes pathologic signaling through G protein βγ subunits and GRK2. Targeting the Gβγ-GRK2 interface after myocardial ischemic injury attenuates disease progression due to a reduction in cardiac fibroblast activation, resulting in favorable changes in both functional parameters and fibrotic remodeling.
TRANSLATIONAL OUTLOOK: Further studies are needed to clarify the specific molecular mechanisms responsible for enhancing isolated cardiomyocyte contractility in the setting of fibroblast-specific GRK2 inhibition before undertaking clinical studies of the therapeutic potential of treatments directed against this signaling pathway in patients with heart failure.
For an expanded Methods section and supplemental figures and tables, please see the online version of this article.
Dr. Blaxall was funded by NIH grants R01 HL132551, R01 HL133695, R01 HL134312, and P01 HL069779. Dr. Molkentin was funded by NIH grant P01 HL069779. Drs. Kamal and Valiente-Alandi were supported by AHA post-doctoral fellowships. Mr. Travers was supported by a pre-doctoral fellowship from the Pharmaceutical Research and Manufacturers of America Foundation.
All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- adrenergic receptor
- cyclic adenosine monophosphate
- G protein−coupled receptor kinase 2
- left ventricle
- myosin heavy chain
- quantitative polymerase chain reaction
- smooth muscle actin
- transforming growth factor
- Received March 3, 2017.
- Revision received May 26, 2017.
- Accepted June 15, 2017.
- 2017 American College of Cardiology Foundation
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- Ostrom R.S.