Abstract 422: Small Molecule and Activated Fibroblast Targeting of the Gβγ-GRK2 Interface After Myocardial Ischemia Attenuates Heart Failure Progression

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Joshua G Travers ◽  
Fadia A Kamal ◽  
Inigo Valiente-Alandi ◽  
Michelle L Nieman ◽  
Michelle A Sargent ◽  
...  
Keyword(s):  
Heart Failure ◽  
Myocardial Ischemia ◽  
G Protein ◽  
Small Molecule ◽  
Therapeutic Potential ◽  
Interstitial Fibrosis ◽  
Cardiac Fibroblasts ◽  
Ischemia Reperfusion ◽  
Heart Failure Progression ◽  
Activated Fibroblasts

Cardiac fibroblasts are a critical cell population responsible for myocardial extracellular matrix homeostasis. Upon injury or pathologic 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, induces pathologic signaling through G protein βγ subunits and their interaction with G protein-coupled receptor kinase 2 (GRK2). We hypothesized that Gβγ-GRK2 inhibition/ablation after myocardial injury would attenuate pathologic myofibroblast activation and cardiac remodeling. 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 possible salutary effects on post-I/R fibroblast activation, pathologic remodeling and cardiac function. Small molecule Gβγ-GRK2 inhibition initiated one week post-injury was cardioprotective in the I/R model of chronic heart failure, including preservation of cardiac contractility and reduction in cardiac fibrotic remodeling. Systemic small molecule Gβγ-GRK2 inhibition initiated one week post-I/R in cardiomyocyte-restricted GRK2 ablated mice (also post-I/R) demonstrated additional cardioprotection, suggesting 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 one 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 heart failure patients. These findings suggest a potential therapeutic role for Gβγ-GRK2 inhibition in limiting pathologic myofibroblast activation, interstitial fibrosis and heart failure progression.

Abstract 428: Pharmacologic and Activated Fibroblast-specific Gβγ-GRK2 Inhibition is Protective Following Ischemia Reperfusion Injury

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Joshua G Travers ◽  
Fadia A Kamal ◽  
Michelle L Nieman ◽  
Michelle A Sargent ◽  
Jeffery D Molkentin ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Function ◽  
Reperfusion Injury ◽  
G Protein ◽  
Knockout Mice ◽  
Ischemia Reperfusion Injury ◽  
Interstitial Fibrosis ◽  
Myocardial Dysfunction ◽  
Ischemia Reperfusion ◽  
Ventricular Compliance

Heart failure is a devastating disease characterized by chamber remodeling, interstitial fibrosis and reduced ventricular compliance. Cardiac fibroblasts are responsible for extracellular matrix homeostasis, however upon injury or pathologic stimulation, these cells transform to a myofibroblast phenotype and play a fundamental role in myocardial fibrosis and remodeling. Chronic sympathetic overstimulation induces excess signaling through G protein βγ subunits and ultimately the pathologic activation of G protein-coupled receptor kinase 2 (GRK2). We hypothesized that Gβγ-GRK2 inhibition plays an important role in the cardiac fibroblast to attenuate pathologic myofibroblast activation and cardiac remodeling. To investigate this hypothesis, mice were subjected to ischemia/reperfusion (I/R) injury and treated with the small molecule Gβγ-GRK2 inhibitor gallein. While animals receiving vehicle demonstrated a reduction in overall cardiac function as measured by echocardiography, mice treated with gallein exhibited nearly complete preservation of cardiac function and reduced fibrotic scar formation. We next sought to establish the cell specificity of this compound by treating inducible cardiomyocyte- and activated fibroblast-specific GRK2 knockout mice post-I/R. Although we observed modest restoration in cardiac function in cardiomyocyte-specific GRK2 null mice, treatment of these mice with gallein resulted in further protection against myocardial dysfunction following injury, suggesting a functional role in other cardiac cell types, including fibroblasts. Activated fibroblast-specific GRK2 knockout mice were also subjected to ischemia/reperfusion injury; these animals displayed preserved myocardial function and reduced collagen deposition compared to littermate controls following injury. Furthermore, systemic Gβγ-GRK2 inhibition by gallein did not appear to confer further protection over activated fibroblast-specific GRK2 ablation alone. In summary, these findings suggest a potential therapeutic role for Gβγ-GRK2 inhibition in limiting pathologic myofibroblast activation, interstitial fibrosis and heart failure progression.

scholarly journals Changes in cardiac resident fibroblast physiology and phenotype in aging

2018 ◽  
Vol 315 (4) ◽  
pp. H745-H755 ◽  
Author(s):  
JoAnn Trial ◽  
Katarzyna A. Cieslik
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Interstitial Fibrosis ◽  
Cardiac Fibroblasts ◽  
Transforming Growth Factor Β ◽  
Tissue Homeostasis ◽  
Apparent Paradox ◽  
Smad Pathway ◽  
Activated Fibroblasts ◽  
Resident Fibroblast

The cardiac fibroblast plays a central role in tissue homeostasis and in repair after injury. With aging, dysregulated cardiac fibroblasts have a reduced capacity to activate a canonical transforming growth factor-β-Smad pathway and differentiate poorly into contractile myofibroblasts. That results in the formation of an insufficient scar after myocardial infarction. In contrast, in the uninjured aged heart, fibroblasts are activated and acquire a profibrotic phenotype that leads to interstitial fibrosis, ventricular stiffness, and diastolic dysfunction, all conditions that may lead to heart failure. There is an apparent paradox in aging, wherein reparative fibrosis is impaired but interstitial, adverse fibrosis is augmented. This could be explained by analyzing the effectiveness of signaling pathways in resident fibroblasts from young versus aged hearts. Whereas defective signaling by transforming growth factor-β leads to insufficient scar formation by myofibroblasts, enhanced activation of the ERK1/2 pathway may be responsible for interstitial fibrosis mediated by activated fibroblasts. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/fibroblast-phenotypic-changes-in-the-aging-heart/ .

Abstract 183: Small Molecule Gβ? Inhibition Reduces Pathologic Activation of Cardiac Fibroblasts

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Joshua G Travers ◽  
Fadia A Kamal ◽  
Burns C Blaxall
Keyword(s):  
Cardiac Hypertrophy ◽  
Small Molecule ◽  
Adrenergic Receptor ◽  
Cellular Proliferation ◽  
Therapeutic Potential ◽  
Cardiac Fibroblasts ◽  
Akt Phosphorylation ◽  
Potential Therapeutic Role ◽  
Protease Activated Receptor 1 ◽  
Chronic Activation

Heart failure (HF) is a devastating disease characterized by cardiac hypertrophy, fibrosis and inflammation. Excess signaling through Gβγ subunits leads to chronic β-adrenergic receptor (β-AR) downregulation, mediated predominantly by GRK2 in complex with PI3Kγ. Our recent work has demonstrated the therapeutic potential of the small molecule Gβγ-GRK2 inhibitor Gallein in limiting HF progression. Chronic activation of cardiac fibroblasts (CF), critical yet underappreciated myocardial cells, is a key contributor to pathologic cardiac remodeling. We hypothesized that Gβγ-GRK2 inhibition may limit pathologic CF activation. CFs were stimulated with Isoproterenol (Iso, β-AR agonist), AngII, or vehicle (V), +/- Gβγ inhibition for 24hr. Gallein treatment attenuated the induction of αSMA expression, a marker of pathologic CF activation, and two inflammatory cytokines, IL-1β and IL-6 in response to these pathologic stimuli (Iso, AngII), as assessed by real time PCR. This data suggest that Gallein treatment may reduce pathologic CF activation. Iso stimulation also enhances the phosphorylation of Akt, a kinase downstream of PI3Kγ known to be involved in cellular proliferation. Gβγ inhibition mitigated this induction, decreasing Akt phosphorylation >60% in response to Iso. This phenomenon was also observed in failing human CFs, in which Gallein decreased Akt phosphorylation >70%. We have recently demonstrated that the protease-activated receptor 1 (PAR1), a GPCR we have implicated in cardiac hypertrophy, is transactivated via chronic β-AR stimulation by induction of MMP-13, a protease we have found to be elevated in HF. Recent data from our lab and others have demonstrated that PAR1 is the most abundantly expressed GPCR in CFs, and that its stimulation in CFs may be pathologic. Interestingly, Gβγ inhibition treatment reduced PAR1 cleavage and activation in response to chronic Iso. In summary, small molecule Gβγ inhibition appears to reduce pathologic CF activation. The reduction in β-AR-mediated PAR1 cleavage reveals an alternative role for Gβγ inhibition in preventing CF activation and proliferation. These data suggest a potential therapeutic role for small molecule Gβγ inhibition in limiting pathologic CF activation and cardiac hypertrophy.

Abstract P295: The Inflammasome Is Involved in Myocardial Ischemia-Reperfusion Injury

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Masafumi Takahashi ◽  
Masanori Kawaguchi ◽  
Fumitake Usui ◽  
Hiroaki Kimura ◽  
Shun'ichiro Taniguchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myocardial Ischemia ◽  
Inflammatory Cell ◽  
Bone Marrow Cells ◽  
Cardiac Fibroblasts ◽  
Ischemia Reperfusion ◽  
Inflammatory Cell Infiltration ◽  
Inflammasome Activation ◽  
Caspase 1 ◽  
Myocardial Ischemia Reperfusion

Background: Accumulating evidence indicates that inflammation is involved in the pathophysiology of myocardial ischemia-reperfusion (I/R) injury. However, the mechanism of I/R-initiated inflammation remains to be determined. The inflammasome is a multiprotein complex consisting of nod-like receptor (NLR), apoptosis-associated speck-like adaptor protein (ASC), and caspase-1, and regulates caspase-1-dependent maturation of IL-1beta and IL-18. In the present study, we investigated the role of inflammasome in myocardial I/R injury. Methods and Results: Wild-type (WT), ASC−/−, and caspase-1−/− mice were subjected to 30 min LAD ligation, followed by reperfusion. ASC and caspase-1 were expressed at the site of myocardial I/R injury. Deficiency of ASC and caspase-1 reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, and subsequent injuries such as infarct development, myocardial fibrosis, and dysfunction in myocardial I/R injury. To determine the contribution of inflammasome in bone marrow cells, we produced bone marrow transplant mice and found that inflammasome activation was critical not only in bone marrow cells but also in myocardial resident cells. Since myocardial damage was observed before the inflammatory cell infiltration after I/R, we hypothesized that myocardial resident cells are responsible for an initial activation of inflammasome. To test this hypothesis, we examined whether hypoxia/reoxygenation (H/R) stimuli could induce inflammasome activation in cardiac fibroblasts and cardiomyocytes in vitro. Interestingly, inflammasome activation was detected only in cardiac fibroblasts, but not in cardiomyocytes, and mediated through reactive oxygen species (ROS) and potassium efflux. Conclusion: These findings indicate that inflammasome activation in cardiac fibroblasts is essential for inflammation and injury after myocardial I/R, and suggest that the inflammasome is a potential novel therapeutic target for myocardial I/R injury.

Abstract 57: Therapeutic Effects of Small Molecule Gβγ Inhibition in Pressure Overload Heart Failure

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Fadia A Kamal ◽  
Alan V Smrcka ◽  
Burns C Blaxall
Keyword(s):  
Heart Failure ◽  
Small Molecule ◽  
Interstitial Fibrosis ◽  
Plasma Catecholamines ◽  
Pressure Overload ◽  
Plasma Catecholamine ◽  
Therapeutic Effects ◽  
Mrna Levels ◽  
Group V ◽  
Plasma Catecholamine Concentrations

Heart failure (HF) is a progressive disease with rapidly increasing rates of morbidity and mortality; it is the leading cause of death worldwide. Elevated sympathetic nervous system activity, a salient feature of HF progression, leads to pathologic attenuation and desensitization of β-adrenergic receptors (β-ARs) due in part to Gβγ-mediated signaling. We recently reported that novel small molecule Gβγ inhibitors selectively block specific Gβγ signals and halt HF progression in pharmacologic and transgenic mouse models of HF. We assessed the hypothesis that the Gβγ inhibitor Gallein could be salutary in treating pre-existing HF in a clinically relevant model. We utilized the pressure-overload HF model of mouse transverse aortic constriction (TAC). Four weeks post-TAC, mice received daily IP injections of vehicle (PBS; group V) or Gallein (10mg/Kg/day; group G) for eight weeks. Gallein treatment improved survival (7 of 9 mice survived vs. 5 of 9 mice in group V) and cardiac function (%EF 75.2 ± 7.5 vs 35.6 ± 17.2 in group V, +dP/dt (mmHg/sec) 7022 ± 485.3 vs. 3584 ± 598.6 in group V), -dP/dt (mmHg/sec) -5826 ± 910.7 vs. -3260 ± 62.3 in group V, LVEDP (mmHg) 11.5 ± 3.7 vs. 29.45 ± 3.6 in group V). In addition, gallein reduced cardiac hypertrophy (HW/BW (mg/g) 5.8 ± 0.3 vs. 8.8 ± 1.1 in group V) and plasma catecholamine concentrations (adrenaline (ng/ml) 1.3 ± 0.3 vs. 6.6 ± 2.8 in group V, noradrenaline (ng/ml) 3.6 ± 0.6 vs. 15.1 ± 3.6 in group V). Reduction of interstitial fibrosis as well as mRNA levels of α-SMA, TNF-α, and IL-6 was observed in the hearts of Gallein treated animals (59.7 ± 14.1%, 43.8 ± 9.3% and 28.5 ± 3.5% relative to group V, respectively). On the molecular level, Gallein treated mice showed less GRK2 and PI3Kγ membrane recruitment, and less Akt activation (42.9 ± 7.1%, 66.7 ± 13.3% and 46.2 ± 7.7% relative to group V, respectively) in myocardial lysates. In conclusion , these data suggest a possible therapeutic role for small molecule Gβγ inhibition in halting the progression of HF, potentially via inhibition of the Gβγ-GRK2-PI3Kγ-Akt pathway. The combined effect of halting HF progression and reducing plasma catecholamines suggests a possible systemic role for small molecule Gβγ inhibition in both the heart and the adrenal gland.

Abstract 319: Small Molecule Gβγ Inhibition Attenuates Cardiac Fibroblast Inflammatory and Pro-Fibrotic Signaling

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Joshua G Travers ◽  
Fadia A Kamal ◽  
Michael S Burhans ◽  
Burns C Blaxall
Keyword(s):  
Myocardial Fibrosis ◽  
Inflammatory Cytokines ◽  
Small Molecule ◽  
Therapeutic Potential ◽  
Interstitial Fibrosis ◽  
Pressure Overload ◽  
Cardiac Fibroblast ◽  
Ventricular Compliance ◽  
Pro Inflammatory Cytokines

Heart failure (HF) is a devastating disease characterized by chamber remodeling, interstitial fibrosis and reduced ventricular compliance. Prolonged sympathetic overstimulation promotes excess signaling through G-protein Gβγ subunits and ultimately results in pathologic GRK2-mediated β-adrenergic receptor (β-AR) downregulation. We have recently demonstrated the therapeutic potential of the small molecule Gβγ-GRK2 inhibitor Gallein in limiting HF progression. Pathologic activation of the cardiac fibroblast (CF) induces the transition to a myofibroblast phenotype, which plays a fundamental role in myocardial fibrosis and remodeling. We hypothesized that Gβγ-GRK2 inhibition plays an important functional role in the CF to attenuate pathologic CF activation, inflammation and interstitial fibrosis. To explore the effect of Gβγ-GRK2 inhibition on inflammation and pro-fibrotic signaling, mice were subjected to 7 days of transverse aortic constriction, a pressure-overload model of HF. In addition to the attenuation in overall cardiac hypertrophy, animals treated with Gallein displayed reduced expression of pro-inflammatory cytokines, including macrophage inflammatory protein 1 alpha (MIP-1α) and MIP-1β, along with Interleukin-6, as assessed by qPCR. Gallein-treated animals also exhibited diminished pro-fibrotic signaling, as evidenced by a reduction in the expression of TGFβ, a major driver of myocardial fibrosis, and decreased expression of collagen. To recapitulate these findings in vitro, primary adult mouse ventricular fibroblasts were pathologically stimulated using Isoproterenol (ISO, β-AR agonist) or Angiotensin II and treated +/- Gallein for 24 hours. CFs treated with Gallein displayed an analogous reduction in the expression of these pro-inflammatory cytokines and collagen. In summary, these data suggest a potential therapeutic role for small molecule Gβγ-GRK2 inhibition in limiting pathologic myofibroblast activation, inflammation and interstitial fibrosis. We believe this fibroblast-targeted approach will lead to the refinement of existing targets and compounds, and possibly the generation of novel therapeutics for the treatment of HF.

scholarly journals A novel mtDNA repair fusion protein attenuates maladaptive remodeling and preserves cardiac function in heart failure

2018 ◽  
Vol 314 (2) ◽  
pp. H311-H321 ◽  
Author(s):  
Jessica M. Bradley ◽  
Zhen Li ◽  
Chelsea L. Organ ◽  
David J. Polhemus ◽  
Hiroyuki Otsuka ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heart Failure ◽  
Mitochondrial Dna ◽  
Myocardial Ischemia ◽  
Fusion Protein ◽  
Ischemia Reperfusion ◽  
Left Ventricular ◽  
Myocardial Infarct Size ◽  
Endonuclease Iii ◽  
Mtdna Repair

Oxidative stress results in mtDNA damage and contributes to myocardial cell death. mtDNA repair enzymes are crucial for mtDNA repair and cell survival. We investigated a novel, mitochondria-targeted fusion protein (Exscien1-III) containing endonuclease III in myocardial ischemia-reperfusion injury and transverse aortic constriction (TAC)-induced heart failure. Male C57/BL6J mice (10–12 wk) were subjected to 45 min of myocardial ischemia and either 24 h or 4 wk of reperfusion. Exscien1-III (4 mg/kg ip) or vehicle was administered at the time of reperfusion. Male C57/BL6J mice were subjected to TAC, and Exscien1-III (4 mg/kg i.p) or vehicle was administered daily starting at 3 wk post-TAC and continued for 12 wk. Echocardiography was performed to assess left ventricular (LV) structure and function. Exscien1-III reduced myocardial infarct size ( P < 0.01) at 24 h of reperfusion and preserved LV ejection fraction at 4 wk postmyocardial ischemia. Exscien1-III attenuated TAC-induced LV dilation and dysfunction at 6–12 wk post-TAC ( P < 0.05). Exscien1-III reduced ( P < 0.05) cardiac hypertrophy and maladaptive remodeling after TAC. Assessment of cardiac mitochondria showed that Exscien1-III localized to mitochondria and increased mitochondrial antioxidant and reduced apoptotic markers. In conclusion, our results indicate that administration of Exscien1-III provides significant protection against myocardial ischemia and preserves myocardial structure and LV performance in the setting of heart failure. NEW & NOTEWORTHY Oxidative stress-induced mitochondrial DNA damage is a prominent feature in the pathogenesis of cardiovascular diseases. In the present study, we demonstrate the efficacy of a novel, mitochondria-targeted fusion protein that traffics endonuclease III specifically for mitochondrial DNA repair in two well-characterized murine models of cardiac injury and failure.

scholarly journals Agrin Promotes Coordinated Therapeutic Processes Leading to Improved Cardiac Repair in Pigs

Circulation ◽  
2020 ◽  
Vol 142 (9) ◽  
pp. 868-881 ◽  
Author(s):  
Andrea Baehr ◽  
Kfir Baruch Umansky ◽  
Elad Bassat ◽  
Victoria Jurisch ◽  
Katharina Klett ◽  
...  
Keyword(s):  
Heart Failure ◽  
Single Dose ◽  
Matrix Protein ◽  
Ischemia Reperfusion Injury ◽  
Therapeutic Potential ◽  
Heart Function ◽  
Heart Diseases ◽  
Ischemia Reperfusion ◽  
Extracellular Matrix Protein ◽  
Number Of Patients

Background: Ischemic heart diseases are leading causes of death and reduced life quality worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a large number of patients with MI develop chronic heart failure over time. We previously reported that a fragment of the extracellular matrix protein agrin promotes cardiac regeneration after MI in adult mice. Methods: To test the therapeutic potential of agrin in a preclinical porcine model, we performed ischemia–reperfusion injuries using balloon occlusion for 60 minutes followed by a 3-, 7-, or 28-day reperfusion period. Results: We demonstrated that local (antegrade) delivery of recombinant human agrin to the infarcted pig heart can target the affected regions in an efficient and clinically relevant manner. A single dose of recombinant human agrin improved heart function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments along with complementary murine studies revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as agrin’s mechanisms of action. Conclusions: A single dose of agrin is capable of reducing ischemia–reperfusion injury and improving heart function, demonstrating that agrin could serve as a therapy for patients with acute MI and potentially heart failure.

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