ß2-Adrenergic Receptors Increase Cardiac Fibroblast Proliferation Through the Gαs/ERK1/2-Dependent Secretion of Interleukin-6.

Fibroblasts are an important resident cell population in the heart involved in maintaining homeostasis and structure during normal conditions. They are also crucial in disease states for sensing signals and initiating the appropriate repair responses to maintain the structural integrity of the heart. This sentinel role of cardiac fibroblasts occurs, in part, through their ability to secrete cytokines. ß-adrenergic receptors (ßAR) are also critical regulators of cardiac function in the normal and diseased state and a major therapeutic target clinically. ßAR are known to influence cytokine secretion in various cell types and they have been shown to be involved in cytokine production in the heart, but their role in regulating cytokine production in cardiac fibroblasts is not well understood. Thus, we hypothesized that ßAR activation on cardiac fibroblasts modulates cytokine production to influence fibroblast function. Using primary fibroblast cultures from neonatal rats and adult mice, increased interleukin (IL)-6 expression and secretion occurred following ß2AR activation. The use of pharmacological inhibitors and genetic manipulations showed that IL-6 elevations occurred through the Gαs-mediated activation of ERK1/2 and resulted in increased fibroblast proliferation. In vivo, a lack of ß2AR resulted in increased infarct size following myocardial infarction and impaired wound closure in a murine dermal wound healing assay. These findings identify an important role for ß2AR in regulating fibroblast proliferation through Gαs/ERK1/2-dependent alterations in IL-6 and may lead to the development of improved heart failure therapies through targeting fibrotic function of ß2AR.

Death receptor 5 contributes to cardiomyocyte hypertrophy through epidermal growth factor receptor transactivation.

Cardiomyocyte survival and death contributes to many cardiac diseases. A common mechanism of cardiomyocyte death is through apoptosis however, numerous death receptors (DR) have been virtually unstudied in the context of cardiovascular disease. Previous studies have identified TNF-related apoptosis inducing ligand (TRAIL) and its receptor, DR5, as being altered in a chronic catecholamine administration model of heart failure, and suggest a role of non-canonical signaling in cardiomyocytes. Furthermore, multiple clinical studies have identified TRAIL or DR5 as biomarkers in the prediction of severity and mortality following myocardial infarction and in heart failure development risk suggesting a role of DR5 signaling in the heart. While TRAIL/DR5 have been extensively studied as a potential cancer therapeutic due to their ability to selectively activate apoptosis in cancer cells, TRAIL and DR5 are highly expressed in the heart where their function is uncharacterized. However, many non-transformed cell types are resistant to TRAIL-induced apoptosis suggesting non-canonical functions in non-cancerous cell types. Our goal was to determine the role of DR5 in the heart with the hypothesis that DR5 does not induce cardiomyocyte apoptosis but initiates non-canonical signaling to promote cardiomyocyte growth and survival. Histological analysis of hearts from mice treated with a DR5 agonists showed increased hypertrophy with no differences in cardiomyocyte death, fibrosis or function. Mechanistic studies in the heart and isolated cardiomyocytes identified ERK1/2 activation with DR5 agonist treatment which contributed to hypertrophy. Furthermore, epidermal growth factor receptor (EGFR) was activated following DR5 agonist treatment through activation of MMP and HB-EGFR cleavage and specific inhibitors of MMP and EGFR prevented DR5-mediated ERK1/2 signaling and hypertrophy. Taken together, these studies identify a previously unidentified role for DR5 in the heart, which does not promote apoptosis but acts through non-canonical MMP-EGFR-ERK1/2 signaling mechanisms to contribute to cardiomyocyte hypertrophy.

Epidermal growth factor receptor-dependent maintenance of cardiac contractility.

AIMS: Epidermal growth factor receptor (EGFR) is essential to the development of multiple tissues and organs and is a target of cancer therapeutics. Due to the embryonic lethality of global EGFR deletion and conflicting reports of cardiac-overexpressed EGFR mutants, its specific impact on the adult heart, normally or in response to chronic stress, has not been established. Using complimentary genetic strategies to modulate cardiomyocyte-specific EGFR expression, we aim to define its role in the regulation of cardiac function and remodelling. METHODS AND RESULTS: A floxed EGFR mouse model with α-myosin heavy chain-Cre-mediated cardiomyocyte-specific EGFR downregulation (CM-EGFR-KD mice) developed contractile dysfunction by 9 weeks of age, marked by impaired diastolic relaxation, as monitored via echocardiographic, haemodynamic, and isolated cardiomyocyte contractility analyses. This contractile defect was maintained over time without overt cardiac remodelling until 10 months of age, after which the mice ultimately developed severe heart failure and reduced lifespan. Acute downregulation of EGFR in adult floxed EGFR mice with adeno-associated virus 9 (AAV9)-encoded Cre with a cardiac troponin T promoter (AAV9-cTnT-Cre) recapitulated the CM-EGFR-KD phenotype, while AAV9-cTnT-EGFR treatment of adult CM-EGFR-KD mice rescued the phenotype. Notably, chronic administration of the ß-adrenergic receptor agonist isoproterenol effectively and reversibly compensated for the contractile dysfunction in the absence of cardiomyocyte hypertrophy in CM-EGFR-KD mice. Mechanistically, EGFR downregulation reduced the expression of protein phosphatase 2A regulatory subunit Ppp2r3a/PR72, which was associated with decreased phosphorylation of phospholamban and Ca2+ clearance, and whose re-expression via AAV9-cTnT-PR72 rescued the CM-EGFR-KD phenotype. CONCLUSIONS: Altogether, our study highlights a previously unrecognized role for EGFR in maintaining contractile homeostasis under physiologic conditions in the adult heart via regulation of PR72 expression.

The Dynamic Interplay Between Cardiac Inflammation and Fibrosis.

Heart failure is a leading cause of death worldwide. While there are multiple etiologies contributing to the development of heart failure, all cause result in impairments in cardiac function that is characterized by changes in cardiac remodeling and compliance. Fibrosis is associated with nearly all forms of heart failure and is an important contributor to disease pathogenesis. Inflammation also plays a critical role in the heart and there is a large degree of interconnectedness between the inflammatory and fibrotic response. This review discusses the cellular and molecular mechanisms contributing to inflammation and fibrosis and the interplay between the two.

Prior ß-blocker treatment decreases leukocyte responsiveness to injury.

Following injury, leukocytes are released from hematopoietic organs and migrate to the site of damage to regulate tissue inflammation and repair, however leukocytes lacking ß2-adrenergic receptor (ß2AR) expression have marked impairments in these processes. ß-blockade is a common strategy for the treatment of many cardiovascular etiologies, therefore the objective of our study was to assess the impact of prior ß-blocker treatment on baseline leukocyte parameters and their responsiveness to acute injury. In a temporal and ßAR isoform-dependent manner, chronic ß-blocker infusion increased splenic vascular cell adhesion molecule-1 (VCAM-1) expression and leukocyte accumulation (monocytes/macrophages, mast cells and neutrophils) and decreased chemokine receptor 2 (CCR2) expression, migration of bone marrow cells (BMC) and peripheral blood leukocytes (PBL), as well as infiltration into the heart following acute cardiac injury. Further, CCR2 expression and migratory responsiveness was significantly reduced in the PBL of patients receiving ß-blocker therapy compared to ß-blocker-naïve patients. These results highlight the ability of chronic ß-blocker treatment to alter baseline leukocyte characteristics that decrease their responsiveness to acute injury and suggest that prior ß-blockade may act to reduce the severity of innate immune responses.

Pepducin-mediated cardioprotection via ß-arrestin-biased ß2-adrenergic receptor-specific signaling.

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. ß-arrestin (ßarr)-biased ß-adrenergic receptor (ßAR) activation promotes survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death at the time of reperfusion to better preserve function. However, a lack of efficacious ßarr-biased orthosteric small molecules has prevented investigation into whether this pathway relays protection against ischemic injury in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of ß2AR, allosterically engaged pro-survival signaling cascades in a ßarr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Methods: Wild-type (WT) C57BL/6, ß2AR knockout (KO), ßarr1KO and ßarr2KO mice received intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min) followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM) were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrial superoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and to investigate the associated mechanism(s). Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a ß2AR- and ßarr-dependent manner, which led to improved contractile function early and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation could be detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability of ICL1-9 effects on ßarr-dependent ß2AR signaling. Conclusion: Pepducin-based allosteric modulation of ßarr-dependent ß2AR signaling represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodeling benefits.