Heart failure is a leading cause of death. Despite the economic and health burden, few recent therapeutic advances have been made and current therapies alleviate the symptoms, but minimally impact mortality, highlighting the need for identifying novel therapeutic targets. Death receptor 5 (DR5) has been studied extensively in cancer for its role in inducing apoptosis in transformed cells. However, DR5 is ubiquitously expressed, including in the heart, where its function is poorly understood. Clinical studies have associated DR5 and its ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), with heart failure due to multiple etiologies. Previous studies in cardiac cells and mouse models have demonstrated that DR5 promotes eccentric cardiac hypertrophy through ERK1/2-dependent mechanisms and the apoptosis of myofibroblasts. ERK1/2 signaling has been associated with prosurvival mechanisms in cardiomyocytes suggesting DR5 agonism may be a novel therapeutic approach to outcomes in heart failure. We hypothesized that activation of DR5 will be protective in heart failure. Using a chronic isoproterenol administration model, mice were administered a DR5 agonist and progression of cardiac dysfunction was monitored by echocardiography. Cardiac remodeling was assessed by histology and prohypertrophic and profibrotic marker expression. Specificity of these responses was confirmed with DR5 knockout and the involvement of ERK1/2 signaling was confirmed using pharmacological inhibitors. DR5 agonists decreased cardiac remodeling and improved contractility in response to isoproterenol, which was prevented by ERK1/2 inhibition. These findings demonstrate that activation of DR5 reduces the progression of cardiac remodeling and dysfunction and may be a novel therapeutic target for heart failure treatment. SIGNIFICANCE STATEMENT: Death receptor 5 (DR5) is expressed in cardiomyocytes where its function is poorly defined and clinically, DR5 has been associated with heart failure development and severity. Previous studies show in healthy cardiomyocytes, DR5 activates ERK1/2 signaling, causing eccentric hypertrophy, which are associated with cardioprotection during heart failure. This study investigates the therapeutic potential of targeting DR5 and demonstrates that, in a chronic isoproterenol-infusion model of cardiac dysfunction, DR5 activation reduces maladaptive cardiac remodeling and preserves function through ERK1/2-dependent mechanisms.

The fibrotic response is involved in nearly all forms of heart failure and dysregulated responses can lead to enhanced cardiac dysfunction. TNF-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor (DR) 5, are associated with multiple forms of heart failure, but their role in the heart is poorly defined. Our previous study identified DR5 expression on cardiac fibroblasts however, the impact of DR5 on fibroblast function remains unexplored. To investigate the role of DR5 in cardiac fibroblasts, a variety of fibroblast functions were examined following treatment with the endogenous ligand, TRAIL, or small molecule agonist, bioymifi. DR5 activation did not induce apoptosis in naïve fibroblasts but activated ERK1/2 signaling to increase proliferation. However, upon activation and differentiation to myofibroblasts, DR5 expression was elevated, and DR5 agonists induced caspase 3 activation resulting in myofibroblast apoptosis. To investigate the impact of DR5 regulation of fibroblasts in vivo, a chronic isoproterenol administration model of heart failure was used. Wild-type (WT) mice receiving isoproterenol had increased hypertrophy, cardiomyocyte death, and fibrosis and decreased contractility compared to vehicle treated animals. DR5 knockout (KO) mice had no overt baseline phenotype however, following isoproterenol infusion, increased cardiomyocyte death and hypertrophy in comparison to isoproterenol treated WT animals was observed. DR5KO mice had an augmented fibrotic response with isoproterenol treatment compared with WT, which corresponded with additional decreases in contractility. These findings identify a dual role for DR5 in cardiac fibroblast function through enhanced naïve fibroblast proliferation, which switches to a pro-apoptotic function upon differentiation to myofibroblasts. This is important in heart failure where DR5 activation suppresses maladaptive remodeling and may represent a novel therapeutic target for the treatment of heart failure.

Immune cell β 2 -adrenergic receptors (β 2 ARs) are important for proinflammatory macrophage infiltration to the heart in a chronic isoproterenol administration model of heart failure. Mice lacking immune cell β 2 AR have decreased immune cell infiltration to their heart, primarily proinflammatory macrophage populations. This decrease culminated to decreased cardiac injury with lessened cardiomyocyte death, decreased interstitial fibrosis and hypertrophy, and improved function demonstrating that β 2 AR regulation of immune responses plays an important role in the heart’s response to persistent βAR stimulation.

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. β-adrenergic receptors (βAR) are also critical regulators of cardiac function in the normal and diseased state and a major therapeutic target clinically. βAR 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 βAR 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 β2AR 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 β2AR 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 β2AR 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 β2AR.

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.

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB‐SAP) selectively kills respiratory motor neurons, and mimics aspects of neuromuscular disorders and neurodegenerative diseases including phrenic motor neuron death, decreased phrenic motor output, and increased microglial cell number. Our previous results demonstrated unexpectedly that respiratory plasticity is not only observed 7 days (d) after CTB‐SAP delivery, but that it is actually enhanced compared to control treatments. At 28d, however, respiratory plasticity is comparable to control levels, suggesting that the underlying mechanisms of plasticity at both time points may differ. Because inflammation is a hallmark of all neurodegenerative diseases, and it is known to prevent the development of acute intermittent hypoxia‐induced phrenic long‐term facilitation (pLTF), we predicted that inflammation would impair pLTF in CTB‐SAP‐treated rats and thus, anti‐inflammatory drugs would enhance pLTF at both time points. Using anesthetized, paralyzed and ventilated CTB‐SAP‐treated adult Sprague Dawley rats, we studied acute intermittent hypoxia (AIH; 3, 5 min bouts of 10.5% O2) induced pLTF following intrathecal C4 delivery of the nonsteroidal anti‐inflammatory drug ketoprofen to rats that received bilateral, intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control) in rats treated 7d or 28d prior. Surprisingly, preliminary results indicate that ketoprofen attenuates pLTF in 7d CTB‐SAP‐treated rats, but enhances it in the 28d CTB‐SAP‐treated rats. These observations suggest that inflammation potentially contributes to pLTF in 7d CTB‐SAP‐treated rats, whereas it may undermine pLTF in 28d CTB‐SAP‐treated rats. Studies are underway to assess cervical spinal levels of both pro‐inflammatory and anti‐inflammatory and neurotrophic molecules at both time points, and their cellular source. Future studies will investigate the potential contributions of microglia to the mechanisms whereby inflammation differentially impacts pLTF in CTB‐SAP‐treated rats. Results from this project will increase our understanding of how inflammation and microglial activities influence respiratory plasticity following motor neuron death, and will have broad implications for therapies restoring ventilatory function in many disorders in which motor neuron death is associated.Support or Funding InformationNIH K99/R00 HL119606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background: Accumulation of multiple subtypes of immune cells in perivascular adipose tissue (PVAT) has been proposed to cause vascular inflammation and dysfunction in type 2 diabetes (T2DM). This study was designed to investigate specific roles for dendritic cells in PVAT in the development of vascular inflammation and impaired PVAT-mediated vasorelaxation in T2DM. Methods and Results: Studies were performed using db/db mice (model of T2DM) and their Db heterozygote (DbHET), lean and normoglycemic controls. Dendritic cell depletion was performed by cross-breeding DbHet with Flt3l(-/-) (null for ligand for FMS-kinase tyrosine kinase) mice. Using PCR, it was found that the majority of dendritic cells (CD11c(+)) were located in PVAT rather than the vascular wall. Flow cytometry similarly showed greater dendritic cell accumulation in adipose tissue from db/db mice than DbHET controls. Adipose tissue from db/db mice displayed increased mRNA levels of proinflammatory cytokines TNF-alpha and IL-6 and decreased mRNA levels of the anti-inflammatory mediator adiponectin, compared to DbHET mice. Depletion of dendritic cells in db(Flt3l-)/db(Flt3l-) (confirmed by flow cytometry) reduced TNF-alpha and IL-6 mRNA levels in diabetic adipose tissue without influencing adiponection expression. Moreover, in mesenteric arteries, dendritic cell depletion improved the ability of PVAT to augment acetylcholine-induced vasorelaxation and anti-contractile activity. Conclusions: In a murine model of T2DM, dendritic cells accumulated predominantly in PVAT, as opposed to the vessel wall, per se. Accumulation of dendritic cells in PVAT was associated with overproduction of pro-inflammatory cytokines, which contributed to an impaired ability of PVAT to augment vasorelaxation and exert anti-contractile activity in T2DM. (C) 2018 Elsevier Inc. All rights reserved.

Amyotrophic lateral sclerosis (ALS) is a devastating disease leading to progressive motor neuron degeneration and death by ventilatory failure. In a rat model of ALS (SOD1G93A), phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is enhanced greater than expected at disease end-stage but the mechanism is unknown. We suggest that one trigger for this enhancement is motor neuron death itself. Intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB-SAP) selectively kill respiratory motor neurons and mimic motor neuron death observed in SOD1G93A rats. This CTB-SAP model allows us to study the impact of respiratory motor neuron death on breathing without many complications attendant to ALS. Here, we tested the hypothesis that phrenic motor neuron death is sufficient to enhance pLTF. pLTF was assessed in anesthetized, paralyzed and ventilated Sprague Dawley rats 7 and 28 days following bilateral intrapleural injections of: 1) CTB-SAP (25 μg), or 2) un-conjugated CTB and SAP (control). CTB-SAP enhanced pLTF at 7 (CTB-SAP: 162 ± 18%, n = 8 vs. Control: 63 ± 3%; n = 8; p < 0.05), but not 28 days post-injection (CTB-SAP: 64 ± 10%, n = 10 vs. Control: 60 ± 13; n = 8; p > 0.05). Thus, pLTF at 7 (not 28) days post-CTB-SAP closely resembles pLTF in end-stage ALS rats, suggesting that processes unique to the early period of motor neuron death enhance pLTF. This project increases our understanding of respiratory plasticity and its implications for breathing in motor neuron disease.

OBJECTIVE:AdipoRon, an adiponectin receptor agonist, was recently proposed for treating insulin resistance and hyperglycemia. As adiponectin is vasoprotective via NO-mediated signaling, it was hypothesized that adipoRon similarly exerts potentially beneficial vasodilator effects. We therefore examined if adipoRon induces vasorelaxation and via what contributing mechanisms.METHODS:Vascular function was assessed in skeletal muscle arteries from rats and cerebral/coronary arteries from mice using pressure and wire myography.RESULTS:Using qPCR, mRNA for adiponectin receptors was demonstrated in skeletal muscle, cerebral and coronary arteries. AdipoRon-caused vasorelaxation was not abolished by compound C (10 μM; AMPK inhibitor). Inhibition of endothelium-dependent relaxation with combinations of l-NAME/indomethacin/apamin/TRAM-34 only slightly reduced adipoRon-mediated vasorelaxation in cerebral and coronary arteries. EC-denuded cremaster arteries showed similar relaxant responses to adipoRon as in intact vessels, suggesting adipoRon directly impacts VSMCs. K(+) currents measured in VSMCs isolated from mouse basilar and LAD arteries were not altered by adiopRon. In cremaster arteries, adipoRon induced vasorelaxation without a marked decrease in VSMC [Ca(2+)]i . Adiponectin, itself, caused vasodilation in intact cremaster arteries while failing to cause significant dilation in EC-denuded arteries, consistent with endothelium dependency of adiponectin.CONCLUSIONS:AdipoRon exerts vasodilation by mechanisms distinct to adiponectin. The dominant mechanism for adipoRon-induced vasorelaxation occurs independently of endothelium-dependent relaxing factors, AMPK activation, K(+) efflux-mediated hyperpolarization and reductions in cytosolic [Ca(2+)]i .

OBJECTIVES:Kv7 channels are considered important regulators of vascular smooth muscle contractility. The present study aimed to examine the hypotheses that (i) Kv7 channels are present in mouse cerebral and coronary arteries and regulate vascular reactivity and (ii) regional differences exist in the activity of these channels.METHODS AND RESULTS:PCR confirmed that basilar, Circle of Willis and LAD arteries express predominantly Kv7.1 and 7.4. Western blot analysis, however, showed greater Kv7.4 protein levels in the cerebral vessels. Relaxation to the Kv7 channel activator, retigabine (1-50 μM) was significantly greater in the basilar artery compared to the LAD artery. Similarly, the Kv7 channel inhibitor, linopirdine (10 μM) caused a stronger contraction of the basilar artery. Furthermore, pre-incubation with linopirdine reduced forskolin (cAMP activator)-induced vasorelaxation in basilar while not altering forskolin-induced vasorelaxation of the LAD, suggesting that Kv7 channels play a more prominent role in the cerebral than in the coronary circulation. Consistent with the vessel data, whole cell Kv7 currents in cerebral VSMCs were potentiated by retigabine and inhibited by linopirdine, while these responses were blunted in coronary VSMCs.CONCLUSIONS:This study provides evidence that mouse Kv7 channels may contribute differently to regulating the functional properties of cerebral and coronary arteries. Such heterogeneity has important implications for developing novel therapeutics for cardiovascular dysfunction.