Following injury, leukocytes are released from hematopoietic organs and migrate to the site of damage to regulate tissue inflammation and repair, however leukocytes lacking β2-adrenergic receptor ...(β2AR) expression have marked impairments in these processes. β-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 β-blocker treatment on baseline leukocyte parameters and their responsiveness to acute injury. In a temporal and βAR isoform-dependent manner, chronic β-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 β-blocker therapy compared to β-blocker-naïve patients. These results highlight the ability of chronic β-blocker treatment to alter baseline leukocyte characteristics that decrease their responsiveness to acute injury and suggest that prior β-blockade may act to reduce the severity of innate immune responses.
Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to Gq/11 ...proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca2+. The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP3 receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca2+ stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Deterioration or inborn malformations of the cardiac conduction system (CCS) interfere with proper impulse propagation in the heart and may lead to sudden cardiac death or heart failure. Patients ...afflicted with arrhythmia depend on antiarrhythmic medication or invasive therapy, such as pacemaker implantation. An ideal way to treat these patients would be CCS tissue restoration. This, however, requires precise knowledge regarding the molecular mechanisms underlying CCS development. Here, we aimed to identify regulators of CCS development. We performed a compound screen in zebrafish embryos and identified tolterodine, a muscarinic receptor antagonist, as a modifier of CCS development. Tolterodine provoked a lower heart rate, pericardiac edema, and arrhythmia. Blockade of muscarinic M3, but not M2, receptors induced transcriptional changes leading to amplification of sinoatrial cells and loss of atrioventricular identity. Transcriptome data from an engineered human heart muscle model provided additional evidence for the contribution of muscarinic M3 receptors during cardiac progenitor specification and differentiation. Taken together, we found that muscarinic M3 receptors control the CCS already before the heart becomes innervated. Our data indicate that muscarinic receptors maintain a delicate balance between the developing sinoatrial node and the atrioventricular canal, which is probably required to prevent the development of arrhythmia.
Heat shock proteins represent an emerging model for the coordinated, multistep regulation of apoptotic signaling events. Although certain aspects of the biochemistry associated with heat shock ...protein cytoprotective effects are known, little information is found describing the regulation of heat shock protein responses to harmful stimuli. During screening for noncanonical beta adrenergic receptor signaling pathways in human urothelial cells, using mass spectroscopy techniques, an agonist-dependent interaction with beta-arrestin and the 27-kDa heat shock protein was observed in vitro. Formation of this beta-arrestin/Hsp27 complex in response to the selective beta adrenergic receptor agonist isoproterenol, was subsequently confirmed in situ by immunofluorescent colocalization studies. Radioligand binding techniques characterized a homogeneous population of the beta2 adrenergic receptor subtype expressed on these cells. Using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunoblot analysis and quantitation of caspase-3 activity to detect apoptosis, preincubation of these cells with isoproterenol was found to be sufficient for protection against programmed cell death initiated by staurosporine. RNA interference strategies confirmed the necessity for Hsp27 as well as both beta-arrestin isoforms to confer this cytoprotective consequence of beta adrenergic receptor activation in this cell model. As a result, these studies represent the first description of an agonist-dependent relationship between a small heat shock protein and beta-arrestin to form a previously unknown antiapoptotic "signalosome."
Label‐free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. β‐adenergic receptors (βAR) ...are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label‐free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous βAR in primary neonatal rat cardiac fibroblasts (NRCF). The EPIC‐BT by Corning was used to measure DMR responses in primary isolated NRCF treated with various βAR and EGFR ligands. Additional molecular assays for cAMP generation and receptor internalization responses were used to correlate the DMR findings with established βAR signaling pathways. Catecholamine stimulation of NRCF induced a concentration‐dependent negative DMR deflection that was competitively blocked by βAR blockade and non‐competitively blocked by irreversible uncoupling of Gs proteins. Subtype‐selective βAR ligand profiling revealed a dominant role for β2AR in mediating the DMR responses, consistent with the relative expression levels of β2AR and β1AR in NRCF. βAR‐mediated cAMP generation profiles revealed similar kinetics to DMR responses, each of which were enhanced via inhibition of cAMP degradation, as well as dynamin‐mediated receptor internalization. Finally, G protein‐independent βAR signaling through epidermal growth factor receptor (EGFR) was assessed, revealing a smaller but significant contribution of this pathway to the DMR response to βAR stimulation. Measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous βAR signaling via both G protein‐dependent and –independent pathways.
e00024
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Following myocardial infarction (MI), immune cell infiltration initiates and regulates cardiac remodeling and repair. Our previous work has shown the importance of β2-adrenergic receptors (β2AR) in ...modulating immune cell infiltration to the heart following injury through the regulation of chemokine receptor expression and splenic retention of bone marrow (BM)-derived leukocyte populations. Pharmacological inhibition of βAR is a common strategy for the treatment of many cardiovascular etiologies, however the impact of β-blocker therapy on immune cell responses following acute cardiac injury is unknown. We hypothesized that prior treatment with β-blockers with distinct isoform selectivity would differentially decrease leukocyte infiltration to the heart following MI. Mice were administered either vehicle, a β1AR-selective (Metoprolol; Met), a β2AR-selective (ICI 118,551; ICI) or a βAR non-selective (Carvedilol; Carv) antagonist for two weeks via minipump. Analysis of mice treated with ICI or Carv showed decreased BM expression of CCR2 and CXCR4, as well as elevated levels of leukocyte populations within the spleen. While these effects were not observed in mice infused with a β1AR-selective dose of Met, a higher concentration of Met (equivalent to moderate doses used in humans) also decreased CCR2 and CXCR4 expression in BM and increased splenic leukocyte retention. To determine the impact of β2AR-selective versus non-selective blockade on immune cell responses following acute cardiac injury, mice from each treatment group underwent sham or MI surgery. In addition to decreased CCR2 and CXCR4 expression in the BM, mice administered ICI, Carv and the non-selective dose of Met had impaired recruitment of leukocyte populations to the heart following MI. Toward clinically relevant ends, we assessed CCR2 and CXCR4 expression in leukocytes isolated from blood samples of healthy and heart failure patients and found that levels of each were decreased in patients having taken β-blockers. In all, these results demonstrate that prior β-blocker treatment negatively regulates immune cell responsiveness to acute cardiac injury.
RATIONALE
Coronary artery disease and subsequent myocardial ischemia (MI) are the most common cause of heart failure (HF) in the US. G protein‐coupled receptor (GPCR) kinase 5 (GRK5) has been shown ...to be upregulated in failing human myocardium. While the canonical role of GRKs is to desensitize receptors via phosphorylation, GRK5 can also locate to the nucleus of cardiomyocytes where it exerts GPCR‐independent effects that promote maladaptive cardiac hypertrophy after hypertrophic stress. Despite numerous data indicating the importance of GRK5 in hypertrophy, it is still unknown if GRK5 has a role in ischemic heart disease.
OBJECTIVE
In this study, we investigated the critical role that GRK5 plays after myocardial ischemic injury with a novel aspect being the regulation of immune and inflammatory responses including recruitment of immune cells to the injured heart.
METHODS AND RESULTS
Cardiac‐specific GRK5 transgenic mice (Tg‐GRK5) and non‐transgenic littermate (NLC) control mice were subjected to MI. Tg‐GRK5‐HF mice showed decreased cardiac function (both global and segmental contractility), augmented left ventricular diameters/volumes and decreased survival rate compared to NLC‐HF mice. Heart weight (HW) to body weight or to tibia length ratios as well as mRNA expression of all major adverse remodeling‐associated biomarkers (ANF, BNP, b‐MHC) were increased in the TgGRK5‐HF compared to NLC‐HF. Cardiac fibrosis in the border zone (BZ) area as well as mRNA levels of Collagen‐1 (Col‐1), Col‐3, MMP2 and CTGF were higher in TgGRK5‐HF compared to NLC‐HF, strongly suggesting increased adverse remodeling. Cardiac inflammatory cytokines (IL‐6 and IL‐1beta), adhesion molecule (VCAM‐1) and chemokines (CCL2, CCL3, CCL5) were augmented in TgGRK5‐HF compared to NLC‐HF and contribute at least in part to increased immune cell recruitment in the heart. In fact, we found neutrophils/macrophages and T‐lymphocytes (T‐cells) respectively augmented at 4 days and 8 weeks after MI in both BZ and infarct zone. Interestingly, cardiac‐specific GRK2 transgenic mice did not show increased cardiac inflammation and chemokine production after MI when compared to NLC‐HF mice.
CONCLUSIONS
Our study shows that cardiac GRK5 has a detrimental effect during ischemic HF. GRK5 overexpression causes reduced cardiac function and increased immune cell recruitment/inflammation. Further, these results suggest GRK5 as a potential therapeutic target to limit HF development after ischemic injury.
Support or Funding Information
This work was supported by National Institutes of Health (NIH P01 HL091799, NIH R37 HL061690, NIH P01 HL075443 Project 2) to W.J.K. and American Heart Association (postdoctoral fellowship 17POST33660942) to C.d.L..
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
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Abstract only β-Adrenergic receptors (βAR) are important regulators of cardiac function in the normal and failing heart. Activation of the β1AR subtype increases contractility and cardiomyocyte death ...whereas the β2AR can promote survival. In response to cardiac injury, increased catecholamines activate and downregulate β1AR thus, β-adrenergic receptor antagonists or β-blockers are commonly prescribed in to restore β1AR expression and preserve contractility. However, promoting the pro-survival effects of β2AR, which are known to occur in part through β-arrestin (ARR)-dependent signaling, may be a beneficial therapeutic strategy. Pepducins have been developed based on the intracellular loop (ICL) domains of β2AR to activate either Gαs- or βARR-dependent β2AR signaling pathways. We hypothesized that pepducin-mediated engagement of βARR-dependent β2AR signaling in the heart would be therapeutically advantageous following ischemia/reperfusion (I/R) injury. To test this, wild-type (WT) C57BL/6 mice received three intracardiac injections of either a βARR-biased pepducin (ICL1-9) or a scrambled control pepducin at the time of ischemia (30 min) followed by reperfusion. Assessment of cardiomyocyte death 24h post-I/R using TUNEL staining showed a decrease in cell death in animals treated with ICL1-9 when compared to scrambled pepducin, correlating with decreased infarct size and improved cardiac function as measured by echocardiography. Assessment of cardiac function at later time points showed that the cardioprotective effects of ICL1-9 were preserved over time and resulted in decreased cardiac remodeling. Although βARR1KO mice displayed similar cell death, infarct size, and contractility changes following I/R as their WT counterparts, they did not manifest a cardioprotective response from ICL1-9 treatment, indicating that βARR signaling is essential in relaying ICL1-9-dependent cardioprotection. These results demonstrate that pharmacologically-mediated activation of βARR-biased β2AR signaling provides a therapeutic benefit in the context of I/R-induced injury and may provide an improved strategy for the treatment of acute cardiac injury.
Abstract only Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. We recently demonstrated that the pepducin ...ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades and enhanced cardiomyocyte contractile function in a βarr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Wild-type (WT) C57BL/6 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 hours, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a β2AR- and βarr-dependent manner, which led to less cardiac fibrosis and improved cardiac function over time. Neonatal rat ventricular myocytes (NRVM) were used in conjunction with serum deprivation or hypoxia/reoxygenation (H/R) models to assess the mechanism by which ICL1-9 promotes cardiomyocyte survival. Notably, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA-dependent manner. ICL1-9 did not alter β2AR density in NRVM or whole heart even up to 24 hr post-treatment, at which timepoint both ICL1-9 localization in cardiomyocytes and RhoA activation were detected, indicating long-lasting presence and effects of ICL1-9 on βarr-dependent β2AR signaling. Thus, βarr-biased β2AR-selective allosteric modulation represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term structural and functional benefits.
Abstract only
β‐Adrenergic receptors (βAR) are important regulators of cardiac function in the normal and failing heart. Activation of the β1AR subtype increases contractility and cardiomyocyte death ...whereas the β2AR can promote survival. In response to cardiac injury, increased catecholamines activate and downregulate β1AR thus, β‐adrenergic receptor antagonists or β‐blockers are commonly prescribed in to restore β1AR expression and preserve contractility. However, promoting the pro‐survival effects of β2AR, which are known to occur in part through β‐arrestin (ARR)‐dependent signaling, may be a beneficial therapeutic strategy. Pepducins have been developed based on the intracellular loop (ICL) domains of β2AR to activate either Gαs‐ or βARR‐dependent β2AR signaling pathways. We hypothesized that pepducin‐mediated engagement of βARR‐dependent β2AR signaling in the heart would be therapeutically advantageous following ischemia/reperfusion (I/R) injury. To test this, wild‐type (WT) C57BL/6 mice received three intracardiac injections of either a βARR‐biased pepducin (ICL1‐9) or a scrambled control pepducin at the time of ischemia (30 min) followed by reperfusion. Assessment of cardiomyocyte death 24h post‐I/R using TUNEL staining showed a decrease in cell death in animals treated with ICL1‐9 when compared to scrambled pepducin, correlating with decreased infarct size and improved cardiac function as measured by echocardiography. Assessment of cardiac function at later time points showed that the cardioprotective effects of ICL1‐9 were preserved over time and resulted in decreased cardiac remodeling. Although βARR1KO mice displayed similar cell death, infarct size, and contractility changes following I/R as their WT counterparts, they did not manifest a cardioprotective response from ICL1‐9 treatment, indicating that βARR signaling is essential in relaying ICL1‐9‐dependent cardioprotection. These results demonstrate that pharmacologically‐mediated activation of βARR‐biased β2AR signaling provides a therapeutic benefit in the context of I/R‐induced injury and may provide an improved strategy for the treatment of acute cardiac injury.
Support or Funding Information
NIH‐HL‐105414‐DGT
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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