Loss of Myeloid Cell-Specific ß2-Adrenergic Receptor Expression Ameliorates Cardiac Function and Remodeling after Acute Ischemia.

Myeloid cells, including neutrophils, monocytes and macrophages, accumulate quickly after ischemic injury in the heart where they play integral roles in the regulation of inflammation and repair. We previously reported that deletion of ß2-adrenergic receptor (ß2AR) in all cells of hematopoietic origin resulted in generalized disruption of immune cell responsiveness to injury, but with unknown impact on myeloid cells specifically. To investigate this, we crossed floxed ß2AR (F/F) mice with myeloid cell-expressing Cre (LysM-Cre) mice to generate myeloid cell-specific ß2AR knockout mice (LB2) and subjected them to myocardial infarction (MI). Via echocardiography and immunohistochemical analyses, LB2 mice displayed better cardiac function and less fibrotic remodeling after MI than the control lines. Despite similar accumulation of myeloid cell subsets in the heart at 1-day post-MI, LB2 mice displayed reduced numbers of Nu by 4 days post-MI, suggesting LB2 hearts have enhanced capacity for Nu efferocytosis. Indeed, bone marrow-derived macrophage (BMDM)-mediated efferocytosis of Nu was enhanced in LB2-versus F/F-derived cells in vitro. Mechanistically, several pro-efferocytosis-related genes were increased in LB2 myeloid cells, with annexin A1 ( Anxa1 ) in particular elevated in several myeloid cell types following MI. Accordingly, shRNA-mediated knockdown of Anxa1 in LB2 bone marrow prior to transplantation into irradiated LB2 mice reduced Mac-induced Nu efferocytosis in vitro and prevented the ameliorative effects of myeloid cell-specific ß2AR deletion on cardiac function and fibrosis following MI in vivo. Altogether, our data reveal a previously unrecognized role for ß2AR in the regulation of myeloid cell-dependent efferocytosis in the heart following injury.

Myeloid cell-specific deletion of epidermal growth factor receptor aggravates acute cardiac injury.

Myeloid cells, including macrophages, play important roles as first responders to cardiac injury and stress. Epidermal growth factor receptor (EGFR) has been identified as a mediator of macrophage responsiveness to select diseases, though its impact on cardiac function or remodeling following acute ischemic injury is unknown. We aimed to define the role of myeloid cell-specific EGFR in the regulation of cardiac function and remodeling following acute myocardial infarction (MI)-induced injury. Floxed EGFR mice were bred with homozygous LysM-Cre (LMC) transgenic mice to yield myeloid-specific EGFR knockout (mKO) mice. Via echocardiography, immunohistochemistry, RNA sequencing and flow cytometry, the impact of myeloid cell-specific EGFR deletion on cardiac structure and function was assessed at baseline and following injury. Compared with LMC controls, myeloid cell-specific EGFR deletion led to an increase in cardiomyocyte hypertrophy at baseline. Bulk RNASeq analysis of isolated cardiac Cd11b+ myeloid cells revealed substantial changes in mKO cell transcripts at baseline, particularly in relation to predicted decreases in neovascularization. In response to myocardial infarction, mKO mice experienced a hastened decline in cardiac function with isolated cardiac Cd11b+ myeloid cells expressing decreased levels of the pro-reparative mediators Vegfa and Il10, which coincided with enhanced cardiac hypertrophy and decreased capillary density. Overall, loss of EGFR qualitatively alters cardiac resident macrophages that promotes a low level of basal stress and a more rapid decrease in cardiac function along with worsened repair following acute ischemic injury.

Effects of maternal hypothyroidism on postnatal cardiomyocyte proliferation and cardiac disease responses of the progeny.

Maternal hypothyroidism (MH) could adversely affect the cardiac disease responses of the progeny. This study tested the hypothesis that MH reduces early postnatal cardiomyocyte (CM) proliferation so that the adult heart of MH progeny has a smaller number of larger cardiac myocytes, which imparts adverse cardiac disease responses following injury. Thyroidectomy (TX) was used to establish MH. The progeny from mice that underwent sham or TX surgery were termed Ctrl (control) or MH (maternal hypothyroidism) progeny, respectively. MH progeny had similar heart weight (HW) to body weight (BW) ratios and larger CM size consistent with fewer CMs at postnatal day 60 (P60) compared with Ctrl (control) progeny. MH progeny had lower numbers of EdU+, Ki67+, and phosphorylated histone H3 (PH3)+ CMs, which suggests they had a decreased CM proliferation in the postnatal timeframe. RNA-seq data showed that genes related to DNA replication were downregulated in P5 MH hearts, including bone morphogenetic protein 10 (Bmp10). Both in vivo and in vitro studies showed Bmp10 treatment increased CM proliferation. After transverse aortic constriction (TAC), the MH progeny had more severe cardiac pathological remodeling compared with the Ctrl progeny. Thyroid hormone (T4) treatment for MH mothers preserved their progeny's postnatal CM proliferation capacity and prevented excessive pathological remodeling after TAC. Our results suggest that CM proliferation during early postnatal development was significantly reduced in MH progeny, resulting in fewer CMs with hypertrophy in adulthood. These changes were associated with more severe cardiac disease responses after pressure overload.NEW & NOTEWORTHY Our study shows that compared with Ctrl (control) progeny, the adult progeny of mothers who have MH (MH progeny) had fewer CMs. This reduction of CM numbers was associated with decreased postnatal CM proliferation. Gene expression studies showed a reduced expression of Bmp10 in MH progeny. Bmp10 has been linked to myocyte proliferation. In vivo and in vitro studies showed that Bmp10 treatment of MH progeny and their myocytes could increase CM proliferation. Differences in CM number and size in adult hearts of MH progeny were linked to more severe cardiac structural and functional remodeling after pressure overload. T4 (synthetic thyroxine) treatment of MH mothers during their pregnancy, prevented the reduction in CM number in their progeny and the adverse response to disease stress.

Pepducin ICL1-9-Mediated ß2-Adrenergic Receptor-Dependent Cardiomyocyte Contractility Occurs in a Gi Protein/ROCK/PKD-Sensitive Manner.

PURPOSE: ß-Adrenergic receptors (ßAR) are essential targets for the treatment of heart failure (HF); however, chronic use of ßAR agonists as positive inotropes to increase contractility in a Gs protein-dependent manner is associated with increased mortality. Alternatively, we previously reported that allosteric modulation of ß2AR with the pepducin intracellular loop (ICL)1-9 increased cardiomyocyte contractility in a ß-arrestin (ßarr)-dependent manner, and subsequently showed that ICL1-9 activates the Ras homolog family member A (RhoA). Here, we aimed to elucidate both the proximal and downstream signaling mediators involved in the promotion of cardiomyocyte contractility in response to ICL1-9. METHODS: We measured adult mouse cardiomyocyte contractility in response to ICL1-9 or isoproterenol (ISO, as a positive control) alone or in the presence of inhibitors of various potential components of ßarr- or RhoA-dependent signaling. We also assessed the contractile effects of ICL1-9 on cardiomyocytes lacking G protein-coupled receptor (GPCR) kinase 2 (GRK2) or 5 (GRK5). RESULTS: Consistent with RhoA activation by ICL1-9, both Rho-associated protein kinase (ROCK) and protein kinase D (PKD) inhibition were able to attenuate ICL1-9-mediated contractility, as was inhibition of myosin light chain kinase (MLCK). While neither GRK2 nor GRK5 deletion impacted ICL1-9-mediated contractility, pertussis toxin attenuated the response, suggesting that ICL1-9 promotes downstream RhoA-dependent signaling in a Gi protein-dependent manner. CONCLUSION: Altogether, our study highlights a novel signaling modality that may offer a new approach to the promotion, or preservation, of cardiac contractility during HF via the allosteric regulation of ß2AR to promote Gi protein/ßarr-dependent activation of RhoA/ROCK/PKD signaling.

The NPXXY Motif Regulates ß-Arrestin Recruitment by the CB1 Cannabinoid Receptor.

Background: Activation of signaling effectors by G-protein coupled receptors (GPCRs) depends on different molecular mechanisms triggered by conserved amino acid residues. Although studies have focused on the G-protein signaling state, the mechanism for ß-arrestin signaling by CB1 is not yet well defined. Studies have indicated that transmembrane helix 7 (TMH7) and the highly conserved NPXXY motif can be subject to different conformational changes in response to biased ligands and could therefore participate in a molecular mechanism to trigger ß-arrestin recruitment. Objective: To investigate the effect of mutations in the NPXXY motif on different signaling pathways activated by the CB1 receptor. Materials and Methods: Point mutations of the NPXXY motif and associated residues were generated in the CB1 receptor using site-directed mutagenesis and transfection into HEK-293 cells. Signaling by wild-type and mutant receptors was analyzed by quantifying inhibition of cAMP, and by ß-arrestin recruitment assays. Results: We found that N7.49 and Y7.53 are essential for ß-arrestin recruitment by CB1. N7.49A and Y7.53F impair ß-arrestin signaling, with no effect on G-protein signaling. We found a regulatory role for residue I2.43; I2.43 interacts with Y7.53, affecting its positioning. Reducing steric bulk at I2.43 (I2.43A) enhances ß-arrestin1 recruitment, while introducing a polar residue (I2.43T) reduces ß-arrestin recruitment. Conclusions: These findings point to a novel mechanism for ß-arrestin recruitment, implicating amino acids in the NPXXY motif as critical for the putative ß-arrestin biased conformational state of Class A GPCRs.

Pathophysiological impact of the adhesion G protein-coupled receptor family.

G protein-coupled receptors (GPCRs) represent one of the most targeted drug classes in the human genome, accounting for greater than 40% of all Food and Drug Administration-approved drugs. However, the second-largest family of GPCRs, known as adhesion GPCRs (aGPCR), have yet to serve as a clinical target despite increasing evidence of their physiological and pathological functions, which suggests an opportunity toward the development of novel therapeutics. To date, the pathophysiological function of aGPCRs is associated with a plethora of diseases including cancer, central nervous system disorders, immunity and inflammation, and others. To highlight their potential as pharmacological targets, we will review three distinct aGPCR members (ADGRG1, ADGRE5, and ADGRF5), highlighting their molecular mechanisms of action and contributions to the development of pathophysiology.

Pepducin-mediated G Protein-Coupled Receptor Signaling in the Cardiovascular System.

ABSTRACT: Pepducins are small-lipidated peptides designed from the intracellular loops of G protein-coupled receptors (GPCRs) that act in an allosteric manner to modulate the activity of GPCRs. Over the past 2 decades, pepducins have progressed initially from pharmacologic tools used to manipulate GPCR activity in an orthosteric site-independent manner to compounds with therapeutic potential that have even been used safely in phase 1 and 2 clinical trials in human subjects. The effect of pepducins at their cognate receptors has been shown to vary between antagonist, partial agonist, and biased agonist outcomes in various primary and clonal cell systems, with even small changes in amino acid sequence altering these properties and their receptor selectivity. To date, pepducins designed from numerous GPCRs have been studied for their impact on pathologic conditions, including cardiovascular diseases such as thrombosis, myocardial infarction, and atherosclerosis. This review will focus in particular on pepducins designed from protease-activated receptors, C-X-C motif chemokine receptors, formyl peptide receptors, and the ß2-adrenergic receptor. We will discuss the historic context of pepducin development for each receptor, as well as the structural, signaling, pathophysiologic consequences, and therapeutic potential for each pepducin class.

G protein-coupled receptor kinase 5 (GRK5) contributes to impaired cardiac function and immune cell recruitment in post-ischemic heart failure.

AIMS: Myocardial infarction (MI) is the most common cause of heart failure (HF) worldwide. G protein-coupled receptor kinase 5 (GRK5) is upregulated in failing human myocardium and promotes maladaptive cardiac hypertrophy in animal models. However, the role of GRK5 in ischemic heart disease is still unknown. In this study, we evaluated whether myocardial GRK5 plays a critical role post-MI in mice and included the examination of specific cardiac immune and inflammatory responses. METHODS AND RESULTS: Cardiomyocyte-specific GRK5 overexpressing transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice as well as cardiomyocyte-specific GRK5 knockout mice (GRK5cKO) and wild type (WT) were subjected to MI and, functional as well as structural changes together with outcomes were studied. TgGRK5 post-MI mice showed decreased cardiac function, augmented left ventricular dimension and decreased survival rate compared to NLC post-MI mice. Cardiac hypertrophy and fibrosis as well as fetal gene expression were increased post-MI in TgGRK5 compared to NLC mice. In TgGRK5 mice, GRK5 elevation produced immuno-regulators that contributed to the elevated and long-lasting leukocyte recruitment into the injured heart and ultimately to chronic cardiac inflammation. We found an increased presence of pro-inflammatory neutrophils and macrophages as well as neutrophils, macrophages and T-lymphocytes at 4-days and 8-weeks respectively post-MI in TgGRK5 hearts. Conversely, GRK5cKO mice were protected from ischemic injury and showed reduced early immune cell recruitment (predominantly monocytes) to the heart, improved contractility and reduced mortality compared to WT post-MI mice. Interestingly, cardiomyocyte-specific GRK2 transgenic mice did not share the same phenotype of TgGRK5 mice and did not have increased cardiac leukocyte migration and cytokine or chemokine production post-MI. CONCLUSIONS: Our study shows that myocyte GRK5 has a crucial and GRK-selective role on the regulation of leucocyte infiltration into the heart, cardiac function and survival in a murine model of post-ischemic HF, supporting GRK5 inhibition as a therapeutic target for HF.

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.

Epidermal growth factor receptor association with ß1-adrenergic receptor is mediated via its juxtamembrane domain.

ß1-adrenergic receptor (ß1AR)-mediated transactivation of epidermal growth factor receptor (EGFR) engages downstream signaling events that impact numerous cellular processes including growth and survival. While association of these receptors has been shown to occur basally and be important for relaying transactivation-specific intracellular events, the mechanism by which they do so is unclear and elucidation of which would aid in understanding the consequence of disrupting their interaction. Using fluorescence resonance energy transfer (FRET) and immunoprecipitation (IP) analyses, we evaluated the impact of C-terminal truncations of EGFR on its ability to associate with ß1AR. While loss of the last 230 amino acid C-terminal phosphotyrosine-rich domain did not disrupt the ability of EGFR to associate with ß1AR, truncation of the entire intracellular domain of EGFR resulted in almost complete loss of its interaction with ß1AR, suggesting that either the kinase domain or juxtamembrane domain (JMD) may be required for this association. Treatment with the EGFR antagonist gefitinib did not prevent ß1AR-EGFR association, however, treatment with a palmitoylated peptide encoding the first 20 amino acids of the JMD domain (JMD-A) disrupted ß1AR-EGFR association over time and prevented ß1AR-mediated ERK1/2 phosphorylation, both in general and specifically in association with EGFR. Conversely, neither a mutated JMD-A peptide nor a palmitoylated peptide fragment consisting of the subsequent 18 amino acids of the JMD domain (JMD-B) were capable of doing so. Altogether, the proximal region of the JMD of EGFR is responsible for its association with ß1AR, and its disruption prevents ß1AR-mediated transactivation, thus providing a new tool to study the functional consequences of disrupting ß1AR-EGFR downstream signaling.

Recent Advances in GPCR-Regulated Leukocyte Responses during Acute Cardiac Injury.

Following acute cardiac injury such as myocardial infarction (MI), the controlled activation and recruitment of various leukocytes to the site of tissue damage significantly impacts chronic changes to cardiac structure and function, and ultimately host survival. While recent research has focused primarily on how leukocytes respond to injury, understanding how to effectively modulate their responsiveness to dampen maladaptive inflammation and promote repair processes is not yet fully understood. The complex spatio-temporal migration and activation of leukocytes are largely controlled by various chemokines and their cognate receptors, belonging to the G protein-coupled receptor (GPCR) family. Beyond chemokine receptors, leukocytes express a host of additional GPCRs that have recently been shown to regulate their responsiveness to cardiac injury. In this minireview, we will briefly discuss the impact of chemokine receptors on leukocyte behaviour, with subsequent focus on the most recent advancements in understanding the impact and therapeutic potential of other GPCR classes on leukocyte responses after acute cardiac injury.

Loss of Protease-Activated Receptor 4 Prevents Inflammation Resolution and Predisposes the Heart to Cardiac Rupture After Myocardial Infarction.

BACKGROUND: Cardiac rupture is a major lethal complication of acute myocardial infarction (MI). Despite significant advances in reperfusion strategies, mortality from cardiac rupture remains high. Studies suggest that cardiac rupture can be accelerated by thrombolytic therapy, but the relevance of this risk factor remains controversial. METHODS: We analyzed protease-activated receptor 4 (Par4) expression in mouse hearts with MI and investigated the effects of Par4 deletion on cardiac remodeling and function after MI by echocardiography, quantitative immunohistochemistry, and flow cytometry. RESULTS: Par4 mRNA and protein levels were increased in mouse hearts after MI and in isolated cardiomyocytes in response to hypertrophic and inflammatory stimuli. Par4-deficient mice showed less myocyte apoptosis, reduced infarct size, and improved functional recovery after acute MI relative to wild-type (WT). Conversely, Par4-/- mice showed impaired cardiac function, greater rates of myocardial rupture, and increased mortality after chronic MI relative to WT. Pathological evaluation of hearts from Par4-/- mice demonstrated a greater infarct expansion, increased cardiac hemorrhage, and delayed neutrophil accumulation, which resulted in impaired post-MI healing compared with WT. Par4 deficiency also attenuated neutrophil apoptosis in vitro and after MI in vivo and impaired inflammation resolution in infarcted myocardium. Transfer of Par4-/- neutrophils, but not of Par4-/- platelets, in WT recipient mice delayed inflammation resolution, increased cardiac hemorrhage, and enhanced cardiac dysfunction. In parallel, adoptive transfer of WT neutrophils into Par4-/- mice restored inflammation resolution, reduced cardiac rupture incidence, and improved cardiac function after MI. CONCLUSIONS: These findings reveal essential roles of Par4 in neutrophil apoptosis and inflammation resolution during myocardial healing and point to Par4 inhibition as a potential therapy that should be limited to the acute phases of ischemic insult and avoided for long-term treatment after MI.

Leukocyte-Dependent Regulation of Cardiac Fibrosis.

Cardiac fibrosis begins as an intrinsic response to injury or ageing that functions to preserve the tissue from further damage. Fibrosis results from activated cardiac myofibroblasts, which secrete extracellular matrix (ECM) proteins in an effort to replace damaged tissue; however, excessive ECM deposition leads to pathological fibrotic remodeling. At this extent, fibrosis gravely disturbs myocardial compliance, and ultimately leads to adverse outcomes like heart failure with heightened mortality. As such, understanding the complexity behind fibrotic remodeling has been a focal point of cardiac research in recent years. Resident cardiac fibroblasts and activated myofibroblasts have been proven integral to the fibrotic response; however, several findings point to additional cell types that may contribute to the development of pathological fibrosis. For one, leukocytes expand in number after injury and exhibit high plasticity, thus their distinct role(s) in cardiac fibrosis is an ongoing and controversial field of study. This review summarizes current findings, focusing on both direct and indirect leukocyte-mediated mechanisms of fibrosis, which may provide novel targeted strategies against fibrotic remodeling.

Loss of dynamic regulation of G protein-coupled receptor kinase 2 by nitric oxide leads to cardiovascular dysfunction with aging.

Nitric oxide (NO) and S-nitrosothiol (SNO) are considered cardio- and vasoprotective substances. We now understand that one mechanism in which NO/SNOs provide cardiovascular protection is through their direct inhibition of cardiac G protein-coupled receptor (GPCR) kinase 2 (GRK2) activity via S-nitrosylation of GRK2 at cysteine 340 (C340). This maintains GPCR homeostasis, including ß-adrenergic receptors, through curbing receptor GRK2-mediated desensitization. Previously, we have developed a knockin mouse (GRK2-C340S) where endogenous GRK2 is resistant to dynamic S-nitrosylation, which led to increased GRK2 desensitizing activity. This unchecked regulation of cardiac GRK2 activity resulted in significantly more myocardial damage after ischemic injury that was resistant to NO-mediated cardioprotection. Although young adult GRK2-C340S mice show no overt phenotype, we now report that as these mice age, they develop significant cardiovascular dysfunction due to the loss of SNO-mediated GRK2 regulation. This pathological phenotype is apparent as early as 12 mo of age and includes reduced cardiac function, increased cardiac perivascular fibrosis, and maladaptive cardiac hypertrophy, which are common maladies found in patients with cardiovascular disease (CVD). There are also vascular reactivity and aortic abnormalities present in these mice. Therefore, our data demonstrate that a chronic and global increase in GRK2 activity is sufficient to cause cardiovascular remodeling and dysfunction, likely due to GRK2's desensitizing effects in several tissues. Because GRK2 levels have been reported to be elevated in elderly CVD patients, GRK2-C340 mice can give insight into the aged-molecular landscape leading to CVD.NEW & NOTEWORTHY Research on G protein-coupled receptor kinase 2 (GRK2) in the setting of cardiovascular aging is largely unknown despite its strong established functions in cardiovascular physiology and pathophysiology. This study uses a mouse model of chronic GRK2 overactivity to further investigate the consequences of long-term GRK2 on cardiac function and structure. We report for the first time that chronic GRK2 overactivity was able to cause cardiac dysfunction and remodeling independent of surgical intervention, highlighting the importance of GRK activity in aged-related heart disease.

Cardiac Expression of Factor X Mediates Cardiac Hypertrophy and Fibrosis in Pressure Overload.

Activated factor X is a key component of the coagulation cascade, but whether it directly regulates pathological cardiac remodeling is unclear. In mice subjected to pressure overload stress, cardiac factor X mRNA expression and activity increased concurrently with cardiac hypertrophy, fibrosis, inflammation and diastolic dysfunction, and responses blocked with a low coagulation-independent dose of rivaroxaban. In vitro, neurohormone stressors increased activated factor X expression in both cardiac myocytes and fibroblasts, resulting in activated factor X-mediated activation of protease-activated receptors and pro-hypertrophic and -fibrotic responses, respectively. Thus, inhibition of cardiac-expressed activated factor X could provide an effective therapy for the prevention of adverse cardiac remodeling in hypertensive patients.

ADP exerts P2Y12 -dependent and P2Y12 -independent effects on primary human T cell responses to stimulation.

Purinergic signaling plays a complex role in inflammation. Nucleotides released by T lymphocytes, endothelial cells, and platelets during inflammation induce cellular responses by binding to receptors that regulate intracellular signaling pathways. Previous studies have found that purinergic signaling can have both proinflammatory and anti-inflammatory effects, but the roles of specific pathways in specific cell types are poorly understood. We investigated the role of the P2Y12 signaling pathway in the activation of T lymphocytes in vitro. We isolated peripheral blood mononuclear cells (PBMCs) from healthy donors and pretreated them with ADP (a P2Y12 agonist), AR-C69931MX (a P2Y12 antagonist), or both. We then stimulated PBMC using phytohemagglutinin (PHA) or anti-CD3/CD28 antibodies. We found that ADP affects T cell responses in term of cell activity and receptor expression through both P2Y12-dependent and P2Y12-independent pathways and other responses (cytokine secretion) primarily through P2Y12 -independent pathways. The ADP-mediated effect changed over time and was stimulus-specific.

Nicotinamide riboside kinase-2 alleviates ischemia-induced heart failure through P38 signaling.

Nicotinamide riboside kinase-2 (NRK-2), a muscle-specific ß1 integrin binding protein, predominantly expresses in skeletal muscle with a trace amount expressed in healthy cardiac tissue. NRK-2 expression dramatically increases in mouse and human ischemic heart however, the specific role of NRK-2 in the pathophysiology of ischemic cardiac diseases is unknown. We employed NRK2 knockout (KO) mice to identify the role of NRK-2 in ischemia-induced cardiac remodeling and dysfunction. Following myocardial infarction (MI), or sham surgeries, serial echocardiography was performed in the KO and littermate control mice. Cardiac contractile function rapidly declined and left ventricular interior dimension (LVID) was significantly increased in the ischemic KO vs. control mice at 2 weeks post-MI. An increase in mortality was observed in the KO vs. control group. The KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric analysis. Consistently, histological assessment revealed an extensive and thin scar and dilated LV chamber accompanied with elevated fibrosis in the KOs post-MI. Mechanistically, we observed that loss of NRK-2 enhanced p38α activation following ischemic injury. Consistently, ex vivo studies demonstrated that the gain of NRK-2 function suppresses the p38α as well as fibroblast activation (α-SMA expression) upon TGF-ß stimulation, and limits cardiomyocytes death upon hypoxia/re­oxygenation. Collectively our findings show, for the first time, that NRK-2 plays a critical role in heart failure progression following ischemic injury. NRK-2 deficiency promotes post-MI scar expansion, rapid LV chamber dilatation, cardiac dysfunction and fibrosis possibly due to increased p38α activation.

Muscarinic receptors promote pacemaker fate at the expense of secondary conduction system tissue in zebrafish.

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.

ß2-adrenergic receptor-mediated mitochondrial biogenesis improves skeletal muscle recovery following spinal cord injury.

In addition to local spinal cord dysfunction, spinal cord injury (SCI) can result in decreased skeletal muscle mitochondrial activity and muscle atrophy. Treatment with the FDA-approved ß2-adrenergic receptor (ADRB2) agonist formoterol has been shown to induce mitochondrial biogenesis (MB) in both the spinal cord and skeletal muscle and, therefore, has the potential to address comprehensive mitochondrial and organ dysfunction following SCI. Female C57BL/6 mice were subjected to moderate contusion SCI (80 Kdyn) followed by daily administration of vehicle or formoterol beginning 8 h after injury, a clinically relevant time-point characterized by a 50% decrease in mtDNA content in the injury site. As measured by the Basso Mouse Scale, formoterol treatment improved locomotor recovery in SCI mice compared to vehicle treatment by 7 DPI, with continued recovery observed through 21 DPI (3.5 v. 2). SCI resulted in 15% body weight loss in all mice by 3 DPI. Mice treated with formoterol returned to pre-surgery weight by 13 DPI, while no weight gain occurred in vehicle-treated SCI mice. Remarkably, formoterol-treated mice exhibited a 30% increase in skeletal muscle mass compared to those treated with vehicle 21 DPI (0.93 v. 0.72% BW), corresponding with increased MB and decreased skeletal muscle atrophy. These effects were not observed in ADRB2 knockout mice subjected to SCI, indicating that formoterol is acting via the ADRB2 receptor. Furthermore, knockout mice exhibited decreased basal spinal cord and skeletal muscle PGC-1α expression, suggesting that ADRB2 may play a role in mitochondrial homeostasis under physiological conditions. These data provide evidence for systemic ADRB2-mediated MB as a therapeutic avenue for the treatment of SCI.