Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy.

BACKGROUND: PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. METHOD: We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1MyHC6). RESULTS: PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 (H9c2 rat myoblast cell line) cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45+), particularly neutrophils (CD11b+ [integrin subunit alpha M], Ly6g+ [lymphocyte antigen 6 family member G]), to the myocardium. CONCLUSIONS: Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure.

JAK1/2 Regulates Synergy Between Interferon Gamma and Lipopolysaccharides in Microglia.

Microglia, the resident immune cells of the brain, regulate neuroinflammation which can lead to secondary neuronal damage and cognitive impairment under pathological conditions. Two of the many molecules that can elicit an inflammatory response from microglia are lipopolysaccharide (LPS), a component of gram-negative bacteria, and interferon gamma (IFNγ), an endogenous pro-inflammatory cytokine. We thoroughly examined the concentration-dependent relationship between LPS from multiple bacterial species and IFNγ in cultured microglia and macrophages. We measured the effects that these immunostimulatory molecules have on pro-inflammatory activity of microglia and used a battery of signaling inhibitors to identify the pathways that contribute to the microglial response. We found that LPS and IFNγ interacted synergistically to induce a pro-inflammatory phenotype in microglia, and that inhibition of JAK1/2 completely blunted the response. We determined that this synergistic action of LPS and IFNγ was likely dependent on JNK and Akt signaling rather than typical pro-inflammatory mediators such as NF-κB. Finally, we demonstrated that LPS derived from Escherichia coli, Klebsiella pneumoniae, and Akkermansia muciniphila can elicit different inflammatory responses from microglia and macrophages, but these responses could be consistently prevented using ruxolitinib, a JAK1/2 inhibitor. Collectively, this work reveals a mechanism by which microglia may become hyperactivated in response to the combination of LPS and IFNγ. Given that elevations in circulating LPS and IFNγ occur in a wide variety of pathological conditions, it is critical to understand the pharmacological interactions between these molecules to develop safe and effective treatments to suppress this process.

Enantiomeric Agonists of the Type 2 Cannabinoid Receptor Reduce Retinal Damage during Proliferative Vitreoretinopathy and Inhibit Hyperactive Microglia In Vitro.

Microglia are resident immune cells of the central nervous system (CNS) and propagate inflammation following damage to the CNS, including the retina. Proliferative vitreoretinopathy (PVR) is a condition that can emerge following retinal detachment and is characterized by severe inflammation and microglial proliferation. The type 2 cannabinoid receptor (CB2) is an emerging pharmacological target to suppress microglial-mediated inflammation when the eyes or brain are damaged. CB2-knockout mice have exacerbated inflammation and retinal pathology during experimental PVR. We aimed to assess the anti-inflammatory effects of CB2 stimulation in the context of retinal damage and also explore the mechanistic roles of CB2 in microglia function. To target CB2, we used a highly selective agonist, HU-308, as well as its enantiomer, HU-433, which is a putative selective agonist. First, ß-arrestin2 and Gαi recruitment was measured to compare activation of human CB2 in an in vitro heterologous expression system. Both agonists were then utilized in a mouse model of PVR, and the effects on retinal damage, inflammation, and cell death were assessed. Finally, we used an in vitro model of microglia to determine the effects of HU-308 and HU-433 on phagocytosis, cytokine release, migration, and intracellular signaling. We observed that HU-308 more strongly recruited both ß-arrestin2 and Gαi compared to HU-433. Stimulation of CB2 with either drug effectively blunted LPS- and IFNγ-mediated signaling as well as NO and TNF release from microglia. Furthermore, both drugs reduced IL-6 accumulation, total caspase-3 cleavage, and retinal pathology following the induction of PVR. Ultimately, this work supports that CB2 is a valuable target for drugs to suppress inflammation and cell death associated with infection or sterile retinopathy, although the magnitude of effector recruitment may not be predictive of anti-inflammatory capacity.

Pannexin 1 Channels Control Cardiomyocyte Metabolism and Neutrophil Recruitment During Non-Ischemic Heart Failure.

Pannexin 1 (PANX1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, a possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1 MyHC6 ). PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism both in vivo and in vitro . In vitro , treatment of H9c2 cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knock-down of PANX1. To investigate non-ischemic heart failure and the preceding cardiac hypertrophy we administered isoproterenol, and we demonstrate that Panx1 MyHC6 mice were protected from systolic and diastolic left ventricle volume increases and cardiomyocyte hypertrophy. Moreover, we found that Panx1 MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45 + ), particularly neutrophils (CD11b + , Ly6g + ), to the myocardium. Together these data demonstrate that PANX1 deficiency in cardiomyocytes impacts glycolytic metabolism and protects against cardiac hypertrophy in non-ischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in heart failure patients.