A role for cardiomyocyte pannexin 1 in non-ischemic heart failure

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Project Summary Cardiac fibrosis is currently untargeted in many cardiac disease settings, yet this is a hallmark of decreased myocardial compliance. Current therapy for patients with heart failure, which has a hallmark of cardiac fibrosis, includes spironolactone, a pannexin 1 (Panx1) channel inhibitor. Despite this, the role for Panx1 in cardiomyocytes in non-ischemic heart failure has not been studied. Additionally, Panx1 plays a role in fibrosis in other diseases, and it has been shown to play roles in inflammation, blood pressure regulation, and myocardial infarction. Furthermore, it can be activated by G-protein subunits during alpha1D and β3-adrenergic stimulation. This evidence provides reason to hypothesize that protection from cardiac dysfunction and decreases pathophysiology including immune cell recruitment and cardiac fibrosis are Panx1 dependent. Preliminary data in a model of non-ischemic heart failure, 14 days of isoproterenol (ISO) injections, shows that male mice of a novel mouse with cardiomyocyte-specific pannexin 1 deletion (Panx1MyHC6) are protected from cardiac hypertrophy and increases in left ventricle volume compared to their Panx1fl/fl counterparts. Additionally, H9c2 cells, a rat myoblast cell line, with a siRNA-mediated knock-down of Panx1 have blunted release of ATP compared to control after β-adrenergic stimulation with ISO. This data and other preliminary studies lead me to address the hypothesis in two aims. In subaim 1A, I will investigate the mechanism of Panx1 activation by β-adrenergic stimulation in cardiomyocytes. I will first determine the mechanism by which ISO activates Panx1 using established Panx1 mutants in Panx1-null H9c2 cells. I hypothesize that the regulation occurs via binding of the G-protein βγ-subunits to Panx1 and have a proposed binding site identified from previous work. I will evaluate this binding site using BioID. In subaim 1B, I will investigate the role of Panx1 channel activation by β-adrenergic stimulation in whole hearts. In subaim 1B, I will perform pressurized contractile studies ex vivo on Langendorff perfused murine hearts to evaluate Panx1’s effect on cardiac contraction. For subaim 1B, I will use our novel Panx1MyHC6 mouse. I will test the role of Panx1 in the induction of cardiac dysfunction in non-ischemic heart failure, using a model of ISO administration for 14 days. Finally, in subaim 2B I will investigate the role of Panx1 in the progression of non-ischemic heart failure. For this aim, I will use another novel mouse model which has inducible deletion of Panx1 under the Tnnt2 promoter. I will use ISO in an osmotic pump for 28 days to induce heart failure and intervene by deleting Panx1 to delineate the role of Panx1 in the onset versus the progression of non-ischemic heart failure. Both aims will evaluate cardiac function using echocardiography with Dr. Wolf. I will use histological analysis for fibrotic content and immune cell infiltration of the myocardium, measure plasma lactate and creatinine as markers of secondary organ damage, and radiotelemetry for blood pressure changes. Together these studies will identify Panx1 as a novel therapeutic target and elucidate whether Panx1 channel activation plays a differential role in heart failure disease states.