Nitro-oxidative stress impairs single cardiomyocyte adrenergic reserve in a murine model of HFpEF

Abstract

Background Exercise intolerance is the central symptom of patients with heart failure and preserved ejection fraction (HFpEF). Underlying reduced cardiac functional reserve in response to adrenergic stimuli (stress testing) has been suggested but the molecular mechanisms are insufficiently understood. In cardiomyocytes, nitric oxide (NO) modifies contractility and is required to achieve a full adrenergic response. Recently, dysregulation of NO release has been described to contribute to HFpEF. In a murine model of HFpEF, we investigated cardiomyocyte's adrenergic functional reserve and the role of NO in cardiomyocyte contractility, calcium handling and adrenergic reserve. Methods Firstly, the effects of NO on sarcomere shortening and calcium handling (Fura-2) were studied in isolated, adult ventricular cardiomyocytes (AMVMs) from 8–10 weeks old, male C57BL/6J mice. Secondly, male C57BL/6J mice (12w) were fed regular CHOW (Sham) or a high fat diet (D12492, Research Diet) and L-NAME (1g/l, via the drinking water) for 15 weeks to induce HFpEF. At week 27, mice underwent echocardiography and exercise testing (treadmill). In AMVMs isolated from HFpEF and Sham mice, we quantified sarcomere shortening, calcium handling (Fura-2), release of NO (CuFL2) and reactive oxygen species (DCF) before and after the addition of isoproterenol (ISO, 1μM), in the absence and presence (40 min preincubation) of an inhibitor of inducible NO Synthase (1400W) or a denitrosylating agent (glutathione). Results In AMVMs, addition of the NO donor SNAP (10μM) increased calcium transient amplitude and accelerated relaxation time. Inhibition of NO synthesis with L-NAME (100μM) impaired adrenergic response upon exposure to ISO. HFpEF mice (evident by diastolic dysfunction (e/e' ratio) and lung edema (wet lung weight / TL)) exhibited significantly reduced exercise capacity (running distance). In AMVMs isolated from HFpEF mice, NO and ROS release were increased at baseline, associated with an increased sarcomere shortening amplitude and faster relaxation and calcium removal as compared to Sham. Strikingly, after the addition of ISO, the adrenergic functional reserve (cellular inotropy and lusitropy) was significantly lower in HFpEF vs. Sham AMVMs. Preincubation with the iNOS inhibitor 1400W or glutathione restored adrenergic inotropic and lusitropic reserve in HFpEF AMVMs. Conclusion In HFpEF, adrenergic reserve is impaired on a single cardiomyocyte level. This is at least partially related to an increase in NO release. Pharmacologic inhibition of iNOS improved adrenergic reserve in HFpEF. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG