Background: Exercise intolerance is the central symptom of patients with heart failure and preserved ejection fraction (HFpEF). An impaired elevation of cardiac output in response to adrenergic stimuli has been suggested but the molecular mechanisms are insufficiently understood. In cardiomyocytes, nitric oxide (NO) modulates calcium handling and contractility. Recently, dysregulation of NO release has been described to contribute to HFpEF. In a murine model of HFpEF, we performed stress echocardiography, investigated cardiomyocyte’s adrenergic functional reserve and the role of NO in cardiomyocyte contractility, calcium handling and adrenergic reserve.

Methods: 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 or stress echocardiography (i.p. injection of isoproterenol (ISO)) and exercise testing (treadmill). In left ventricular cardiomyocytes (LVCM) 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 addition of ISO (1µM), in the absence and presence (40 min preincubation) of an inhibitor of inducible NO Synthase (1400W), neural NO Synthase (SMTC) or a de-nitrosylating agent (glutathione).

Results: HFpEF mice (preserved LVEF, increased E/e’ and lung edema (wet lung weight / TL)) exhibited significantly reduced exercise capacity (running distance) and deficits on stress echocardiography (trend towards an ISO-mediated reduction of stroke volume and thus a loss of ISO-mediated augmentation of cardiac output in HFpEF). Surprisingly, sarcomere shortening in LVCM isolated from HFpEF mice showed a higher amplitude, slower peak and faster relaxation compared to Sham. After the addition of ISO, there were no differences of absolute values between HFpEF and Sham mice in sarcomere shortening amplitude or kinetics. Accordingly, relative, adrenergic, inotropic and lusitropic reserve in HFpEF LVCM was reduced. Increased contractility was not caused by an increase of calcium transient amplitudes but rather a shift of sarcomere sensitivity (ΔCa/Δsarc length at 50% relaxation). Calcium decay was accelerated in HFpEF possibly explaining for the faster cellular relaxation. In LVCM from HFpEF mice, cytosolic ROS release was increased. NO release at baseline was unaltered, but HFpEF LVCM’s showed increased NO release with ISO. Preincubation with the NOS inhibitors (1400W, SMTC) or glutathione selectively attenuated the augmentation in shortening amplitude and sarcomere relaxation and partially restored adrenergic reserve in HFpEF LVCM. 

Conclusion: In this two-hit metabolic HFpEF model the adrenergic functional reserve is reduced. Increased contractility at baseline, partially mediated by NO, reduces the HFpEF cardiomyocytes‘ intrinsic inotropic and lusitropic reserve upon adrenergic stimulation.  Enhanced LVCM contractility despite unchanged systolic function in vivo at rest may reflect an increase in cellular afterload in HFpEF.