Neprilysin is a metallopeptidase and the target of sacubitril, an FDA-approved therapy for heart failure (HF). While the benefits of neprilysin inhibition are generally attributed to preservation of circulating natriuretic peptides, emerging studies suggest that neprilysin inhibition may also have direct cardioprotective effects. In the present study, we found that cardiac neprilysin is significantly upregulated in cardiac tissue from patients with dilated cardiomyopathy (DCM), and it is also upregulated in a mouse pressure overload model of HF. Using immunofluorescence, we showed that neprilysin localizes to the sarcoplasmic reticulum in cardiomyocytes. In a live cell model, neprilysin directly cleaved the C terminus of PLB, a regulator of the sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase (SERCA). PLB cleavage by neprilysin resulted in a loss of membrane anchoring and diffusion of cleaved PLB into the cytoplasm. Fluorescence energy transfer experiments and functional measurements showed that mislocalization of PLB resulted in loss of dynamic regulation of SERCA. Importantly, we found that a V49A mutation of PLB prevented cleavage, highlighting the importance of this locus for recognition and proteolysis by neprilysin. To test the functional impact of neprilysin in cardiomyocytes, we overexpressed neprilysin in neonatal rat ventricular cardiomyocytes. This resulted in increased Ca 2+ transient amplitudes and accelerated decay kinetics, consistent with loss of negative regulation of SERCA by PLB. These findings uncover a novel molecular target of neprilysin in the heart and suggest a previously unrecognized mechanism of action of neprilysin inhibition therapy. Together, our results support targeting neprilysin through sacubitril or other inhibitors as a strategy to restore calcium handling in HF.
Cunningham et al. (Sun,) studied this question.