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bioRxiv · 10.64898/2026.06.23.732949

Neprilysin mediated cleavage of phospholamban dysregulates SERCA in heart failure

Cunningham, J. D.·Phillips, T. A.·Mazzenga, A. R.·Nagrani, K. N.·Bui, T. H.·Edassery, S.·Barefield, D. Y.·Robia, S. L.
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Abstract

BackgroundNeprilysin (NEP) is a zinc-dependent metalloprotease targeted in heart failure therapy to prevent it degrading circulating cardioprotective vasoactive peptides. NEP can also cleave sarcolipin (SLN), the skeletal- and atrial muscle-specific micropeptide regulator of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). A direct pathophysiological role of NEP in ventricular muscle has not been established. MethodsProteomics and immunoblot analysis of human myocardial specimens were used to quantify NEP abundance in failing and non-failing hearts. Heterologous protein expression and biochemical binding assays assessed NEP-mediated cleavage of phospholamban (PLB) and its impact on PLB-SERCA interactions. Functional consequences of NEP expression or inhibition were evaluated in neonatal rat ventricular myocytes and in a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of heart failure. ResultsWe observed increased NEP abundance in failing human myocardium relative to non-failing controls. We demonstrated that NEP cleaves phospholamban (PLB), disrupting PLB-SERCA interactions. Mutation of PLB (V49A), prevented NEP cleavage and preserved PLB-SERCA binding, indicating V49 is critical for NEP substrate recognition. In neonatal rat ventricular myocytes, NEP expression was associated with faster Ca2+ transient decay kinetics and increased SR Ca2+ load, consistent with reduced SERCA inhibition. Inhibition of NEP in a hiPSC-CM heart failure model attenuated the hypertrophic transcriptional responses and reversed Ca2+-transport dysregulation. ConclusionsThese findings implicate increased NEP expression in the sarcoplasmic reticulum of cardiomyocytes as previously unrecognized maladaptive consequence of heart failure contributing to cardiac dysfunction. In this novel pathophysiological mechanism, increased NEP results in PLB cleavage and loss of regulation of SERCA. While this may relieve SERCA inhibition and augment cellular Ca2+ handling, loss of PLB chronically disrupts hearts dynamic response to adrenergic stress, changing heart rate, or other physiological challenges. The data provide new insight into the cardioprotective effects of pharmacological NEP inhibition in clinical practice, reveal a novel mechanism of action of neprilysin inhibition in cardiomyocytes and may help inform future therapeutic strategies for patients with heart failure. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/732949v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@5ddc5dorg.highwire.dtl.DTLVardef@2e746corg.highwire.dtl.DTLVardef@1dd8273org.highwire.dtl.DTLVardef@19f5635_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Cunningham, J. D., Phillips, T. A., Mazzenga, A. R., Nagrani, K. N., Bui, T. H., Edassery, S., Barefield, D. Y., Robia, S. L.. 2026-06-29. Neprilysin mediated cleavage of phospholamban dysregulates SERCA in heart failure. https://doi.org/10.64898/2026.06.23.732949

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