Neprilysin mediated cleavage of phospholamban dysregulates SERCA in heart failure

Figures

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  Figure 1.. A) Schematic of human heart tissue membrane protein enrichment and delipidification workflow prior to mass spectrometry. B) MS results of differentially expressed proteins in HF versus NF comparison. C) Quantification from the mass spectrometry data specifically for NEP (gene name: MME). D) Western blot for NEP for non-failing (NF) and heart failure (HF) left ventricle samples. E) Summary data from data in C (N=5). p values were calculated via t-test or adjusted t-test. F) Immunofluorescence microscopy of NF and HF human ventricular myocytes revealed neprilysin (green), SERCA (blue), and actin (red). Boxes 1, 2 in the HF, Merged image are enlarged at right. Arrows indicate NEP localized at the intercalated disc (Region 1) or overlapping with SR-localized SERCA in intermyofibrillar regions (Region 2) of failing human cardiomyocytes.

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  Figure 2.. A) HEK293T cells transfected with mCer-SERCA, YFP-PLB (bottom row) NEP. B) Cell-by-cell FRET binding curve between mCer3-SERCA (donor) and YFP-PLB (acceptor). Apparent FRET efficiency is plotted as a function of acceptor expression. C) As in B, with NEP co-expression. The red oval highlights cells with markedly low FRET. D) FRET-based binding curve between mCer3-SERCA and V49A-YFP-PLB E) NEP co-expression did not affect FRET from SERCA to V49A-PLB. F) FRET binding curve between mCer3-SERCA and YFP-SLN, G) NEP co-expression reduced the apparent affinity of SLN for SERCA. H) Summary of apparent dissociation constants (KD) for all SERCA-binding micropeptides with and without NEP co-expression. Data represent 3 independent transfections, ∼1000 cells per transfection.

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  Figure 3.. A) Fluorescence microscopy of cells before (intact) and after (+Sap) plasma membrane permeabilization with saponin revealed whether PLB was membrane anchored or soluble. B) Quantification of residual fluorescence after permeabilization (N/n = 3–6/7–9). C) Automated quantification of the loss of fluorescence of 150–250 cells after permeabilization. Only the truncated mutant I48X and PLB coexpressed with NEP were lost from permeabilized cells. Statistical significance was determined using one-way ANOVA followed by a Bonferroni post-hoc test. D) Schematic illustrating expected FRAP profiles of membrane-bound proteins versus freely-diffusible cytoplasmic proteins. E) TIRF image of YFP-PLB in ER membrane before and after spot photobleaching, and after 60 s of recovery. F) WT-PLB exhibited slow fluorescence recovery (τ ≈ 12 s), consistent with stable membrane association. G) The truncation mutant I48X-PLB showed rapid recovery (τ ≈ 1.7 s), reflecting increased lateral mobility. H) Coexpression of NEP with wild-type PLB produced biphasic recovery kinetics, with a fast component (τ1 ≈ 1.0 s) indicative of a mobile, cleaved species, and a slower component (τ2 ≈ 11 s) corresponding to…

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  Figure 4.. NEP enhances baseline Ca2+ cycling but reduces frequency-dependent SERCA modulation in NRVMs. A) NRVMs expressing RFP-α-actinin to mark sarcomere structures. Scale bar, 20 μm. B) Representative Ca2+ transients (F340/F380) at 0.5 Hz pacing. C) Ca2+ transient amplitude (ΔF340/F380) at 0.5 Hz. N/n = 8–14/3. D) Ca2+ transient decay time constant (τ) of cells paced at 0.5 Hz, fit by single-exponential decay. E) Ratio of decay τ at 1 Hz to decay τ at 0.5 Hz; larger values indicate increased frequency-dependent acceleration of Ca2+ reuptake. Differences were determined by two-way ANOVA with Tukey’s post hoc test (C, D) or unpaired t-test (E).

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  Figure 5.. A) Summary of experimental design. B) Volcano plots showing differential gene expression changes for Ctrl vs ET-1 (left) and ET-1 vs ET-1 + Sac (right). ET-1 induces widespread transcriptional remodeling that was partially reversed by NEP inhibition. Significantly up- and downregulated genes (adjusted P < 0.05) are shown in red and blue, respectively. C) Gene ontology biological process (GO-BP) analysis. D) Relative expression of representative hypertrophic and Ca2+-handling genes. ET-1 induces fetal gene expression (e.g., increases in NPPA, NPPB, MYH7, decreases in JPH2, BIN1) and remodeling markers, whereas NEP inhibition partially normalizes these changes and preserves expression of Ca2+-handling components.

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  Figure 6.. Proposed model for NEP-mediated dysregulation of SERCA in HF. A) PLB (red) anchored in the SR bilayer inhibits SERCA (gray); NEP cleaves PLB at its C-terminal valine, releasing the cytosolic fragment and yielding a dysregulated SERCA (blue) no longer subject to PLB-mediated control. B) A model of intracellular NEP activity in HF: cardiac stress drives NEP upregulation in cardiomyocytes and cleavage of PLB at the SR, acutely disinhibiting SERCA and increasing contractility (i, green) while progressively depleting the functional PLB pool, blunting β-adrenergic responsiveness (ii) and the force–frequency response (FFR) (iii) and reducing contractile reserve (iv), reinforcing cardiac stress in a maladaptive feedback loop.