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Design of a proteolytically stable sodium-calcium exchanger 1 (NCX1)-phospholemman (PLM) disruptor peptide for in vivo studies

ABSTRACT: The cardiac sodium–calcium exchanger (NCX1) is important for normal Na+ and Ca2+ homeostasis and cardiomyocyte relaxation and contraction. NCX1 activity is reduced by its endogenous inhibitor phosphorylated phospholemman (pSer68-PLM). Disrupting the pSer68-PLM-NCX1 interaction is therefore a potential therapeutic strategy to reverse NCX1 inhibition in cardiac disease. We have previously designed a high affinity NCX1-PLM disruptor peptide (OPT) that reverses the inhibitory pSer68-PLM-NCX1 interaction in HEK293 cells. In the present study, we performed N and C-terminal truncation analyses of OPT and identified PYKEIEQLIELANYQV as the minimal pSer68-PLM binding sequence. To increase peptide stability in human serum, we replaced the proline in its N-terminus with an N-methyl-proline (NOPT) after identification of N-terminus as substitution tolerant by two dimensional peptide array analysis. Mass spectrometry analysis revealed that the half-life of the novel NOPT peptide was increased 10-fold compared to that of OPT. Biotinylated NOPT pulled down endogenous PLM from rat left ventricle lysate and exhibited pSer68-PLM binding in an ELISA-based assay. Importantly, NOPT reduced the PLM-NCX1 interaction in an ELISA-based assay and cell permeable NOPT-TAT increased the NCX1 activity in adult cardiomyocytes isolated from both wild type (WT) and HF animals. Finally, we found that NOPT interacted with pSer68-PLM with a KD value of 2.7±3.8 μM, whereas the NCX1 cytoplasmic part interacted with PLM and pSer68-PLM with KD values of 14±2 nM and 24±14 nM. In conclusion, we have here developed a proteolytically stable NCX1-PLM disruptor peptide that upregulates NCX1 activity in WT and HF cardiomyocytes.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tuula Nyman

LAB HEAD: Tuula Nyman

PROVIDER: PXD022797 | Pride | 2021-09-09

REPOSITORIES: Pride

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Design of a Proteolytically Stable Sodium-Calcium Exchanger 1 Activator Peptide for In Vivo Studies.

Wanichawan Pimthanya P Skogestad Jonas J Lunde Marianne M Støle Thea Parsberg TP Stensland Maria M Nyman Tuula A TA Sjaastad Ivar I Sejersted Ole M OM Aronsen Jan Magnus JM Carlson Cathrine Rein CR

Frontiers in pharmacology 20210607

The cardiac sodium-calcium exchanger (NCX1) is important for normal Na+- and Ca2+-homeostasis and cardiomyocyte relaxation and contraction. It has been suggested that NCX1 activity is reduced by phosphorylated phospholemman (pSer68-PLM); however its direct interaction with PLM is debated. Disruption of the potentially inhibitory pSer68-PLM-NCX1 interaction might be a therapeutic strategy to increase NCX1 activity in cardiac disease. In the present study, we aimed to analyze ...[more]

PMID: 34163352

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Project description:Acute occlusion of a coronary artery results in swift tissue necrosis. Bordering areas of the infarcted myocardium may also experience impaired blood supply and reduced oxygen delivery leading to altered metabolic and mechanical processes. While transcriptional changes in hypoxic cardiomyocytes are well-studied, little is known about the proteins that are actively secreted from these cells. We established a novel secretome analysis of cardiomyocytes by combining stable isotope labeling and click chemistry with subsequent mass spectrometry analysis. Further functional validation experiments included ELISA measurement of human samples, murine LAD ligation and adeno-associated virus (AAV) 9-mediated in vivo overexpression in mice. The presented approach is feasible for the analysis of the secretome of primary cardiomyocytes without serum starvation. 1026 proteins were identified to be secreted within 24 hours, indicating a 5-fold increase in detection compared to former approaches. Among them, a variety of proteins have so far not been explored in the context of cardiovascular pathologies. One of the most strongly upregulated secreted factors upon hypoxia was proprotein convertase subtilisin/kexin type 6 (PCSK6). Validation experiments revealed an increase of PCSK6 on mRNA and protein level in hypoxic cardiomyocytes. PCSK6 expression was elevated in hearts of mice following 3 days of ligation of the left anterior descending artery, a finding confirmed by immunohistochemistry. ELISA measurements in human serum also indicate distinct kinetics for PCSK6 in patients suffering from acute myocardial infarction, with a peak on day 3 post-infarction. Transfer of PCSK6-depleted cardiomyocyte secretome resulted in decreased expression of collagen I and III in fibroblasts compared to control treated cells, and siRNA mediated knockdown of PCSK6 in cardiomyocytes impacted transforming growth factor-β activation and mothers against decapentaplegic homolog 3 (SMAD3) translocation in fibroblasts. An Adeno-associated virus (AAV) 9-mediated, cardiomyocyte-specific overexpression of PCSK6 in mice resulted in increased collagen expression and cardiac fibrosis as well as decreased left ventricular function after myocardial infarction. In conclusion, a novel mass spectrometry-based approach allows the investigation of the secretome of primary cardiomyocytes. Analysis of hypoxia-induced secretion led to the identification of PCSK6 to be crucially involved in cardiac remodeling after acute myocardial infarction. Secretome analysis was performed on neonatal rat ventricular cardiomyocytes (NRVCMs) which were incubated under hypoxic conditions (1.5% O2, 5% CO2, 93.5% N2) for 12 (Hypoxia 0-12h), 24 (Hypoxia 0-24h) and 30 (Hypoxia 24-30h) hours. Furthermore, knockdown (KD) of PCSK6 in vitro mediated by small interfering RNA (siRNA) was performed to investigate changes in the secretome of cardiomyocytes with PCSK6 KD vs. control (control siRNA) during 24 hours of hypoxia (PCSK6 KD 0-24h). Cells were pulse-labeled with AHA (L-azidohomoalanine) and SILAC (stable isotope labeling with amino acids in cell culture) for 12 (Hypoxia 0-12h), 24 (Hypoxia 0-24h/PCSK6 KD 0-24h) and 6 hours (Hypoxia 24-30h). For Hypoxia 0-12h 3 replicates, Hypoxia 0-24h 6 replicates, PCSK6 KD 0-24h 3 Replicates and Hypoxia 24-30h 2 replicates were performed with label-swap.

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Dataset Information

Design of a proteolytically stable sodium-calcium exchanger 1 (NCX1)-phospholemman (PLM) disruptor peptide for in vivo studies

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Publications

Design of a Proteolytically Stable Sodium-Calcium Exchanger 1 Activator Peptide for <i>In Vivo</i> Studies.

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