Targeting N-Myristoylation Through NMT2 Prevents Cardiac Hypertrophy and Heart Failure

Title: Targeting N-Myristoylation Through NMT2 Prevents Cardiac Hypertrophy and Heart Failure

Journal: JACC. Basic to translational science

Published: 2023

Background

Protein N-myristoylation is a lipid post-translational modification in which a myristoyl group is added to the N-terminal glycine of proteins, increasing proteomic diversity and regulating protein function. In cardiovascular disease, aberrant post-translational modifications contribute to pathological cardiac remodeling and heart failure, yet the role and substrates of N-myristoylation in this context have not been fully explored. Identification of myristoylated targets and understanding how N-myristoylation influences hypertrophy and heart failure progression could uncover new therapeutic strategies.

Materials & Methods

Click chemistry for labeling N-myristoylated proteins

For click metabolic labeling,18,19 Click-iT myristic acid azide (Thermo Fisher Scientific) was introduced to the cell culture. After cells were lysed, protein was incubated with alkyne-agarose resin with 50% slurry by Click-iT protein enrichment technology. Trypsin-digested peptides were introduced to subsequent analysis.

Liquid chromatography–tandem mass spectrometry

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed using an Easy-nLC1000 system (Thermo Fisher Scientific) coupled to a Q Exactive mass spectrometer 1000 (Thermo Fisher Scientific). An ion target value for mass spectrometry (MS) was set to 106, tandem MS to 105, and the intensity threshold was set to 1.8 × 103.

Proteomics data analysis

All raw data acquired from LC-MS/MS spectra were processed with Proteome Discoverer version 1.4. The amino acid sequences of the peptides were identified using MASCOT and Sequest-HT search engines against Rat database in the SwissProt and Uniprot. Scaffold 5 (Proteome Software, Inc) was used to report tandem MS–based peptide. The label-free quantification of LC-MS/MS was applied to determine protein abundance in each sample by measuring the intensity of the corresponding MS spectrum features of the protein. Data were exported from Scaffold for statistical analysis of the differences in mean intensities among multiple replicate samples.

Results

Figure 1. Global Profiling of Substrate Proteins of N-Myristoylation in Cardiac Myocytes by Click Chemistry–Based Quantitative Proteomics.

(A) A schematic diagram for the workflow of proteomic strategy using click chemistry. Neonatal rat cardiomyocytes (NRCMs) and H9c2 myocytes were incorporated with myristic acid azide for the labeling. N-myristoylated proteins were selectively captured by Click-iT protein enrichment technology using a click reaction with alkyne-agarose resin and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). According to quantitative proteomics, 103 and 195 N-myristoylated–enriched proteins are identified in (B) NRCM and (C) H9c2 myocytes, respectively. The log quantitative value is plotted for each N-myristoylated substrate. The light green bars show proteins with the MG motif at the N-terminal, and the black bars show those with no MG motif. Data are presented as mean values from 5 and 3 independent experiments performed in triplicate, respectively. (D and E) Scatterplots for log quantitative value and log percentage of total spectra for N-myristoylated proteins according to the containing motif in (D) NRCM and (E) H9c2 myocytes. (F) Biological functions of 40 N-myristoylated proteins commonly detected in NRCM and H9c2 myocytes according to gene ontology annotation.

Myristoylation Figure 2. Alteration in N-Myristoylated Levels in Response to Ang II in Cardiac Myocytes.

(A) The workflow for chemical proteomics. NMT2 expressions were inhibited in NRCM by Ad5-shNMT2 or in H9c2 myocytes by siNMT2, and then cells were stimulated with angiotensin II (Ang II, 1 μmol/L) for 24 hours before collection for click chemistry–based LC-MS/MS proteomics. (B to E) Volcano plots of the difference of quantitative value for N-myristoylated protein levels and log P value followed with the presence and absence of Ang II in Ad5-shCTRL– or Ad5-shNMT2–infected NRCM and in siCTRL- or siNMT2-transfected H9c2 myocytes from 3 independent experiments performed in triplicate. (F and G) Quantification of N-myristoylated MARCKS in (F) NRCM and (G) H9c2 myocytes.

Conclusions

The study demonstrates that N-myristoylation mediated by N-myristoyltransferase 2 (NMT2) plays a protective role against pathological cardiac hypertrophy and heart failure in a pressure overload murine model. Using click chemistry–based quantitative chemical proteomics, N-myristoylated substrates were identified in cardiac myocytes, revealing that myristoylation of key proteins such as MARCKS modulates hypertrophic signaling pathways through regulation of kinases and histone modifications. Knockdown of NMT2 worsened cardiac dysfunction and remodeling, while gene transfer of NMT2 alleviated these effects, suggesting that targeting N-myristoylation through modulation of NMT2 activity has therapeutic potential in preventing cardiac remodeling and heart failure.

Reference

Tomita, Y., Anzai, F., Misaka, T., Ogawara, R., Ichimura, S., Wada, K., Kimishima, Y., Yokokawa, T., Ishida, T., & Takeishi, Y. (2023). Targeting N-Myristoylation Through NMT2 Prevents Cardiac Hypertrophy and Heart Failure. JACC. Basic to translational science, 8(10), 1263–1282. https://doi.org/10.1016/j.jacbts.2023.06.006

* For Research Use Only. Not for use in diagnostic procedures.

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