Phosphorylation in Novel Mitochondrial Creatine Kinase Tyrosine Residues Render Cardioprotection against Hypoxia/Reoxygenation Injury

Figures

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  Fig. 1. Phosphoproteome analysis of cardiac proteins during control and IPC. (A) Schematic diagram illustrates the experimental workflow for rat heats, (B, C) after which total number of phosphopeptides are identified using liquid chromatography-tandem mass spectroscopy. (D) Phosphorylation of CKMT2 during perfusion and IPC compared to ischemia only and I/R. (E) CKMT2 mRNA level of healthy and non-responding human patients (responding n=12, non-responding n=8) (F) GEO data analysis (GSE3585) of CKMT2 mRNA in patients experiencing dilated cardiomyopathy (each group, n=9). Values are mean±standard error of the mean. (A, B) Two-tailed unpaired Student's t-tests. I/R, ischemia/reperfusion; IPC, ischemic preconditioning; NS, statistically not significant; mRNA, messenger RNA.

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  Fig. 2. CKMT2 overexpression protects against H/R in cardiac AC16 cells. (A) Schematic diagram of MTS control and CKMT2. (B) CKMT2-GFP and MTS-GFP were transfected into AC16 cells for 24 hours. verification of GFP-CKMT2 expression in transfected cells and co-localization with mitochondrial TMRE staining. Images were acquired by confocal microscopy, using a 60× objective. Green corresponds to GFP; red: TMRE; yellow: overlap of TMRE and CKMT2 within mitochondria (merged). Representative images are shown (scale bar=10 μm). (C, D) MTS-GFP and CKMT2-GFP were transfected into AC16 cells for 24 hours. Transfected cells were incubated in 2% O2 for 18 hours to simulate hypoxia condition, followed by 2 hours re-oxygenation at 18% O2, before conducting cell viability and intracellular ATP content analysis. (E, F) MTS-GFP vector and CKMT2 were transfected into AC16 cells for 24 hours. After hypoxia/re-oxygenation, cells were stained with 200 nM TMRE and 1 μM Mito-SOX for 30 minutes and analyzed by flow cytometry. Data were normalized to MTS-GFP. The data are expressed as the mean±standard error of the mean of 3 to 4 independent experiments, where applicable. (C-F) Two-way analysis of variance, post…

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  Fig. 3. Phosphorylation of human CKMT2 occurs at various conserved sites. (A) Cartoon representation of CKMT2 and (B) frequency of CKMT2 phosphorylation sites based on a cross-literature review of high throughput paper at Phsophosite.org, and quantitative analysis of residue conservation at the 3 most referenced CKMT2 phosphorylation sites as calculated by ConSurf. (C) Average percent confidence of 7 prediction servers regarding the deleterious effect of phosphomutations, as calculated by HOPE.

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  Fig. 4. Single mutation in CKMT2 phosphorylation sites reduces mitochondria function under H/R condition. (A) Schematic diagram of CKMT2 and CKMT2 alanine substitution mutants in Tyr159 (Y159A), Tyr255 (Y255A), and Tyr368 (Y368A). (B) CKMT2-GFP and CKMT2 substitution mutant-GFP were transfected into AC16 cells for 24 hours. Mitochondria were labeled with TMRE (red). Cells were visualized using confocal microscopy. (C) CKMT2 isoenzyme activity assays from the HIS-purified lysates of pcDNA6.1 or pcDNA6.1-CKMT2, and pcDNA6.1-CKMT2 substitution mutants. The horizontal axis shows the transgene and each graph corresponds to individual isoenzymes CKMT2/CKMT2 substitution mutants. (D, E) CKMT2-GFP and CKMT2 substitution mutant-GFP were transfected into AC16 cells for 24 hours. Transfected cells were incubated in 2% O2 for 18 hours to simulate hypoxia condition, followed by 2 hours re-oxygenation at 18% O2, before conducting cell viability and intracellular ATP content analysis. (F, G) CKMT2-GFP and CKMT2 substitution mutant-GFP were transfected into AC16 cells for 24 hours. After hypoxia/re-oxygenation, cells were stained with 200 nM TMRE and 1 μM Mito-SOX for 30 minutes and analyzed by flow…

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  Fig. 5. CKMT2 influence mitochondria protein expression. (A) Representative figures and densitometric ratios of (B) PGC-1α, ERRα, and PHB1 during single substitution mutation under normoxic and H/R conditions. CKMT2-GFP and CKMT2 substitution mutant-GFP were transfected into AC16 cells for 24 hours. Ratios of proteins to GAPDH were densitometrically quantified using ImageJ. (B) One-way analysis of variance, post hoc Dunnett or Holm-Sidak multiple comparisons test. PGC-1α, proliferator-activated receptor γ coactivator-1α; ERRα, estrogen-related receptor-α; PHB1, prohibitin 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H/R, hypoxia/reoxygenation. *p<0.01, †p<0.01, ‡p<0.001.

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  Fig. 6. Phosphorylation of CKMT2 in Y159, Y255, and Y368 confer cardioprotection against H/R injury in AC16 cells. Schematic diagram indicating the specific tyrosine residues affected during H/R, which led to the regulation of creatine kinase activity and mitochondrial function. Created with BioRender.com. H/R, hypoxia/reoxygenation; ATP, adenosine triphosphate; Δψm, mitochondrial membrane potential; mtROS, mitochondrial reactive oxygen species.