Research ArticleCardiovascular Biology

Restricting mitochondrial GRK2 post-ischemia confers cardioprotection by reducing myocyte death and maintaining glucose oxidation

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Science Signaling  11 Dec 2018:
Vol. 11, Issue 560, eaau0144
DOI: 10.1126/scisignal.aau0144
  • Fig. 1 A normal cardiac phenotype in GRK2-S670A KI mice and mitochondrial translocation of GRK2 post-IR injury.

    (A) GRK2 immunoprecipitates (IP) from WT or KI cardiac lysates were immunoblotted (IB) for total GRK2 or phospho (P)–Ser670 (n = 6 hearts per genotype). (B) Whole cardiac lysates from WT and KI animals were immunoblotted for total GRK2 and β-actin as a loading control. (C) Quantification of (B) (n = 8 hearts per genotype). (D) Heart weight–to–body weight (HW/BW) parameters in KI and WT mice (n = 13 to 20 hearts per genotype). (E) In vivo cardiac function as assessed by ejection fraction percentage determined by echocardiography (n = 25 to 26 hearts per genotype). (F) Left ventricular (LV) fractional shortening percentage determined by echocardiography (n = 25 to 26 hearts per genotype). (G) Radioligand binding results of myocardial sarcolemmal βAR levels in WT and KI cardiac lysates (n = 7 hearts per genotype). (H) Representative transmission electron microscopic images of cardiac tissue from WT and KI (n = 3 hearts per genotype). Scale bars, 1 μm. (I) Whole-cell lysates from the AAR in WT and KI hearts post-IR injury (24 hours) were immunoblotted for total GRK2 levels (n = 4 to 6 hearts per group). Quantification is shown in fig. S3A. (J) Purified mitochondrial fractions from the AAR in WT and KI hearts were immunoblotted for GRK2. Whole-cell lysates from mouse embryonic fibroblasts derived from WT and GRK2 knockout mice were used as positive and negative control for the blots. (K) Quantification of protein immunoblots shown in (J) (n = 5 to 8 hearts per group). (L) Sections from the AAR in WT and KI hearts post-IR injury were immunogold labeled for GRK2. GRK2 immunogold labeling of sections from transgenic mice overexpressing GRK2 was used as control. Representative images are shown in fig. S3B (n = 20 to 24 images from three hearts per genotype). Statistical significance was determined by Student’s t test or analysis of variance (ANOVA). Data are shown as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.005. a.u., arbitrary units.

  • Fig. 2 Limiting mitochondrial GRK2 translocation post-IR injury significantly reduces LV infarct size through diminished myocyte cell death.

    (A) Representative Evans blue staining of hearts 24 hours after IR injury of WT and KI mice. Blue area is the area not at risk (ANAR), red is the AAR, and white represents the infarct. (B) Quantification of Evans blue staining (n = 19 to 20 hearts per group). (C) %AAR or the area subjected to injury in WT and KI mice (n = 19 to 20 hearts per group). (D) Serum troponin I levels were measured 24 hours post-IR injury in WT and KI mice (n = 5 to 6 mice per group). (E) TUNEL (red) analysis of cardiac tissue from WT and KI mice counterstained with α-actinin (green) to visualize cardiomyocytes; 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear marker (blue). Scale bar, 100 μm. (F) TUNEL quantification of images in (E) (n = 3 to 5 hearts per group). (G) Echocardiography ejection fraction percentage in WT and KI mice 24 hours post-IR injury reveals improved cardiac function in the KI animals (n = 19 to 20 mice per group). (H) LV fractional shortening percentage as determined by echocardiography (n = 19 to 20 mice per group). Statistical significance was determined by ANOVA. Data are shown as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.005.

  • Fig. 3 Adult cardiomyocytes from GRK2-S670A KI mice post-IR injury maintain mitochondrial respiration and a normal glucose respiratory response.

    (A) OCRs measured using a Seahorse flux analyzer in cardiomyocytes isolated from post-IR injury (24 hours) WT and KI mice (n = 44 to 47 wells per group; data from three hearts per genotype and all data normalized to total protein content). Oligo, oligomycin; Rot, rotenone; Ant A, antimycin A. (B) Quantification of basal respiration from data in (A); n same as in (A). (C) Quantification of ATP production from data in (A); n same as in (A). (D) Seahorse tracings of OCRs in cardiomyocytes from WT and KI mice post-IR injury normalized to protein content (n = 27 to 36 wells from three hearts per genotype). Eto, etomoxir. (E) Quantification of glucose-mediated respiration shown in (D); n values same as in (D). (F) Quantification of palmitate-mediated respiration shown in (D); n values same as in (D), ANOVA. Data are shown as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.005.

  • Fig. 4 Defective mitochondrial GRK2 translocation post-IR injury improves glucose respiration by modulating PDH activity and glucose oxidation.

    (A) Radioligand binding studies of βAR in cardiac sarcolemmal membranes from WT or KI mice post-IR injury (n = 4 hearts per group). (B and C) Representative Western blots for plasma membrane GLUT4 in the AAR (B) and quantification (C). Gβ was used as loading control for the plasma membrane preparations (n = 4 hearts per group). (D and E) Representative Western blot for plasma membrane GLUT1 in the AAR (D) and quantification (E). Gβ was used as loading control for the plasma membrane preparations (n = 4 hearts per group). (F) PDH activity assay in pulverized cardiac tissue from the AAR (n = 7 to 9 hearts per group). (G) Radioactive glucose oxidation measurements from AAR samples (n = 6 to 7 hearts per genotype). CPM, counts per minute. ANOVA and Student’s t test. Data are shown as means ± SEM; *P < 0.05.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/560/eaau0144/DC1

    Fig. S1. Targeting strategy for the GRK2-S670A KI mice, screening, and baseline characteristics.

    Fig. S2. Adult fibroblast migration is not affected by the S670A mutation in GRK2.

    Fig. S3. Total GRK2 levels in the AAR are not altered post-IR injury, and immunogold electron microscopy suggests a role for phosphorylation of Ser670 in GRK2 for mitochondrial translocation.

    Fig. S4. Raw baseline OCR recordings from adult cardiomyocytes derived from WT and GRK2-S670A KI mice subjected to an in vitro ischemia protocol.

  • This PDF file includes:

    • Fig. S1. Targeting strategy for the GRK2-S670A KI mice, screening, and baseline characteristics.
    • Fig. S2. Adult fibroblast migration is not affected by the S670A mutation in GRK2.
    • Fig. S3. Total GRK2 levels in the AAR are not altered post-IR injury, and immunogold electron microscopy suggests a role for phosphorylation of Ser670 in GRK2 for mitochondrial translocation.
    • Fig. S4. Raw baseline OCR recordings from adult cardiomyocytes derived from WT and GRK2-S670A KI mice subjected to an in vitro ischemia protocol.

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