Mitochondrial accumulation of GRK2 as a protective mechanism against hypoxia-induced endothelial dysfunction

Abstract

Hypoxia, a condition characterized by a temporary lack of oxygen, causes mitochondrial damage, which in turn leads to endothelial dysfunction. G-protein-coupled receptor kinase 2 (GRK2) plays a key role in vascular homeostasis and remodeling, influencing endothelial function through various pathways. GRK2 moves within the cellular compartments and is linked to mitochondrial function and biogenesis, promoting ATP production and protecting against oxidative stress and cell death. The present study examined how mitochondrial GRK2 accumulation affects vascular reactivity and endothelial function in transient hypoxic conditions. Using a cloning strategy, we employed a small peptide (10aa) TAT-conjugated based on the pleckstrin homology domain of GRK2 to redirect GRK2 from the plasma membrane to the mitochondria. Mitochondrial accumulation of GRK2 increases vasodilatory responses in isolated swine artery segments, indicating potential therapeutic applications for cardiovascular disorders. Furthermore, in endothelial cells, GRK2 accumulation within mitochondria protects membrane potential, mitochondrial mass and prevents oxidative damage and cell death caused by transient hypoxia. Our findings show that GRK2 accumulation in mitochondria represents a potential therapeutic target to prevent transient hypoxia-induced damage.

Fig. 1. Mitochondrial localization of GRK2 in Endothelial Cells.

In A, a representative immunoblot shows (i) the expression of G-protein-coupled receptor kinase 2 (GRK2) and ATP synthase β subunit in scramble and PH1-treated cells. The molecular markers are indicated on the left. Quantification (ii) shows band pixels between GRK2 and GAPDH for the cytosolic fraction and whole lysates, GRK2, and β subunit for the mitochondrial fraction. Data are the mean ± SEM of 3 independent experiments. Two-way ANOVA, *p < 0.05. In B, Immunofluorescence images showing co-localization of GRK2 (green) with mitochondria stained with MitoTracker (red) in ECs. Nuclei were counterstained with DAPI (blue). Merged images reveal areas of overlap between GRK2 and mitochondria (yellow), indicating mitochondrial localization of GRK2. (ii) Immunofluorescence analysis showing Pearson correlation of GRK2 (green) with mitochondria (red) in and (iii) GRK2 expression mean intensity fluorescence. Data are the mean of 3 independent experiments ± SEM. *p < 0,05, ***0,001 unpaired test t-Student.

Fig. 2. GRK2 improves vasodilation under hypoxia.

In A, Vascular reactivity of the isolated femoral vessel in response to increasing doses of acetylcholine (Ach). Concentration–response curves for CTR and PH1-treated vessels are generated for normoxic and hypoxic conditions. Data are the mean of 3 independent experiments ± SEM. Two-way ANOVA, *p < 0.05, **p < 0,01.

Fig. 3. PH1 reduces vascular ROS in hypoxia.

In A (i), representative images of ROS-positive cells stained with DHE, and the nucleus stained with DAPI. Quantification (ii) shows the red fluorescence (DHE) intensity in vessels under hypoxia conditions. Data are the mean of 3 independent experiments ± SEM. *p < 0,05 unpaired test t-Student.

Fig. 4. GRK2 limits hypoxia-induced cell death.

In A, a representative image of CD31-positive cells demonstrating a high purity of ECs isolation. In B, the representative immunoblot shows (i) the expression of Hypoxia-inducible factor 1-alpha (HIF-1-α). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a loading control in CTR and PH1-treated cells in normoxic and hypoxic conditions. The molecular markers are indicated on the left. Quantification (ii) shows band pixels between HIF-1-α and GAPDH as fold of control (mean ± SEM of at least 3 independent experiments). Two-way ANOVA, *p < 0.05. In C, Annexin V/PI assay shows apoptotic levels in CTR and PH1-treated cells in normoxic and hypoxic conditions, with the representative plot (i). Histograms show (ii) the mean quantification ± SEM of apoptotic annexin V-positive cells (expressed as fold of control). Data are the mean ± SEM of at least 3 independent experiments. Two-way ANOVA, *p < 0.05.

Fig. 5. GRK2 preserves viability and promotes migration.

In A, the growth curve of CTR and PH1-treated cells in normoxic and hypoxic conditions. Cells are detached and counted at 0, 24, 48, and 96 h. Data are the mean ± SEM of 3 independent experiments. In B, quantitative analysis of cell viability from the CCK-8 assay in normoxic and hypoxic conditions. The histograms represent the mean ± SEM of at least three independent experiments. In C, D, scratch test (i) demonstrates cell migration in CTR and PH1-treated cells in normoxic and hypoxic conditions, respectively. Histograms show (ii) the mean quantification of wound healing expressed as % of the area at 0 and 6 h post-hypoxia. Data are the mean ± SEM of at least 3 independent experiments. Two-way ANOVA, *p < 0.05.

Fig. 6. GRK2 reduces ROS and preserves mitochondria.

In A, MitoSox shows mitochondrial superoxide production in CTR and PH1-treated cells in normoxic and hypoxic conditions. In B, the Dihydrorhodamine 123 (DHR) assay detects intracellular reactive oxygen species (ROS) in CTR and PH1-treated cells in normoxic and hypoxic conditions. Data are the mean of 3 independent experiments ± SEM. Two-way ANOVA, *p < 0.05, ** p < 0,01. In C, western blotting shows (i) the expression of mitochondrial superoxide dismutase (SOD-2), and in D, xanthine oxidase (xanthine ox). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a loading control in CTR and PH1-treated cells in normoxic and hypoxic conditions. The molecular marker is indicated on the left. Quantification (ii) shows band pixels between SOD-2, xanthine oxidase, and GAPDH (mean ± SEM of at least 3 independent experiments). In E, histograms show TMRM fluorescence monitoring the mitochondrial membrane potential in control and treated cells expressed as % of control. In F, the % of Nonyl acridine orange positive cells is a marker of mitochondrial mass. Data are the mean of at least 3 independent experiments ± SEM. Two-way ANOVA, *p < 0.05.