Myocardial GRK2 Reduces Fatty Acid Metabolism and β-Adrenergic Receptor-Mediated Mitochondrial Responses

Abstract

G-protein coupled receptor (GPCR) kinase 2 (GRK2) is upregulated in heart failure (HF) patients and mouse models of cardiac disease. GRK2 is a regulator of β-adrenergic receptors (βARs), a GPCR involved in ionotropic and chronotropic responses. We and others have recently reported GRK2 to be localized in the mitochondria, although its function in the mitochondria and/or metabolism remain not clearly defined. We hypothesized that upregulation of GRK2 reduced mitochondrial respiratory function and responses to βAR activation. Utilizing isolated mouse primary adult cardiomyocytes (ACMs), we investigated the role of glucose, palmitate, ketone bodies, and BCAAs in mediating cell survival. Our results showed that myocyte upregulation of GRK2 promotes palmitate-induced cell death. Isotopologue labeling and mass spectrometry showed that the upregulation of GRK2 reduces β-hydroxybutyryl CoA generation. Next, using isoproterenol (ISO), a non-selective βAR-agonist, we determined mitochondrial function in mouse and human primary ACMs. Upregulation of GRK2 impaired ISO-mediated mitochondrial functional responses, which we propose is important for metabolic adaptations in pathological conditions. Increased cardiac levels of GRK2 reduced fatty acid-specific catabolic pathways and impaired ISO-stimulated mitochondrial function. Our data support the notion that GRK2 participates in bioenergetic remodeling and may be an important avenue for the development of novel pharmacological strategies in HF.

Keywords: GRK2; beta-adrenergic receptors; cardiomyocytes; metabolism; mitochondria.

Figure 1

Upregulation of GRK2 in the myocyte enhances palmitate-driven cell death. (A) Digitonin treatment in ACMs induced cell death and labeling for EthD-1. Calcein-AM (green) labeled live cells. Scale bar is 50 µm. (B) Representative confocal images using calcein-AM (green, live cells) and ethidium homodimer-1 (red, dead cells) in ACMs in the presence of specific substrates. (Scale bar is 50 μm). (C) Quantification of B displayed as mean ± SEM. (Glucose: 0.205 0.031 Grk2TG; 0.120 ± 0.019 NLC; palmitate/carnitine: 0.433 ± 0.060 Grk2TG; 0.237 ± 0.050 NLC; ketone: 0.201 ± 0.029 Grk2TG; 0.178 ± 0.030 NLC; BCAAs: 0.244 ± 0.027 Grk2TG; 0.146 ± 0.029 NLC; n = 10 images/condition in 3 hearts/genotype; *** p < 0.0001).

Figure 2

GRK2 negatively impact fatty-acid catabolism. (A) Palmitate-derived isotopically labeled malonyl-CoA, acetyl-CoA, and 3-Hydroxybutyryl-CoA in Grk2TG ACMs normalized to NLC. (B) Same as A but glucose-derived acyl-CoAs. Data were normalized to NLC (n = 3 hearts/condition, mean ± SEM; * p < 0.05).

Figure 3

Chronic βAR stimulation and GRK2 levels in cellular compartments is altered in Grk2TG hearts (A) Left ventricular ejection fraction and (B) fractional shortening were measured by echocardiography (n= 6 independent mice/group). (C) Representative immunoblots of GRK2 expression level in mitochondria (left) and cytoplasm (right). (D) Mitochondrial and (E) cytosolic GRK2 protein quantification (n= 8 independent mice/group; mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.005).

Figure 4

Mitochondrial function responses are altered in response to βAR stimulation and GRK2 expression. (A) Overall mitochondrial OCR traces are shown for NLC saline (NLC S; red), NLC ISO (black), Grk2TG + saline (Grk2TG S; blue), and Grk2TG ISO (green) during a mitochondrial stress test (n = 114 to 144 wells/condition in 3 independent hearts/condition). (B) Quantification of basal respiration. (C) ADP-linked respiration, and (D) maximal respiration are shown from data in panel A (same n value). (E) Representative image of hACMs (scale bar is 100 um). (F) Mitochondrial OCR in hACMs from the same donor in the presence or absence of ISO, (n = 20–25 wells/condition) (mean ± SEM, * p < 0.05, *** p < 0.005 and **** p < 0.0001).