GRK2 participates in myocardial fatty acid metabolic responses and [beta]-adrenergic receptor-mediated mitochondrial respiration
Authors:   , Zhai Ruxu , Mathiasen Joanne R. 
Published: September 2022

Heart disease is the leading cause of death in the US and worldwide. One person dies every 37s in the United States from cardiovascular disease (CVD), globally claiming an estimated 17.9 million lives per year. Projections released by the American Heart Association are alarming with an anticipated 46% increase in diagnosis in 30-years (2000 to 2030). Although heart research has greatly contributed to the discovery of various pharmacological targets (e.g. [beta]-blocker therapy), there is no cure for CVD. The [beta]-adrenergic receptor ([beta]-AR), a member of the G-protein coupled receptor (GPCR) superfamily, is an important regulator of cardiac contraction as it provides increased chronotropic (heart rate) and ionotropic (contractility) responses. GPCR-kinases (GRKs) canonically initiate receptor endocytic internalization processes. GRK2 and GRK5 are the main GRK isoforms in the heart, and GRK2 has been shown to be upregulated in human heart failure (HF). Genetic-mouse models of GRK2 have confirmed a detrimental cardiac impact of GRK2 upregulation, especially post-myocardial injury. Recently, we and others have shown that GRK2 can also localize to the cardiac mitochondria where it regulates metabolism, particularly during cellular stress. As the heart utilizes a variety of metabolites for energy production and subsequent muscle contraction, understanding the link between metabolic substrate utilization and [beta]AR-signaling is vital for the development of new pharmacological strategies to treat heart disease. We hypothesized that GRK2 upregulation decreases substrate-specific metabolic utilization promoting cardiac remodeling and decreasing [beta]AR-mediated mitochondrial responses to receptor stimulation. Using a cardiac-specific GRK2 overexpressing mouse model (grk2TG), we isolated adult cardiomyocytes (ACMs) to test our hypothesis. Live-Dead assays showed a trend towards a decrease in cell survival when grk2TG ACMs were cultured exclusively with palmitate (WT 76.2%+/-16.3, vs grk2TG 57.3%+/-16.5, p=0.5778). Carbon-13 palmitate tracing also suggested that grk2TG cells have an impairment in fatty acid utilization, leading to an overall decrease in fatty acid metabolites, with a trend towards an increase of [beta]-hydroxybutyrate. Moreover, our data suggest that protein remodeling, especially the localization of connexin 43 (Cx43) in ACMs may be altered as a result of the upregulation of GRK2 when cells are restricted to palmitate as their energy source. Lastly, we tested how [beta]AR-stimulation altered mitochondrial respiratory responses in ACMs. Isoproterenol (ISO) was delivered via osmotic minipumps for 6 days and electron transport chain function measured via Seahorse Analyzer. Our data shows that WT ACMs stimulated with ISO increased basal mitochondrial respiration that was coupled to increased ADP-linked respiration and decreased reserve capacity (basal respiration in pmolO2/min/mg protein: WT Saline 255.4+/-57.0, WT-ISO 597.9+/-44.2 p = 0.0039, n = 114-144 recordings/heart in 3 hearts/group). In grk2TG ACMs, ISO-stimulation led to no statistical differences in basal respiration nor ADP-linked respiration when compared to WT-saline or grk2TG-saline ACMs. Moreover, there was a statistically significant decrease in basal respiration and ADP-linked responses when grk2TG-ISO was compared to WT-ISO ACMs (basal respiration in pmolO2/min/mg protein: grk2TG ISO: 292.9+/-28.1, WT-ISO: 597.9+/-44.2, p = 0.0119, WT-ISO n = 114-144 recordings/condition in n = 3 hearts, grk2TG ISO n= 76-96 recordings/condition in n = 2 hearts). This data suggests that ISO-stimulated mitochondrial electron transport chain activity in grk2TG ACMs is reduced and linked to decreased ADP-linked respiration, which may contribute to cardiac contractile dysfunction. In conclusion, our studies support the notion that the upregulation of GRK2 in the heart is detrimental to cardiac function as it reduces substrate-specific metabolic pathways, promotes protein remodeling, and decreases [beta]AR-mediated mitochondrial responses. Further experiments will delineate the precise mechanisms by which the non-canonical signaling of GRK2 impacts cardiac pathological conditions.