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  • br Material and methods br

    2024-04-13


    Material and methods
    Results
    Discussion GRK2 activity has increasingly been shown to contribute to the progression of numerous diseases and organ dysfunction [5], [6]. For instance, a large body of work over the last two decades has identified GRK2 as a major regulator of cardiac dysfunction, wherein genetic ablation or inhibition of GRK2 is sufficient to protect the heart from ischemic insult, prevent adverse cardiac remodeling during chronic HF and prevent βAR desensitization to enhance cardiomyocyte contractility [4], [14], [15], [16], [17]. Thus the identification and validation of small molecules inhibitors of GRK2 remains an important priority and has led to the discovery of a number of several types of antagonists, including RNA aptamers, peptides and various small molecules with distinct chemical and structural properties [6], though generally with poor potency, selectivity or physicochemical properties unfit for therapeutics. However, the Food and Drug Administration-approved SSRI paroxetine was identified using a high throughput aptamer displacement assay to bind directly to GRK2 to inhibit its ability to phosphorylate rhodopsin, tubulin and thyrotropin-releasing hormone receptor, while increasing βAR-stimulated cardiomyocyte (in vitro) and cardiac (in vivo) contractility [7]. Subsequently, paroxetine has been shown to reverse cardiac dysfunction induced by either ischemia [8] or high fat diet [18], inhibit T cell activation [19] and decrease gedatolisib cytokine production [20]. Despite the increasing use of paroxetine in studies in which its effects are attributed to inhibition of receptor desensitization processes classically engaged by GRK2, such as βarr recruitment and GPCR internalization, no studies have confirmed whether it actually attenuates these responses. The mechanistic impact of paroxetine on proximal βAR signaling is of particular interest in light of the aforementioned studies which detailed its beneficial effects on cardiac structure and function following injury [8], [18], suggestive of a GRK2 inhibition-mediated reversal of βAR desensitization. Thus, using a variety of molecular approaches we have determined that, consistent with GRK2 inhibition, paroxetine indeed attenuates proximal βAR signaling processes, including βarr recruitment, receptor phosphorylation and internalization. An unexpected result observed in our study was the impact of the maximal concentration of fluoxetine on β2AR phosphorylation and βarr2 recruitment to β2AR following ISO stimulation. Although fluoxetine did not block ISO-mediated β2AR phosphorylation and βarr2 recruitment as effectively as paroxetine, a moderate level of inhibition was achieved. Fluoxetine was previously shown not to impact βAR-attributable effects, including cardiac contractility and post-ischemic cardiac remodeling in vivo at concentrations equimolar to paroxetine [8], thus providing an in-class SSRI negative control for testing against paroxetine's additional GRK2 inhibitory effects. Since βAR-mediated effects on cardiomyocyte contractility and maladaptive cardiac remodeling following injury are attributed to β1AR signaling in particular [2], and β1AR signaling was unaffected by fluoxetine in our study, it is possible that the effects of fluoxetine on GRK-dependent β2AR signaling we observed are not functionally relevant in vivo, at least in the heart. Alternatively, it has been demonstrated that different cell types express unique cohorts of GRK isoforms such that, beyond GRK2, β2AR has been shown to be predominantly sensitive to GRK6 in HEK 293 cells and to GRK3 in U2OS cells [10], [11]. Thus, although fluoxetine did not inhibit GRK-mediated phosphorylation activity in vitro or downstream functional effects on cardiac contractility in vivo [7], [8], whether it has affinity for and can partially inhibit GRK3 in U2OS cells has not been established. As such, the applicability of using fluoxetine as a negative control for paroxetine-mediated effects may need to be validated on a cell-by-cell basis. Regardless, and despite its proximal effects on β2AR phosphorylation and βarr2 recruitment in U2OS cells, ultimately fluoxetine did not inhibit ligand-stimulated β2AR internalization, suggesting that even if high concentrations of fluoxetine can negatively impact proximal GRK-dependent β2AR signaling responses, the threshold for inhibiting subsequent downstream processes including receptor internalization has not been achieved. In all, our results suggest that while the use of fluoxetine as a negative control for comparative studies with paroxetine should be validated in a receptor- and cell type-dependent manner, its lack of effect on proximal β1AR signaling responses are consistent with its previous use in cardiac studies.