👤 Russell Desnoyer

Plasma accumulation of the gut microbial metabolite 4-ethylphenylsulfate (4EPS), derived from dietary amino acid, tyrosine, has been associated with cardiovascular, renal, metabolic, and neurological disorders. AngII (angiotensin II) infusion increases circulating 4EPS in mice, suggesting a potential mechanistic role. We hypothesized that 4EPS modulates AngII-regulated pathophysiology and disease progression by directly inhibiting AT1R (angiotensin II type 1 receptor). This hypothesis was tested by combining AT1R pharmacology, cell signaling assays, ex vivo vascular studies, an AngII-induced aortic aneurysm growth model, and plasma proteomics analysis. in vitro, 4EPS reduced the binding of both AngII and the antagonist candesartan to AT1R and suppressed AngII-induced calcium signaling. Ex vivo, 4EPS attenuated AngII-mediated vasoconstriction. In vivo, high-fat diet-fed ApoE-null mice coinfused with AngII and 4EPS showed significant blunting of blood pressure elevation and a marked reduction in aortic aneurysm-related mortality compared with mice infused with AngII alone. Analysis of aortic remodeling revealed increased elastin preservation and decreased thickening of the intimal and medial layers in 4EPS-treated animals. Plasma proteomics indicated alterations in actin-cytoskeletal signaling pathways consistent with reduced activation of ERK (extracellular-regulated kinase) 1/2, filamin-A, and proteins involved in vascular smooth muscle cell motility. These findings identify 4EPS as a benign, endogenous AT1R antagonist that diminishes AngII-mediated hemodynamic and vascular pathology. By suppressing cytoskeletal signaling associated with vascular remodeling, 4EPS provides significant protection against hypertension and aortic aneurysm progression in mice, revealing a previously unrecognized protective role for a gut microbial metabolite in modulating renin-angiotensin system activity. Show less

Compartmentalization of cAMP-dependent protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) targets PKA to distinct subcellular locations in many cell types. However, the question of whether AKAP-mediated PKA anchoring in the heart regulates cardiac contractile function has not been addressed. We disrupted AKAP-mediated PKA anchoring in cardiac myocytes by introducing, via adenovirus-mediated gene transfer, Ht31, a peptide that binds the PKA regulatory subunit type II (RII) with high affinity. This peptide competes with endogenous AKAPs for RII binding. Ht31P (a proline-substituted derivative), which does not bind RII, was used as a negative control. We then investigated the effects of Ht31 expression on RII distribution, Ca(2+) cycling, cell shortening, and PKA-dependent substrate phosphorylation. By confocal microscopy, we showed redistribution of RII from the perinuclear region and from periodic transverse striations in Ht31P-expressing cells to a diffuse cytosolic localization in Ht31-expressing cells. In the presence of 10 nmol/L isoproterenol, Ht31-expressing myocytes displayed an increased rate and amplitude of cell shortening and relaxation compared with control cells (uninfected and Ht31P-expressing myocytes); with isoproterenol stimulation we observed decreased time to 90% decline in Ca(2+) but no significant difference between Ht31-expressing and control cells in the rate of Ca(2+) cycling or amplitude of the Ca(2+) transient. The increase in PKA-dependent phosphorylation of troponin I and myosin binding protein C on isoproterenol stimulation was significantly reduced in Ht31-expressing cells compared with controls. Our results demonstrate that, in response to beta-adrenergic stimulation, cardiomyocyte function and substrate phosphorylation by PKA is regulated by targeting of PKA by AKAPs. Show less