| Author |
DelloStritto, Daniel J. Sinharoy, Pritam Connell, Patrick J. Fahmy, Joseph N. Cappelli, Holly C. Thodeti, Charles K. Geldenhuys, Werner J. Damron, Derek S. Bratz, Ian N. |
| Description |
Author Affiliation: DelloStritto DJ ( Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA. Electronic address: ddellostritto@neomed.edu.); Sinharoy P ( Department of Biological Sciences, Kent State University, 256 Cunningham Hall, Kent, OH 44242, USA. Electronic address: psinharo@kent.edu.); Connell PJ ( Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA. Electronic address: pconnell1@neomed.edu.); Fahmy JN ( Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA. Electronic address: jfahmy@neomed.edu.); Cappelli HC ( Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA); Thodeti CK ( Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA. Electronic address: cthodeti@neomed.edu.); Geldenhuys WJ ( Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, P.O. Box 9500, Morgantown, WV 26506, USA. Electronic address: werner.geldenhuys@hsc.wvu.edu.); Damron DS ( Department of Biological Sciences, Kent State University, 256 Cunningham Hall, Kent, OH 44242, USA. Electronic address: ddamron@kent.edu.); Bratz IN ( Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA. Electronic address: ibratz@neomed.edu.) |
| Abstract |
We demonstrated previously that TRPV1-dependent regulation of coronary blood flow (CBF) is disrupted in diabetes. Further, we have shown that endothelial TRPV1 is differentially regulated, ultimately leading to the inactivation of TRPV1, when exposed to a prolonged pathophysiological oxidative environment. This environment has been shown to increase lipid peroxidation byproducts including 4-Hydroxynonenal (4-HNE). 4-HNE is notorious for producing protein post-translation modification (PTM) via reactions with the amino acids: cysteine, histidine and lysine. Thus, we sought to determine if 4-HNE mediated post-translational modification of TRPV1 could account for dysfunctional TRPV1-mediated signaling observed in diabetes. Our initial studies demonstrate 4-HNE infusion decreases TRPV1-dependent coronary blood flow in C57BKS/J (WT) mice. Further, we found that TRPV1-dependent vasorelaxation was suppressed after 4-HNE treatment in isolated mouse coronary arterioles. Moreover, we demonstrate 4-HNE significantly inhibited TRPV1 currents and Ca entry utilizing patch-clamp electrophysiology and calcium imaging respectively. Using molecular modeling, we identified potential pore cysteines residues that, when mutated, could restore TRPV1 function in the presence of 4-HNE. Specifically, complete rescue of capsaicin-mediated activation of TRPV1 was obtained following mutation of pore Cysteine 621. Finally, His tag pull-down of TRPV1 in HEK cells treated with 4-HNE demonstrated a significant increase in 4-HNE binding to TRPV1, which was reduced in the TRPV1 C621G mutant. Taken together these data suggest that 4-HNE decreases TRPV1-mediated responses, at both the in vivo and in vitro levels and this dysfunction can be rescued via mutation of the pore Cysteine 621. Our results show the first evidence of an amino acid specific modification of TRPV1 by 4-HNE suggesting this 4-HNE-dependent modification of TRPV1 may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes. |
