4-Hydroxynonenal dependent alteration of TRPV1-mediated coronary microvascular signaling.
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 Ca2+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.
Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA. Electronic address: ddellostritto@neomed.edu.
Daniel J
DJ
DelloStritto
Department of Biological Sciences, Kent State University, 256 Cunningham Hall, Kent, OH 44242, USA. Electronic address: psinharo@kent.edu.
Pritam
P
Sinharoy
Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA. Electronic address: pconnell1@neomed.edu.
Patrick J
PJ
Connell
Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA. Electronic address: jfahmy@neomed.edu.
Joseph N
JN
Fahmy
Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA; Department of Biomedical Sciences, Kent State University, 256 Cunningham Hall, Kent, OH 44242, USA. Electronic address: hcapelli@neomed.edu.
Holly C
HC
Cappelli
Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA. Electronic address: cthodeti@neomed.edu.
Charles K
CK
Thodeti
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.
Werner J
WJ
Geldenhuys
Department of Biological Sciences, Kent State University, 256 Cunningham Hall, Kent, OH 44242, USA. Electronic address: ddamron@kent.edu.
Derek S
DS
Damron
Department of Integrative Medical Sciences, Northeast Ohio Medical University, 4209 St. Rt. 44, Rootstown, OH 44272, USA. Electronic address: ibratz@neomed.edu.
Ian N
IN
Bratz
D000200:Action Potentials / Q000187:drug effects; D000447:Aldehydes / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001783:Blood Flow Velocity; D020013:Calcium Signaling / Q000187:drug effects; D002211:Capsaicin / Q000494:pharmacology*; D002317:Cardiovascular Agents / Q000494:pharmacology*; D003326:Coronary Circulation / Q000187:drug effects; D003331:Coronary Vessels / Q000378:metabolism / Q000503:physiopathology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003920:Diabetes Mellitus / Q000188:drug therapy / Q000378:metabolism* / Q000503:physiopathology; D004195:Disease Models, Animal; D005263:Femoral Artery / Q000378:metabolism / Q000503:physiopathology; D057809:HEK293 Cells; D006801:Humans; D015227:Lipid Peroxidation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018408:Patch-Clamp Techniques; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*; D014664:Vasodilation / Q000187:drug effects
D000447:Aldehydes; D002317:Cardiovascular Agents; D050916:TRPV Cation Channels; C493921:TRPV1 protein, mouse; C027576:4-hydroxy-2-nonenal; D003545:Cysteine; D002211:Capsaicin
4-Hydroxynonenal; Coronary regulation; Lipid peroxidation; Post-translational modification; Reactive oxygen species; TRPV1
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