ABSTRACT The Role of TRPV Ion Channels in Heat-induced Sympatholysis of Human Skeletal Muscle Feed Arteries Jayson Gifford 1,2 , Stephen J. Ives 1,3 , Songyoung Park 1,2 , Robert H. I. Andtbacka 4,5 , Michelle T. Mueller 4,5 , Gerald S. Treiman 4,5 , Christopher Ward 4,5 , Joel D. Trinity 2,4 , and Russell S. Richardson 1,2,4 1 Department of Exercise & Sport Science, University of Utah, Salt Lake City, UT; 2 Geriatric Research, Education, and Clinical Center, Salt Lake City VAMC, 3 Department of Health and Exercise Sciences, Skidmore College, Saratoga Springs, NY; 4 UT Department of Internal Medicine, University of Utah, Salt Lake City, UT; 5 Division of Surgery, Salt Lake VAMC; Salt Lake City, UT METHODS BACKGROUND AND PURPOSE  Heating isolated human skeletal muscle feed arteries (SMFA; Figure 1) to temperatures observed during exercise results in attenuated α 1 -adrenergic vasocontraction 1,2 . How this sympatholytic response to heat is initiated and whether it affects α 2 -adrenergic vasocontraction in human SMFA is unknown.  The purpose of this study was to determine if heat also inhibits α 2 -adrenergic vasocontraction in SMFA and determine if the temperature-sensitive vanilloid type transient receptor potential (TRPV) ion channels mediate this sympatholytic response. METHODS  α 1 (phenylephrine, PE) and α 2 (dexmedetomidine, DEX)-induced contractions were assessed with and without the TRPV inhibitor ruthenium red (RR) at temperatures representative of skeletal muscle during rest (37°C) and moderate-intensity exercise (39°C) using wire myography.  Endothelial function (acetylcholine, ACh) and smooth muscle function (sodium nitroprusside, SNP and potassium chloride, KCL) were also assessed under these conditions. RESULTS  At 37°C DEX elicited a small contractile response compared to PE (Figure 2).  Heating to 39°C completely prevented α 2 -induced contractions and significantly attenuated α 1 -induced contractions (Figure 2B).  Inhibition of TRPV channels restored α 1 and α 2 -induced contraction at 39°C (Figure 2). Interestingly, such inhibition potentiated α 2 , but not α 1 -induced contraction at 37°C (Figure 3).  Inhibition of TRPV channels blunted ACh relaxation without affecting smooth muscle function as assessed by KCL and SNP (Figure 4). CONCLUSIONS  Heating SMFA to temperatures common during moderate intensity exercise completely prevented the small vasocontractile response to the α 2 -agonist DEX.  Inhibition of the TRPV ion channels restores both α 1 and α 2- adrenergic contraction in the heat while blunting ACh-induced dilation.  Thus, it appears that the temperature-sensitive TRPV ion channels sense the increase in temperature to initiate the sympatholytic effect of heat, possibly in an endothelium- dependent mechanism. RESULTS  Isolated SMFA from the axillary and inguinal regions (Figure 1) were harvested from 26 subjects (Table 1) undergoing surgery related to the diagnosis of melanoma, distant to the affected skin.  Prior to the main experiment, arteries were divided into eight segments, mounted on a wire myograph (DMT, Denmark) and stretched to the length that elicited the greatest tension response to 100 mM KCL at 37°C (LTmax)  Half of the arterial segments were incubated in the TRPV inhibitor RR (30 μM) Vital Characteristics Mean ± SEM Age 53 ± 3 Male/Female (n) 18/8 Body Mass Index (kg/m 2 ) 29 ± 1 Systolic Blood Pressure (mmHg) 129 ± 4 Diastolic Blood Pressure (mmHg) 78 ± 3 Mean Arterial Blood Pressure (mmHg) 95 ± 5 Cardiovascular Medication Users/n Diuretic 3/26 Ca 2+ Channel Blocker 3/26 β-Blocker 2/26 Angiotensin Blocker 1/26 Angiotensin Converting Enzyme Inhibitor 5/26 Statin 9/26 Artery Characteristics Mean ± SEM Internal Diameter (μm) 515 ± 40 Axillary (n/total) 22 Inguinal (n/total) 4 Table 1: Subject Characteristics and Artery Characteristics DEX Dose (logM) -10 -9 -8 -7 -6 -5 -4 -3 Vasocontraction (% LTmax) 0 2 4 6 37°C Con (n= 8) 39°C Con (n= 8) 39°C RR (n= 8) † * PE Dose (logM) -9 -8 -7 -6 -5 -4 -3 Vasocontraction (% LTmax) 0 20 40 60 37°C Con (n= 11) 39°C Con (n= 11) 39°C RR (n= 11) * * * * * † A B DEX Dose (logM) -10 -9 -8 -7 -6 -5 -4 -3 Vasocontraction (% LTmax) 0 2 4 6 8 37°C Con (n=8) 37°C RR (n=8) † * * † PE Dose (logM) -9 -8 -7 -6 -5 -4 -3 Vasocontraction (% LTmax) 0 20 40 60 80 37°C Con (n=17) 37°C RR (n=17) A B * * * * KCL Dose (mM) 0 20 40 60 80 100 Vasocontraction (% LTMax) 0 20 40 60 80 100 120 37°C Con (n=6) 39°C Con (n=6) 39°C RR (n=6) SNP Dose (logM) -10 -9 -8 -7 -6 -5 -4 Vasorelaxation (%) 0 20 40 60 80 100 37°C Con (n=9) 39°C Con (n=9) 39°C RR (n=9) B C -8 -7 -6 -5 -4 -3 Vasorelaxation (%) 0 20 40 60 80 37°C Con (n=11) 39°C Con (n=11) 39°C RR (n=11) † * * ACh Dose (logM) Pre * * * A Pre Figure 2: Effect of TRPV Inhibition on Heat-induced Sympatholysis: A.) α 1 vasocontraction with PE was reduced when heated to 39ºC compared to 37ºC. Inhibition of the TRPV ion channels restored the α 1 vasocontraction at 39ºC. B.) α 2 vasocontraction with DEX elicited much smaller contractions than observed with α 1 vasocontraction. Nevertheless, heating to 39ºC completely prevented α 2 vasocontraction. Inhibition of the TRPV ion channels restored α 2 vasocontraction at 39ºC. ✝Response of condition significantly different than 37°C control condition (p<0.05). * Response to dose significantly different than 37°C control condition (p<0.05). Figure 3: Effect of TRPV Inhibition on Adrenergic Vasocontraction at 37ºC: A.) α 1 vasocontraction with PE at 37°C was not significantly affected by TRPV inhibition (P=0.24). B.) α 2 vasocontraction with DEX was significantly augmented at when the TRPV ion channels were inhibited at 37°C. ✝Response of condition significantly different than 37°C control condition (p<0.05). * Response to dose significantly different than 37°C control condition (p<0.05). Figure 4: Effect of TRPV Inhibition and Heat on Endothelial and Smooth Muscle Function: A.) Endothelial-dependent vasorelaxation with ACh was significantly attenuated with TRPV inhibition (P<0.05). B.) Endothelial-independent vasorelaxation was not significantly affected by heat or TRPV inhibition (P>0.05). C.) Receptor-independent vasocontraction with KCL was not significantly affected by heat and TRPV inhibition (P>0.05).  As has been postulated to be the case for larger, proximal arteries, the α 2 adrenoreceptors appear to play a minor role in adrenergic vasocontraction in SMFA in comparison to the α 1 adrenoreceptors 4 .  α 2 –adrenergic vasocontraction, like α 1 –adrenergic vasocontraction, is attenuated when SMFA are heated to temperatures common to skeletal muscles during moderate-intensity exercise. In fact, heat completely prevented α 2 –adrenergic vasocontraction in response to DEX, suggesting that the α 2 vasocontraction may be more potently affected by heat-induced sympatholysis than α 1 contraction.  As inhibiting the TRPV ion channels with RR restored both α 1 and α 2 vasocontraction at 39ºC and inhibited endothelial-dependent dilation, it appears that these ion channels may be involved in sensing the rise in temperature and initiating the sympatholytic effect of heat in an endothelial-dependent mechanism.  The TRPV ion channels appear to modulate vascular function, particularly α 2 vasocontraction, at normothermic temperatures (i.e. 37ºC) as inhibition of the channels resulted in significantly augmented α 2 vasocontraction. 1. Ives SJ, Andtbacka RHI, Kwon SH, ShiuY-T, Ruan T, Noyes RD, Zhang Q-J, Symons JD, and Richardson RS. Journal of Applied Physiology 113: 2012. 2. Ives SJ, Andtbacka RHI, Noyes RD, McDaniel J, Amann M, Witman MAH, Symons JD, Wray DW, and Richardson RS. Experimental Physiology 96: 2011. 3. Ives SJ, Andtbacka RHI, Park SY, Donato AJ, Gifford JR, Noyes RD, Lesniewski LA, and Richardson RS. Acta Physiologica 206: 135-141, 2012. 4. Faber JE. . Circulation Research 62: 37-50, 1988. Figure 1: Muscle Feed Artery 3 Funded in part by NIH grant PO1 HL 091830 and VA Merit Grant (E6910R) Correspondence: Jayson.Gifford@Utah.edu  α 1 (PE, 10 -9 -10 -3 Log M), and α 2 agonist (DEX, 10 -10 -10 -3 Log M ) concentration response curves were performed at 37°C and 39°C  Endothelial-dependent and independent relaxation were assessed in preconstricted arteries with Ach (10 -7 -10 -3 Log M) and SNP (10 -9 -10 -4 Log M) at 37°C and 39°C  Vasocontraction data are represented as a percentage of LTmax  Data were analyzed using one way ANOVA and repeated-measures ANOVA followed by Dunnet’s post hoc test. Data represent mean ± SEM. REFERENCES DISCUSSION