0022-3565/10/3352-451–457$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 335, No. 2 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 172700/3637304 JPET 335:451–457, 2010 Printed in U.S.A.

Increased Blood Pressure and Hyperdynamic Cardiovascular Responses in Carriers of a Common Hyperfunctional Variant of Adenylyl 6

Gary J. Hodges, Robert Gros, Robert A. Hegele, Stan Van Uum, J. Kevin Shoemaker, and Ross D. Feldman School of Kinesiology (G.J.H.) and Departments of Physiology and Pharmacology (R.G., J.K.S., R.D.F.) and Medicine (R.G., R.A.H., S.V.U., R.D.F.), University of Western Ontario, London, Ontario, Canada; and Vascular Biology Research Groups, Robarts Research Institute, London, Ontario, Canada (R.G., R.A.H., R.D.F.)

Received July 9, 2010; accepted August 17, 2010 Downloaded from

ABSTRACT (ADCY) is a critical regulator of metabolic and dilator responsiveness), and with lower body negative pressure

cardiovascular function. We have identified a genetic variant [to test forearm vasoconstrictor and heart rate (HR) responsive- jpet.aspetjournals.org (A674S) in ADCY isoform 6 (ADCY6). Subsequent studies dem- ness] in a subsample of 21 subjects. At rest, cardiac output and onstrated that the expression of this ADCY6 variant paralleled blood pressure were higher in carriers of ADCY6 A674S. This an increase in adenylyl cyclase-mediated functions. However, was paralleled by an increase in plasma renin activity, but not in the impact of this hyperfunctional variant on cardiovascular plasma norepinephrine. During handgrip exercise, FBF and function is unknown. Therefore, we evaluated the hemody- vasodilator responses were greater in carriers of ADCY6 namic profile of carriers of ADCY6 A674S. The association of A674S. Responses to reactive hyperemia were not different ADCY6 A674S with anthropometric and hemodynamic param- between genotypes. With lower body negative pressure, the eters was assessed in 364 healthy white subjects. The allele HR response to this orthostatic stress was markedly higher in at ASPET Journals on March 6, 2015 encoding this variant was present in 6.9% of the subjects, and carriers of ADCY6 A674S. These data indicate that the relatively those individuals had increased blood pressure. To determine common hyperfunctional ADCY6 A674S variant underlies a the hemodynamic basis for increased blood pressure in carriers hyperdynamic cardiovascular response and increased blood of ADCY6 A674S, we assessed forearm blood flow (FBF) and pressure. cardiac output at rest, during handgrip exercise (to test vaso-

Introduction functional effects including vascular reactivity, cellular growth, hypertrophy, and apoptosis (Gros et al., 2006). Fur- The adenylyl (ADCYs) are a ubiquitously ex- thermore, alterations in the regulation of adenylyl cyclase pressed family of that catalyze the generation of activity have been implicated in the pathogenesis of hyper- cAMP from ATP. They regulate a broad range of cellular tension, heart failure, and diabetes (Moxham and Malbon, functions (Patel et al., 2001) and are critical effectors for a 1996; Roth et al., 1999, 2002; Tepe and Liggett, 1999; Ma- number of G protein-coupled receptors (GPCRs). Adenylyl tsumoto et al., 2005). cyclase activation has been suggested to be the rate-limiting Nine membrane-bound isoforms of adenylyl cyclase have step in the GPCR signaling cascade (Ostrom et al., 2000). In been cloned, grouped into three major subfamilies: group 1, vascular cells, activity of adenylyl cyclase regulates acute ADCY1, ADCY3, ADCY8; group 2, ADCY2, ADCY4, ADCY7; and group 3, ADCY5, ADCY6 (Patel et al., 2001; Ludwig and This study was supported by the Heart and Stroke Foundation of Ontario Seuwen, 2002). In addition, ADCY9 has been characterized [Grant T6624] (to R.D.F.), the Natural Sciences and Engineering Research as a distinct (and atypical) isoform (Sunahara and Taussig, Council of Canada (J.K.S.), and the Canadian Institutes of Health Research 2002) with restricted expression, and a soluble adenylyl cy- (J.K.S). R.G. is supported by a New Investigator Award from the Heart and Stroke Foundation of Canada. clase has been characterized that is the predominant form in G.J.H. and R.G. contributed equally to this work. mammalian sperm (Wuttke et al., 2001). Each isoform has a Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. specific pattern of tissue/organ distribution and a specific doi:10.1124/jpet.110.172700. pattern of regulation by G proteins, calcium/calmodulin, and

ABBREVIATIONS: ADCY, adenylyl cyclase; FBF, forearm blood flow; HR, heart rate; FMD, flow-mediated dilation; LBNP, lower body negative pressure; TPR, total peripheral resistance; GPCR, G protein-coupled receptor; BSA, body surface area. 451 452 Hodges et al. protein kinases (Wuttke et al., 2001; Sunahara and Taussig, on local advertising and e-mail invitations for volunteers within the 2002; Wang and Brown, 2004). Robarts Research Institute and the University of Western Ontario. Variability in cAMP synthesis was thought to be deter- Of the 25 individuals that we identified as expressing the ADCY6 mined predominantly either by the extent of adenylyl cyclase genetic variant we were able to recruit seven individuals (three expression, the specific characteristics of the GPCRs linked males) all of whom were heterozygotic carriers of ADCY6 A674S (hereafter referred to as the ADCY6 variant group) for more detailed to activation, or variation in the concentration of the analysis of cardiovascular responsiveness. Fourteen individuals “regulatory factors” (i.e., G proteins, protein kinases, ions) (seven males) who did not express the genetic variant were recruited (Feldman and Gros, 1998). The importance of “genetic” vari- as a control group. The mean Ϯ S.E.M. ages of the control and ability has been unappreciated, that is, the expression of ADCY6 variant groups were 26 Ϯ 1 and 26 Ϯ 1 years, respectively. genetic structural variants of the enzyme that differ by func- By self-report, all participants were nonsmokers who were free of tion and/or activity. However, studies by us and others have cardiovascular and neurological disease. Participants reported to the suggested that variability in the expression of ADCY genetic laboratory after at least a 3-h fast and having abstained from caf- variants may be an important regulator of adenylyl cyclase- feine, alcohol, and exercise for a minimum of 12 h. Before experi- mediated responses (Ikoma et al., 2003; Small et al., 2003; mentation, participants were encouraged to maintain typical water Nordman et al., 2008). consumption and sleeping behaviors. Although the menstrual phase Several genetic variants have been described for a range of was not assessed, data between males and females displayed no perceptible difference and were, therefore, pooled. Informed consent G proteins and GPCRs linked to adenylyl cyclase (Rana et al., was obtained for all analyses, with approval from the University of 2001; Siffert, 2003). Expression of these variants leads to Western Ontario Research Ethics Review Board. Downloaded from alterations in receptor-mediated activation of adenylyl cy- Measurements. During the initial screening, the five measure- clase and alterations in downstream effector pathways. The ments of seated blood pressure and heart rate were averaged and identification of dysfunctional genetic variants of ADCY recorded (BP Tru, Vancouver, British Columbia, Canada). A 10-ml presently is limited to those in ADCY6, ADCY3, and ADCY9, blood sample was taken for biochemical and genetic determinations. of which the latter has a much more restricted distribution Data on sex, weight, height, waist circumference, and smoking sta- than other isoforms (Small et al., 2003). A single-nucleotide tus were also obtained. Waist circumferences were normalized by polymorphism in intron 17 of encoding the very widely sex, based on sex-specific Adult Treatment Panel III-recommended jpet.aspetjournals.org expressed ADCY6 isoform (Wang and Brown, 2004) was upper limits [National Cholesterol Education Program (NCEP) Ex- pert Panel on Detection, Evaluation, and Treatment of High Blood identified in a Japanese population (Nordman et al., 2008). Cholesterol in Adults (Adult Treatment Panel II]. However, the impact of this variant on adenylyl cyclase func- In the follow-up study of cardiovascular responsiveness, all tion is currently unknown. In our initial studies, we discov- measurements were performed with the participants in the supine ered a relatively common (ϳ7% in whites) missense single- position in a quiet, darkened room kept at a relatively constant nucleotide polymorphism in ADCY6, with the trivial name of temperature (21–23°C). Heart rate was recorded via a three-lead ADCY6 A674S (Gros et al., 2005). In a mammalian vascular ECG (Pilot 9200; Colin Medical Instruments, San Antonio, TX), at ASPET Journals on March 6, 2015 cell system, specifically rat vascular smooth muscle cells and blood pressure was measured continuously by finger photop- transfected using adenoviral constructs, we have shown that lethysmography (Finapres 2300; Ohmeda, Englewood, CO) and expression of the ADCY6 A674S variant isoform resulted in validated intermittently via sphygmomanometry. Brachial artery enhanced adenylyl cyclase activity and function compared mean blood velocity (4.7 MHz) and diameter (10-MHz imaging transducer) were assessed by pulsed-wave Doppler ultrasound with expression of wild-type ADCY6. Further, in humans (echo ultrasound imaging; GE Vingmed System Five; GE Health- expression of the ADCY6 A674S variant correlates with care, Little Chalfont, Buckinghamshire, UK). Artery images were 1) increased adenylyl cyclase enzymatic activity and 2) recorded on a S-VHS videocassette recorder (SVO-9500 MDP; enhanced adenylyl cyclase-mediated vascular responses Sony, Tokyo, Japan). Cardiac output was derived on a beat-by- (Gros et al., 2007). However, whether there were detect- beat basis from the transfer function of the finger pulsatile pres- able alterations in cardiovascular or phenotypic character- sure wave form, as validated for these maneuvers (Shoemaker et istics associated with expression of this variant in humans al., 2007; Frances et al., 2008). All analog data were sampled at was unknown. 1000 Hz by using a data-acquisition system (PowerLab; ADInstru- Consequently, the present studies were performed to as- ments, Colorado Springs, CO). Cardiac output was normalized to body surface area (BSA) (Q ) by using the equation of DuBois and sess the association of the ADCY6 A674S variant with phe- c DuBois (1916): BSA (m2) ϭ 0.007184 ϫ weight (kg)0.425 ϫ height notypic characteristics within a large group of healthy, (cm)0.725. Stroke volume (corrected for BSA) was determined from younger human subjects. In addition, in a subset of these the quotient of cardiac output and HR. Forearm blood flow (FBF) subjects we examined the impact of expression of this genetic was calculated as FBF ϭ blood velocity ϫ r2 ϫ 60/V, where r is the variant on hemodynamic responses both at rest and with brachial artery radius, 60 is to convert flow from s to min, and V exercise and orthostatic stress. Data demonstrate that carri- is volume of the forearm. ers of the ADCY6 A674S genetic variant have increased blood Handgrip Exercise. Adenylyl cyclase activation by a range of pressure related to a hyperdynamic profile consistent with metabolic factors is an important determinant of the vasodilator the effect of increased adenylyl cyclase function. response to exercise. To assess whether those subjects expressing the ADCY6 genetic variant demonstrated an exaggerated vasodilatory response to exercise we performed handgrip protocols. Baseline mea- Materials and Methods sures were recorded for 60 s, followed by three repeated 5-s handgrip contractions with 60 s of recovery after each trial. The handgrip Participants. A total of 364 healthy white volunteers (age range contraction intensity was 40% of the maximal voluntary contraction 18 to 50 years) were screened. Exclusion criteria included, but were force, determined before this protocol from three to five trials of not limited to, history of cardiovascular events, average alcohol in- voluntary maximal hand gripping. The dilator response to this con- take Ͼ2 units per day, pregnancy, and use of antihypertensive/blood traction was assessed over a 5-s average immediately after each pressure-altering drugs or anticoagulants. Recruitment was based contraction and averaged to produce the mean FBF responses. Effect of ADCY6 Variant on Blood Pressure, Vascular Function 453

Flow-Mediated Dilation. Beyond adenylyl cyclase activation, Results vasodilator responses to exercise may also be mediated by endothe- lial factors (e.g., nitric oxide) in response to enhanced endothelial Genotype and Allele Frequencies of ADCY6 A674S shear stress (Clifford and Hellsten, 2004). This flow-mediated dila- Variant. In the population of 364 healthy white volunteers tion (FMD) is mediated predominantly by endothelial responses. we identified 25 individuals (24 heterozygotes and 1 homozy- Thus, to determine whether any alterations seen in handgrip re- gote) who had ADCY6 p.A674S (g.2714G Ͼ T) variant in sponses might be related to endothelial factors a reactive hyperemia their genomes. The genotype frequencies for 2714G/G, and FMD protocol was performed. While the subjects were supine, a 2714G/T, and 2714T/T were 0.931, 0.066, and 0.003, respec- rapid inflation/deflation pneumatic cuff (D.E. Hokanson Inc., Belle- tively. The 2714T allele frequency was 0.035, and there was vue, WA) was placed around the right forearm immediately distal to no deviation of genotype frequencies from Hardy-Weinberg the olecranon process to provide forearm ischemia and avoid exces- expectations. These findings were similar to our previously sive myogenic contributions to brachial artery dilation after ischemia reported frequency of ADCY6 A674S in a white population (Corretti et al., 2002; Betik et al., 2004). Ultrasound parameters (Gros et al., 2007). were set to optimize longitudinal, B-mode images of the lumen/ arterial wall interface. Specifically, central and forearm hemody- ADCY6 Variant Associations. Participants were classi- Ͼ namics were recorded during 5 min of baseline, forearm cuff inflation fied according to g.2714G T genotype with the single to Ͼ200 mm Hg for 5 min, and then for 3 min after cuff deflation. 2714T/T homozygote added to the 2714G/T heterozygote Lower Body Negative Pressure. To determine whether any group (S674 subjects), whereas the 2714G/G homozygotes alterations in vasodilator responses between groups might be related formed the comparator group (A674 subjects). Seated systolic to a generalized vascular “hyper-reactivity” characterized by both blood pressure was increased in ADCY6 S674 subjects com- Downloaded from exaggerated vasodilator and vasoconstrictor responses we assessed pared with ADCY6 A674 subjects (p Ͻ 0.05; Table 1). Dia- blood flow and cardiac hemodynamic responses after lower body stolic blood pressure also tended to be higher in the ADCY6 negative pressure (LBNP). The legs and hips were sealed in an variant group, although this difference was of borderline air-tight container connected to a vacuum source (Kimmerly and significance (p ϭ 0.05) (Table 1). Shoemaker, 2002). Data collection for each test commenced after 5 Waist circumference was reduced in the ADCY6 S674 vari- min of undisturbed rest in the supine position. An initial 5-min ant group compared with the ADCY6 A674-expressing indi- jpet.aspetjournals.org baseline period was followed by 5 min of LBNP at Ϫ40 mm Hg and 5 min of rest at atmospheric pressure. viduals (controls) (Table 1). In contrast, mean body mass Blood Processing and Analysis. Blood samples were obtained index did not differ according to genotype. from an antecubital fossa vein at baseline and during the LBNP Baseline Hemodynamic and Biochemical Assess- protocol. A 3-ml sample of whole blood for determination of cat- ments. Of the 25 individuals that we identified as expressing echolamines was mixed with 75 ␮l of EGTA-glutathione anticoagu- the ADCY6 genetic variant we were able to recruit seven lant and was centrifuged at 4°C for 15 min. A 6-ml sample of whole individuals (three males) who were heterozygotic carriers of blood for determination of renin activity was collected in two 3-ml ADCY6 A674S (hereafter referred to as the ADCY6 variant at ASPET Journals on March 6, 2015 samples in 4-ml BD vacutainers (BD Biosciences, San Jose, CA), group) for more detailed analysis of cardiovascular respon- each containing 7.2 mg of EDTA, and centrifuged at 4°C for 15 min. siveness. Fourteen individuals (seven males) who did not Catecholamines were extracted from the plasma by adding acid- express the genetic variant were recruited as a control group. washed alumina and mixing by inversion. The pellet was washed Baseline assessments were performed after approximately with distilled water, and the catecholamines were released into 0.1 20 min of quite supine rest. Systolic, mean arterial, and M perchloric acid and separated by centrifugation. The concentra- tions of norepinephrine were determined in duplicate by high-per- diastolic blood pressure all were significantly increased in formance liquid chromatography with electrochemical detection the ADCY6 variant group (Table 2). In addition, compared (2465 ElectroChemical Detector; Waters, Milford, MA) and quanti- with the control group, those expressing the ADCY6 vari- fied by determining the area under the peak. Working and internal ant had increased mean heart rate and cardiac output standards were used to correct the measurements. Renin activity (both p Ͻ 0.05). assays were performed commercially by ␥-Dynacare Medical Labo- Under baseline conditions, plasma renin activity was ratories (London, Ontario, Canada). greater in the ADCY6 variant group (0.63 Ϯ 0.11 ng/liter/s) DNA Analysis. Genomic DNA was extracted from whole blood as (p Ͻ 0.05) compared with control subjects (0.31 Ϯ 0.11 ng/ described previously (Gros, et. al, 2005). In brief, genotyping of the liter/s). In contrast, baseline levels of plasma norepinephrine ADCY6 A674S variant was performed by using exon-specific DNA amplification followed by purification using shrimp alkaline phos- TABLE 1 phatase (Roche Diagnostics, Mannheim, Germany) and exonuclease Anthorpometric and hemodynamic profiles of individuals in screening I (New England Biolabs, Ipswich, MA) and capillary pheresis in an study automated DNA sequencer as described previously (Cao and Hegele, Values are mean Ϯ standard error. Waist circumference was gender-corrected by 2003). using the Adult Treatment Panel III “upper limit of normal” standards of 88 cm for Data Analysis. For the initial population screening, the statisti- females and 102 cm for males (National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in cal significance of differences in quantitative variables between con- Adults (Adult Treatment Panel II). trol and ADCY6 variant groups was determined by Student’s t test Characteristic Control ADCY6 Variant for unpaired data (Prism 4.0; GraphPad Software Inc., San Diego, CA). In the subsample studied for cardiovascular responsiveness, the Number (male/female) 339 (105/234) 25 (8/17) Ϯ Ϯ associations of genotype on baseline values and responsiveness (de- Age (yr) 28 1262 Waist circumference 86 Ϯ 183Ϯ 1* fined as the change in each variable from rest) were assessed by (%, gender-corrected) using a mixed one-way repeated measures analysis of variance (SAS Body mass index (kg ⅐ mϪ2) 24 Ϯ 124Ϯ 1 version 9.1; SAS Institute, Cary, NC). Post hoc analysis of significant Systolic blood pressure (mm Hg) 110 Ϯ 1 113 Ϯ 1* interactions was assessed by using Bonferroni analysis. Data are Diastolic blood pressure (mm Hg) 69 Ϯ 172Ϯ 1 ⅐ Ϫ1 Ϯ Ϯ presented as mean Ϯ standard error, and statistical significance was Heart rate (beats min ) 74 1753 assumed when p Ͻ 0.05. * p Ͻ 0.05 vs. control (wild-type). 454 Hodges et al.

TABLE 2 Baseline anthropometric and cardiovascular status in participants of hemodynamic studies Values are mean Ϯ standard error.

Characteristics Control ADCY6 Variant Number (male/female) 14 (7/7) 7 (3/4) Age (yr) 26 Ϯ 126Ϯ 1 Heart rate (beats ⅐ minϪ1) 64 Ϯ 470Ϯ 4* Mean arterial pressure (mm Hg) 81 Ϯ 590Ϯ 6* Systolic blood pressure (mm Hg) 110 Ϯ 5 124 Ϯ 5* Diastolic blood pressure (mm Hg) 67 Ϯ 376Ϯ 3* ⅐ Ϫ1 ⅐ 2 Ϯ Ϯ Qc (L min m ) 3.01 0.11 3.66 0.28* ⅐ Ϫ2 ⅐ Ϫ1 Ϯ Ϯ SVc (L m beat ) 0.047 0.001 0.052 0.003 ⅐ Ϫ1 ⅐ Ϫ1 ⅐ 2 Ϯ Ϯ TPRc (mm Hg L min m ) 27.1 0.9 25.5 1.8

Qc, cardiac output corrected for body surface area; SVc, stroke volume corrected for body surface area; TPRc, TPR corrected for body surface area. * p Ͻ 0.05 vs. control (wild type). were similar between groups (control ϭ 1.28 Ϯ 0.25 nM; ADCY6 variant ϭ 1.38 Ϯ 0.24 nM) (p Ͼ 0.1). Fig. 2. The mean FBF responses from the control (filled bars) and ADCY6 variant (empty bars) groups during baseline, reactive hyperemia, and Downloaded from p Ͻ 0.05 from baseline. Data represent the ,ء .Handgrip Exercise Responses. Baseline measures of flow-mediated dilation FBF did not differ between genotypic groups (p Ͼ 0.05). With mean Ϯ standard error. exercise, FBF increased in both ADCY6 variant and control groups. However, compared with control subjects, the exer- 0.05). Thus, endothelial-mediated vasodilatory responses cise-induced increase in FBF was almost four times greater were not enhanced in individuals expressing the ADCY6 in those expressing the ADCY6 variant [Fig. 1; increased variant.

ϳ30% in the control subjects (p Ͻ 0.05) versus ϳ120% in the Lower Body Negative Pressure. During LBNP, both jpet.aspetjournals.org ADCY6 variant group (p Ͻ 0.001)]. groups demonstrated reductions in FBF (p Ͻ 0.05; Fig. 3A). Reactive Hyperemia and Flow-Mediated Dilation. To However, the forearm vascular response to LBNP was not determine whether the increase in FBF seen after handgrip greater in the ADCY6 variant group. In fact, the increase in exercise was caused by a generalized enhancement of vaso- changes in total peripheral resistance (TPRc) in response to dilatory responses not specific to adenylyl cyclase-mediated LBNP was greater in the control than the ADCY6 variant effects, we next assessed the (endothelial-dependent) effect of group (Fig. 3B; p Ͻ 0.05). In contrast to the unchanged or reactive hyperemia on FBF. Compared with control subjects, blunted vascular responses to LBNP, the cardiac chrono- at ASPET Journals on March 6, 2015 the ADCY6 variant group displayed no difference in baseline tropic responses to LBNP, mediated indirectly via barorecep- FBF (p ϭ 0.181; Fig. 2) before the FMD procedure. In addi- tor unloading, were exaggerated in the ADCY6 variant tion, the initial hyperemic response to the deflation of the cuff group. Compared with the insignificant increase in HR seen was unaffected by genetic status (p Ͼ 0.05; Fig. 2). The extent in controls after LBNP, in ADCY6 variants, HR was signifi- of FMD in the brachial artery (between 45 and 90 s after cuff cantly increased (p Ͻ 0.05) (Fig. 4). The increase in HR was Ϫ1 deflation) did not differ between the two groups, with the 14 beats ⅐ min greater in the ADCY6 variant group during increases in the diameters of the control group being 8.7 Ϯ LBNP versus controls (p Ͻ 0.05) (Fig. 4). It is noteworthy 0.5% and the ADCY6 variant group being 9.2 Ϯ 0.7% (p Ͼ that cardiac output in the ADCY6 variant group was signif-

Fig. 3. Effect of LBNP on forearm blood flow and total peripheral resis- tance. A, changes in forearm blood flow during LBNP. There were no differences between the control (filled bars) and ADCY6 variant (empty bars) groups at baseline. Both groups show significant vasoconstriction and comparable FBF responses to LBNP and recovered to values not p Ͻ 0.05 from baseline. Data represent the ,ء .different from baseline Ϯ mean standard error. B, changes in total peripheral resistance (TPRc) Fig. 1. FBF responses in the handgrip exercise study. The three trials of during LBNP. Note the large increase in TPRc in the control group with exercise were combined for the control (filled bars) and ADCY6 S674 LBNP versus the nonsignificant increase in the ADCY6 variant group. p Ͻ 0.05 between groups. †, p Ͻ 0.05 from baseline. Data represent the ,ء p Ͻ 0.05 between groups. †, p Ͻ 0.05 from ,ء .empty bars) variant groups) baseline within groups. Data represent the mean Ϯ standard error. mean Ϯ standard error. Effect of ADCY6 Variant on Blood Pressure, Vascular Function 455

unknown. The present studies have confirmed that the ADCY6 A674S variant is present in ϳ7% of whites and further demonstrate that its expression is associated with increased blood pressure and hyperdynamic cardiovascular responses characterized by 1) increased cardiac output and heart rate, 2) elevated plasma renin activity, 3) a markedly greater vasodilator response to handgrip exercise, and 4) augmented heart rate response to baroreceptor unloading (LBNP) without impact on reflex-mediated sympathetic fore- arm vasoconstriction. Together, the findings indicate that the expression of the ADCY6 A674S genetic variant leads to a hyperdynamic cardiovascular system both under baseline conditions and in response to stressors that activate adenylyl cyclase. Determinants of Increased Blood Pressure in Sub- jects Expressing the ADCY6 A674S Genetic Variant. It could be reasonably argued that the major functional deter- Fig. 4. Increases in heart rate during LBNP in the control (filled bars) minants of cAMP-mediated regulation of blood pressure re- and ADCY6 variant (empty bars) groups. Note the large HR response in Downloaded from the ADCY6 variant group compared with the modest response in the late to the effects of adenylyl cyclase activation on 1) cardiac p Ͻ 0.05 between groups. †, p Ͻ 0.05 from baseline. Data contractility/heart rate, 2) the renin-angiotensin system axis ,ء .control group represent the mean Ϯ standard error. via cAMP-mediated regulation of renin release, and 3) vas- icantly higher than controls both at rest and with LBNP (but cular reactivity predominantly via vasodilator mechanisms. not during the recovery phase; Fig. 5A). However, the mag- In regard to the impact of expression of the ADCY6 variant on cardiac function, under baseline conditions cardiac output nitude of fall in cardiac output during LBNP was similar in jpet.aspetjournals.org both groups (p Ͼ 0.05) (Fig. 5B). was higher in the ADCY6 variant group. This effect seems to With LBNP, the increase in plasma norepinephrine with have been related primarily to the increased heart rate, LBNP did not differ between groups (control, ϩ0.86 Ϯ 0.10 because stroke volume did not differ significantly between nm; ADCY6, ϩ0.93 Ϯ 0.13 nM) (p Ͼ 0.05). Plasma renin groups. The observation that cardiac output was elevated, activity was essentially unaltered with acute orthostatic without appreciable reductions in afterload (higher systolic stress, and the extent of change was similar between the pressure and normal vascular resistance) in this group, sug- Ϫ Ϯ gests that cardiac filling pressures were also sustained or control ( 0.05 0.03 ng/liter/s) and ADCY6 variant at ASPET Journals on March 6, 2015 (Ϫ0.07 Ϯ 0.02 ng/liter/s) groups. elevated. In regard to the potential effect of expression of the ADCY6 variant on vascular reactivity, total peripheral resistance Discussion was not reduced (nor was baseline FBF increased). Acutely Our studies had identified a relatively common amino acid increased adenylyl cyclase activation (as would be seen with variant of ADCY6, namely A674S (Gros et al., 2005, 2007). infusion of the nonselective ␤-adrenergic agonist isoprotere- Furthermore, we have shown previously that this variant is nol) is associated with decreased peripheral resistance re- hyperfunctional in vitro in the context of enzymatic activity lated to both direct and baroreflex-mediated responses. It is and adenylyl cyclase-mediated vascular responses (Gros et noteworthy that in our initial studies subjects carrying the al., 2007). However, the significance of its expression in re- A674S ADCY6 genetic variant demonstrated enhanced ad- gard to phenotypic characteristics and in vivo effects on re- enylyl cyclase-mediated vasodilator responses as determined sponses to acute exercise and vasoconstrictor stimuli were by the extent of isoproterenol-mediated vasorelaxation as-

Fig. 5. Cardiac output responses during LBNP in the ADCY6 variant (empty bars) and control (filled bars) groups. A, the data during rest (baseline), LBNP, and re- Ͻ ء covery. B, the percentage change in Qc during LBNP. , p 0.05 between groups. Data represent the mean Ϯ standard error. 456 Hodges et al. sessed by linear variable differential transformer techniques cular disease and/or complications related to visceral obesity (Gros et al., 2007). However, chronically, the direct effects of (the metabolic syndrome and diabetes) remains to be deter- adenylyl cyclase activation on vascular reactivity would be mined. However, these studies indicate that genetic regula- expected to be modulated by its actions in regulating renin tion of GPCR-mediated adenylyl cyclase activation is an im- release. In the ADCY6 variant group, plasma renin activity portant determinant of human cardiovascular function. was increased compared with the control group. Thus, the failure to detect a reduced total peripheral resistance in Acknowledgments subjects expressing the ADCY6 variant might reflect the We thank the subjects for their participation in these studies, chronic effects of increased renin activity, leading to en- Nancy Schmidt and Amanda Rothwell for help with subject recruit- hanced aldosterone-mediated effects on intravascular filling ment, and Arlene Fleischhauer for assistance with data collection. and/or increased ambient angiotensin II-mediated effects. However, regardless of the countervailing balance of en- References hanced adenylyl cyclase-mediated vasodilation versus en- Betik AC, Luckham VB, and Hughson RL (2004) Flow-mediated dilation in human brachial artery after different circulatory occlusion conditions. Am J Physiol Heart hanced adenylyl cyclase-mediated renin release on periph- Circ Physiol 286:H442–H448. eral resistance, altogether, our findings support the Cao H and Hegele RA (2003) LMNA is mutated in Hutchinson-Gilford progeria (MIM 176670) but not in Wiedemann-Rautenstrauch progeroid syndrome (MIM 264090). hypothesis that the increased blood pressure in the ADCY6 J Hum Genet 48:271–274. variant group is related primarily to increased cardiac Clifford PS and Hellsten Y (2004) Vasodilatory mechanisms in contracting skeletal output and increased plasma renin activity. muscle. J Appl Physiol 97:393–403. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager Downloaded from Notably the hemodynamic responses to handgrip exer- MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, et al. (2002) cise support the hypothesis that subjects with the ADCY6 Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery A674S variant have exaggerated adenylyl cyclase-medi- Reactivity Task Force. J Am Coll Cardiol 39:257–265. ated hemodynamic responses. Acute exercise causes rapid DuBois D and DuBois EF (1916) Clinical calorimetry: tenth paper–a formula to estimate the approximate surface area if height and weight be known. Arch Int and large changes in muscle perfusion (Shoemaker et al., Med 17:863–871. 1997a,b,c, 1998). Among the multiple mechanisms mediat- Feldman RD and Gros R (1998) Impaired vasodilator function in hypertension: the

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