Adenylyl cyclase (ADCY) is a critical regulator of metabolic and cardiovascular function. We have identified a genetic variant (A674S) in ADCY isoform 6 (ADCY6). Subsequent studies demonstrated that the expression of this ADCY6 variant paralleled an increase in adenylyl cyclase-mediated functions. However, the impact of this hyperfunctional variant on cardiovascular function is unknown. Therefore, we evaluated the hemodynamic profile of carriers of ADCY6 A674S. The association of ADCY6 A674S with anthropometric and hemodynamic parameters was assessed in 364 healthy white subjects. The allele encoding this variant was present in 6.9% of the subjects, and those individuals had increased blood pressure. To determine the hemodynamic basis for increased blood pressure in carriers of ADCY6 A674S, we assessed forearm blood flow (FBF) and cardiac output at rest, during handgrip exercise (to test vasodilator responsiveness), and with lower body negative pressure [to test forearm vasoconstrictor and heart rate (HR) responsiveness] in a subsample of 21 subjects. At rest, cardiac output and blood pressure were higher in carriers of ADCY6 A674S. This was paralleled by an increase in plasma renin activity, but not in plasma norepinephrine. During handgrip exercise, FBF and vasodilator responses were greater in carriers of ADCY6 A674S. Responses to reactive hyperemia were not different between genotypes. With lower body negative pressure, the HR response to this orthostatic stress was markedly higher in carriers of ADCY6 A674S. These data indicate that the relatively common hyperfunctional ADCY6 A674S variant underlies a hyperdynamic cardiovascular response and increased blood pressure.
The adenylyl cyclases (ADCYs) are a ubiquitously expressed family of enzymes that catalyze the generation of cAMP from ATP. They regulate a broad range of cellular functions (Patel et al., 2001) and are critical effectors for a number of G protein-coupled receptors (GPCRs). Adenylyl cyclase activation has been suggested to be the rate-limiting step in the GPCR signaling cascade (Ostrom et al., 2000). In vascular cells, activity of adenylyl cyclase regulates acute functional effects including vascular reactivity, cellular growth, hypertrophy, and apoptosis (Gros et al., 2006). Furthermore, alterations in the regulation of adenylyl cyclase activity have been implicated in the pathogenesis of hypertension, heart failure, and diabetes (Moxham and Malbon, 1996; Roth et al., 1999, 2002; Tepe and Liggett, 1999; Matsumoto et al., 2005).
Nine membrane-bound isoforms of adenylyl cyclase have been cloned, grouped into three major subfamilies: group 1, ADCY1, ADCY3, ADCY8; group 2, ADCY2, ADCY4, ADCY7; and group 3, ADCY5, ADCY6 (Patel et al., 2001; Ludwig and Seuwen, 2002). In addition, ADCY9 has been characterized as a distinct (and atypical) isoform (Sunahara and Taussig, 2002) with restricted expression, and a soluble adenylyl cyclase has been characterized that is the predominant form in mammalian sperm (Wuttke et al., 2001). Each isoform has a specific pattern of tissue/organ distribution and a specific pattern of regulation by G proteins, calcium/calmodulin, and protein kinases (Wuttke et al., 2001; Sunahara and Taussig, 2002; Wang and Brown, 2004).
Variability in cAMP synthesis was thought to be determined predominantly either by the extent of adenylyl cyclase expression, the specific characteristics of the GPCRs linked to enzyme activation, or variation in the concentration of the “regulatory factors” (i.e., G proteins, protein kinases, ions) (Feldman and Gros, 1998). The importance of “genetic” variability has been unappreciated, that is, the expression of genetic structural variants of the enzyme that differ by function and/or activity. However, studies by us and others have suggested that variability in the expression of ADCY genetic variants may be an important regulator of adenylyl cyclase-mediated responses (Ikoma et al., 2003; Small et al., 2003; Nordman et al., 2008).
Several genetic variants have been described for a range of G proteins and GPCRs linked to adenylyl cyclase (Rana et al., 2001; Siffert, 2003). Expression of these variants leads to alterations in receptor-mediated activation of adenylyl cyclase and alterations in downstream effector pathways. The identification of dysfunctional genetic variants of ADCY presently is limited to those in ADCY6, ADCY3, and ADCY9, of which the latter has a much more restricted distribution than other isoforms (Small et al., 2003). A single-nucleotide polymorphism in intron 17 of genes encoding the very widely expressed ADCY6 isoform (Wang and Brown, 2004) was identified in a Japanese population (Nordman et al., 2008). However, the impact of this variant on adenylyl cyclase function is currently unknown. In our initial studies, we discovered a relatively common (∼7% in whites) missense single-nucleotide polymorphism in ADCY6, with the trivial name of ADCY6 A674S (Gros et al., 2005). In a mammalian vascular cell system, specifically rat vascular smooth muscle cells transfected using adenoviral constructs, we have shown that expression of the ADCY6 A674S variant isoform resulted in enhanced adenylyl cyclase activity and function compared with expression of wild-type ADCY6. Further, in humans expression of the ADCY6 A674S variant correlates with 1) increased adenylyl cyclase enzymatic activity and 2) enhanced adenylyl cyclase-mediated vascular responses (Gros et al., 2007). However, whether there were detectable alterations in cardiovascular or phenotypic characteristics associated with expression of this variant in humans was unknown.
Consequently, the present studies were performed to assess the association of the ADCY6 A674S variant with phenotypic characteristics within a large group of healthy, younger human subjects. In addition, in a subset of these subjects we examined the impact of expression of this genetic variant on hemodynamic responses both at rest and with exercise and orthostatic stress. Data demonstrate that carriers of the ADCY6 A674S genetic variant have increased blood pressure related to a hyperdynamic profile consistent with the effect of increased adenylyl cyclase function.
Materials and Methods
A total of 364 healthy white volunteers (age range 18 to 50 years) were screened. Exclusion criteria included, but were not limited to, history of cardiovascular events, average alcohol intake >2 units per day, pregnancy, and use of antihypertensive/blood pressure-altering drugs or anticoagulants. Recruitment was based on local advertising and e-mail invitations for volunteers within the Robarts Research Institute and the University of Western Ontario.
Of the 25 individuals that we identified as expressing the ADCY6 genetic variant we were able to recruit seven individuals (three males) all of whom were heterozygotic carriers of ADCY6 A674S (hereafter referred to as the ADCY6 variant group) for more detailed analysis of cardiovascular responsiveness. Fourteen individuals (seven males) who did not express the genetic variant were recruited as a control group. The mean ± S.E.M. ages of the control and ADCY6 variant groups were 26 ± 1 and 26 ± 1 years, respectively. By self-report, all participants were nonsmokers who were free of cardiovascular and neurological disease. Participants reported to the laboratory after at least a 3-h fast and having abstained from caffeine, alcohol, and exercise for a minimum of 12 h. Before experimentation, participants were encouraged to maintain typical water consumption and sleeping behaviors. Although the menstrual phase was not assessed, data between males and females displayed no perceptible difference and were, therefore, pooled. Informed consent was obtained for all analyses, with approval from the University of Western Ontario Research Ethics Review Board.
During the initial screening, the five measurements of seated blood pressure and heart rate were averaged and recorded (BP Tru, Vancouver, British Columbia, Canada). A 10-ml blood sample was taken for biochemical and genetic determinations. Data on sex, weight, height, waist circumference, and smoking status were also obtained. Waist circumferences were normalized by sex, based on sex-specific Adult Treatment Panel III-recommended upper limits [National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II].
In the follow-up study of cardiovascular responsiveness, all measurements were performed with the participants in the supine position in a quiet, darkened room kept at a relatively constant temperature (21–23°C). Heart rate was recorded via a three-lead ECG (Pilot 9200; Colin Medical Instruments, San Antonio, TX), and blood pressure was measured continuously by finger photoplethysmography (Finapres 2300; Ohmeda, Englewood, CO) and validated intermittently via sphygmomanometry. Brachial artery mean blood velocity (4.7 MHz) and diameter (10-MHz imaging transducer) were assessed by pulsed-wave Doppler ultrasound (echo ultrasound imaging; GE Vingmed System Five; GE Healthcare, Little Chalfont, Buckinghamshire, UK). Artery images were recorded on a S-VHS videocassette recorder (SVO-9500 MDP; Sony, Tokyo, Japan). Cardiac output was derived on a beat-by-beat basis from the transfer function of the finger pulsatile pressure wave form, as validated for these maneuvers (Shoemaker et al., 2007; Frances et al., 2008). All analog data were sampled at 1000 Hz by using a data-acquisition system (PowerLab; ADInstruments, Colorado Springs, CO). Cardiac output was normalized to body surface area (BSA) (Qc) by using the equation of DuBois and DuBois (1916): BSA (m2) = 0.007184 × weight (kg)0.425 × height (cm)0.725. Stroke volume (corrected for BSA) was determined from the quotient of cardiac output and HR. Forearm blood flow (FBF) was calculated as FBF = blood velocity × r2 × 60/V, where r is the brachial artery radius, 60 is to convert flow from s to min, and V is volume of the forearm.
Adenylyl cyclase activation by a range of metabolic factors is an important determinant of the vasodilator 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 measures were recorded for 60 s, followed by three repeated 5-s handgrip contractions with 60 s of recovery after each trial. The handgrip contraction intensity was 40% of the maximal voluntary contraction force, determined before this protocol from three to five trials of voluntary maximal hand gripping. The dilator response to this contraction was assessed over a 5-s average immediately after each contraction and averaged to produce the mean FBF responses.
Beyond adenylyl cyclase activation, vasodilator responses to exercise may also be mediated by endothelial factors (e.g., nitric oxide) in response to enhanced endothelial shear stress (Clifford and Hellsten, 2004). This flow-mediated dilation (FMD) is mediated predominantly by endothelial responses. Thus, to determine whether any alterations seen in handgrip responses might be related to endothelial factors a reactive hyperemia and FMD protocol was performed. While the subjects were supine, a rapid inflation/deflation pneumatic cuff (D.E. Hokanson Inc., Bellevue, WA) was placed around the right forearm immediately distal to the olecranon process to provide forearm ischemia and avoid excessive myogenic contributions to brachial artery dilation after ischemia (Corretti et al., 2002; Betik et al., 2004). Ultrasound parameters were set to optimize longitudinal, B-mode images of the lumen/arterial wall interface. Specifically, central and forearm hemodynamics were recorded during 5 min of baseline, forearm cuff inflation to >200 mm Hg for 5 min, and then for 3 min after cuff deflation.
Lower Body Negative Pressure.
To determine whether any alterations in vasodilator responses between groups might be related to a generalized vascular “hyper-reactivity” characterized by both exaggerated vasodilator and vasoconstrictor responses we assessed blood flow and cardiac hemodynamic responses after lower body negative pressure (LBNP). The legs and hips were sealed in an air-tight container connected to a vacuum source (Kimmerly and Shoemaker, 2002). Data collection for each test commenced after 5 min of undisturbed rest in the supine position. An initial 5-min baseline period was followed by 5 min of LBNP at −40 mm Hg and 5 min of rest at atmospheric pressure.
Blood Processing and Analysis.
Blood samples were obtained from an antecubital fossa vein at baseline and during the LBNP protocol. A 3-ml sample of whole blood for determination of catecholamines was mixed with 75 μl of EGTA-glutathione anticoagulant and was centrifuged at 4°C for 15 min. A 6-ml sample of whole blood for determination of renin activity was collected in two 3-ml samples in 4-ml BD vacutainers (BD Biosciences, San Jose, CA), each containing 7.2 mg of EDTA, and centrifuged at 4°C for 15 min.
Catecholamines were extracted from the plasma by adding acid-washed alumina and mixing by inversion. The pellet was washed with distilled water, and the catecholamines were released into 0.1 M perchloric acid and separated by centrifugation. The concentrations of norepinephrine were determined in duplicate by high-performance liquid chromatography with electrochemical detection (2465 ElectroChemical Detector; Waters, Milford, MA) and quantified by determining the area under the peak. Working and internal standards were used to correct the measurements. Renin activity assays were performed commercially by γ-Dynacare Medical Laboratories (London, Ontario, Canada).
Genomic DNA was extracted from whole blood as described previously (Gros, et. al, 2005). In brief, genotyping of the ADCY6 A674S variant was performed by using exon-specific DNA amplification followed by purification using shrimp alkaline phosphatase (Roche Diagnostics, Mannheim, Germany) and exonuclease I (New England Biolabs, Ipswich, MA) and capillary pheresis in an automated DNA sequencer as described previously (Cao and Hegele, 2003).
For the initial population screening, the statistical significance of differences in quantitative variables between control and ADCY6 variant groups was determined by Student's t test for unpaired data (Prism 4.0; GraphPad Software Inc., San Diego, CA). In the subsample studied for cardiovascular responsiveness, the associations of genotype on baseline values and responsiveness (defined as the change in each variable from rest) were assessed by using a mixed one-way repeated measures analysis of variance (SAS version 9.1; SAS Institute, Cary, NC). Post hoc analysis of significant interactions was assessed by using Bonferroni analysis. Data are presented as mean ± standard error, and statistical significance was assumed when p < 0.05.
Genotype and Allele Frequencies of ADCY6 A674S Variant.
In the population of 364 healthy white volunteers we identified 25 individuals (24 heterozygotes and 1 homozygote) who had ADCY6 p.A674S (g.2714G > T) variant in their genomes. The genotype frequencies for 2714G/G, 2714G/T, and 2714T/T were 0.931, 0.066, and 0.003, respectively. The 2714T allele frequency was 0.035, and there was no deviation of genotype frequencies from Hardy-Weinberg expectations. These findings were similar to our previously reported frequency of ADCY6 A674S in a white population (Gros et al., 2007).
ADCY6 Variant Associations.
Participants were classified according to g.2714G > T genotype with the single 2714T/T homozygote added to the 2714G/T heterozygote group (S674 subjects), whereas the 2714G/G homozygotes formed the comparator group (A674 subjects). Seated systolic blood pressure was increased in ADCY6 S674 subjects compared with ADCY6 A674 subjects (p < 0.05; Table 1). Diastolic blood pressure also tended to be higher in the ADCY6 variant group, although this difference was of borderline significance (p = 0.05) (Table 1).
Waist circumference was reduced in the ADCY6 S674 variant group compared with the ADCY6 A674-expressing individuals (controls) (Table 1). In contrast, mean body mass index did not differ according to genotype.
Baseline Hemodynamic and Biochemical Assessments.
Of the 25 individuals that we identified as expressing the ADCY6 genetic variant we were able to recruit seven individuals (three males) who were heterozygotic carriers of ADCY6 A674S (hereafter referred to as the ADCY6 variant group) for more detailed analysis of cardiovascular responsiveness. Fourteen individuals (seven males) who did not express the genetic variant were recruited as a control group.
Baseline assessments were performed after approximately 20 min of quite supine rest. Systolic, mean arterial, and diastolic blood pressure all were significantly increased in the ADCY6 variant group (Table 2). In addition, compared with the control group, those expressing the ADCY6 variant had increased mean heart rate and cardiac output (both p < 0.05).
Under baseline conditions, plasma renin activity was greater in the ADCY6 variant group (0.63 ± 0.11 ng/liter/s) (p < 0.05) compared with control subjects (0.31 ± 0.11 ng/liter/s). In contrast, baseline levels of plasma norepinephrine were similar between groups (control = 1.28 ± 0.25 nM; ADCY6 variant = 1.38 ± 0.24 nM) (p > 0.1).
Handgrip Exercise Responses.
Baseline measures of FBF did not differ between genotypic groups (p > 0.05). With exercise, FBF increased in both ADCY6 variant and control groups. However, compared with control subjects, the exercise-induced increase in FBF was almost four times greater in those expressing the ADCY6 variant [Fig. 1; increased ∼30% in the control subjects (p < 0.05) versus ∼120% in the ADCY6 variant group (p < 0.001)].
Reactive Hyperemia and Flow-Mediated Dilation.
To determine whether the increase in FBF seen after handgrip exercise was caused by a generalized enhancement of vasodilatory responses not specific to adenylyl cyclase-mediated effects, we next assessed the (endothelial-dependent) effect of reactive hyperemia on FBF. Compared with control subjects, the ADCY6 variant group displayed no difference in baseline FBF (p = 0.181; Fig. 2) before the FMD procedure. In addition, the initial hyperemic response to the deflation of the cuff was unaffected by genetic status (p > 0.05; Fig. 2). The extent of FMD in the brachial artery (between 45 and 90 s after cuff deflation) did not differ between the two groups, with the increases in the diameters of the control group being 8.7 ± 0.5% and the ADCY6 variant group being 9.2 ± 0.7% (p > 0.05). Thus, endothelial-mediated vasodilatory responses were not enhanced in individuals expressing the ADCY6 variant.
Lower Body Negative Pressure.
During LBNP, both groups demonstrated reductions in FBF (p < 0.05; Fig. 3A). However, the forearm vascular response to LBNP was not greater in the ADCY6 variant group. In fact, the increase in changes in total peripheral resistance (TPRc) in response to LBNP was greater in the control than the ADCY6 variant group (Fig. 3B; p < 0.05). In contrast to the unchanged or blunted vascular responses to LBNP, the cardiac chronotropic responses to LBNP, mediated indirectly via baroreceptor unloading, were exaggerated in the ADCY6 variant group. Compared with the insignificant increase in HR seen in controls after LBNP, in ADCY6 variants, HR was significantly increased (p < 0.05) (Fig. 4). The increase in HR was 14 beats · min−1 greater in the ADCY6 variant group during LBNP versus controls (p < 0.05) (Fig. 4). It is noteworthy that cardiac output in the ADCY6 variant group was significantly higher than controls both at rest and with LBNP (but not during the recovery phase; Fig. 5A). However, the magnitude of fall in cardiac output during LBNP was similar in both groups (p > 0.05) (Fig. 5B).
With LBNP, the increase in plasma norepinephrine with LBNP did not differ between groups (control, +0.86 ± 0.10 nm; ADCY6, +0.93 ± 0.13 nM) (p > 0.05). Plasma renin activity was essentially unaltered with acute orthostatic stress, and the extent of change was similar between the control (−0.05 ± 0.03 ng/liter/s) and ADCY6 variant (−0.07 ± 0.02 ng/liter/s) groups.
Our studies had identified a relatively common amino acid variant of ADCY6, namely A674S (Gros et al., 2005, 2007). Furthermore, we have shown previously that this variant is hyperfunctional in vitro in the context of enzymatic activity and adenylyl cyclase-mediated vascular responses (Gros et al., 2007). However, the significance of its expression in regard to phenotypic characteristics and in vivo effects on responses to acute exercise and vasoconstrictor stimuli were unknown. The present studies have confirmed that the ADCY6 A674S variant is present in ∼7% 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 forearm 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 Subjects Expressing the ADCY6 A674S Genetic Variant.
It could be reasonably argued that the major functional determinants of cAMP-mediated regulation of blood pressure relate to the effects of adenylyl cyclase activation on 1) cardiac contractility/heart rate, 2) the renin-angiotensin system axis via cAMP-mediated regulation of renin release, and 3) vascular reactivity predominantly via vasodilator mechanisms. In regard to the impact of expression of the ADCY6 variant on cardiac function, under baseline conditions cardiac output was higher in the ADCY6 variant group. This effect seems to have been related primarily to the increased heart rate, because stroke volume did not differ significantly between groups. The observation that cardiac output was elevated, without appreciable reductions in afterload (higher systolic pressure and normal vascular resistance) in this group, suggests that cardiac filling pressures were also sustained or elevated.
In regard to the potential effect of expression of the ADCY6 variant on vascular reactivity, total peripheral resistance was not reduced (nor was baseline FBF increased). Acutely increased adenylyl cyclase activation (as would be seen with infusion of the nonselective β-adrenergic agonist isoproterenol) is associated with decreased peripheral resistance related to both direct and baroreflex-mediated responses. It is noteworthy that in our initial studies subjects carrying the A674S ADCY6 genetic variant demonstrated enhanced adenylyl cyclase-mediated vasodilator responses as determined by the extent of isoproterenol-mediated vasorelaxation assessed by linear variable differential transformer techniques (Gros et al., 2007). However, chronically, the direct effects of adenylyl cyclase activation on vascular reactivity would be expected to be modulated by its actions in regulating renin release. In the ADCY6 variant group, plasma renin activity was increased compared with the control group. Thus, the failure to detect a reduced total peripheral resistance in subjects expressing the ADCY6 variant might reflect the chronic effects of increased renin activity, leading to enhanced aldosterone-mediated effects on intravascular filling and/or increased ambient angiotensin II-mediated effects. However, regardless of the countervailing balance of enhanced adenylyl cyclase-mediated vasodilation versus enhanced adenylyl cyclase-mediated renin release on peripheral resistance, altogether, our findings support the hypothesis that the increased blood pressure in the ADCY6 variant group is related primarily to increased cardiac output and increased plasma renin activity.
Notably the hemodynamic responses to handgrip exercise support the hypothesis that subjects with the ADCY6 A674S variant have exaggerated adenylyl cyclase-mediated hemodynamic responses. Acute exercise causes rapid and large changes in muscle perfusion (Shoemaker et al., 1997a,b,c, 1998). Among the multiple mechanisms mediating this effect is the role of metabolic factors released by working muscles. The mediators of exercise hyperemia that are released from contracting skeletal muscle include adenosine and prostaglandins that act through GPCRs to affect intracellular calcium levels in vascular smooth muscle (Betik et al., 2004; Marshall, 2007) via an adenylyl cyclase-regulated pathway. The pronounced difference in the FBF to handgrip exercise in the ADCY6 variant group is consistent with the suggestion of this genetic variant producing a “hyperfunctional” phenotype. These alterations in vasodilator responses were not paralleled by significant differences in flow-mediated vasodilator processes as assessed by the effects of reactive hyperemia. This latter response is mediated predominantly by endothelial factors that are (largely) independent of adenylyl cyclase activation. Thus, these findings in aggregate would suggest that the alterations in hand grip responses in individuals expressing the ADCY6 variant are not related to a generalized enhancement of vasodilator responses.
It is noteworthy that the ADCY6 variant group had ∼4-cm decreased waist circumference reflecting a 10% decrease in abdominal girth, without a significant difference in body mass index. Waist circumference has been increasingly appreciated as a surrogate for abdominal fat and, more importantly, as a highly predictive risk factor for the development of the metabolic syndrome and, ultimately, atherosclerotic complications (Pouliot et al., 1994). The observation that waist circumference, but not body mass index, was reduced in healthy, younger ADCY6 variant subjects suggests that the difference in waist circumference reflected a difference in abdominal fat, but not a difference in “whole-body” fat.
In summary, these data indicate that the expression of a novel, relatively common genetic variant of ADCY6 is associated with an increase in adenylyl cyclase function that parallels increased blood pressure and cardiac output and exaggerated adenylyl cyclase-mediated responses to exercise and altered distribution of body fat. Whether these findings have implications in regard to the development of cardiovascular disease and/or complications related to visceral obesity (the metabolic syndrome and diabetes) remains to be determined. However, these studies indicate that genetic regulation of GPCR-mediated adenylyl cyclase activation is an important determinant of human cardiovascular function.
We thank the subjects for their participation in these studies, Nancy Schmidt and Amanda Rothwell for help with subject recruitment, and Arlene Fleischhauer for assistance with data collection.
This study was supported by the Heart and Stroke Foundation of Ontario [Grant T6624] (to R.D.F.), the Natural Sciences and Engineering Research Council of Canada (J.K.S.), and the Canadian Institutes of Health Research (J.K.S). R.G. is supported by a New Investigator Award from the Heart and Stroke Foundation of Canada.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- adenylyl cyclase
- forearm blood flow
- heart rate
- flow-mediated dilation
- lower body negative pressure
- total peripheral resistance
- G protein-coupled receptor
- body surface area
- Received July 9, 2010.
- Accepted August 17, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics