Abstract
Objective. The 1936G AKAP10 allele is associated with increased adult basal heart rate (HR) and decreased variability, markers of low cholinergic/vagus sensitivity associated with hypertension. Blood pressure (BP) values in newborns are important measurable markers of cardiovascular risk later in life. The question was whether decreased vagal function-related 1936A > G AKAP10 is associated with newborn BP. Study design. 114 healthy Polish newborns born after 37th gestational week to healthy women with uncomplicated pregnancies. At birth, newborn cord blood obtained for isolation of genomic DNA. BP and HR measured on days 1 and 3 after delivery. Results. Diastolic BP on day 3 and absolute and relative differences between diastolic BP values, as well as between mean BP values on day 3 and on day 1 after birth, in carriers of 1936G AKAP10 allele, were significantly higher as compared with wild-type homozygotes. Conclusion. Results demonstrate possible association between 1936G AKAP10 variant and BP in Polish newborns.
Introduction
The cAMP-dependent protein kinase (PKA, protein kinase A) is a multisubstrate serine/threonine protein kinase responsible for modulating many physiological processes (Citation1). A-Kinase anchoring proteins (AKAPs) coordinate the specificity of PKA signaling by localizing the kinase to subcellular sites through binding to a specific docking domain at the N-terminus of the R subunits (Citation2–5). The I646V mutation alters binding of AKAP10 to PKA RIα and the targeting of the RIα subunit to the mitochondria (Citation6). A study by Kammerer et al. (Citation6) indicated that this transition was found statistically significantly less often in older subjects. Further studies revealed that 1936G (V646) AKAP10 was associated in adults with increased basal heart rate (HR) and decreased heart rate variability (HRV), which are markers of low cholinergic/vagus nerve sensitivity (Citation7,Citation8).
Using a broad range of indicators of vagal function, including HR and HRV, Thayer et al. (Citation9) have shown that decreased vagal function is associated with an increased risk for cardiovascular morbidity and mortality. The findings from large epidemiological studies provided strong evidence that vagal tone, as measured by HRV, is lower in subjects with hypertension than in normotensive persons even after adjustment for a range of covariates (Citation10,Citation11). Lower HRV, reduced vagal tone and higher sympathetic function have also been associated with increased blood pressure (BP) values putting subjects at a higher risk of developing hypertension (Citation12).
These studies support the thesis that the autonomic nervous system in involved in the development of hypertension, and that decreases in vagal tone may precede the development of this critical risk factor for cardiovascular disease (Citation9–13). Cardiovascular events that occur in adults find their roots in risk factors operating early in life. Among the factors influencing cardiovascular risk, BP values in newborns represent an important measurable marker of the level of potential cardiovascular risk later in life, because the levels are both the cause and the consequence of early vascular alterations (Citation14,Citation15). It should be noted that there are no studies that we are aware of which provide measurements of HRV in newborns and association with future hypertension.
Therefore, this raises the question of whether a decrease in the vagal function-related 1936A > G AKAP10 polymorphism is associated with BP values recorded in full-term newborns. In addition to the factors mentioned above it should be noted that phenotype–genotype correlation studies using newborns seem to be more plausible than those with older individuals due to exclusion of confounding environmental influences such as diet, lifestyle, smoking or disease (Citation16).
Materials and methods
Study subjects
The study was approved by the ethics committee at the Pomeranian Medical University in Szczecin, and parents gave informed consent. At the Department of Neonatology at the Pomeranian Medical University we prospectively recruited 114 consecutive healthy Polish newborns (55 females and 59 males), born after the end of the 37th week of gestation to healthy women with uncomplicated pregnancies. No newborn had a history of cardiovascular disease, or disease known to affect the autonomic nervous system. All newborns were breast-fed and free of medication (i.e. without medicines known to influence HR and/or BP). Twins, newborns of mothers with pre-eclampsia, hypertension of any cause, diabetes, history of illicit substance use or antenatal steroid therapy were excluded. Other exclusion criteria were congenital infection, intra-uterine growth restriction (i.e. below the 10th percentile birth mass, length or head circumference), chromosomal aberrations or congenital malformations. Congenital malformations of heart and kidneys were excluded on the basis of ultrasound images.
At birth, cord blood of newborns was obtained for isolation of genomic DNA. The gender of the newborn, birth weight, body length and head circumference were taken from standard hospital records. Body surface area (BSA) was calculated as the square root of [length (cm) × weight (kg)/3600] according to Mosteller (Citation17). Blood electrolyte concentrations were not quantified.
The measurement of blood pressure
A Diascope Artema oscillometer (Survivalink Corporation, Minnetonka, Minneapolis, MN, USA) was used to determine systolic and diastolic BP (SBP or DBP, respectively), and only one of the investigators (BL) performed all of the BP measurements using a standardized protocol (Citation18). Subsequently, the mean BP (MBP) and pulse pressure (PP) were calculated using the following formulas: MBP = 1/3(SBP–DBP) + DBP or PP = SBP − DBP. An appropriately sized BP cuff (with the inflatable portion of the cuff encircling 75% or greater of the limb circumference, and the length of the cuff at least two-thirds of the length of the upper limb segment) was used on one arm, with the newborn in a resting state: awake or asleep. BPs were measured in the supine position on days 1 and 3 after delivery, at least 1.5 h following their last feeding or medical intervention. The cuff was applied to the right upper arm and the newborn was then left undisturbed for at least 15 min or until the newborn was sleeping or in a quiet awake state. If the newborns did not become quiet by themselves they were given 5% glucose to drink to quieten them. Three successive BP measurements were taken at 3-min intervals. Before each BP measurement, HR was measured as a 1-min mean before the air pump was started. Statistical analyses of the BP measurements included derivatives such as absolute and relative differences (ΔV or ΔV [%], respectively) between values on day 3 and on day 1 after birth (V3 – V1 or V – V1/V1, where V3 = value on day 3 and V1 = value on day 1, respectively) for SBP, DBP, MBP and PP.
Determination of AKAP10 genotype
Genomic DNA from cord blood was isolated with the QIAamp Blood DNA Mini Kit (QIAGEN, Hilden, Germany). To genotype the A-to-G transition at nucleotide 1936 of the AKAP10 gene, we used our own PCR–RFLP method as described previously (Citation19). Briefly, genomic DNA was amplified by PCR with primers flanking the polymorphic region: 5′-TCggTgTTAggTATCCAgTagTTg-3′ as sense primer, and 5′-TAAgAAggTAATCC CCA CAgCAgT-3′ as antisense primer. These primers yielded a PCR product of 437 bp in length. Subsequently, the products of amplification were incubated at 65°C for 3 h with 1 U of restriction enzyme Tai I (MBI Fermentas, Vilnius, Lithuania), and separated using 3% agarose gel electrophoresis, and stained with ethidium bromide. The “wild-type” sequence (1936A allele) was identified by lack of the Tai I restriction site, while the 1936G allele was digested into 269- and 168-bp restriction fragments. Results were recorded by photographing gels under UV light. All samples were independently genotyped using a blind method in duplicate, i.e. all samples were anonymously labeled by one person and then genotyped by the second person.
Statistical analysis
The AKAP10 genotype distribution conformed to the expected Hardy–Weinberg equilibrium (assessed using a two-tailed χ2 test) and the distribution of all quantitative variables in our cohort approached normality (skewness < 2 for all variables), allowing us to assess association between AKAP10 genotype and each outcome variable by one-way analysis of variance (ANOVA). Quantitative data were presented as means ± SD. In addition, HR and BP values, with respect to dominant and recessive mode of inheritance of the 1936G AKAP10 minor allele, were analyzed by multiple regression with adjustment for confounding variables:
All dependent variables were adjusted for gender because gender was found to have borderline significance in a χ2 test (p = 0.049); variables were also adjusted for gestational age and/or birth weight if the p-value for univariate correlations between gestational age or birth weight and dependent variables were found to be less than 0.1. Statistical significance was defined as p < 0.05. All data were analyzed using STATISTICA (data analysis software system, version 8.0, StatSoft Inc., Tulsa, OK, USA, 2007; www.statsoft.com).
Results
All PCR–RFLP samples were genotyped twice and a concordance rate of 100% was attained. There were 41 AA AKAP10 homozygotes (36.0%), 59 AG heterozygotes (51.7%) and 14 GG AKAP10 homozygotes (12.3%). The frequency of the 1936G AKAP10 minor allele was 38.2% (35.6% in newborn males and 40.9% in newborn females). The mean body weight was 3409 ± 530 g, body length 56.2 ± 3.3 cm, head circumference 33.7 ± 1.5 cm and BSA 0.23 ± 0.02 m2. No significant differences in anthropometric variables with respect to AKAP10 genotype were found (p-values: for birth weight 0.61, gestational age 0.11 and gender 0.049). No significant differences in HR and BP, with respect to AKAP10 genotype assessed by one-way ANOVA, were found except absolute and relative changes in DBP between day 1 and day 3 after birth (ΔDBP and ΔDBP [%], respectively) (). Multiple regression analysis with adjustment for gender, gestational age and birth weight revealed that DBP3, ΔDBP, ΔDBP [%], ΔMBP, ΔMBP [%] in 1936G variant carriers (AG + GG, dominant mode of inheritance) were significantly higher when compared with wild-type homozygotes (AA AKAP10) (p-values 0.03, 0.003, 0.01, 0.03 and 0.04 respectively; ).
Table I. Heart rate and blood pressure characteristics (means ± SD) in newborns with respect to AKAP10 genotypes.
Discussion
The results of our study show an association between the 1936A > G AKAP10 genotype and BP in Polish newborns born full term. DBP, absolute and relative differences between DBP values, as well as between MBP values on day 3 and on day 1 after birth, in carriers of the minor 1936G AKAP10 allele were significantly higher than in wild-type homozygotes. It is noteworthy that BP values in our newborns are similar to those reported previously by other authors, and that the higher BP at day 3 after birth (relative to those at day 1) is a significant physiological phenomenon (Citation20–24). This phenomenon is possibly connected to vascular alterations and changes in function of the autonomic nervous system (Citation25). It is considered that autonomic regulatory mechanisms, including baro- and chemoreceptors, are important modulators of BP and circulatory function in the newborn. Maturation of baroreflex function may be associated with maturational changes in parasympathetic activity: especially as, in newborns, baroreflex depends rather on parasympathetic modulation than on vascular tone modulated by the sympathetic system (Citation26).
To the best of our knowledge, this report is the first study that has examined the association of a functional AKAP10 polymorphism with BP, and the first study that has examined the association of genetic polymorphism with BP in a cohort of newborns within a few days after birth. To minimize the impact of adverse factors that could affect the outcome of BP measurements, the study was conducted in a carefully selected, homogeneous group of newborns, which met all the criteria adopted for inclusion. Measurements of BP using an oscillometric method were performed, which are commonly recommended for newborns (Citation27), according to protocols in the literature that give reproducible results (Citation18).
Limitations to the study are: (i) a small study cohort; (ii) no association studied between HRV and polymorphism. However, it should be stressed that our study was performed on a cohort of healthy newborns, in which the risk of cardiovascular diseases was not predicted, and we therefore avoided the use of an aggravating procedure such as extended ECG recordings due to technical/ethical problems. Usually HRV is computed as the standard deviation of all normal RR intervals (SDNN) from a 5-min stationary period selected from 24-h Holter electrocardiographic recordings – but here three spot HR measurements were taken over a period of 2 min (Citation3). It should be noted that corrections for multiple comparisons have not been used, and therefore the results should be interpreted with caution.
Previously, Kammerer et al. (Citation6) explored possible disease associations between AKAP10 polymorphism and 97 traits using a sample of 417 white twin pairs from the adult twin registry at St Thomas’ Hospital (London, UK). However, of the traits analyzed, including hypertension, cardiovascular diseases, diabetes, obesity and osteoporosis, only the PR interval in electrocardiography was statistically significant at a nominal level of 0.05 (Citation6). Additionally, so far only a few studies evaluating the association of genetic polymorphisms and BP in early childhood with adjustment for confounding obstetric and perinatal variables (e.g. gestational age) have been published (Citation28,Citation29). In contrast to our report, results of these studies were based on measurements of BP at the earliest at 24 months of age.
In 2007, Tingley et al. (Citation7) generated Akap10-mutant mice. The Akap10 mutation truncated the protein C terminus and eliminated the last 51 amino acids that include its PKA-binding domain. Compared with wild-type littermates (with the G allele (V646) Akap10), A allele Akap10-mutant mice had stronger baroreflex (greater HR slowing), indicating higher vagus nerve sensitivity. As the mouse Akap10 mutation, which lacked PKA binding increased vagus nerve sensitivity, the authors predicted and confirmed that the minor human allele, V646, which has a high binding affinity for PKA, would decrease vagus nerve sensitivity. Indeed the V646 variant has been associated with low cholinergic/vagus nerve sensitivity expressed as increased basal HR and decreased HRV (Citation7,Citation8).
In contrast to the results of the work Tingley et al., and confirmed later by Neumann et al. (Citation7,Citation8), we have not found a significant association between AKAP10 gene polymorphism and HR in the newborns studied by us. However, when comparing the results obtained by us and the results from the work of Tingley et al., extreme caution should be taken because of significant differences in the study design. Our study included 114 newborns in whom HR was calculated as the average of 3-min measurements recorded during their resting/sleeping. Tingley et al. (Citation7) used 24-h continuous ECG recording in 122 participants (mean age 64 years) in the Heart and Soul Study, which allowed the measurement of HR as well as HRV. In contrast to our study, Tingley et al. (Citation7) did not present HR results for the genotypes of AKAP10, and only showed that carriers of the 1936G (V646) variant AKAP10 were more likely to have a mean HR (over 24 h) > 75 beats/min, which is an independent risk factor for sudden cardiac death in the adult population (Citation30). This indicator is a marker in the general population for adults, but for newborns, all of our HR values at the day 1 and day 3 were > 75 beats/min, and hence we decided to distinguish test groups according to the average value of HR at days 1 and 3 (122.0 and 118.3 beats/min, respectively). We found no significant differences in the prevalence of genotypes of AKAP10 between subgroups of newborns with HR higher or lower than the mean values at both at day 1 and day 3 after birth. The physiological phenomenon of baroreceptor reflex maturation in the neonatal period (Citation31,Citation32) and the above discrepancies, do not allow the conclusion that the resulting higher BP (increased in neonatal 1936G allele carriers) is linked exclusively with AKAP10-associated reduced sensitivity of the vagus nerve. An additional, indirect argument against the exclusivity of such a link is established by our lack of association between polymorphism AKAP10 and pulse pressure values. In 2004, Virtanen et al. (Citation33) demonstrated that an increased pulse pressure is associated with reduced baroreflex sensitivity. It is also worth noting that a relationship has been shown between the AKAP10 minor G allele and clinical phenotypes unrelated to the cholinergic system (Citation4,Citation34).
The underlying molecular mechanism for the relationship between the AKAP10 polymorphism and neonatal BP is also unclear. Evolutionary conservation of AKAP10, including the PKA binding domain, suggests that the AKAP10 serves a critical function in diverse species (Citation7). Interestingly, the 15 available non-human vertebrate AKAP10 sequences, including chimp and mice, all encode valine at position 646, suggesting that I646 is unique to humans (among invertebrata isoleucine has been found at position 646 only in Anopheles gambiae) (Citation6). The V646 AKAP10 variant binds with three- fold greater affinity in vitro and results in increased compartmentalization and targeting of PKA to the mitochondria (Citation6). Compartmentalization of AKAPs is known to play an important role in the specificity and regulation of PKA by cAMP-mediated signaling (Citation2,Citation3). AKAP10, which interacts at its carboxyl terminus with PKA and PDZ domain proteins, contains two tandem regulator of G-protein signalling (RGS) domains. However, so far AKAP10 has demonstrated no specific interactions with heterotrimeric G proteins, and no ability to activate them, raising the possibility of non-traditional targets (Citation35,Citation36). Recently, Eggers et al. (Citation36) reported that the RGS domains of AKAP10 interact with the small GTPases Rab4 and Rab11, which regulate endocytic recycling. Therefore, the authors suggested that 1936G AKAP10-associated disease phenotypes could be the result of altered surface expression of various receptors or channels (Citation35), including adrenergic receptors involved in the BP regulation (Citation37,Citation38). Consideration should also be give to the idea that PKA–AKAPs pathway is involved in water and electrolyte homeostasis, e.g. via modulation of the epithelial sodium channel (ENaC) or aquaporin 2 (Citation39–41).
In conclusion, our results demonstrate a possible association between the 1936G AKAP10 variant and BP in a Caucasian newborn population.
Acknowledgements
We wish to acknowledge helpful advice from Prof. Timothy Spector and Juliette Harris, Ph.D., from the Department of Twin Research & Genetic Epidemiology, King's College London, UK. This study was funded entirely by the Pomeranian Medical University, Szczecin, Poland.
Disclosure
The authors declare no conflict of interest.
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