Peter A. Robbins

Natural selection on EPAS1 (HIF2α) associated with low hemoglobin concentration in Tibetan highlanders

By impairing both function and survival, the severe reduction in oxygen availability associated with high-altitude environments is likely to act as an agent of natural selection. We used genomic and candidate gene approaches to search for evidence of such genetic selection. First, a genome-wide allelic differentiation scan (GWADS) comparing indigenous highlanders of the Tibetan Plateau (3,200–3,500 m) with closely related lowland Han revealed a genome-wide significant divergence across eight SNPs located near EPAS1. This gene encodes the transcription factor HIF2α, which stimulates production of red blood cells and thus increases the concentration of hemoglobin in blood. Second, in a separate cohort of Tibetans residing at 4,200 m, we identified 31 EPAS1 SNPs in high linkage disequilibrium that correlated significantly with hemoglobin concentration. The sex-adjusted hemoglobin concentration was, on average, 0.8 g/dL lower in the major allele homozygotes compared with the heterozygotes. These findings were replicated in a third cohort of Tibetans residing at 4,300 m. The alleles associating with lower hemoglobin concentrations were correlated with the signal from the GWADS study and were observed at greatly elevated frequencies in the Tibetan cohorts compared with the Han. High hemoglobin concentrations are a cardinal feature of chronic mountain sickness offering one plausible mechanism for selection. Alternatively, as EPAS1 is pleiotropic in its effects, selection may have operated on some other aspect of the phenotype. Whichever of these explanations is correct, the evidence for genetic selection at the EPAS1 locus from the GWADS study is supported by the replicated studies associating function with the allelic variants.

Indexes of Flow and Cross-sectional Area of the Middle Cerebral Artery Using Doppler Ultrasound During Hypoxia and Hypercapnia in Humans

BACKGROUND AND PURPOSE This study examined changes in cross-sectional area of the middle cerebral artery as assessed by changes in Doppler signal power during hypoxia and hypercapnia. In addition, it examined the degree of consistency among three indexes of cerebral blood flow and velocity: the velocity spectral outline (VP), the intensity-weighted mean velocity (VIWM), and an index of middle cerebral artery flow (P. VIWM). P. VIWM was calculated as the product of VIWM multiplied by the total power signal. Power is proportional to cross-sectional area of the vessel; this calculation therefore allows for any changes in this variable. METHODS Four protocols were used, each repeated six times for six healthy adults aged 20.8 +/- 1.7 years (mean +/- SD). The first was a control protocol (A) with end-tidal PO2 (ETPO2) maintained at 100 mm Hg and ETPCO2 at 1 to 2 mm Hg above eucapnia throughout. The second was a hypoxic step protocol (B) with ETPO2 lowered from control values to 50 mm Hg for 20 minutes. The third was a hypercapnic step protocol (C) with ETPCO2 elevated from control by 7.5 mm Hg for 20 minutes. The fourth was a combined hypoxic and hypercapnic step protocol (D) lasting 20 minutes. A dynamic end-tidal forcing system was used to control ETPCO2 and ETPO2. Doppler data were collected and stored every 10 milliseconds, and mean values were determined later on a beat-by-beat basis. VP, VIWM, power, and P.VIWM were expressed as a percentage of the average value over a 3-minute period before the step. RESULTS In protocols A and B, there were no changes in power and there were no differences between VP, VIWM, and P.VIWM. In C, at the relief from hypercapnia, there was a transient nonsignificant increase in power and a transient nonsignificant decrease in both VP and VIWM compared with P.VIWM. In D, during the stimulus period, VP was significantly higher than VIWM (paired t test, P < .05), but both indexes were not different from P.VIWM. In the period that followed relief from hypoxia and hypercapnia, the Doppler power signal was significantly increased by 3.8%. During this period, VP and VIWM were significantly lower than P.VIWM. CONCLUSIONS At the levels of either hypoxia or hypercapnia used in this study, there were no changes in cross-sectional area of the middle cerebral artery, and changes in both VP and VIWM accurately reflect changes in P.VIWM. With combined hypoxia and hypercapnia, however, at the relief from the stimuli when there is a very large and rapid decrease in P.VIWM, power is increased, suggesting an increase in the cross-sectional area. During this period, changes in VP and VIWM underestimate the changes in P.VIWM.

The IUPS human physiome project

Abstract. The Physiome Project of the International Union of Physiological Sciences (IUPS) is attempting to provide a comprehensive framework for modelling the human body using computational methods which can incorporate the biochemistry, biophysics and anatomy of cells, tissues and organs. A major goal of the project is to use computational modelling to analyse integrative biological function in terms of underlying structure and molecular mechanisms. To support that goal the project is establishing web-accessible physiological databases dealing with model-related data, including bibliographic information, at the cell, tissue, organ and organ system levels. Here we discuss the background and goals of the project, the problems of modelling across multiple spatial and temporal scales, and the development of model ontologies and markup languages at all levels of biological function.

The human side of hypoxia-inducible factor.

When humans are exposed to hypoxia, systemic and intracellular changes operate together to minimise hypoxic injury and restore adequate oxygenation. Emerging evidence indicates that the hypoxia‐inducible factor (HIF) family of transcription factors plays a central regulatory role in these homeostatic changes at both the systemic and cellular levels. HIF was discovered through its action as the transcriptional activator of erythropoietin, and has subsequently been found to control intracellular hypoxic responses throughout the body. HIF is primarily regulated by specific prolyl hydroxylase‐domain enzymes (PHDs) that initiate its degradation via the von Hippel‐Lindau tumour suppressor protein (VHL). The oxygen and iron dependency of PHD activity accounts for regulation of the pathway by both cellular oxygen and iron status. Recent studies conducted in patients with rare genetic diseases have begun to uncover the wider importance of the PHD‐VHL‐HIF axis in systems‐level human biology. These studies indicate that, in addition to regulating erythropoiesis, the system plays an important role in cardiopulmonary regulation. This article reviews our current understanding of the importance of HIF in human systems‐level physiology, and is modelled around the classic physiological response to high‐altitude hypoxia.

Nonlinear modeling of the dynamic effects of arterial pressure and CO/sub 2/ variations on cerebral blood flow in healthy humans

The effect of spontaneous beat-to-beat mean arterial blood pressure fluctuations and breath-to-breath end-tidal CO2 fluctuations on beat-to-beat cerebral blood flow velocity variations is studied using the Laguerre-Volterra network methodology for multiple-input nonlinear systems. The observations made from experimental measurements from ten healthy human subjects reveal that, whereas pressure fluctuations explain most of the high-frequency blood flow velocity variations (above 0.04 Hz), end-tidal CO2 fluctuations as well as nonlinear interactions between pressure and CO2 have a considerable effect in the lower frequencies (below 0.04 Hz). They also indicate that cerebral autoregulation is strongly nonlinear and dynamic (frequency-dependent). Nonlinearities are mainly active in the low-frequency range (below 0.04 Hz) and are more prominent in the dynamics of the end-tidal CO2-blood flow velocity relationship. Significant nonstationarities are also revealed by the obtained models, with greater variability evident for the effects of CO2 on blood flow velocity dynamics.

Effects of iron supplementation and depletion on hypoxic pulmonary hypertension: two randomized controlled trials.

CONTEXT Hypoxia is a major cause of pulmonary hypertension in respiratory disease and at high altitude. Recent work has established that the effect of hypoxia on pulmonary arterial pressure may depend on iron status, possibly acting through the transcription factor hypoxia-inducible factor, but the pathophysiological and clinical importance of this interaction is unknown. OBJECTIVE To determine whether increasing or decreasing iron availability modifies altitude-induced hypoxic pulmonary hypertension. DESIGN, SETTING, AND PARTICIPANTS Two randomized, double-blind, placebo-controlled protocols conducted in October-November 2008. In the first protocol, 22 healthy sea-level resident men (aged 19-60 years) were studied over 1 week of hypoxia at Cerro de Pasco, Peru (altitude 4340 m). In the second protocol, 11 high-altitude resident men (aged 30-59 years) diagnosed with chronic mountain sickness were studied over 1 month of hypoxia at Cerro de Pasco, Peru. INTERVENTION In the first protocol, participants received intravenous infusions of Fe(III)-hydroxide sucrose (200 mg) or placebo on the third day of hypoxia. In the second protocol, patients underwent staged isovolemic venesection of 2 L of blood. Two weeks later, patients received intravenous infusions of Fe(III)-hydroxide sucrose (400 mg) or placebo, which were subsequently crossed over. MAIN OUTCOME MEASURE Effect of varying iron availability on pulmonary artery systolic pressure (PASP) assessed by Doppler echocardiography. RESULTS In the sea-level resident protocol, approximately 40% of the pulmonary hypertensive response to hypoxia was reversed by infusion of iron, which reduced PASP by 6 mm Hg (95% confidence interval [CI], 4-8 mm Hg), from 37 mm Hg (95% CI, 34-40 mm Hg) to 31 mm Hg (95% CI, 29-33 mm Hg; P = .01). In the chronic mountain sickness protocol, progressive iron deficiency induced by venesection was associated with an approximately 25% increase in PASP of 9 mm Hg (95% CI, 4-14 mm Hg), from 37 mm Hg (95% CI, 30-44 mm Hg) to 46 mm Hg (95% CI, 40-52 mm Hg; P = .003). During the subsequent crossover period, no acute effect of iron replacement on PASP was detected. CONCLUSION Hypoxic pulmonary hypertension may be attenuated by iron supplementation and exacerbated by iron depletion. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00952302.

Regulation of human metabolism by hypoxia- inducible factor

The hypoxia-inducible factor (HIF) family of transcription factors directs a coordinated cellular response to hypoxia that includes the transcriptional regulation of a number of metabolic enzymes. Chuvash polycythemia (CP) is an autosomal recessive human disorder in which the regulatory degradation of HIF is impaired, resulting in elevated levels of HIF at normal oxygen tensions. Apart from the polycythemia, CP patients have marked abnormalities of cardiopulmonary function. No studies of integrated metabolic function have been reported. Here we describe the response of these patients to a series of metabolic stresses: exercise of a large muscle mass on a cycle ergometer, exercise of a small muscle mass (calf muscle) which allowed noninvasive in vivo assessments of muscle metabolism using 31P magnetic resonance spectroscopy, and a standard meal tolerance test. During exercise, CP patients had early and marked phosphocreatine depletion and acidosis in skeletal muscle, greater accumulation of lactate in blood, and reduced maximum exercise capacities. Muscle biopsy specimens from CP patients showed elevated levels of transcript for pyruvate dehydrogenase kinase, phosphofructokinase, and muscle pyruvate kinase. In cell culture, a range of experimental manipulations have been used to study the effects of HIF on cellular metabolism. However, these approaches provide no potential to investigate integrated responses at the level of the whole organism. Although CP is relatively subtle disorder, our study now reveals a striking regulatory role for HIF on metabolism during exercise in humans. These findings have significant implications for the development of therapeutic approaches targeting the HIF pathway.

The increase in pulmonary arterial pressure caused by hypoxia depends on iron status

Hypoxia is a major cause of pulmonary hypertension. Gene expression activated by the transcription factor hypoxia‐inducible factor (HIF) is central to this process. The oxygen‐sensing iron‐dependent dioxygenase enzymes that regulate HIF are highly sensitive to varying iron availability. It is unknown whether iron similarly influences the pulmonary vasculature. This human physiology study aimed to determine whether varying iron availability affects pulmonary arterial pressure and the pulmonary vascular response to hypoxia, as predicted biochemically by the role of HIF. In a controlled crossover study, 16 healthy iron‐replete volunteers undertook two separate protocols. The ‘Iron Protocol’ studied the effects of an intravenous infusion of iron on the pulmonary vascular response to 8 h of sustained hypoxia. The ‘Desferrioxamine Protocol’ examined the effects of an 8 h intravenous infusion of the iron chelator desferrioxamine on the pulmonary circulation. Primary outcome measures were pulmonary artery systolic pressure (PASP) and the PASP response to acute hypoxia (ΔPASP), assessed by Doppler echocardiography. In the Iron Protocol, infusion of iron abolished or greatly reduced both the elevation in baseline PASP (P < 0.001) and the enhanced sensitivity of the pulmonary vasculature to acute hypoxia (P= 0.002) that are induced by exposure to sustained hypoxia. In the Desferrioxamine Protocol, desferrioxamine significantly elevated both PASP (P < 0.001) and ΔPASP (P= 0.01). We conclude that iron availability modifies pulmonary arterial pressure and pulmonary vascular responses to hypoxia. Further research should investigate the potential for therapeutic manipulation of iron status in the management of hypoxic pulmonary hypertensive disease.

Changes in arterial K+ and ventilation during exercise in normal subjects and subjects with McArdle's syndrome.

1. We have examined the relationship between ventilation (VE), lactate (La) and arterial plasma K+ concentrations [( K+]a) during incremental exercise in six normal subjects and in four subjects with McArdles syndrome (myophosphorylase deficiency) who do not become acidotic during exercise. 2. In normal subjects, [K+]a rose to ca 7 mM at the point of exhaustion. The time courses of the increases in VE, La and [K+]a were all similar during the exercise period. La reached its peak concentration during the recovery from exercise when both VE and [K+]a were returning to resting levels. 3. McArdles subjects, like normal subjects, had a non‐linear ventilatory response during incremental exercise. Their [K+]a was closely related to VE throughout exercise and recovery. 4. The arterial pH of McArdles subjects, rather than remaining constant, actually rose from the onset of exercise. 5. For a given level of exercise, the levels of VE and [K+]a were greater in the McArdles subjects than in normal subjects. 6. These findings are consistent with the idea that hyperkalaemia may contribute significantly to the drive to breathe, especially during heavy exercise.

Genetic Signatures Reveal High-Altitude Adaptation in a Set of Ethiopian Populations

The Tibetan and Andean Plateaus and Ethiopian highlands are the largest regions to have long-term high-altitude residents. Such populations are exposed to lower barometric pressures and hence atmospheric partial pressures of oxygen. Such “hypobaric hypoxia” may limit physical functional capacity, reproductive health, and even survival. As such, selection of genetic variants advantageous to hypoxic adaptation is likely to have occurred. Identifying signatures of such selection is likely to help understanding of hypoxic adaptive processes. Here, we seek evidence of such positive selection using five Ethiopian populations, three of which are from high-altitude areas in Ethiopia. As these populations may have been recipients of Eurasian gene flow, we correct for this admixture. Using single-nucleotide polymorphism genotype data from multiple populations, we find the strongest signal of selection in BHLHE41 (also known as DEC2 or SHARP1). Remarkably, a major role of this gene is regulation of the same hypoxia response pathway on which selection has most strikingly been observed in both Tibetan and Andean populations. Because it is also an important player in the circadian rhythm pathway, BHLHE41 might also provide insights into the mechanisms underlying the recognized impacts of hypoxia on the circadian clock. These results support the view that Ethiopian, Andean, and Tibetan populations living at high altitude have adapted to hypoxia differently, with convergent evolution affecting different genes from the same pathway.