Prolonged Exposure to Microgravity Reduces Cardiac Contractility and Initiates Remodeling in Drosophila.

Understanding the effects of microgravity on human organs is crucial to exploration of low-earth orbit, the moon, and beyond. Drosophila can be sent to space in large numbers to examine the effects of microgravity on heart structure and function, which is fundamentally conserved from flies to humans. Flies reared in microgravity exhibit cardiac constriction with myofibrillar remodeling and diminished output. RNA sequencing (RNA-seq) in isolated hearts revealed reduced expression of sarcomeric/extracellular matrix (ECM) genes and dramatically increased proteasomal gene expression, consistent with the observed compromised, smaller hearts and suggesting abnormal proteostasis. This was examined further on a second flight in which we found dramatically elevated proteasome aggregates co-localizing with increased amyloid and polyQ deposits. Remarkably, in long-QT causing sei/hERG mutants, proteasomal gene expression at 1g, although less than the wild-type expression, was nevertheless increased in microgravity. Therefore, cardiac remodeling and proteostatic stress may be a fundamental response of heart muscle to microgravity.

Reduced Cardiac Calcineurin Expression Mimics Long-Term Hypoxia-Induced Heart Defects in Drosophila.

BACKGROUND: Hypoxia is often associated with cardiopulmonary diseases, which represent some of the leading causes of mortality worldwide. Long-term hypoxia exposures, whether from disease or environmental condition, can cause cardiomyopathy and lead to heart failure. Indeed, hypoxia-induced heart failure is a hallmark feature of chronic mountain sickness in maladapted populations living at high altitude. In a previously established Drosophila heart model for long-term hypoxia exposure, we found that hypoxia caused heart dysfunction. Calcineurin is known to be critical in cardiac hypertrophy under normoxia, but its role in the heart under hypoxia is poorly understood. METHODS AND RESULTS: In the present study, we explore the function of calcineurin, a gene candidate we found downregulated in the Drosophila heart after lifetime and multigenerational hypoxia exposure. We examined the roles of 2 homologs of Calcineurin A, CanA14F, and Pp2B in the Drosophila cardiac response to long-term hypoxia. We found that knockdown of these calcineurin catalytic subunits caused cardiac restriction under normoxia that are further aggravated under hypoxia. Conversely, cardiac overexpression of Pp2B under hypoxia was lethal, suggesting that a hypertrophic signal in the presence of insufficient oxygen supply is deleterious. CONCLUSIONS: Our results suggest a key role for calcineurin in cardiac remodeling during long-term hypoxia with implications for diseases of chronic hypoxia, and it likely contributes to mechanisms underlying these disease states.

Ibuprofen Blunts Ventilatory Acclimatization to Sustained Hypoxia in Humans.

Ventilatory acclimatization to hypoxia is a time-dependent increase in ventilation and the hypoxic ventilatory response (HVR) that involves neural plasticity in both carotid body chemoreceptors and brainstem respiratory centers. The mechanisms of such plasticity are not completely understood but recent animal studies show it can be blocked by administering ibuprofen, a nonsteroidal anti-inflammatory drug, during chronic hypoxia. We tested the hypothesis that ibuprofen would also block the increase in HVR with chronic hypoxia in humans in 15 healthy men and women using a double-blind, placebo controlled, cross-over trial. The isocapnic HVR was measured with standard methods in subjects treated with ibuprofen (400 mg every 8 hrs) or placebo for 48 hours at sea level and 48 hours at high altitude (3,800 m). Subjects returned to sea level for at least 30 days prior to repeating the protocol with the opposite treatment. Ibuprofen significantly decreased the HVR after acclimatization to high altitude compared to placebo but it did not affect ventilation or arterial O2 saturation breathing ambient air at high altitude. Hence, compensatory responses prevent hypoventilation with decreased isocapnic ventilatory O2-sensitivity from ibuprofen at this altitude. The effect of ibuprofen to decrease the HVR in humans provides the first experimental evidence that a signaling mechanism described for ventilatory acclimatization to hypoxia in animal models also occurs in people. This establishes a foundation for the future experiments to test the potential role of different mechanisms for neural plasticity and ventilatory acclimatization in humans with chronic hypoxemia from lung disease.

Cardiac responses to hypoxia and reoxygenation in Drosophila.

An adequate supply of oxygen is important for the survival of all tissues, but it is especially critical for tissues with high-energy demands, such as the heart. Insufficient tissue oxygenation occurs under a variety of conditions, including high altitude, embryonic and fetal development, inflammation, and thrombotic diseases, often affecting multiple organ systems. Responses and adaptations of the heart to hypoxia are of particular relevance in human cardiovascular and pulmonary diseases, in which the effects of hypoxic exposure can range in severity from transient to long-lasting. This study uses the genetic model system Drosophila to investigate cardiac responses to acute (30 min), sustained (18 h), and chronic (3 wk) hypoxia with reoxygenation. Whereas hearts from wild-type flies recovered quickly after acute hypoxia, exposure to sustained or chronic hypoxia significantly compromised heart function upon reoxygenation. Hearts from flies with mutations in sima, the Drosophila homolog of the hypoxia-inducible factor alpha subunit (HIF-α), exhibited exaggerated reductions in cardiac output in response to hypoxia. Heart function in hypoxia-selected flies, selected over many generations for survival in a low-oxygen environment, revealed reduced cardiac output in terms of decreased heart rate and fractional shortening compared with their normoxia controls. Hypoxia-selected flies also had smaller hearts, myofibrillar disorganization, and increased extracellular collagen deposition, consistent with the observed reductions in contractility. This study indicates that longer-duration hypoxic insults exert deleterious effects on heart function that are mediated, in part, by sima and advances Drosophila models for the genetic analysis of cardiac-specific responses to hypoxia and reoxygenation.

Identification of genes underlying hypoxia tolerance in Drosophila by a P-element screen.

Hypoxia occurs in physiologic conditions (e.g. high altitude) or during pathologic states (e.g. ischemia). Our research is focused on understanding the molecular mechanisms that lead to adaptation and survival or injury to hypoxic stress using Drosophila as a model system. To identify genes involved in hypoxia tolerance, we screened the P-SUP P-element insertion lines available for all the chromosomes of Drosophila. We screened for the eclosion rates of embryos developing under 5% O(2) condition and the number of adult flies surviving one week after eclosion in the same hypoxic environment. Out of 2187 lines (covering ~1870 genes) screened, 44 P-element lines representing 44 individual genes had significantly higher eclosion rates (i.e. >70%) than those of the controls (i.e. ~7-8%) under hypoxia. The molecular function of these candidate genes ranged from cell cycle regulation, DNA or protein binding, GTP binding activity, and transcriptional regulators. In addition, based on pathway analysis, we found these genes are involved in multiple pathways, such as Notch, Wnt, Jnk, and Hedgehog. Particularly, we found that 20 out of the 44 candidate genes are linked to Notch signaling pathway, strongly suggesting that this pathway is essential for hypoxia tolerance in flies. By employing the UAS/RNAi-Gal4 system, we discovered that genes such as osa (linked to Wnt and Notch pathways) and lqf (Notch regulator) play an important role in survival and development under hypoxia in Drosophila. Based on these results and our previous studies, we conclude that hypoxia tolerance is a polygenic trait including the Notch pathway.

Sodium nitrite mitigates ventilator-induced lung injury in rats.

BACKGROUND: Nitrite (NO2) is a physiologic source of nitric oxide and protects against ischemia-reperfusion injuries. We hypothesized that nitrite would be protective in a rat model of ventilator-induced lung injury and sought to determine if nitrite protection is mediated by enzymic catalytic reduction to nitric oxide. METHODS: Rats were anesthetized and mechanically ventilated. Group 1 had low tidal volume ventilation (LVT) (6 ml/kg and 2 cm H2O positive end-expiratory pressure; n=10); group 2 had high tidal volume ventilation (HVT) (2 h of 35 cm H2O inspiratory peak pressure and 0 cm H2O positive end-expiratory pressure; n=14); groups 3-5: HVT with sodium nitrite (NaNO2) pretreatment (0.25, 2.5, 25 µmol/kg IV; n=6-8); group 6: HVT+NaNO2+nitric oxide scavenger 2-(4-carboxyphenyl)-4,5dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3oxide(n=6); group 7: HVT+NaNO2+nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (n=7); and group 8: HVT+NaNO2+xanthine oxidoreductase inhibitor allopurinol (n=6). Injury assessment included physiologic measurements (gas exchange, lung compliance, lung edema formation, vascular perfusion pressures) with histologic and biochemical correlates of lung injury and protection. RESULTS: Injurious ventilation caused statistically significant injury in untreated animals. NaNO2 pretreatment mitigated the gas exchange deterioration, lung edema formation, and histologic injury with maximal protection at 2.5 µmol/kg. Decreasing nitric oxide bioavailability by nitric oxide scavenging, nitric oxide synthase inhibition, or xanthine oxidoreductase inhibition abolished the protection by NaNO2. CONCLUSIONS: Nitrite confers protection against ventilator-induced lung injury in rats. Catalytic reduction to nitric oxide and mitigation of ventilator-induced lung injury is dependent on both xanthine oxidoreductase and nitric oxide synthases.