Membrane remodelling triggers maturation of excitation-contraction coupling in 3D-shaped human-induced pluripotent stem cell-derived cardiomyocytes.

The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the Ca2+-dependent excitation-contraction (EC) coupling mechanism. Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca2+ channels critical for EC coupling in close proximity, the L-type Ca2+ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca2+-dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca2+ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches.

nNOS-derived NO modulates force production and iNO-derived NO the excitability in C2C12-derived 3D tissue engineering skeletal muscle via different NO signaling pathways.

Nitric oxide (NO) is a bioactive gas produced by one of the three NO synthases: neuronal NOS (nNOS), inducible (iNOS), and endothelial NOS (eNOS). NO has a relevant modulatory role in muscle contraction; this takes place through two major signaling pathways: (i) activation of soluble guanylate cyclase and, thus, protein kinase G or (ii) nitrosylation of sulfur groups of cysteine. Although it has been suggested that nNOS-derived NO is the responsible isoform in muscle contraction, the roles of eNOS and iNOS and their signaling pathways have not yet been clarified. To elucidate the action of each pathway, we optimized the generation of myooids, an engineered skeletal muscle tissue based on the C2C12 cell line. In comparison with diaphragm strips from wild-type mice, 180 myooids were analyzed, which expressed all relevant excitation-contraction coupling proteins and both nNOS and iNOS isoforms. Along with the biochemical results, myooids treated with NO donor (SNAP) and unspecific NOS blocker (L-NAME) revealed a comparable NO modulatory effect on force production as was observed in the diaphragm strips. Under the effects of pharmacological tools, we analyzed the myooids in response to electrical stimulation of two possible signaling pathways and NO sources. The nNOS-derived NO exerted its negative effect on force production via the sGG-PKG pathway, while iNOS-derived NO increased the excitability in response to sub-threshold electrical stimulation. These results strengthen the hypotheses of previous reports on the mechanism of action of NO during force production, showed a novel function of iNOS-derived NO, and establish the myooid as a novel and robust alternative model for pathophysiological skeletal muscle research.

Substrate Stiffness Influences Structural and Functional Remodeling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Novel treatment strategies for cardiac tissue regeneration are heading for the use of engineered cardiac tissue made from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Despite the proven cardiogenic phenotype of these cells, a significant lack of structural and functional properties of mature myocytes prevents safe integration into the diseased heart. To date, maturation processes of cardiomyocytes remain largely unknown but may comprise biophysical cues from the immediate cell environment. Mechanosensing is one critical ability of cells to react to environmental changes. Accordingly, the surrounding substrate stiffness, comprised of extracellular matrix (ECM), cells, and growth surface, critically influences the myocyte's physiology, as known from deleterious remodeling processes in fibrotic hearts. Conversely, the mechanical properties during culture of iPSC-CMs may impact on their structural and functional maturation. Here, we tested the hypothesis that the environmental stiffness influences structural and functional properties of iPSC-CMs and investigated the effect of different substrate stiffnesses on cell contractility, excitation-contraction (EC) coupling, and intercellular coupling. Culture surfaces with defined stiffnesses ranging from rigid glass with 25GPa to PDMS of physiological softness were coated with ECM proteins and seeded with murine iPSC-CMs. Using confocal imaging, cardiac protein expression was assessed. Ca2+ handling and contractile properties were analyzed on different substrate stiffnesses. Intercellular coupling via gap junctions was investigated by fluorescence recovery after photobleaching (FRAP). Our data revealed greater organization of L-type Ca2+ channels and ryanodine receptors and increased EC-coupling gain, demonstrating structural and functional maturation in cells grown on soft surfaces. In addition, increased shortening and altered contraction dynamics revealed increased myofilament Ca2+ sensitivity in phase-plane loops. Moreover, connexin 43 expression was significantly increased in iPSC-CMs grown on soft surfaces leading to improved intercellular coupling. Taken together, our results demonstrate that soft surfaces with stiffnesses in the physiological range improve the expression pattern and interaction of cardiac proteins relevant for EC-coupling. In parallel, soft substrates influence contractile properties and improve intercellular coupling in iPSC-CMs. We conclude that the mechanical stiffness of the cell environment plays an important role in driving iPSC-CMs toward further maturation by inducing adaptive responses.

Cardiomyocyte depolarization triggers NOS-dependent NO transient after calcium release, reducing the subsequent calcium transient.

Cardiac excitation-contraction coupling and metabolic and signaling activities are centrally modulated by nitric oxide (NO), which is produced by one of three NO synthases (NOSs). Despite the significant role of NO in cardiac Ca2+ homeostasis regulation under different pathophysiological conditions, such as Duchenne muscular dystrophy (DMD), no precise method describes the production, source or effect of NO through two NO signaling pathways: soluble guanylate cyclase-protein kinase G (NO-sGC-PKG) and S-nitrosylation (SNO). Using a novel strategy involving isolated murine cardiomyocytes loaded with a copper-based dye highly specific for NO, we observed a single transient NO production signal after each electrical stimulation event. The NO transient signal started 67.5 ms after the beginning of Rhod-2 Ca2+ transient signal and lasted for approximately 430 ms. Specific NOS isoform blockers or NO scavengers significantly inhibited the NO transient, suggesting that wild-type (WT) cardiomyocytes produce nNOS-dependent NO transients. Conversely, NO transient in mdx cardiomyocyte, a mouse model of DMD, was dependent on inducible NOS (iNOS) and endothelial (eNOS). In a consecutive stimulation protocol, the nNOS-dependent NO transient in WT cardiomyocytes significantly reduced the next Ca2+ transient via NO-sGC-PKG. In mdx cardiomyocytes, this inhibitory effect was iNOS- and eNOS-dependent and occurred through the SNO pathway. Basal NO production was nNOS- and iNOS-dependent in WT cardiomyocytes and eNOS- and iNOS-dependent in mdx cardiomyocytes. These results showed cardiomyocyte produces NO isoform-dependent transients upon membrane depolarization at the millisecond time scale activating a specific signaling pathway to negatively modulate the subsequent Ca2+ transient.

Mepivacaine reduces calcium transients in isolated murine ventricular cardiomyocytes.

BACKGROUND: The potential mechanism of mepivacaine's myocardial depressant effect observed in papillary muscle has not yet been investigated at cellular level. Therefore, we evaluated mepivacaine's effects on Ca2+ transient in isolated adult mouse cardiomyocytes. METHODS: Single ventricular myocytes were enzymatically isolated from wild-type C57Bl/6 mice and loaded with 10 µM fluorescent Ca2+ indicator Fluo-4-AM to record intracellular Ca2+ transients upon electrical stimulation. The mepivacaine effects at half-maximal inhibitory concentration (IC50) was determined on calibrated cardiomyocytes' Ca2+ transients by non-parametric statistical analyses on biophysical parameters. Combination of mepivacaine with NCX blockers ORM-10103 or NiCl2 were used to test a possible mechanism to explain mepivacaine-induced Ca2+ transients' reduction. RESULTS: A significant inhibition at mepivacaine's IC50 (50 µM) on Ca2+ transients was measured in biophysical parameters such as peak (control: 528.6 ± 73.61 nM vs mepivacaine: 130.9 ± 15.63 nM; p < 0.05), peak area (control: 401.7 ± 63.09 nM*s vs mepivacaine: 72.14 ± 10.46 nM*s; p < 0.05), slope (control: 7699 ± 1110 nM/s vs mepivacaine: 1686 ± 226.6 nM/s; p < 0.05), time to peak (control: 107.9 ± 8.967 ms vs mepivacaine: 83.61 ± 7.650 ms; p < 0.05) and D50 (control: 457.1 ± 47.16 ms vs mepivacaine: 284.5 ± 22.71 ms; p < 0.05). Combination of mepivacaine with NCX blockers ORM-10103 or NiCl2 showed a significant increase in the baseline of [Ca2+] and arrhythmic activity upon electrical stimulation. CONCLUSION: At cellular level, mepivacaine blocks Na+ channels, enhancing the reverse mode activity of NCX, leading to a significant reduction of Ca2+ transients. These results suggest a new mechanism for the mepivacaine-reduction contractility effect.

Augmentation of myocardial If dysregulates calcium homeostasis and causes adverse cardiac remodeling.

HCN channels underlie the depolarizing funny current (If) that contributes importantly to cardiac pacemaking. If is upregulated in failing and infarcted hearts, but its implication in disease mechanisms remained unresolved. We generated transgenic mice (HCN4tg/wt) to assess functional consequences of HCN4 overexpression-mediated If increase in cardiomyocytes to levels observed in human heart failure. HCN4tg/wt animals exhibit a dilated cardiomyopathy phenotype with increased cellular arrhythmogenicity but unchanged heart rate and conduction parameters. If augmentation induces a diastolic Na+ influx shifting the Na+/Ca2+ exchanger equilibrium towards 'reverse mode' leading to increased [Ca2+]i. Changed Ca2+ homeostasis results in significantly higher systolic [Ca2+]i transients and stimulates apoptosis. Pharmacological inhibition of If prevents the rise of [Ca2+]i and protects from ventricular remodeling. Here we report that augmented myocardial If alters intracellular Ca2+ homeostasis leading to structural cardiac changes and increased arrhythmogenicity. Inhibition of myocardial If per se may constitute a therapeutic mechanism to prevent cardiomyopathy.

Chronic hypoxia changes gene expression profile of primary rat carotid body cells: consequences on the expression of NOS isoforms and ET-1 receptors.

Sustained chronic hypoxia (CH) produces morphological and functional changes in the carotid body (CB). Nitric oxide (NO) and endothelin-1 (ET-1) play a major role as modulators of the CB oxygen chemosensory process. To characterize the effects of CH related to normoxia (Nx) on gene expression, particularly on ET-1 and NO pathways, primary cultures of rat CB cells were exposed to 7 days of CH. Total RNA was extracted, and cDNA-32P was synthesized and hybridized with 1,185 genes printed on a nylon membrane Atlas cDNA Expression Array. Out of 324 differentially expressed genes, 184 genes were upregulated, while 140 genes were downregulated. The cluster annotation and protein network analyses showed that both NO and ET-1 signaling pathways were significantly enriched and key elements of each pathway were differentially expressed. Thus, we assessed the effect of CH at the protein level of nitric oxide synthase (NOS) isoforms and ET-1 receptors. CH induced an increase in the expression of endothelial NOS, inducible NOS, and ETB. During CH, the administration of SNAP, a NO donor, upregulated ETB. Treatment with Tezosentan (ET-1 receptor blocker) during CH upregulated all three NOS isoforms, while the NOS blocker L-NAME induced upregulation of iNOS and ETB and downregulated the protein levels of ETA. These results show that CH for 7 days changed the cultured cell CB gene expression profile, the NO and ET-1 signaling pathways were highly enriched, and these two signaling pathways interfered with the protein expression of each other.

High-fat diet suppresses the positive effect of creatine supplementation on skeletal muscle function by reducing protein expression of IGF-PI3K-AKT-mTOR pathway.

High-fat (HF) diets in combination with sedentary lifestyle represent one of the major public health concerns predisposing to obesity and diabetes leading to skeletal muscle atrophy, decreased fiber diameter and muscle mass with accumulation of fat tissue resulting in loss of muscle strength. One strategy to overcome the maleficent effects of HF diet is resistance training, a strategy used to improve muscle mass, reverting the negative effects on obesity-related changes in skeletal muscle. Together with resistance training, supplementation with creatine monohydrate (CrM) in the diet has been used to improve muscle mass and strength. Creatine is a non-essential amino acid that is directly involved in the cross-bridge cycle providing a phosphate group to ADP during the initiation of muscle contraction. Besides its antioxidant and anti-inflammatory effects CrM also upregulates IGF-1 resulting in hyperthophy with an increase in muscle function. However, it is unknown whether CrM supplementation during resistance training would revert the negative effects of high-fat diet on the muscle performance. During 8 weeks we measured muscle performance to climb a 1.1m and 80° ladder with increasing load on trained rats that had received standard diet or high-fat diet, supplemented or not with CrM. We observed that the CrM supplementation up-regulated IGF-1 and phospho-AKT protein levels, suggesting an activation of the IGF1-PI3K-Akt/PKB-mTOR pathway. Moreover, despite the CrM supplementation, HF diet down-regulated several proteins of the IGF1-PI3K-Akt/PKB-mTOR pathway, suggesting that diet lipid content is crucial to maintain or improve muscle function during resistance training.

Alcohol reduces muscle fatigue through atomistic interactions with nicotinic receptors.

Alcohol consumption affects many organs and tissues, including skeletal muscle. However, the molecular mechanism of ethanol action on skeletal muscle remains unclear. Here, using molecular dynamics simulations and single channel recordings, we show that ethanol interacts with a negatively charged amino acid within an extracellular region of the neuromuscular nicotinic acetylcholine receptor (nAChR), thereby altering its global conformation and reducing the single channel current amplitude. Charge reversal of the negatively charged amino acid abolishes the nAChR-ethanol interaction. Moreover, using transgenic animals harboring the charge-reversal mutation, ex vivo measurements of muscle force production show that ethanol counters fatigue in wild type but not homozygous αE83K mutant animals. In accord, in vivo studies of motor coordination following ethanol administration reveal an approximately twofold improvement for wild type compared to homozygous mutant animals. Together, the converging results from molecular to animal studies suggest that ethanol counters muscle fatigue through its interaction with neuromuscular nAChRs.

NO-sGC Pathway Modulates Ca2+ Release and Muscle Contraction in Zebrafish Skeletal Muscle.

Vertebrate skeletal muscle contraction and relaxation is a complex process that depends on Ca2+ ions to promote the interaction of actin and myosin. This process can be modulated by nitric oxide (NO), a gas molecule synthesized endogenously by (nitric oxide synthase) NOS isoforms. At nanomolar concentrations NO activates soluble guanylate cyclase (sGC), which in turn activates protein kinase G via conversion of GTP into cyclic GMP. Alternatively, NO post-translationally modifies proteins via S-nitrosylation of the thiol group of cysteine. However, the mechanisms of action of NO on Ca2+ homeostasis during muscle contraction are not fully understood and we hypothesize that NO exerts its effects on Ca2+ homeostasis in skeletal muscles mainly through negative modulation of Ca2+ release and Ca2+ uptake via the NO-sGC-PKG pathway. To address this, we used 5-7 days-post fecundation-larvae of zebrafish, a well-established animal model for physiological and pathophysiological muscle activity. We evaluated the response of muscle contraction and Ca2+ transients in presence of SNAP, a NO-donor, or L-NAME, an unspecific NOS blocker in combination with specific blockers of key proteins of Ca2+ homeostasis. We also evaluate the expression of NOS in combination with dihydropteridine receptor, ryanodine receptor and sarco/endoplasmic reticulum Ca2+ ATPase. We concluded that endogenous NO reduced force production through negative modulation of Ca2+ transients via the NO-sGC pathway. This effect could be reversed using an unspecific NOS blocker or sGC blocker.

Extraction Protocols for Individual Zebrafish's Ventricle Myosin and Skeletal Muscle Actin for In vitro Motility Assays.

The in vitro motility assay (IVMA) is a technique that enables the measurement of the interaction between actin and myosin providing a relatively simple model to understand the mechanical muscle function. For actin-myosin IVMA, myosin is immobilized in a measurement chamber, where it converts chemical energy provided by ATP hydrolysis into mechanical energy. The result is the movement of fluorescently labeled actin filaments that can be recorded microscopically and analyzed quantitatively. Resulting sliding speeds and patterns help to characterize the underlying actin-myosin interaction that can be affected by different factors such as mutations or active compounds. Additionally, modulatory actions of the regulatory proteins tropomyosin and troponin in the presence of calcium on actin-myosin interaction can be studied with the IVMA. Zebrafish is considered a suitable model organism for cardiovascular and skeletal muscle research. In this context, straightforward protocols for the isolation and use of zebrafish muscle proteins in the IVMA would provide a useful tool in molecular studies. Currently, there are no protocols available for the mentioned purpose. Therefore, we developed fast and easy protocols for characterization of zebrafish proteins in the IVMA. Our protocols enable the interested researcher to (i) isolate actin from zebrafish skeletal muscle and (ii) extract functionally intact myosin from cardiac and skeletal muscle of individual adult zebrafish. Zebrafish tail muscle actin is isolated after acetone powder preparation, polymerized, and labeled with Rhodamine-Phalloidin. Myosin from ventricles of adult zebrafish is extracted directly into IVMA flow-cells. The same extraction protocol is applicable for comparably small tissue pieces as from zebrafish tail, mouse and frog muscle. After addition of the fluorescently labeled F-actin from zebrafish-or other origin-and ATP, sliding movement can be visualized using a fluorescence microscope and an intensified CCD camera. Taken together, we introduce a method for functional analysis in zebrafish cardiac and skeletal muscle research to study mutations at the molecular level of thick or thin filament proteins. Additionally, preliminary data indicate the usefulness of the presented method to perform the IVMA with myosin extracted from muscles of other animal models.

Essential light chain S195 phosphorylation is required for cardiac adaptation under physical stress.

AIMS: Regulatory proteins of the sarcomere are pivotal for normal heart function and when affected by mutations are frequently causing cardiomyopathy. The exact function of these regulatory proteins and how mutations in these translate into distinct cardiomyopathy phenotypes remains poorly understood. Mutations in the essential myosin light chain (ELC) are linked to human cardiomyopathy characterized by a marked variability in disease phenotypes and high incidences of sudden death. Here we studied the role of the highly conserved S195 phosphorylation site of ELC using heterozygous adult zebrafish lazy susan (laz(m647)) in regulating contractile function in normal physiology and disease. METHODS AND RESULTS: Echocardiography revealed signs of systolic dysfunction in otherwise phenotypically unremarkable heterozygote mutants. However, after physical stress, heart function of laz heterozygous zebrafish severely deteriorated causing heart failure and sudden death. Mechanistically, we show that upon physical stress, ELCs become phosphorylated and lack of S195 dominant-negatively impairs ELC phosphorylation. In vitro motility analysis with native myosin from adult heterozygous hearts demonstrates that S195 loss, specifically following physical stress, results in altered acto-myosin sliding velocities and myosin binding cooperativity, causing reduced force generation and organ dysfunction. CONCLUSION: Using adult heterozygous zebrafish, we show that ELC S195 phosphorylation is pivotal for adaptation of cardiac function to augmented physical stress and we provide novel mechanistic insights into the pathogenesis of ELC-linked cardiomyopathy.

Functional second harmonic generation microscopy probes molecular dynamics with high temporal resolution.

Second harmonic generation (SHG) microscopy is a powerful tool for label free ex vivo or in vivo imaging, widely used to investigate structure and organization of endogenous SHG emitting proteins such as myosin or collagen. Polarization resolved SHG microscopy renders supplementary information and is used to probe different molecular states. This development towards functional SHG microscopy is calling for new methods for high speed functional imaging of dynamic processes. In this work we present two approaches with linear polarized light and demonstrate high speed line scan measurements of the molecular dynamics of the motor protein myosin with a time resolution of 1 ms in mammalian muscle cells. Such a high speed functional SHG microscopy has high potential to deliver new insights into structural and temporal molecular dynamics under ex vivo or in vivo conditions.

Ebf factors and MyoD cooperate to regulate muscle relaxation via Atp2a1.

Myogenic regulatory factors such as MyoD and Myf5 lie at the core of vertebrate muscle differentiation. However, E-boxes, the cognate binding sites for these transcription factors, are not restricted to the promoters/enhancers of muscle cell-specific genes. Thus, the specificity in myogenic transcription is poorly defined. Here we describe the transcription factor Ebf3 as a new determinant of muscle cell-specific transcription. In the absence of Ebf3 the lung does not unfold at birth, resulting in respiratory failure and perinatal death. This is due to a hypercontractile diaphragm with impaired Ca(2+) efflux-related muscle functions. Expression of the Ca(2+) pump Serca1 (Atp2a1) is downregulated in the absence of Ebf3, and its transgenic expression rescues this phenotype. Ebf3 binds directly to the promoter of Atp2a1 and synergises with MyoD in the induction of Atp2a1. In skeletal muscle, the homologous family member Ebf1 is strongly expressed and together with MyoD induces Atp2a1. Thus, Ebf3 is a new regulator of terminal muscle differentiation in the diaphragm, and Ebf factors cooperate with MyoD in the induction of muscle-specific genes.

Ventilatory chemosensory drive is blunted in the mdx mouse model of Duchenne Muscular Dystrophy (DMD).

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the DMD gene resulting in an absence of dystrophin in neurons and muscle. Respiratory failure is the most common cause of mortality and previous studies have largely concentrated on diaphragmatic muscle necrosis and respiratory failure component. Here, we investigated the integrity of respiratory control mechanisms in the mdx mouse model of DMD. Whole body plethysmograph in parallel with phrenic nerve activity recordings revealed a lower respiratory rate and minute ventilation during normoxia and a blunting of the hypoxic ventilatory reflex in response to mild levels of hypoxia together with a poor performance on a hypoxic stress test in mdx mice. Arterial blood gas analysis revealed low PaO2 and pH and high PaCO2 in mdx mice. To investigate chemosensory respiratory drive, we analyzed the carotid body by molecular and functional means. Dystrophin mRNA and protein was expressed in normal mice carotid bodies however, they are absent in mdx mice. Functional analysis revealed abnormalities in Dejours test and the early component of the hypercapnic ventilatory reflex in mdx mice. Together, these results demonstrate a malfunction in the peripheral chemosensory drive that would be predicted to contribute to the respiratory failure in mdx mice. These data suggest that investigating and monitoring peripheral chemosensory drive function may be useful for improving the management of DMD patients with respiratory failure.

Cardiac and respiratory dysfunction in Duchenne muscular dystrophy and the role of second messengers.

Duchenne muscular dystrophy (DMD) affects young boys and is characterized by the absence of dystrophin, a large cytoskeletal protein present in skeletal and cardiac muscle cells and neurons. The heart and diaphragm become necrotic in DMD patients and animal models of DMD, resulting in cardiorespiratory failure as the leading cause of death. The major consequences of the absence of dystrophin are high levels of intracellular Ca(2+) and the unbalanced production of NO that can finally trigger protein degradation and cell death. Cytoplasmic increase in Ca(2+) concentration directly and indirectly triggers different processes such as necrosis, fibrosis, and activation of macrophages. The absence of the neuronal isoform of nitric oxide synthase (nNOS) and the overproduction of NO by the inducible isoform (iNOS) further increase the intracellular Ca(2+) via a hypernitrosylation of the ryanodine receptor. NO overproduction, which further induces the expression of iNOS but decreases the expression of the endothelial isoform (eNOS), deregulates the muscle tissue blood flow creating an ischemic situation. The high levels of Ca(2+) in dystrophic muscles and the ischemic state of the muscle tissue would culminate in a positive feedback loop. While efforts continue toward optimizing cardiac and respiratory care of DMD patients, both Ca(2+) and NO in cardiac and respiratory muscle pathways have been shown to be important to the etiology of the disease. Understanding the mechanisms behind the fine regulation of Ca(2+) -NO may be important for a noninterventional and noninvasive supportive approach to treat DMD patients, improving the quality of life and natural history of DMD patients.

Expression profiling reveals novel hypoxic biomarkers in peripheral blood of adult mice exposed to chronic hypoxia.

Hypoxia induces a myriad of changes including an increase in hematocrit due to erythropoietin (EPO) mediated erythropoiesis. While hypoxia is of importance physiologically and clinically, lacunae exist in our knowledge of the systemic and temporal changes in gene expression occurring in blood during the exposure and recovery from hypoxia. To identify these changes expression profiling was conducted on blood obtained from cohorts of C57Bl-10 wild type mice that were maintained at normoxia (NX), exposed for two weeks to normobaric chronic hypoxia (CH) or two weeks of CH followed by two weeks of normoxic recovery (REC). Using stringent bioinformatic cut-offs (0% FDR, 2 fold change cut-off), 230 genes were identified and separated into four distinct temporal categories. Class I) contained 1 transcript up-regulated in both CH and REC; Class II) contained 202 transcripts up-regulated in CH but down-regulated after REC; Class III) contained 9 transcripts down-regulated both in CH and REC; Class IV) contained 18 transcripts down-regulated after CH exposure but up-regulated after REC. Profiling was independently validated and extended by analyzing expression levels of selected genes as novel biomarkers from our profile (e.g. spectrin alpha-1, ubiquitin domain family-1 and pyrroline-5-carboxylate reductase-1) by performing qPCR at 7 different time points during CH and REC. Our identification and characterization of these genes define transcriptome level changes occurring during chronic hypoxia and normoxic recovery as well as novel blood biomarkers that may be useful in monitoring a variety of physiological and pathological conditions associated with hypoxia.