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Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfs1 (mitochondrial Rieske iron-sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined, and glucose utilization increased. Simultaneously, the hearts underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy. Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident. In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA sequencing revealed upregulation of genes associated with cardiac development and proliferation. Metabolomic analysis revealed a decrease in α-ketoglutarate (required for TET-mediated demethylation) and an increase in S-adenosylmethionine (required for methyltransferase activity). Analysis revealed an increase in methylated CpGs near gene transcriptional start sites. Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes, resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation.

Aims: Therapeutic approaches to treat familial dilated cardiomyopathy (DCM), which is characterized by depressed sarcomeric tension and susceptibility to Ca(2+)-related arrhythmias, have been generally unsuccessful. Our objective in the present work was to determine the effect of the angiotensin II type 1 receptor (AT1R) biased ligand, TRV120023, on contractility of hearts of a transgenic mouse model of familial DCM with mutation in tropomyosin at position 54 (TG-E54K). Our rationale is based on previous studies, which have supported the hypothesis that biased G-protein-coupled receptor ligands, signalling via β-arrestin, increase cardiac contractility with no effect on Ca(2+) transients. Our previous work demonstrated that the biased ligand TRV120023 is able to block angiotensin-induced hypertrophy, while promoting an increase in sarcomere Ca(2+) response., Methods and Results: We tested the hypothesis that the depression in cardiac function associated with DCM can be offset by infusion of the AT1R biased ligand, TRV120023. We intravenously infused saline, TRV120023, or the unbiased ligand, losartan, for 15 min in TG-E54K and non-transgenic mice to obtain left ventricular pressure-volume relations. Hearts were analysed for sarcomeric protein phosphorylation. Results showed that the AT1R biased ligand increases cardiac performance in TG-E54K mice in association with increased myosin light chain-2 phosphorylation., Conclusion: Treatment of mice with an AT1R biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R biased ligands may prove to be a novel inotropic approach in familial DCM., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Rationale: Chronic hypoxia induces pulmonary vascular remodeling, pulmonary hypertension, and right ventricular hypertrophy. At present, little is known about mechanisms driving these responses. Hypoxia-inducible factor-1α (HIF-1α) is a master regulator of transcription in hypoxic cells, up-regulating genes involved in energy metabolism, proliferation, and extracellular matrix reorganization. Systemic loss of a single HIF-1α allele has been shown to attenuate hypoxic pulmonary hypertension, but the cells contributing to this response have not been identified., Objectives: We sought to determine the contribution of HIF-1α in smooth muscle on pulmonary vascular and right heart responses to chronic hypoxia., Methods: We used mice with homozygous conditional deletion of HIF-1α combined with tamoxifen-inducible smooth muscle-specific Cre recombinase expression. Mice received either tamoxifen or vehicle followed by exposure to either normoxia or chronic hypoxia (10% O2) for 30 days before measurement of cardiopulmonary responses., Measurements and Main Results: Tamoxifen-induced smooth muscle-specific deletion of HIF-1α attenuated pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, right ventricular hypertrophy was unchanged despite attenuated pulmonary pressures., Conclusions: These results indicate that HIF-1α in smooth muscle contributes to pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, loss of HIF-1 function in smooth muscle does not affect hypoxic cardiac remodeling, suggesting that the cardiac hypertrophy response is not directly coupled to the increase in pulmonary artery pressure.

Rationale: The role of reactive oxygen species (ROS) signaling in the O(2) sensing mechanism underlying acute hypoxic pulmonary vasoconstriction (HPV) has been controversial. Although mitochondria are important sources of ROS, studies using chemical inhibitors have yielded conflicting results, whereas cellular models using genetic suppression have precluded in vivo confirmation. Hence, genetic animal models are required to test mechanistic hypotheses., Objectives: We tested whether mitochondrial Complex III is required for the ROS signaling and vasoconstriction responses to acute hypoxia in pulmonary arteries (PA)., Methods: A mouse permitting Cre-mediated conditional deletion of the Rieske iron-sulfur protein (RISP) of Complex III was generated. Adenoviral Cre recombinase was used to delete RISP from isolated PA vessels or smooth muscle cells (PASMC)., Measurements and Main Results: In PASMC, RISP depletion abolished hypoxia-induced increases in ROS signaling in the mitochondrial intermembrane space and cytosol, and it abrogated hypoxia-induced increases in [Ca(2+)](i). In isolated PA vessels, RISP depletion abolished hypoxia-induced ROS signaling in the cytosol. Breeding the RISP mice with transgenic mice expressing tamoxifen-activated Cre in smooth muscle permitted the depletion of RISP in PASMC in vivo. Precision-cut lung slices from those mice revealed that RISP depletion abolished hypoxia-induced increases in [Ca(2+)](i) of the PA. In vivo RISP depletion in smooth muscle attenuated the acute hypoxia-induced increase in right ventricular systolic pressure in anesthetized mice., Conclusions: Acute hypoxia induces superoxide release from Complex III of smooth muscle cells. These oxidant signals diffuse into the cytosol and trigger increases in [Ca(2+)](i) that cause acute hypoxic pulmonary vasoconstriction.

AMP-activated protein kinase (AMPK) is an energy sensor activated by increases in [AMP] or by oxidant stress (reactive oxygen species [ROS]). Hypoxia increases cellular ROS signaling, but the pathways underlying subsequent AMPK activation are not known. We tested the hypothesis that hypoxia activates AMPK by ROS-mediated opening of calcium release-activated calcium (CRAC) channels. Hypoxia (1.5% O(2)) augments cellular ROS as detected by the redox-sensitive green fluorescent protein (roGFP) but does not increase the [AMP]/[ATP] ratio. Increases in intracellular calcium during hypoxia were detected with Fura2 and the calcium-calmodulin fluorescence resonance energy transfer (FRET) sensor YC2.3. Antioxidant treatment or removal of extracellular calcium abrogates hypoxia-induced calcium signaling and subsequent AMPK phosphorylation during hypoxia. Oxidant stress triggers relocation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, to the plasma membrane. Knockdown of STIM1 by short interfering RNA (siRNA) attenuates the calcium responses to hypoxia and subsequent AMPK phosphorylation, while inhibition of L-type calcium channels has no effect. Knockdown of the AMPK upstream kinase LKB1 by siRNA does not prevent AMPK activation during hypoxia, but knockdown of CaMKKβ abolishes the AMPK response. These findings reveal that hypoxia can trigger AMPK activation in the apparent absence of increased [AMP] through ROS-dependent CRAC channel activation, leading to increases in cytosolic calcium that activate the AMPK upstream kinase CaMKKβ.

Cellular oxygen sensing is required for hypoxia-inducible factor-1alpha stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Here we find that terpestacin, a small molecule previously identified in a screen of microbial extracts, binds to the 13.4-kDa subunit (UQCRB) of mitochondrial Complex III, resulting in inhibition of hypoxia-induced reactive oxygen species generation. Consequently, such inhibition blocks hypoxia-inducible factor activation and tumor angiogenesis in vivo, without inhibiting mitochondrial respiration. Overexpression of UQCRB or its suppression using RNA interference demonstrates that it plays a crucial role in the oxygen sensing mechanism that regulates responses to hypoxia. These findings provide a novel molecular basis of terpestacin targeting UQCRB of Complex III in selective suppression of tumor progression.

Phase I of the hypoxic pulmonary vasoconstriction (HPV) response begins upon transition to hypoxia and involves an increase in cytosolic calcium ([Ca(2+)](i)). Phase II develops during prolonged hypoxia and involves increases in constriction without further increases in [Ca(2+)](i), suggesting an increase in Ca(2+) sensitivity. Prolonged hypoxia activates RhoA and RhoA kinase, which may increase Ca(2+) sensitivity, but the mechanism is unknown. We previously found that reactive oxygen species (ROS) trigger Phase I. We therefore asked whether ROS generation during prolonged hypoxia activates RhoA in PA smooth muscle cells (PASMCs) and endothelial cells (PAECs) during Phase II. By using a cytosolic redox sensor, RoGFP, we detected increased oxidant signaling in prolonged hypoxia in PASMCs (29.8 +/- 1.3% to 39.8 +/- 1.4%) and PAECs (25.9 +/- 2.1% to 43.7.9 +/- 3.5%), which was reversed on the return to normoxia and was attenuated with EUK-134 in both cell types. RhoA activity increased in PASMCs and PAECs during prolonged hypoxia (6.4 +/- 1.2-fold and 5.8 +/- 1.6-fold) and with exogenous H(2)O(2) (4.1- and 2.3-fold, respectively). However, abrogation of the ROS signal in PASMCs or PAECs with EUK-134 or anoxia failed to attenuate the increased RhoA activity. Thus, the ROS signal is sustained during prolonged hypoxia in PASMCs and PAECs, and this is sufficient but not required for RhoA activation.

Rationale: Recent studies have implicated mitochondrial reactive oxygen species (ROS) in regulating hypoxic pulmonary vasoconstriction (HPV), but controversy exists regarding whether hypoxia increases or decreases ROS generation., Objective: This study tested the hypothesis that hypoxia induces redox changes that differ among subcellular compartments in pulmonary (PASMCs) and systemic (SASMCs) smooth muscle cells., Methods and Results: We used a novel, redox-sensitive, ratiometric fluorescent protein sensor (RoGFP) to assess the effects of hypoxia on redox signaling in cultured PASMCs and SASMCs. Using genetic targeting sequences, RoGFP was expressed in the cytosol (Cyto-RoGFP), the mitochondrial matrix (Mito-RoGFP), or the mitochondrial intermembrane space (IMS-RoGFP), allowing assessment of oxidant signaling in distinct intracellular compartments. Superfusion of PASMCs or SASMCs with hypoxic media increased oxidation of both Cyto-RoGFP and IMS-RoGFP. However, hypoxia decreased oxidation of Mito-RoGFP in both cell types. The hypoxia-induced oxidation of Cyto-RoGFP was attenuated through the overexpression of cytosolic catalase in PASMCs., Conclusions: These results indicate that hypoxia causes a decrease in nonspecific ROS generation in the matrix compartment, whereas it increases regulated ROS production in the IMS, which diffuses to the cytosol of both PASMCs and SASMCs.

Nitric oxide (NO) has been implicated as a cardioprotective agent during ischemia/reperfusion (I/R), but the mechanism of protection is unknown. Oxidant stress contributes to cell death in I/R, so we tested whether NO protects by attenuating oxidant stress. Cardiomyocytes and murine embryonic fibroblasts were administered NO (10-1200 nM) during simulated ischemia, and cell death was assessed during reperfusion without NO. In each case, NO abrogated cell death during reperfusion. Cells overexpressing endothelial NO synthase (NOS) exhibited a similar protection, which was abolished by the NOS inhibitor N(omega)-nitro-l-arginine methyl ester. Protection was not mediated by guanylate cyclase or the mitochondrial K(ATP) channel, as inhibitors of these systems failed to abolish protection. NO did not prevent decreases in mitochondrial potential, but cells protected with NO demonstrated recovery of potential at reperfusion. Measurements using C11-BODIPY reveal that NO attenuates lipid peroxidation during ischemia and reperfusion. Measurements of oxidant stress using the ratiometric redox sensor HSP-FRET demonstrate that NO attenuates protein oxidation during ischemia. These findings reveal that physiological levels of NO during ischemia can attenuate oxidant stress both during ischemia and during reperfusion. This response is associated with a remarkable attenuation of cell death, suggesting that ischemic cell death may be a regulated event.

Mitochondria have been implicated as a potential site of O(2) sensing underlying hypoxic pulmonary vasoconstriction (HPV), but 2 disparate models have been proposed to explain their reaction to hypoxia. One model proposes that hypoxia-induced increases in mitochondrial reactive oxygen species (ROS) generation activate HPV through an oxidant-signaling pathway, whereas the other proposes that HPV is a result of decreased oxidant signaling. In an attempt to resolve this debate, we use a novel, ratiometric, redox-sensitive fluorescence resonance energy transfer (HSP-FRET) probe, in concert with measurements of reduced/oxidized glutathione (GSH/GSSG), to assess cytosolic redox responses in cultured pulmonary artery smooth muscle cells (PASMCs). Superfusion of PASMCs with hypoxic media increases the HSP-FRET ratio and decreases GSH/GSSG, indicating an increase in oxidant stress. The antioxidants pyrrolidinedithiocarbamate and N-acetyl-l-cysteine attenuated this response, as well as the hypoxia-induced increases in cytosolic calcium ([Ca(2+)](i)), assessed by the Ca(2+)-sensitive FRET sensor YC2.3. Adenoviral overexpression of glutathione peroxidase or cytosolic or mitochondrial catalase attenuated the hypoxia-induced increase in ROS signaling and [Ca(2+)](i). Adenoviral overexpression of cytosolic Cu, Zn-superoxide dismutase (SOD-I) had no effect on the hypoxia-induced increase in ROS signaling and [Ca(2+)](i), whereas mitochondrial matrix-targeted Mn-SOD (SOD-II) augmented [Ca(2+)](i). The mitochondrial inhibitor myxothiazol attenuated the hypoxia-induced changes in the ROS signaling and [Ca(2+)](i), whereas cyanide augmented the increase in [Ca(2+)](i). Finally, simultaneous measurement of ROS and Ca(2+) signaling in the same cell revealed that the initial increase in these 2 signals could not be distinguished temporally. These results demonstrate that hypoxia triggers increases in PASMC [Ca(2+)](i) by augmenting ROS signaling from the mitochondria.

Mechanical stretch activates a number of signaling pathways in endothelial cells, and it elicits a variety of functional responses including increases in the phosphorylation of focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in integrin-mediated signal transduction. Stretch also triggers an increase in the generation of reactive oxygen species (ROS), which may function as second messengers in the signal transduction cascades that activate cellular responses to strain. Mitochondria represent an important source of ROS in the cell, and these organelles may release ROS in response to strain by virtue of their attachment to cytoskeletal proteins. We therefore tested whether cyclic stretch increases FAK phosphorylation at Tyr397 through a mitochondrial ROS signaling pathway in bovine pulmonary artery endothelial cells (BPAEC). Oxidant signaling, measured using 2'7'-dichlorofluorescin (DCFH), increased 152 +/- 16% during 1.5 h of cyclic strain relative to unstrained controls. The mitochondrial inhibitors diphenylene iodonium (5 microM) or rotenone (2 microM) attenuated this increase, whereas L-nitroarginine (100 microM), allopurinol (100 microM), or apocynin (30 microM) had no effect. The antioxidants ebselen (5 microM) and dithiodidiethyldithiocarbamate (1 mM) inhibited the strain-induced increase in oxidant signaling, but Hb (5 microM) had no effect. These results indicate that strain induces oxidant release from mitochondria. Treatment with cytochalasin D (5 microM) abrogated strain-induced DCFH oxidation in BPAEC, indicating that actin filaments were required for stretch-induced mitochondrial ROS generation. Cyclic strain increased FAK phosphorylation at Tyr397, but this was abolished by mitochondrial inhibitors as well as by antioxidants. Strain-induced FAK phosphorylation was abrogated by inhibition of protein kinase C (PKC) with Ro-31-8220 or Gö-6976. These findings indicate that mitochondrial oxidants generated in response to endothelial strain trigger FAK phosphorylation through a signaling pathway that involves PKC.

We hypothesized that mitochondria function as the O2 sensors underlying hypoxic pulmonary vasoconstriction by releasing reactive oxygen species (ROS) from complex III of the electron transport chain (ETC). We have previously found that antioxidants or inhibition of the proximal region of the ETC attenuates hypoxic pulmonary vasoconstriction in rat lungs and blocks hypoxia-induced contraction of isolated pulmonary arterial (PA) myocytes. To determine whether the hypoxia-induced increases in mitochondrial ROS act to trigger calcium increases, we measured changes in cytosolic calcium ([Ca2+]i) using fura 2-AM (fluorescence at 340/380 nm) during perfusion with hypoxic media (PO2 12 mm Hg). Hypoxia caused an increase in fura 2 fluorescence, indicating an increase in [Ca2+]i. In superfused PA myocytes, diphenyleneiodonium, rotenone, and myxothiazol, which inhibit the proximal region of the ETC, attenuated hypoxia-induced calcium increases. Antimycin A and cyanide, which inhibit the distal region of the ETC, failed to abolish hypoxia-induced [Ca2+]i increases. To test whether mitochondrial H2O2 is required to trigger [Ca2+]i increases, catalase was overexpressed in PA myocytes with the use of a recombinant adenovirus. Catalase overexpression attenuated hypoxia-induced increases in [Ca2+]i, suggesting that H2O2 acts upstream from calcium increases during hypoxia. These results support the conclusion that mitochondria function as O2 sensors during hypoxia and demonstrate that ROS generated in the proximal region of the ETC act as second messengers to trigger calcium increases in PA myocytes during acute hypoxia.

Endothelial cells increase their secretion of the cytokine interleukin-6 (IL-6) during hypoxia, which then acts in an autocrine fashion to increase the permeability of cell monolayers. These responses are attenuated by antioxidants, suggesting that reactive oxygen species (ROS) participate in signaling in hypoxic endothelium. We tested whether mitochondria are responsible for these ROS in human umbilical vein endothelial cells exposed to hypoxia. Oxidation of the probe 2', 7'-dichlorodihydrofluorescein to fluorescent dichlorofluorescein or the probe dihydroethidium was used to assess oxidant signaling, whereas permeability was assessed by using transendothelial electrical resistance. Hypoxia elicited increases in dichlorofluorescein and dihydroethidium fluorescence that were abrogated by the mitochondrial electron transport (ET) inhibitors rotenone (2 micromol/L) and diphenyleneiodonium (5 micromol/L). The same ET inhibitors also attenuated hypoxia-induced increases in nuclear factor-kappaB (NF-kappaB) activation, although they did not abrogate NF-kappaB activation in response to endotoxin (lipopolysaccharide). ET inhibition also abolished the hypoxia-induced increases in IL-6 mRNA expression, hypoxia-stimulated IL-6 secretion into the media, and the hypoxia-induced increases in transendothelial electrical resistance of human umbilical vein endothelial cell monolayers. By contrast, the above responses to hypoxia were not significantly affected by treatment with the NAD(P)H oxidase inhibitor apocynin (30 micromol/L), the xanthine oxidase inhibitor allopurinol (100 micromol/L), or the NO synthase inhibitor N-nitro-L-arginine (100 micromol/L). We conclude that ROS signals originating from the mitochondrial ET chain trigger the increase in NF-kappaB activation, the transcriptional activation of IL-6, the secretion of IL-6 into the cell culture media, and the increases in endothelial permeability observed during hypoxia.

Hypoxia is a strong stimulus for the transcription of a set of genes, including erythropoietin and vascular endothelial growth factor. Here we report on the cloning, functional significance, and expression of a complementary DNA (cDNA) that is involved in hypoxia-mediated expression of these 2 genes. The full-length cDNA encodes a predicted protein of 806 amino acids that contains a leucine zipper motif. This protein, termed HAF for hypoxia-associated factor, binds to a 17-base pair (bp) region of the erythropoietin promoter, which was shown earlier to participate in hypoxia-induced expression of the erythropoietin gene. In Hep3B cells, clones modified to express HAF antisense RNA showed an attenuated response to hypoxia-mediated induction of both erythropoietin and vascular endothelial growth factor transcription. HAF showed sequence-specific interaction with a DNA element in the 5' untranslated region of VEGF gene. The HAF 2.6-kilobase (kb) messenger RNA (mRNA) is expressed in most adult tissues. The highest expression occurs in fetal liver and the least in adult liver. HAF is the murine homolog of Sart-1, a 125-kd human protein expressed in the nuclei of normal and malignant cells. (Blood. 2000;96:491-497)