MFN2-driven mitochondria-to-nucleus tethering allows a non-canonical nuclear entry pathway of the mitochondrial pyruvate dehydrogenase complex.

The mitochondrial pyruvate dehydrogenase complex (PDC) translocates into the nucleus, facilitating histone acetylation by producing acetyl-CoA. We describe a noncanonical pathway for nuclear PDC (nPDC) import that does not involve nuclear pore complexes (NPCs). Mitochondria cluster around the nucleus in response to proliferative stimuli and tether onto the nuclear envelope (NE) via mitofusin-2 (MFN2)-enriched contact points. A decrease in nuclear MFN2 levels decreases mitochondria tethering and nPDC levels. Mitochondrial PDC crosses the NE and interacts with lamin A, forming a ring below the NE before crossing through the lamin layer into the nucleoplasm, in areas away from NPCs. Effective blockage of NPC trafficking does not decrease nPDC levels. The PDC-lamin interaction is maintained during cell division, when lamin depolymerizes and disassembles before reforming daughter nuclear envelopes, providing another pathway for nPDC entry during mitosis. Our work provides a different angle to understanding mitochondria-to-nucleus communication and nuclear metabolism.

A nuclear pyruvate dehydrogenase complex is important for the generation of acetyl-CoA and histone acetylation.

DNA transcription, replication, and repair are regulated by histone acetylation, a process that requires the generation of acetyl-coenzyme A (CoA). Here, we show that all the subunits of the mitochondrial pyruvate dehydrogenase complex (PDC) are also present and functional in the nucleus of mammalian cells. We found that knockdown of nuclear PDC in isolated functional nuclei decreased the de novo synthesis of acetyl-CoA and acetylation of core histones. Nuclear PDC levels increased in a cell-cycle-dependent manner and in response to serum, epidermal growth factor, or mitochondrial stress; this was accompanied by a corresponding decrease in mitochondrial PDC levels, suggesting a translocation from the mitochondria to the nucleus. Inhibition of nuclear PDC decreased acetylation of specific lysine residues on histones important for G1-S phase progression and expression of S phase markers. Dynamic translocation of mitochondrial PDC to the nucleus provides a pathway for nuclear acetyl-CoA synthesis required for histone acetylation and epigenetic regulation.

TRIM35 Monoubiquitinates H2B in Cardiac Cells, Implications for Heart Failure.

BACKGROUND: The tumor suppressor and proapoptotic transcription factor P53 is induced (and activated) in several forms of heart failure, including cardiotoxicity and dilated cardiomyopathy; however, the precise mechanism that coordinates its induction with accessibility to its transcriptional promoter sites remains unresolved, especially in the setting of mature terminally differentiated (nonreplicative) cardiomyocytes. METHODS: Male and female control or TRIM35 (tripartite motif containing 35) overexpression adolescent (aged 1-3 months) and adult (aged 4-6 months) transgenic mice were used for all in vivo experiments. Primary adolescent or adult mouse cardiomyocytes were isolated from control or TRIM35 overexpression transgenic mice for all in vitro experiments. Adenovirus or small-interfering RNA was used for all molecular experiments to overexpress or knockdown, respectively, target genes in primary mouse cardiomyocytes. Patient dilated cardiomyopathy or nonfailing left ventricle samples were used for translational and mechanistic insight. Chromatin immunoprecipitation and DNA sequencing or quantitative real-time polymerase chain reaction (qPCR) was used to assess P53 binding to its transcriptional promoter targets, and RNA sequencing was used to identify disease-specific signaling pathways. RESULTS: Here, we show that E3-ubiquitin ligase TRIM35 can directly monoubiquitinate lysine-120 (K120) on histone 2B in postnatal mature cardiomyocytes. This epigenetic modification was sufficient to promote chromatin remodeling, accessibility of P53 to its transcriptional promoter targets, and elongation of its transcribed mRNA. We found that increased P53 transcriptional activity (in cardiomyocyte-specific Trim35 overexpression transgenic mice) was sufficient to initiate heart failure and these molecular findings were recapitulated in nonischemic human LV dilated cardiomyopathy samples. CONCLUSIONS: These findings suggest that TRIM35 and the K120Ub-histone 2B epigenetic modification are molecular features of cardiomyocytes that can collectively predict dilated cardiomyopathy pathogenesis.

A miR-208-Mef2 axis drives the decompensation of right ventricular function in pulmonary hypertension.

RATIONALE: Right ventricular (RV) failure is a major cause of morbidity and mortality in pulmonary hypertension, but its mechanism remains unknown. Myocyte enhancer factor 2 (Mef2) has been implicated in RV development, regulating metabolic, contractile, and angiogenic genes. Moreover, Mef2 regulates microRNAs that have emerged as important determinants of cardiac development and disease, but for which the role in RV is still unclear. OBJECTIVE: We hypothesized a critical role of a Mef2-microRNAs axis in RV failure. METHODS AND RESULTS: In a rat pulmonary hypertension model (monocrotaline), we studied RV free wall tissues from rats with normal, compensated, and decompensated RV hypertrophy, carefully defined based on clinically relevant parameters, including RV systolic and end-diastolic pressures, cardiac output, RV size, and morbidity. Mef2c expression was sharply increased in compensating phase of RVH tissues but was lost in decompensation phase of RVH. An unbiased screening of microRNAs in our model resulted to a short microRNA signature of decompensated RV failure, which included the myocardium-specific miR-208, which was progressively downregulated as RV failure progressed, in contrast to what is described in left ventricular failure. With mechanistic in vitro experiments using neonatal and adult RV cardiomyocytes, we showed that miR-208 inhibition, as well as tumor necrosis factor-α, activates the complex mediator of transcription 13/nuclear receptor corepressor 1 axis, which in turn promotes Mef2 inhibition, closing a self-limiting feedback loop, driving the transition from compensating phase of RVH toward decompensation phase of RVH. In our model, serum tumor necrosis factor-α levels progressively increased with time while serum miR-208 levels decreased, mirroring its levels in RV myocardium. CONCLUSIONS: We describe an RV-specific mechanism for heart failure, which could potentially lead to new biomarkers and therapeutic targets.

A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth.

The unique metabolic profile of cancer (aerobic glycolysis) might confer apoptosis resistance and be therapeutically targeted. Compared to normal cells, several human cancers have high mitochondrial membrane potential (DeltaPsim) and low expression of the K+ channel Kv1.5, both contributing to apoptosis resistance. Dichloroacetate (DCA) inhibits mitochondrial pyruvate dehydrogenase kinase (PDK), shifts metabolism from glycolysis to glucose oxidation, decreases DeltaPsim, increases mitochondrial H2O2, and activates Kv channels in all cancer, but not normal, cells; DCA upregulates Kv1.5 by an NFAT1-dependent mechanism. DCA induces apoptosis, decreases proliferation, and inhibits tumor growth, without apparent toxicity. Molecular inhibition of PDK2 by siRNA mimics DCA. The mitochondria-NFAT-Kv axis and PDK are important therapeutic targets in cancer; the orally available DCA is a promising selective anticancer agent.

Phosphatidylethanolamine deficiency in Mammalian mitochondria impairs oxidative phosphorylation and alters mitochondrial morphology.

Mitochondrial dysfunction is implicated in neurodegenerative, cardiovascular, and metabolic disorders, but the role of phospholipids, particularly the nonbilayer-forming lipid phosphatidylethanolamine (PE), in mitochondrial function is poorly understood. Elimination of mitochondrial PE (mtPE) synthesis via phosphatidylserine decarboxylase in mice profoundly alters mitochondrial morphology and is embryonic lethal (Steenbergen, R., Nanowski, T. S., Beigneux, A., Kulinski, A., Young, S. G., and Vance, J. E. (2005) J. Biol. Chem. 280, 40032-40040). We now report that moderate <30% depletion of mtPE alters mitochondrial morphology and function and impairs cell growth. Acute reduction of mtPE by RNAi silencing of phosphatidylserine decarboxylase and chronic reduction of mtPE in PSB-2 cells that have only 5% of normal phosphatidylserine synthesis decreased respiratory capacity, ATP production, and activities of electron transport chain complexes (C) I and CIV but not CV. Blue native-PAGE analysis revealed defects in the organization of CI and CIV into supercomplexes in PE-deficient mitochondria, correlated with reduced amounts of CI and CIV proteins. Thus, mtPE deficiency impairs formation and/or membrane integration of respiratory supercomplexes. Despite normal or increased levels of mitochondrial fusion proteins in mtPE-deficient cells, and no reduction in mitochondrial membrane potential, mitochondria were extensively fragmented, and mitochondrial ultrastructure was grossly aberrant. In general, chronic reduction of mtPE caused more pronounced mitochondrial defects than did acute mtPE depletion. The functional and morphological changes in PSB-2 cells were largely reversed by normalization of mtPE content by supplementation with lyso-PE, a mtPE precursor. These studies demonstrate that even a modest reduction of mtPE in mammalian cells profoundly alters mitochondrial functions.

Attenuating endoplasmic reticulum stress as a novel therapeutic strategy in pulmonary hypertension.

BACKGROUND: Evidence suggestive of endoplasmic reticulum (ER) stress in the pulmonary arteries of patients with pulmonary arterial hypertension has been described for decades but has never been therapeutically targeted. ER stress is a feature of many conditions associated with pulmonary arterial hypertension like hypoxia, inflammation, or loss-of-function mutations. ER stress signaling in the pulmonary circulation involves the activation of activating transcription factor 6, which, via induction of the reticulin protein Nogo, can lead to the disruption of the functional ER-mitochondria unit and the increasingly recognized cancer-like metabolic shift in pulmonary arterial hypertension that promotes proliferation and apoptosis resistance in the pulmonary artery wall. We hypothesized that chemical chaperones known to suppress ER stress signaling, like 4-phenylbutyrate (PBA) or tauroursodeoxycholic acid, will inhibit the disruption of the ER-mitochondrial unit and prevent/reverse pulmonary arterial hypertension. METHODS AND RESULTS: PBA in the drinking water both prevented and reversed chronic hypoxia-induced pulmonary hypertension in mice, decreasing pulmonary vascular resistance, pulmonary artery remodeling, and right ventricular hypertrophy and improving functional capacity without affecting systemic hemodynamics. These results were replicated in the monocrotaline rat model. PBA and tauroursodeoxycholic acid improved ER stress indexes in vivo and in vitro, decreased activating transcription factor 6 activation (cleavage, nuclear localization, luciferase, and downstream target expression), and inhibited the hypoxia-induced decrease in mitochondrial calcium and mitochondrial function. In addition, these chemical chaperones suppressed proliferation and induced apoptosis in pulmonary artery smooth muscle cells in vitro and in vivo. CONCLUSIONS: Attenuating ER stress with clinically used chemical chaperones may be a novel therapeutic strategy in pulmonary hypertension with high translational potential.

Targeting cell motility in pulmonary arterial hypertension.

Pulmonary artery smooth muscle cells (PASMC), in pulmonary arterial hypertension (PAH), contribute to obliterative vascular remodelling and are characterised by enhanced proliferation, suppressed apoptosis and, a much less studied, increased migration potential. One of the major proteins that regulate cell migration is focal adhesion kinase (FAK), but its role in PAH is not fully understood. We hypothesised that targeting cell migration by FAK inhibition may be a new therapeutic strategy in PAH. In vivo, inhalation of FAK-siRNA (n=5) or oral delivery of PF-228 (FAK inhibitor PF-573 228; n=5) inhibited rat monocrotaline induced PAH, improving the haemodynamics, vascular remodelling (media thickness), and right ventricular hypertrophy. In vitro, FAK was activated in PAH human lungs (n=8) or PASMC when compared to those form healthy subjects (Western blot, n=5), in a Src-dependent manner, as it was reversed by the specific Src inhibitor PP2. The degree of FAK phosphorylation at Y576 correlated positively with pulmonary vascular resistance in PAH patients. FAK inhibition (siRNA, PF-228 and PP2) in PAH-PASMCs induced a fivefold increase in apoptosis (percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling), a 2.5-fold decrease in proliferation (%Ki67), an 18% decrease in cell migration (colorimetric assay) and a 50% decrease in cell invasion (wound healing). Suppressing PASMC migration by FAK inhibition inhibits PAH progression and may open a new therapeutic window in PAH.

TRIM35-mediated degradation of nuclear PKM2 destabilizes GATA4/6 and induces P53 in cardiomyocytes to promote heart failure.

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that translocates to the nucleus to regulate transcription factors in different tissues or pathologic states. Although studied extensively in cancer, its biological role in the heart remains unresolved. PKM1 is more abundant than the PKM2 isoform in cardiomyocytes, and thus, we speculated that PKM2 is not genetically redundant to PKM1 and may be critical in regulating cardiomyocyte-specific transcription factors important for cardiac survival. Here, we showed that nuclear PKM2 (S37P-PKM2) in cardiomyocytes interacts with prosurvival and proapoptotic transcription factors, including GATA4, GATA6, and P53. Cardiomyocyte-specific PKM2-deficient mice (Pkm2 Mut Cre+) developed age-dependent dilated cardiac dysfunction and had decreased amounts of GATA4 and GATA6 (GATA4/6) but increased amounts of P53 compared to Control Cre+ hearts. Nuclear PKM2 prevented caspase-1-dependent cleavage and degradation of GATA4/6 while also providing a molecular platform for MDM2-mediated reduction of P53. In a preclinical heart failure mouse model, nuclear PKM2 and GATA4/6 were decreased, whereas P53 was increased in cardiomyocytes. Loss of nuclear PKM2 was ubiquitination dependent and associated with the induction of the E3 ubiquitin ligase TRIM35. In mice, cardiomyocyte-specific TRIM35 overexpression resulted in decreased S37P-PKM2 and GATA4/6 along with increased P53 in cardiomyocytes compared to littermate controls and similar cardiac dysfunction to Pkm2 Mut Cre+ mice. In patients with dilated left ventricles, increase in TRIM35 was associated with decreased S37P-PKM2 and GATA4/6 and increased P53. This study supports a previously unrecognized role for PKM2 as a molecular platform that mediates cell signaling events essential for cardiac survival.

Loss of the fructose transporter SLC2A5 inhibits cancer cell migration.

Metastasis is the primary cause of cancer patient death and the elevation of SLC2A5 gene expression is often observed in metastatic cancer cells. Here we evaluated the importance of SLC2A5 in cancer cell motility by silencing its gene. We discovered that CRISPR/Cas9-mediated inactivation of the SLC2A5 gene inhibited cancer cell proliferation and migration in vitro as well as metastases in vivo in several animal models. Moreover, SLC2A5-attenuated cancer cells exhibited dramatic alterations in mitochondrial architecture and localization, uncovering the importance of SLC2A5 in directing mitochondrial function for cancer cell motility and migration. The direct association of increased abundance of SLC2A5 in cancer cells with metastatic risk in several types of cancers identifies SLC2A5 as an important therapeutic target to reduce or prevent cancer metastasis.

A reversible metabolic stress-sensitive regulation of CRMP2A orchestrates EMT/stemness and increases metastatic potential in cancer.

An epithelial-to-mesenchymal transition (EMT) phenotype with cancer stem cell-like properties is a critical feature of aggressive/metastatic tumors, but the mechanism(s) that promote it and its relation to metabolic stress remain unknown. Here we show that Collapsin Response Mediator Protein 2A (CRMP2A) is unexpectedly and reversibly induced in cancer cells in response to multiple metabolic stresses, including low glucose and hypoxia, and inhibits EMT/stemness. Loss of CRMP2A, when metabolic stress decreases (e.g., around blood vessels in vivo) or by gene deletion, induces extensive microtubule remodeling, increased glutamine utilization toward pyrimidine synthesis, and an EMT/stemness phenotype with increased migration, chemoresistance, tumor initiation capacity/growth, and metastatic potential. In a cohort of 27 prostate cancer patients with biopsies from primary tumors and distant metastases, CRMP2A expression decreases in the metastatic versus primary tumors. CRMP2A is an endogenous molecular brake on cancer EMT/stemness and its loss increases the aggressiveness and metastatic potential of tumors.

SNPs for Genes Encoding the Mitochondrial Proteins Sirtuin3 and Uncoupling Protein 2 Are Associated With Disease Severity, Type 2 Diabetes, and Outcomes in Patients With Pulmonary Arterial Hypertension and This Is Recapitulated in a New Mouse Model Lacking Both Genes.

Background Isolated loss-of-function single nucleotide polymorphisms (SNPs) for SIRT3 (a mitochondrial deacetylase) and UCP2 (an atypical uncoupling protein enabling mitochondrial calcium entry) have been associated with both pulmonary arterial hypertension (PAH) and insulin resistance, but their collective role in animal models and patients is unknown. Methods and Results In a prospective cohort of patients with PAH (n=60), we measured SNPs for both SIRT3 and UCP2, along with several clinical features (including invasive hemodynamic data) and outcomes. We found SIRT3 and UCP2 SNPs often both in the same patient in a homozygous or heterozygous manner, correlating positively with PAH severity and associated with the presence of type 2 diabetes and 10-year outcomes (death and transplantation). To explore this mechanistically, we generated double knockout mice for Sirt3 and Ucp2 and found increasing severity of PAH (mean pulmonary artery pressure, right ventricular hypertrophy/dilatation and extensive vascular remodeling, including inflammatory plexogenic lesions, in a gene dose-dependent manner), along with insulin resistance, compared with wild-type mice. The suppressed mitochondrial function (decreased respiration, increased mitochondrial membrane potential) in the double knockout pulmonary artery smooth muscle cells was associated with apoptosis resistance and increased proliferation, compared with wild-type mice. Conclusions Our work supports the metabolic theory of PAH and shows that these mice exhibit spontaneous severe PAH (without environmental or chemical triggers) that mimics human PAH and may explain the findings in our patient cohort. Our study offers a new mouse model of PAH, with several features of human disease that are typically absent in other PAH mouse models.

Dehydroepiandrosterone reverses systemic vascular remodeling through the inhibition of the Akt/GSK3-{beta}/NFAT axis.

BACKGROUND: The remodeled vessel wall in many vascular diseases such as restenosis after injury is characterized by proliferative and apoptosis-resistant vascular smooth muscle cells. There is evidence that proproliferative and antiapoptotic states are characterized by a metabolic (glycolytic phenotype and hyperpolarized mitochondria) and electric (downregulation and inhibition of plasmalemmal K(+) channels) remodeling that involves activation of the Akt pathway. Dehydroepiandrosterone (DHEA) is a naturally occurring and clinically used steroid known to inhibit the Akt axis in cancer. We hypothesized that DHEA will prevent and reverse the remodeling that follows vascular injury. METHODS AND RESULTS: We used cultured human carotid vascular smooth muscle cell and saphenous vein grafts in tissue culture, stimulated by platelet-derived growth factor to induce proliferation in vitro and the rat carotid injury model in vivo. DHEA decreased proliferation and increased vascular smooth muscle cell apoptosis in vitro and in vivo, reducing vascular remodeling while sparing healthy tissues after oral intake. Using pharmacological (agonists and antagonists of Akt and its downstream target glycogen-synthase-kinase-3beta [GSK-3beta]) and molecular (forced expression of constitutively active Akt1) approaches, we showed that the effects of DHEA were mediated by inhibition of Akt and subsequent activation of GSK-3beta, leading to mitochondrial depolarization, increased reactive oxygen species, activation of redox-sensitive plasmalemmal voltage-gated K(+) channels, and decreased [Ca(2+)](i). These functional changes were accompanied by sustained molecular effects toward the same direction; by decreasing [Ca(2+)](i) and inhibiting GSK-3beta, DHEA inhibited the nuclear factor of activated T cells transcription factor, thus increasing expression of Kv channels (Kv1.5) and contributing to sustained mitochondrial depolarization. These results were independent of any steroid-related effects because they were not altered by androgen and estrogen inhibitors but involved a membrane G protein-coupled receptor. CONCLUSIONS: We suggest that the orally available DHEA might be an attractive candidate for the treatment of systemic vascular remodeling, including restenosis, and we propose a novel mechanism of action for this important hormone and drug.

Airway delivery of mesenchymal stem cells prevents arrested alveolar growth in neonatal lung injury in rats.

RATIONALE: Bronchopulmonary dysplasia (BPD) and emphysema are characterized by arrested alveolar development or loss of alveoli; both are significant global health problems and currently lack effective therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) prevent adult lung injury, but their therapeutic potential in neonatal lung disease is unknown. OBJECTIVES: We hypothesized that intratracheal delivery of BMSCs would prevent alveolar destruction in experimental BPD. METHODS: In vitro, BMSC differentiation and migration were assessed using co-culture assays and a modified Boyden chamber. In vivo, the therapeutic potential of BMSCs was assessed in a chronic hyperoxia-induced model of BPD in newborn rats. MEASUREMENTS AND MAIN RESULTS: In vitro, BMSCs developed immunophenotypic and ultrastructural characteristics of type II alveolar epithelial cells (AEC2) (surfactant protein C expression and lamellar bodies) when co-cultured with lung tissue, but not with culture medium alone or liver. Migration assays revealed preferential attraction of BMSCs toward oxygen-damaged lung versus normal lung. In vivo, chronic hyperoxia in newborn rats led to air space enlargement and loss of lung capillaries, and this was associated with a decrease in circulating and resident lung BMSCs. Intratracheal delivery of BMSCs on Postnatal Day 4 improved survival and exercise tolerance while attenuating alveolar and lung vascular injury and pulmonary hypertension. Engrafted BMSCs coexpressed the AEC2-specific marker surfactant protein C. However, engraftment was disproportionately low for cell replacement to account for the therapeutic benefit, suggesting a paracrine-mediated mechanism. In vitro, BMSC-derived conditioned medium prevented O(2)-induced AEC2 apoptosis, accelerated AEC2 wound healing, and enhanced endothelial cord formation. CONCLUSIONS: BMSCs prevent arrested alveolar and vascular growth in part through paracrine activity. Stem cell-based therapies may offer new therapeutic avenues for lung diseases that currently lack efficient treatments.

Myocardial Functional Decline During Prolonged Ex Situ Heart Perfusion.

BACKGROUND: Myocardial function declines in a time-dependent fashion during ex situ heart perfusion. Cell death and metabolic alterations may contribute to this phenomenon, limiting the safe perfusion period and the potential of ex situ heart perfusion to expand the donor pool. Our aim was to investigate the etiology of myocardial functional decline in ex situ perfused hearts. METHODS: Cardiac function, apoptosis, effectors and markers of cell death, and metabolic function were assessed in healthy pig hearts perfused for 12 hours. These hearts were perfused in nonworking mode or working mode. RESULTS: Cardiac function declined during ex situ heart perfusion regardless of perfusion mode but was significantly better preserved in the hearts perfused in working mode (11-hour cardiac index/1-hour cardiac index: working mode, 33%; nonworking mode, 10%; p = 0.025). The rate of apoptosis was higher in the ex situ perfused hearts compared with in vivo samples (apoptotic cells: in vivo, 0.13%; working mode, 0.54%; nonworking mode, 0.88%; p < 0.001), but the absolute values were low and out of proportion to the decline in function in either group. Myocardial dysfunction at the end of the perfusion interval was partially rescued by delivery of a pyruvate bolus. CONCLUSIONS: A significant decline in myocardial function occurs over time in hearts preserved ex situ that is out of proportion to the magnitude of myocyte cell death present in dysfunctional hearts. Alterations in myocardial substrate utilization during prolonged ex situ heart perfusion may contribute to this phenomenon and represent an avenue to improve donor heart preservation.

Oxygen activates the Rho/Rho-kinase pathway and induces RhoB and ROCK-1 expression in human and rabbit ductus arteriosus by increasing mitochondria-derived reactive oxygen species: a newly recognized mechanism for sustaining ductal constriction.

BACKGROUND: Constriction of the ductus arteriosus (DA) is initiated at birth by inhibition of O2-sensitive K+ channels in DA smooth muscle cells. Subsequent membrane depolarization and calcium influx through L-type calcium channels initiates functional closure. We hypothesize that Rho-kinase activation is an additional mechanism that sustains DA constriction. METHODS AND RESULTS: The effect of increased PO2 on the activity and expression of Rho-kinase was assessed in DAs from neonates with hypoplastic left-heart syndrome (n=15) and rabbits (339 term and 99 preterm rabbits). Rho-kinase inhibitors (Y-27632 and fasudil) prevent and reverse O2 constriction. Heterogeneity exists in the sensitivity of constrictors (PO2=endothelin=phenylephrine>KCl) and of fetal vessels (DA=pulmonary artery>aorta) to Rho-kinase inhibition. Inhibition of L-type calcium channels (nifedipine) or removal of extracellular calcium inhibits approximately two thirds of O2 constriction. Residual DA constriction reflects calcium sensitization, which persists after removal of extracellular calcium and blocking of sarcoplasmic reticulum Ca2+-ATPase. In term DA, an increase in PO2 activates Rho-kinase and thereby increases RhoB and ROCK-1 expression. Activation of Rho-kinase in DA smooth muscle cells is initiated by a PO2-dependent, rotenone-sensitive increase in mitochondrion-derived reactive O2 species. O2 effects on Rho-kinase are mimicked by exogenous H2O2. In preterm DAs, immaturity of mitochondrial reactive oxygen species generation is associated with reduced and delayed O2 constriction and lack of PO2-dependent upregulation of Rho-kinase expression. CONCLUSIONS: O2 activates Rho-kinase and increases Rho-kinase expression in term DA smooth muscle cells by a redox-regulated, positive-feedback mechanism that promotes sustained vasoconstriction. Conversely, Rho-kinase inhibitors may be useful in maintaining DA patency, as a bridge to congenital heart surgery.

Phosphodiesterase type 5 is highly expressed in the hypertrophied human right ventricle, and acute inhibition of phosphodiesterase type 5 improves contractility.

BACKGROUND: Sildenafil was recently approved for the treatment of pulmonary arterial hypertension. The beneficial effects of phosphodiesterase type 5 (PDE5) inhibitors in pulmonary arterial hypertension are thought to result from relatively selective vasodilatory and antiproliferative effects on the pulmonary vasculature and, on the basis of early data showing lack of significant PDE5 expression in the normal heart, are thought to spare the myocardium. METHODS AND RESULTS: We studied surgical specimens from 9 patients and show here for the first time that although PDE5 is not expressed in the myocardium of the normal human right ventricle (RV), mRNA and protein are markedly upregulated in hypertrophied RV (RVH) myocardium. PDE5 also is upregulated in rat RVH. PDE5 inhibition (with either MY-5445 or sildenafil) significantly increases contractility, measured in the perfused heart (modified Langendorff preparation) and isolated cardiomyocytes, in RVH but not normal RV. PDE5 inhibition leads to increases in both cGMP and cAMP in RVH but not normal RV. Protein kinase G activity is suppressed in RVH, explaining why the PDE5 inhibitor-induced increase in cGMP does not lead to inhibition of contractility. Rather, it leads to inhibition of the cGMP-sensitive PDE3, explaining the increase in cAMP and contractility. This is further supported by our findings that, in RVH protein kinase A, inhibition completely inhibits PDE5-induced inotropy, whereas protein kinase G inhibition does not. CONCLUSIONS: The ability of PDE5 inhibitors to increase RV inotropy and to decrease RV afterload without significantly affecting systemic hemodynamics makes them ideal for the treatment of diseases affecting the RV, including pulmonary arterial hypertension.

Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by genetic and acquired abnormalities that suppress apoptosis and enhance cell proliferation in the vascular wall, including downregulation of the bone morphogenetic protein axis and voltage-gated K+ (Kv) channels. Survivin is an "inhibitor of apoptosis" protein, previously thought to be expressed primarily in cancer cells. We found that survivin was expressed in the pulmonary arteries (PAs) of 6 patients with PAH and rats with monocrotaline-induced PAH, but not in the PAs of 3 patients and rats without PAH. Gene therapy with inhalation of an adenovirus carrying a phosphorylation-deficient survivin mutant with dominant-negative properties reversed established monocrotaline-induced PAH and prolonged survival by 25%. The survivin mutant lowered pulmonary vascular resistance, RV hypertrophy, and PA medial hypertrophy. Both in vitro and in vivo, inhibition of survivin induced PA smooth muscle cell apoptosis, decreased proliferation, depolarized mitochondria, caused efflux of cytochrome c in the cytoplasm and translocation of apoptosis-inducing factor into the nucleus, and increased Kv channel current; the opposite effects were observed with gene transfer of WT survivin, both in vivo and in vitro. Inhibition of the inappropriate expression of survivin that accompanies human and experimental PAH is a novel therapeutic strategy that acts by inducing vascular mitochondria-dependent apoptosis.

An abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: similarities to human pulmonary arterial hypertension.

BACKGROUND: The cause of pulmonary arterial hypertension (PAH) was investigated in humans and fawn hooded rats (FHR), a spontaneously pulmonary hypertensive strain. METHODS AND RESULTS: Serial Doppler echocardiograms and cardiac catheterizations were performed in FHR and FHR/BN1, a consomic control that is genetically identical except for introgression of chromosome 1. PAH began after 20 weeks of age, causing death by &60 weeks. FHR/BN1 did not develop PAH. FHR pulmonary arterial smooth muscle cells (PASMCs) had a rarified reticulum of hyperpolarized mitochondria with reduced expression of electron transport chain components and superoxide dismutase-2. These mitochondrial abnormalities preceded PAH and persisted in culture. Depressed mitochondrial reactive oxygen species (ROS) production caused normoxic activation of hypoxia inducible factor (HIF-1alpha), which then inhibited expression of oxygen-sensitive, voltage-gated K+ channels (eg, Kv1.5). Disruption of this mitochondrial-HIF-Kv pathway impaired oxygen sensing (reducing hypoxic pulmonary vasoconstriction, causing polycythemia), analogous to the pathophysiology of chronically hypoxic Sprague-Dawley rats. Restoring ROS (exogenous H2O2) or blocking HIF-1alpha activation (dominant-negative HIF-1alpha) restored Kv1.5 expression/function. Dichloroacetate, a mitochondrial pyruvate dehydrogenase kinase inhibitor, corrected the mitochondrial-HIF-Kv pathway in FHR-PAH and human PAH PASMCs. Oral dichloroacetate regressed FHR-PAH and polycythemia, increasing survival. Chromosome 1 genes that were dysregulated in FHRs and relevant to the mitochondria-HIF-Kv pathway included HIF-3alpha (an HIF-1alpha repressor), mitochondrial cytochrome c oxidase, and superoxide dismutase-2. Like FHRs, human PAH-PASMCs had dysmorphic, hyperpolarized mitochondria; normoxic HIF-1alpha activation; and reduced expression/activity of HIF-3alpha, cytochrome c oxidase, and superoxide dismutase-2. CONCLUSIONS: FHRs have a chromosome 1 abnormality that disrupts a mitochondria-ROS-HIF-Kv pathway, leading to PAH. Similar abnormalities occur in idiopathic human PAH. This study reveals an intersection between oxygen-sensing mechanisms and PAH. The mitochondria-ROS-HIF-Kv pathway offers new targets for PAH therapy.

A Leukocyte Filter Does Not Provide Further Benefit During Ex Vivo Lung Perfusion.

Normothermic ex vivo lung perfusion (EVLP) allows for assessment and reconditioning of donor lungs. Although a leukocyte filter (LF) is routinely incorporated into the EVLP circuit; its efficacy remains to be determined. Twelve pig lungs were perfused and ventilated ex vivo in a normothermic state for 12 hours. Lungs (n = 3) were allocated to four groups according to perfusate composition and the presence or absence of a LF in the circuit (acellular ± LF, cellular ± LF). Acceptable physiologic lung parameters were achieved during EVLP; however, increased amounts of pro-inflammatory cytokines (TNF-α and IL-6) and leukocytes in the perfusate were observed despite the presence or absence of a LF. Analysis of cells washed off the LF demonstrates that it trapped leukocytes although being ineffective throughout perfusion as it became saturated over 12 hours of EVLP. We conclude that there is no objective evidence to support the routine incorporation of a LF during EVLP as it does not provide further benefit and its removal does not appear to cause harm. The lack of hypothesized benefit to a LF may be because of the saturation of the LF with donor leukocytes, leading to similar amounts of circulating leukocytes still present in the perfusate with and without a LF.