Lack of Contribution of p66shc to Pressure Overload-Induced Right Heart Hypertrophy.

The leading cause of death in pulmonary arterial hypertension (PAH) is right ventricular (RV) failure (RVF). Reactive oxygen species (ROS) have been suggested to play a role in the development of RV hypertrophy (RVH) and the transition to RVF. The hydrogen peroxide-generating protein p66shc has been associated with left ventricular (LV) hypertrophy but its role in RVH is unclear. The purpose of this study was to determine whether genetic deletion of p66shc affects the development and/or progression of RVH and RVF in the pulmonary artery banding (PAB) model of RV pressure overload. The impact of p66shc on mitochondrial ROS formation, RV cardiomyocyte function, as well as on RV morphology and function were studied three weeks after PAB or sham operation. PAB in wild type mice did not affect mitochondrial ROS production or RV cardiomyocyte function, but induced RVH and impaired cardiac function. Genetic deletion of p66shc did also not alter basal mitochondrial ROS production or RV cardiomyocyte function, but impaired RV cardiomyocyte shortening was observed following PAB. The development of RVH and RVF following PAB was not affected by p66shc deletion. Thus, our data suggest that p66shc-derived ROS are not involved in the development and progression of RVH or RVF in PAH.

Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing.

RATIONALE: Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. OBJECTIVES: To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. METHODS AND RESULTS: Isolated ventilated and perfused lungs from Cox4i2-/- mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2-/- mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2-/- PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2-/- PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2-/- PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. CONCLUSIONS: Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion to hydrogen peroxide, contributes to cellular membrane depolarization and HPV. These findings provide a new model for oxygen-sensing processes in the lung and possibly also in other organs.

Impact of the mitochondria-targeted antioxidant MitoQ on hypoxia-induced pulmonary hypertension.

Increased mitochondrial reactive oxygen species (ROS), particularly superoxide have been suggested to mediate hypoxic pulmonary vasoconstriction (HPV), chronic hypoxia-induced pulmonary hypertension (PH) and right ventricular (RV) remodelling.We determined ROS in acute, chronic hypoxia and investigated the effect of the mitochondria-targeted antioxidant MitoQ under these conditions.The effect of MitoQ or its inactive carrier substance, decyltriphenylphosphonium (TPP+), on acute HPV (1% O2 for 10 minutes) was investigated in isolated blood-free perfused mouse lungs. Mice exposed for 4 weeks to chronic hypoxia (10% O2) or after banding of the main pulmonary artery (PAB) were treated with MitoQ or TPP+ (50 mg/kg/day).Total cellular superoxide and mitochondrial ROS levels were increased in pulmonary artery smooth muscle cells (PASMC), but decreased in pulmonary fibroblasts in acute hypoxia. MitoQ significantly inhibited HPV and acute hypoxia-induced rise in superoxide concentration. ROS was decreased in PASMC, while it increased in the RV after chronic hypoxia. Correspondingly, MitoQ did not affect the development of chronic hypoxia-induced PH, but attenuated RV remodelling after chronic hypoxia as well as after PAB.Increased mitochondrial ROS of PASMC mediate acute HPV, but not chronic hypoxia-induced PH. MitoQ may be beneficial under conditions of exaggerated acute HPV.

Mitochondrial hyperpolarization in pulmonary vascular remodeling. Mitochondrial uncoupling protein deficiency as disease model.

Alterations of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitochondrial respiration are possible triggers of pulmonary vascular remodeling in pulmonary hypertension (PH). We investigated the role of MMP in PH and hypothesized that deletion of the mitochondrial uncoupling protein 2 (UCP2) increases MMP, thus promoting pulmonary vascular remodeling and PH. MMP was measured by JC-1 in isolated pulmonary arterial smooth muscle cells (PASMCs) of patients with PH and animals with PH induced by exposure to monocrotaline (MCT) or chronic hypoxia. PH was quantified in vivo in UCP2-deficient (UCP2(-/-)) mice by hemodynamics, morphometry, and echocardiography. ROS were measured by electron spin resonance spectroscopy and proliferation by thymidine incorporation. Mitochondrial respiration was investigated by high-resolution respirometry. MMP was increased in PASMCs of patients and in animal models of PH. UCP2(-/-) mice exhibited pulmonary vascular remodeling and mild PH compared with wild-type (WT) mice. PASMCs of UCP2(-/-) mice showed increased proliferation, MMP, and ROS release. Increased proliferation of UCP2(-/-) PASMCs could be attenuated by ROS inhibitors and inhibited by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which decreased MMP to the level of WT mice. Mitochondrial respiration was altered in PASMCs from MCT rats and PASMCs exposed to hypoxia but not in isolated pulmonary mitochondria of UCP2(-/-) mice or PASMCs after treatment with small interfering RNA for UCP2. Our data suggest that increased MMP causes vascular remodeling in UCP2(-/-) mice partially via increased ROS. In chronic hypoxia and MCT-induced PH, additional pathomechanisms such as decreased respiration may play a role.

Genetic deletion of p66shc and/or cyclophilin D results in decreased pulmonary vascular tone.

AIMS: The pulmonary vascular tone and hypoxia-induced alterations of the pulmonary vasculature may be regulated by the mitochondrial membrane permeability transition pore (mPTP) that controls mitochondrial calcium load and apoptosis. We thus investigated, if the mitochondrial proteins p66shc and cyclophilin D (CypD) that regulate mPTP opening affect the pulmonary vascular tone. METHODS AND RESULTS: Mice deficient for p66shc (p66shc-/-), CypD (CypD-/-), or both proteins (p66shc/CypD-/-) exhibited decreased pulmonary vascular resistance (PVR) compared to wild-type mice determined in isolated lungs and in vivo. In contrast, systemic arterial pressure was only lower in CypD-/- mice. As cardiac function and pulmonary vascular remodelling did not differ between genotypes, we determined alterations of vascular contractility in isolated lungs and calcium handling in pulmonary arterial smooth muscle cells (PASMC) as underlying reason for decreased PVR. Potassium chloride (KCl)-induced pulmonary vasoconstriction and KCl-induced cytosolic calcium increase determined by Fura-2 were attenuated in all gene-deficient mice. In contrast, KCl-induced mitochondrial calcium increase determined by the genetically encoded Mito-Car-GECO and calcium retention capacity were increased only in CypD-/- and p66shc/CypD-/- mitochondria indicating that decreased mPTP opening affected KCl-induced intracellular calcium peaks in these cells. All mouse strains showed a similar pulmonary vascular response to chronic hypoxia, while acute hypoxic pulmonary vasoconstriction was decreased in gene-deficient mice indicating that CypD and p66shc regulate vascular contractility but not remodelling. CONCLUSIONS: We conclude that p66shc specifically regulates the pulmonary vascular tone, while CypD also affects systemic pressure. However, only CypD acts via regulation of mPTP opening and mitochondrial calcium regulation.

Protection against pressure overload-induced right heart failure by uncoupling protein 2 silencing.

AIMS: The role of uncoupling protein 2 (UCP2) in cardiac adaptation to pressure overload remains unclear. In a classical model of left ventricular pressure overload genetic deletion of UCP2 (UCP2-/-) protected against cardiac hypertrophy and failure. However, in UCP2-/- mice increased proliferation of pulmonary arterial smooth muscle cells induces mild pulmonary hypertension, right ventricular (RV) hypertrophy, and reduced cardiac output. This suggests a different role for UCP2 in RV and left ventricular adaptation to pressure overload. To clarify this situation in more detail UCP2-/- and wild-type mice were exposed to pulmonary arterial banding (PAB). METHODS AND RESULTS: Mice were analysed (haemodynamics, morphometry, and echocardiography) 3 weeks after PAB or sham surgery. Myocytes and non-myocytes were isolated and analysed separately. Cell shortening of myocytes and fura-2 loading of cardiomyocytes were used to characterize their function. Brd assay was performed to study fibroblast proliferation. Isolated mitochondria were analysed to investigate the role of UCP2 for reactive oxygen species (ROS) production. UCP2 mRNA was 2.7-fold stronger expressed in RV myocytes than in left ventricular myocytes and stronger expressed in non-myocytes compared with myocytes. Three weeks after PAB, cardiac output was reduced in wild type but preserved in UCP2-/- mice. UCP2-/- had increased RV wall thickness, but lower RV internal diameters and displayed a significant stronger fibrosis. Cardiac fibroblasts from UCP2-/- had reduced proliferation rates but higher collagen-1 expression. Myocytes isolated from mice after PAB banding showed preserved function that was further improved by UCP2-/-. Mitochondrial ROS production and respiration was similar between UCP2-/- or wild-type hearts. CONCLUSION: Despite a mild pulmonary hypertension in UCP2-/- mice, hearts from these mice are well preserved against additional pressure overload (severe pulmonary hypertension). This-at least in part-depends on different behaviour of non-myocytes (fibroblasts).