PI3K as Mediator of Apoptosis and Contractile Dysfunction in TGFß1-Stimulated Cardiomyocytes.

BACKGROUND: TGFß1 is a growth factor that plays a major role in the remodeling process of the heart by inducing cardiomyocyte dysfunction and apoptosis, as well as fibrosis thereby restricting heart function. TGFß1 mediates its effect via the TGFß receptor I (ALK5) and the activation of SMAD transcription factors, but TGFß1 is also known as activator of phosphoinositide-3-kinase (PI3K) via the non-SMAD signaling pathway. The aim of this study was to investigate whether PI3K is also involved in TGFß1-induced cardiomyocytes apoptosis and contractile dysfunction. METHODS AND RESULTS: Incubation of isolated ventricular cardiomyocytes with TGFß1 resulted in impaired contractile function. Pre-incubation of cells with the PI3K inhibitor Ly294002 or the ALK5 inhibitor SB431542 attenuated the decreased cell shortening in TGFß1-stimulated cells. Additionally, TGFß-induced apoptosis was significantly reduced by the PI3K inhibitor Ly294002. Administration of a PI3Kγ-specific inhibitor AS605240 abolished the TGFß effect on apoptosis and cell shortening. This was also confirmed in cardiomyocytes from PI3Kγ KO mice. Induction of SMAD binding activity and the TGFß target gene collagen 1 could be blocked by the PI3K inhibitor Ly294002, but not by the specific PI3Kγ inhibitor AS605240. CONCLUSIONS: TGFß1-induced SMAD activation, cardiomyocyte apoptosis, and impaired cell shortening are mediated via both, the ALK5 receptor and PI3K, in adult cardiomyocytes. PI3Kγ specifically contributes to apoptosis induction and impairment of contractile function independent of SMAD signaling.

Cardiomyocytes-specific deletion of monoamine oxidase B reduces irreversible myocardial ischemia/reperfusion injury.

Monoamine oxidase B (MAO-B), a protein localized at the outer mitochondrial membrane, catalyzes the oxidative deamination of biogenic amines thereby producing reactive oxygen species (ROS). Increased ROS formation contributes to myocardial ischemia/reperfusion (I/R); however, the importance of different ROS producing enzymes for increased I/R-induced ROS formation and the subsequent I/R injury is still a matter of debate. Here we describe the first cardiomyocytes-specific MAO-B knockout mouse and test the hypothesis that lack of cardiomyocyte MAO-B protects the heart from I/R injury. A cardiac-specific and tamoxifen-inducible MAO-B knockout mouse (MAO-B KO) was generated using the Cre/lox system; Cre-negative MAO-Bfl/fl littermates served as controls (WT). Lack of MAO-B was verified by Western blot and immunohistochemistry. Cardiac function of MAO-B KO and WT was analyzed by echocardiography, quantification of mitochondrial ROS production, and measurement of myocardial infarct size (in % of ventricle) in hearts exposed to global I/R using the Langendorff technique. MAO-B protein expression was significantly down-regulated in MAO-B KO mice after two weeks of tamoxifen feeding followed by ten weeks of feeding with normal chow. ROS formation stimulated by the MAO-B-specific substrate ß-phenylethylamin (PEA; 250 µM) was significantly lower in mitochondria isolated from MAO-B KO compared to WT hearts (WT 4.5 ± 0.8 a. u.; MAO-B KO 1.2 ± 0.3 a. u.). Echocardiography revealed no significant differences in LV dimensions as well as ejection fraction (EF) between WT and MAO-B KO mice (EF: WT 67.3 ± 8.8%; MAO-B KO 67.7 ± 6.5%). After I/R, infarct size was significantly lower in MAO-B KO hearts (WT 69.3 ± 15.1%; MAO-B KO 46.8 ± 12.0%). CONCLUSION: Lack of cardiomyocytes-specific MAO-B reduces infarct size suggesting that MAO-B activity contributes to acute reperfusion injury.

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.

P66shc and its role in ischemic cardiovascular diseases.

Oxidative stress caused by an imbalance in the formation and removal of reactive oxygen species (ROS) plays an important role in the development of several cardiovascular diseases. ROS originate from various cellular origins; however, the highest amount of ROS is produced by mitochondria. One of the proteins contributing to mitochondrial ROS formation is the adaptor protein p66shc, which upon cellular stresses translocates from the cytosol to the mitochondria. In the present review, we focus on the role of p66shc in longevity, in the development of cardiovascular diseases including diabetes, atherosclerosis and its risk factors, myocardial ischemia/reperfusion injury and the protection from it by ischemic preconditioning. Also, the contribution of p66shc towards cerebral pathologies and the potential of the protein as a therapeutic target for the treatment of the aforementioned diseases are discussed.

JDP2 overexpression provokes cardiac dysfunction in mice.

The transcriptional regulator JDP2 (Jun dimerization protein 2) has been identified as a prognostic marker for patients to develop heart failure after myocardial infarction. We now performed in vivo studies on JDP2-overexpressing mice, to clarify the impact of JDP2 on heart failure progression. Therefore, during birth up to the age of 4 weeks cardiac-specific JDP2 overexpression was prevented by doxycycline feeding in transgenic mice. Then, JDP2 overexpression was started. Already after 1 week, cardiac function, determined by echocardiography, decreased which was also resembled on the cardiomyocyte level. After 5 weeks blood pressure declined, ejection fraction and cardiac output was reduced and left ventricular dilatation developed. Heart weight/body weight, and mRNA expression of ANP, inflammatory marker genes, collagen and fibronectin increased. Collagen 1 protein expression increased, and fibrosis developed. As an additional sign of elevated extracellular matrix remodeling, matrix metalloproteinase 2 activity increased in JDP2 mice. Thus, JDP2 overexpression is deleterious to heart function in vivo. It can be concluded that JDP2 overexpression provokes cardiac dysfunction in adult mice that is accompanied by hypertrophy and fibrosis. Thus, induction of JDP2 is a maladaptive response contributing to heart failure development.

Phosphoinositide 3-Kinase Gamma Inhibition Protects From Anthracycline Cardiotoxicity and Reduces Tumor Growth.

BACKGROUND: Anthracyclines, such as doxorubicin (DOX), are potent anticancer agents for the treatment of solid tumors and hematologic malignancies. However, their clinical use is hampered by cardiotoxicity. This study sought to investigate the role of phosphoinositide 3-kinase γ (PI3Kγ) in DOX-induced cardiotoxicity and the potential cardioprotective and anticancer effects of PI3Kγ inhibition. METHODS: Mice expressing a kinase-inactive PI3Kγ or receiving PI3Kγ-selective inhibitors were subjected to chronic DOX treatment. Cardiac function was analyzed by echocardiography, and DOX-mediated signaling was assessed in whole hearts or isolated cardiomyocytes. The dual cardioprotective and antitumor action of PI3Kγ inhibition was assessed in mouse mammary tumor models. RESULTS: PI3Kγ kinase-dead mice showed preserved cardiac function after chronic low-dose DOX treatment and were protected against DOX-induced cardiotoxicity. The beneficial effects of PI3Kγ inhibition were causally linked to enhanced autophagic disposal of DOX-damaged mitochondria. Consistently, either pharmacological or genetic blockade of autophagy in vivo abrogated the resistance of PI3Kγ kinase-dead mice to DOX cardiotoxicity. Mechanistically, PI3Kγ was triggered in DOX-treated hearts, downstream of Toll-like receptor 9, by the mitochondrial DNA released by injured organelles and contained in autolysosomes. This autolysosomal PI3Kγ/Akt/mTOR/Ulk1 signaling provided maladaptive feedback inhibition of autophagy. PI3Kγ blockade in models of mammary gland tumors prevented DOX-induced cardiac dysfunction and concomitantly synergized with the antitumor action of DOX by unleashing anticancer immunity. CONCLUSIONS: Blockade of PI3Kγ may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

AP39, a mitochondria-targeting hydrogen sulfide (H2 S) donor, protects against myocardial reperfusion injury independently of salvage kinase signalling.

BACKGROUND AND PURPOSE: H2 S protects myocardium against ischaemia/reperfusion injury. This protection may involve the cytosolic reperfusion injury salvage kinase (RISK) pathway, but direct effects on mitochondrial function are possible. Here, we investigated the potential cardioprotective effect of a mitochondria-specific H2 S donor, AP39, at reperfusion against ischaemia/reperfusion injury. EXPERIMENTAL APPROACH: Anaesthetized rats underwent myocardial ischaemia (30 min)/reperfusion (120 min) with randomization to receive interventions before reperfusion: vehicle, AP39 (0.01, 0.1, 1 µmol·kg-1 ), or control compounds AP219 and ADT-OH (1 µmol·kg-1 ). LY294002, L-NAME or ODQ were used to investigate the involvement of the RISK pathway. Myocardial samples harvested 5 min after reperfusion were analysed for RISK protein phosphorylation and isolated cardiac mitochondria were used to examine the direct mitochondrial effects of AP39. KEY RESULTS: AP39, dose-dependently, reduced infarct size. Inhibition of either PI3K/Akt, eNOS or sGC did not affect this effect of AP39. Western blot analysis confirmed that AP39 did not induce phosphorylation of Akt, eNOS, GSK-3ß or ERK1/2. In isolated subsarcolemmal and interfibrillar mitochondria, AP39 significantly attenuated mitochondrial ROS generation without affecting respiratory complexes I or II. Furthermore, AP39 inhibited mitochondrial permeability transition pore (PTP) opening and co-incubation of mitochondria with AP39 and cyclosporine A induced an additive inhibitory effect on the PTP. CONCLUSION AND IMPLICATIONS: AP39 protects against reperfusion injury independently of the cytosolic RISK pathway. This cardioprotective effect could be mediated by inhibiting PTP via a cyclophilin D-independent mechanism. Thus, selective delivery of H2 S to mitochondria may be therapeutically applicable for employing the cardioprotective utility of H2 S.

NOX4 in Mitochondria: Yeast Two-Hybrid-Based Interaction with Complex I Without Relevance for Basal Reactive Oxygen Species?

NADPH oxidases (NOXs) represent the only known dedicated source of reactive oxygen species (ROS) and thus a prime therapeutic target. Type 4 NOX is unique as it produces H2O2, is constitutively active, and has been suggested to localize to cardiac mitochondria, thus possibly linking mitochondrial and NOX-derived ROS formation. The aim of this study was to identify NOX4-binding proteins and examine the possible physiological localization of NOX4 to mitochondria and its impact on mitochondrial ROS formation. We here provide evidence that NOX4 can, in principle, enter protein-protein interactions with mitochondrial complex I NADH dehydrogenase subunits, 1 and 4L. However, under physiological conditions, NOX4 protein was neither detectable in the kidney nor in cardiomyocyte mitochondria. The NOX inhibitor, GKT136901, slightly reduced ROS formation in cardiomyocyte mitochondria, but this effect was observed in both wild-type and Nox4(-/-) mice. NOX4 may thus associate with mitochondrial complex I proteins, but in cardiac and renal mitochondria under basal conditions, expression is beyond our detection limits and does not contribute to ROS formation.