Does Cell-Type-Specific Silencing of Monoamine Oxidase B Interfere with the Development of Right Ventricle (RV) Hypertrophy or Right Ventricle Failure in Pulmonary Hypertension?

Increased mitochondrial reactive oxygen species (ROS) formation is important for the development of right ventricular (RV) hypertrophy (RVH) and failure (RVF) during pulmonary hypertension (PH). ROS molecules are produced in different compartments within the cell, with mitochondria known to produce the strongest ROS signal. Among ROS-forming mitochondrial proteins, outer-mitochondrial-membrane-located monoamine oxidases (MAOs, type A or B) are capable of degrading neurotransmitters, thereby producing large amounts of ROS. In mice, MAO-B is the dominant isoform, which is present in almost all cell types within the heart. We analyzed the effect of an inducible cardiomyocyte-specific knockout of MAO-B (cmMAO-B KO) for the development of RVH and RVF in mice. Right ventricular hypertrophy was induced by pulmonary artery banding (PAB). RV dimensions and function were measured through echocardiography. ROS production (dihydroethidium staining), protein kinase activity (PamStation device), and systemic hemodynamics (in vivo catheterization) were assessed. A significant decrease in ROS formation was measured in cmMAO-B KO mice during PAB compared to Cre-negative littermates, which was associated with reduced activity of protein kinases involved in hypertrophic growth. In contrast to littermates in which the RV was dilated and hypertrophied following PAB, RV dimensions were unaffected in response to PAB in cmMAO-B KO mice, and no decline in RV systolic function otherwise seen in littermates during PAB was measured in cmMAO-B KO mice. In conclusion, cmMAO-B KO mice are protected against RV dilatation, hypertrophy, and dysfunction following RV pressure overload compared to littermates. These results support the hypothesis that cmMAO-B is a key player in causing RV hypertrophy and failure during PH.

Cardiomyocyte maturation alters molecular stress response capacities and determines cell survival upon mitochondrial dysfunction.

Cardiomyocyte maturation during pre- and postnatal development requires multiple intertwined processes, including a switch in energy generation from glucose utilization in the embryonic heart towards fatty acid oxidation after birth. This is accompanied by a boost in mitochondrial mass to increase capacities for oxidative phosphorylation and ATP generation required for efficient contraction. Whether cardiomyocyte differentiation is paralleled by augmented capacities to deal with reactive oxygen species (ROS), physiological byproducts of the mitochondrial electron transport chain (ETC), is less clear. Here we show that expression of genes and proteins involved in redox homeostasis and protein quality control within mitochondria increases after birth in the mouse and human heart. Using primary embryonic, neonatal and adult mouse cardiomyocytes in vitro we investigated how excessive ROS production induced by mitochondrial dysfunction affects cell survival and stress response at different stages of maturation. Embryonic and neonatal cardiomyocytes largely tolerate inhibition of ETC complex III by antimycin A (AMA) as well as ATP synthase (complex V) by oligomycin but are susceptible to complex I inhibition by rotenone. All three inhibitors alter the intracellular distribution and ultrastructure of mitochondria in neonatal cardiomyocytes. In contrast, adult cardiomyocytes treated with AMA undergo rapid morphological changes and cellular disintegration. At the molecular level embryonic cardiomyocytes activate antioxidative defense mechanisms, the integrated stress response (ISR) and ER stress but not the mitochondrial unfolded protein response upon complex III inhibition. In contrast, adult cardiomyocytes fail to activate the ISR and antioxidative proteins following AMA treatment. In conclusion, our results identified fundamental differences in cell survival and stress response in differentiated compared to immature cardiomyocytes subjected to mitochondrial dysfunction. The high stress tolerance of immature cardiomyocytes might allow outlasting unfavorable intrauterine conditions thereby preventing fetal or perinatal heart disease and may contribute to the regenerative capacity of the embryonic and neonatal mammalian heart.

The Role of the Redox Enzyme p66Shc in Biological Aging of the Lung.

The mitochondrial adaptor protein p66Shc has been suggested to control life span in mice via the release of hydrogen peroxide. However, the role of p66Shc in lung aging remains unsolved. Thus, we investigated the effects of p66Shc-/- on the aging of the lung and pulmonary circulation. In vivo lung and cardiac characteristics were investigated in p66Shc-/- and wild type (WT) mice at 3, 12, and 24 months of age by lung function measurements, micro-computed tomography (µCT), and echocardiography. Alveolar number and muscularization of small pulmonary arteries were measured by stereology and vascular morphometry, respectively. Protein and mRNA levels of senescent markers were measured by western blot and PCR, respectively. Lung function declined similarly in WT and p66Shc-/- mice during aging. However, µCT analyses and stereology showed slightly enhanced signs of aging-related parameters in p66Shc-/- mice, such as a decline of alveolar density. Accordingly, p66Shc-/- mice showed higher protein expression of the senescence marker p21 in lung homogenate compared to WT mice of the corresponding age. Pulmonary vascular remodeling was increased during aging, but aged p66Shc-/- mice showed similar muscularization of pulmonary vessels and hemodynamics like WT mice. In the heart, p66Shc-/- prevented the deterioration of right ventricular (RV) function but promoted the decline of left ventricular (LV) function during aging. p66Shc-/- affects the aging process of the lung and the heart differently. While p66Shc-/- slightly accelerates lung aging and deteriorates LV function in aged mice, it seems to exert protective effects on RV function during aging.

Monoamine Oxidase A Contributes to Serotonin-But Not Norepinephrine-Dependent Damage of Rat Ventricular Myocytes.

Serotonin effects on cardiac hypertrophy, senescence, and failure are dependent either on activation of specific receptors or serotonin uptake and serotonin degradation by monoamine oxidases (MAOs). Receptor-dependent effects are specific for serotonin, but MAO-dependent effects are nonspecific as MAOs also metabolize other substrates such as catecholamines. Our study evaluates the role of MAO-A in serotonin- and norepinephrine-dependent cell damage. Experiments were performed in vivo to study the regulation of MAOA and MAOB expression and in vitro on isolated cultured adult rat ventricular cardiomyocytes (cultured for 24 h) to study the function of MAO-A. MAOA but not MAOB expression increased in maladaptive hypertrophic stages. Serotonin and norepinephrine induced morphologic cell damage (loss of rod-shaped cell structure). However, MAO-A inhibition suppressed serotonin-dependent but not norepinephrine-dependent damages. Serotonin but not norepinephrine caused a reduction in cell shortening in nondamaged cells. Serotonin induced mitochondria-dependent oxidative stress. In vivo, MAOA was induced during aging and hypertension but the expression of the corresponding serotonin uptake receptor (SLC6A4) was reduced and enzymes that reduce either oxidative stress (CAT) or accumulation of 5-hydroxyindolacetaldehyde (ALDH2) were induced. In summary, the data show that MAO-A potentially affects cardiomyocytes' function but that serotonin is not necessarily the native substrate.

Sex Difference in Cardioprotection against Acute Myocardial Infarction in MAO-B Knockout Mice In Vivo.

The cardiomyocyte-specific knockout (KO) of monoamine oxidase (MAO)-B, an enzyme involved in the formation of reactive oxygen species (ROS), reduced myocardial ischemia/reperfusion (I/R) injury in vitro. Because sex hormones have a strong impact on MAO metabolic pathways, we analyzed the myocardial infarct size (IS) following I/R in female and male MAO-B KO mice in vivo. METHOD AND RESULTS: To induce the deletion of MAO-B, MAO-B KO mice (Myh6 Cre+/MAO-Bfl/fl) and wild-type (WT, Cre-negative MAO-Bfl/fl littermates) were fed with tamoxifen for 2 weeks followed by 10 weeks of normal mice chow. Myocardial infarction (assessed by TTC staining and expressed as a percentage of the area at risk as determined by Evans blue staining)) was induced by 45 min coronary occlusion followed by 120 min of reperfusion. RESULTS: The mortality following I/R was higher in male compared to female mice, with the lowest mortality found in MAO-B KO female mice. IS was significantly higher in male WT mice compared to female WT mice. MAO-B KO reduced IS in male mice but had no further impact on IS in female MAO-B KO mice. Interestingly, there was no difference in the plasma estradiol levels among the groups. CONCLUSION: The cardiomyocyte-specific knockout of MAO-B protects male mice against acute myocardial infarction but had no effect on the infarct size in female mice.

YB-1 Is a Novel Target for the Inhibition of α-Adrenergic-Induced Hypertrophy.

Cardiac hypertrophy resulting from sympathetic nervous system activation triggers the development of heart failure. The transcription factor Y-box binding protein 1 (YB-1) can interact with transcription factors involved in cardiac hypertrophy and may thereby interfere with the hypertrophy growth process. Therefore, the question arises as to whether YB-1 influences cardiomyocyte hypertrophy and might thereby influence the development of heart failure. YB-1 expression is downregulated in human heart biopsies of patients with ischemic cardiomyopathy (n = 8), leading to heart failure. To study the impact of reduced YB-1 in cardiac cells, we performed small interfering RNA (siRNA) experiments in H9C2 cells as well as in adult cardiomyocytes (CMs) of rats. The specificity of YB-1 siRNA was analyzed by a miRNA-like off-target prediction assay identifying potential genes. Testing three high-scoring genes by transfecting cardiac cells with YB-1 siRNA did not result in downregulation of these genes in contrast to YB-1, whose downregulation increased hypertrophic growth. Hypertrophic growth was mediated by PI3K under PE stimulation, as well by downregulation with YB-1 siRNA. On the other hand, overexpression of YB-1 in CMs, caused by infection with an adenovirus encoding YB-1 (AdYB-1), prevented hypertrophic growth under α-adrenergic stimulation with phenylephrine (PE), but not under stimulation with growth differentiation factor 15 (GDF15; n = 10-16). An adenovirus encoding the green fluorescent protein (AdGFP) served as the control. YB-1 overexpression enhanced the mRNA expression of the Gq inhibitor regulator of G-protein signaling 2 (RGS2) under PE stimulation (n = 6), potentially explaining its inhibitory effect on PE-induced hypertrophic growth. This study shows that YB-1 protects cardiomyocytes against PE-induced hypertrophic growth. Like in human end-stage heart failure, YB-1 downregulation may cause the heart to lose its protection against hypertrophic stimuli and progress to heart failure. Therefore, the transcription factor YB-1 is a pivotal signaling molecule, providing perspectives for therapeutic approaches.

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.

Mitochondria Isolated from Hearts Subjected to Ischemia/Reperfusion Benefit from Adenine Nucleotide Translocase 1 Overexpression.

Reperfusion is the only feasible therapy following myocardial infarction, but reperfusion has been shown to damage mitochondrial function and disrupt energy production in the heart. Adenine nucleotide translocase 1 (ANT1) facilitates the transfer of ADP/ATP across the inner mitochondrial membrane; therefore, we tested whether ANT1 exerts protective effects on mitochondrial function during ischemia/reperfusion (I/R). The hearts of wild-type (WT) and transgenic ANT1-overexpressing (ANT1-TG) rats were exposed to I/R injury using the standard Langendorff technique, after which mitochondrial function, hemodynamic parameters, infarct size, and components of the contractile apparatus were determined. ANT1-TG hearts expressed higher ANT protein levels, with reduced levels of oxidative 4-hydroxynonenal ANT modifications following I/R. ANT1-TG mitochondria isolated from I/R hearts displayed stable calcium retention capacity (CRC) and improved membrane potential stability compared with WT mitochondria. Mitochondria isolated from ANT1-TG hearts experienced less restricted oxygen consumption than WT mitochondria after I/R. Left ventricular diastolic pressure (Pdia) decreased in ANT1-TG hearts compared with WT hearts following I/R. Preserved diastolic function was accompanied by a decrease in the phospho-lamban (PLB)/sarcoplasmic reticulum calcium ATPase (SERCA2a) ratio in ANT1-TG hearts compared with that in WT hearts. In addition, the phosphorylated (P)-PLB/PLB ratio increased in ANT1-TG hearts after I/R but not in WT hearts, which indicated more effective calcium uptake into the sarcoplasmic reticulum in ANT1-TG hearts. In conclusion, ANT1-TG rat hearts coped more efficiently with I/R than WT rat hearts, which was reflected by preserved mitochondrial energy balance, diastolic function, and calcium dynamics after reperfusion.

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.

Connexin 43 phosphorylation by casein kinase 1 is essential for the cardioprotection by ischemic preconditioning.

Myocardial connexin 43 (Cx43) forms gap junctions and hemichannels, and is also present within subsarcolemmal mitochondria. The protein is phosphorylated by several kinases including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and casein kinase 1 (CK1). A reduction in Cx43 content abrogates myocardial infarct size reduction by ischemic preconditioning (IPC). The present study characterizes the contribution of Cx43 phosphorylation towards mitochondrial function, hemichannel activity, and the cardioprotection by IPC in wild-type (WT) mice and in mice in which Cx43-phosphorylation sites targeted by above kinases are mutated to non-phosphorylatable residues (Cx43MAPKmut, Cx43PKCmut, and Cx43CK1mut mice). The amount of Cx43 in the left ventricle and in mitochondria was reduced in all mutant strains compared to WT mice and Cx43 phosphorylation was altered at residues not directly targeted by the mutations. Whereas complex 1 respiration was reduced in all strains, complex 2 respiration was decreased in Cx43CK1mut mice only. In Cx43 epitope-mutated mice, formation of reactive oxygen species and opening of the mitochondrial permeability transition pore were not affected. The hemichannel open probability was reduced in Cx43PKCmut and Cx43CK1mut but not in Cx43MAPKmut cardiomyocytes. Infarct size in isolated saline-perfused hearts after ischemia/reperfusion (45 min/120 min) was comparable between genotypes and was significantly reduced by IPC (3 × 3 min ischemia/5 min reperfusion) in WT, Cx43MAPKmut, and Cx43PKCmut, but not in Cx43CK1mut mice, an effect independent from the amount of Cx43 and the probability of hemichannel opening. Taken together, our study shows that alterations of Cx43 phosphorylation affect specific cellular functions and highlights the importance of Cx43 phosphorylation by CK1 for IPC's cardioprotection.

Purinergic Regulation of Endothelial Barrier Function.

Increased vascular permeability is a hallmark of several cardiovascular anomalies, including ischaemia/reperfusion injury and inflammation. During both ischaemia/reperfusion and inflammation, massive amounts of various nucleotides, particularly adenosine 5'-triphosphate (ATP) and adenosine, are released that can induce a plethora of signalling pathways via activation of several purinergic receptors and may affect endothelial barrier properties. The nature of the effects on endothelial barrier function may depend on the prevalence and type of purinergic receptors activated in a particular tissue. In this review, we discuss the influence of the activation of various purinergic receptors and downstream signalling pathways on vascular permeability during pathological conditions.

Matrix Metalloproteinases Repress Hypertrophic Growth in Cardiac Myocytes.

PURPOSE: Matrix metalloproteinases (MMPs) are identified as modulators of the extracellular matrix in heart failure progression. However, evidence for intracellular effects of MMPs is emerging. Pro- and anti-hypertrophic cardiac effects are described. This may be due to the various sources of different MMPs in the heart tissue. Therefore, the aim of the present study was to determine the role of MMPs in hypertrophic growth of isolated rat ventricular cardiac myocytes. METHODS: Cardiomyocytes were isolated form ventricular tissues of the rat hearts by collagenase perfusion. RT-qPCR, western blots, and zymography were used for expression and MMP activity analysis. Cross-sectional area and the rate of protein synthesis were determined as parameters for hypertrophic growth. RESULTS: MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 mRNAs were detected in cardiomyocytes, and protein expression of MMP-2, MMP-9, and MMP-14 was identified. Hypertrophic stimulation of cardiomyocytes did not enhance, but interestingly decreased expression of MMPs, indicating that downregulation of MMPs may promote hypertrophic growth. Indeed, the nonselective MMP inhibitors TAPI-0 or TIMP2 and the MMP-2-selective ARP-100 enhanced hypertrophic growth. Furthermore, TAPI-0 increased phosphorylation and thus activation of extracellular signaling kinase (ERK) and Akt (protein kinase B), as well as inhibition of glycogen synthase 3ß (GSK3ß). Abrogation of MEK/ERK- or phosphatidylinositol-3-kinase(PI3K)/Akt/GSK3ß-signaling with PD98059 or LY290042, respectively, inhibited hypertrophic growth under TAPI-0. CONCLUSION: MMPs' inhibition promotes hypertrophic growth in cardiomyocytes in vitro. Therefore, MMPs in the healthy heart may be important players to repress cardiac hypertrophy.

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.

Structural, Pro-Inflammatory and Calcium Handling Remodeling Underlies Spontaneous Onset of Paroxysmal Atrial Fibrillation in JDP2-Overexpressing Mice.

BACKGROUND: Cardiac-specific JDP2 overexpression provokes ventricular dysfunction and atrial dilatation in mice. We performed in vivo studies on JDP2-overexpressing mice to investigate the impact of JDP2 on the predisposition to spontaneous atrial fibrillation (AF). METHODS: JDP2-overexpression was started by withdrawal of a doxycycline diet in 4-week-old mice. The spontaneous onset of AF was documented by ECG within 4 to 5 weeks of JDP2 overexpression. Gene expression was analyzed by real-time RT-PCR and Western blots. RESULTS: In atrial tissue of JDP2 mice, besides the 3.6-fold increase of JDP2 mRNA, no changes could be detected within one week of JDP2 overexpression. Atrial dilatation and hypertrophy, combined with elongated cardiomyocytes and fibrosis, became evident after 5 weeks of JDP2 overexpression. Electrocardiogram (ECG) recordings revealed prolonged PQ-intervals and broadened P-waves and QRS-complexes, as well as AV-blocks and paroxysmal AF. Furthermore, reductions were found in the atrial mRNA and protein level of the calcium-handling proteins NCX, Cav1.2 and RyR2, as well as of connexin40 mRNA. mRNA of the hypertrophic marker gene ANP, pro-inflammatory MCP1, as well as markers of immune cell infiltration (CD68, CD20) were increased in JDP2 mice. CONCLUSION: JDP2 is an important regulator of atrial calcium and immune homeostasis and is involved in the development of atrial conduction defects and arrhythmogenic substrates preceding paroxysmal AF.

Adenine Nucleotide Translocase 1 Expression is Coupled to the HSP27-Mediated TLR4 Signaling in Cardiomyocytes.

The cardiac-specific overexpression of the adenine nucleotide translocase 1 (ANT1) has cardioprotective effects in various experimental heart disease models. Here, we analyzed the link between ANT1 expression and heat shock protein 27 (HSP27)-mediated toll-like receptor 4 (TLR4) signaling, which represents a novel communication pathway between mitochondria and the extracellular environment. The interaction between ANT1 and HSP27 was identified by co-immunoprecipitation from neonatal rat cardiomyocytes. ANT1 transgenic (ANT1-TG) cardiomyocytes demonstrated elevated HSP27 expression levels. Increased levels of HSP27 were released from the ANT1-TG cardiomyocytes under both normoxic and hypoxic conditions. Extracellular HSP27 stimulated TLR4 signaling via protein kinase B (AKT). The HSP27-mediated activation of the TLR4 pathway was more pronounced in ANT1-TG cardiomyocytes than in wild-type (WT) cardiomyocytes. HSP27-specific antibodies inhibited TLR4 activation and the expression of HSP27. Inhibition of the HSP27-mediated TLR4 signaling pathway with the TLR4 inhibitor oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) reduced the mitochondrial membrane potential (∆ψm) and increased caspase 3/7 activity, which are both markers for cell stress. Conversely, treating cardiomyocytes with recombinant HSP27 protein stimulated TLR4 signaling, induced HSP27 and ANT1 expression, and stabilized the mitochondrial membrane potential. The activation of HSP27 signaling was verified in ischemic ANT1-TG heart tissue, where it correlated with ANT1 expression and the tightness of the inner mitochondrial membrane. Our study shows a new mechanism by which ANT1 is part of the cardioprotective HSP27-mediated TLR4 signaling.

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.

Endothelial Mesenchymal Transition in Hypoxic Microvascular Endothelial Cells and Paracrine Induction of Cardiomyocyte Apoptosis Are Mediated via TGFß1/SMAD Signaling.

Cardiac remodeling plays a crucial role in the development of heart failure after mycocardial infarction. Besides cardiomyocytes, endothelial cells are recognized to contribute to cardiac remodeling. We now investigated processes of endothelial mesenchymal transition (EndoMT) in microvascular endothelial cells of rat (MVEC) under hypoxia and paracrine effects on ventricular cardiomyocytes of adult rat. Exposure of MVECs to hypoxia/reoxygenation enhanced TGFß/SMAD signaling, since phosphorylation, and thus activation, of SMAD1/5 and SMAD2 increased. This increase was blocked by inhibitors of TGFß receptor types ALK1 or ALK5. Exposure of ventricular cardiomyocytes to conditioned medium from hypoxic/reoxygenated MVECs enhanced SMAD2 phosphorylation and provoked apoptosis in cardiomyoyctes. Both were blocked by ALK5 inhibition. To analyze autocrine effects of hypoxic TGFß signaling we investigated EndoMT in MVECs. After 3 days of hypoxia the mesenchymal marker protein α-smooth muscle actin (α-SMA), and the number of α-SMA- and fibroblast specific protein 1 (FSP1)-positive cells increased in MVECs cultures. This was blocked by ALK5 inhibition. Similarly, TGFß1 provoked enhanced expression of α-SMA and FSP1 in MVECs. In conclusion, hypoxia provokes EndoMT in MVECs via TGFß1/SMAD2 signaling. Furthermore, release of TGFß1 from MVECs acts in a paracrine loop on cardiomyocytes and provokes apoptotic death. Thus, in myocardial infarction hypoxic endothelial cells may contribute to cardiac remodeling and heart failure progression by promotion of cardiac fibrosis and cardiomyocytes death.

Effect of nitric oxide deficiency on the pulmonary PTHrP system.

Nitric oxide (NO) deficiency is common in pulmonary diseases, but its effect on pulmonary remodelling is still controversial. As pulmonary parathyroid hormone-related protein (PTHrP) expression is a key regulator of pulmonary fibrosis and development, the effect of chronic NO deficiency on the pulmonary PTHrP system and its relationship with oxidative stress was addressed. NO bioavailability in adult rats was reduced by systemic administration of L-NAME via tap water. To clarify the role of NO synthase (NOS)-3-derived NO on pulmonary expression of PTHrP, NOS-3-deficient mice were used. Captopril and hydralazine were used to reduce the hypertensive effect of L-NAME treatment and to interfere with the pulmonary renin-angiotensin system (RAS). Quantitative RT-PCR and immunoblot techniques were used to characterize the expression of key proteins involved in pulmonary remodelling. L-NAME administration significantly reduced pulmonary NO concentration and caused oxidative stress as characterized by increased pulmonary nitrite concentration and increased expression of NOX2, p47phox and p67phox. Furthermore, L-NAME induced the pulmonary expression of PTHrP and of its corresponding receptor, PTH-1R. Expression of PTHrP and PTH-1R correlated with the expression of two well-established PTHrP downstream targets, ADRP and PPARγ, suggesting an activation of the pulmonary PTHrP system by NO deficiency. Captopril reduced the expression of PTHrP, profibrotic markers and ornithine decarboxylase, but neither that of PTH-1R nor that of ADRP and PPARγ. All transcriptional changes were confirmed in NOS-3-deficient mice. In conclusion, NOS-3-derived NO suppresses pulmonary PTHrP and PTH-1R expression, thereby modifying pulmonary remodelling.