Doxorubicin-induced and trastuzumab-induced cardiotoxicity in mice is not prevented by metoprolol.

AIMS: Our objectives were to validate a murine model of chronic cardiotoxicity induced by Doxorubicin (Dox) and Trastuzumab (Trast) and to test the potential cardio-protective effect of metoprolol. METHODS AND RESULTS: Male C57Bl6 mice were intraperitoneally injected during 2 weeks with Dox (24 mg/kg) or saline, and then with Trast (10 mg/kg) or saline for two more weeks. Half of the mice received metoprolol (100 mg/kg). Cardiotoxicity was defined by a decline in left ventricular ejection fraction (LVEF) ≥ 10 points. At Day 42, Dox + Trast-treated mice exhibited a 13-points decline in LVEF (74 ± 2.6% vs. 87 ± 0.8% for control mice, P < 0.001) and a severe cardiac atrophy (heart weight: 105 ± 2.7 mg vs. 119 ± 3.9 mg for control mice, P < 0.01). This cardiac atrophy resulted from an excess of cardiac necrosis (assessed by plasma cardiac troponin I level: 3.2 ± 0.4 ng/L vs. 1.3 ± 0.06 ng/L for control mice, P < 0.01), an increase in apoptosis (caspase 3 activity showing a six-fold increase for Dox + Trast-treated mice vs. controls, P < 0.001), and cardiomyocyte atrophy (myocyte size: 0.67 ± 0.08 µm2 vs. 1.36 ± 0.10 µm2 for control mice, P < 0.001). In addition, Dox + Trast-treated mice were shown to have an increased cardiac oxidative stress (164 ± 14 dihydroethidine-marked nuclei per area vs. 56 ± 9.5 for control mice, P < 0.01) and increased cardiac fibrosis (the semi-quantitative fibrosis score was three-fold higher for Dox + Trast-treated mice as compared with controls, P < 0.01). Metoprolol was not able to prevent either the decrease in LVEF or the severe cardiac atrophy, the cardiac necrosis, and the cardiac remodelling induced by chemotherapies. CONCLUSION: A murine model of chronic cardiotoxicity induced by Dox and Trast was characterized by a decrease in cardiac function, a cardiac apoptosis and necrosis leading to cardiomyocyte atrophy. Metoprolol did not prevent this cardiotoxicity.

Adrecizumab, a non-neutralizing anti-adrenomedullin antibody, improves haemodynamics and attenuates myocardial oxidative stress in septic rats.

BACKGROUND: Sepsis still represents a major health issue, with persistent high morbidity and mortality rates. Cardiovascular dysfunction occurs frequently during sepsis. Adrenomedullin has been identified as a key mediator in vascular tone regulation. A non-neutralizing anti-adrenomedullin antibody, Adrecizumab, may improve haemodynamic dysfunction during caecal ligation and puncture-induced septic shock in a murine model. Our objective was to determine the role of Adrecizumab on haemodynamics in a rat model of sepsis. METHODS: For the induction of sepsis, caecal ligation and puncture were performed in Wistar male rats. Single blinded administration of Adrecizumab (2 mg/kg) or placebo was injected i.v. 24 h after the surgery, and norepinephrine was infused as the standard of care. There were > 7 animals per group. Invasive blood pressure and cardiac function (by echocardiography) were assessed until 3 h after Adrecizumab injection. RESULTS: A single therapeutic injection of Adrecizumab in septic rats induced rapid haemodynamic benefits with an increase in systolic blood pressure in septic-Adrecizumab rats versus untreated-septic rats (p = 0.049). The shortening fraction did not differ between the untreated-septic and septic-Adrecizumab groups. However, cardiac output increased during the 3 h after a single dose of Adrecizumab compared to untreated septic rats (p = 0.006). A single dose of Adrecizumab resulted in similar haemodynamics to the continuous administration of norepinephrine. Three hours after a single injection of Adrecizumab, there was no change in the inflammatory phenotype (TNFα, IL-10) in the hearts of the septic rats. By contrast, 3 h after a single Adrecizumab injection, free-radical production decreased in the hearts of septic-Adrecizumab vs untreated septic rats (p < 0.05). CONCLUSIONS: In a rat model of sepsis, a single therapeutic injection of Adrecizumab rapidly restored haemodynamic parameters and blunted myocardial oxidative stress. Currently, a proof-of-concept and dose-finding phase II trial (Adrenoss-2) is ongoing in patients with septic shock and elevated concentrations of circulating bio-adrenomedullin.

Effects of hypoestrogenism and/or hyperaldosteronism on myocardial remodeling in female mice.

We investigated the potential adverse effects of hyperaldosteronism and/or hypoestrogenism on cardiac phenotype, and examined their combined effects in female mice overexpressing cardiac aldosterone synthase (AS). We focused on some signaling cascades challenging defensive responses to adapt and/or to survive in the face of double deleterious stresses, such as Ca2+ -homeostasis, pro/anti-hypertrophic, endoplasmic reticulum stress (ER stress), pro- or anti-apoptotic effectors, and MAP kinase activation, and redox signaling. These protein expressions were assessed by immunoblotting at 9 weeks after surgery. Female wild type (FWT) and FAS mice were fed with phytoestrogen-free diet; underwent ovariectomy (Ovx) or sham-operation (Sham). Ovx increased gain weight and hypertrophy index. Transthoracic echocardiograghy was performed. Both Ovx-induced heart rate decrease and fractional shortening increase were associated with collagen type III shift. Cardiac estrogen receptor (ERα, ERß) protein expression levels were downregulated in Ovx mice. Hypoestrogenism increased plasma aldosterone and MR protein expression in FAS mice. Both aldosterone and Ovx played as mirror effects on up and downstream signaling effectors of calcium/redox homeostasis, apoptosis, such as concomitant CaMKII activation and calcineurin down-regulation, MAP kinase inhibition (ERK1/2, p38 MAPK) and Akt activation. The ratio Bcl2/Bax is in favor to promote cell survivor. Finally, myocardium had dynamically orchestrated multiple signaling cascades to restore tolerance to hostile environment thereby contributing to a better maintenance of Ca2+ /redox homeostasis. Ovx-induced collagen type III isoform shift and its upregulation may be important for the biomechanical transduction of the heart and the recovery of cardiac function in FAS mice. OVX antagonized aldosterone signaling pathways.

QSOX1, a novel actor of cardiac protection upon acute stress in mice.

QSOX1, a sulfhydryl oxidase, was shown to be upregulated in the heart upon acute heart failure (AHF). The aim of the study was to unravel QSOX1 roles during AHF. We generated and characterized mice with QSOX1 gene deletion. The QSOX1-/- mice were viable but adult male exhibited a silent dilated cardiomyopathy. The QSOX1-/- hearts were characterized by low protein SERCA2a levels associated with a calcium homeostasis alteration, high levels of the endoplasmic reticulum (ER) chaperone proteins Grp78/Bip, and of the ER apoptosis sensor CHOP, indicating a chronic unfolded protein response (UPR). Importantly the QSOX1invalidation led to overexpression of two ER oxidases, ERO1-α and PRDX4. Acute stress was induced by isoproterenol injection (ISO, 300 mg/kg/12 h) for 2 days. In both groups, the PERK UPR pathway was transiently activated 6 h after the first ISO injection as indicated by eIF2 phosphorylation. By day-3 after the onset of stress, both WT and QSOX1-/- mice exhibited AHF profile but while high cardiac QSOX1 level was induced in WT hearts, ERO1-α and PRDX4 levels drop down in QSOX1-/-. At that time, QSOX1-/- hearts exhibited an enhanced inflammation (CD68+ cells and Galectin-3 expression) and oxidative stress (DHE staining and oxyblot) when compared to WT ones. In conclusion, the lack of QSOX1 promotes the upregulation of two ER oxidases ERO1α and PRDX4 that likely rescues oxidative protein folding in the hearts. However, signs of chronic ER stress remained present and were associated with a dilated cardiomyopathy. The superimposition of acute stress allowed us to propose that QSOX1 participate to the early response to cardiac stress but not to immediate UPR response. Taken altogether, the data indicated that QSOX1 is required 1) for a proper protein folding in the endo/sarcoplasmic reticulum (ER/SR) and 2) for resolution and protective response during acute stress.

Loss of Notch3 Signaling in Vascular Smooth Muscle Cells Promotes Severe Heart Failure Upon Hypertension.

Hypertension, which is a risk factor of heart failure, provokes adaptive changes at the vasculature and cardiac levels. Notch3 signaling plays an important role in resistance arteries by controlling the maturation of vascular smooth muscle cells. Notch3 deletion is protective in pulmonary hypertension while deleterious in arterial hypertension. Although this latter phenotype was attributed to renal and cardiac alterations, the underlying mechanisms remained unknown. To investigate the role of Notch3 signaling in the cardiac adaptation to hypertension, we used mice with either constitutive Notch3 or smooth muscle cell-specific conditional RBPJκ knockout. At baseline, both genotypes exhibited a cardiac arteriolar rarefaction associated with oxidative stress. In response to angiotensin II-induced hypertension, the heart of Notch3 knockout and SM-RBPJκ knockout mice did not adapt to pressure overload and developed heart failure, which could lead to an early and fatal acute decompensation of heart failure. This cardiac maladaptation was characterized by an absence of media hypertrophy of the media arteries, the transition of smooth muscle cells toward a synthetic phenotype, and an alteration of angiogenic pathways. A subset of mice exhibited an early fatal acute decompensated heart failure, in which the same alterations were observed, although in a more rapid timeframe. Altogether, these observations indicate that Notch3 plays a major role in coronary adaptation to pressure overload. These data also show that the hypertrophy of coronary arterial media on pressure overload is mandatory to initially maintain a normal cardiac function and is regulated by the Notch3/RBPJκ pathway.

Akt-mediated cardioprotective effects of aldosterone in type 2 diabetic mice.

Studies have shown that aldosterone would have angiogenic effects and therefore would be beneficial in the context of cardiovascular diseases. We thus investigated the potential involvement of aldosterone in triggering a cardiac angiogenic response in the context of type-2 diabetes and the molecular pathways involved. Male 3-wk-old aldosterone synthase (AS)-overexpressing mice and their control wild-type (WT) littermates were fed a standard or high-fat, high-sucrose (HFHS) diet. After 6 mo of diet treatment, mice were euthanized, and cardiac samples were assayed by RT-PCR, immunoblotting, and immunohistology. HFHS diet induced type-2 diabetes in WT (WT-D) and AS (AS-D) mice. VEGFa mRNAs decreased in WT-D (-43%, P<0.05 vs. WT) and increased in AS-D mice (+236%, P< 0.01 vs. WT-D). In WT-D mouse hearts, the proapoptotic p38MAPK was activated (P<0.05 vs. WT and AS-D), whereas Akt activity decreased (-64%, P<0.05 vs. WT). The AS mice, which exhibited a cardiac up-regulation of IGF1-R, showed an increase in Akt phosphorylation when diabetes was induced (P<0.05 vs. WT and AS-D). Contrary to WT-D mice, AS-D mouse hearts did not express inflammatory markers and exhibited a normal capillary density (P<0.05 vs. WT-D). To our knowledge, this is the first study providing new insights into the mechanisms whereby aldosterone prevents diabetes-induced cardiac disorders.

Crucial role for endoplasmic reticulum stress during megakaryocyte maturation.

OBJECTIVE: Apoptotic-like phase is an essential step for the platelet formation from megakaryocytes. How controlled is this signaling pathway remained poorly understood. The aim of this study was to determine whether endoplasmic reticulum (ER) stress-induced apoptosis occurs during thrombopoiesis. APPROACH AND RESULTS: Investigation of ER stress and maturation markers in different models of human thrombopoiesis (CHRF, DAMI, MEG-01 cell lines, and hematopoietic stem cells: CD34(+)) as well as in immature pathological platelets clearly indicated that ER stress occurs transiently during thrombopoiesis. Direct ER stress induction by tunicamycin, an inhibitor of N-glycosylation, or by sarco/endoplasmic reticulum Ca(2+) ATPase type 3b overexpression, which interferes with reticular calcium, leads to some degree of maturation in megakaryocytic cell lines. On the contrary, exposure to salubrinal, a phosphatase inhibitor that prevents eukaryotic translation initiation factor 2α-P dephosphorylation and inhibits ER stress-induced apoptosis, decreased both expression of maturation markers in MEG-01 and CD34(+) cells as well as numbers of mature megakaryocytes and proplatelet formation in cultured CD34(+) cells. CONCLUSIONS: Taken as a whole, our research suggests that transient ER stress activation triggers the apoptotic-like phase of the thrombopoiesis process.

Aldosterone inhibits the fetal program and increases hypertrophy in the heart of hypertensive mice.

BACKGROUND: Arterial hypertension (AH) induces cardiac hypertrophy and reactivation of "fetal" gene expression. In rodent heart, alpha-Myosin Heavy Chain (MyHC) and its micro-RNA miR-208a regulate the expression of beta-MyHC and of its intronic miR-208b. However, the role of aldosterone in these processes remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: RT-PCR and western-blot were used to investigate the genes modulated by arterial hypertension and cardiac hyperaldosteronism. We developed a model of double-transgenic mice (AS-Ren) with cardiac hyperaldosteronism (AS mice) and systemic hypertension (Ren). AS-Ren mice had increased (x2) angiotensin II in plasma and increased (x2) aldosterone in heart. Ren and AS-Ren mice had a robust and similar hypertension (+70%) versus their controls. Anatomical data and echocardiography showed a worsening of cardiac hypertrophy (+41%) in AS-Ren mice (P<0.05 vs Ren). The increase of ANP (x 2.5; P<0.01) mRNA observed in Ren mice was blunted in AS-Ren mice. This non-induction of antitrophic natriuretic peptides may be involved in the higher trophic cardiac response in AS-Ren mice, as indicated by the markedly reduced cardiac hypertrophy in ANP-infused AS-Ren mice for one month. Besides, the AH-induced increase of ßMyHC and its intronic miRNA-208b was prevented in AS-Ren. The inhibition of miR 208a (-75%, p<0.001) in AS-Ren mice compared to AS was associated with increased Sox 6 mRNA (x 1.34; p<0.05), an inhibitor of ßMyHC transcription. Eplerenone prevented all aldosterone-dependent effects. CONCLUSIONS/SIGNIFICANCE: Our results indicate that increased aldosterone in heart inhibits the induction of atrial natriuretic peptide expression, via the mineralocorticoid receptor. This worsens cardiac hypertrophy without changing blood pressure. Moreover, this work reveals an original aldosterone-dependent inhibition of miR-208a in hypertension, resulting in the inhibition of ß-myosin heavy chain expression through the induction of its transcriptional repressor Sox6. Thus, aldosterone inhibits the fetal program and increases cardiac hypertrophy in hypertensive mice.

Aldosterone inhibits antifibrotic factors in mouse hypertensive heart.

The renin-angiotensin-aldosterone system is involved in the arterial hypertension-associated cardiovascular remodeling. In this context, the development of cardiac fibrosis results from an imbalance between profibrotic and antifibrotic pathways, in which the role of aldosterone is yet not established. To determine the role of intracardiac aldosterone in the development of myocardial fibrosis during hypertension, we used a double transgenic model (AS-Ren) of cardiac hyperaldosteronism (AS) and systemic hypertension (Ren). The 9-month-old hypertensive mice had cardiac fibrosis, and hyperaldosteronism enhanced the fibrotic level. The mRNA levels of connective tissue growth factor and transforming growth factor-ß1 were similarly increased in Ren and AS-Ren mice compared with wild-type and AS mice, respectively. Hyperaldosteronism combined with hypertension favored the macrophage infiltration (CD68(+) cells) in heart, and enhanced the mRNA level of monocyte chemoattractant protein 1, osteopontin, and galectin 3. Interestingly, in AS-Ren mice the hypertension-induced increase in bone morphogenetic protein 4 mRNA and protein levels was significantly inhibited, and B-type natriuretic peptide expression was blunted. The mineralocorticoid receptor antagonist eplerenone restored B-type natriuretic peptide and bone morphogenetic protein 4 levels and decreased CD68 and galectin 3 levels in AS-Ren mice. Finally, when hypertension was induced by angiotensin II infusion in wild-type and AS mice, the mRNA profiles did not differ from those observed in Ren and AS-Ren mice, respectively. The aldosterone-induced inhibition of B-type natriuretic peptide and bone morphogenetic protein 4 expression was confirmed in vitro in neonatal mouse cardiomyocytes. Altogether, we demonstrate that, at the cardiac level, hyperaldosteronism worsens hypertension-induced fibrosis through 2 mineralocorticoid receptor-dependent mechanisms, activation of inflammation/galectin 3-induced fibrosis and inhibition of antifibrotic factors (B-type natriuretic peptide and bone morphogenetic protein 4).

Role of L-type calcium channel blocking in epidermal growth factor receptor-independent activation of extracellular signal regulated kinase 1/2.

OBJECTIVE: Vascular smooth muscle cell (VSMC) differentiation, growth and survival, key events in the development of cardiovascular diseases, are under the control of signaling enzymes including extracellular signal regulated kinase 1/2 (ERK 1/2), Akt and epidermal growth factor receptor (EGFR) activation. EGFR trans-activation is known to mediate thrombin- or angiotensin II (AII)-stimulated ERK 1/2 activation. However, our laboratory has demonstrated, in thrombin-stimulated VSMC, that the prevention of intracellular Ca2+ elevation ([Ca2+]i) by BAPTA-AM pretreatment unveiled EGFR-independent ERK 1/2 activation. Since calcium channel blockers (CCBs) also impair agonist-induced [Ca2+]i elevation, we investigated whether EGFR-independent ERK 1/2 activation could occur in VSMCs treated by CCBs such as amlodipine, isradipine and verapamil, and examined the possible role of Akt. METHODS: Cultured VSMCs were pretreated or not with CCBs and with various inhibitors of the signaling pathways under study, prior to stimulation by thrombin or AII, and the phosphorylation/activation status of EGFR, Akt and ERK 1/2 was determined by Western blotting using phospho-specific antibodies. RESULTS AND CONCLUSION: Unlike BAPTA, CCBs did not impair stimulus-induced EGFR trans-activation, hence ERK1/2 phosphorylation. However, when EGFR kinase was inhibited, CCBs and BAPTA dose-dependently protected stimulus-induced ERK1/2 phosphorylation. The effect of amlodipine could not be mimicked by its R+ enantiomer, which is devoid of CCB activity, suggesting that the effects of CCBs were accounted for by their L-type Ca2+ channel-blocking property. Altogether, our results indicated that in G-protein-coupled receptor (GPCR)-stimulated VSMCs, the prevention of [Ca2+]i elevation by CCBs unmasked an EGFR kinase-independent phosphorylation of ERK 1/2. Since EGFR kinase inhibitors are supposed to be efficient in the treatment of some cancers, such a mechanism might be clinically relevant in hypertensive patients with cancer.

Evidence for ERK1/2 activation by thrombin that is independent of EGFR transactivation.

Thrombin is involved in abnormal proliferation of vascular smooth muscle cells (VSMCs) associated with pathogenic vascular remodeling. Thrombin stimulation results in extracellular signal-regulated kinase (ERK)1/2 activation through transactivation of the epidermal growth factor receptor (EGFR). Here, using specific antibodies and inhibitors, we investigated the thrombin-induced phosphorylation of Src family kinases, nonreceptor proline-rich tyrosine kinase (Pyk2), EGFR, and ERK1/2. Our results show that Src and Pyk2 are involved upstream of the EGFR transactivation that is required for ERK1/2 phosphorylation. The investigation of the role of intracellular calcium concentration ([Ca2+]i) and calcium mobilization with the Ca2+ chelator BAPTA and thapsigargin, respectively, indicated that thrombin- and thapsigargin-induced phosphorylation of the EGFR but not ERK1/2 is dependent on an increase in [Ca2+]i. Moreover, only after BAPTA-AM pretreatment was thrombin-induced activation of ERK1/2 partially preserved from the effects of EGFR and PKC inhibition but not Src family kinase inhibition. These results suggest that BAPTA, by preventing [Ca2+]i elevation, unmasks a new pathway of Src family kinase-dependent thrombin-stimulated ERK1/2 phosphorylation that is independent of EGFR and PKC activation.