The lysyl oxidase inhibitor (ß-aminopropionitrile) reduces leptin profibrotic effects and ameliorates cardiovascular remodeling in diet-induced obesity in rats.

Lysyl oxidase (LOX) is an extracellular matrix (ECM)-modifying enzyme that has been involved in cardiovascular remodeling. We explore the impact of LOX inhibition in ECM alterations induced by obesity in the cardiovascular system. LOX is overexpressed in the heart and aorta from rats fed a high-fat diet (HFD). ß-Aminopropionitrile (BAPN), an inhibitor of LOX activity, significantly attenuated the increase in body weight and cardiac hypertrophy observed in HFD rats. No significant differences were found in cardiac function or blood pressure among any group. However, HFD rats showed cardiac and vascular fibrosis and enhanced levels of superoxide anion (O2(-)), collagen I and transforming growth factor ß (TGF-ß) in heart and aorta and connective tissue growth factor (CTGF) in aorta, effects that were attenuated by LOX inhibition. Interestingly, BAPN also prevented the increase in circulating leptin levels detected in HFD fed animals. Leptin increased protein levels of collagen I, TGF-ß and CTGF, Akt phosphorylation and O2(-) production in both cardiac myofibroblasts and vascular smooth muscle cells in culture, while LOX inhibition ameliorated these alterations. LOX knockdown also attenuated leptin-induced collagen I production in cardiovascular cells. Our findings indicate that LOX inhibition attenuates the fibrosis and the oxidative stress induced by a HFD on the cardiovascular system. The reduction of leptin levels by BAPN in vivo and the ability of this compound to inhibit leptin-induced profibrotic mediators and ROS production in cardiac and vascular cells suggest that interactions between leptin and LOX regulate downstream events responsible for myocardial and vascular fibrosis in obesity.

Exposure to low mercury concentration in vivo impairs myocardial contractile function.

Increased cardiovascular risk after mercury exposure has been described but cardiac effects resulting from controlled chronic treatment are not yet well explored. We analyzed the effects of chronic exposure to low mercury concentrations on hemodynamic and ventricular function of isolated hearts. Wistar rats were treated with HgCl2 (1st dose 4.6 µg/kg, subsequent dose 0.07 µg/kg/day, im, 30 days) or vehicle. Mercury treatment did not affect blood pressure (BP) nor produced cardiac hypertrophy or changes of myocyte morphometry and collagen content. This treatment: 1) in vivo increased left ventricle end diastolic pressure (LVEDP) without changing left ventricular systolic pressure (LVSP) and heart rate; 2) in isolated hearts reduced LV isovolumic systolic pressure and time derivatives, and ß-adrenergic response; 3) increased myosin ATPase activity; 4) reduced Na+-K+ ATPase (NKA) activity; 5) reduced protein expression of SERCA and phosphorylated phospholamban on serine 16 while phospholamban expression increased; as a consequence SERCA/phospholamban ratio reduced; 6) reduced sodium/calcium exchanger (NCX) protein expression and α-1 isoform of NKA, whereas α-2 isoform of NKA did not change. Chronic exposure for 30 days to low concentrations of mercury does not change BP, heart rate or LVSP but produces small but significant increase of LVEDP. However, in isolated hearts mercury treatment promoted contractility dysfunction as a result of the decreased NKA activity, reduction of NCX and SERCA and increased PLB protein expression. These findings offer further evidence that mercury chronic exposure, even at small concentrations, is an environmental risk factor affecting heart function.

Immunocytochemical detection of synaptophysin in C57BL/6 mice cochlea during aging process.

Aged mammals frequently exhibit a bilateral, progressive, and symmetric deafness related to the degeneration of auditory receptor. However, little is still known about aging effects on synapses in this receptor. Synaptophysin (Syp) is a 38 kDa Ca2+ binding glycoprotein widely found in presynaptic membrane and vesicles. The Syp has been found in presynaptic buttons of efferent auditory fibers, within the developing and adult auditory receptor. The detection of Syp in aged cochleae could provide relevant information about synaptic changes and receptor degeneration process observed in old animals. This paper focuses on aging linked changes related to the presence of Syp in cochleae of C57BL/6J mice (from 1 to 24 months old). Results showed that during the first months of age, no significant changes were observed in the Syp distribution under the basal pole of inner (IHCs) neither the outer (OHCs) hair cells. At six months of age, a significant decrease of Syp immunocytochemical detection appeared in fibers under the most external row of OHCs, but restricted to the cochlear basal coil. Only a very scarce reduction of Syp was noted under the IHC and the other OHC rows, also at the basal coil. From mice 9 months old on, a progressive decrease of the presence of Syp was found under IHC and all OHC rows starting at the basal coil and reaching the apical coil in the oldest mice. All these data could indicate that the cochlea aging process early affects to presynaptic membrane proteins of efferent endings fibers. This early alteration of cochleae efferent synapses could be involved in the whole degeneration of the Corti's organ.

The Crosstalk between Cardiac Lipotoxicity and Mitochondrial Oxidative Stress in the Cardiac Alterations in Diet-Induced Obesity in Rats.

The impact of the mitochondria-targeted antioxidant MitoQ was evaluated in the cardiac alterations associated with obesity. Male Wistar rats were fed either a high fat diet (HFD, 35% fat) or a standard diet (CT, 3.5% fat) for 7 weeks and treated with MitoQ (200 µM). The effect of MitoQ (5 nM) in rat cardiac myoblasts treated for 24 h with palmitic acid (PA, 200 µM) was evaluated. MitoQ reduced cardiac oxidative stress and prevented the development of cardiac fibrosis, hypertrophy, myocardial 18-FDG uptake reduction, and mitochondrial lipid remodeling in HFD rats. It also ameliorated cardiac mitochondrial protein level changes observed in HFD: reductions in fumarate hydratase, complex I and II, as well as increases in mitofusin 1 (MFN1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha, and cyclophilin F (cycloF). In vitro, MitoQ prevented oxidative stress and ameliorated alterations in mitochondrial proteins observed in palmitic acid (PA)-stimulated cardiac myoblasts: increases in carnitine palmitoyltransferase 1A, cycloF, and cytochrome C. PA induced phosphorylation of extracellular signal-regulated kinases and nuclear factor-κB p65. Therefore, the data show the beneficial effects of MitoQ in the cardiac damage induced by obesity and suggests a crosstalk between lipotoxicity and mitochondrial oxidative stress in this damage.

Simultaneous HPLC quantification of monoamines and metabolites in the blood-free rat cochlea.

Monoamine quantification in peripheral sensory receptors, such as the cochlea, is of major interest since monoamines could play a role in neurotransmission. A three-step biochemical protocol was developed to analyze monoamine content within the cochlea. Removal of the blood by aortic perfusion was carried out with an anticoagulant solution prior to the dissection of the cochlea from the temporal bone. The cochlear monoamines and some of their metabolites were then quantified, from homogenated cochlear tissue, by a new application of high performance liquid chromatography coupled to electrochemical detection. This method demonstrated enough sensitivity to detect norepinephrine (NE), dopamine (DA), serotonin (5-HT) and some of their metabolites (3,4-dihydroxyphenylacetic acid, DOPAC; homovanillic acid, HVA; and 5-hydroxyindole-3-acetic acid, 5-HIAA). Furthermore, it enabled the demonstration of noise-induced changes in the cochlear concentrations of NE, DA, DOPAC and HVA. In addition, the aortic perfusion allowed removal of the blood-borne 5-HT from the cochlea without inducing systemic alterations or monoamine degradation, as shown by the absence of effects on NE, DA, DOPAC, HVA or 5-HIAA concentrations. The present methodology may constitute a useful strategy to analyze monoamine turnover in the cochlea and other peripheral sensory receptors.

Leptin induces cardiac fibrosis through galectin-3, mTOR and oxidative stress: potential role in obesity.

OBJECTIVE: Leptin acts as a cardiac profibrotic factor. However, the mechanisms underlying this effect are unclear. Therefore, we sought to elucidate the mediators involved in this process and the potential role of leptin in cardiac fibrosis associated with obesity. METHODS: Male Wistar rats were fed either a high-fat diet (HFD; 33.5% fat), or a standard diet (3.5% fat) for 6 weeks. RESULTS: HFD animals show cardiac hypertrophy, fibrosis and an increase in O2- production as evaluated by dihydroethidium. Echocardiographic parameters of cardiac structure and systolic function were similar in both groups. Cardiac levels of leptin, collagen I, galectin-3 and transforming growth factor ß (TGF-ß) were higher in HFD than in controls. In cardiac myofibroblasts, leptin (10-100 ng/ml) increased O2-, collagen I, galectin-3, TGF-ß and connective tissue growth factor production (CTGF). These effects were prevented by the presence of either melatonin (10 mmol/l) or the inhibitor of mTOR, rapamycin (10 mmol/l). Blockage of galectin-3 activity by N-acetyllactosamine (LacNac 10 mmol/l) reduced both collagen I and O2(*-) production induced by leptin. The p70S6 kinase activation/phosphorylation, the downstream mediator of mTOR, induced by leptin was not modified by melatonin. Leptin reduced the metalloproteinase (MMP) 2 activity and the presence of melatonin, rapamycin or LacNac were unable to prevent it. CONCLUSION: The data suggest that leptin locally produced in the heart could participate in the fibrosis observed in HFD by affecting collagen turnover. Collagen synthesis induced by leptin seems to be mediated by the production of galectin-3, TGF-ß and CTGF through oxidative stress increased by activation of mTOR pathway.

DIOL triterpenes block profibrotic effects of angiotensin II and protect from cardiac hypertrophy.

BACKGROUND: The natural triterpenes, erythrodiol and uvaol, exert anti-inflammatory, vasorelaxing and anti-proliferative effects. Angiotensin II is a well-known profibrotic and proliferative agent that participates in the cardiac remodeling associated with different pathological situations through the stimulation and proliferation of cardiac fibroblasts. Therefore, the aim of the study was to investigate the preventive effects of the natural triterpenes erythrodiol and uvaol on the proliferation and collagen production induced by angiotensin II in cardiac myofibroblasts. Their actions on cardiac hypertrophy triggered by angiotensin II were also studied. METHODOLOGY/PRINCIPAL FINDINGS: The effect of erythrodiol and uvaol on angiotensin II-induced proliferation was evaluated in cardiac myofibroblasts from adult rats in the presence or the absence of the inhibitors of PPAR-γ, GW9662 or JNK, SP600125. The effect on collagen levels induced by angiotensin II was evaluated in cardiac myofibroblasts and mouse heart. The presence of low doses of both triterpenes reduced the proliferation of cardiac myofibroblasts induced by angiotensin II. Pretreatment with GW9662 reversed the effect elicited by both triterpenes while SP600125 did not modify it. Both triterpenes at high doses produced an increase in annexing-V binding in the presence or absence of angiotensin II, which was reduced by either SP600125 or GW9662. Erythrodiol and uvaol decreased collagen I and galectin 3 levels induced by angiotensin II in cardiac myofribroblasts. Finally, cardiac hypertrophy, ventricular remodeling, fibrosis, and increases in myocyte area and brain natriuretic peptide levels observed in angiotensin II-infused mice were reduced in triterpene-treated animals. CONCLUSIONS/SIGNIFICANCE: Erythrodiol and uvaol reduce cardiac hypertrophy and left ventricle remodeling induced by angiotensin II in mice by diminishing fibrosis and myocyte area. They also modulate growth and survival of cardiac myofibroblasts. They inhibit the angiotensin II-induced proliferation in a PPAR-γ-dependent manner, while at high doses they activate pathways of programmed cell death that are dependent on JNK and PPAR-γ.

Effects of aging on C57BL/6J mice: an electrophysiological and morphological study.

Presbycusis is a progressive hearing loss associated with aging that manifests as deafness linked to cochlear morphological degeneration. The effects of aging on the auditory system were studied in C57BL/6J mice using electrophysiological (brainstem auditory evoked potentials; BAEP) and morphological techniques. Cochleae of animals aged 1, 6, 9, 12, 15, 18, 21, or 24 months old were used for that purpose. The BAEP showed a progressive increase in latency and a reduction in amplitude. Morphological studies demonstrated total degeneration of the organ of Corti, which was replaced by a single epithelial layer. An affinity histochemistry study demonstrated minor modifications of glycoconjugates in the organ of Corti during the aging process.