Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1.

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.

Up-regulation of c-jun mRNA in cardiac myocytes requires the extracellular signal-regulated kinase cascade, but c-Jun N-terminal kinases are required for efficient up-regulation of c-Jun protein.

Cardiac hypertrophy, an important adaptational response, is associated with up-regulation of the immediate early gene, c- jun, which encodes the c-Jun transcription factor. c-Jun may feed back to up-regulate its own transcription and, since the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) phosphorylate c-Jun(Ser-63/73) to increase its transactivating activity, JNKs are thought to be the principal factors involved in c- jun up-regulation. Hypertrophy in primary cultures of cardiac myocytes is induced by endothelin-1, phenylephrine or PMA, probably through activation of one or more of the MAPK family. These three agonists increased c- jun mRNA with the rank order of potency of PMA approximately endothelin-1>phenylephrine. Up-regulation of c- jun mRNA by endothelin-1 was attenuated by inhibitors of protein kinase C (GF109203X) and the extracellular signal-regulated kinase (ERK) cascade (PD98059 or U0126), but not by inhibitors of the JNK (SP600125) or p38-MAPK (SB203580) cascades. Hyperosmotic shock (0.5 M sorbitol) powerfully activates JNKs, but did not increase c- jun mRNA. These data suggest that ERKs, rather than JNKs, are required for c- jun up-regulation. However, endothelin-1 and phenylephrine induced greater up-regulation of c-Jun protein than PMA and phosphorylation of c-Jun(Ser-63/73) correlated with the level of c-Jun protein. Up-regulation of c-Jun protein by endothelin-1 was attenuated by inhibitors of protein kinase C and the ERK cascade, probably correlating with a primary input of ERKs into transcription. In addition, SP600125 inhibited the phosphorylation of c-Jun(Ser-63/73), attenuated the increase in c-Jun protein induced by endothelin-1 and increased the rate of c-Jun degradation. Thus whereas ERKs are the principal MAPKs required for c- jun transcription, JNKs are necessary to stabilize c-Jun for efficient up-regulation of the protein.

Endothelin-1 promotes phosphorylation of CREB transcription factor in primary cultures of neonatal rat cardiac myocytes: implications for the regulation of c-jun expression.

Cardiac myocyte hypertrophy is associated with an increase in expression of immediate early genes (e.g. c-jun) via activation of pre-existing transcription factors. The activity of CREB transcription factor is regulated through phosphorylation of Ser-133 by one of several protein kinases (e.g. protein kinase A (PKA), p90 ribosomal S6 kinases (RSKs) and the related kinase, MSK1). A cell-permeable form of cAMP, hypertrophic agonists (endothelin-1 (ET-1), phenylephrine (PE)) and hyperosmotic shock all promoted phosphorylation of CREB(Ser-133) in rat neonatal cardiac myocytes. The response to endothelin-1 required the extracellular signal-regulated kinase cascade which stimulates both RSKs and MSK1. Phosphorylation of CREB(Ser-133) in response to ET-1 was not associated with any increase in DNA binding to a consensus cAMP-response element (CRE). The rat c-jun promoter contains elements which may bind either c-Jun/ATF2 or CREB/ATF1 dimers. Using extracts from rat cardiac myocytes, we identified at least two complexes which bind to the most proximal of these elements, one of which contained CREB and the other c-Jun. Thus, phosphorylation and activation of CREB in cardiac myocytes may be effected by a range of different stimuli to influence the expression of immediate early genes such as c-jun.

Phenylephrine and endothelin-1 upregulate connective tissue growth factor in neonatal rat cardiac myocytes.

Cardiac hypertrophy is associated with hypertrophic growth of cardiac myocytes and increased fibrosis. Much is known of the stimuli which promote myocyte hypertrophy and the changes associated with the response, but the links between the two are largely unknown. Using subtractive hybridization, we identified three genes which are acutely (<1 h) upregulated in neonatal rat ventricular myocytes exposed to the alpha-adrenergic agonist, phenylephrine. One represented connective tissue growth factor (CTGF) which is implicated in fibrosis and promotes hypertrophy in other cells. We further examined the expression of CTGF mRNA and protein in cardiac myocytes using quantitative PCR and immunoblotting, confirming that phenylephrine increased CTGF mRNA (maximal within 1 h) and protein (increased over 4 - 24 h). Endothelin-1 promoted a greater, though transient, increase in CTGF mRNA, but the increase in CTGF protein was sustained over 8 h. Neither agonist increased CTGF mRNA in cardiac non-myocytes. By increasing the expression of CTGF in cardiac myocytes, hypertrophic agonists such as phenylephrine and endothelin-1 may promote fibrosis. CTGF may also propagate the hypertrophic response initiated by these agonists.

Using U0126 to dissect the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade in the regulation of gene expression by endothelin-1 in cardiac myocytes

The hypertrophic agonist endothelin-1 rapidly but transiently activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade (and other signalling pathways) in cardiac myocytes, but the events linking this to hypertrophy are not understood. Using Affymetrix rat U34A microarrays, we identified the short-term (2-4 h) changes in gene expression induced in neonatal myocytes by endothelin-1 alone or in combination with the ERK1/2 cascade inhibitor, U0126. Expression of 15 genes was significantly changed by U0126 alone, and expression of an additional 78 genes was significantly changed by endothelin-1. Of the genes upregulated by U0126, four are classically induced through the aryl hydrocarbon receptor (AhR) by dioxins suggesting that U0126 activates the xenobiotic response element in cardiac myocytes potentially independently of effects on ERK1/2 signalling. The 78 genes showing altered expression with endothelin-1 formed five clusters: (i) three clusters showing upregulation by endothelin-1 according to time course (4 h > 2 h; 2 h > 4 h; 2 h approximately 4 h) with at least partial inhibition by U0126; (ii) a cluster of 11 genes upregulated by endothelin-1 but unaffected by U0126 suggesting regulation through signalling pathways other than ERK1/2; (iii) a cluster of six genes downregulated by endothelin-1 with attenuation by U0126. Thus, U0126 apparently activates the AhR in cardiac myocytes (which must be taken into account in protracted studies), but careful analysis allows identification of genes potentially regulated acutely via the ERK1/2 cascade. Our data suggest that the majority of changes in gene expression induced by endothelin-1 are mediated by the ERK1/2 cascade.

Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure

Impaired mechanosensing leads to heart failure and we have previously shown that a decreased ratio of cytoplasmic to nuclear CSRP3/Muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. Here we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 μM blebbistatin resulted in a ∼3 fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signaling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme-oxygenase1 (HO-1) activity with PPZII blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea pig model of angiotensin II infusion (400 ng/kg/min) over 12 weeks. Using subcellular fractionation we showed that the MLP ratio increased 88% (n = 4, P < 0.01) during compensated hypertrophy, but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01 n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO-1 and HDAC signaling.

Diabetes mellitus-related morphoquantitative changes in the celiac ganglion neurons of the dog

Diabetes mellitus is the most common endocrine disturbance of domestic carnivores and can cause autonomic neurological disorders, although these are still poorly understood in veterinary medicine. There is little information available on the quantitative adaptation mechanisms of the sympathetic ganglia during diabetes mellitus in domestic mammals. By combining morphometric methods and NADPH-diaphorase staining (as a possible marker for nitric oxide producing neurons), type I diabetes mellitus-related morphoquantitative changes were investigated in the celiac ganglion neurons in dogs. Twelve left celiac ganglia from adult female German shepherd dogs were examined: six ganglia were from non-diabetic and six from diabetic subjects. Consistent hypertrophy of the ganglia was noted in diabetic animals with increase of 55% in length, 53% in width, and 61.5% in thickness. The ordinary microstructure of the ganglia was modified leading to an uneven distribution of the ganglionic units and a more evident distribution of axon fascicles. In contrast to non-diabetic dogs, there was a lack of NADPH-diaphorase perikarial labelling in the celiac ganglion neurons of diabetic animals. The morphometric study showed that both the neuronal and nuclear sizes were significantly larger in diabetic dogs (1.3 and 1.39 times, respectively). The profile density and area fraction of NADPH-diaphorase-reactive celiac ganglion neurons were significantly larger (1.35 and 1.48 times, respectively) in non-diabetic dogs compared to NADPH-diaphorase-non-reactive celiac ganglion neurons in diabetic dogs. Although this study suggests that diabetic neuropathy is associated with neuronal hypertrophy, controversy remains over the possibility of ongoing neuronal loss and the functional interrelationship between them. It is unclear whether neuronal hypertrophy could be a compensation mechanism for a putative neuronal loss during the diabetes mellitus. (C) 2007 Elsevier Ltd. All rights reserved.

Lifetime Overproduction of Circulating Angiotensin-(1-7) Attenuates Deoxycorticosterone Acetate-Salt Hypertension-Induced Cardiac Dysfunction and Remodeling

We evaluated the development of arterial hypertension, cardiac function, and collagen deposition, as well as the level of components of the renin-angiotensin system in the heart of transgenic rats that overexpress an angiotensin (Ang)-(1-7)-producing fusion protein, TGR(A1-7)3292 (TG), which induces a lifetime increase in circulating levels of this peptide. After 30 days of the induction of the deoxycorticosterone acetate (DOCA)-salt hypertension model, DOCA-TG rats were hypertensive but presented a lower systolic arterial pressure in comparison with DOCA-Sprague-Dawley (SD) rats. In contrast to DOCA-SD rats that presented left ventricle (LV) hypertrophy and diastolic dysfunction, DOCA-TG rats did not develop cardiac hypertrophy or changes in ventricular function. In addition, DOCA-TG rats showed attenuation in mRNA expression for collagen type I and III compared with the increased levels of DOCA-SD rats. Ang II plasma and LV levels were reduced in SD and TG hypertensive rats in comparison with normotensive animals. DOCA-TG rats presented a reduction in plasma Ang-(1-7) levels; however, there was a great increase in Ang-(1-7) (approximate to 3-fold) accompanied by a decrease in mRNA expression of both angiotensin-converting enzyme and angiotensin-converting enzyme 2 in the LV. The mRNA expression of Mas and Ang II type 1 receptors in the LV was not significantly changed in DOCA-SD or DOCA-TG rats. This study showed that TG rats with increased circulating levels of Ang-(1-7) are protected against cardiac dysfunction and fibrosis and also present an attenuated increase in blood pressure after DOCA-salt hypertension. In addition, DOCA-TG rats showed an important local increase in Ang-(1-7) levels in the LV, which might have contributed to the attenuation of cardiac dysfunction and prefibrotic lesions. (Hypertension. 2010;55:889-896.)

Ace gene dosage influences the development of renovascular hypertension

P>1. Clinical and experimental evidence highlights the importance of the renin-angiotensin system in renovascular hypertension. Furthermore, genetic factors affecting angiotensin-converting enzyme (ACE) could influence the development of renovascular hypertension. 2. To test the effect of small gene perturbations on the development of renovascular hypertension, mice harbouring two or three copies of the Ace gene were submitted to 4 weeks of two-kidney, one-clip (2K1C) hypertension. Blood pressure (BP), cardiac hypertrophy, baroreflex sensitivity and blood pressure and heart rate variability were assessed and compared between the different groups. 3. The increase in BP induced by 2K1C was higher in mice with three copies of the Ace gene compared with mice with only two copies (46 vs 23 mmHg, respectively). Moreover, there was a 3.8-fold increase in the slope of the left ventricle mass/BP relationship in mice with three copies of the Ace gene. Micewith three copies of the Ace gene exhibited greater increases in cardiac and serum ACE activity than mice with only two copies of the gene. Both baroreflex bradycardia and tachycardia were significantly depressed in mice with three copies of the Ace gene after induction of 2K1C hypertension. The variance in basal systolic BP was greater in mice with three copies of the Ace gene after 2K1C hypertension compared with those with only two copies of the gene (106 vs 54%, respectively). In addition, the low-frequency component of the pulse interval was higher mice with three copies of the Ace gene after 2K1C hypertension compared with those with only two (168 vs 86%, respectively). Finally, in mice with three copies of the Ace gene, renovascular hypertension induced a 6.1-fold increase in the sympathovagal balance compared with a 3.2-fold increase in mice with only two copies of the gene. 4. Collectively, these data provide direct evidence that small genetic disturbances in ACE levels per se have an influence on haemodynamic, cardiac mass and autonomic nervous system responses in mice under pathological perturbation.

Homocysteine Thiolactone Induces Cardiac Dysfunction: Role of Oxidative Stress

This study investigates the cardiac functioning in male Wistar rats after treatments with methionine and homocysteine thiolactone (HcyT). The rats were distributed into 3 groups and treated for 8 weeks. Group I was the control (CO) group, given water, group II was treated with methionine, and group III with HcyT (100 mg/kg). Morphometric and functional cardiac parameters were evaluated by echocardiography. Superoxide dismutase (SOD), catalase, and glutathione S-transferase activities, chemiluminescence, thiobarbituric acid reactive substances, and immunocontent were measured in the myocardium. Hyperhomocysteinemia was observed in rats submitted to the both treatments. The results showed diastolic function was compromised in HcyT group, seen by the increase of E/A (peak velocity of early (E) and late (A) diastolic filling) ratio, decrease in deceleration time of E wave and left ventricular isovolumic relaxation time. Myocardial performance index was increased in HcyT group and was found associated with increased SOD immunocontent. HcyT group demonstrated an increase in SOD, catalase, and glutatione S-transferase activity, and chemiluminescence and thiobarbituric acid reactive substances. Overall, these results indicated that HcyT induces a cardiac dysfunction and could be associated with oxidative stress increase in the myocardium.

Expression of Circadian Clock and Melatonin Receptors within Cultured Rat Cardiomyocytes

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demostrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor ROR alpha have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and ROR alpha and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORa were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erb alpha expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erb alpha mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erb alpha mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity. (Author correspondence: cipolla@icb.usp.br)

Thyroid hormones alter the adenine nucleotide hydrolysis in adult rat blood serum

The effects of ATP, ADP, and adenosine in the processes of platelet aggregation, vasodilatation, and coronary flow have been known for many years. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes the main system for rapid inactivation of circulating adenine nucleotides. Thyroid disorders affect a number of biological factors including adenosine levels in different fractions. Then, we intend to investigate if the soluble nucleotidases responsible for the ATP, ADP, and AMP hydrolysis are affected by variations in the thyroid hormone levels in blood serum from adult rats. Hyperthyroidism was induced by daily intraperitoneal injections of L-thyroxine (T4) (2.5 and 10.0 mu g/100 g body weight, respectively) for 7 or 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water during 7 or 14 days. The treatments efficacy was confirmed by determination of hemodynamic parameters and cardiac hypertrophy evaluation. T4 treatment predominantly inhibited, and hypothyroidism (14 days after thyroidectomy) predominantly increased the ATP, ADP, and AMP hydrolysis in rat blood serum. These results suggest that both excess and deficiency of thyroid hormones can modulate the ATP diphosphohydrolase and 5`-nucleotidase activities in rat blood serum and consequently modulate the effects mediated by these enzymes and their products in vascular system. (C) 2010 International Union of Biochemistry and Molecular Biology, Inc.

Redox-sensitive prosurvival and proapoptotic protein expression in the myocardial remodeling post-infarction in rats

In this study, we investigated the oxidative stress influence in some prosurvival and proapoptotic proteins after myocardial infarction (MI). Male Wistar rats were divided in two groups: Sham-operated (control) and MI. MI was induced by left coronary artery occlusion. 28-days after surgery, echocardiographic, morphometric, and hemodynamic parameters were evaluated. Redox status (reduced to oxidized glutathione ratio, GSH/GSSG) and hydrogen peroxide levels (H(2)O(2)) were measured in heart tissue. The p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK-3 beta/GSK-3 beta ratios, as well as apoptosis-inducing factor (AIF) myocardial protein expression were quantified by Western blot. MI group showed an increase in cardiac hypertrophy (23%) associated with a decrease in ejection fraction (38%) and increase in left ventricular end-diastolic pressure (82%) when compared to control, characterizing ventricular dysfunction. Redox status imbalance was seen in MI animals, as evidenced by the decrease in the GSH/GSSG ratio (30%) and increased levels of H(2)O(2) (45%). This group also showed an increase in the ERK phosphorylation and a reduction of Akt and mTOR phosphorylation when compared to control. Moreover, we showed a reduction in the GSK-3 beta phosphorylation and an increase in AIF protein expression in MI group. Taken together, our results show increased H(2)O(2) levels and cellular redox imbalance associated to a higher p-ERK and AIF immunocontent, which would contribute to a maladaptive hypertrophy phenotype.

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

Aims Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. Methods and results NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO(2)) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 mu M). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. Conclusion Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Chronic resistance training decreases MuRF-1 and Atrogin-1 gene expression but does not modify Akt, GSK-3 beta and p70S6K levels in rats

Long-term adaptation to resistance training is probably due to the cumulative molecular effects of each exercise session. Therefore, we studied in female Wistar rats the molecular effects of a chronic resistance training regimen (3 months) leading to skeletal muscle hypertrophy in the plantaris muscle. Our results demonstrated that muscle proteolytic genes MuRF-1 and Atrogin-1 were significantly decreased in the exercised group measured 24 h after the last resistance exercise session (41.64 and 61.19%, respectively; P < 0.05). Nonetheless, when measured at the same time point, 4EBP-1, GSK-3 beta and eIF2B epsilon mRNA levels and Akt, GSK-3 beta and p70S6K protein levels (regulators of translation initiation) were not modified. Such data suggests that if gene transcription constitutes a control point in the protein synthesis pathway this regulation probably occurs in early adaptation periods or during extreme situations leading to skeletal muscle remodeling. However, proteolytic gene expression is modified even after a prolonged resistance training regimen leading to moderate skeletal muscle hypertrophy.