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.

Ras: the stress and the strain

The last 10–15 years have seen an expansion in the understanding of the intracellular signalling pathways activated in cardiac myocytes in response to hypertrophic or lethal stimuli. The mitogen-activated protein kinases (MAPKs) were identified as potential key mediators of cardiac myocyte responses in the early to mid-1990's, with the extracellular signal-regulated kinases 1/2 (ERK1/2) being potently activated by heterotrimeric Gq protein-coupled receptor (GqPCR) agonists, and the c-Jun N-terminal kinases (JNKs) and p38-MAPKs being potently activated by cell stresses.

Inflame my heart (by p38-MAPK).

Although many studies have explored the stimuli which promote hypertrophic growth or death in cardiac myocytes and the signaling pathways which they activate, the mechanisms by which these pathways promote the pathophysiological responses are still obscure. The mitogen-activated protein kinase (MAPK) cascades (in which MAPKs are phosphorylated and activated by upstream MAPK kinases [MKKs] which are, in turn, phosphorylated and activated by MKK kinases [MKKKs]) were identified in the early- to mid-1990s as potentially key regulatory pathways in cardiac myocyte pathophysiology.1,2 The principal MAPKs investigated in cardiac myocytes are the extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs), and p38-MAPKs. ERK1/2 are potently activated by hypertrophic stimuli, whereas JNKs and p38-MAPKs are potently activated by cellular stresses (eg, oxidative stress). However, there is cross-talk such that JNKs and p38-MAPKs are activated by hypertrophic stimuli and ERK1/2 are activated by cellular stresses, and the contribution of each pathway to the overall cardiac myocyte response is not entirely clear. MAPKs phosphorylate a number of known transcription factors to alter their transactivating activities thus, presumably, influencing gene expression to elicit the cellular response.3 Nevertheless, the immediate consequences (ie, the transcription factors which are phosphorylated) and downstream consequences (ie, genes with altered expression) of MAPK signaling in the heart or specifically in cardiac myocytes are still largely unknown. To start to address this issue for the p38-MAPK pathway in the (rat) heart (Figure), Tenhunen et al4 directly injected adenoviruses encoding wild-type (WT) p38-MAPKα together …

Oxidative stress and growth-regulating intracellular signaling pathways in cardiac myocytes

The toxic effects of oxidative stress on cells (including cardiac myocytes, the contractile cells of the heart) are well known. However, an increasing body of evidence has suggested that increased production of reactive oxygen species (ROS) promotes cardiac myocyte growth. Thus, ROS may be 'second messenger' molecules in their own right, and growth-promoting neurohumoral agonists might exert their effects by stimulating production of ROS. The authors review the principal growth-promoting intracellular signaling pathways that are activated by ROS in cardiac myocytes, namely the mitogen-activated protein kinase cascades (extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinases, and p38-mitogen-activated protein kinases) and the phosphoinositide 3-kinase/protein kinase B (Akt) pathway. Possible mechanisms are discussed by which these pathways are activated by ROS, including the oxidation of active site cysteinyl residues of protein and lipid phosphatases with their consequent inactivation, the potential involvement of protein kinase C or the apoptosis signal-regulating kinase 1, and the current models for the activation of the guanine nucleotide binding protein Ras.

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.

Tobacco smoke-induced left ventricular remodelling is not associated with metalloproteinase-2 or -9 activation

Aim: To investigate the role of MMP-2 and MMP-9 in cardiac remodelling induced by tobacco smoke exposure in rats.Methods: Rats were allocated into two groups: C (n = 9): control animals; ETS (n = 9): exposed to tobacco smoke. After 4months, the animals underwent echocardiography, morphometric study and determination of MMP-2 and MMP-9 activity.Results: ETS rats had larger diastolic (C= 15.6 +/- 1.2 mm/kg, ETS = 18.0 +/- 0.9 mm/kg; p < 0.001) and systolic (C= 7.3 +/- 1.2 mm/kg, ETS = 9.2 0.9 mm/kg; p = 0.001) ventricular diameters adjusted for body weight. Fractional shortening (C= 53 +/- 4.8%, ETS = 48 +/- 3.3%; p = 0.031) and ejection fraction (C= 0. 89 +/- 0.03 5 ETS = 0. 86 +/- 0.02; p = 0.03 0) were smaller in the ETS group. Myocyte cross-sectional area (C= 245 8 mu m(2), ETS=253 8 mu m(2); p = 0.028) was higher in ETS rats. There were no differences in MNtP-2 (C=50 +/- 14%; ETS 43 +/- 11%, p 0.22 +/- 8) or MMP-9 (C=0.36 +/- 0.3%; ETS=0.62 +/- 0.3%, p=0.630) activity between the groups.Conclusion: MMP-2 and MMP-9 did not participate in the remodelling process induced by tobacco smoke exposure. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Myocardial remodeling and dysfunction are induced by chronic food restriction in spontaneously hypertensive rats

Several studies have shown alterations in hearts from animals subjected to food restriction (FR). However, few experiments in hearts evaluating pressure overload have been reported. We examined the effects of chronic FR on myocardial function and morphology in spontaneously hypertensive rats (SHR). Sixty-day-old SHR were fed a control (C) or a restricted diet (daily intake reduced to 50% of amount of food consumed by the control group) for 90 days. Myocardial performance was studied in isolated left ventricular (LV) papillary muscle. Food restriction decreased body weight and LV weight; LV weight/body-weight ratio was lower in the food-restricted group (SHR-C, 2.84 +/- 0.21 mg/g; SHR-FR, 2.56 +/- 0.24 mg/g; P <.05). Food restriction did not change arterial systolic blood pressure. Myocyte surface area was lower in the food-restricted group (P <.01). Food restriction induced myocardial ultrastructural alterations including reduced sarcoplasm content, reduced and disorganized myofilaments, disorganized Z line, dilated sarcoplasmic reticulum, and deep infoldings of plasma membrane. Myocardial hydroxyproline concentration was increased in the restricted rats. Peak developed tension (P <.05) and maximum rate of tension development (P <.01) were decreased in the SHR-FR group. In conclusion, myocardium of SHR subjected to chronic FR presents attenuation of hypertrophy development, ultrastructural changes, increased collagen content, and systolic dysfunction. (c) 2006 Elsevier B.V. All rights reserved.

Myocardial fibrosis rather than hypertrophy induces diastolic dysfunction in renovascular hypertensive rats

The aim of the present study was to evaluate the-effect of interstitial fibrosis alone or associated with hypertrophy. on diastolic myocardial function in renovascular hypertensive rats. Myocardial function was evaluated in isolated papillary muscle from renovascular hypertensive Wistar rats (RHT, n = 14), renovascular hypertensive rats treated with the angiotensin converting enzyme inhibitor (ACEI) ramipril, 20 mg.kg(-1).day(-1) (RHT RAM, n = 14), and age-matched unoperated and untreated Wistar rats (CONT, n = 12). The ACEI treatment for 3 weeks allowed the regression of myocyte mass and the maintenance of interstitial fibrosis. Myocardial passive stiffness was analyzed by the resting tension - length relationship. The myocardial fibrosis was evaluated by measuring myocardial hydroxyproline (Hyp) concentration and by histological studies of the myocardium stained with hematoxylin and eosin or picrosirius red. Left ventricular weight was significantly higher in RHT (0.97 +/- 0.12 g) compared with CONT (0.66 +/- 0.06 g) and RHT RAM (0.69 +/- 0.14 g). The Hyp levels were 2.9 +/- 0.4, 3.4 +/- 0.3, and 3.8 +/- 0.4 mu g/mg of dry tissue for the CONT, RHT, and RHT RAM, respectively. Perivascular and interstitial fibrosis were observed in RHT and RHT RAM groups. There were lymphomononuclear inflammatory exudate and edema around arteries, involving adjacent myocytes in the RHT group. There was an increased passive stiffness in RHT and RHT RAM groups compared with the CONT group. In conclusion, our results indicate that the Impaired diastolic function in the renovascular hypertensive rats is related to interstitial fibrosis rather than to myocardial hypertrophy.

Alterations in myocardial collagen content affect rat papillary muscle function

We investigated the influence of myocardial collagen volume fraction (CVF, %) and hydroxyproline concentration (mu g/mg) on rat papillary muscle function. Collagen excess was obtained in 10 rats with unilateral renal ischemia for 5 wk followed by 3-wk treatment with ramipril (20 mg . kg(-1) . day(-1)) (RHTR rats; CVF = 3.83 +/- 0.80, hydroxyproline = 3.79 +/- 0.50). Collagen degradation was induced by double infusion of oxidized glutathione (GSSG rats; CVF 5 2.45 +/- 0.52, hydroxyproline = 2.85 +/- 0.18). Nine untreated rats were used as controls (CFV = 3.04 +/- 0.58, hydroxyproline = 3.21 +/- 0.30). Active stiffness (AS; g . cm(-2) . %L-max(-1)) and myocyte cross-sectional area (MA; mu m(2)) were increased in the GSSG rats compared with controls [AS 5.86 vs. 3.96 (P< 0.05); MA 363 +/- 59 vs. 305 +/- 28 (P< 0.05)]. In GSSG and RHTR groups the passive tension-length curves were shifted downwards, indicating decreased passive stiffness, and upwards, indicating increased passive stiffness, respectively. Decreased collagen content induced by GSSG is related to myocyte hypertrophy, decreased passive stiffness, and increased AS, and increased collagen concentration causes myocardial diastolic dysfunction with no effect on systolic function.

Miocardiopatia diabética

A miocardiopatia diabética é uma doença do músculo cardíaco causada pelo diabetes mellitus e não relacionada às patologias vascular e valvular ou à hipertensão arterial sistêmica. Observações experimentais e clínicas têm demonstrado hipertrofia, necrose, apoptose e aumento do tecido intersticial miocárdico. Acredita-se que a miocardiopatia diabética seja decorrente de anormalidades metabólicas como hiperlipidemia, hiperinsulinemia e hiperglicemia, e de alterações do metabolismo cardíaco. Tais alterações podem causar aumento do estresse oxidativo, fibrose intersticial, perda celular e comprometimento do trânsito intracelular de íons e da homeostase do cálcio. Clinicamente, é possível a detecção de disfunção diastólica assintomática na fase inicial. No momento em que surgem os sinais e sintomas de insuficiência cardíaca, observamos disfunção diastólica isolada, sendo que o comprometimento da função sistólica, habitualmente, é tardio. O tratamento da miocardiopatia diabética com insuficiência cardíaca não difere das miocardiopatias de outras etiologias e deve seguir as diretrizes de acordo com o comprometimento da função ventricular, se diastólica isolada ou diastólica e sistólica.

Remodelação miocárdica após infarto agudo do miocárdio experimental em ratos: efeito do bloqueio do sistema renina angiotensina aldosterona

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A exposição à fumaça de cigarro intensifica a remodelação ventricular após o infarto agudo do miocárdio

OBJETIVO: Analisar os efeitos da exposição à fumaça de cigarro (EFC) na remodelação ventricular após o infarto agudo do miocárdio (IAM). MÉTODOS: Ratos foram infartados e distribuídos em dois grupos: C (controle, n = 31) e F (EFC: 40 cigarros/dia, n = 22). Após seis meses, foi realizado ecocardiograma, estudo funcional com coração isolado e morfometria. Para comparação, foi utilizado o teste t (com média ± desvio padrão) ou teste de Mann-Whitney (com mediana e percentis 25 e 75). RESULTADOS: Os animais EFC apresentaram tendência a maiores áreas ventriculares diastólicas (C = 1,5 ± 0,4 mm², F = 1,9 ± 0,4 mm²; p = 0,08) e sistólicas (C = 1,05 ± 0,3 mm², F = 1,32 ± 0,4 mm²; p = 0,08) do VE. A função sistólica do VE, avaliada pela fração de variação de área, tendeu a ser menor nos animais EFC (C = 31,9 ± 9,3 %, F = 25,5 ± 7,6 %; p = 0,08). Os valores da - dp/dt dos animais EFC foram estatisticamente inferiores (C = 1474 ± 397 mmHg, F = 916 ± 261 mmHg; p = 0,02) aos animais-controle. Os animais EFC apresentaram maior peso do VD, ajustado ao peso corporal (C = 0,8 ± 0,3 mg/g, F = 1,3 ± 0,4 mg/g; p = 0,01), maior teor de água nos pulmões (C = 4,8 (4,3-4,8)%, F = 5,4 (5,1-5,5); p = 0,03) e maior área seccional do miócito do VE (C = 239,8 ± 5,8 µm², F = 253,9 ± 7,9 µm²; p = 0,01). CONCLUSÃO: A exposição à fumaça de cigarro intensifica a remodelação ventricular após IAM.

Efeitos do betacaroteno e do tabagismo sobre a remodelação cardíaca pós-infarto do miocárdio

OBJETIVO: Analisar os efeitos do betacaroteno no processo de remodelação ventricular após o infarto agudo do miocárdio (IAM), em ratos expostos à fumaça do cigarro. MÉTODOS: Após o IAM, os animais foram divididos em quatro grupos: 1) grupo C, 24 animais que receberam dieta-padrão; 2) grupo BC, 26 animais que receberam betacaroteno; 3) grupo EFC, 26 animais que receberam dieta-padrão e foram expostos à fumaça de cigarro; e 4) grupo BC+EFC, 20 animais que receberam betacaroteno e foram expostos à fumaça de cigarro. Após seis meses, foi realizado estudo morfofuncional. Utilizou-se significância de 5%. RESULTADOS: em relação às áreas diastólicas (AD) e sistólicas (AS), os valores do grupo BC foram maiores que os do grupo C. Considerando a AD/peso corporal (PC) e AS/PC, os valores do grupo BC+EFC foram maiores que os valores de C. em relação à fração de variação de área, foram observadas diferenças significativas entre EFC (valores menores) e C (valores maiores) e entre BC (valores menores) e C (valores maiores). Não foram observadas diferenças entre os grupos em relação ao tamanho do infarto. O grupo EFC apresentou valores maiores da área seccional dos miócitos (ASM) que os animais-controle. em adição, o grupo BC+EFC apresentou maiores valores de ASM que BC, EFC e C. CONCLUSÃO: Após o infarto do miocárdio, o tabagismo e o betacaroteno promoveram intensificação do processo de remodelação cardíaca; houve potencialização dos efeitos deletérios no processo de remodelação com os dois tratamentos em conjunto.

O bloqueio do sistema renina-angiotensina atenua a remodelação cardíaca de ratos submetidos a estenose aórtica

OBJETIVO: Avaliar o papel do bloqueador dos receptores AT1 e do inibidor da enzima conversora da angiotensina na remodelação cardíaca induzida por estenose aórtica em ratos. MÉTODOS: Ratos Wistar foram divididos em 4 grupos: controle (C, n=13), estenose aórtica (EAo, n=11), EAo com lisinopril, 20 mg/kg/dia (LIS, n=11) e EAo com losartan, 40 mg/kg/dia (LOS, n=9). Os tratamentos foram iniciados 3 dias antes da cirurgia. Após 6 semanas, os animais foram submetidos ao estudo ecocardiográfico, quantificação da concentração de hidroxiprolina e da área seccional (CSA) miocitária do ventrículo esquerdo (VE). RESULTADOS: A EAo induziu aumento da espessura da parede do VE. Os animais LIS e LOS não apresentaram diferença em relação aos animais controles. Os ratos EAo e LIS apresentaram maiores diâmetros do átrio esquerdo que os ratos controles, enquanto nos animais LOS não houve diferença. Os animais com EAo apresentaram maiores valores da porcentagem de encurtamento que os controle. Esse fato não foi modificado com LIS ou LOS. A CSA dos animais do grupo EAo foi maior que a dos controle. Entretanto, o tratamento com LOS e com LIS atenuou o aumento da área induzida pela EAo. A EAo resultou em aumento na concentração de HOP, enquanto o grupo LOS não apresentou diferença em relação ao grupo controle. CONCLUSÃO: O bloqueio do sistema renina-angiotensina, com bloqueador AT1 e com IECA, pode atenuar o desenvolvimento de hipertrofia cardíaca, porém só o bloqueio dos receptores AT1 atenua a fibrose intersticial do VE.

Follow-up study of morphology and cardiac function in rats undergoing induction of supravalvular aortic stenosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)