Differential regulation of collagen secretion by kinin receptors in cardiac fibroblast and myofibroblast.

UNLABELLED: Kinins mediate their cellular effects through B1 (B1R) and B2 (B2R) receptors, and the activation of B2R reduces collagen synthesis in cardiac fibroblasts (CF). However, the question of whether B1R and/or B2R have a role in cardiac myofibroblasts remains unanswered. METHODS: CF were isolated from neonate rats and myofibroblasts were generated by an 84 h treatment with TGF-ß1 (CMF). B1R was evaluated by western blot, immunocytochemistry and radioligand assay; B2R, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and cyclooxygenases 1 and 2 (COX-1, and COX-2) were evaluated by western blot; intracellular Ca⁺² levels were evaluated with Fluo-4AM; collagen secretion was measured in the culture media using the picrosirius red assay kit. RESULTS: B2R, iNOS, COX-1 and low levels of B1R but not eNOS, were detected by western blot in CF. Also, B1R, B2R, and COX-2 but not iNOS, eNOS or COX-1, were detected by western blot in CMF. By immunocytochemistry, our results showed lower intracellular B1R levels in CF and higher B1R levels in CMF, mainly localized on the cell membrane. Additionally, we found B1R only in CMF cellular membrane through radioligand displacement assay. Bradykinin (BK) B2R agonist increased intracellular Ca²âº levels and reduced collagen secretion both in CF and CMF. These effects were blocked by HOE-140, and inhibited by L-NAME, 1400 W and indomethacin. Des-Arg-kallidin (DAKD) B1R agonist did not increase intracellular Ca²âº levels in CF; however, after preincubation for 1h with DAKD and re-stimulation with the same agonist, we found a low increase in intracellular Ca²âº levels. Finally, DAKD increased intracellular Ca²âº levels and decreased collagen secretion in CMF, being this effect blocked by the B1R antagonist des-Arg9-Leu8-kallidin and indomethacin, but not by L-NAME or 1400 W. CONCLUSION: B1R, B2R, iNOS and COX-1 were expressed differently between CF and CMF, and collagen secretion was regulated differentially by kinin receptor agonists in cultured CF and CMF.

Simvastatin disrupts cytoskeleton and decreases cardiac fibroblast adhesion, migration and viability.

Statins reduce the isoprenoids farnesyl and geranylgeranyl pyrophosphate, essential intermediates, which control a diversity of cellular events such as cytoskeleton integrity, adhesion, migration and viability. Cardiac fibroblasts are the major non-myocyte cell constituent in the normal heart, and play a key role in the maintenance of extracellular matrix. The effects of simvastatin on cardiac fibroblast processes previously mentioned remain unknown. Our aims were to investigate the effects of simvastatin on cytoskeleton structure and focal adhesion complex assembly and their relationships with cell adhesion, migration and viability in cultured cardiac fibroblasts. To this end, cells were treated with simvastatin for 24 h and changes in actin cytoskeleton, levels of vimentin and paxillin as well as their subcellular localization were analyzed by Western blot and immunocytochemistry, respectively. Cell adhesion to plastic or collagen coated dishes, migration in Transwell chambers, and cell viability were analyzed after simvastatin treatment. Our results show that simvastatin disrupts actin cytoskeleton and focal adhesion complex evaluated by phalloidin stain and immunocytochemistry for paxillin and vinculin. All these effects occurred by a cholesterol synthesis-independent mechanism. Simvastatin decreased cell adhesion, migration and viability in a concentration-dependent manner. Finally, simvastatin decreased angiotensin II-induced phospho-paxillin levels and cell adhesion. We concluded that simvastatin disrupts cytoskeleton integrity and focal adhesion complex assembly in cultured cardiac fibroblasts by a cholesterol-independent mechanism and consequently decreases cell migration, adhesion and viability.

Attenuation of endoplasmic reticulum stress using the chemical chaperone 4-phenylbutyric acid prevents cardiac fibrosis induced by isoproterenol.

Increasing evidence indicates that endoplasmic reticulum (ER) stress is involved in various diseases. In the human heart, ischemia/reperfusion has been correlated to ER stress, and several markers of the unfolded protein response (UPR) participate during cardiac remodeling and fibrosis. Here, we used isoproterenol (ISO) injection as a model for in vivo cardiac fibrosis. ISO induced significant cardiomyocyte loss and collagen deposition in the damaged areas of the endocardium. These responses were accompanied by an increase in the protein levels of the luminal ER chaperones BIP and PDI, as well as an increase in the UPR effector CHOP. The use of the chemical chaperone 4-phenylbutyric acid (4-PBA) prevented the activation of the UPR, the increase in luminal chaperones and also, leads to decreased collagen deposition, cardiomyocyte loss into the damaged zones. Our results suggest that cardiac damage and fibrosis induced in vivo by the beta-adrenergic agonist ISO are tightly related to ER stress signaling pathways, and that increasing the ER luminal folding capacity with exogenously administrated 4-PBA is a powerful strategy for preventing the development of cardiac fibrosis. Additionally, 4-PBA might prevent the loss of cardiomyocytes. Our data suggests that the attenuation of ER stress pathways with pharmacological compounds such as the chemical chaperone 4-PBA can prevent the development of cardiac fibrosis and adverse remodeling.

Simvastatin induces apoptosis by a Rho-dependent mechanism in cultured cardiac fibroblasts and myofibroblasts.

UNLABELLED: Several clinical trials have shown the beneficial effects of statins in the prevention of coronary heart disease. Additionally, statins promote apoptosis in vascular smooth muscle cells, in renal tubular epithelial cells and also in a variety of cell lines; yet, the effects of statins on cardiac fibroblast and myofibroblast, primarily responsible for cardiac tissue healing are almost unknown. Here, we investigated the effects of simvastatin on cardiac fibroblast and myofibroblast viability and studied the molecular cell death mechanism triggered by simvastatin in both cell types. METHODS: Rat neonatal cardiac fibroblasts and myofibroblasts were treated with simvastatin (0.1-10µM) up to 72h. Cell viability and apoptosis were evaluated by trypan blue exclusion method and by flow cytometry, respectively. Caspase-3 activation and Rho protein levels and activity were also determined by Western blot and pull-down assay, respectively. RESULTS: Simvastatin induces caspase-dependent apoptosis of cardiac fibroblasts and myofibroblasts in a concentration- and time-dependent manner, with greater effects on fibroblasts than myofibroblasts. These effects were prevented by mevalonate, farnesylpyrophosphate and geranylgeranylpyrophosphate, but not squalene. These last results suggest that apoptosis was dependent on small GTPases of the Rho family rather than Ras. CONCLUSION: Simvastatin triggered apoptosis of cardiac fibroblasts and myofibroblasts by a mechanism independent of cholesterol synthesis, but dependent of isoprenilation of Rho protein. Additionally, cardiac fibroblasts were more susceptible to simvastatin-induced apoptosis than cardiac myofibroblasts. Thus simvastatin could avoid adverse cardiac remodeling leading to a less fibrotic repair of the damaged tissues.

Beta(2)-adrenergic receptor regulates cardiac fibroblast autophagy and collagen degradation.

Autophagy is a physiological degradative process key to cell survival during nutrient deprivation, cell differentiation and development. It plays a major role in the turnover of damaged macromolecules and organelles, and it has been involved in the pathogenesis of different cardiovascular diseases. Activation of the adrenergic system is commonly associated with cardiac fibrosis and remodeling, and cardiac fibroblasts are key players in these processes. Whether adrenergic stimulation modulates cardiac fibroblast autophagy remains unexplored. In the present study, we aimed at this question and evaluated the effects of b(2)-adrenergic stimulation upon autophagy. Cultured adult rat cardiac fibroblasts were treated with agonists or antagonists of beta-adrenergic receptors (b-AR), and autophagy was assessed by electron microscopy, GFP-LC3 subcellular distribution, and immunowesternblot of endogenous LC3. The predominant expression of b(2)-ARs was determined and characterized by radioligand binding assays using [(3)H]dihydroalprenolol. Both, isoproterenol and norepinephrine (non-selective b-AR agonists), as well as salbutamol (selective b(2)-AR agonist) increased autophagic flux, and these effects were blocked by propanolol (b-AR antagonist), ICI-118,551 (selective b(2)-AR antagonist), 3-methyladenine but not by atenolol (selective b(1)-AR antagonist). The increase in autophagy was correlated with an enhanced degradation of collagen, and this effect was abrogated by the inhibition of autophagic flux. Overall, our data suggest that b(2)-adrenergic stimulation triggers autophagy in cardiac fibroblasts, and that this response could contribute to reduce the deleterious effects of high adrenergic stimulation upon cardiac fibrosis.

Phospholipase C/protein kinase C pathway mediates angiotensin II-dependent apoptosis in neonatal rat cardiac fibroblasts expressing AT1 receptor.

Cardiac fibroblasts are the major non-myocyte cell constituent in the myocardium, and they are involved in heart remodeling. Angiotensin II type 1 receptor (AT1R) mediates the established actions of angiotensin II (Ang II), and changes in its expression have been reported in cardiac fibroblasts after myocardial infarction. However, the AT1R-dependent signaling pathways involved in cardiac fibroblast death remain unknown. Using adenovirus, we ectopically expressed AT1R in cultured neonatal rat cardiac fibroblasts and investigated the role of the phospholipase (PLC)/protein kinase C (PKC) pathway on Ang II-dependent death. Ang II induced cardiac fibroblast death characterized by an early loss of mitochondrial membrane potential, increased Bax/Bcl-2 ratio, caspase-3 activation, and DNA fragmentation. All these effects were prevented by the AT1R antagonist losartan, PLC inhibitor U73122, and PKC inhibitor Gö6976. We conclude that Ang II stimulates the intrinsic apoptotic pathway in cultured cardiac fibroblasts by the AT1R/PLC/PKC signaling pathway.

Enalapril attenuates downregulation of Angiotensin-converting enzyme 2 in the late phase of ventricular dysfunction in myocardial infarcted rat.

The early and long-term effects of coronary artery ligation on the plasma and left ventricular angiotensin-converting enzyme (ACE and ACE2) activities, ACE and ACE2 mRNA levels, circulating angiotensin (Ang) levels [Ang I, Ang-(1-7), Ang-(1-9), and Ang II], and cardiac function were evaluated 1 and 8 weeks after experimental myocardial infarction in adult Sprague Dawley rats. Sham-operated rats were used as controls. Coronary artery ligation caused myocardial infarction, hypertrophy, and dysfunction 8 weeks after surgery. At week 1, circulating Ang II and Ang-(1-9) levels as well as left ventricular and plasma ACE and ACE2 activities increased in myocardial-infarcted rats as compared with controls. At 8 weeks post-myocardial infarction, circulating ACE activity, ACE mRNA levels, and Ang II levels remained higher, but plasma and left ventricular ACE2 activities and mRNA levels and circulating levels of Ang-(1-9) were lower than in controls. No changes in plasma Ang-(1-7) levels were observed at any time. Enalapril prevented cardiac hypertrophy and dysfunction as well as the changes in left ventricular ACE, left ventricular and plasmatic ACE2, and circulating levels of Ang II and Ang-(1-9) after 8 weeks postinfarction. Thus, the decrease in ACE2 expression and activity and circulating Ang-(1-9) levels in late ventricular dysfunction post-myocardial infarction were prevented with enalapril. These findings suggest that in this second arm of the renin-angiotensin system, ACE2 may act through Ang-(1-9), rather than Ang-(1-7), as a counterregulator of the first arm, where ACE catalyzes the formation of Ang II.

Effects of carvedilol on oxidative stress and chronotropic response to exercise in patients with chronic heart failure.

BACKGROUND: Our previous studies suggest that the increase in heart rate from rest to peak exercise is reduced in patients with chronic heart failure (CHF) and this is associated with increased oxidative stress, as determined by malondialdehyde (MDA) plasma levels. AIM: To investigate the effects of carvedilol on the heart rate response to exercise and oxidative stress in patients with CHF. METHODS AND RESULTS: Thirty stable NYHA classes II-III CHF patients received carvedilol therapy for 6 months, at a mean maintenance dose of 25 mg (range 6.25-50 mg/day). After treatment, the patients showed a significant improvement in their functional NYHA class (p=0.013), increased left ventricular ejection fraction (LVEF) (24+/-1.4% to 31+/-2.3%, p=0.003) and 6-min walk distance (499+/-18 to 534+/-18 m, p=0.03), without changes in the peak VO2. At baseline, norepinephrine (NE) plasma levels increased with exercise (510+/-51 to 2513+/-230 pg/mL, p<0.001), and these levels were not affected by carvedilol. Chronotropic responsiveness index (increase in heart rate divided by the increase in NE from rest to peak exercise) was not changed by carvedilol (0.049+/-0.001 to 0.042+/-0.001, p=0.6). MDA levels of CHF patients decreased after treatment with carvedilol (2.4+/-0.2 to 1.1+/-0.2 microM, p<0.001), without changes in antioxidant enzyme activities. CONCLUSIONS: Carvedilol treatment in patients with CHF results in reduced oxidative stress without restoration of the chronotropic responsiveness index.

Effects of carvedilol upon intra- and interventricular synchrony in patients with chronic heart failure.

Radionuclide isotopic ventriculography with phase analysis was performed in 30 patients with stable heart failure (HF), determining left ventricular (LV) and interventricular contraction synchrony at baseline and after 6 months of treatment with maximal tolerated doses of carvedilol. Patients with HF had significant ventricular dyssynchrony compared with a normal population. The 50th percentile of patients with the greatest dyssynchrony at baseline showed significant improvement in ventricular synchrony after receiving carvedilol, and this was correlated positively with a reduction in end-diastolic LV volumes.

Efectos del Carvedilol sobre la sincronía ventricular en pacientes con insuficiencia cardíaca congestiva / Effects of Carvedilol on ventricular synchrony in patients with congestive heart failure

Introducción: Carvedilol es un bloqueador adrenérgico que mejora el remodelamiento ventricular izquierdo y reduce la morbi-mortalidad de los pacientes con insuficiencia cardíaca congestiva (ICC). Esto podría estar relacionado con una corrección de la sincronía ventricular. Objetivo: Evaluar el efecto de Carvedilol sobre la sincronía en la contracción ventricular en pacientes con ICC. Métodos: Se estudiaron 30 pacientes con ICC estable, capacidad funcional NYHA (CF) II-III, fracción de eyección (FE) < 40 por ciento, los cuales estaban tratados en forma habitual. Se excluyeron pacientes usuarios de betabloqueadores o de marcapaso. Se realizó ventriculografía radioisotópica de equilibrio, al inicio y posterior a la terapia con Carvedilol por 6 meses, para evaluar la función sistólica y la sincronía ventricular. La sincronía interventricular fue calculada mediante la diferencia de promedio de fase de ambos ventrículos y la intraventricular usando la desviación estándar del análisis de fase. Resultados: La edad fue 55 ± 13 años, 71 por ciento hombres, 35 por ciento de etiología isquémica y 29 por ciento con bloqueo completo de rama izquierda (BCRI). Posterior a la terapia con Carvedilol (dosis promedio de 22 mg, rango de 6.25 – 50 mg/día) hubo una mejoría en la CF y en la distancia recorrida en 6 min (499 ± 18 m a 534 ± 17 m ). La FE mejoró de 24 ± 8.3 por ciento a 31 ± 11. 3 por ciento (p<0.001). En los pacientes con peor sincronía, bajo el percentil 50, mejoró la sincronía intraventricular (113 ± ms vs. 94 ± 38 ms, p=0.02) e interventricular (62.8 ± 7 ms vs. 39.4 ± 9 ms, p=0.02). Los pacientes con etiología no isquémica tuvieron una mejoría en la sincronía intraventricular (103.8 ± 7 ms vs 78.3 ± 12 ms, p=0.04) e interventricular (68.1 ± 9 ms vs. 35.3 ± 12 ms, p=0.02). En aquellos sin BCRI mejoró la sincronía intraventricular (112.1 ± 8 ms vs. 88.5 ± 11.2 ms, p=0.01). No hubo cambios significativos en pacientes con causa isquémica o con BCRI. Conclusiones: En pacientes con IC y disfunción ventricular izquierda, Carvedilol mejora la sincronía intra e interventricular. Estos efectos podrían estar relacionados a una acción favorable sobre el remodelamiento cardíaco.

Polimorfismos de los receptores -β adrenérgicos y su relación con el riesgo de insuficiencia cardiaca, etiología y prevalencia de infarto al miocardio / Polymorphysm of beta-adrenergic receptors and risk of heart failure, etiology and prevalence of myocardial infarction

Introducción: Los polimorfismos de los receptores ß-adrenérgicos (AR) influencian el grado de actividad del receptor. Los ßAR tienen un rol importante en la regulación de la contractilidad y podrían tener implicancias en el riesgo de desarrollar insuficiencia cardiaca (IC), como en su pronóstico y respuesta terapéutica. Objetivo: Evaluar los genotipos e interacciones genéticas entre los polimorfismos del ß1 y ßAR como predictores de riesgo de desarrollar IC, su relación con la etiología de la IC y prevalencia de infarto. Métodos: Se genotipificaron 80 pacientes con IC y 88 sujetos sanos por edad y sexo. Los pacientes con IC tenían FE<35 por ciento y CF II-IV de la NYHA. Los polimorfismos se determinaron amplificando por la Reacción de Polimerización en Cadena (PCR) los genes de los ßAR y analiz¨¢ndolos con enzimas de restricción (PCR-RFLP). Los datos se analizaron mediante los tests estadísticos c2, Fisher, regresión loguística y razón de disparidad. Los datos se ajustaron por edad y sexo. Las interacciones entre los polimorfismos ß1AR Arg389 →Gly, ß2Ar Gln27 →Glu y ß2AR Thr 164 →lle se evaluaron en función del riesgo a desarrollar ICC. Resultados: Las frecuencias de los genotipos ß2AR Gln27→Glu y 1 AR Arg389→Gly fueron diferentes en los sujetos con IC comparados con los controles. La presencia del ß2AR Glu27Glu pero no de la variante 1 AR Gly389Gly fue predictor de ICC (OR ajustado=2,81; Cl=1,49 a 5,31 para el 2AR Glu27Glu; p=0,001 y OR ajustado=0,58; Cl=0,13 a 2,53; p=0,466 para b1AR Gly389Gly). Se encontró una interacción entre los polimorfismos ß1AR Arg389Arg y los polimorfismos del ß2AR Arg16Arg, Gln27Gln, y Thr164Thr. Estas interacciones se asociaron a una reducción en el riesgo de IC (OR=0,25, Cl=0,09 a 0,69; p=0,009; OR=0,18, Cl=0,07 a 0,46, p<0,001 y OR=0,48, Cl=0,25 a 0,91, p=0,026, respectivamente). Además, en los pacientes con IC, la frecuencia del polimorfismo 2AR Glu27Glu se asoció con una mayor incidencia de infarto al miocardio (con infarto: 0,534, sin = 0,313, p=0,01). Conclusiones: La variante Glu27Glu del ß2AR fue un predictor de IC, los polimorfismos del ß2AR Arg16Arg, Gln27Gln, y Thr164Thr y 1AR Arg389Arg se asociaron a una disminución del riesgo de ICC. El genotipo y frecuencia del alelo ß2AR Gln27 ----- Glu se relacionó a la etiología de la ICC y con la prevalencia de infarto al miocardio. Estos hallazgos pueden ser relevantes en la predicción de riesgo de ICC, su pronóstico y respuesta terapéutica.