The mKATP Channels and protein-kinase C Are Involved in the Cardioprotective Effects of Genistein on Estrogen-Deficient Rat Hearts Exposed to Ischemia/Reperfusion: Energetic Study.

Estrogenic deficiency is considered a risk of coronary disease in women. The phytoestrogen genistein could be a safe preventive strategy. The first aim of this work was to validate a model of cardiac stunning in which natural estrogenic deficiency rats, ie, adult young male (YM) and aged female (AgF), are compared with young female rats (YF). The second aim was to study whether the in vivo administration of genistein prevents the stunning in estrogenic deficiency rats. The third aim was to evaluate whether in our estrogenic deficiency model exists a synergy between genistein and estradiol. The fourth aim was to characterize the underlying mechanisms of genistein. Stunning was induced by ischemia/reperfusion (I/R) in isolated hearts inside a calorimeter. The left ventricular pressure (P) and total heat rate (Ht) were simultaneously measured, while diastolic contracture and muscle economy (P/Ht) were calculated. During R, P/Ht and P recovered less in AgF and YM than in YF rat hearts. Genistein through i.p. (GST-ip) improved P and P/Ht in AgF and YM, but not in YF. In YM, the cardioprotections of GST-ip and estradiol were synergistic. After ischemia, GST-ip increased SR Ca leak causing diastolic contracture. The GST-ip cardioprotection neither was affected by blockade of PI3K-Akt, NO synthases, or phosphatases, but it was sensitive to blockade of protein-kinase C and mKATP channels. Results suggest that (1) estrogenic deficiency worsens cardiac stunning, (2) GST-ip was more cardioprotective in estrogenic deficiency and synergistic with estradiol, and (3) cardioprotection of GST-ip depends on the protein-kinase C and mKATP channel pathway activation.

Cardioprotective Mechanisms of Hypothyroidism on Ischemia/Reperfusion in Rats and Effects of Carvedilol: Energetic Study.

Hypothyroidism is considered a cardiac risk factor, but there is controversial evidence about its effects on coronary disease. The aim of this work was to evaluate the influence of hypothyroidism in rat hearts exposed to 2 degrees of stunning due to ischemia and reperfusion (I/R) as well as the underlying mechanisms. Hypothyroid (HypoT) rats were obtained by drinking 0.02% methimazole during 15 days. Isolated hearts were perfused and introduced in a flow calorimeter to measure contractile performance (P), total heat rate (Ht), and muscle economy (P/Ht). Hearts were exposed to 2 models of I/R, moderate and severe (respectively 20 or 30 minutes I/45 minutes R). Moreover, free cytosolic and mitochondrial calcium changes were measured by confocal fluorometry on cardiomyocytes. Comparison to euthyroid (EuT) hearts was done. Hypothyroidism was cardioprotective, but HypoT hearts were more sensitive than EuT hearts to the preischemic blockade of mitochondrial transporters mNCX and mKATP channels. Moreover, the postischemic recovery of P and P/Ht in HypoT hearts was strongly reduced by inhibition of the cellular pathways of PI3K/Akt and protein kinase C (PKC), and it was increased by nitric oxide synthase (NOS) inhibition. However, physiological concentrations of adrenaline reduced the cardioprotection of HypoT, but oral treatment with 20 mg/kg/day carvedilol prevented it. Results show that hypothyroidism reduces the mitochondrial Ca2+ overload during I/R by mKATP channel activation and Ca2+ extrusion through mNCX, while the PI3K/Akt and PKC pathways are involved in that cardioprotection. Contrarily, NOS activation and adrenaline blunt such cardioprotection, but carvedilol prevented the adrenergic dysfunction. These results would explain why hypothyroidism is a clinical risk factor in angor patients under adrenergic exacerbation but reduced the incidence of acute episodes of coronary syndrome in hospitalized patients. Results suggest that a treatment with carvedilol could be a potential therapeutic agent to prevent cardiac postischemic dysfunction in hypothyroid patients.

Antitussive, antispasmodic, bronchodilating and cardiac inotropic effects of the essential oil from Blepharocalyx salicifolius leaves.

ETHNOPHARMACOLOGY RELEVANCE: Blepharocalyx salicifolius (Kunth) O. Berg (Myrtaceae) is a tree native to Argentina and Uruguay that grows and is cultivated along the riverside of the Rio de la Plata. The leaves of this plant species, locally known as "anacahuita" are used in South America to prepare infusions for the empiric treatment of cough and bronchospasm, as well as diarrhoea and other intestinal disorders. Although previous phytochemical studies have been performed with the essential oil extracted from Blepharocalyx salicifolius, pharmacological evidence supporting its traditional use is still lacking. AIM OF THE STUDY: To experimentally evaluate the pharmacological properties of Blepharocalyx salicifolius based on its traditional use. The studies were performed with tincture (T-Bs) and essential oil (EO-Bs) prepared from its leaves, in isolated rat trachea, intestine and heart preparations. METHODS: The ex-vivo effects of T-Bs and EO-Bs were evaluated with the agonists carbachol (CCh) and calcium chloride (Ca2+) in the contractile concentration-response curves (CRC) of the isolated intestine. The muscle relaxant effect of EO-Bs was evaluated in the isolated trachea and compared with the effect achieved with papaverine as a positive control. The T-Bs and EO-Bs cardiac effects were analysed by perfusion of an isolated rat heart before a period of ischemia/reperfusion (stunning model). The antitussive effect of both T-Bs and EO-Bs was evaluated in mice exposed to ammonia using codeine as a positive control. RESULTS: Both T-Bs and EO-Bs induced a non-competitive inhibition of the CCh-CRC in the rat intestine, with IC50 values of 170.3 ± 48.5µg T-Bs/mL (n = 6) and 5.9 ± 1.6µg EO-Bs/mL (n = 6), respectively. EO-Bs also inhibited non-competitively the Ca2+-CRC, with IC50 value of 1.8 ± 0.3µg EO-Bs/mL (n = 8). A similar effect was obtained with the main active component of the EO-Bs 1,8-cineole. In isolated trachea, EO-Bs induced the relaxation of the CCh-contracted tissue (1.7 ± 0.2µg EO-Bs/mL, n = 11) up to a maximal relaxation that was 1.9 times higher than that of papaverine. In the isolated heart, EO-Bs induced a poor negative inotropic response, and did not improve the contractile and energetic recovery after ischemia and reperfusion. In the mouse cough model, EO-Bs (90mg/Kg) was as effective as codeine (30mg/Kg) in reducing cough frequency. CONCLUSIONS: The results indicate that the preparations from Blepharocalyx salicifolius leaves were effective as central antitussive, bronchodilating and antispasmodic agents, suggestive of a mechanism associated with the inhibition of Ca2+ influx into smooth muscle. The EO-Bs displayed only a poor ability to reduce cardiac inotropism, and was devoid of any cardioprotective properties. Thus, the present study validates the traditional use of this South American plant for asthma, cough and bronchospasm, shedding new light into its potency and putative mechanism of action.

Effects of pyruvate on the energetics of rat ventricles stunned by ischemia-reperfusion.

Pyruvate (Pyr) was proposed as an additive to cold high-K(+)-low-Ca(2+) cardioplegia (CPG) to protect the heart during surgery. We explored whether Pyr and CPG would work synergistically to protect rat hearts from stunning during ischemia-reperfusion (I/R). We measured the heat release and contractility of perfused ventricles during I/R, and the cytosolic and mitochondrial [Ca(2+)] in cardiomyocytes by confocal microscopy. We found that under cold-CPG (30 °C), 10 mmol·L(-1) Pyr reduced the post-ischemic contractile recovery (PICR) as well as muscle economy, when added either before ischemia or during I/R, which was reversed by blockade of UCam. In noncardioplegic hearts, Pyr was cardioprotective when it was present during I/R, more so at 37 °C than at 30 °C, with improved economy. In cardiomyocytes, the addition of Pyr to CPG slightly increased the mitochondrial [Ca(2+)] but decreased cytosolic [Ca(2+)]. The results suggest that Pyr only protects hearts from stunning when present before ischemia and during reperfusion, and that it dampens the cardioprotective properties of CPG. The mechanisms underlying such different behavior depend on the dynamic balance between Pyr stimulation of the energetic state and mitochondrial Ca(2+) uptake. Our results support the use of Pyr in stunned hearts, but not in cold high-K(+) cardioplegia.

Effect of carticaine on the sarcoplasmic reticulum Ca2+-adenosine triphosphatase. II. Cations dependence.

Ca2+-ATPase is a major intrinsic protein in the sarcoplasmic reticulum (SR) from skeletal muscles. It actively transports Ca2+ from the cytoplasm to the SR lumen, reducing cytoplasmic [Ca2+] to promote muscle relaxation. Carticaine is a local anesthetic widely used in operative dentistry. We previously showed that carticaine inhibits SR Ca2+-ATPase activity and the coupled Ca(2+) uptake by isolated SR vesicles, and increases the rate of Ca2+ efflux from preloaded vesicles. We also found that these effects were antagonized by divalent cations, and concluded that they were mainly due to the direct interaction of carticaine with the Ca2+-ATPase protein. Here we present additional results on the modulation of the above effects of carticaine by Ca2+ and Mg2+. The activating effect of Ca2+ on the ATPase activity is competitively inhibited by carticaine, indicating a decreased Ca2+ binding to the high affinity Ca2+ transport sites. The activating effect of Mg2+ on the phosphorylation of Ca2+-ATPase by orthophosphate is also inhibited by carticaine. The anesthetic does not affect the reaction mechanism of the cations acting as cofactors of ATP in the catalytic site. On the basis of the present and our previous results, we propose a model that describes the effect of carticaine on the Ca2+-ATPase cycle.

Energética del comportamiento iónico en la contracción muscular cardíaca: aspectos fisiológicos y fisiopatológicos / Energetics of ionic behavior in heart muscle contraction: physiologic and physiopathologic aspects

Es aceptado que los movimientos iónicos a través de diferentes sistemas membranosos (sarcolema, retículo, sarcoplásmico y mitocondria) juegan un papel importante en el metabolismo del músculo cardíaco. Por otra parte, tanto su participación relativa como el gasto de energía asociado a dichos movimientos, no han sido definitivamente establecidos. Mediciones biofísicas y bioquímicas de los diferentes mecanismos de intercambio iónico, han provisto datos que llevaron a postular diferentes modelos funcionales para el metabolismo de reposo y el metabolismo activo del músculo cardíaco. El presente trabalho analisa, desde un punto de vista energético, datos bioquímicos y biofísicos extraídos de la literatura calculando el rango del consumo de energia que sería atribuible a cada mecanismo. Particularmente, son analizados los movimientos de sodio, potasio y calcio durante el estado de reposo y/o el estado activo y se discute la participación fraccional de las distintas organelas (sarcolema, retículo sarcoplásmico y mitocondria). Con este análisis y a partir de la cantidad conocida de energía liberada ( o la cantidad de oxígeno consumido) por el músculo es posible determinar la existencia de suficiente energía para un modelo dado de intercambio iónico durante el proceso de excitación-contracción. Además del análisis mencionado, se presenta una revisión de estudios energéticos realizados en condiciones patológicas. En particular, se analizan patologías con compromiso energético directo tal como la hipertrofia cardíaca, la isquemia y la anoxia en las que la alteración de los mecanismo de transporte iónico parecen jugar un papel crucial

Caffeine effects on heart muscle energetics: species differences

The effects of caffeine (1mM) on energy expenditure and mechanical parameters in rat and toad perfused heart ventricles were examined at various stimulation frequencies. While in rat muscles caffeine significantly depressed developed tension and maximal rates of contraction and relaxation at all frequencies tested, in toad ventricle a slight positive inotropic effect was observed. Even though caffeine did not alter total contraction time in both preparations, in the rat ventricle the last part of relaxation was prolonged. In rat ventricle in the presence of caffeine, the ratios between active heat production per beat and either developed tension or tension time integral increased at all frequencies tested (+303 +/- 47 microJ.mN-1 x g-1 and +1.21 +/- 0.13 mJ.mN-1 x s-1 x g-1 respectively) indicating a decrease in contractile economy. In toad ventricle no changes on these ratios were observed. The fact that only in rat ventricle caffeine decreased muscle economy suggests that caffeine affects a system that is active in rat ventricle but it is not operative in toad ventricle. This gives support to the hypothesis that if in rat ventricle SR-Ca pump (1 ATP hydrolyzed/2 Ca transported) is inhibited by caffeine cytosolic Ca would have to be removed by alternative mechanisms such as Na-Ca exchanger or sarcolemmal Ca pump both with a higher rate of ATP hydrolysis (1 ATP hydrolyzed/Ca transported) with the consequent decrease in muscle economy. Resting heat production was increased by caffeine in both preparations and the magnitude of the increment (+3.0 +/- 0.6 mW.g-1 and +0.75 +/- 0.21 mW.g-1 for rat and toad ventricle respectively) also correlates with the different degree of SR activity in both species

Caffeine influence on the dependence of myocardial response to extracellular calcium

En la aurícula izquierda aislada de rata, el aumento de la concentración extracellular de Ca ... induce un aumento de la tensión desarrollada hasta un máximo de 5.1 ñ 0.5 mN. El desarrollo de tensión como función de la [Ca] fue evaluado por un análisis de Hill, del que se obtuvo un K0,5 = 0.79 (0.55-1.13) mmol. l**-1 y un coeficiente de Hill n= 1.70 (1.46-1.94). La cafeína (1 mmol. 1**-1) disminuyó la fuerza máxima inducida por el aumento de la [Ca] a 3.2 ñ 0.3 mN (p<0.05). El análisis de Hill mostró que tanto el K0.5=0.22 (0.14-0.34) mmol. 1**-1 como el coeficiente de Hill n= 1.21 (0.86-1.56) mmol45 l**l habían disminuído en presencia de cafeína. El eflujo de Ca (realizado a una [Ca] de 1.3 mmol. l**l) disminuyó en presencia de cafeína en alrededor de un 27% (0.38 micronmolgm peso húmedo**l). La disminución del coeficiente de Hill observado en presencia de cafeína sugiere la desaparición de un compartimiento o de un proceso cooperativo. Los experimentos de eflujo de **45Ca sugieren la existencia de un compartimiento que tendría cuatro veces la cantidad de Ca requerida para una contracción máxima y que puede ser deplecionado por la cafeína. En ausencia de este compartimiento (i.e., en presencia de cafeína), las fuentes remanentes de Ca poderían proveer suficiente calcio como para desarrollar más del 70% de la actividad contráctil máxima