Cobalt chloride postconditioning as myoprotective therapy in cardiac ischemia-reperfusion.

Since damage induced by ischemia-reperfusion (I/R) involves alterations in Ca2+ homeostasis and is reduced by ischemic postconditioning (IP) and that CoCl2 can trigger changes resembling the response to a hypoxic event in normoxia and its blockade on Ca2+ current in heart muscle, our aim was to evaluate CoCl2 as an IP therapeutic tool. Mechanic and energetic parameters of isolated and arterially perfused male Wistar rat heart ventricles were simultaneously analyzed in a model of I/R in which 0.23 mmol/L CoCl2 was introduced upon reperfusion and kept or withdrawn after 20 min or introduced after 20 min of reperfusion. The presence of CoCl2 did not affect diastolic pressure but increased post-ischemic contractile recovery, which peaked at 20 min and decreased at the end of reperfusion. This decrease was prevented when CoCl2 was removed at 20 min of reperfusion. Total heat release increased throughout reperfusion, while economy increased between 15 and 25 min. No effect was observed when CoCl2 was introduced at 20 min of reperfusion. In addition, both the area under the contracture curve evoked by 10 mmol/L caffeine-36 mmol/L Na+ and the contracture tension relaxation rate were higher with CoCl2.Furthermore, CoCl2 decreased the number of arrhythmias during reperfusion and the ventricular damaged area. The presence of CoCl2 in reperfusion induces cardioprotection consistent with the improvement in cellular calcium handling. The use of CoCl2 constitutes a potential cardioprotective tool of clinical relevance.

Angiotensin II type 1 receptor is involved in hypertension and vascular alterations caused by environmental toxicant hexachlorobenzene.

Environmental hexachlorobenzene (HCB) increases blood pressure (BP) in female rats, causing alterations in arterial structure and function. Here we study the role of Angiotensin II receptor type 1 (AT1) in HCB-induced hypertension through the use of AT1 antagonist losartan. HCB-treated male rats showed a 22.7% increase in BP which was prevented by losartan. Losartan blocked HCB-induced changes in arterial morphology (decreased aorta cell number and increased wall thickness). Losartan also prevented HCB-induced alterations in artery relaxation by acetylcholine and nitroprusside but not the reduction in the maximum contraction by phenylephrine. Losartan rescued arterial molecular alterations caused by HCB (i.e. an increase in TGF-ß1 and AT1 expression and a decrease in eNOS expression and nitrite levels) and reduced hydrogen sulfide plasma concentration. In conclusion: in this work we demonstrate that AT1 activity is involved in HCB effects on the vascular system leading to hypertension.

Acute hypobaric hypoxia and cardiac energetic response in prepubertal rats: Role of nitric oxide.

NEW FINDINGS: What is the central question of this study? In adult rat hearts, exposure to hypobaric hypoxia increases tolerance to hypoxia-reoxygenation, termed endogenous cardioprotection. The mechanism involves the nitric oxide system and modulation of mitochondrial oxygen consumption. What is the cardiac energetic response in prepubertal rats exposed to hypobaric hypoxia? What is the main finding and its importance? Prepubertal rats, unlike adult rats, did not increase tolerance to hypoxia-reoxygenation in response acute exposure to hypobaric hypoxia, which impaired cardiac contractile economy. This finding could be related to a failure to increase nitric oxide synthase expression, hence modulation of mitochondrial oxygen consumption and ATP production. ABSTRACT: Studies in our laboratory showed that exposure of rats to hypobaric hypoxia (HH) increased the tolerance of the heart to hypoxia-reoxygenation (H/R), involving mitochondrial and cytosolic nitric oxide synthase (NOS) systems. The objective of the present study was to evaluate how the degree of somatic maturation could alter this healthy response. Prepubertal male rats were exposed for 48 h to a simulated altitude of 4400 m in a hypobaric chamber. The mechanical energetic activity in perfused hearts and the contractile functional capacity of NOS in isolated left ventricular papillary muscles were evaluated during H/R. Cytosolic nitric oxide (NO), production of nitrites/nitrates (Nx), expression of NOS isoforms, mitochondrial O2 consumption and ATP production were also evaluated. The left ventricular pressure during H/R was not improved by HH. However, the energetic activity was increased. Thus, the contractile economy (left ventricular pressure/energetic activity) decreased in HH. Nitric oxide did not modify papillary muscle contractility after H/R. Cytosolic p-eNOS-Ser1177 and inducible NOS expression were decreased by HH, but no changes were observed in NO production. Interestingly, HH increased Nx levels, but O2  consumption and ATP production in mitochondria were not affected by HH. Prepubertal rats exposed to HH preserved cardiac contractile function, but with a high energetic cost, modifying contractile economy. Although this could be related to the decreased NOS expression detected, cytosolic NO production was preserved, maybe through the Nx metabolic pathway, without modification of mitochondrial ATP production and O2  consumption. In this scenario, the treatment was unable to increase tolerance to H/R as observed in adult animals.

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.

Cardioprotection of stevioside on stunned rat hearts: A mechano-energetical study.

BACKGROUND: The sweetener and hypoglycemic properties of stevioside (STV) are well known, as the main component of the plant Stevia rebaudiana. Given its extensive use in diabetic patients, it was of interest to evaluate its effects on the most frequent cardiovascular disease, the coronary insufficiency. PURPOSE: To study whether STV could be cardioprotective against ischemia-reperfusion (I/R) in a model of "stunning" in rat hearts. STUDY DESIGN: A preclinical study was performed in isolated hearts from rats in the following groups: non-treated rats whose hearts were perfused with STV 0.3 mg/ml and their controls (C) exposed to either moderate stunning (20 min I/45 min R) or severe stunning (30 min I/45 min R), and a group of rats orally treated with STV 25 mg/kg/day in the drink water during 1 week before the experiment of severe stunning in the isolated hearts were done. METHODS: The mechano-calorimetrical performance of isolated beating hearts was recorded during stabilization period with control Krebs perfusion inside a calorimeter, with or without 0.3 mg/ml STV before the respective period of I/R. The left ventricular maximal developed pressure (P) and total heat rate (Ht) were continuously measured. RESULTS: Both, orally administered and perfused STV improved the post-ischemic contractile recovery (PICR, as % of initial control P) and the total muscle economy (P/Ht) after the severe stunning, but only improved P/Ht in moderate stunning. However, STV increased the diastolic pressure (LVEDP) during I/R in both stunning models. For studying the mechanism of action, ischemic hearts were reperfused with 10 mM caffeine-36 mM Na+-Krebs to induce a contracture dependent on sarcorreticular Ca2+ content, whose relaxation mainly depends on mitochondrial Ca2+ uptake. STV at 0.3 mg/ml increased the area-under-curve of the caffeine-dependent contracture (AUC-LVP). Moreover, at room temperature STV increased the mitochondrial Ca2+ uptake measured by Rhod-2 fluorescence in rat cardiomyocytes, but prevented the [Ca2+]m overload assessed by caffeine-dependent SR release. CONCLUSIONS: Results suggest that STV is cardioprotective against I/R under oral administration or direct perfusion in hearts. The mechanism includes the regulation of the myocardial calcium homeostasis and the energetic during I/R in several sites, mainly reducing mitochondrial Ca2+ overload and increasing the sarcorreticular Ca2+ store.

Mitochondrial Bioenergetics During Ischemia and Reperfusion.

During ischemia and reperfusion (I/R) mitochondria suffer a deficiency to supply the cardiomyocyte with chemical energy, but also contribute to the cytosolic ionic alterations especially of Ca2+. Their free calcium concentration ([Ca2+]m) mainly depends on mitochondrial entrance through the uniporter (UCam) and extrusion in exchange with Na+ (mNCX) driven by the electrochemical gradient (ΔΨm). Cardiac energetic is frequently estimated by the oxygen consumption, which determines metabolism coupled to ATP production and to the maintaining of ΔΨm. Nevertheless, a better estimation of heart energy consumption is the total heat release associated to ATP hydrolysis, metabolism, and binding reactions, which is measurable either in the presence or the absence of oxygenation or perfusion. Consequently, a mechano-calorimetrical approach on isolated hearts gives a tool to evaluate muscle economy. The mitochondrial role during I/R depends on the injury degree. We investigated the role of the mitochondrial Ca2+ transporters in the energetic of hearts stunned by a model of no-flow I/R in rat hearts. This chapter explores an integrated view of previous and new results which give evidences to the mitochondrial role in cardiac stunning by ischemia o hypoxia, and the influence of thyroid alterations and cardioprotective strategies, such as cardioplegic solutions (high K-low Ca, pyruvate) and the phytoestrogen genistein in both sex. Rat ventricles were perfused in a flow-calorimeter at either 30 °C or 37 °C to continuously measure the left ventricular pressure (LVP) and total heat rate (Ht). A pharmacological treatment was done before exposing to no-flow I and R. The post-ischemic contractile (PICR as %) and energetical (Ht) recovery and muscle economy (Eco: P/Ht) were determined during stunning. The functional interaction between mitochondria (Mit) and sarcoplasmic reticulum (SR) was evaluated with selective mitochondrial inhibitors in hearts reperfused with Krebs-10 mM caffeine-36 mM Na+. The caffeine induced contracture (CIC) was due to SR Ca2+ release, while relaxation mainly depends on mitochondrial Ca2+ uptake since neither SL-NCX nor SERCA are functional under this media. The ratio of area-under-curves over ischemic values (AUC-ΔHt/AUC-ΔLVP) estimates the energetical consumption (EC) to maintain CIC. Relaxation of CIC was accelerated by inhibition of mNCX or by adding the aerobic substrate pyruvate, while both increased EC. Contrarily, relaxation was slowed by cardioplegia (high K-low Ca Krebs) and by inhibition of UCam. Thus, Mit regulate the cytosolic [Ca2+] and SR Ca2+ content. Both, hyperthyroidism (HpT) and hypothyroidism (HypoT) reduced the peak of CIC but increased EC, in spite of improving PICR. Both, CIC and PICR in HpT were also sensitive to inhibition of mNCX or UCam, suggesting that Mit contribute to regulate the SR store and Ca2+ release. The interaction between mitochondria and SR and the energetic consequences were also analyzed for the effects of genistein in hearts exposed to I/R, and for the hypoxia/reoxygenation process. Our results give evidence about the mitochondrial regulation of both PICR and energetic consumption during stunning, through the Ca2+ movement.

Cardioprotective effect of hyperthyroidism on the stunned rat heart during ischaemia-reperfusion: energetics and role of mitochondria.

NEW FINDINGS: What is the central question of this study? Hyperthyroidism is a cardiac risk factor, but thyroid therapy is used on myocardial stunning. What is the consequence of hyperthyroidism for mitochondrial metabolism and Ca(2+) handling of the postischaemic stunned heart? What is the main finding and its importance? Hyperthyroidism reduced stunning and improved muscle economy of the postischaemic rat heart. The activities of the mitochondrial sodium-calcium exchanger and mitochondrial K(+) channel in hyperthyroid rat hearts were different from those in the euthyroid rat hearts. These findings contribute to the understanding of mitochondrial bioenergetics in pathology and support thyroid therapy in the stunning induced by ischaemia. Transient ischaemia and hyperthyroidism are cardiovascular risk factors. Nevertheless, 3,5,3'-triiodothyronine/thyroxine therapy has been used to revert myocardial stunning. We studied the influence of hyperthyroidism on the role played by mitochondria in myocardial stunning consequent to ischaemia-reperfusion. Rats were injected s.c. daily with 20 µg kg(-1) triiodothyronine for 15 days (HpT group). Isolated ventricles from either HpT or euthyroid (EuT) rats were perfused in a calorimeter, and left intraventricular pressure (in millimetres of mercury) and heat release (Ht; in milliwatts per gram) were measured. Stunning was evoked by 20 min of no-flow ischaemia and 45 min reperfusion. The HpT hearts developed higher postischaemic contractile recovery (PICR) and improved total muscle economy (P/Ht) with lower diastolic contracture (ΔLVEDP) than EuT hearts. Release of Ca(2+) from the sarcoplasmic reticulum during reperfusion with 10 mm caffeine in low-[Na(+) ] Krebs solution evoked a higher contracture in EuT than in HpT hearts. Blockade of the mitochondrial sodium-calcium exchanger with clonazepam increased ΔLVEDP and reduced P/Ht and PICR in HpT but not in EuT hearts. The clonazepam-induced dysfunction in HpT hearts was reduced by ciclosporin, suggesting a dependance on activation of the mitochondrial permeability transition pore. Blockade of the mitochondrial Ca(2+) uniporter with Ru360 reduced P/Ht and PICR to ∼10% in both HpT and EuT hearts. Blockade of mitochondrial K(+) channels with 5-hydroxydecanoate increased LVEDP and reduced PICR and P/Ht in HpT hearts, while it only increased LVEDP in EuT hearts. The results suggest that hyperthyroidism prevents the stunning with high dependence on the mitochondrial sodium-calcium exchanger and mitochondrial K(+) channels. Both HpT and EuT hearts showed a similar and critical role of the uniporter. The HpT hearts have a slow sarcoplasmic reticulum Ca(2+) loss and low mitochondrial Ca(2+) uptake.

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.

Energetics of Ca2+ homeostasis during ischemia-reperfusion on neonatal rat hearts under high-[K+] cardioplegia.

The mechanocalorimetric consequences and mechanisms involved in Ca2+ homeostasis during ischemia-reperfusion (I/R) as well as the protective role of cardioplegic pretreatment with high [K+] (25 mmol/L) and low or near-normal [Ca2+] (0.5 or 2 mmol/L) were evaluated in a model of neonatal rat heart. Beating hearts from 10-12-day-old rats were perfused with Krebs solution (2 mmol/L Ca2+) under both isotonic and isometric conditions. During pretreatment, hearts were exposed for 20 min to either Krebs (control) or cardioplegia (CPG) before 15 min ischemia and 45 min reperfusion while being continuously measured for either contractility or total heat rate (Ht) in a flow calorimeter. Contractile recovery after reperfusion in hearts exposed to ischemia only (control) was higher in the isometric hearts under optimal length (87.9% +/- 8.1%) than in the isotonic hearts (57.3% +/- 10.6%). This same behavior was found in hearts pretreated with CPG-0.5 mmol/L Ca2+. Ht in controls was reduced from 11.5 +/- 0.8 mW/g in the initial beating condition to 1.11 +/- 0.33 mW/g during ischemia and was increased to 13.02 +/- 0.93 mW/g (113.8% +/- 5.0% of preischemic) after reperfusion. Hearts pretreated with CPG-0.5 mmol/L Ca2+ showed the same behavior. However, when extracellular calcium ([Ca]o) was increased to 2 mmol/L under CPG, isotonic hearts, but not isometric hearts, significantly increased the contractile recovery to a maximum of 88.7% +/- 10.8% of preischemic levels. Ht was recovered to 92.1% +/- 4.3% of preischemic, suggesting that contractile recovery was less energetically expensive after CPG-2 mmol/L Ca2+ than it was in postischemic hearts exposed to control or CPG-0.5 mmol/L Ca2+. The role of the sarcoplasmic reticulum store was evaluated by pretreating hearts with 10 mmol/L caffeine, which reduced contractile recovery only under isometric conditions or after increasing [Ca]o in CPG under isotonic conditions, suggesting that the contribution of the sarcoplasmic reticulum was dependent on the fibre length or the [Ca]o. The inhibition of the reverse mode of the sarcolemmal Na/Ca exchanger (NCX) and the mitochondrial Ca uniporter (CaU) by KB-R7943 (KBR) at 5 micromol/L in CPG-0.5 mmol/L Ca2+ improved contractile recovery of isotonic hearts, whereas it decreased Ht at the start of reperfusion, suggesting that mitochondria could uptake Ca2+ vía the mitochondrial CaU. Neither the positive inotropism nor Ht were changed by inhibiting the mitochondrial NCX with 10 micromol/L clonazepam in CPG-0.5 mmol/L Ca2+ + 5 micromol/L KBR, which suggests that the mitochondrial NCX does not have a role. Finally, the role of the forward mode of the sarcolemmal NCX was evidenced by the fall in contractile recovery with increased Ht when KBR was increased to 20 micromol/L and added to CPG-2 mmol/L Ca2+ + 10 mmol/L caffeine before I/R. Thus the sarcolemmal NCX was essential for removing the diastolic Ca2+ during the periods of CPG and I/R. In summary, Ca2+ homeostasis during I/R of neonatal rat hearts is different from that of adult rats. High-[K+] CPG protected neonatal hearts only under isotonic conditions, at a near-normal [Ca]o, or by exposure to KBR. Mitochondria were able to uptake Ca2+ via the mitochondrial CaU and reduce the Ca2+ available for contractile recovery. Nevertheless, after increasing [Ca]o in CPG, the sarcoplasmic reticulum had a main role in restoring contractility during reperfusion, as it does in adults. Thus, the degree of maturation of the heart must be taken into account to evaluate the effects of CPG and drugs on I/R.

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 (AU)

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 (Au)

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 (AU)

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