Mechanisms of cardiodepression by an Na+-H+ exchange inhibitor methyl-N-isobutyl amiloride (MIA) on the heart: lack of beneficial effects in ischemia-reperfusion injury.

Although Na+-H+ exchange (NHE) inhibitors such as methyl-N-isobutyl amiloride (MIA) are known to depress the cardiac function, the mechanisms of their negative inotropic effect are not completely understood. In this study, isolated rat hearts were perfused with MIA to study its action on cardiac performance, whereas isolated subcellular organelles such as sarcolemma, myofibrils, sarcoplasmic reticulum, and mitochondria were treated with MIA to determine its effect on their function. The effect of MIA on intracellular Ca2+ mobilization was examined in fura-2-AM-loaded cardiomyocytes. MIA was observed to depress cardiac function in a concentration-dependent manner in HCO3- -free buffer. On the other hand, MIA had an initial positive inotropic effect followed by a negative inotropic effect in HCO3-containing buffer. MIA increased the basal concentration of intracellular Ca2+ ([Ca2+]i) and augmented the KCl-mediated increase in [Ca2+]i. MIA did not show any direct effect on myofibrils, sarcolemma, and sarcoplasmic reticulum ATPase activities; however, this agent was found to decrease the intracellular pH, which reduced the myofibrils Ca2+-stimulated ATPase activity. MIA also increased Ca2+ uptake by mitochondria without having any direct effect on sarcoplasmic reticulum Ca2+ uptake. In addition, MIA did not protect the hearts subjected to mild Ca2+ paradox as well as ischemia-reperfusion-mediated injury. These results suggest that the increase in [Ca2+]i in cardiomyocytes may be responsible for the initial positive inotropic effect of MIA, but its negative inotropic action may be due to mitochondrial Ca2+ overloading as well as indirect depression of myofibrillar Ca2+ ATPase activity. Thus the accumulation of [H+]i as well as occurrence of intracellular and mitochondrial Ca2+ overload may explain the lack of beneficial effects of MIA in preventing the ischemia-reperfusion-induced myocardial injury.

Potential role and mechanisms of subcellular remodeling in cardiac dysfunction due to ischemic heart disease.

Several studies have revealed varying degrees of changes in sarcoplasmic reticular and myofibrillar activities, protein content, gene expression and intracellular Ca-handling during cardiac dysfunction due to ischemia-reperfusion (I/R); however, relatively little is known about the sarcolemmal and mitochondrial alterations, as well as their mechanisms in the I/R hearts. Because I/R is associated with oxidative stress and intracellular Ca-overload, it has been indicated that changes in subcellular activities, protein content and gene expression due to I/R are related to both oxidative stress and Ca-overload. Intracellular Ca-overload appears to induce changes in subcellular activities, protein contents and gene expression (subcellular remodeling) by activation of proteases and phospholipases, as well as by affecting the genetic apparatus, whereas oxidative stress is considered to cause oxidation of functional groups of different subcellular proteins in addition to modifying the genetic machinery. Ischemic preconditioning, which is known to depress the development of both intracellular Ca-overload and oxidative stress due to I/R, was observed to attenuate the I/R-induced subcellular remodeling and improve cardiac performance. It is suggested that a combination therapy with antioxidants and interventions, which reduce the development of intracellular Ca-overload, may improve cardiac function by preventing or attenuating the occurrence of subcellular remodeling due to ischemic heart disease. It is proposed that defects in the activities of subcellular organelles may serve as underlying mechanisms for I/R-induced cardiac dysfunction under acute conditions, whereas subcellular remodeling due to alterations in gene expression may explain the impaired cardiac performance under chronic conditions of I/R.

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase.

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.

Role of oxidative stress in alterations of mitochondrial function in ischemic-reperfused hearts.

To study the mechanisms of mitochondrial dysfunction due to ischemia-reperfusion (I/R) injury, rat hearts were subjected to 20 or 30 min of global ischemia followed by 30 min of reperfusion. After recording both left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) to monitor the status of cardiac performance, mitochondria from these hearts were isolated to determine respiratory and oxidative phosphorylation activities. Although hearts subjected to 20 min of ischemia failed to generate LVDP and showed a marked increase in LVEDP, no changes in mitochondrial respiration and phosphorylation were observed. Reperfusion of 20-min ischemic hearts depressed mitochondrial function significantly but recovered LVDP completely and lowered the elevated LVEDP. On the other hand, depressed LVDP and elevated LVEDP in 30-min ischemic hearts were associated with depressions in both mitochondrial respiration and oxidative phosphorylation. Reperfusion of 30-min ischemic hearts elevated LVEDP, attenuated LVDP, and decreased mitochondrial state 3 and uncoupled respiration, respiratory control index, ADP-to-O ratio, as well as oxidative phosphorylation rate. Alterations of cardiac performance and mitochondrial function in I/R hearts were attenuated or prevented by pretreatment with oxyradical scavenging mixture (superoxide dismutase and catalase) or antioxidants [N-acetyl-L-cysteine or N-(2-mercaptopropionyl)-glycine]. Furthermore, alterations in cardiac performance and mitochondrial function due to I/R were simulated by an oxyradical-generating system (xanthine plus xanthine oxidase) and an oxidant (H(2)O(2)) either upon perfusing the heart or upon incubation with mitochondria. These results support the view that oxidative stress plays an important role in inducing changes in cardiac performance and mitochondrial function due to I/R.

Dependence of changes in beta-adrenoceptor signal transduction on type and stage of cardiac hypertrophy.

To examine whether cardiac hypertrophy is associated with changes in beta-adrenoceptor signal transduction mechanisms, pressure overload (PO) was induced by occlusion of the abdominal aorta and volume overload (VO) by creation of an aortocaval shunt for 4 and 24 wk in rats. After hemodynamic assessment of the animals, the left ventricular (LV) particulate fraction was isolated for measurement of beta(1)-adrenoceptors and adenylyl cyclase activity, and cardiomyocytes were isolated for monitoring of the intracellular Ca(2+) concentration. Although PO and VO produced cardiac hypertrophy and increased LV end-diastolic pressure at 4 wk, cardiac function was increased in animals subjected to PO but remained unaltered in animals subjected to VO. Cardiac hypertrophy and increased LV end-diastolic pressure were associated with depressed cardiac function at 24 wk of PO or VO, but clinical signs of congestive heart failure were evident only in animals subjected to VO. Isoproterenol-induced increases in cardiac function, activation of adenylyl cyclase activity, and increase in intracellular Ca(2+) concentration, as well as beta(1)-adrenoceptor density, were unaltered by PO at 4 wk, augmented by VO at 4 wk, and attenuated by PO and VO at 24 wk. These results suggest that alterations in beta(1)-adrenoceptor signal transduction are dependent on the type and stage of cardiac hypertrophy.

Differential changes in beta-adrenoceptor signal transduction in left and right ventricles of infarcted rats.

Although different experimental and clinical studies have revealed varying degrees of defects in beta-adrenoceptors (beta-ARs) during the development of heart failure, the mechanisms for differences in beta-AR signal transduction between the left (LV) and right ventricle (RV) are not understood. Because biochemical alterations in the myocardium depend on the stage of heart disease, this study was undertaken to assess the status of beta-ARs in the LV and RV at different stages of heart failure. Myocardial infarction was induced in rats by occluding the left coronary artery for 8 and 24 weeks. The beta-AR signal transduction was monitored by measuring beta1-AR density, the isoproterenol-induced positive inotropic effect, the increase in [Ca2+]i in cardiomyocytes, and the activation of adenylyl cyclase. The beta-AR signal transduction parameters in the 8- and 24-week failing LV were depressed, whereas the RV showed upregulation at 8 weeks and downregulation at 24 weeks of these mechanisms. These results suggest that beta-AR-mediated signal transduction in the LV and RV are differentially regulated and are dependent upon the stage of development of congestive heart failure due to myocardial infarction.

MAPK activation and apoptotic alterations in hearts subjected to calcium paradox are attenuated by taurine.

OBJECTIVE: The Ca2+ -paradox is an important phenomenon to study cell injury induced by Ca2+ -overload in myocardium. Although intracellular Ca2+ -overload acts as a trigger and modulator of cell death due to apoptosis under various pathophysiological conditions, the presence of apoptosis in hearts subjected to Ca2+ -paradox has not been demonstrated. Since taurine attenuates the changes in cardiac function due to Ca2+ -paradox, this study investigated the occurrence and mechanisms of apoptosis in Ca2+ -paradoxic hearts treated in the absence and presence of taurine. METHODS: Ca2+ -paradox was induced by perfusing the isolated rat heart with Ca2+ -free medium for 5 min followed by reperfusion with Ca2+ -containing medium for 30 min. Apoptosis related signal transduction mechanisms were determined in Ca2+ -paradoxic hearts perfused with or without 10 mM taurine. RESULTS: Marked alterations in cardiac function and the presence of apoptosis were seen in Ca2+ -paradoxic hearts reperfused for 30 min. Unlike the total protein contents in hearts subjected to Ca2+ -paradox, the contents of phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase (ERK)1, ERK2 and c-jun amino-terminal kinase were increased by 125 +/- 8.6%, 296 +/- 14.3%, 213 +/- 8.5% and 133 +/- 4.2%, respectively vs. control. Caspase-3 and phosphorylated Bcl-2 contents were also increased by 193 +/- 10.2% and 134 +/- 5.0% vs. control whereas phosphorylated Bad and the ratio of Bcl-2/Bad were depressed by 32 +/- 10.8% and 0.23 +/- 0.5% vs. control in Ca2+ -paradoxic hearts. The apoptosis as seen in Ca2+ -paradoxic hearts reperfused for 30 min was not evident in hearts at 10 min Ca2+ -repletion but was similar to hearts subjected to 60 min Ca2+ -repletion. These changes in the apoptotic pathway in cardiomyocytes subjected to Ca2+ -paradox were prevented by taurine. Furthermore, taurine attenuated the KCl- or ATP-induced increase in intracellular concentration of Ca2+ in cardiomyocytes. CONCLUSIONS: This study suggests that cardiac dysfunction due to Ca2+ -paradox may be associated with apoptosis. In addition, the beneficial effects of taurine on cardiac function may be related to the attenuation of changes in MAPK and apoptotic signal transduction mechanisms in Ca2+ -paradoxic hearts.

Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger.

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.

Involvement of Na+/Ca2+ exchanger in catecholamine-induced increase in intracellular calcium in cardiomyocytes.

Although sarcolemmal (SL) Na+/Ca2+ exchanger is known to regulate the intracellular Ca2+ concentration ([Ca2+]i), its involvement in catecholamine-induced increase in [Ca2+]i is not fully understood. To gain some information in this regard, isolated rat cardiomyocytes were treated with different agents, which are known to modify Ca2+ movements, in the absence or presence of a beta-adrenoceptor agonist, isoproterenol, and [Ca2+]i in cardiomyocytes was determined spectrofluorometrically with fura-2 AM. Treatment with isoproterenol did not alter [Ca2+]i in quiescent cardiomyocytes, whereas the ATP (purinergic receptor agonist)-induced increase in [Ca2+]i was significantly potentiated by isoproterenol. Unlike ryanodine and cyclopiazonic acid, which affect the sarcoplasmic reticulum function, SL L-type Ca2+ channel blockers verapamil and diltiazem, as well as a SL Ca2+-pump inhibitor, vanadate, caused a significant depression in the isoproterenol-induced increase in [Ca2+]i. The SL Na+/Ca2+ exchange blockers amiloride, Ni2+, and KB-R7943 also attenuated the isoproterenol-mediated increase in [Ca2+]i. Combination of KB-R7943 and verapamil showed additive inhibitory effects on the isoproterenol-induced increase in [Ca2+]i. The isoproterenol-induced increase in [Ca2+]i in KCl-depolarized cardiomyocytes was augmented by low Na+; this augmentation was significantly depressed by treatment with KB-R7943. The positive inotropic action of isoproterenol in isolated hearts was also reduced by KB-R7943. These data suggest that in addition to SL L-type Ca2+ channels, SL Na+/Ca2+ exchanger seems to play an important role in catecholamine-induced increase in [Ca2+]i in cardiomyocytes.

Pharmacological basis of different targets for the treatment of atherosclerosis.

The development of atherosclerotic plaque is a highly regulated and complex process which occurs as a result of structural and functional alterations in endothelial cells, smooth muscle cells (SMCs), monocytes/macrophages, T-lymphocytes and platelets. The plaque formation in the coronary arteries or rupture of the plaque in the peripheral vasculature in latter stages of atherosclerosis triggers the onset of acute ischemic events involving myocardium. Although lipid lowering with statins has been established as an important therapy for the treatment of atherosclerosis, partially beneficial effects of statins beyond decreasing lipid levels has shifted the focus to develop newer drugs that can affect directly the process of atherosclerosis. Blockade of renin angiotensin system, augmentation of nitric oxide availability, reduction of Ca(2+) influx, prevention of oxidative stress as well as attenuation of inflammation, platelet activation and SMC proliferation have been recognized as targets for drug treatment to control the development, progression and management of atherosclerosis. A major challenge for future drug development is to formulate a combination therapy affecting different targets to improve the treatment of atherosclerosis.

Mechanisms of lysophosphatidic acid-induced increase in intracellular calcium in vascular smooth muscle cells.

Although lysophosphatidic acid (LPA) is known to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMCs), the mechanisms of [Ca2+]i mobilization by LPA are not fully understood. In the present study, the effect of LPA on [Ca2+]i mobilization in cultured A10 VSMCs was examined by Fura-2 fluorescence technique. The expression of LPA receptors was studied by immunostaining. LPA was observed to increase [Ca2+]i in a concentration-dependent manner; this increase was dependent on the concentration of extracellular Ca2+. Both sarcolemmal (SL) Na(+)-Ca2+ exchange inhibitors (amiloride, Ni2+ and KB-R7943) and Na(+)-H+ exchange inhibitor (MIA) as well as SL store-operated Ca2+ channel (SOC) antagonists (SK&F 96365, tyrphostin A9 and gadolinium), unlike SL Ca2+ channel antagonists (verapamil and diltiazem), inhibited the LPA-induced increase in [Ca2+]i. In addition, sarcoplasmic reticulum (SR) Ca2+ channel blocker (ryanodine), SR Ca2+ channel opener (caffeine), SR Ca2+ pump ATPase inhibitor (thapsigargin) and inositol 1,4,5-trisphosphate (InsP3) receptor antagonists (xestospongin and 2-aminoethoxydiphenyl borate) were found to inhibit the LPA-induced Ca2+ mobilization. Furthermore, phospholipase C (PLC) inhibitor (U 73122) and protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) attenuated the LPA-induced increase in [Ca2+]i. These results indicate that Ca2+ mobilization by LPA involves extracellular Ca2+ entry through SL Na(+)-Ca2+ exchanger, Na(+)-H+ exchanger and SL SOCs. In addition, ryanodine-sensitive and InsP(3)-sensitive intracellular Ca2+ pools may be associated with the LPA-induced increase in [Ca2+]i. Furthermore, the LPA-induced [Ca2+]i mobilization in VSMCs seems to be due to the activation of both PLC and PKC.

Prevention of remodeling in congestive heart failure due to myocardial infarction by blockade of the renin-angiotensin system.

Ventricular remodeling subsequent to myocardial infarction (MI) is a complex process and is considered to be a major determinant of the clinical course of congestive heart failure (CHF). Emerging evidence suggests that activation of the renin-angiotensin system (RAS) plays an important role in post-MI ventricular remodeling; however, it is becoming clear that this is one of several neurohumoral systems that are activated in CHF. Blockade of RAS by angiotensin-converting enzyme inhibitors or angiotensin II type 1 receptor antagonists attenuates the ventricular dysfunction, but the effects of individual drugs in reducing the morbidity and mortality in CHF patients are variable. Furthermore, there is a difference of opinion as to the time of initiation of therapy with RAS blockers after the onset of MI. Since blockade of RAS partially improves cardiac function, it is suggested that a combination therapy involving RAS blockers (angiotensin-converting enzyme inhibitors or angiotensin II type 1 receptor antagonists) and agents that affect other neurohumoral systems may prove useful for improved treatment of CHF. Although activation of RAS has been shown to promote oxidative stress in experimental studies, the use of antioxidant therapy in CHF patients is controversial. Recent experimental studies have shown that ventricular remodeling in CHF is associated with remodeling of subcellular organelles such as sarcolemma, sarcoplasmic reticulum, myofibrils and extracellular matrix in terms of their molecular structure and composition. Since attenuation of remodeling in one and/or more subcellular organelles by different agents may prevent the progression of CHF, it is a challenge to develop specific drugs affecting molecular mechanisms associated with subcellular remodeling for the improved therapy of CHF.

Selenium improves cardiac function by attenuating the activation of NF-kappaB due to ischemia-reperfusion injury.

Although selenium, an essential trace element and a component of glutathione peroxidase, is known to protect the heart from ischemia-reperfusion (I/R)-induced injury, the mechanisms of this protection are not fully understood. For this purpose, isolated rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion; sodium selenite (25-1,000 nM) was added in the perfusion medium 10 min prior to ischemia, as well as during reperfusion. Selenium caused a dose-dependent improvement in cardiac performance and attenuated the decrease in the ratio of reduced glutathione to oxidized glutathione, as well as the increased level of malondialdehyde in I/R heart. Elevated ratios of nuclear factor-kappaB (NF-kappaB) in particulate and cytosolic fraction and of phosphorylated NF-kappaB and total NF-kappaB in I/R hearts were reduced by selenium. Cardiac dysfunction in hearts perfused with xanthine plus xanthine oxidase mixture, as well as hydrogen peroxide, or subjected to Ca2+ paradox was also attenuated by selenium. These data suggest that selenium protects the heart against I/R injury due to its action on the redox state and deactivation of NF-kappaB in I/R hearts.

Prenatal exposure to maternal undernutrition induces adult cardiac dysfunction.

An adverse environmental experience of the growing fetus may lead to permanent changes in the structure and function of organs that may predispose the individual to chronic diseases in later life; however, nothing is known about the occurrence and mechanisms of heart failure. We employed a rat model in which pregnant dams were fed diets containing either 180 g (normal) or 90 g (low) casein/kg for 2 weeks before mating and throughout pregnancy. The ejection fraction (EF) of the pups exposed to the low-protein (LP) diet was severely depressed in the first 2 weeks of life and was associated with an increase in cardiomyocyte apoptosis. This early depressed cardiac function was followed by progressive recovery and normalization of the EF of the offspring in the LP group. The left ventricular (LV) internal diameters were increased between 24 h and 84 d (12 weeks) of age in the LP-exposed group. Although between 3 d and 2 weeks of age the LV wall of the heart in the LP group was thinner, a progressive increase in LV wall thickness was seen. At 40 weeks of age, although the EF was normal, a two-fold elevation in LV end-diastolic pressure, reduced cardiac output, decreased maximum rates of contraction and relaxation, and reduced mean arterial pressure were observed. Our findings demonstrate that exposure of the developing fetus to a maternal LP diet programs cardiac dysfunction in the offspring in later life.

Modification of alterations in cardiac function and sarcoplasmic reticulum by vanadate in ischemic-reperfused rat hearts.

To study the cardioprotective effects of vanadate on ischemia-reperfusion (I/R) injury, isolated rat hearts perfused at constant flow were subjected to global ischemia for 30 min followed by reperfusion for 30 min. In this experimental model, I/R markedly decreased ventricular developed pressure and increased end-diastolic pressure. Pretreatment of hearts with 4 microM vanadate attenuated I/R-induced cardiac dysfunction. The reduction in sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+ release, as well as SR protein contents for Ca2+-pump ATPase and Ca2+-release channel, was also prevented by vanadate. Pretreatment of hearts with an antioxidant mixture containing superoxide dismutase + catalase exerted effects similar to those of vanadate in I/R hearts. Postischemic treatment of hearts with vanadate or superoxide dismutase + catalase also had beneficial effects on I/R-induced changes in cardiac performance and SR function. Alterations in cardiac function and SR Ca2+ transport due to an oxyradical-generating system (xanthine + xanthine oxidase) or an oxidant (H2O2) were attenuated by treatment with vanadate. These results suggest that vanadate may exert beneficial effects on cardiac performance and SR function in I/R hearts because of its antioxidant action.

Pentoxifylline attenuates cardiac dysfunction and reduces TNF-alpha level in ischemic-reperfused heart.

Although pentoxifylline (PTXF), a phosphodiesterase inhibitor, has been reported to exert beneficial effects in cardiac bypass surgery, its effect and mechanisms against ischemia-reperfusion (I/R) injury in heart are poorly understood. Because I/R is known to increase the level of tumor necrosis factor (TNF)-alpha in myocardium and PTXF has been shown to depress the production of TNF-alpha in failing heart, this study examined the hypothesis that PTXF may attenuate cardiac dysfunction and reduce TNF-alpha content in I/R heart. For this purpose, isolated rat hearts were subjected to global ischemia for 30 min followed by reperfusion for 2-30 min. Although cardiac dysfunction due to ischemia was not affected, the recovery of heart function upon reperfusion was markedly improved by PTXF treatment. This cardioprotective effect of PTXF was dose dependent; maximal effect was seen at a concentration of 125 microM. TNF-alpha, nuclear factor-kappaB (NF-kappaB), and phosphorylated NF-kappaB contents were decreased in ischemic heart but were markedly increased within 2 min of starting reperfusion. The ratio of cytosolic-to-homogenate NF-kappaB was decreased, whereas the ratio of particulate-to-homogenate NF-kappaB was increased in I/R hearts. These changes in TNF-alpha and NF-kappaB protein contents as well as in NF-kappaB redistribution due to I/R were significantly attenuated by PTXF treatment. The results of this study indicate that the cardioprotective effects of PTXF against I/R injury may be due to reductions in the activation of NF-kappaB and the production of TNF-alpha content.

Attenuation of extracellular ATP response in cardiomyocytes isolated from hearts subjected to ischemia-reperfusion.

Extracellular ATP is known to augment cardiac contractility by increasing intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes; however, the status of ATP-mediated Ca2+ mobilization in hearts undergoing ischemia-reperfusion (I/R) has not been examined previously. In this study, therefore, isolated rat hearts were subjected to 10-30 min of global ischemia and 30 min of reperfusion, and the effect of extracellular ATP on [Ca2+]i was measured in purified cardiomyocytes by fura-2 microfluorometry. Reperfusion for 30 min of 20-min ischemic hearts, unlike 10-min ischemic hearts, revealed a partial depression in cardiac function and ATP-induced increase in [Ca2+]i; no changes in basal [Ca2+]i were evident in 10- or 20-min I/R preparations. On the other hand, reperfusion of 30-min ischemic hearts for 5, 15, or 30 min showed a marked depression in both cardiac function and ATP-induced increase in [Ca2+]i and a dramatic increase in basal [Ca2+]i. The positive inotropic effect of extracellular ATP was attenuated, and the maximal binding characteristics of 35S-labeled adenosine 5'-[gamma-thio]triphosphate with crude membranes from hearts undergoing I/R was decreased. ATP-induced increase in [Ca2+]i in cardiomyocytes was depressed by verapamil and Cibacron Blue in both control and I/R hearts; however, this response in I/R hearts, unlike control hearts, was not affected by ryanodine. I/R-induced alterations in cardiac function and ATP-induced increase in [Ca2+]i were attenuated by treatment with an antioxidant mixture and by ischemic preconditioning. The observed changes due to I/R were simulated in hearts perfused with H2O2. The results suggest an impairment of extracellular ATP-induced Ca2+ mobilization in I/R hearts, and this defect appears to be mediated through oxidative stress.

Upregulation of beta-adrenergic receptors in heart failure due to volume overload.

To examine the mechanisms of changes in beta-adrenergic signal transduction in heart failing due to volume overload, we studied the status of beta-adrenoceptors (beta-ARs), G protein-coupled receptor kinase (GRK), and beta-arrestin in heart failure due to aortocaval shunt (AVS). Heart failure in rats was induced by creating AVS for 16 wk, and beta-AR binding, GRK activity, as well as their protein content, and mRNA levels were determined in both left and right ventricles. The density and protein content for beta1-ARs, unlike those for beta2-ARs, were increased in the failing hearts. Furthermore, protein contents for GRK isoforms and beta-arrestin-1 were decreased in membranous fractions and increased in cytosolic fractions from the failing hearts. On the other hand, steady-state mRNA levels for beta1-ARs and GRK2, as well as protein content for Gbetagamma-subunits, did not change in the failing heart. Basal cardiac function was depressed; however, both in vivo and ex vivo positive inotropic responses of the failing hearts to isoproterenol were augmented. Treatment of AVS animals with imidapril (1 mg.kg(-1).day(-1)) or losartan (20 mg.kg(-1).day(-1)) retarded the progression of heart failure; partially prevented changes in beta1-ARs, GRKs, and beta-arrestin-1 in the failing myocardium; and attenuated the increase in positive inotropic effect of isoproterenol. These results indicate that upregulation of beta1-ARs is associated with subcellular redistribution of GRKs and beta-arrestin-1 in the failing heart due to volume overload. Furthermore, attenuation of alterations in beta-adrenergic system by imidapril or losartan may be due to blockade of the renin-angiotensin system in the AVS model of heart failure.

TNF-alpha as a potential mediator of cardiac dysfunction due to intracellular Ca2+-overload.

TNF-alpha has been shown to be involved in cardiac dysfunction during ischemia/reperfusion injury; however, no information regarding the status of TNF-alpha production in myocardial injury due to intracellular Ca2+-overload is available in the literature. The intracellular Ca2+-overload was induced in the isolated rat hearts subjected to 5 min Ca2+-depletion and 30 min Ca2+-repletion (Ca2+-paradox). The Ca2+-paradox hearts exhibited a dramatic depression in left ventricular developed pressure, a marked elevation in left ventricular end diastolic pressure, and more than a 4-fold increase in TNF-alpha content. The ratio of cytosolic to homogenate nuclear factor-kappaB (NFkappaB) was decreased whereas the ratio of phospho-NFkappaB to total NFkappaB was increased in the Ca2+-paradox hearts. All these changes due to Ca2+-paradox were significantly attenuated upon treating the hearts with 100 microM pentoxifylline. These results suggest that activation of NFkappaB and increased production of TNF-alpha may play an important role in cardiac injury due to intracellular Ca2+-overload.

Imidapril treatment improves the attenuated inotropic and intracellular calcium responses to ATP in heart failure due to myocardial infarction.

1. Adenosine 5'-triphosphate (ATP) is known to augment cardiac contractile activity and cause an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in isolated cardiomyocytes. However, no information regarding the ATP-mediated signal transduction in the myocardium in congestive heart failure (CHF) is available. 2. CHF due to myocardial infarction (MI) in rats was induced by the occlusion of the left coronary artery for 8 weeks. The positive inotropy due to ATP was depressed in failing hearts. Treatment of 3 weeks infarcted animals with imidapril (1 mg kg(-1) day(-1)) for a period of 5 weeks improved the left ventricle function and decreased the attenuation of inotropic response to ATP. 3. ATP-induced increase in [Ca(2+)](i) was significantly depressed in cardiomyocytes isolated from the failing heart and this change was partially attenuated by imidapril treatment. However, the binding characteristics of (35)S-labeled adenosine 5'-(gamma-thio) triphosphate in sarcolemma isolated from the failing heart remained unaltered. 4. ATP-induced increase in [Ca(2+)](i) was depressed by verapamil and cibacron blue in both control and failing heart cardiomyocytes; however, the ATP response in the failing hearts, unlike the control preparations, was not decreased by ryanodine. This insensitivity to ryanodine was attenuated by imidapril treatment. 5. Treatment of infarcted rats with enalapril and losartan produced effects similar to imidapril. 6. These findings indicate that the positive inotropic response to ATP and ATP-induced increase in [Ca(2+)](i) in cardiomyocytes are impaired in heart failure. Furthermore, blockade of renin angiotensin system prevented the impairment of the ATP-mediated inotropic and [Ca(2+)](i) responses in the failing heart.