Interrelationship between cellular calcium homeostasis and free radical generation in myocardial reperfusion injury

Chemico-Biological Interactions 104 (1997) 65 – 85

Mini-review

Interrelationship between cellular calcium homeostasis and free radical generation in myocardial reperfusion injury Debasis Bagchi a, Gerold J. Wetscher 1,a, Manashi Bagchi a, Paul R Hinder a, Galen Perdikis a, Sidney J. Stohs a, Ronald A. Hinder a, Dipak K. Das b,* a

Departments of Surgery, Pharmaceutical Sciences and Pharmacology, Creighton Uni6ersity, Omaha, NE, USA b Cardio6ascular Di6ision, Department of Surgery, Uni6ersity of Connecticut School of Medicine, Farmington, CT, USA Received in revised form 9 January 1997; accepted 10 January 1997

Abstract This review describes the interrelationship between two important biological factors, intracellular calcium overloading and oxygen-derived free radicals, which play a crucial role in the pathogenesis of myocardial ischemic reperfusion injury. Free radicals are generated during the reperfusion of ischemic myocardium, and polyunsaturated fatty acids in the membrane phospholipids are the likely targets of the free radical attack. On the other hand, activation of phospholipases can provoke the breakdown of membrane phospholipids which results in the activation of arachidonate cascade leading to the generation of protaglandins, and oxygen free radicals can be produced during the interconversion of the prostaglandins. In conclusion, it has been emphasized that the two seemingly different causative factors of reperfusion injury, intracellular calcium overloading and free radical generation are, in fact, highly interrelated. © 1997 Elsevier Science Ireland Ltd.

* Corresponding author. Tel.: + 1 860 6793687 fax: + 1 860 6792451. 1 Present address: Second Department of Surgery, University of Innsbruck, Austria. 0009-2797/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 0 9 - 2 7 9 7 ( 9 7 ) 0 3 7 6 6 - 6

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1. Introduction Since the demonstration by Shen and Jennings [1] that myocardial reperfusion following temporary coronary artery occlusion accelerated an accumulation of intracellular Ca2 + in canine heart, the role of Ca2 + in ischemia reperfusion injury has been a subject of intensive investigation. In normal myocardial cells, resting levels of cytosolic Ca2 + are maintained at submicromolar concentrations by three different mechanisms; (i) limiting Ca2 + infiltration into the myocytes; (ii) sequestering Ca2 + in the intracellular compartments; and (iii) extruding Ca2 + out of the cells against a 10 000-fold ionic gradient. Prolonged ischemia as well as reperfusion of ischemic heart cause intracellular Ca2 + overloading due to massive Ca2 + entry and dysfunction of Ca2 + sequestration machanisms contributing to the perturbation of intracellular Ca2 + homeostasis. Such dysregulation of Ca2 + homeostasis results in the alteration of a cascade of Ca2 + -dependent cellular events which are the characteristics of myocardial ischemia reperfusion injury. A growing body of evidence now supports the role of Ca2 + as an intracellular second messenger that regulates a large number of intracellular events to maintain normal cellular function. In general, the calcium signaling system is highly complex and intimately related with an excitation-contraction coupling mechanism. The mammalian heart requires a 1:1 coupling between the action potential generated by electrical excitation and the mechanical activity related to twitch contraction. Both voltage clamp and fluorescent indicator studies revealed that Ca2 + release is approximately parallel to the calcium current over a wide range of voltages [2]. A transient rise in the intracellular [Ca2 + ]i potentiates the signal linking electrical excitation of the membrane to mechanical activity of the cell. A small amount of Ca2 + penetrating through the voltage-dependent Ca2 + channel during ischemia may trigger a signal that is probably amplified several-fold by the Ca2 + induced Ca2 + release potentiated by mechanisms discussed in this review. Massive intracellular Ca2 + overloading occurs during reperfusion through Na + /Ca2 + exchange and/or Ca2 + –calmudolin/protein kinase C dependent pathways. Ca2 + infiltration during ischemia may predict the severity of Ca2 + influx during reperfusion leading to reperfusion injury. The focus of this review will be the intracellular signaling mechanism of Ca2 + in the ischemic reperfusion myocardium. The three key factors, free radicals, phospholipids and Ca2 + , which in combination play a major role in myocardial reperfusion injury, are highly interrelated. Much attention will be paid to discuss this interrelationship in an attempt to provide a better understanding of the role of Ca2 + in reperfusion injury. There cannot be any doubt that Ca2 + antagonism alone cannot amiliorate the reperfusion injury. The coordinated regulation of Ca2 + homeostasis must be clearly understood before Ca2 + blockers may be used succesfully for patient care for the prevention of ischemia reperfusion injury.

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2. Ca2 + and ischemia reperfusion injury: interrelationship with oxygen free radicals and sarcolemmal phospholipids Although the precise mechanism of myocardial ischemia reperfusion injury is still not clearly understood, three interrelated factors are believed to play a key role in the pathophysiology of reperfusion injury. These include: (i) loss of sarcolemmal phospholipids leading to the accumulation of lysophosphoglycerides and free fatty acids [3,4]; (ii) generation of oxygen free radicals and partial loss of several intracellular antioxidant enzymes [5–8]; and (iii) intracellular Ca2 + overloading that ultimately results in cell death and tissue injury [9–12]. Among these events, Ca2 + homeostasis is perhaps the most critical factor in determining the biochemical basis of ultimate cell death [13]. There is a tight and coordinated relationship between Ca2 + influx and free radical generation in the ischemic reperfused heart. The interrelationship between Ca2 + overload and oxygen free radical generation that contributes to myocardial

Fig. 1. Interrelationship between free radical generation and Ca2 + .

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reperfusion injury is shown in Fig. 1. While the exact sequences of the cascade of the events remain a matter of debate, there is no doubt that oxygen free radicals are generated during the reperfusion of ischemic myocardium [5–8,14–17]. The generated free radicals cause membrane lipid peroxidation leading to the formation of lipid degradation products which by virtue of their ionophoretic properties can promote Ca2 + influx [18]. It is known that polyunsaturated fatty acids in the membrane phospholipids are targets of free radical attack which results in the lipid peroxidation, forming lipid hydroperoxidides and endoperoxides. The phospholipid hydroperoxides possess a ligand environment capable of interacting with Ca2 + , facilitating Ca2 + transport to the myocardial membrane [19]. These hydroperoxides are weak acids in which the OOH group is covalently attached to a nonpolar fatty acid residue of a phospholipid molecule, thus rendering a charge at physiologic pH which may be neutralized in the presence of Ca2 + , a necessary step for the hydrophobization of Ca2 + . Degradation of these phospholipid hydroperoxides leads to formation of stable products such as hydroxy derivatives (ROH) and dialkylperoxides (ROOR) which are incapable of promoting Ca2 + transport [20]. Therefore, it appears that lipid peroxidation products are capable of switching Ca2 + transport on and off. In addition, free radicals, specifically hydroxyl radical (OH · ), can facilitate the entry of Na + into the cell by oxidizing Na + -channels [21]. Increased intracellular Na + leads to an increase in Na + /Ca2 + exchange. Thus, it is apparent that a factor to be considered in Ca2 + accumulation is free radical generation. On the other hand, Ca2 + may enter via a number of other pathways and activate phospholipases [22,23] which, in turn, leads to phospholipid degradation [2,4]. Inositol 1,4,5-triphosphate (IP3), a product of receptor activated hydrolysis of phosphatidylinositol 4,5-bisphosphate, plays a role as a second messenger by promoting mobilization of intracellular calcium [24]. IP3 not only releases Ca2 + from the sarcoplasmic reticulum, but also can increase cytosolic Ca2 + by altering flux [25]. Moreover, IP3 can inhibit the membrane-bound Na + /Ca2 + exchanger, and polyunsaturated fatty acids released by degradation of membrane phospholipids can mobilize the IP3-sensitive Ca2 + pool, causing Ca2 + overload [26,27]. Furthermore, reperfusion of an ischemic heart enhances phosphodiesterase degradation and phosphoinositide turnover [28]. A related study has showed that inhibition of phospholipase C attenuates myocardial ischemic-reperfusion injury [29]. Diacylglycerol formed by the action of phospholipase C could potentiate the slow Ca2 + channel by activation of protein kinase C [30]. It is tempting to speculate that enhanced formation of IP3 and diacylglycerol can act synergistically to increase intracellular Ca2 + . The activation of phospholipases results in the accumulation of free fatty acids, especially arachidonic acid, which generates prostaglandins (PG) and thromboxanes by the cyclooxygenase pathway [31]. Oxygen free radicals can be produced during the conversion of PGG2 into PGH2 [18,32,33], which can further result in the breakdown of sarcolemmal phospholipids. Another important interdependency between free radicals and Ca2 + homeostasis can be found from the documentation that xanthine oxidase is stimulated during

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the ischemic episode. One of the major mechanisms for superoxide generation is by the action of xanthine oxidase on xanthine or hypoxanthine in the presence of molecular oxygen. Evidence supports the notion that xanthine dehydrogenase present in the myocardium is converted into xanthine oxidase during ischemia by the action of Ca2 + -dependent proteolytic enzymes. Ischemia also induces the progressive degradation of myocardial ATP into xanthine and hypoxanthine. The moment the third substrate, molecular oxygen, becomes available during reperfusion, superoxide radicals are produced in the reperfused myocardium [34]. This hypothesis originally put forward by McCord [34] was challanged by a number of subsequent reports. The amount of XO is usually very low in mammalian heart, and thus can not account for the amount of reactive oxygen species produced during the reperfusion of ischemic myocardium [35]. A growing body of evidence indicates that activated polymorphonuclear (PMN) leukocytes may be a potential source of oxygen free radicals in the reperfused myocardium. Activation of PMN requires various chemotactic factors such as compliments, eicosanoids or platelet activating factor (PAF). Some of these compliments are generated within the cells by activation of Ca2 + -activated neutral proteases under Ca2 + overloading conditions [36]. Ca2 + may also cause PMN activation through the activation of phospholipase A2 and generation of cyclooxygenase products such as the eicosanoids and PAF It has been shown that ibuprofen, a nonsteroidal anti-inflammatory cyclooxygenase blocker, can prevent PMN accumulation in the ischemic reperfused myocardium [37]. The precise coordination between Ca2 + and oxygen free radicals remains highly controversial. It is quite possible that Ca2 + overloading occurs first which then induces free radical generation, as discussed above. Alternatively, oxygen free radical generation could precede Ca2 + overloading and pave the way for intracellular Ca2 + influx. For example, reactive oxygen species are potential mediators of sarcoplasmic reticular dysfunction. The uptake of Ca2 + is significantly affected by the free radicals [38]. Na + /K + -ATPase is extremely susceptible to free radical attack, and inactivation of this enzyme could become a major cause of Ca2 + infiltration [39]. Furthermore, as discussed earlier, sarcolemmal phospholipids which are rich in polyunsaturated fatty acids, are targets for free radical attack. Such attack renders the membrane susceptible to injury by altering the microviscosity [40,41] and causing lipid peroxidation [42] that facilitates Ca2 + influx through the leaky membrane.

3. Ca2 + paradox — its relationship with O2 paradox, pH paradox and antioxidant paradox Ischemic myocardium presumably contains little Ca2 + , no O2, an acidic pH and reduced antioxidants. Under such circumstances, when an ischemic myocardium is reperfused with buffer or blood having a pH of 7.4 and contains normal amounts (relatively higher amounts than those present in the ischemic heart) of Ca2 + , O2, and antioxidants, it results in a variety of cellular events including intracellular

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Ca2 + overloading and generation of free radicals, generally termed as Ca2 + paradox, O2 paradox, pH paradox and antioxidant paradox [43–45]. The role of intracellular pH in myocardial preservation and its relationship to intracellular Ca2 + concentration has not been adequately defined. It has been shown that post-ischemic Ca2 + gain is highly pH sensitive [43–45]. Reperfusion with an acidotic buffer (pH 6.4–6.6) attenuates Ca2 + gain while with an alkalotic madium (pH 7.9), Ca2 + gain is exacerbated [44]. Maneuvers that allow continued intracellular acidosis, such as by inhibition of Na + /H + exchange, prolong survival. Conversely, manipulations that prevent a decrease in pH reduce cell survival. Ca2 + overload results in the inhibition of mitochondrial O2 consumption [46,47]. Thus, it is likely that there is a relationship between the shift in redox potential, the formation of free radicals, intracellular pH, and Ca2 + overload in the genesis of ischemia reperfusion injury, and therefore, a close relationship between O2 paradox, Ca2 + paradox and pH paradox in the genesis of reperfusion injury.

4. Evidence for Ca2 + overload in ischemic reperfused myocardium As mentioned earlier, the first documentation of intracellular Ca2 + overloading was provided by Shen and Jennings in 1972 [1]. Such documentation was primarily based on morphological evidence obtained from electron microscopy and microincineration. Most of the infiltrated Ca2 + was localized in dense bodies within the mitochondrial matrix space, usually adjacent to a ceista. These mitochondrial dense bodies, often referred to as granular dense bodies, are indicators of intracellular Ca2 + overload, because they are morphologically identical to those found in mitochondria. These observations were subsequently supported by many investigators who also provided similar evidence for Ca2 + overload in reperfused myocardium [48]. It is now known that massive Ca2 + influx in the mitochondria is a characteristic feature of reperfusion in the early phase of reperfusion injury. When myocardial cells are subjected to up to 40 min of ischemia, major morphological changes occur which include intermyofibrilar edema and mitochondrial swelling with amorphous densities and prominant I bands typical of myofiber relaxation. At this stage, no indication of Ca2 + influx into the mitochondria is found. However, at the onset of reperfusion, sudden extension of ultrastructural damage occurs involving the development of contraction bands, disruption of myofibrils and sarcolemma, explosive cell swelling, and the appearance of intramitochondrial dense bodies, the latter signalling the intracellular Ca2 + overloading. Studies using radioactive calcium also supports the morphological data. Significant uptake of 45Ca2 + occurs only after 60 min of ischemia. To the contrary, 40 min of ischemia followed by 10 min of reperfusion was found to be associated with an 18-fold increase in 45Ca2 + uptake [49]. Ca2 + uptake measurements using 47 Ca2 + also demonstrated a large Ca2 + gain in the reperfused myocardium [50]. Isotopic Ca2 + data indicate that such Ca2 + overload occurs due to enhanced influx rather than decreased efflux.

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With the advent of advanced techniques, it is now known that Ca2 + overload can also occur prior to the onset of lethal myocardial injury. For example, using fluorescent Ca2 + indicators such as Indo-1 and Fura-2, it was shown that an increase in cytosolic Ca2 + can occur as early as 90 s of ischemia [51]. Temporary elevation of cytosolic Ca2 + occurs during early reperfusion after a brief period of ischemia and exhibits a progressive and steady increase after 30 min of ischemia [52]. NMR spectroscopy using 19F also reveals an increase in cytosolic Ca2 + between 10 – 20 min of ischemia and its gradual decline during reperfusion [53]. Another recent study using a fluorescence probe showed a significant rise of Ca2 + in rabbit heart during the first 2 min of ischemia [54]. In another study using an intracellular calcium analyzer in conjunction with Fura-2 AM, a consistent rise in intracellular [Ca2 + ]i for up to 30 min of ischemia and followed by a slow and progressive reduction during the reperfusion has been demonstrated [55]. In this study, an intracellular Ca2 + analyzer was used to examine [Ca2 + ]i transient during ischemia and reperfusion. This instrument permits monitoring of the beat-to-beat change of [Ca2 + ]i that occurrs during each cardiac cycle as shown in Fig. 2. The F340/F380 ratio indicates the change in the cytosolic free Ca2 + . In this study, the [Ca2 + ]i increased immediately after the induction of ischemia (10–15% increase of the F340/F380 ratio over baseline), rose steadily and progressively during 30 min of ischemia, peaked at the first few min of reperfusion, and then dropped. It should be noted that cytosolic Ca2 + measurements contradict the morphological results which do not demonstrate any rise in [Ca2 + ]i during the early phase of ischemia, suggesting that mechanisms of Ca2 + overloading during ischemia are different from those during reperfusion. It is believed that the rise in [Ca2 + ]i during ischemia is primarily due to the failure of intracellular compartments to sequestrate cytosolic Ca2 + , while although Ca2 + influx is increased during reperfusion, elevated cytosolic Ca2 + tends to be mitigated by concomitant restoration of Ca2 + buffering ability between various intracellular compartments.

5. Mechanisms of intracellular Ca2 + overloading Intracellular Ca2 + accumulation is an essential feature of ischemia reperfusion injury. There are a number of possible mechanisms by which Ca2 + can gain access to the myocytes. The major mechanisms by which Ca2 + may enter into the myocytes are discussed below.

5.1. Voltage-dependent Ca 2 + channels Ca2 + may enter through the voltage-sensitive (slow) channels which are activated during ischemia by changes in membrane action potential, by action of specific neurotransmitters (e.g. norepinephrine), or by specific cell surface receptors [56]. These slow channels play an essential role in the excitation-contraction coupling in the cardiac muscle. Ca2 + infiltration through these channels during the plateau phase of action potential triggers Ca2 + release from the sarcoplasmic reticulum by

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Fig. 2. (a) Schematic diagram of the untracellular Ca2 + analyzer.(b) Recordings showing the effects of ischemia and reperfusion on ECG, fluorescence at 340 and 380 nm, and F340/F380 fluorescence ratio.

the mechanism commonly known as Ca2 + -induced Ca2 + release. However, the amounts of Ca2 + entering through the channels are very small, accounting for only less than one-tenth of that required to fully activate myofilaments upon contraction. Evidence that supports the role of Ca2 + channels in reperfusion injury is based on the observations that a number of Ca2 + slow channel blockers are able to reduce myocardial ischemia reperfusion injury. These slow channel blockers specifically interact with the a1 subunit of L-type voltage-operated Ca2 + channels. A list of first and second generation channel blockers are shown in Table 1.

Diltiazem Clentiazem

Cinnarizine Flunarizine

Amlodipine and lacidipine were included in the third generation category in a recent paper. [Mayer, O. Pharmacokinetics and pharmaodynamics of calcium blockers. Vnitr-Lek 1995,41:164-16].

a

Third generation

Nifedipine Nimodipine, nisoldipine nitrendipine, felodipine, amlodipinea, manindipine, isradipine, lacidipinea, niguldipine, mepirodipine, ludipine, 8663S, CD 349, MDl 72567 Amlodipine, lecidipine

First generation Second generation

Verapamil Anipami, falipamil gallopamil, tiapamil, RO-5967

Calcium blockers

Generation

Table 1 A list of first, second and third generation of selected calcium blockers

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A wide variety of Ca2 + channel antagonists including varapamil [57], nifedipine [58], diltiazem [59] and lidoflazine [60] have been found to reduce myocardial reperfusion injury (Table 1). The second generation dihydropyridine Ca2 + channel blocker, nisoldipine, when administered to hearts 60 min prior to global ischemia, was found to inhibit the increase in end-diastolic pressure during ischemia [61]. In addition, this agent improved the recovery of left ventricular developed pressure and coronary flow during reperfusion in a concentration-dependent manner. The maximal cardioprotective effect was observed in 10 − 8 M nisoldipine. Other studies have also shown beneficial effects of adding nisoldipine to the coronary perfusate prior to ischemia [62]. In this study, nisoldipine reduced the [Ca2 + ]i during both ischemia and reperfusion, and attenuated ischemia reperfusion injury as evidenced by better recovery of left ventricular functions. At 64 and 100 nM concentrations, nisoldipine reduced the formation of lipid peroxidation products, suggesting that this dihydropyridine was also able to reduce the oxidative stress which developed during reperfusion of the ischemic myocardium. In another study, nisoldipine was administered as a 4.5 mg/kg intravenous bolus over 3 min followed by 0.2 mg/kg over 60 min to six patients with symptoms of an acute myocardial infarction [63]. The authors of this study concluded that nisoldipine improved global and regional left ventricular function in patients with acute myocardial infarction within the first 24 h. Most of the studies using Ca2 + channel blockers indicate that such blockers are effective only if given prior to ischemia. The use of Ca2 + channel blockers after ischemia has not met with success [64]. It has now become apparent that the beneficial effects of Ca2 + channel blockers are primarily due to rapid cessation of electrical and contractile activities at the time of ischemia which helps to conserve energy for cellular reparative processes following ischemic insult. This contention has been substantiated by several reports [65]. In addition, Ca2 + channel blockers are unable to prevent intracellular Ca2 + overload when added during reperfusion [66]. Nevertheless, free radical scavenging activities of some Ca2 + channel blockers as observed is demonstrated in Fig. 3 and may warrant their use in the face of reperfusion.

5.2. Na + /Ca 2 + exchanger Substantial evidence exists to support the notion that the Na + /Ca2 + exchanger serves as the major physiological route of Ca2 + entry during reperfusion. Significant Na + influx is known to occur during ischemia in exchange for H + ions. The transport of Ca2 + involves a carrier-mediated transmembrane exchange of Na + for Ca2 + . During ischemia, acidosis induced by aerobic metabolism leads to enhanced exchange of intracellular H + for Na + . In addition, inhibition of Na + /K + -ATPase by energy depletion can also cause accumulation of intracellular Na + . More than 25 years ago, Glitsch et al. recognized Na + /Ca2 + exchange as an important route for Ca2 + influx into the cell [67]. More recent studies reveal that three Na + ions are exchanged for each Ca2 + in this exchange system, favoring Ca2 + influx at positive membrane potential and Ca2 + efflux at negative membrane

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potential. Myocardial ischemia and reperfusion cause an increase in intracellular Na + concentration [68]. The same author demonstrated that the Na + pump is inhibited during hypoxia and sustained during reperfusion; and this system could account for Na + load and massive Ca2 + influx via Na + /Ca2 + exchange [69]. Increases in Na + influx can also contribute towards the intracellular Ca2 + accumulation. For example, fast Na + channels are opened upon excitation of cell membranes in conjunction with Na + pump inhibition during ischemia/reperfusion. Ventricular tachycardia and ventricular fibrillation associated with the reperfusion of ischemic myocardium may cause Na + accumulation by this mechanism. Another and perhaps the more important route of Na + infiltration is through the exchange of Na + for H + ion. Glycogenolysis and ATP hydrolysis during ischemia lead to the accumulation of lactic acid, causing intracellular acidosis. However, sufficient pH gradient cannot develop during ischemia because of the concomitant reduction of extracellular pH due to a lack of H + washout. At the onset of reperfusion when adequate coronary flow is established, extracellular pH becomes normalized, causing the development of a large transmembrane pH gradient, thereby providing an optimum condition for Na + influx through Na + /H + exchange.

Fig. 3. Effect of Ca2 + slow channel blockers on the free radical generation during reperfusion of the ischemic myocardium.

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A number of studies have been conducted to inhibit Na + /Ca2 + exchange in an attempt to reduce myocardial ischemia reperfusion injury. The most effective drug for this purpose is amiloride, a potassium sparing diuretic that inhibits Na + influx. Most of the studies using amiloride have been found to be effective in reducing reperfusion injury. For example, amiloride at a 0.174 mM concentration significantly improved systolic contractility during reperfusion of rat heart following 30 – 60 min of global ischemia [70]. In a similar study, 0.1 mM of amiloride was able to improve both systolic and diastolic functions in the hypoxic-reoxygenated rat heart simultaneously, reducing the intracellular Ca + overload [71]. Similar studies have supported these reports and further demonstrated that 0.25 mM amiloride results in better recovery of left ventricular compliance and contractile functions in concert with reduction of creatine kinase release and improvement of myocardial segment shortening during post-ischemic reperfusion [72]. Recently, attempts have been made to use analogs of amiloride in reducing reperfusion injury. For example, 5-(N-ethyl-N-isopropyl) amiloride at a 3 mM concentration produced negative chronotropic and inotropic effects, but unlike amiloride increased coronary vascular resistance. As compared to amiloride, this commpound provided a lesser degree of protection from the reperfusion injury. Another amiloride analog, dichlorobenzamil, was also found to be effective in reducing Ca2 + overload in cultured chick embryo heart cells [73]. Recent evidence suggests that it is the Na + /H + exchange inhibition that mediates myocardial protection from ischemic reperfusion injury. It was suggested that return of physiologic pH upon reperfusion may activate Na + /H + exchange leading to net Ca2 + accumulation [44]. This hypothesis received support from the observation that reperfusion of the cardiomyocytes with dimethylamiloride, a Na + /H + exchange inhibitor, blocked the increase of pH after simulated reperfusion simultaneously amiliorating the reperfusion injury. On the other hand, dichlorobenzamil, a Na + /Ca2 + exchange inhibitor, caused the reduction of free Ca2 + during reperfusion, but failed to prevent reperfusion injury. Taken together these observations suggest that it is the pH paradox rather than changes in free Ca2 + that plays a significant role in reperfusion injury.

5.3. Ca 2 + /Calmodulin As mentioned earlier, ATP production by mitochondria is instrumental in potentiating intracellular Ca2 + accumulation during reperfusion. Accumulating evidence suggests that certain protein kinases play a role in the energy-requiring processes leading to Ca2 + influx. During ischemia, protein kinase C is translocated to the plasmalemmal membrane from the cytosol, and activated with the formation of a quarternary complex with phospholipids, diacylglycerol and calcium [74]. Activation of protein kinase C can cause Ca2 + influx into the cell by stimulating the Na + /H + exchange mechanism [75]. Protein kinase C can also alter Ca2 + influx through Ca2 + channels because these voltage-dependent channels are targets for phosphorylation by protein kinase C.

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Two different protein kinases are known to exist that are strictly dependent on Ca2 + . These two kinases are protein kinase C and Ca2 + /calmodulin-dependent protein kinase. Calmodulin is abundant in heart, and it is generally believed that many intracellular calcium activities are mediated by this calcium-receptor protein. This hypothesis is supported by the fact that Ca2 + /calmodulin antagonism is beneficial for myocardial preservation. For example, the antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) has been shown to protect ischemic myocardium from reperfusion injury [76]. Other calmodulin antagonists such as chlorpromazine and trifluoperazine have also been found to be effective in reducing myocardial injury associated with calcium overload [77]. The fact that ischemia-reperfusion induced acceleration of membrane phospholipid degradation is inhibited by Ca2 + /calmodulin antagonists chlorpromazine and trifluoperazine [78] suggests that Ca2 + /calmodulin activation of phospholipase A2 may be involved in the phospholipid breakdown process. A study has demonstrated the efficacy of Ca2 + /calmodulin antagonism in preventing reperfusion injury in a pig heart model undergoing coronary revascularization [79]. However, most of the calmodulin receptor bockers are also inhibitors of protein kinase C. In another related study, a highly specific calmodulin blocker, CGS 9343B, enhanced post-ischemic myocardial recovery in isolated pig heart as judged by improved regional as well as global myocardial functions, better preservation of high energy phosphate compounds, and reduced release of creatine kinase [80].

5.4. NO and Ca 2 + Recent evidence suggests that NO may play a significant role in regulation of myocardial contractility. For example, NO has been shown to reduce the contraction of isolated cardiomyocytes [81]. An increase in intracellular Ca2 + is crucial for the activation of NO synthase in endothelial cells in the presence of calmodulin [82]. Under these conditions, an increase in Ca2 + in the endothelium is likely to induce production of NO from L-arginine and cause relaxation of the coronary arteries via an increase in cGMP levels. Recently, it has been proposed that NO and cGMP may play key regulatory roles for Ca2 + entry into cells [83]. By using NOS and guanylate cyclase inhibitors, these authors demonstrated that the Ca2 + content of the internal stores apparently regulated NOS activity to generate NO, and NO activated guanylate cyclase to produce cGMP, which had a biphasic action on restoring Ca2 + entry after it had been blocked by NOS/guanylate cyclase inhibitors. It is generally believed that NOS can transfer electrons from NADPH to molecular oxygen via FAD or FMN to form superoxide anion and H2O2 in the presence of Ca2 + [84]. Ca2 + requirement for in vitro NOS expression was first shown in late nineties [85]. Various calmodulin antagonists and calmodulin-binding proteins can inhibit the catalytic activity of NOS [86]. In another recent study, Ca2 + was found to regulate NO production at the mRNA level [87]. These authors clearly demonstrated that NOS mRNA induction was inhibited by calcium ionophore, A23187, ionomycin and thapsigargin that cause an increase in [Ca2 + ]i.

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Another related study showed that NO donor compounds that can generate NO in aqueous solutions inhibit Ca2 + release from isolated sarcoplasmic reticulum [88]. In skeletal muscle, Ca2 + release activity is decreased when the medium was supplemented with L-arginine. This effect of L-arginine was prevented by L-NAME, an inhibitor of NOS. Their results further suggested that NO directly affects the sarcoplasmic reticulum Ca2 + release machinery because, exogenously added cGMP did not block the Ca2 + release. Such effect of NO on sarcoplasmic reticulum Ca2 + release could account for the NO-induced force reduction in cardiac muscle [89].

6. Ca2 + blockers in patient care Due to the vasodilatory properties of the Ca2 + channel blockers, these drugs were thought to be potentially useful for patients suffering from acute ischemia. However, the results of clinical trials with Ca2 + blockers have not been encouraging. For many patients, calcium antagonism have been found to be harmful, prinicipally because these drugs acutely lower blood pressure. As a result, the renin-angiotensin system is stimulated, leading to the increase in Na + and water retention. Additionally, some of these Ca2 + channel blockers exert a negative inotropic effect and cause tachycardia. Some of the second generation Ca2 + channel blockers possess minimal negative inotropic action on hearts, and have been found to be of some use for ischemic patients. For example, 2 mg/kg and 4 mg/kg nisoldipine was used over a period of 3 min to study the hemodynamic effects of 12 patients with acute myocardial infarction [90]. A 20–30% reduction in vascular resistance in conjunction with a significant rise in cardiac index were observed in this study for patients with both normal and abnormal left ventricular filling pressure, suggesting that this dihydropyrine Ca2 + channel blocker may be beneficial for patients with and without left ventricular failure. In another study, nisoldipine improved global and regional left ventricular functions in patients with acute myocardial infarction within the first 24 h. On the contrary, Wilson et al. [91] observed that nisoldipine at a bolus dose of 4 mg/kg resulted in undue tachycardia or hypotension to the patients with acute myocardial infarction. Clinical trials using the first generation of Ca2 + channel blockers have proven unsuccessful. For example, there have been several studies with nifedipine for the treatment of acute myocardial infarction [92]. A Norwegian multicenter trial found the use of nifedipine to be ineffective for the reduction of myocardial ischemic reperfusion injury [93]. Four large clinical trials, TRENT (trial of early nifedipine in acute myocardial infarction) [94], SPRINT (secondary prevention reinfarction Israeli nifedipine trial [95], MDPIT (multicenter diltiazem postinfarction trial) [96] and DAVIT II (Danish verapamil in myocardial infarction trial II) [97], all failed to show any beneficial effects for the patients. In another study, nifedipine, diltiazem and verapamil were found to create increased risk of myocardial infarction both among subjects with CVD and among those without CVD [98]. Results of many secondary prevention randomized clinical trials comparing Ca2 + channel blockers have proven to be harmful or without any effect [99].

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Fig. 4. Intracellular signaling of Ca2 + in ischemic reperfused myocardium.

These results suggest that Ca2 + antagonism may not be useful in treating patients with acute myocardial infarction. On the other hand, Ca2 + channel blockers may be of some use for the stunned myocardium where the contractile functions of the heart are reduced after revascularization. It is believed that Ca2 + channel blockers may be useful for selected groups of patients including those with small hearts and angina.

7. Summary and conclusion It should be clear from this review that intracellular Ca2 + overloading plays a central role in the pathogenesis of myocardial ischemia reperfusion injury. A small amount of Ca2 + may gain access into the cell during ischemia resulting from ischemic membrane depolarization and opening of voltage sensitive and ligandgated Ca2 + slow channels. This Ca2 + influx may or may not increase the cytosolic Ca2 + level appreciably depending on the balance between the magnitude of Ca2 + influx and efficiency of intracellular compartments to sequester and extrude Ca2 + out of the cytosol. However, even this low amount of Ca2 + may serve as a signal to potentiate several cascades of metabolic events, including massive intracellular Ca2 + overloading during reperfusion, and may influence the ultimate severity of reperfusion injury. As mentioned earlier, Ca2 + serves as a second messenger in the cardiac cell leading to the activation of a large number of intracellular enzymes and regulatory proteins. A relatively larger amount of Ca2 + may gain access into the myocytes through Na + /Ca2 + exchange and calmodulin receptor mediated transport mechanism. The precise intracellular signaling mechanism of Ca2 + during ischemia reperfusion is not completely understood (Fig. 4). The high level of intracellular Ca2 + can very easily activate a number of intracellular enzymes including Ca2 + -sensitive proteases and phospholipases. Activation of phospholipases A2 and C can result in the formation of free fatty acids, predominantly arachidonic acid. Arachidonic acid

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may not only disrupt the membrane by virtue of its detergent-like properties, it may also act as an ionophoretic agent to pull more Ca2 + . Furthermore, arachidonic acid is a well known substrate for cyclooxygenase. Activation of the arachidonic acid cascade results in the formation of protaglandins and thromboxanes, the former being a substrate for oxygen free radical production. On the other hand, phospholipase C-catalyzed hydrolysis of phosphoinositides generates IP3 and diacylglycerol. The former can mobilize intracellular Ca2 + , while the latter can activate protein kinase C that is indirectly involved in exacerbation of Ca2 + overload via the Ca2 + channel or via activation of Na + /H + exchange. Activated proteases can also cause damage to the heart during reperfusion. It has been hypothesized that activation of such proteases can lead to the conversion of xanthine dehydrogenase into xanthine oxidase which in turn catalyzes the formation of hypoxanthine and xanthine. Both hypoxanthine and xanthine are necessary substrates for the formation of oxygen free radicals during reperfusion of ischemic myocardium. Thus, Ca2 + may be instrumental in the formation of oxygen free radicals that contribute to reperfusion injury. Electrophysiological dysrrangements during reperfusion have been attributed to intracellular Ca2 + overloading. Ca2 + can directly lead to ventricular arrhythmias by provoking random diastolic release of Ca2 + from the sarcoplasmic reticulum or indirectly by forming oxygen free radicals that are known to cause electrophysiological abnormalities. Interference with the events of intracellular Ca2 + overloading presumably attenuates myocardial ischemia reperfusion injury. Thus, inhibition of Ca2 + slow channel, Ca2 + /calmodulin antagonism and inhibition of Na + /Ca2 + exchange all have been found to amiliorate reperfusion injury in various degrees. However, the exact metabolic events of the Ca2 + signaling mechanism are far from clear. Better understanding of intracellular Ca2 + signaling during ischemia and reperfusion will facilitate clinical application of Ca2 + channel blockers in patient care.

Acknowledgements This work was partially supported by the National Heart, Lung and Blood Institutes Grants HL 22559 and HL 33889.

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