Improvement of Cardiac Function by Polyclonal Antibodies Against Ca2+/Mg2+ ecto-ATPase in Hearts Subjected to Ischemia-Reperfusion

TL;DR

This study shows that antibodies against a specific enzyme can improve heart function after injury by reducing calcium overload.

Contribution

The novel finding is that inhibiting Ca2+/Mg2+ ecto-ATPase with antibodies improves recovery after heart injury.

Findings

Pretreatment with antibodies improved recovery of cardiac function after ischemia-reperfusion injury.

Antibodies reduced Ca2+/Mg2+ ecto-ATPase activity and attenuated intracellular calcium overload.

Treatment with antibodies decreased ATP-induced calcium increases in heart cells.

Abstract

Delayed reperfusion of an ischemic heart is known to impair the recovery of cardiac function, and the occurrence of intracellular Ca2+ overload in the myocardium is considered to play a critical role in the development of ischemia-reperfusion (I/R) injury. Since Ca2+/Mg2+ ecto-ATPase, which is activated by millimolar concentrations of Ca2+ or Mg2+, has been shown to serve as a Ca2+ gating mechanism for the entry of Ca2+ and subsequent development of intracellular Ca2+ overload, we investigated the role of depression in Ca2+/Mg2+ ecto-ATPase activity by polyclonal antibodies against Ca2+/Mg2+ ecto-ATPase in promoting the recovery of cardiac function in isolated perfused rat hearts upon subjection to I/R injury. Incubation of sarcolemma (SL) membranes with immune serum or purified IgG antibody fraction was found to depress both Ca2+-ATPase and Mg2+-ATPase activities. Pretreatment of hearts with immune serum or purified antibodies was observed to improve the recovery of cardiac function and depress the SL Ca2+/Mg2+ ecto-ATPase activities in hearts subjected to I/R injury. A marked increase in myocardial Ca2+ content in I/R hearts was also attenuated by immune serum treatment. Furthermore, treatment of cardiomyocytes from normal hearts with immune serum or purified antibodies reduced the ATP-induced increase in intracellular Ca2+ concentration. These results suggest that improvement in the recovery of cardiac function in hearts subjected to I/R injury by polyclonal Ca2+/Mg2+ ecto-ATPase antibodies may be due to the attenuation of intracellular Ca2+ overload.

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1. Introduction

Reperfusion of an ischemic heart is essential for the salvage of ischemic myocardium; however, delayed reperfusion is known to impair the recovery of cardiac function in the ischemic heart. This phenomenon is associated with alterations in myocardial energy metabolism as well as ultrastructural defects and is termed as ischemia-reperfusion (I/R) injury [1,2,3,4,5,6,7,8,9,10]. I/R injury is invariably observed in clinical procedures such as angioplasty, thrombolysis, coronary by-pass surgery and cardiac transplantation [11,12,13,14,15,16,17]. Although mechanisms for the occurrence of cardiac dysfunction due to I/R injury are of complex nature [18,19,20,21,22], it is generally held that the development of intracellular Ca^2+^ overload plays a critical role in inducing cell damage, changes in myocardial metabolism and subcellular abnormalities in the heart [23,24,25,26,27,28,29]. This view is supported by the fact that inhibitors of various sarcolemma (SL) sites, such as voltage-dependent Ca^2+^ channels, Na^+^- Ca^2+^ exchange, and store-operated Ca^2+^ channels, have been shown to reduce the entry of Ca^2+^ into cardiomyocytes and exert varying degrees of cardioprotective effects in hearts subjected to I/R injury [30,31,32,33,34,35,36,37]. Different antioxidants and blockers of purinergic P2Y receptor activation were also observed to depress Ca^2+^ influx and attenuate I/R injury in the heart [23,34,38,39].

Since Ca^2+^/Mg^2+^ ecto-ATPase, which is maximally activated by 4 mM Ca^2+^ or Mg^2+^, has been reported to serve as a Ca^2+^ gating mechanism in the SL membrane for the entry of Ca^2+^ [40,41,42], it is likely that inhibitory interventions for Ca^2+^/Mg^2+^ ecto-ATPase activity may exert beneficial effects in hearts subjected to I/R injury. It is pointed out that we have isolated and purified Ca^2+^/Mg^2+^ ecto-ATPase from cardiac sarcolemma and have prepared immune serum and polyclonal antibodies fractions, which were found to inhibit this enzyme markedly [43,44,45,46]. Accordingly, this study was undertaken to investigate the effects of an immune serum and a purified polyclonal immunoglobulin G (IgG) antibody fraction against Ca^2+^/Mg^2+^ ecto-ATPase on cardiac function in the isolated perfused hearts subjected to I/R injury. In addition, the effects of treatments with the immune serum and/or antibody fraction were studied on SL Ca^2+^/Mg^2+^ ecto-ATPase activities and myocardial Ca^2+^ content in hearts with or without I/R injury. Furthermore, the effects of the immune serum or purified antibody fraction were examined on the ATP-induced increase in the intracellular concentration of free Ca^2+^ [Ca^2+^]i in cardiomyocytes isolated from normal rat hearts.

2. Results

Although the immune serum or polyclonal antibody IgG fraction has been reported to inhibit the purified cardiac Ca^2+^/Mg^2+^ ecto-ATPase activity [45], this study tested whether these interventions affect the Ca^2+^/Mg^2+^ ecto-ATPase enzyme, which is embedded in the SL membrane, isolated by the hypotonic shock-LiBr method [47]. The results in Table 1 show that SL Ca^2+^/Mg^2+^ ecto-ATPase activities in the presence of 4 mM Ca^2+^ or 4 mM Mg^2+^ were decreased by immune serum in a concentration-dependent manner. Incubation of SL membranes with the 10 μg/mL antibody fraction also inhibited Ca^2+^/Mg^2+^ ecto-ATPase activities. In another set of experiments, the incubation of the plasma membrane, isolated by the sucrose density gradient method [44], with non-immune and immune (mg/mL) preparations showed that Ca^2+^/Mg^2+^ ecto-ATPase activities (in the presence of 4 mM Ca^2+^) were: control 150 ± 6.9, non-immune serum 142 ± 5.5 and immune serum 36 ± 3.8 (μmol Pi/mg protein/h)

In order to test if the ATPase activity in the SL membrane is affected by purinergic receptor inhibitors or Ca^2+^ antagonists, SL membranes, isolated by the hypotonic shock- LiBr method [47], were also treated with pyridoxal 5′-phosphate (25 μM) and Cibacron Blue (100 μM), purinergic P2Y receptor inhibitors, and verapamil (10 μM), a Ca^2+^ antagonist [38,39]. The Ca^2+^/Mg^2+^ ecto-ATPase activities (in the presence of 4 mM Ca^2+^) were: control 40 ± 2.6; pyridoxal 5′-phosphate 41 ± 3.0; Cibacron Blue 38 ± 2.7 and verapamil– 39 ± 3.1 (μmol Pi/mg protein/h).

2.1. Effects of Immune Serum or Antibody Fractions on Cardiac Function

In order to examine the effects of immune preparations on the I/R-induced recovery of cardiac function, isolated perfused hearts were treated with non-immune and immune serum (0.5 mg/mL) for 10 min before the induction of global ischemia for 30 min followed by reperfusion for a period of 60 min. From the results shown in Figure 1, it can be seen that reperfusion of the ischemia hearts treated with non-immune serum showed depressed LVDP and ±dP/dt values as well as elevated LVEDP. On the other hand, treatment with immune serum against Ca^2+^/Mg^2+^ ecto-ATPase showed a marked recovery of LVDP and ±dP/dt values, as well as a significant depression in the elevated LVEDP upon reperfusion of the ischemic hearts. It should be mentioned that in a separate set of experiments both non-immune and immune serum treatments were found to exert no significant effects on the contractile activity of normal hearts. Furthermore, the pattern of changes in reperfusion-induced recovery of cardiac function in ischemic hearts with or without non-immune serum treatment was not different from each other.

In order to investigate if the reperfusion-induced changes in the recovery of cardiac function are associated with alterations in Ca^2+^/Mg^2+^ ecto-ATPase activity, SL membranes were isolated from the I/R hearts (30 min ischemia followed by 30 min reperfusion) at the termination of reperfusion period. Treatments of I/R hearts with 0.3 to 1.0 mg/mL concentrations of immune serum showed concentration-dependent improvement in LVDP, a slight but significant decrease in elevated LVEDP and a depression in SL Ca^2+^/Mg^2+^ ecto-ATPase activities (Table 2). Treatment with 0.1 mg/mL immune serum showed no significant effect on any parameters. Improved cardiac function and depressed SL Ca^2+^/Mg^2+^ ecto-ATPase activities were also seen in I/R hearts upon treatment with different concentrations of the purified antibody IgG fraction (Table 2).

From the data in Table 3, it is evident that reperfusion for different intervals of the ischemic hearts (treated with non-immune serum) showed impaired recovery of LVDP, elevated LVEDP and no changes in Ca^2+^/Mg^2+^ ecto-ATPase activity. On the other hand, a progressive and improved recovery of LVDP and an increase at 3 min followed by a significant decrease at 5 to 20 min in LVEDP in I/R hearts treated with immune serum were shown (Table 4). Reperfusion of the ischemic heart treated with immune serum showed no change at 3 min but produced significant depression in ecto-ATPase activity at 5 to 30 min (Table 4). A comparison of data for different parameters in Table 3 and Table 4 reveals that reperfusion of ischemic hearts treated with immune serum produced no significant recovery of LVDP or change in Ca^2+^/Mg^2+^ ecto-ATPase activity at 3 min compared to alterations induced in ischemic hearts treated with non-immune serum. However, ischemic hearts treated with immune serum showed a marked recovery of LVDP, significant depression in the elevated LVEDP and a reduction in the ecto-ATPase activity at 5 to 30 min of reperfusion.

In another set of experiments, cardiac function and myocardial Ca^2+^ content were determined in control, ischemic and reperfused hearts. Reperfusion (30 min) of the ischemic hearts (30 min ischemia) was carried out in preparations treated with non-immune and immune serum, and myocardial Ca^2+^ content was determined at the termination of the experiment. The results in Table 5 show that impaired recovery of cardiac function was associated with an increased level of myocardial Ca^2+^ content in I/R hearts treated with non-immune serum. On the other hand, improved recovery of cardiac function and a depression in myocardial Ca^2+^ content were observed in I/R hearts treated with immune serum (Table 5).

2.2. Effects of Immune Preparations on Cardiomyocyte [Ca2+]i

Since Ca^2+^/Mg^2+^ ecto-ATPase is known to be activated by ATP for promoting the entry of Ca^2+^ and is considered to contribute to the occurrence of intracellular Ca^2+^ overload in cardiomyocytes [40,41,42], inhibition of ATP-induced increase in [Ca^2+^]i by immune preparations is expected to prevent the occurrence of intracellular Ca^2+^ overload. Accordingly, the effects of non-immune and immune serum as well as non-immune IgG fraction and antibody IgG fraction were examined on ATP-induced changes in [Ca^2+^]i in cardiomyocytes isolated from normal hearts. It can be seen from Figure 2 that treatment of cardiomyocytes with immune serum, unlike non-immune serum, depressed the ATP-induced increase in [Ca^2+^]i in a concentration-dependent manner. The depressant effect of immune serum on the ATP-induced increase in [Ca^2+^]i was of a specific nature because the treatment of cardiomyocytes with immune serum did not affect the increase in [Ca^2+^]i due to depolarization with KCl or stimulation with the Ca^2+^ receptor agonist Bay-K8644 (Figure 2). The data in Table 6 also show that pretreatment of cardiomyocytes with polyclonal antibodies, unlike the non-immune IgG fraction, inhibited the ATP-induced increase in [Ca^2+^]i in a concentration-dependent manner without affecting the basal [Ca^2+^]i levels in cardiomyocytes.

In order to show whether the depression in the ATP-induced increase in [Ca^2+^]i by polyclonal antibodies is of a specific nature, the effects of some purinergic receptor inhibitors and a Ca^2+^ antagonist [34,38] on the ATP-induced increase in [Ca^2+^]i in cardiomyocytes were studied. Treatments of cardiomyocytes with Cibacron Blue (100 μM), pyridoxal 5′- phosphate (25 μM) and verapamil (10 μM) were found to depress the ATP-induced increase in [Ca^2+^]I, as the values for the control and Cibacron Blue-, pyridoxal 5′-phosphate- and verapamil-treated preparations were 46 ± 2.4, 10 ± 3.4, 17 ± 2.6 and 20 ± 2.5 nM Ca^2+^, respectively. No changes in basal [Ca^2+^]i were observed by these treatments.

3. Discussion

In this study we have demonstrated that pretreatment of the ischemic heart with immune serum or purified polyclonal antibodies against Ca^2+^/Mg^2+^ ecto-ATPase improved the recovery of cardiac function upon reperfusion. This cardioprotective effect of the immune serum/antibody fraction on the ischemic hearts was evident at 3 to 60 min of reperfusion period and appears to be a consequence of preventing metabolic changes in the myocardium due to I/R injury. In this regard, it is pointed out that several studies have revealed that an increase in ATP concentration occurs in the extracellular space of myocardium during the development of I/R injury [48,49,50,51,52]. Furthermore, the extracellular ATP is known to activate Ca^2+^/Mg^2+^ ecto-ATPase in the SL membrane and promote the entry of Ca^2+^ into cardiomyocytes for the occurrence of intracellular Ca^2+^ overload [40,41,42,53]. It should be noted that the incubation of SL membranes with immune serum/antibody fractions was observed to inhibit Ca^2+^/Mg^2+^ ecto-ATPase activity, and pretreatment of the ischemic hearts with these interventions was also found to inhibit Ca^2+^/Mg^2+^ ecto-ATPase activity in SL membranes isolated from the I/R hearts. Such a depression in the ATP-induced activation of Ca^2+^/Mg^2+^ ecto-ATPase in the hearts subjected to I/R injury can be seen to reduce Ca^2+^ entry and prevent the occurrence of intracellular Ca^2+^ overload. This view is supported by our observation that the elevated level of myocardial Ca^2+^ in I/R hearts was markedly reduced upon pretreatment with immune serum. It is noteworthy that the development of intracellular Ca^2+^ overload in hearts subjected to the Ca^2+^ paradox has been reported to be associated with a depression in SL Ca^2+^/Mg^2+^ ecto-ATPase activity [47,54]. The generation of superoxide radicals as well as H_2_O_2_ and hydroxyl radicals have also been shown to depress SL Ca^2+^/Mg^2+^ ecto-ATPase, respectively [55]. Thus, the depression in SL Ca^2+^/Mg^2+^ ecto-ATPase observed in this study may be due to the excessive formation of oxyradicals and the development of oxidative stress due to I/R injury [56]. In view of the effect of oxidative stress on SL Ca^2+^/Mg^2+^ ecto-ATPase activity, it is possible that the addition of an oxidant to the antibody preparation used in this study may modify its cardioprotective action. However, we did not carry out any experiment in this regard.

It was observed that the immune serum or antibody preparation not only decreased the SL Ca^2+^/Mg^2+^ ecto-ATPase but also attenuated the ATP-induced increase in [Ca^2+^]i in cardiomyocytes. Such an action of polyclonal antibodies on the ATP-induced increase in [Ca^2+^]i can be seen to inhibit Ca^2+^ entry into cardiomyocytes, reduce the development of intracellular Ca^2+^ overload and increase the recovery of cardiac function in I/R hearts. Since purinergic receptor inhibitors (pyridoxal 5′-phosphate and Cibacrone Blue) have also been reported to attenuate the ATP-induced increase in [Ca^2+^]i cardiomyocytes and improve cardiac function in I/R hearts [34,38,49], it can be argued that the cardioprotective effects of polyclonal antibodies may be mediated through P2Y receptor inhibition. However, this seems unlikely because pyridoxal 5′-phosphate and Cibacrone Blue were observed to exert no effect on the ATP-induced activation of SL Ca^2+^/Mg^2+^ ecto-ATPase or the ATP-induced increase in [Ca^2+^]i in cardiomyocytes. Since purinergic receptors other than P2Y receptors are also present in the myocardium [50], extensive work needs to be carried out to rule out the role of purinergic receptor inhibition in the action of polyclonal antibodies. It is, however, pointed out that the inhibitory effect of the polyclonal antibody fraction was specific, as it did not modify the depolarization-induced increase in [Ca^2+^]i by KCl or activation by the Ca^2+^ receptor agonist Bay-K 8644 [34,38]. It is possible that ATP may induce an increase in [Ca^2+^]i by two separate mechanisms: one by activating the Ca^2+^ gating (Ca^2+^/Mg^2+^ ecto-ATPase) mechanism directly and the other by activating Ca^2+^ channels through purinergic P2Y receptors. Thus, the ability of polyclonal antibodies or immune serum to improve cardiac function in I/R hearts may be related to their direct action on Ca^2+^/Mg^2+^ ecto-ATPase for preventing the occurrence of Ca^2+^ overload.

Irrespective of the exact mechanism of the beneficial effect of the polyclonal antibodies in improving the recovery of cardiac function, the present study is the first to demonstrate that SL Ca^2+^/Mg^2+^ ecto-ATPase is a potential target for drug development for the treatment of cardiac complications due to ischemia-reperfusion injury. This view is consistent with our previous observation indicating the involvement of SL Ca^2+^/Mg^2+^ ecto-ATPase activation in the development of intracellular Ca^2+^ overload in the diseased heart [40,41,42]. It is also pointed out that a humanized monoclonal antibody targeting Ectonucleotidase ENPP1 has recently been shown to rescue cardiac metabolism and heart function after myocardial infarction [57], and the inhibitors of ecto-nucleoside triphosphate diphosphohydrolase are recommended for the treatment of various cardiovascular diseases [58]. A recombinant soluble form of human CD3/Ectonucleotidase is considered to be a potential therapeutic agent for thrombotic disease [59]. Thus, it is becoming apparent that several antibodies and other preparations interacting with different targets for ATP-induced activation or ATP degradation are becoming available for the future development of interventions for the treatment of cardiovascular diseases.

In view of the complex pathophysiology of cardiac dysfunction due to I/R injury [60,61,62,63,64], different interventions have been reported to attenuate the SL, sarcoplasmic reticulum and mitochondrial defects for improving the recovery of cardiac function in I/R hearts [65,66,67,68,69,70,71,72,73,74,75,76,77,78]. It should be mentioned that several pharmacological and non-pharmacological approaches including antioxidants, endothelin-1 antagonists, urotensin-II receptor antagonists and PAR-1 antagonists have been indicated in the past for preventing I/R-induced injury [23,79,80,81,82,83,84]. However, it is our contention that strategies for employing antibody interventions against specific SL targets for the control of Ca^2+^ movements may prove more beneficial.

4. Materials and Methods

The protocols (# 96-022) used in this study were approved by the University of Manitoba Animal Care Committee in accordance with guidelines of the Canadian Council on Animal Care.

4.1. Isolated Perfused Rat Heart Preparations

Male Sprague–Dawley rats (250–300 g each) were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (9 mg/kg). The isolated hearts were mounted on a Langendorff apparatus and perfused with Krebs–Henseleit medium at (37° C) gassed with a mixture of 95% oxygen and 5% carbon dioxide at a flow rate of 10 mL/min. The hearts were electrically stimulated at 300 beats/min, and the left ventricular developed pressure (LVDP), the left ventricular end-diastolic pressure (LVEDP) and the rate of change in pressure (±dP/dt) were recorded by a transducer (model 1050 BP, Biopac System, Goleta, CA, USA), which was connected with a water-filled latex balloon inserted into the left ventricle. After a stabilization period of 20 min, the hearts were made ischemic by stopping the coronary flow for 30 min, whereas reperfusion was carried out for 30 to 60 min by switching on the perfusion flow. The procedures for the isolated perfused heart preparations as well as for the induction of ischemia-reperfusion (I/R) were similar to those described earlier [29,34]. Treatments of the heart with different concentrations of non-immune and immune serum or non-immune IgG and immune antibody IgG fractions were carried out by infusion in the perfusion stream for a period of 10 min before inducing global ischemia. Control hearts were perfused for a comparable period without any treatment.

4.2. Preparation of Immune Serum and Polyclonal Antibody Fraction

Both non-immune serum and immune serum against purified Ca2+/Mg2+ ecto-ATPase [43,44] were generated in New Zealand white rabbits by Cedarlane Laboratories Limited (Burlington, ON, Canada) according to the procedure described elsewhere [85]. The method for preparing purified immunoglobulin G (IgG) fractions from non-immune/immune serum was also the same as described earlier [85]. Both these antiserum and IgG antibody fractions were found to inhibit Ca2+/Mg2+ ecto-ATPase specifically [45,46].

4.3. Measurement of SL Ca2+/Mg2+ Ecto-ATPase Activities and Myocardial Ca2+ Content

The SL membranes were isolated from control, ischemic and I/R hearts by the hypotonic shock-LiBr treatment technique as described earlier [47]. The measurement of Ca2+/Mg2+ ecto-ATPase activities was carried out by incubating the SL membrane in a medium containing 50 mM Tris-HCL, pH 7.4 at 37° C in the presence of 4 mM Ca2+ or 4 mM Mg2+. The ATPase reaction was initiated by the addition of 4 mM ATP. The concentration of inorganic phosphate (Pi) released in the protein-free supernatant was assayed by the method described before [47]. The Ca2+/Mg2+ ecto-ATPase activities were also determined upon isolating SL membranes from the normal unperfused hearts. For studying the effects of non-immune and immune serum or polyclonal antibody fraction on ATPase activities in vitro, SL membranes were pretreated with these interventions for 5 min before initiating the reaction with ATP. Myocardial Ca2+ content of the control, ischemic and I/R hearts with non-immune or immune serum treatment was also determined according to the procedure described elsewhere [54].

4.4. Measurement of [Ca2+]i in Cardiomyocytes

Purified cardiomyocytes were isolated from the normal unperfused hearts (ventricles) by the method described previously [29,34]. For determination of [Ca2+]i, cardiomyocytes were incubated with 5 μM Fura-2 AM for 40 min and washed to remove the extracellular dye. The concentration of [Ca2+]i in cardiomyocytes was calculated by monitoring the intensity of fluorescence by a SLM DMX-1100 dual-wavelength spectrofluorometer (SLM Instruments, Urbana, IL, USA) as described earlier in detail [29,34]. Alterations in [Ca2+]i due to different agents, which are known to promote Ca2+ entry, were studied; ATP (50 μM)-induced, KCl (30 mM)-induced or Bay-K8644 (2 μM)-induced increases in [Ca2+]i were examined in Fura-2 AM-loaded cardiomyocytes with or without different treatments [29,34]. Pretreatments of cardiomyocytes were carried out by incubation with different concentrations of non-immune and immune serum or polyclonal antibodies for 5 min before monitoring [Ca2+]i.

4.5. Statistical Analysis

The results were expressed as mean ± SE. The difference between two groups was evaluated by the Student’s t-test. Values showing p < 0.05 were considered significant. Since different concentrations of non-immune preparations did not show any significant effect either on Ca2+- or Mg2+-ATPase, their values were grouped together.

5. Conclusions

It is now well known that I/R injury is a major health hazard, as there occurs an impaired recovery of cardiac function upon delayed reperfusion of the ischemic heart. In this study, we have reported the beneficial effects of an immune serum and/or polyclonal antibodies against SL Ca^2+^/Mg^2+^ ecto-ATPase in improving cardiac function of I/R hearts. Since the activation of Ca^2+^/Mg^2+^ ecto-ATPase has been shown to promote Ca^2+^ entry and cause intracellular Ca^2+^ overload for inducing myocardial cell damage and impairing cardiac function, treatment of the ischemic heart with polyclonal antibodies was observed to depress the activation of Ca^2+^/Mg^2+^ ecto-ATPase, attenuate the ATP-induced increase in [Ca^2+^]i, reduce the myocardial Ca^2+^ content and promote cardiac function in I/R hearts. On the basis of these results, it is suggested that polyclonal antibodies against Ca^2+^/Mg^2+^ ecto-ATPase improve the recovery of cardiac function in I/R hearts by depressing the activation of SL Ca^2+^/Mg^2+^ ecto-ATPase and preventing the occurrence of Ca^2+^ overload. We believe that polyclonal antibodies against Ca^2+^/Mg^2+^ ecto-ATPase are an excellent intervention for developing a highly viable treatment of ischemic heart disease.