Indolylmaleimide Derivative IM-17 Shows Cardioprotective Effects in Ischemia-Reperfusion Injury.

We previously developed IM-54 as a novel type of inhibitor of hydrogen-peroxide-induced necrotic cell death. Here, we examined its cell death inhibition profile. IM-54 was found to selectively inhibit oxidative stress-induced necrosis, but it did not inhibit apoptosis induced by various anticancer drugs or Fas ligand, or necroptosis. IM-17, an IM derivative having improved water-solubility and metabolic stability, was developed and confirmed to retain necrosis-inhibitory activity. IM-17 showed cardioprotective effects in an isolated rat heart model and an in vivo arrhythmia model, suggesting that IM derivatives may have therapeutic potential.

In recent decades, cell death has been recognized to play an important role in the development and maintenance of multicellular organisms. Originally, cell death was divided into two major categories, apoptosis and necrosis according to the morphological features.[1] Apoptosis involves characteristic and regulated changes, such as membrane blebbing, nuclear condensation, and formation of apoptotic bodies. On the other hand, necrosis is associated with cellular swelling and rupture of the cellular membrane, which could be induced by physical damage. Therefore, necrosis is thought to be accidental and unregulated cell death, and only apoptosis is considered as naturally occurring cell suicide, so-called “programmed cell death”. Most cell death research has focused on apoptosis, and the molecular mechanisms and physiological importance of apoptosis have been well characterized.[2] In particular, a family of proteases, called caspase, was identified as mediators and executors of apoptotic signals, and caspase-mediated apoptosis was found to play an important role in the selection of lymphocytes[3,4] and in cellular immunity.[5] Inhibition of apoptosis is thought to be involved in the pathogenesis of various diseases, such as cancer, autoimmune disease, and AIDS.

On the other hand, abnormal acceleration of cell death is known to cause various diseases.[2] In some cases, such as neurodegenerative diseases[6] or infarction,[7,8] necrosis was found to contribute to critical damage leading to lethality. Rupture of the cell membrane, a typical hallmark of necrosis, causes the release of various factors from cells, including Ca2+, glutamate, and proteases, which cause damage to surrounding cells and expand the damaged area. Therefore, the reduction of necrosis-induced damage may be a potent therapeutic approach for diseases involving acceleration of cell death.

We previously developed a bisindolylmaleimide derivative MS-1 (Figure )[9] from a well-known PKC inhibitor, BM I, and reported that it inhibited necrotic cell death induced by H2O2 through PKC-independent mechanisms. This molecule was also found to reduce the area of myocardial infarction in an in vivo rat ischemia-reperfusion injury model,[10] in which oxidative-stress-induced necrosis is thought to be involved.[11,12] This fact suggested that MS-1 could be a novel therapeutic lead. However, MS-1 showed cytotoxicity and inhibitory activities toward several kinases at high concentrations.[9] Further work led to the development of IM-54,[13−15] which shows strong inhibition of H2O2-induced necrosis (comparable to MS-1), with greatly reduced cytotoxicity.[13] In addition IM-54 did not show significant inhibitory activities against a panel of 467 kinases (Tables S1 and S2). Therefore, IM-54 is also expected to have a therapeutic effect on ischemia-reperfusion injury. Here, we report the cell death inhibition profile of IM-54, as well as the protective effect of a new water-soluble IM derivative against ischemia-reperfusion injury in rat heart.

Figure 1

Structures of MS-1 and IM-54.

First, we examined the effects of IM-54 on various types of cell death (Figure ). HL-60 cells were treated with various cell death inducers in the presence or absence of IM-54 or Z-VAD, a general caspase inhibitor. Cell viability was determined by AlamarBlue assay (Figure A), and morphological changes were observed by phase-contrast imaging (Figure B). As shown in Figure B, HL-60 cells showed typical morphological changes of apoptosis (blebbing and formation of apoptotic bodies) and necrosis (swelling and rupture of the cell membrane). We found that IM-54 inhibited necrosis induced by oxidative stress (TBHP and H2O2), whereas Z-VAD did not. On the other hand, IM-54 did not inhibit apoptosis induced by anticancer drugs (actinomycin D, camptothecin, and etoposide) or physiological death ligand (Fas ligand), which was strongly inhibited by Z-VAD in each case. Interestingly, at a low concentration, H2O2 was found to induce both apoptotic and necrotic cell death (Figures C). In this case, apoptotic cell death was inhibited by Z-VAD, and necrotic cell death was inhibited by IM-54, and cotreatment with Z-VAD and IM-54 completely inhibited both apoptotic and necrotic cell death (Figures C and 2D). These results imply a complementary character of IM-54 and Z-VAD as cell death inhibitors. In our previous study, IM-54 itself did not react directly with H2O2, and the data in Figure C also support the idea that IM-54 is not a sacrificial antioxidant, which could inhibit both apoptotic and necrotic cell death.

Figure 2

Cell death inhibition profile of IM-54. (A, B) Effects of IM-54 and Z-VAD on various types of cell death. HL-60 cells were treated with various stimuli, actinomycin D (1 μM, 6 h), camptothecin (1 μM, 6 h), etoposide (100 μM, 4 h), Fas ligand (FasL) (100 ng/mL, 18 h), H2O2 (100 μM, 3 h), or tert-butyl hydroperoxide (TBHP) (30 μM, 3 h) in the presence or absence of IM-54 (10 μM) or Z-VAD (100 μM). Cell viability was determined by AlamarBlue assay (A), and morphological changes were observed by means of phase-contrast imaging (B). (C, D) HL-60 cells were exposed to a low concentration of H2O2 (30 μM, 4 h) in the presence or absence of IM-54 (10 μM) or Z-VAD (100 μM). Cell viability was determined by AlamarBlue assay (C), and morphological changes were observed by means of phase-contrast imaging (D). Apoptotic (red arrows) and necrotic (blue arrows) morphologies were observed.

In recent studies, nonapoptotic molecular mechanisms were proposed for some types of necrosis;[16] thus, it may be possible to reduce necrosis by employing inhibitors of necrosis signaling. Indeed, inhibitors of necroptosis, which was defined as regulated necrosis induced by physiological death ligand, showed therapeutic activity in various disease models, including an ischemia-reperfusion injury model.[17,18] Therefore, we also examined the effects of IM-54 on necroptosis induced by Fas ligand, cycloheximide, and Z-VAD in Jurkat cells, using a reported procedure.[17] We found that IM-54 had no effect on necroptosis, which was inhibited by Nec-1,[17] a necroptosis inhibitor (Figures A and 3B). We also examined the effects on cell death induced by Fas ligand alone (Figure C and 3D). As in the case of HL-60 cells, Z-VAD inhibited the Fas-ligand-induced Jurkat cell death, whereas IM-54 did not (Figure C). Interestingly, the morphological changes of Jurkat cells induced by Fas ligand were not the same as in the case of HL-60. They seemed rather similar to the necrotic morphology of H2O2-treated HL-60 cells, the appearance of which was inhibited by Z-VAD (Figure D). These results imply that IM-54 has no effect on either apoptotic or necrotic cell death induced by death ligand.

Figure 3

Comparison of IM-54 with well-known cell death inhibitors. (A, B) Necroptosis was induced in Jurkat cells. Jurkat cells were treated with FasL (100 ng/mL, 20 h) in the presence of Z-VAD (100 μM) or cycloheximide (CHX) (5 μM). The effects of IM-54 (10 μM) or Nec-1 (30 μM) on necroptosis were evaluated by examination of cell viability (A) and morphology (B). (C, D) Jurkat cells were treated with FasL (100 ng/mL, 18 h) in the presence or absence of IM-54 (10 μM) or Z-VAD (100 μM). Cell viability (C) and morphological changes (D) were examined. (E, F) HL-60 cells were treated with H2O2 (100 μM, 3 h) in the presence or absence of various cell death inhibitors, cyclosporine A (a cyclophilin D inhibitor), DPQ (a PARP-1 inhibitor), Nec-1, and 3-methyladenine (3-MA, an autophagy inhibitor). Cell viability was determined by AlamarBlue assay (A, C, E, F), and morphological changes were observed by means of phase-contrast imaging (B, D).

In addition to Nec-1, we examined the effects of well-known cell death inhibitors, cyclosporine A (CsA, a cyclophilin D inhibitor), DPQ (a PARP-1 inhibitor), and 3-methyladenine (3-MA, an autophagy inhibitor) on oxidative stress-induced necrosis. Although CsA[19] and DPQ[20] have been reported to show therapeutic effects on myocardial infarction, none of the tested molecules inhibited H2O2-induced necrosis of HL-60 cells (Figure E and 3F). These results imply the involvement of a unique mechanism in cell death inhibition by IM derivatives.

We next planned an in vivo study of IM-54, but its water-solubility was too low. To overcome this problem, we designed and synthesized more water-soluble IM derivatives. Using a procedure similar to the one we reported before,[13,15] we introduced various hydroxyl or amino groups into IM derivatives (Scheme S1) and examined the necrosis-inhibitory activity of the obtained compounds. For quantitative estimation of the effect of each compound on necrosis, we used the lactate dehydrogenase (LDH) assay (Table ). In this assay, rupture of the cellular membrane, a typical hallmark of necrosis, is quantified in terms of LDH release from the cytosol. By using this method, we determined the IC50 values for necrotic cell death induced by H2O2. As previously reported,[13] the effect of alkyl chain length was also examined in this assay system with IM-20, IM-12, IM-13, IM-54, and IM-25. IM-54 having the C5 alkyl chain showed the greatest activity among the aminoalkyl derivatives. Introduction of a hydroxyl or amino group into the side chain generally decreased the activity (IM-17, IM-18, IM-19, IM-27, IM-90, and IM-91), regardless of the length of the alkyl chain. These results indicate that the hydrophobicity of the aminoalkyl chain is important for the cell death-inhibitory activity. However, among several hydrophilic-chain-containing derivatives, IM-17 showed reasonably good activity and was easily converted into the water-soluble HCl salt by treatment with an ethereal solution of HCl (Scheme S2). Moreover, IM-17 showed the higher stability to metabolism in the rat liver S9 fraction than IM-12 and IM-54 (Figure S1). Therefore, IM-17 was selected for further investigation.

Table 1

Cell Death-Inhibitory Activities of IM Derivatives against HL-60 Cells Treated with H2O2

Since HL-60 is a leukemia cell line, we next examined the cytoprotective activity of IM derivatives using a cardiac cell line before moving on to study the effect in a rat heart in vivo model. Rat cardiomyoblast H9c2 cells were reported to show necrotic cell death induced by TBHP, which is thought to mimic the oxidative stress in ischemia-reperfusion injury.[21] Therefore, the effects on TBHP-induced necrotic cell death might provide a measure of the therapeutic potential of compounds in ischemia-reperfusion injury. In the same manner as described for HL-60 cells, H9c2 cells were treated with TBHP in the absence or presence of a test compound, and necrotic cells were quantified by LDH assay (Figure A). All compounds showed cytoprotective activity, suggesting that they would have cardioprotective activity. MS-1 showed the strongest activity at low concentration, but it also showed cytotoxicity at high concentration, as observed in HL-60 cells. Among the IM derivatives, IM54, IM-12, and IM-17 also showed strong cytoprotective effects, and no cytotoxicity was observed even at 30 μM.

Figure 4

Cardioprotective effects of IM derivatives. (A) Cell death-inhibitory activities of IM derivatives against H9c2 cells treated with TBHP. Rat cardiomyoblast H9c2 cells were treated with TBHP (300 μM) in the presence or absence of IM derivatives. Cell death-inhibitory activity was determined by using an LDH assay. (B, C) Cardioprotective effects of IM-17 in a Langendorff rat heart ischemia-reperfusion injury model. IM-17 HCl salt (3 μM) was added to the perfusion buffer 10 min before ischemia. No-flow ischemia was maintained for 30 min, and reperfusion was accomplished by restoring flow for 60 min. Cardioprotective effects of IM-17 were examined based on recovery of LVDP (left ventricular developed pressure) (B) and release of CK (creatine kinase) (C).

With these promising results in hand, we next tested the potency of IM-17 in a Langendorff isolated rat heart model (Figure B, C). According to the reported method,[22] rat hearts were isolated and perfused in the Langendorff mode with Krebs-Henseleit buffer. After IM-17 treatment (3 μM) for 10 min, isolated rat heart was subjected to 30 min of global ischemia followed by 60 min of reperfusion. The left ventricular developed pressure (LVDP) was measured as a marker of heart function (Figure B). Creatine kinase (CK) released from myocardial cells was monitored as a marker of cell membrane integrity, which is rapidly impaired in necrosis (Figure C). The results were obtained from three independent experiments. As shown in Figures B and 4C, IM-17 clearly improved the recovery of LVDP and suppressed the release of CK, indicating that it can ameliorate the damage to rat heart cells caused by ischemia-reperfusion. Moreover, another IM derivative IM-12 was also effective in this model (Figure S2). These results suggest that IM derivatives have therapeutic potential.

Encouraged by the results in the Langendorff model, we finally examined the in vivo cardioprotective effects of IM-17. In some cases, ischemia-reperfusion injury induces ventricular arrhythmia leading to a sudden death within minutes to hours.[23−25] Thus, it is important to overcome ischemia-reperfusion-injury-induced arrhythmia as well as myocardial infarction. Although the precise mechanism is not fully understood, reactive oxygen species (ROS) are thought to be involved,[26,27] as in the case of myocardial infarction. Therefore, we examined the protective effects of IM-17 against ischemia-reperfusion-induced arrhythmia in a rat model according to reported methods.[28,29] In this model, myocardial ischemia was achieved by tightening the coronary snare, and at 5 min after ischemia, reperfusion was started by releasing the snare. IM-17 was intravenously injected at 5 min before ischemia (preischemia treatment) or at 1 min before reperfusion (postischemia treatment). The total duration of ventricular fibrillation (Vf) was calculated as the sum of the duration of episodes occurring within 10 min after reperfusion. As shown in Table , 60–75% of rats of the control group died of ischemia-reperfusion-induced arrhythmia within 10 min after reperfusion. In the case of preischemia treatment experiments, the incidence and the total duration of ischemia-reperfusion-induced Vf after reperfusion was reduced from 100% to 60% and from ca. 95.3 s to ca. 38.2 s, respectively, by treatment with 1 mg/kg of IM-17. Furthermore, treatment with 3 mg/kg IM-17 completely abolished Vf and reduced mortality to 0%. Even in the case of postischemia treatment, 3 mg/kg of IM-17 significantly reduced the mortality (to 20%) and the Vf duration (to ca. 57.0 s). These results suggest that IM-17 reduced the death rate by inhibiting ischemia-reperfusion-induced arrhythmia. In industrialized countries, ischemic disorders, such as heart attack or cerebral ischemia, are major causes of death, and better therapeutic strategies are still needed.[30] Due to the severe damage induced by reperfusion, simple recanalization is not so effective. In this study, IM-17 was found to show cardioprotective effects in ischemia-reperfusion injury in vivo as well as at the organ level. Therefore, IM derivatives could be promising leads for innovative therapeutic agents to treat ischemic diseases. Further structural development studies are in progress, together with studies of possible therapeutic applications and examination of the molecular mechanism of action of IM derivatives.

Table 2

Effects of IM-17 on the Ischemia/Reperfusion-Induced Ventricular Fibrillation and Mortality in Ratsa

			ventricular fibrillation
	dose (mg/kg)	mortality (%)	incidence (%)	duration (sec)	n
preischemia treatment	0	60	100	95.3 ± 13.0	5
	1	20	60	38.2 ± 21.7	5
	3	0	0	0	5
postischemia treatmen	0	75	75	96.3 ± 24.1	8
	3	20	100	57.0 ± 25.5	5

IM-17 HCl salt was injected i.v. at 5 min before ischemia (preischemia treatment) or 1 min before reperfusion (postischemia treatment) over 1 min. N indicates the number of rats used in each experiment.