Pathways Involved in Interleukin-1β–Mediated Murine Cardiomyocyte Apoptosis

Interleukin-1 (IL-1), a multifunctional proinflammatory cytokine, was originally described as the first endogenous pyrogen. It can exert complex biological effects by modulating gene expression in a wide variety of cell types and inflammatory conditions.^1–3 Interleukin-1 consists of 2 distinct ligands (IL-1α and IL-1β) with high-sequence homology; however, only IL-1β can be released from cytosol to exocellular space after activation.^4,5 In addition, IL-1 is consistently induced and activated after tissue injury, and it appears to mediate reparative responses.^6,7 In the pathogenesis of cardiovascular diseases, IL-1 signaling promotes the formation of atheromatous lesions, increases vascular inflammation, and triggers plaque destabilization.^8,9 After myocardial infarction, IL-1 signaling critically regulates the inflammatory response and is involved in the development of adverse remodeling by increasing the expression of matrix metalloproteinases.^7,10 Emerging evidence suggests that IL-1 signaling has an essential role in heart failure by suppressing cardiac function, promoting myocardial hypertrophy, and even inducing cardiomyocyte apoptosis.^7,11

Interleukin-1β has induced rat cardiomyocyte apoptosis that was associated with activation of Bcl-2 family members.¹¹ Although the activation of pro-apoptotic pathways mediated by IL-1 stimulation during cardiomyocyte injury has been confirmed, the mechanism involved in this process is poorly defined. Coincidentally, a marked upregulation of IL-1 has been found in ischemic hearts.^12–14 Therefore, understanding the mechanism of IL-1β–mediated cardiomyocyte apoptosis is important in the designing of optimal therapeutic approaches to counter heart failure. In the current study, we used cardiomyocytes isolated from neonatal mice and explored the molecular pathways involved in IL-1β–mediated cardiomyocyte apoptosis in vitro, including caspase-dependent pathways, caspase-independent pathways, and inhibitor of apoptosis proteins (IAPs).

Materials and Methods

We used the neonatal (1–3 d after birth) C57BL/6J mouse in accordance with our institution's guidelines for the care and use of animals. We referred to standard methods for the primary culture of neonatal rat cardiomyocytes and modified them for the neonatal mouse.^11,15 First, each mouse heart was gently removed into a Petri dish that contained cold Gibco^® phosphate-buffered saline solution (Life Technologies, part of Thermo Fisher Scientific Inc.; Grand Island, NY). The atria were discarded. The ventricles were dissected into small pieces with use of fine scissors and were placed into a new 50-mL tube (Corning Life Sciences; Tewksbury, Mass) that contained 0.25% Gibco trypsin-EDTA solution (Life Technologies) and 0.2% type II collagenase solution (Sigma-Aldrich Co. LLC; St. Louis, Mo). The tube was incubated at a temperature of 37 °C in a water bath for approximately 30 min, with gentle stirring.

The neonatal mouse cardiomyocytes were isolated by means of digestion with collagenase and trypsin. During the first 3 days of culture, we added fibroblast growth inhibitor to the medium, to purify the cardiac myocytes from the isolated population. The digestion was stopped by the addition of a Gibco minimal essential medium that contained 10% Gibco fetal bovine serum (Life Technologies).

After being washed twice with the medium in a centrifuge for 5 min at 250 ×g at a temperature of 4 °C, the isolated cells were placed into 6-well plates (Corning Costar Corporation; Corning, NY) at a density of 2 × 10⁵ cells/cm² in a humidified 5% C incubator at a temperature of 37 °C. The next day, the unattached cells were removed and fresh medium was added, supplemented with fibroblast growth inhibitor (0.1 mmol/L of bromodeoxyuridine). The culture medium was changed daily until the cells were used; fibroblast growth inhibitor was added only on the first 3 days. By means of our method, the purity of the cardiomyocytes exceeded 90%, as determined from the results of cardiac troponin I immunostaining (Abcam; Cambridge, Mass).

After routine culture in minimal essential medium and 10% fetal bovine serum for another 3 to 5 days, the cardiomyocytes were transferred into a new dish (concentration, 1 × 10⁶ cells/mL) and were incubated with the addition of 10 ng/mL of recombinant murine IL-1β (PeproTech; Rocky Hill, NJ) for 72 hr. The cardiomyocytes were then dispersed into single cells with use of trypsin and were analyzed by means of flow cytometry under annexin V staining.

Statistical Analysis

Results are presented as mean ± SD. Significance between groups was evaluated by means of the Student t test with use of SPSS 20.0 for Windows (IBM Corporation; Armonk, NY). A P value less than 0.05 was considered statistically significant.

TABLE I. Primers Used to Synthesize Complementary DNA of Cardiomyocytes by Means of Real-Time Quantitative Polymerase Chain Reaction

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Results

In comparison with the control samples (which did not undergo IL-1β incubation), the IL-1β–incubated groups had a significantly higher cell apoptotic ratio (36.7% ± 8.4% vs 5.5% ± 2.3%, P=0.0034) (Fig. 1). Interleukin-1β therefore induced apoptosis of murine cardiomyocytes in vitro.

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Endonuclease G and HtrA2/Omi. Endonuclease G and HtrA2/Omi were involved in caspase-independent pathways during IL-1β–mediated cardiomyocyte apoptosis. To explore the molecular mechanisms of this apoptosis, we checked principal effectors involved in the caspase-dependent and caspase-independent pathways. The IL-1β–incubated cardiomyocytes had significantly higher levels of cytosolic cytochrome c (31 ± 4.4 vs 6 ± 0.4 ng/mL per 10⁶ cells; P=0.0014) than did the unincubated control samples, as determined by means of ELISA analysis. The released cytochrome c in cytoplasm triggered the formation of the apoptosome complex that leads to cell apoptosis via a caspase-dependent pathway. The results of Western blot analysis revealed that the cardiomyocytes had significantly higher active (cleaved) caspase-3 expression after IL-1β incubation than did the control samples (Fig. 2). As a central executioner caspase, caspase 3 was activated in the cardiomyocytes under IL-1β treatment. These results indicate that IL-1β signaling triggers the caspase-dependent apoptotic pathway in cardiomyocytes.

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In regard to the caspase-independent pathways, the results of real-time quantitative polymerase chain reaction (PCR) analysis revealed that the IL-1β–incubated cardiomyocytes had higher endonuclease G and HtrA2/Omi mRNA expression than did the control samples (endonuclease G, P=0.0084; and HtrA2/Omi, P=0.0483). However, we found no significant difference in Smac/Diablo or AIF expressions of mRNA between the groups, with or without IL-1β incubation (Fig. 3). Interleukin-1β signaling also triggered the caspase-independent apoptotic pathways, which were mediated by endonuclease G and HtrA2/Omi.

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Interleukin-1β Signaling and Inhibition of Survivin and XIAP during Apoptosis. Further, we wanted to know whether anti-apoptotic molecules such as IAPs were involved in IL-1β–mediated cardiomyocyte apoptosis. The results of real-time quantitative PCR analysis indicated that IL-1β–incubated cardiomyocytes had lower survivin and XIAP mRNA expression than did the control samples (survivin, P=0.0001; and XIAP, P=0.0069). We unexpectedly found that c-IAP1 and c-IAP2, two other IAPs, had higher mRNA levels after IL-1β incubation (c-IAP1, P=0.0227; and c-IAP2, P=0.0003) (Fig. 4). Therefore, IL-1β signaling simultaneously mediated the inhibition of IAP family members, including survivin and XIAP during cardiomyocyte apoptosis.

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Discussion

The results of abundant studies of cardiac biology suggest that the actions of IL-1β are highly complex, depending on cell type.^7,16,17 To better define the mechanisms involved in cytokine-induced cardiomyocyte apoptosis, Ing and colleagues¹¹ examined the effects of IL-1β with and without the combination of other inflammatory cytokines, such as interferon-γ and tumor necrosis factor-α. The investigators found that IL-1β alone was sufficient to induce apoptosis of neonatal rat cardiomyocytes, which was associated with expression of nitric oxide and Bcl-2 family members. In that study, pro- and anti-apoptotic genes (Bak and Bcl-xL) of cardiomyocytes were upregulated after IL-1β stimulation.¹¹ Coincidentally, we found apoptosis in response to IL-1β treatment on murine cardiomyocytes in our experiments. A statistically significant increase in the apoptosis of cardiomyocytes was achieved after 72-hr incubation with IL-1β, whereas there was no significant difference between these samples and the unincubated control samples at 24 or 48 hr (data not shown). This suggests that IL-1β triggers other intracellular signaling associated with cardiomyocyte apoptosis, instead of priming cell-death programs directly. The results of other studies to determine the molecular mechanisms of this process have revealed that cardiomyocytes express a higher level of active caspase 3, a principal effector in caspase-dependent pathways, after IL-1β incubation in vitro. The apoptotic signals from the death receptor and mitochondrion usually converge to caspase 3. As a central-executioner caspase, caspase 3 can be activated by the upstream cascades, which results in the orderly dismantling and removal of cells.¹⁸ Accordingly, the apoptotic process of cardiomyocytes cannot be reversed after caspase pathways are activated by IL-1β stimulation.

Apoptosis is dependent on caspase activation, which leads to substrate cleavage and ultimately cell death.¹⁹ However, in some processes, cells undergo apoptosis without caspase activation, and this is called caspase-independent cell death. Caspase-independent cell death occurs in response to most intrinsic apoptotic cues and shares some characteristics with the caspase-dependent process, such as mitochondrial outer-membrane permeabilization.^20,21 This last process leads to the release of various proteins from the mitochondrial intermembrane space, including cytochrome c, AIF, endonuclease G, HtrA2/Omi, and Smac/Diablo. ^21–26 The released cytochrome c interacts with other molecules (such as Apaf-1 and caspase 9) and forms the apoptosome complex in cytoplasm, which leads to activation of the apoptotic program via a caspase-dependent pathway.²⁷ After the IL-1β treatment, we found that the cardiomyocytes released cytochrome c into cytoplasm. This finding became further evidence of the activation of caspase-dependent apoptotic pathways in cardiomyocytes mediated by IL-1β signaling (Fig. 5).

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In addition, cytochrome c, AIF, endonuclease G, HtrA2/Omi, and Smac/Diablo have been shown to induce caspase-independent cell death after mitochondrial release. Our results of quantitative PCR analysis showed that IL-1β signaling in cardiomyocytes triggered the caspase-independent apoptotic pathways that were mediated by endonuclease G and HtrA2/Omi (Fig. 5). Endonuclease G is a nuclear-encoded mitochondrial nuclease that reportedly functions in apoptosis.^28,29 During cell apoptosis, endonuclease G can be released from the mitochondria, translocate to the nucleus, and cooperate with other nucleases to trigger DNA fragmentation.^23,30,31 In addition, endonuclease G expression can be promoted under certain initial stimuli during cell apoptosis.^32–34 Our results illuminated the fact that IL-1β signaling initiated the death program in cardiomyocytes by increasing the expression of endonuclease G.

The serine protease HtrA2/Omi is found in the mitochondria and can be released during cell apoptosis.^24,35 Activated (cleaved) HtrA2/Omi exposes an amino terminal motif resembling that of the IAP inhibitor Smac/Diablo. The interaction between HtrA2/Omi and IAP family members antagonizes their inhibition of caspase activity and protection from apoptosis.^36,37 In our study, IL-1β signaling significantly increased HtrA2/Omi expression, which might amplify the antagonistic action on IAP family members and promote cardiomyocyte apoptosis.

In addition to the pro-apoptotic molecules discussed above, we explored anti-apoptotic factors. The IAPs are structurally related anti-apoptotic proteins that were initially identified in baculoviruses. Mammalian IAPs, such as c-IAP1, c-IAP2, survivin, and XIAP, are able to block apoptosis either by binding and inhibiting caspases or through caspase-independent mechanisms.^38–40 This family of proteins has become increasingly prominent in the apoptotic process. The results of our quantitative PCR analysis indicated that IL-1β signaling mediated the inhibition of survivin and XIAP in cardiomyocyte apoptosis. The modulation of survivin and XIAP would attenuate their inhibitory function on apoptosis, which would consequently promote the pro-apoptotic progression of cardiomyocytes. Nevertheless, we found higher levels of both c-IAP1 and c-IAP2 on the mRNA level after IL-1β stimulation. This suggests that IL-1β signaling precisely modulates the apoptotic process of cardiomyocytes by inducing or inhibiting specific gene expression.

A limitation of this study is that we observed the activation of various molecules without providing information on the interactions of various pathways that result in a net increase in cardiomyocyte apoptosis. In future studies, we expect to identify the dominant pathways that result in cardiomyocyte apoptosis, and we plan to explore the interactions of the molecules involved in the various pathways. Another limitation is that this study was performed with the use of cultured cardiomyocytes. To confirm the findings from in vitro experiments, an animal model of heart failure should include IL-1 neutralization and IL-1 receptor antagonism.

Extensive experimental evidence has already suggested an important role for IL-1 signaling in cardiovascular diseases.^7,8,13,41,42 Interleukin-1 signaling seems to be a promising therapeutic target in a variety of cardiovascular conditions, including heart failure. The inhibition of IL-1 signaling might reduce the progression of cardiomyocyte apoptosis and preserve heart function. The current study has revealed that IL-1β signaling induces the activation of caspase-dependent and -independent pathways and the inhibition of IAPs during cardiomyocyte apoptosis in vitro. Understanding the molecular mechanisms involved in IL-1β–mediated cardiomyocyte apoptosis might assist in the design of optimal therapeutic approaches for cardiomyocyte survival.