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(Circulation. 1995;91:1520-1524.)
© 1995 American Heart Association, Inc.

Articles

Hypoxic Stress Induces Cardiac Myocyte–Derived Interleukin-6

Keiko Yamauchi-Takihara, MD, PhD; Yoshiji Ihara, MD; Atsushi Ogata, MD, PhD; Kazuyuki Yoshizaki, MD, PhD; Junichi Azuma, MD; Tadamitsu Kishimoto, MD, PhD

From the Department of Medicine III, Osaka University Medical School, Suita, Osaka, Japan.

Correspondence to Keiko Yamauchi-Takihara, MD, PhD, Department of Medicine III, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan.

	Abstract

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Background Hypoxic and ischemic stresses cause a series of well-documented changes in myocardial cells and tissues, including loss of contractility, changes in lipid and fatty acid metabolism, and irreversible membrane damage leading to eventual cellular death. Activated neutrophils are considered to be involved in this myocardial cellular injury. By stimulation of the neutrophils with chemotactic factors, canine neutrophils can be induced to adhere to isolated cardiac myocytes only if the myocytes have been previously exposed to cytokines such as tumor necrosis factor–, interleukin (IL)-1, and IL-6.

Methods and Results To examine the possible involvement of IL-6 in ischemia-reperfusion injury, we used cultured rat neonatal cardiac myocytes to study the effects of hypoxic stress on the production of IL-6 by cardiac myocytes. Unstimulated cardiac myocytes (3x10⁵ cells per dish) produced 320 pg IL-6 over 4 hours in vitro (ie, biological activity equal to 320 pg recombinant IL-6, as detected by bioassay using the MH-60.BSF2 cell line). The incubation of cardiac myocytes under hypoxic conditions for 4 hours induced significantly increased production of IL-6 compared with normoxic conditions (2.82±0.49 versus 1.64±0.18 U/mL, P<.05). Furthermore, reoxygenation for 2 hours after 2 hours of hypoxic stress significantly augmented the production of IL-6 by cardiac myocytes (4.34±0.52 U/mL, P<.05). These responses to hypoxia and reoxygenation were not observed in fibroblasts isolated from the same tissue. Although unstimulated cardiac myocytes lacked IL-6 mRNA expression detectable by Northern blot analysis, hypoxic stress induced the expression of IL-6 mRNA in the cardiac myocytes. Several pathophysiologically relevant factors also augmented IL-6 release from cultured cardiac myocytes, including IL-1ß, ionomycin, and epinephrine.

Conclusions Cardiac myocytes respond to hypoxic stress to augment the production of IL-6, and the IL-6 derived from cardiac myocytes may play an important role in the progression of myocardial dysfunction observed in cardiac ischemia-reperfusion injury.

Key Words: ischemia • heart diseases • reperfusion injury

	Introduction

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Cellular elements of the immune system have been suggested to play a role in mediating the global myocardial dysfunction observed in septic shock, cardiac allograft rejection, ischemic heart disease, and some forms of idiopathic cardiomyopathy. However, recent reports have pointed out the importance of proinflammatory cytokines, which have direct effects on the contractility of the mammalian heart¹ and on neutrophil-myocyte adhesion.²

Interleukin-6 (IL-6) has been shown to have pleiotropic functions, including strong activities not only as a B-cell–stimulating factor³ but also as a hepatocyte-stimulating factor inducing the production of a series of acute-phase proteins by hepatocytes.⁴ IL-6 is produced by several kinds of cell lineages, such as macrophages,⁵ lymphocytes,³ endothelial cells,⁶ and fibroblasts,⁷ that have important roles in inflammation. Elevated levels of serum IL-6 have been demonstrated not only in patients with inflammations resulting from bacterial or viral infections⁸ but also in patients with acute myocardial infarction,⁹ suggesting that IL-6 may play an important role in the pathogenesis of ischemic heart disease.

To elucidate the molecular mechanisms of the ischemia-reperfusion injury, we examined the effects of hypoxia-reoxygenation on cultured cardiac myocytes and proved that this stress can induce the production of IL-6 in cardiac myocytes.

	Methods

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Materials
Medium-199 (Flow Laboratories, Inc), newborn calf serum ([NCS] GIBCO), bovine pancreas insulin, human transferrin, bromodeoxyuridine, trypsin, collagenase (Sigma Chemical Co), and vitamin B₁₂ (Wako) were used for myocardial cell culture. Epinephrine (1 mg/mL) from Daiichi Pharmaceuticals Co Ltd, ionomycin from Sigma Chemical, and human recombinant IL-1ß (100 µg/mL in 20 mmol/L phosphate buffer) kindly provided by Ohtsuka Pharmaceuticals Co Ltd were used in the present study.

Cell Culture
Primary cultures of cardiac myocytes were prepared from the ventricles of 1-day-old Wistar rats, essentially according to the method of Simpson.¹⁰ Trypsinization was performed with 0.125% trypsin–0.025% collagenase. Culture was enriched for myocardial cells by preplating for 60 minutes to deplete the population of nonmyocardial cells (NMC). Nonattached cells were then suspended in medium-199 (adjusted to pH 7.4 with 20 mmol/L sodium bicarbonate [M-199]), supplemented with 10% NCS, plated at a concentration of 3x10⁵ cells/mL onto 35-mm plastic culture dishes, and cultured at 37°C in 95% air–5% CO₂. The culture medium was changed to M-199; supplemented with insulin (10 µg/mL), transferrin (10 µg/mL), 1.5 µmol/L vitamin B₁₂, penicillin (50 U/mL), and bromodeoxyuridine (0.1 mmol/L) 24 hours after seeding; and maintained for 2 days. On the day of experiments, the medium was changed again.

Incubation of Myocytes
To establish the kinetics of hypoxia-induced IL-6 production by myocytes, either cardiac myocyte–rich culture or NMC-rich culture was exposed to varying durations of hypoxic conditions or normoxic conditions. These conditions were achieved by flooding the sealed modular chambers with gas mixtures of either 95% N₂–5% CO₂ (PO₂, 30 to 40 mm Hg) or 95% air–5% CO₂. For each of the conditions, pH of the medium was unchanged and cell viability was confirmed by continuous contraction. The culture medium was then isolated and analyzed for IL-6.

IL-6 Assay
The MH-60.BSF2 cell line, which is an IL-6–dependent murine B-cell hybrid cell line, was used for the measurement of IL-6 activity in the cultured medium as previously described.¹¹ Briefly 10⁴ cells were cultured in 200 µL of medium for 48 hours in 96-well microtiter plates (Falcon 3072; Becton Dickinson Co) with cultured medium that had been heated at 56°C for 30 minutes. The cells were pulsed with [³H]thymidine (0.5 µCi per well) for the last 6 hours of culture. A standard proliferation curve was obtained with recombinant IL-6 (rIL-6). With this assay, the lower limit of detection of IL-6 activity is 0.001 U/mL.¹² One unit of IL-6 activity is equal to 200 pg of rIL-6.¹³

RNA Preparation
At the termination of experiments, cells were rapidly rinsed three times with ice-cold phosphate-buffered saline (PBS) and lysed in situ, and total cellular RNA was isolated by the acid guanidinium thiocyanate–phenol–chloroform method.¹⁴

Twenty micrograms of total RNA were size-fractionated by formaldehyde–agarose gel electrophoresis and transferred to a nitrocellulose filter (Schleicher & Schnell, Inc) in the presence of 20x standard saline citrate (SSC) (1x SSC is 300 mmol/L sodium chloride, 30 mmol/L sodium citrate, pH 7.0). The filter was baked at 80°C in a vacuum oven for 2 hours and prehybridized at 42°C for 4 to 6 hours in a solution containing 5x SSPE (20x SSPE is 3 mol/L sodium chloride, 0.2 mol/L monobasic sodium phosphate, and 0.02 mol/L EDTA disodium salt), 5x Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS), 25 µg/mL sheared herring sperm DNA, and formamide at a final concentration of 50%.

A 1.1-kb murine IL-6 cDNA fragment from pHP1B5¹⁵ labeled with [-³²P]dCTP (Amersham) by random priming was used as probe. After hybridization for 24 hours at 42°C, filters were washed with 3x SSC and 0.1% SDS at 42°C, washed twice with 1x SSC and 0.1% SDS at 42°C, air dried, and exposed to x-ray film (Kodak XAR-5) for 72 hours using intensifying screens at -70°C.

Statistical Analysis
Results are presented as mean±SEM values. Statistical analysis was performed by Student's t test; a value of P<.05 was considered significant.

	Results

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IL-6 Production by Cardiac Myocytes
The initial experiments were performed to examine the production of IL-6 by cultured cardiac myocytes. Rat neonatal cardiac myocytes were cultured in 95% air–5% CO₂ for 4 hours. Thereafter, the culture medium was isolated and examined for IL-6 activity. As shown in Fig 1, the IL-6 activities in the cultured medium from the cardiac myocytes cultured in the absence or presence of 10% NCS, as measured by bioassay using the MH-60.BSF2 cell line, were 0.80±0.15 and 1.64±0.18 U/mL, respectively. These findings show that rat neonatal cardiac myocytes in culture produced detectable amounts of IL-6 continuously under normal culture conditions.

View larger version (29K): [in this window] [in a new window]   Figure 1. Bar graph of effects of hypoxia and reoxygenation on the production of interleukin-6 (IL-6) by cultured cardiac myocytes. Cardiac myocytes were cultured with 2 mL of medium-199 adjusted to pH 7.4 (M-199) with or without 10% newborn calf serum (NCS). Culture medium was isolated after 4 hours of incubation under normoxia (95% air–5% CO2) (Normoxia) or hypoxia (95% N2–5% CO2) (Hypoxia) or after 2 hours of Hypoxia followed by 2 hours of normoxia (Reoxy). IL-6 activity in the culture medium was measured by bioassay using the MH-60.BSF2 cell line. Values are given as mean±SEM of four or five samples. Similar results were observed in four independent experiments. *P<.05 compared with Normoxia. P<.05 compared with Hypoxia.

We next examined whether cardiac myocytes express IL-6 when these myocytes are challenged with hypoxic stress. The data presented in Fig 1 show the effect of hypoxia on the production of cardiac myocyte–derived IL-6; the production of IL-6 was markedly augmented by 4 hours of hypoxic stress (2.82±0.49 U/mL, P<.05 versus normoxia at 4 hours). To examine the effects of reoxygenation, we exposed the cardiac myocytes to hypoxia followed by normoxia. The supernatant of the cultured cardiac myocytes was isolated after 2 hours of hypoxia followed by 2 hours of normoxia. IL-6 production by cardiac myocytes after reoxygenation differed significantly from that resulting from normoxia or hypoxia alone (4.34±0.52 U/mL, P<.05).

Because various concomitant factors have been well documented as manifesting myocardial reperfusion injury associated with vigorous neutrophil infiltration, we next studied the effects of cytokine, calcium, and epinephrine on IL-6 production by cardiac myocytes (Fig 2). Cardiac myocytes were cultured with M-199 plus 10% NCS with the addition of ionomycin (5x10^-5 mol/L), epinephrine (10^-5 mol/L), or IL-1ß (1 ng/mL) for 4 hours. The results were standardized to the control IL-6 production at 4 hours of normoxia and expressed as a percentage of the control value. As shown in Fig 2, hypoxia, ionomycin, epinephrine, and IL-1ß significantly augmented the IL-6 production by cardiac myocytes by 170%, 160%, 504%, and 540%, respectively (P<.05), suggesting that not only well-known IL-6–inducing factors but also Ca²⁺ ionophore and catecholamine induce the production of IL-6 in cardiac myocytes.

View larger version (47K): [in this window] [in a new window]   Figure 2. Bar graph of effects of Ca2+ ionophore, catecholamine, and interleukin-1ß (IL-1ß) on interleukin-6 production by cultured cardiac myocytes. Cardiac myocytes were cultured with medium-199 adjusted to pH 7.4 and 10% newborn calf serum and treated with ionomycin (5x10-5 mol/L), epinephrine (10-5 mol/L), or IL-1ß (1 ng/mL) for 4 hours. Results are given as a percentage of basal interleukin-6 production under normoxic conditions for 4 hours (ie, control). Values are given as mean±SEM of four or five samples. Similar results were observed in four independent experiments. *P<.05 compared with control.

Effects of Hypoxia on IL-6 Production by NMC
To exclude the possibility that the results described were induced by contaminated NMC, additional experiments were conducted with an NMC-rich culture. After trypsinization, the cells were incubated for 60 minutes at 37°C in 95% air–5% CO₂ to allow the NMC to adhere to the culture dishes. Nonattached cells were collected to culture in other dishes and considered myocyte-rich culture; they were then incubated according to the protocol described in "Methods." Also, the attached cells were incubated in 2 mL of M-199 plus 10% NCS and used for the experiments at a concentration of 1.8x10⁵ cells/mL. This culture yielded a preparation of cells consisting of more than 95% of fibroblasts and was considered a NMC-rich culture. Although the NMC-rich culture under normoxic conditions also revealed some IL-6 activity (2.13±0.25 U/mL in 4 hours), neither hypoxic incubation for 4 hours nor reoxygenation resulted in enhanced IL-6 production by these cells (Fig 3).

View larger version (22K): [in this window] [in a new window]   Figure 3. Bar graph of comparison of interleukin-6 (IL-6) production by myocyte-rich culture and nonmyocardial cell (NMC)–rich culture. Myocyte-rich culture (Myocytes) and NMC-rich culture (Non-Myocytes) were incubated in medium-199 adjusted to pH 7.4 and 10% newborn calf serum under normoxia for 4 hours (Normoxia), hypoxia for 4 hours (Hypoxia), or hypoxia for 2 hours followed by normoxia for 2 hours (Reoxy). Culture medium was isolated at the end of the each experiment period and assayed for IL-6. Results are given as a percentage of basal IL-6 production under normoxic condition for 4 hours (Normoxia). Values are given as mean±SEM of four or five samples. Similar results were observed in four independent experiments. *P<.05 compared with Normoxia.

Northern Blot Analysis of mRNA Derived From Cardiac Myocytes
To investigate whether augmented IL-6 production is regulated at the level of translation or mRNA, we performed Northern blot analysis. Unstimulated cardiac myocytes lacked detectable IL-6 mRNA expression (Fig 4). However, hypoxic stress induced expression of IL-6 mRNA as early as 30 minutes and reached a maximal level at 60 minutes, followed by a decrease to undetectable levels by 90 minutes. Incubation with IL-1ß for 60 minutes also induced the expression of IL-6 mRNA in cardiac myocytes (Fig 4). In superinduction experiments with the protein synthesis inhibitor cycloheximide, the IL-6 signal was markedly accentuated as a result of hypoxic stimulation (Fig 5).

View larger version (67K): [in this window] [in a new window]   Figure 4. Northern blot analysis of interleukin-6 (IL-6) gene expression in cardiac myocytes. Total RNA from cultured cardiac myocytes was isolated after 30, 60, and 90 minutes of hypoxic stimulation or 60 minutes of interleukin-1ß (1 ng/mL) stimulation. Twenty micrograms per lane was separated by 1.2% formaldehyde–agarose gel electrophoresis, blotted to a nylon membrane. Filters were directly visualized by ultraviolet transillumination (RNA) or hybridized with IL-6 cDNA probe. Data are representative of three separate experiments.

View larger version (48K): [in this window] [in a new window]   Figure 5. Northern blot analysis of superinduction of interleukin-6 gene in cardiac myocytes. Cardiac myocytes were incubated under normoxic condition in the presence of 100 nmol/L cyclohexamide (CHX) or under hypoxic condition for 60 minutes in the presence or absence of 100 nmol/L CHX. Total RNA was isolated and treated as described in Fig 4.

	Discussion

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We investigated whether cardiac myocytes subjected to hypoxic stress followed by reoxygenation simulating ischemia-reperfusion would actively respond by producing IL-6. Our first series of experiments involved exposure of cardiac myocytes to normoxia or hypoxia. Cardiac myocytes incubated under hypoxic conditions for 4 hours showed a significant increase in IL-6 production without alteration in cellular viability.

Next, hypoxic and normoxic conditions were imposed sequentially on the in vitro cardiac myocyte system. Using this model, we demonstrated that hypoxic preconditioning significantly augmented IL-6 production in cardiac myocytes. The evidence that the myocardial cells respond to hypoxic stress to induce IL-6 production could in part account for the elevated serum levels in patients with acute myocardial infarction.⁹ Recently, we demonstrated that the patients with angina pectoris who score positive on the exercise ECG test had elevated serum IL-6 levels soon after the exercise test (unpublished observations). IL-6 is reported to be secreted by various kinds of cells such as endothelial cells,⁶ vascular smooth muscle cells,¹⁶ and activated monocytes and macrophages,⁵ which may also be potential sources of elevated serum IL-6 levels in acute myocardial infarction.

In cardiac myocytes, as in other cell types,^{16 17} IL-1ß appears to be one of the most effective stimuli for IL-6 production and gene expression (Figs 2 and 4). Furthermore, cardiac myocytes produce IL-6 in response to epinephrine or ionomycine, which induces intracellular Ca²⁺ elevation. Stimulation of cardiac myocytes with these concomitant factors is also observed in the ischemic myocardium.

Numerous studies indicate that neutrophils contribute to myocardial cell injury on reperfusion. Entman et al^{2 18 19} recently demonstrated that neutrophils adhere to the cardiac myocytes if the myocytes are stimulated to express intercellular adhesion molecule–1 (ICAM-1) and the neutrophils are stimulated to enhance CD18-dependent adhesion. Stimulation of myocytes with IL-1, tumor necrosis factor– (TNF-), IL-6, and postischemic cardiac lymph was reported to induce expression of ICAM-1 on the cell surface.^{2 20}

Another interesting finding of the present study was the unresponsiveness of the fibroblasts to hypoxic stress, although IL-6 production is consistently seen under normoxic conditions. Similar results were reported in a recent study by Webster et al.²¹ The c-fos and c-jun expressions were induced by hypoxia in cultured cardiac myocytes, but they were not induced in nonmuscle primary fibroblasts. These findings may also be compatible with the hypothesis that induced expression of c-fos and c-jun gene products may be responsible for the IL-6 induction in cardiac myocytes exposed to hypoxia.

Both transcriptional induction of c-fos and c-jun genes and nuclear accumulation of these gene products have been reported to occur several hours before energy depletion or cell damage in neonatal rat cardiac myocytes exposed to hypoxia.²¹ The transcription factor AP-1, consisting of fos and jun family gene products, regulates the expression of a number of genes by binding to specific DNA sequence elements contained in the target gene promoters (reviewed in References 22 and 23). Although IL-1 induces IL-6 gene expression mediated by nuclear factor (NF)-B²⁴ and NF-IL-6,^{25 26} which are potent transcription factors binding to the promoter of the IL-6 gene, the specific DNA sequence that binds to AP-1 is also present in this promoter.^{27 28} Thus, the AP-1 complex may interact and activate expression of the IL-6 gene in myocardial cells under hypoxic conditions. The molecular signals that mediate the responses of mammalian cells and tissues to hypoxia or ischemia remain to be identified. The induced expression of the IL-6 gene by cardiac myocytes under hypoxic conditions may provide an invaluable method of examining the molecular mechanisms of the cardiac responses to hypoxia.

Although the effects of IL-6 on the cardiovascular system are not well known, Finkel et al¹ reported that several recombinant cytokines, including TNF-, IL-2, and IL-6, had a negative inotropic effect on hamster papillary muscle. The production of IL-6 induced by hypoxic stress in cardiac myocytes may also be involved in contractile dysfunction and in the myocardial reperfusion injury observed in ischemic heart disease. The association between hypoxic stress and augmented production of IL-6 in the cardiac myocytes would support the role of IL-6 not only as a potential mediator in the migration and activation of neutrophils but also in the progression of myocardial dysfunction provided in ischemia-reperfusion injury.

	Acknowledgments

 
This work was supported in part by grants from the Ministry of Education, Culture and Science, Japan. We thank Ms Y. Yamaguchi for excellent secretarial assistance.

Received July 13, 1994; revision received September 19, 1994; accepted October 5, 1994.

	References

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