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Archives of cardiovascular diseases
Volume 111, n° 10
pages 545-554 (octobre 2018)
Doi : 10.1016/j.acvd.2017.12.006
Received : 8 Mars 2017 ;  accepted : 6 December 2017
Clinical research

Mechanism of interleukin-1 receptor antagonist protection against myocardial ischaemia/reperfusion-induced injury
Mécanismes de la protection par l’antagoniste des récepteurs de l’interleukine I pour lutter contre les lésions d’ischémie myocardique/reperfusion
 

Wenhao Qian a, , Changyun Zhao b, Dongye Li b, Rui Dai b
a Department of Cardiology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221002, China 
b Department of ICU, Zhejiang Hospital, Hangzhou, Zhejiang 310000, China 

Corresponding author. Research Institute of Cardiovascular Diseases, Xuzhou Medical College, 84, West Huaihai road, Xuzhou, Jiangsu 221002, China.Research Institute of Cardiovascular Diseases, Xuzhou Medical College, 84, West Huaihai road, Xuzhou, Jiangsu 221002, China.
Summary
Background

New therapeutic targets are required for ischaemic heart disease; our study was designed to assess the theoretical foundation and experimental basis underlying the use of a new anticytokine agent, interleukin-1 receptor antagonist (IL-1ra), in this setting.

Aim

To investigate the cardioprotective properties of IL-1ra in terms of inhibition of apoptosis and improvement in systolic and diastolic functions of ischaemia/reperfusion (I/R)-injured cardiomyocytes, via a reduction in the inositol 1,4,5-trisphosphate (IP3) receptor-mediated Ca2+ overload induced by myocardial I/R injury.

Methods

For in vivo animal experiments, 30 adult male Sprague Dawley rats were anaesthetized and randomized into sham, I/R, and IL-1ra+I/R groups (n =10 in each). All rats except the sham group were subjected to 30minutes of myocardial ischaemia, followed by 2hours of reperfusion. At a cellular level, healthy male Sprague Dawley rats under pentobarbital sodium anaesthesia underwent heart removal and isolation of individual ventricular cardiomyocytes using enzymatic hydrolysis, which were randomized into five groups: dimethyl sulphoxide; I/R; IL-1ra+I/R; 2-aminoethoxydiphenyl borate (2-APB)+I/R; and 2-APB+IL-1ra+I/R.

Results

In the IL-1ra+I/R group, exacerbation of myocardial infarct size and I/R-induced injury was inhibited. At the cellular level, in the I/R group, peak shortening (% cell length) and maximal velocities of shortening and relengthening were significantly decreased and intracellular Ca2+ transient amplitude (measured as fura-fluorescence intensity change) was diminished by electric stimulation, with the decay time constant of Ca2+ transients increased versus the dimethyl sulphoxide group. Compared with the I/R group, statistically ameliorated variables were achieved in the IL-1ra+I/R, 2-APB+I/R and 2-APB+IL-1ra+I/R groups, with the 2-APB+IL-1ra+I/R group presenting more significant improvement, while there was no statistical difference between the IL-1ra+I/R and 2-APB+I/R groups.

Conclusions

The downregulation of IP3 receptors by IL-1ra attenuates Ca2+ overload and the systolic and diastolic dysfunctions of hypoxia/reoxygenation-injured cardiomyocytes, which contributes to inhibition of apoptosis in I/R-injured cardiomyocytes and reduction of myocardial infarct size in vivo.

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Résumé
Objectif

Déterminer si l’antagoniste des récepteurs de l’interleukine I (IL-1ra) pourrait protéger contre l’ischémie/reperfusion myocardique (IR) par downregulation des récepteurs inositol 1, 4, 5 triphosphates (IP3R) et la réduction de la surcharge calcique. Ainsi, l’apoptose des cardiomyocytes de rats a été inhibée avec une amélioration significative des fonctions systolique et diastolique ainsi qu’une possible relation entre ces mécanismes moléculaires.

Méthode

Des rats males sains anesthésiés du pentabarbital ont eu une ablation du cœur et l’isolation des cardiomyocytes ventriculaires en utilisant une hydrolyse enzymatique. Ils ont été randomisés en 5 groupes : dimethyl sulfoxide (DMSO), lésion d’ischémie/reperfusion, interleukine-1ra+ischémie/reperfusion, 2-amino-ethoxydiphenyl borate+ischémie/reperfusion et 2-amino-ethoxydiphenyl borate+inhibition aux antagonistes des récepteurs à l’interleukine 1+lésion d’ischémie/reperfusion. Dans le groupe DMSO, les cardiomyocytes ont été incubés avec du DMSO seul pendant 19heures, tandis que les autres groupes ont eu une incubation de 14heures, suivie d’une hypoxie de 3heures puis d’une réoxygénation de deux heures avec ou sans adjonction d’antagonistes aux récepteurs à l’interleukine 2/APB comme prétraitement. Les index systolique et diastolique et le courant calcique des cardiomyocytes isolés ont été mesurés.

Résultats

Dans le groupe ischémie/reperfusion, le pic de raccourcissement (pourcentage de raccourcissement cellulaire) et la vélocité maximale de raccourcissement, ainsi que son ré-allongement (±dl/dt) étaient significativement réduits (p <0,001 et p <0,001), et l’amplitude du calcium intracellulaire diminuait par la stimulation électrique (<0,001) avec une constante de décroissance du transit calcique (Tau) augmentée de façon significative (p <0,001) versus le groupe DMSO. Comparativement au groupe ischémie/reperfusion les paramètres statistiquement améliorés de façon significative concernent les groupes IL-1ra+I/R, 2-APB+I/rand 2-APB+IL-1ra+I/R, tandis qu’il n’y avait pas de différence statistique significative entre le groupe IL-1ra+I/R et le groupe 2-APB+I/R (p >0,05).

Conclusion

La dowregulation des récepteurs IP3 atténue la surcharge calcique et les dysfonctions systolo-diastoliques des lésions observées sur les cardiomyocytes au décours d’une hypoxie/réoxygénation, ce qui pourrait contribuer à l’inhibition de l’apoptose des cardiomyocytes sièges de lésions d’ischémie reperfusion.

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Keywords : Interleukin-1 receptor antagonist, Myocardium, Calcium overload, IP3 receptor, Ischaemia/reperfusion-induced injury

Mots clés : Antagoniste des récepteurs à l’interleukine I, Myocarde, Surcharge calcique, Récepteur IP3, Lésions ischémie/reperfusion myocardique

Abbreviations : 2-APB, +dL/dt, −dL/dt, DMSO, ΔFFI, IL-1, IL-1ra, IP3, I/R, LAD, Tau, TBST, TTC


Background

Ischaemic heart disease is the leading cause of death worldwide [1]. Lack of blood flow to the heart leads to an imbalance between O2 demand and supply, resulting in acute myocardial infarction. The restoration of blood flow to the ischaemic myocardium, termed reperfusion, by either thrombolytic therapy or primary percutaneous coronary intervention, is the top strategy for reduction of myocardial infarct size and improvement of clinical outcomes [2]. Unfortunately, myocardial ischaemia/reperfusion (I/R) is a double-edged sword, which can also induce injury and lead to a paradoxical combination of events, characterized by apoptosis, inflammation, infarction, Ca2+ overload and contractile impairment [3].

Ca2+ overload is reportedly an important contributor to the pathogenesis of myocardial I/R injury [4]. Along with other physiological derangements, abnormal elevation of cytosolic Ca2+ concentration leads to arrhythmias and myocardial systolic and diastolic disorders, as well as cellular apoptosis, necrosis and death [5, 6, 7]. In normal physiological processes, Ca2+ adenosine triphosphatase in the sarcoplasmic reticulum (SERCA) pumps Ca2+ into the endoplasmic reticulum, while inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors release Ca2+ from the organelle, thus maintaining intracellular Ca2+ homeostasis [8]. During reperfusion, IP3 receptor-mediated Ca2+ release is an important contributor to Ca2+ overload [9, 10]. 2-aminoethoxydiphenyl borate (2-APB), a cell-permeable antagonist of the IP3 receptor, can effectively block Ca2+ release via the IP3 pathway [11].

Recombinant human interleukin-1 (IL-1) receptor antagonist (IL-1ra) is a competitive inhibitor of IL-1α and IL-1β signalling, by binding to the IL-1 receptor [12, 13]. IL-1ra and IL-1 blockade can reportedly reduce myocardial infarct size and inhibit cardiomyocytic apoptosis by interrupting the inflammatory reactions during myocardial I/R injury, and exert potential benefit in terms of remodelling acutely infarcted myocardium [14, 15]. However, it remains unclear whether IL-1ra can reduce Ca2+ overload, thereby improving the systolic and diastolic functions of cardiomyocytes and ultimately reducing apoptosis in I/R-injured cardiomyocytes.

Therefore, this study was designed to assess the cardioprotective properties of IL-1ra, in terms of inhibition of apoptosis and improvement in systolic and diastolic functions of I/R-injured cardiomyocytes, via a reduction in the IP3 receptor-mediated Ca2+ overload induced by myocardial I/R injury.

Methods
Experimental protocol

All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Committee on Animal Care of Xuzhou Medical College.

Establishment of animal model of myocardial I/R-induced injury

Healthy adult male Sprague Dawley rats (n =30), weighing 220–250g (Experimental Animal Centre, Xuzhou Medical College, Jiangsu, China) underwent anaesthesia under pentobarbital sodium (50mg/kg, intraperitoneally) and catheterization via the left jugular vein. An electrocardiogram and heart rate were recorded simultaneously on a polygraph. With left thoracotomy and pericardotomy, the left anterior descending artery (LAD) was exposed and occluded by ligation with a 6–0 Prolene® (Ethicon Inc., Somerville, NJ, USA) suture over PE10 tubing [16]. At the end of 30minutes of ischaemia, the PE10 tubing was removed for 2hours of reperfusion of the myocardium. Successful occlusion of the LAD was confirmed by the presence of ST-segment elevation on the electrocardiogram and ventricular myocardium discoloration from bright red to dark red or paleness. Adequate reperfusion was confirmed by epicardial hyperaemia, electrocardiogram modifications and resolution of ST-segment elevation by>50% [17]. The rats were randomized into three groups (n =10in each): sham group, subjected to the surgical procedures without occlusion of the LAD; I/R group, subjected to I/R and administration of NaCl0.9% (1mL/kg, intravenously); IL-1ra+I/R group, with identical procedures to the I/R group except for administration of IL-1ra (2mg/kg, intravenously; Shanghai Biomabs Pharmaceuticals Co. Ltd., Shanghai, China) for 5minutes before reperfusion.

Evaluation of myocardial infarct size

At the end of 2hours of reperfusion, myocardial infarct size was assessed by triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, St. Louis, MO, USA) staining [18]. The left ventricle was frozen at −20°C for 2hours, and was sliced longitudinally (starting with the cardiac apex) into five 2mm-thick sections, which were weighed and incubated in 1% TTC at 37°C for 30minutes. Subsequently, the sections were placed in normal saline containing 10% formaldehyde for 1hour, followed by image scanning and measurements of infarct size, using ImageTool software (UTHSCSA, San Antonio, TX, USA) to calculate the percentage of infarct size.

Western blot analyses

Whole homogenates of frozen left ventricle tissue were separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in phosphate-buffered saline. The protein concentration in each sample was determined using a bicinchoninic acid (BCA) protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Samples were transferred to polyvinylidene difluoride (PVDF)-Plus membranes [19]. With the addition of 5% albumin bovine fraction V for blockage in Tris-buffered saline containing 0.1% Tween® 20 (TBST) (Sigma-Aldrich), membranes were immunoblotted at 4°C overnight with primary antibodies, as follows: IP3 receptor (1:1000; Santa Cruz, CA, USA), Bcl-2 and Bax (1:500; Santa Cruz), caspase-3 (1:500; Sigma-Aldrich) and β-actin (1:1000; Zhongshan Golden Bridge, Beijing, China). Subsequently, the membranes were retrieved, rinsed three times in TBST and transferred into 15mL conical tubes containing the corresponding secondary antibodies (1:1000; Zhongshan Golden Bridge), for incubation at room temperature for 1hour. Protein bands were visualized by nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate. With the membranes scanned, the relative intensities of bands were analysed by the ImageJ 3.0 system (NIH, Bethesda, MD, USA). The optical density of the bands in the control group was considered as 1 arbitrary densitometry unit.

Isolation of cardiomyocytes

Ventricular cardiomyocytes were isolated from the heart in Sprague Dawley rats [20] using collagenase (type II) (Gibco-BRL; Life Technologies, Grand Island, NY, USA) and Ca2+-free buffer. The cells were suspended three times in Krebs bicarbonate solution (pH 7.2) (inmmol/L: 15NaCl, 85KCl, 30KH2 PO4 , 5MgSO4 , 5sodium pyruvate, 5creatine, 20taurine, 2L-glutamic acid, 20HEPES, 20glucose, 0.2CaCl2 and 0.5EGTA), gassed with 100% O2 at 37°C. Overall, 80–87% of the viable cardiomyocytes were quiescent, and were subsequently cultured in Dulbecco's minimal essential medium containing 1% penicillin-streptomycin at a density of 2×104 cells in 12-well plates. Finally, the myocytes were transferred to a CO2 incubator (Heracell™ 150i; Heraeus, Hanau, Germany) in an atmosphere of 95%O2 and 5%CO2 at 37°C.

Protocol for simulated I/R

At the end of 1hour of equilibration in the incubator, cardiomyocytes were exposed to an “ischaemic buffer” consisting of the following (inmmol/L): 118NaCl, 24NaHCO3 , 1.0NaH2 PO4 , 2.5CaCl2 -2H2 O, 1.2MgCl2 , 20sodium lactate, 16KCl and 102 deoxyglucose (pH adjusted to 6.2) [21]. The cells were incubated at 37°C in a triple-gas incubator in a 1%O2 , 5% CO2 and 94% N2 atmosphere for 3hours throughout the ischaemic simulation, followed by transference to a normal cell medium under normoxic conditions for 2hours of reperfusion.

Cell treatments with IL-1ra and 2-APB

In the IL-1ra and 2-APB+IL-1ra groups, cells were cultured with IL-1ra at a concentration of 2.5μg/mL for 12hours before simulated I/R [22]. In the 2-APB+I/R and 2-APB+IL-1ra+I/R groups, 2-APB (50μmol/L; Sigma-Aldrich) [23] was added to the cells 30minutes before the IL-1ra pretreatment, followed by simulated I/R. In the I/R group, cardiomyocytes in medium were incubated in a cell culture chamber for 14hours, followed by simulated ischaemia for 3hours and reperfusion for 2hours. In the dimethyl sulphoxide (DMSO) group, cardiomyocytes were incubated with vehicle alone. The harvested cardiomyocytes were rinsed with phosphate-buffered saline for IP3 receptor western blot analysis.

Measurement of the shortening amplitude of cardiomyocytes

At the end of reperfusion in each group (except the DMSO group), medium containing ventricular myocytes was added dropwise to an open chamber on the stage of an inverted microscope (Olympus, Tokyo, Japan). With the spontaneous attachment to the bottom of the chamber, cardiomyocytes were superfused at 2mL/min with Krebs–Henseleit (KH) buffer (containing 2.0mm Ca2+ and 100nm isoprenaline) at 37°C, adjusted to pH 7.4 by equilibration, in a 95% O2 and 5% CO2 atmosphere. Rod-shaped ventricular cardiomyocytes with clear sarcomeres were screened for electrical stimulation at 0.5Hz. At least 10 cardiomyocytes per heart from each group were evaluated, with all the procedures recorded with a video recorder (Panasonic Corporation, Osaka, Japan) [24, 25] connected to a computer for data processing. Detection indicators included: resting cell length; peak shortening (% cell length, contraction amplitude); +dL/dt (maximal velocity of shortening); and −dL/dt (maximal velocity of relengthening).

Measurement of Ca2+ transients

By recalcification at a gradient to 1.8mmol/L, cardiomyocytes were loaded with 0.5μmol/L Fura 2-AM (Dojindo Laboratories, Kumamoto, Japan) and maintained for 30minutes at 25°C in darkness [26]. The fluorescence ratio measurements were recorded with a dual-excitation fluorescence photomultiplier system (IonOptix LLC, Milton, MA, USA). The cells were rinsed and resuspended in normal Tyrode's solution. Thereafter, the cardiomyocytes were loaded in the cell chamber on the inverted microscope for stimulation at 0.5Hz for 10ms, and imaged under a Fluor×40 objective lens. Cells were exposed to light under a 75W Xenon lamp at wavelengths of 340nm and 380nm, alternatively. Fluorescence emission was detected at 510nm [27]. Variations in intracellular free Ca2+ concentrations were reflected by the ratio of the two wavelengths of fluorescence. Detection indicators included: resting intracellular Ca2+ concentration (F0); Ca2+ transient amplitude (ΔFFI); decay time constant of Ca2+ transients (Tau); fluorescence ratio (RF340/F380) values normalized to the resting value (F/F0).

Statistical analyses

For each experimental series, data are presented as means±standard deviations. Statistical analyses were performed with GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA). Statistical significance (P <0.05) for each variable was estimated by one-way or two-way analysis of variance, followed by Bonferroni post hoc tests.

Results
IL-1ra reduced myocardial infarct size

Ligation of the LAD for 30minutes, followed by 2hours of reperfusion, resulted in a substantial transmural infarction. Representative slices of myocardium from sham, I/R and IL-1ra+I/R groups are shown in Figure 1, Compared with the sham group, myocardial infarct size was increased at the end of I/R (43.59±3.46% vs. 0.00±0.00; P <0.001), but was attenuated by pretreatment with IL-1ra (P <0.001). Compared with the I/R group, the magnitude of the myocardial infarction in the IL-1ra+I/R group was diminished (34.66±3.07% vs. 43.59±3.46%; P <0.05).



Figure 1


Figure 1. 

Infarct size in each group. A. Cross-sectional photomicrographs of triphenyltetrazolium chloride (TTC)-stained (white) infarcted regions. B. Percentage of infarcted area in the left ventricle in ischaemia/reperfusion (I/R) and interleukin-1 receptor antagonist (IL-1ra)+I/R rats. Data are expressed as mean±standard deviation; n =3. *P <0.05, **P <0.01, ***P <0.001 versus the sham group. # P <0.05, ## P <0.01 versus the I/R group.

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Effects of IL-1ra on cardiomyocytic apoptosis

As shown in Figure 2, western blot analyses revealed that expression of proapoptotic proteins (cleaved caspase-3 and Bax) were significantly upregulated after I/R, while expression of the antiapoptotic protein Bcl-2 was downregulated (P <0.001 versus the sham group). Compared with the I/R group, expression of cleaved caspase-3 and Bax was partly inhibited (P <0.001 and P <0.01 versus the I/R group, respectively), and Bcl-2 was upexpressed after IL-1ra pretreatment (P <0.05 versus the I/R group).



Figure 2


Figure 2. 

Effects of the interleukin-1 receptor antagonist (IL-1ra) on the expression of Bax (A, B), Bcl-2 (A, C), Bax/Bcl-2 (D) and caspase-3 (E, F). At the end of 2hours of reperfusion, the whole homogenates from frozen left ventricular tissue were analysed for protein expression by western blot analyses. Data are expressed as mean±standard deviation; n =3. *P <0.05, **P <0.01, ***P <0.001 versus the sham group. # P <0.05, ## P <0.01, ### P <0.001 versus the ischaemia/reperfusion (I/R) group.

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Effects of IL-1ra on IP3 receptor expression

Western blot analyses revealed that expression of the IP3 receptor was upregulated in the I/R group versus the sham group (P <0.01), while IL-1ra treatment downregulated IP3 expression (P <0.05 versus the I/R group) (Figure 3).



Figure 3


Figure 3. 

Effects of interleukin-1 receptor antagonist (IL-1ra) on the expression of the inositol 1,4,5-trisphosphate receptor (IP3R). Data are expressed as mean±standard deviation; n =3. *P <0.05, **P <0.01, ***P <0.001 versus the sham group. # P <0.05, ## P <0.01 versus the ischaemia/reperfusion (I/R) group.

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Effects of IL-1ra on the contractile properties of isolated ventricular cardiomyocytes

We evaluated the effects of IL-1ra on the contractile properties of isolated cardiomyocytes under normal and I/R conditions. As shown in Figure 4, at the end of simulated I/R, cardiomyocytes in the I/R group displayed markedly impaired peak shortening and reduced±dL/dt, while time to 90% relengthening was increased versus the DMSO group (all P <0.001). Administration of IL-1ra or 2-APB alone, under I/R conditions, led to an increase in peak shortening (P <0.001 and P <0.05, respectively, versus the I/R group), +dL/dt (P <0.001 and P <0.01, respectively, versus the I/R group) and–dL/dt (both P <0.001 versus the I/R group), with a decrease in time to 90% relengthening (both P <0.001 versus the I/R group), but there was no significant difference between the two groups. The same was also true of the 2-APB+IL-1ra+I/R group (peak shortening,±dL/dt and time to 90% relengthening: P <0.001 versus the I/R group), but compared with the IL-1ra+I/R and 2-APB+I/R groups, the 2-APB+IL-1ra+I/R group had higher values for peak shortening (P <0.05 and P <0.001, respectively), +dL/dt (P <0.05 and P <0.001, respectively) and–dL/dt (P <0.01 and P <0.001, respectively), and a shorter time to 90% relengthening (P <0.05 and P <0.001, respectively). There were no differences in resting cell length and time to peak.



Figure 4


Figure 4. 

Effects of the interleukin-1 receptor antagonist (IL-1ra) on the contractile properties of isolated ventricular cardiomyocytes. A. Resting cell length. B. Peak shortening (normalized to cell length); C. Maximal velocity of shortening (+dL/dt). D. Maximal velocity of relengthening (−dL/dt). E. Time to peak shortening (TPS). F. TR (90) (time to 90% relengthening). Data are expressed as mean±standard deviation; n =10–15 cells each. *P <0.05, ** P <0.01, ***P <0.001 versus the dimethyl sulphoxide (DMSO) group. # P <0.05, ## P <0.01, ### P <0.001 versus the ischaemia/reperfusion (I/R) group. $ P <0.05, $$ P <0.01, $$$ P <0.001 versus the IL-1ra+I/R group. % P <0.05, %% P <0.01, %%% P <0.001 versus the 2-aminoethoxydiphenyl borate (2-APB)+I/R group.

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Effects of IL-1ra on the Ca2+ transients in isolated ventricular cardiomyocytes

To explore whether IL-1ra could reduce Ca2+ overload in I/R, we evaluated intracellular Ca2+ homeostasis using the fluorescence dye Fura 2-AM. Results (Figure 5), showed that Ca2+ transient indicators were diminished at the end of simulated I/R, i.e. ΔFFI was significantly reduced (P <0.001 versus the DMSO group) and Tau values were significantly prolonged (P <0.001 versus the DMSO group), while pretreatment with either IL-1ra or 2-APB alone partially restored ΔFFI and Tau values, with no differences between the two groups. In the 2-APB+IL-1ra+I/R group, ΔFFI was increased and Tau values were reduced versus the I/R group, with more significance compared with the IL-1ra and 2-APB groups. Intergroup differences in resting intracellular Ca2+ concentrations were insignificant (P >0.05).



Figure 5


Figure 5. 

Effects of the interleukin-1 receptor antagonist (IL-1ra) on the Ca2+ transients of isolated ventricular cardiomyocytes. A. Representative recordings expressed as F/F0. B. Resting intracellular Ca2+. C. Ca2+ transient amplitude (ΔFFI). D. Decay time constants of the Ca2+ transients (Tau). Data are expressed as mean±standard deviation; n =10–15 cells each. *P <0.05, **P <0.01, ***P <0.001 versus the dimethyl sulphoxide (DMSO) group. # P <0.05, ## P <0.01, ### P <0.001 versus the ischaemia/reperfusion (I/R) group. $ P <0.05, $$ P <0.01, $$$ P <0.001 versus the IL-1ra+I/R group. % P <0.05, %% P <0.01, %%% P <0.001 versus the 2-aminoethoxydiphenyl borate (2-APB)+I/R group.

Zoom

Effects of IL-1ra and 2-APB on IP3 receptor expression in cardiomyocytes

Western blot analyses revealed that IP3 receptor expression was upregulated in the I/R group versus the DMSO group (P <0.001), while IP3 receptor expression was downregulated in the IL-1ra group (P <0.01 versus the I/R group) (Figure 6), the 2-APB+I/R group and the 2-APB+IL-1ra group. IP3 receptor expression was downregulated in the 2-APB+IL-1ra group and in the other two treatment groups.



Figure 6


Figure 6. 

Effects of the interleukin-1 receptor antagonist (IL-1ra) and 2-aminoethoxydiphenyl borate (2-APB) on the expression of the inositol 1,4,5-trisphosphate receptor (IP3R) in cardiomyocytes. Data are expressed as mean±standard deviation; n =3. *P <0.05, **P <0.01, ***P <0.001 versus the dimethyl sulphoxide (DMSO) group. # P <0.05, ## P <0.01, ### P <0.001 versus the ischaemia/reperfusion (I/R) group. $ P <0.05, $$ P <0.01, $$$ P <0.001 versus the IL-1ra+I/R group. % P <0.05, %% P <0.01, %%% P <0.001 versus the 2-APB+I/R group.

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Discussion

Our TTC staining showed that IL-1ra treatment reduced I/R-induced myocardial infarct size, and our western blot results demonstrated that IL-1ra treatment upregulated expression of the antiapoptotic protein Bcl-2, and downregulated expression of the proapoptotic proteins Bax and cleaved casepase-3, thereby increasing the Bcl-2/Bax ratio. Research into IL-1ra gene transfection of a myocardial I/R injury model confirmed that overexpression of IL-1ra, by reducing the IL-1-induced apoptosis associated with inflammation, reduces myocardial infarct size, thus reducing I/R injury and providing myocardial protection [28]. Despite the consensus on this schema of cardiomyocytic protection by IL-1ra, there could be alternative mechanisms.

In our experiment, the IP3 receptor protein was upregulated subsequent to myocardial I/R, while intervention with IL-1ra downregulates the expression of the IP3 receptor, which suggests that the IP3 receptor is involved in the myocardial I/R injury. IP3 receptor-mediated Ca2+ release is critical to Ca2+ overload, with the risk of Ca2+ overload increasing with IP3 receptor-released Ca2+ [29]. Persistent elevation of intracellular Ca2+ concentrations is reported to be a signal for apoptosis activation, and an increase in Ca2+ release can undermine the homeostasis of cells per se, which is a key contributor to the system of apoptosis [30]. We thus hypothesize that IL-1ra-reduced myocardial infarct size and myocardial apoptosis in I/R are related to a reduction in Ca2+ overload, and that this reduction is mediated by IP3 receptors.

Our test results for individual cardiomyocytes showed the benefits of IL-1ra in terms of systolic and diastolic functions, which not only improve systolic peak, but also increase maximum reduction rate and complex long rates, with a reduction in myocardial relaxation time, indicating that IL-1ra improves single contractile cardiomyocytes and diastolic function, with the IP3 receptor inhibitor 2-APB having similar effects on cardiac systolic and diastolic functions. Meanwhile, the combination of 2-APB and IL-1ra has a synergistic effect on systolic and diastolic functions. Variation in Ca2+ load in myocardiocytes is the main determinant in cell excitation-contraction coupling and systolic/diastolic function [31]. In each myocardial contraction, Ca2+-mediated excitation-contraction coupling serves as an initial link, accompanied by elevation and restoration of intracellular Ca2+ concentrations. This time effect of Ca2+ concentrations creates intracellular Ca2+ transients, which correspond to the variations in the contractile force of the myocardial cells [32].

Our subsequent evaluation of intracellular Ca2+ transients with the fluorescence dye Fura 2-AM showed that, after myocardial I/R injury, Ca2+ transient amplitudes were significantly reduced and the diastolic Ca2+ attenuation process was prolonged, i.e. myocytes in a state of Ca2+ overload, with Ca2+ transients diminished and attenuation retarded. Similar to 2-APB, IL-1ra can increase the Ca2+ transient peak and shorten the duration of diastolic Ca2+ attenuation, with I/R myocardial Ca2+ load greatly ameliorated; this can be further enhanced by its combination with 2-APB. These effects indicated a strong resemblance between IL-1ra and 2-APB in the protection of the cardiomyocytes, which is achieved by a reduction in Ca2+ overload via inhibition of IP3 receptor expression, at least in part. The determination results of in vitro IP3 receptor expression at the cellular level were identical to ex vivo tests, both indicating that myocardial I/R injury upregulated IP3 receptor expression, which was downregulated by IL-1ra.

Conclusions

In this study, we observed that the combination of 2-APB and IL-1ra as pretreatment before I/R compared with 2-APB alone was more potent in terms of improvement in systolic and diastolic functions, Ca2+ transients and apoptosis-related indicators after I/R. Even in the case of inhibited IP3 receptor, IL-1ra still played a protective role, which was entirely independent of the IP3 receptor. In addition to the downregulation of IP3 receptor, the protective effects of IL-1ra might rest on the involvement of other pathways, as we speculated, presumably L-type Ca2+ channels, which bring novel prospects for our future studies. Our experiment was flawed, in that we only focused on the downregulation of the IP3 receptor by IL-1ra and the achieved reduction in Ca2+ overload, without research into effective approaches to the downregulation of the IP3 receptor, which requires further research in terms of its underlying molecular biological mechanism.

Funding

None.

Contribution of authors

Wenhao Qian and Changyun Zhao contributed equally to this work.

Disclosure of interest

The authors declare that they have no competing interest.

References

Lawlor M., Gersh B., Opie L., Gaziano T.A. The global perspective of ischemic heart disease Chronic coronary artery disease: a companion to braunwald's heart disease Philadelphia: PA: Elsevier (2018).  16-32
Frank A., Bonney M., Bonney S., Weitzel L., Koeppen M., Eckle T. Myocardial ischemia reperfusion injury: from basic science to clinical bedside Semin Cardiothorac Vasc Anesth 2012 ;  16 : 123-132 [cross-ref]
Hausenloy D.J., Yellon D.M. Myocardial ischemia-reperfusion injury: a neglected therapeutic target J Clin Invest 2013 ;  123 : 92-100 [cross-ref]
Sun S.L., Guo L., Ren Y.C., and al. Anti-apoptosis effect of polysaccharide isolated from the seeds of Cuscuta chinensis Lam on cardiomyocytes in aging rats Mol Biol Rep 2014 ;  41 : 6117-6124 [cross-ref]
Garcia-Dorado D., Ruiz-Meana M., Inserte J., Rodriguez-Sinovas A., Piper H.M. Calcium-mediated cell death during myocardial reperfusion Cardiovasc Res 2012 ;  94 : 168-180 [cross-ref]
Janczewski A.M., Lakatta E.G. Modulation of sarcoplasmic reticulum Ca(2+) cycling in systolic and diastolic heart failure associated with aging Heart Fail Rev 2010 ;  15 : 431-445 [cross-ref]
Li P.C., Yang Y.C., Hwang G.Y., Kao L.S., Lin C.Y. Inhibition of reverse-mode sodium-calcium exchanger activity and apoptosis by levosimendan in human cardiomyocyte progenitor cell-derived cardiomyocytes after anoxia and reoxygenation PLoS One 2014 ;  9 : e85909
Samanta K., Kar P., Chakraborti T., Chakraborti S. An overview of endoplasmic reticulum calpain system New York: NY: Springer (2013).  3-19
Bartok A., Golenár T., Bánsághi S., and al. Study of the capacity of each IP3 receptor isoform to support er-mitochondrial calcium transfer Biophys J 2016 ;  110 : [312a].
Matecki S., Fauconnier J., Lacampagne A. Reactive oxygen species and muscular dystrophy Systems biology of free radicals and antioxidants Berlin: Heidelberg: Springer-Verlag (2014).  3055-3079
Ewart M.A., Ugusman A., Vishwanath A., and al. Changes in IP3 receptor expression and function in aortic smooth muscle of atherosclerotic mice J Vasc Res 2017 ;  54 : 68-78 [cross-ref]
Dinarello C.A. Interleukin-1 and interleukin-1 antagonism Blood 1991 ;  77 : 1627-1652
Galea J., Ogungbenro K., Hulme S., and al. Reduction of inflammation after administration of interleukin-1 receptor antagonist following aneurysmal subarachnoid hemorrhage: results of the Subcutaneous Interleukin-1Ra in SAH (SCIL-SAH) study J Neurosurg 2018 ;  128 : 515-523 [cross-ref]
Abbate A., Kontos M.C., Grizzard J.D., and al. Interleukin-1 blockade with anakinra to prevent adverse cardiac remodeling after acute myocardial infarction (Virginia Commonwealth University Anakinra Remodeling Trial [VCU-ART] pilot study) Am J Cardiol 2010 ;  105 : [1371e1–1377e1].
Grothusen C., Hagemann A., Attmann T., and al. Impact of an interleukin-1 receptor antagonist and erythropoietin on experimental myocardial ischemia/reperfusion injury Sci World J 2012 ;  2012 : 737585
Li G., Qian W., Zhao C. Analyzing the anti-ischemia-reperfusion injury effects of ginsenoside Rb1 mediated through the inhibition of P 38alpha MAPK Can J Physiol Pharmacol 2016 ;  94 : 97-103 [cross-ref]
Stumpner J., Redel A., Kellermann A., and al. Differential role of Pim-1 kinase in anesthetic-induced and ischemic preconditioning against myocardial infarction Anesthesiology 2009 ;  111 : 1257-1264 [cross-ref]
Fang F., Li D., Pan H., and al. Luteolin inhibits apoptosis and improves cardiomyocyte contractile function through the PI3K/Akt pathway in simulated ischemia/reperfusion Pharmacology 2011 ;  88 : 149-158 [cross-ref]
Patterson S.D., Hess D., Yungwirth T., Aebersold R. High-yield recovery of electroblotted proteins and cleavage fragments from a cationic polyvinylidene fluoride-based membrane Anal Biochem 1992 ;  202 : 193-203 [cross-ref]
Qi L., Pan H., Li D., Fang F., Chen D., Sun H. Luteolin improves contractile function and attenuates apoptosis following ischemia-reperfusion in adult rat cardiomyocytes Eur J Pharmacol 2011 ;  668 : 201-207 [cross-ref]
Burley D.S., Ferdinandy P., Baxter G.F. Cyclic GMP and protein kinase-G in myocardial ischaemia-reperfusion: opportunities and obstacles for survival signaling Br J Pharmacol 2007 ;  152 : 855-869 [cross-ref]
Abbate A., Salloum F.N., Vecile E., and al. Anakinra, a recombinant human interleukin-1 receptor antagonist, inhibits apoptosis in experimental acute myocardial infarction Circulation 2008 ;  117 : 2670-2683 [cross-ref]
Paillard M., Tubbs E., Thiebaut P.A., and al. Depressing mitochondria-reticulum interactions protects cardiomyocytes from lethal hypoxia-reoxygenation injury Circulation 2013 ;  128 : 1555-1565 [cross-ref]
Hao Y., Sun Y., Xu C., and al. Improvement of contractile function in isolated cardiomyocytes from ischemia-reperfusion rats by ginkgolide B pretreatment J Cardiovasc Pharmacol 2009 ;  54 : 3-9 [cross-ref]
Wu Q., Zhao Z., Sun H., Hao Y.L., Yan C.D., Gu S.L. Oestrogen changed cardiomyocyte contraction and beta-adrenoceptor expression in rat hearts subjected to ischaemia-reperfusion Exp Physiol 2008 ;  93 : 1034-1043 [cross-ref]
Turdi S., Schamber R.M., Roe N.D., Chew H.G., Culver B., Ren J. Acute methamphetamine exposure inhibits cardiac contractile function Toxicol Lett 2009 ;  189 : 152-158 [cross-ref]
Lovelock J.D., Monasky M.M., Jeong E.M., and al. Ranolazine improves cardiac diastolic dysfunction through modulation of myofilament calcium sensitivity Circ Res 2012 ;  110 : 841-850 [cross-ref]
Suzuki K., Murtuza B., Smolenski R.T., and al. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts Circulation 2001 ;  104 : [I207–I212].
Domeier T.L., Zima A.V., Maxwell J.T., Huke S., Mignery G.A., Blatter L.A. IP3 receptor-dependent Ca2+ release modulates excitation-contraction coupling in rabbit ventricular myocytes Am J Physiol Heart Circ Physiol 2008 ;  294 : [H596–H604].
Cao Z., Liu D., Zhang Q., Sun X., Li Y. Aluminum chloride induces osteoblasts apoptosis via disrupting calcium homeostasis and activating Ca(2+)/CaMKII signal pathway Biol Trace Elem Res 2016 ;  169 : 247-253 [cross-ref]
Ito Y., Suko J., Chidsey C.A. Intracellular calcium and myocardial contractility. V. Calcium uptake of sarcoplasmic reticulum fractions in hypertrophied and failing rabbit hearts J Mol Cell Cardiol 1974 ;  6 : 237-247 [cross-ref]
Langer G.A. The role of calcium in the control of myocardial contractility: an update J Mol Cell Cardiol 1980 ;  12 : 231-239 [cross-ref]



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