Original articleHigh-mobility group box 1 (HMGB1) impaired cardiac excitation–contraction coupling by enhancing the sarcoplasmic reticulum (SR) Ca2 + leak through TLR4–ROS signaling in cardiomyocytes
Introduction
Inflammatory response and cytokine elaboration are integral components of the host response to multiple pathological stimulations and play a particularly active role in cardiac injury [1], [2]. Numerous myocardial cytokines including TNF-α, IL-1, IL-6 and IL-10, have been demonstrated to severely impair cardiac function [3], [4], [5]. Recently, another inflammatory cytokine, high-mobility group box 1 (HMGB1) has aroused much attention for its important role in the pathogenesis of cardiac dysfunction in many diseases, such as ischemic heart diseases, sepsis, and diabetic cardiomyopathy [6], [7], [8], [9]. HMGB1 is a nuclear factor released passively by necrotic cells and actively by activated immune cells. Once released into the extracellular milieu, HMGB1 activates inflammatory responses, serving as a late mediator of systemic inflammation [10]. It has been demonstrated that the serum HMGB1 level was significantly increased in patients with ST-elevation myocardial infarction (MI). Strikingly, the serum HMGB1 level was associated with the severity of heart injury. A high peak serum HMGB1 level was associated with pump failure, cardiac rupture, and in-hospital cardiac death in patients with MI [11]. In addition, the plasma concentration of HMGB1 was correlated with the degree of organ dysfunction during septic shock [12] and the severity of heart failure (HF) in both diabetic and non-diabetic patients [13]. Passive immunization with either neutralizing anti-HMGB1 antibodies or HMGB1-specific blockage via box A treatment prevented cardiac dysfunction in mice with ischemia–reperfusion injury [14], sepsis [15], and diabetic cardiomyopathy [8]. All the evidence supports the importance of HMGB1 in the pathogenesis of cardiac dysfunction in these diseases.
Of note, despite the deleterious effects, HMGB1 has long-term beneficial effects due to the induction of myocardial regeneration and attenuation of left ventricular remodeling, especially in ischemic heart diseases [11], [16]. Thus, extracellular HMGB1 may activate signals that promote either cardiac adaptation/protection or maladaptive responses. In this consideration, elucidating the mechanisms which specifically induce the deleterious effects of HMGB1 is of particular importance for the design of effective therapeutic strategy preventing the deleterious effects of HMGB1 without affecting its cardioprotective activity.
The cardiac ryanodine receptor (RyR2) plays an essential role in cardiac excitation–contraction coupling (EC coupling) by gating Ca2 + release from the sarcoplasmic reticulum (SR) via the Ca2 +-induced Ca2 + release (CICR) mechanism [17], [18]. There is evidence that the SR leaks calcium through RyR2 even in the absence of stimulation at resting state, which may have a protective role against an excessive increase of SR Ca2 + content [19]. However, if RyR2 becomes excessively active, it will reduce SR Ca2 + content by enhancing diastolic SR Ca2 + leak, resulting in a decrease of systolic Ca2 + transient and hence contractile dysfunction in cardiomyocytes [20], [21], [22]. Furthermore, enhancement of SR Ca2 + leak during diastole could initiate Ca2 + waves through CICR mechanism and trigger cardiac arrhythmias [23], [24]. Evidence from various models of heart failure (HF) suggests that cardiac RyR2s became excessively active i.e., “leaky”, which plays an important role in promoting the pathogenesis of HF [20], [22], [25]. Recently, it has been shown that inflammatory factor TNF-α caused SR Ca2 + leak, which contributes to arrhythmia, cardiac infarction and myocardial remodeling in cardiac ischemia–reperfusion injury [26].
It is well recognized that RyR2 acts as a cellular redox sensor due to the rich free thiol groups in its structure [27], [28]. In particular, the oxidation of its cysteine groups facilitates RyR opening and SR Ca2 + leak. Yano et al. has demonstrated that oxidative stress of RyR2 produced SR Ca2 + leak by destabilizing the interdomain interactions within RyR2, which accounts for SR Ca2 + leak in HF [25]. Research from Gyorke's lab has also shown that redox modification of RyR2s increased the open probability of the Ca2 + releasing channels, leading to SR Ca2 + leak in chronic HF [20]. Furthermore, intracellular oxidative stress has been shown to activate Ca2 +/calmodulin dependent protein kinase II (CaMKII) by oxidizing the enzyme and consequently increased RyR activity by phosphorylation of RyR at Ser2814 [29], [30].
It has been suggested that HMGB1 causes TLR4-dependent activation of NAD(P)H oxidase as well as increased reactive oxygen species (ROS) production through both MyD88–IRAK4–p38 MAPK and MyD88–IRAK4–Akt signaling pathways [31], [32]. We thus proposed that HMGB1 induces SR Ca2 + leak and hence negative inotropic effect in cardiomyocytes. To test this hypothesis, we investigated the effects of HMGB1 on Ca2 + handling and myocyte contractility in isolated adult rat ventricular myocytes. The results show that HMGB1 enhanced Ca2 + spark-mediated SR leak by activation of TLR4/ROS signaling pathway. The enhancement of SR leak decreased SR Ca2 + content, which consequently diminished systolic Ca2 + transient and impaired cardiac contractile function.
Section snippets
Isolation of adult rat ventricular myocytes
Adult Sprague–Dawley rats of either sex, weighing 200 to 250 g from animal center of Southern Medical University were used throughout the study. All animal experiments were performed in accordance with ethical standards as formulated in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996) and approved by Institutional Animal Care and Use Committee of Shenzhen University. Adult rat ventricular myocytes
HMGB1 dose-dependently increased the frequency of Ca2 + sparks in cardiomyocytes
Ca2 + sparks as the elementary Ca2 + release events reflect RyR activities and many features of SR function in intact cells. To investigate whether HMGB1 regulates RyR activity and SR function, we measured the rate and characteristics of Ca2 + spark in isolated adult rat ventricular myocytes (ARVMs). The cells were incubated with HMGB1 (50–200 ng/ml) for 30 min before the experiments. We found that HMGB1 dose-dependently increased the rate of Ca2 + sparks (Figs. 1A, B). The average frequency of Ca2 +
Discussion
In this study, we demonstrated for the first time that HMGB1 impaired cardiac EC coupling by enhancing Ca2 + spark-mediated SR Ca2 + leak. The enhancement of SR Ca2 + leak caused partial depletion of SR Ca2 + content, which consequently decreased systolic Ca2 + transient and hence cardiac contractility. Furthermore, we revealed that oxidation and CaMKII-induced hyperphosphorylation of RyR2 (at Ser2814) underlie the hyperactive Ca2 + sparks upon HMGB1 stimulation. The intracellular oxidative stress
Conflict of interest statement
None.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 31171096, 31371159 and 81200122) and Basic Research Foundation of Shenzhen (Nos. JC201005250059A, JCYJ20120613115535998 and JCYJ20130326112207234).
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These authors contributed equally to this work.