Original articleTachycardia-induced CD44/NOX4 signaling is involved in the development of atrial remodeling
Introduction
Atrial fibrillation (AF), the most common sustained arrhythmia, is characterized by atrial electrical, contractile, and structural remodeling [1]. Accumulating evidence suggests that intracellular Ca2+ overload and handling abnormality contribute considerably to the pathogenesis of AF [[1], [2], [3]]. The elevation of diastolic [Ca2+] through an increase of sarcoplasmic reticulum (SR) Ca2+ spark frequency is recognized as a crucial mechanism in AF genesis [[1], [2], [3], [4]]. Ca2+ sparks are known to participate in the initiation and propagation of Ca2+ waves in atrial myocytes, thus contributing to the development of delayed afterdepolarizations [[1], [2], [3], [4], [5]]. It has also been indicated that Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation (Ser2814) of ryanodine receptor type 2 (RyR2) may lead to increased diastolic SR Ca2+ leak [[5], [6], [7], [8]]. Therefore, the expression of CaMKII-dependent p-RyR2 and the frequency of Ca2+ spark could be recognized as vital markers for Ca2+-handling abnormalities in atrial myocytes.
Numerous clinical and experimental studies have demonstrated the association of oxidative stress with AF [[9], [10], [11], [12], [13], [14], [15]]. Potential sources of oxidative stress in atrial myocytes include the mitochondrial electron transport chains, xanthine oxidases, “uncoupled” nitric oxide synthases, and NADPH oxidases (NOXs) [[9], [10], [11], [12], [13], [14], [15]]. Our prior studies have suggested that reactive oxygen species (ROS) derived from NOXs, especially NOX2 and 4 subunits, may mediate tachypacing-induced myofibril degradation in atrial myocytes and fibrotic response in atrial fibroblasts [11,13]. Recently, oxidized CaMKII (ox-CaMKII) has been identified as a crucial signal for connecting increased atrial ROS to AF [14]. We and others have revealed increased ox-CaMKII expression in atrial tissues from AF patients and in rapid atrial pacing models [14,15]. In this study, we further clarified the role of NOXs and other related signaling in Ca2+-handling abnormalities of atrial myocytes.
CD44, a transmembrane receptor for hyaluronan (HA), is implicated in regulating various cellular processes [16]. It has been indicated that HA is a signaling connector between TGF-β and CD44 in some cells [[17], [18], [19], [20], [21], [22]]. Our recent study found that CD44 suppression with an anti-CD44 blocking antibody could not only attenuate atrial fibrosis but also diminish AF occurrence, implicating the critical role of CD44 in the pathogenesis of atrial fibrosis and fibrillation [23]. Furthermore, a recent study in pulmonary fibrosis indicated that CD44 is involved in TGF-β/SMAD3-mediated NOX4 induction and ROS production [24]. Accordingly, the present study further evaluated the contributing roles of TGF-β/SMAD3 (a canonical downstream target of TGF-β) [25], HA/CD44, and NOX4/ROS signaling in atrial remodeling, especially Ca2+-handling abnormalities in myocytes. We utilized a previously characterized system (HL-1 cell line) to assess atrial remodeling in myocytes [26]. The potential relevance of findings obtained from in vitro pacing was verified in CD44 knock-out mice using ex vivo and in vivo tachypacing models.
Section snippets
Atrial-derived myocytes (HL-1 cell line) culture
HL-1 atrial myocytes were maintained in Claycomb medium and subjected to field stimulation as described elsewhere [11,15,27,28]. To induce tachycardia, HL-1 cells (≥1 × 106 cells) were cultured on 4-well rectangular dishes (Nuclon, Netherlands) and placed into C-Dish 100TM-Culture Dishes (IonOptix, MA). Cells were paced with 10-ms stimuli of 40-V intensity at selected frequencies (C-Pace EP culture pacer, IonOptix). >90% capture efficiency was confirmed by microscopic examination and by the
Tachypacing induces HAS/HA/CD44 signaling
Prior studies have demonstrated that tachypacing in cultured HL-1 myocytes may reflect the feature of tachycardia-induced remodeling in vivo [11,15,27,28]. Accordingly, we utilized this atrium-derived system to evaluate the effect of tachypacing on HAS/HA expression in vitro. Time- and frequency-dependent experiments revealed that tachypacing in HL-1 cells resulted in a higher expression of HAS2 mRNA, with the most evident change occurring at the sixth hour (Fig. 1A). Secreted HA was measured
Discussion
In this study, we have proposed an CD44-related mechanism by linking NOX4/ROS signaling to the pathogenesis of atrial remodeling and AF, especially Ca2+-handling abnormalities in atrial myocytes. We utilize in vitro, ex vivo, and in vivo tachypacing models to explore the involvement of CD44/NOX4 signaling in AF development. This study follows our prior work regarding the contributing role of CD44 in AF genesis to demonstrate that tachypacing induces a higher HAS/HA/CD44 expression in atrial
Declaration of Competing Interest
US patent was issued for the method in treating and/or preventing AF patients with a pharmaceutical composition containing an anti-CD44 neutralizing antibody or an antigen binding portion.
Acknowledgments
We thank Mr. Chih-Chun Chen for his technical assistance in confocal microscopy. This work was supported by Chang Gung Research Grant Foundation [CMRPG 3F0991-3, 3F2281-3, 3G1371-3 to WJC]; and Ministry of Science and Technology, Taiwan [104-2314-B-182-052-MY3, 107-2314-B-182-072-MY3, and 107-2314-B-182-073-MY3 to WJC].
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The first two authors contribute equally to this work.