TRPM7 regulates angiotensin II-induced sinoatrial node fibrosis in sick sinus syndrome rats by mediating Smad signaling

Heart and Vessels https://doi.org/10.1007/s00380-018-1146-0 ORIGINAL ARTICLE TRPM7 regulates angiotensin II‑induced sinoatrial node fibrosis in sick sinus syndrome rats by mediating Smad signaling Hongbin Zhong1 · Tingjun Wang1 · Guili Lian1 · Changsheng Xu1 · Huajun Wang1 · Liangdi Xie1 Received: 12 December 2017 / Accepted: 2 March 2018 © The Author(s) 2018. This article is an open access publication Abstract Sinoatrial node fibrosis is involved in the pathogenesis of sinus sick syndrome (SSS). Transient receptor potential (TRP) subfamily M member 7 (TRPM7) is implicated in cardiac fibrosis. However, the mechanisms underlying the regulation of sinoatrial node (SAN) fibrosis in SSS by TRPM7 remain unknown. The aim of this study was to investigate the role of angiotensin II (Ang II)/TRPM7/Smad pathway in the SAN fibrosis in rats with SSS. The rat SSS model was established with sodium hydroxide pinpoint pressing permeation. Forty-eight rats were randomly divided into six groups: normal control (ctrl), sham operation (sham), postoperative 1-, 2-, 3-, and 4-week SSS, respectively. The tissue explant culture method was used to culture cardiac fibroblasts (CFs) from rat SAN tissues. TRPM7 siRNA or encoding plasmids were used to knock down or overexpress TRPM7. Collagen (Col) distribution in SAN and atria was assessed using PASM–Masson staining. Ang II, Col I, and Col III levels in serum and tissues or in CFs were determined by ELISA. TRPM7, smad2 and p-smad2 levels were evaluated by real-time PCR, and/or western blot and immunohistochemistry. SAN and atria in rats of the SSS groups had more fibers and higher levels of Ang II, Col I and III than the sham rats. Similar findings were obtained for TRPM7 and pSmad2 expression. In vitro, Ang II promoted CFs collagen synthesis in a dose-dependent manner, and potentiated TRPM7 and p-Smad2 expression. TRPM7 depletion inhibited Ang II-induced p-Smad2 expression and collagen synthesis in CFs, whereas increased TRPM7 expression did the opposite. SAN fibrosis is regulated by the Ang II/TRPM7/Smad pathway in SSS, indicating that TRPM7 is a potential target for SAN fibrosis therapy in SSS. Keywords Sick sinus syndrome · Sinoatrial node · Angiotensin II · Collagen · TRPM7 · Smad2 Abbreviation Ang II Angiotensin II CFs Cardiac fibroblasts CMFs Cardiac myofibroblasts SAN Sinoatrial node SD Sprague–Dawley SSS Sinus sick syndrome TRP Transient receptor potential TRPM Transient receptor potential melastatin TRPM7 Transient receptor potential subfamily M member 7 * Liangdi Xie 1 Fujian Hypertension Research Institute, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China Introduction Sick sinus syndrome (SSS), a common clinical arrhythmia accounting for approximately 50% of permanent pacemakers worldwide [1], manifests as bradycardia, sinus arrest, sinoatrial blockage, or bradycardia-tachycardia syndrome [2, 3]. SSS is also an independent risk for severe cardiovascular disorders and mortality [4]. Studies on anatomical morphology of sinoatrial node (SAN) have shown that fibrous tissues in SAN play an important role in the maintenance of normal pacing and conduction of SAN, while the fibrosis of SAN tissues affects the generation of SAN action potential and conduction, resulting in SSS [5–7]. In addition, the atrium in sinus node disease due to extensive atrial fibrosis can lead to an atrial standstill [8]. Mechanistically, abnormal ion channels responsible for the initiation and/or conduction of cardiac action potentials are considered the main electrophysiological mechanism underlying the occurrence of SSS [9, 10]. For instance, increased C a 2+ flux promoted the pathogenesis of SSS 13 Vol.:(0123456789) Heart and Vessels [11] and the suppression of muscarinic-gated K + channel by chemicals or genetic deletion reduced SSS in a mouse SSS model [12]. In addition, angiotensin II (Ang II) is well documented to promote the formation of cardiac myoblasts (CFs) into cardiac myofibroblasts (CMFs), which is a key process in the pathogenesis of cardiac fibrosis [13] and intracellular C a2+ flux plays an important role in this transformation process [14]. The transient receptor potential (TRP) family is a group of highly conserved genes that encode membrane proteins that act as ion channels including Ca2+ and Mg2+ ions [15] and is involved in the mediation of a variety of cellular events and in the pathophysiology of numerous human diseases including cardiovascular disorders [16]. The TRP family is currently divided into seven subfamilies, including the transient receptor potential melastatin (TRPM) subfamily [15]. Among this subfamily of 8 members, TRPM7 is localized in both the plasma membrane and intracellular organelles and exhibits both protein kinase activity and ion channel functions [17]. TRPM7 is highly expressed in SAN, and the knockout of TRPM7 in both zebrafish and mice interfered with cardiac automaticity [18], indicating the importance of TRPM7 in SAN homeostasis. In addition, TRPM7 signaling appears to functionally interact with Ang II signaling. For example, Ang II stimulation upregulated TRPM7 expression in vascular smooth muscle cells [19], while TRPM7 regulated the downstream molecular phenotypes of CFs induced by Ang II [20]. In the clinic, TRPM and its mediated C a2+-influx signal in CFs of SSS patients have been shown to play a key role in the transformation of CFs into CMFs [21], which was supported by the findings that TRPM7 contributed to the Ang II-mediated progression of atrial fibrosis through the regulation of influx of C a2+ and M g2+ [22]. It has been well recognized that the Ang II-mediated TGFβ1/Smad pathway plays an important role in promoting CFs to secrete extracellular matrix and in myocardial collagen deposition [23–25]. The molecular basis underlying how TRPM7 is incorporated into Ang II-mediated TGFβ1/Smad signaling to direct the development of SSS has not been well defined, although one study proposed that TRPM7 was potentially required in TGF-β-induced fibrogenesis in human atrial fibrillation [26] and another study showed that TRPM7 mediated TGF-β1-elicited collagen expression in hepatic stellate cells [27]. In the present study, we established a rat SSS model with different degrees of SAN fibrosis and then investigated the changes in the levels of Ang II, TRPM7 and Smad2 in these rats in vivo. We also employed gain- and loss-of-function approaches to further examine the effects of TRPM7 on Smad2 signaling and fibrosis in Ang IIinduced transformation of CFs into CMFs in vitro. 13 Materials and methods Animal A total of 48 Sprague–Dawley (SD) rats (12-week-old males, weighed 250 ± 10 g) were purchased from the Shanghai SLACCAS Laboratory Animal Co., Ltd (Shanghai, China; Certificate No. 20120005). Four rats were housed per cage with free access to tap water and food. The room was automatically controlled under a constant temperature of 22 ± 2 °C (...truncated)