ClC is a member of the

ClC-3 is a member of the voltage-gated chloride channel superfamily and has many roles in cell proliferation, apoptosis, CC-115 receptor progression and so on (Dai et al., 2005). A growing number of studies showed that ClC-3 encoded a volume-regulated Cl− channel (ICl,Vol) in heart, that could contribute to the regulation of the cardiomyocyte volume (Nagasaki et al., 2000). Activation of ClC-3-related volume-sensitive outwardly-rectifying anion channels displayed the protection of myocardium from infarction through regulating cardiac volume decrease when cell swells during ischemia and reperfusion (Bozeat et al., 2011, Yu et al., 2016). Recent studies demonstrated that cardiac-specific inducible ClC-3 gene deletion eliminated native ICl,Vol and promoted myocardial hypertrophy in adult mice (Duan, 2011, Xiong et al., 2010). Isoprenaline was found to consistently reduce ICl,vol in Ca-free hypotonic bath solutions with strong intracellular Ca buffering in canine atrial myocytes (Nagasaki et al., 2000). All these results suggested that ClC-3 may play a role in cardiac hypertrophy induced by isoprenaline. Therefore, our present study intends to study whether ClC-3 is associated with cardiac hypertrophy induced by isoprenaline. Rat cardiomyoblast cell line H9c2 cells, rat primary neonatal cardiomyocyte cells, wild C57/BL/6 mice and mice treated with adeno-associated virus 9 (AAV9) carrying ClC-3 were used as models in vitro and in vivo respectively. Cell volume, heart mass index, histological analysis and echocardiography were detected to evaluate the cardiac hypertrophy of models. Changes in the expression levels of ANF, β-MHC and ClC-3 in the models in the absence or presence of propanolol, an antagonist β-adrenergic receptor, were assessed by real-time PCR, immunofluorescence techniques, western blot and immunohistochemical staining.
Materials and methods
Results
Discussion Cardiac hypertrophy is characterized by an increased cardiomyocyte size, excess protein synthesis, and thickened ventricular walls. Although myocardiac hypertrophy initially might be adaptive and compensatory, a sustained pathological hypertrophic response is deleterious and eventually develops with heart failure, such as dilated cardiomyopathy and myocardial infarction (Accornero et al., 2011, Aggarwal et al., 2014, Ai et al., 2010). Isoprenaline, a good activator of β-adrenergic receptors in heart, is believed to evoke myocardial hypertrophy in many studies in vitro and in vivo (Chen et al., 2012, Odashiro et al., 2002, Szabo et al., 1975). In line with these findings, our present study demonstrated that isoprenaline could significantly induce an increase in cell size of H9c2 cells, primary rat neonatal cardiomyocytes and cardiomyocytes of C57/BL/6 mice, an augment of heart mass index and a thickening of interventricular septum and left ventricular posterior wall in mice, associated with a rise in ANF and β-MHC mRNA expression in primary rat neonatal cardiomyocytes and C57/BL/6 mice. Meanwhile, our results showed that isoprenaline down-regulated ClC-3 expression in hypertrophic cardiomyocytes and mice myocardium, and this down-regulation of ClC-3 expression elicited by isoprenaline could be prevented by the pretreatment with propranolol. ClC-3, a large gene family of chloride channels, was initially found to be expressed in rat brain neuronal cells as a neuronal anion channel (Kawasaki et al., 1994). A large number of studies has documented that ClC-3 is critical for adaptive remodeling in response to physiological and pathophysiological stress correlated with cell volume perturbations in different cell types, such as vascular smooth muscle cells (Ganapathi et al., 2013, Guan et al., 2006), human nonpigmented ciliary epithelial cells (Coca-Prados et al., 1996), human prostate cancer epithelial cells (Lemonnier et al., 2004), nasopharyngeal carcinoma cells (Huang et al., 2014, Mao et al., 2008), Hela cells (Hermoso et al., 2002). However, the functional role of ClC-3 associated with cell volume in cardiomyocytes is currently little known. Nagasaki, M et al. found that chloride currents activated by changes in cell volume (volume-activated chloride currents, ICl,vol) obviously increased in cardiac ClC-3 expression, and thought that ClC-3 might be a ubiquitous volume-regulated chloride channel in myocardium (Nagasaki et al., 2000). Hiramatsu et al. observed that ClC-3 mRNA levels in rat hypertrophic heart induced by banding the abdominal aorta decreased in the early phase after banding, whereas increased in the late phase (Hiramatsu et al., 2002). Xiong et al. found that cardiac-specific inducible ClC-3 knock-out mice exhibited myocardial hypertrophy and heart failure (Xiong et al., 2010). In the present study, we presented several lines of evidence in vitro and in vivo to support the idea that the mRNA and protein expression levels of ClC-3 were reduced in hypertrophic cardiomyocytes or cardiac hypertrophy mice induced by isoprenaline, and this reduction could be reversed by propranolol-pretreatment. These suggest that the down-regulation of ClC-3 mRNA and protein expression induced by isoprenaline may be involved in cardiac hypertrophy through reducing volume-activated chloride currents. However, volume-activated chloride currents are double regulated by the levels of ClC-3 protein expression and the activity of single ClC-3 chloride channel (Gong et al., 2004, Guo et al., 2016). In addition to reducing the expression of ClC-3 mRNA and protein expression, does isoprenaline also affect the activity of ClC-3 chloride channels? Further study is needed.