MicroRNA-145 suppresses ROS-induced Ca2+ overload of cardiomyocytes by targeting CaMKIIδ
Introduction
Myocardial infarction (MI) is associated with increased reactive oxygen species (ROS) production, heart failure, and increased mortality [1]. The calcium/calmodulin dependent protein kinase II (CaMKII) has emerged as a MI- and ROS-activated signaling molecule that regulates expression of apoptotic genes and affects adverse outcomes after MI [2], [3], [4]. Cardiac-specific transgenic overexpression of CaMKII results in cardiac hypertrophy, heart failure, and premature death [5]. CaMKII is constitutively activated by threonine-287 phosphorylation, and constitutively active CaMKII can lead to many of these MI-related adverse effects [6], [7]. In recent in vitro experiments, we described an apoptotic pathway that involves increases in ROS produced by diesel exhaust particles, and activation of CaMKII [8].
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by targeting the 3′ untranslated region (3′UTR) of target mRNAs. As a result, translation is suppressed or target mRNA is rapidly degraded [9], [10]. Recent studies showed that miRNAs are involved in cardiac physiology and pathology [11], [12]. Many such studies have demonstrated that miRNAs can regulate cardiac apoptosis [13]. Myocardial-specific miR-1 and miR-133a may play an important role in cardiac apoptosis. miR-1 regulates cardiomyocyte apoptosis via post-transcriptional repression of IGF-1, Hsp60, and Bcl-2 [14], [15], [16]. A miR-133a mimic down-regulated caspase-9 protein expression and attenuated ischemia–reperfusion (I/R)-induced apoptosis [17]. miR-21, the miR-30 family, and miR-199a are anti-apoptotic miRNAs, and miR-195 and miR-320 are pro-apoptotic miRNAs [13], [18].
A previous study demonstrated that miR-145 is abundantly expressed in smooth muscle [19]. One of the targets of miR-145 in vascular smooth muscle cells (VSMC) is CaMKII [20]. miR-145 induces smooth muscle cell (SMC) proliferation and differentiation [20], [21]. Although miR-145 initiates apoptosis in cancer cells [22], [23], [24], the role of miR-145 in cardiomyocytes remains unclear. Therefore, our research focused on the effect of miR-145 expression in Ca2+ overload by activation of CaMKII in cardiomyocytes and determined whether miR-145 regulates ROS-induced cardiomyocyte apoptosis. Our data suggest that miR-145 may be a powerful therapeutic target for ischemic heart diseases.
Section snippets
Isolation of rat ventricular cardiomyocytes
Animals were handled in compliance with the Guiding Principles in the Care and Use of Animals. All experimental procedures for animal studies were approved by the Committee for the Care and Use of Laboratory Animals, Yonsei University College of Medicine, and performed in accordance with the Committee’s Guidelines and Regulations for Animal Care (NIH Publication No. 85-23, revised 1996). We isolated and purified neonatal rat cardiomyocytes. Briefly, 1- to 2-day-old Sprague Dawley rat pups were
H2O2 induces CaMKIIδ -mediated Ca2+ overload
To test H2O2-mediated induction of phosphorylated CaMKIIδ (p-CaMKIIδ), cardiomyocytes were treated with H2O2. H2O2 induced p-CaMKIIδ; however, the induction was markedly reduced by KN93 (a CaMKII selective inhibitor) and NAC (a ROS scavenger) (Fig. 1A). Cells treated with the analogue KN92 (which does not inhibit CaMKII) also exhibited induction of Ca2+, but KN92 did not protect against H2O2-induced Ca2+ overload. However, KN93 and NAC-treated cells exhibited reduced Ca2+ overload (Fig. 1B). To
Discussion
ROS are generated in MI, reperfusion injury, and mediated cell death [1], [25]. As powerful regulators of gene expression, miRNAs are now acknowledged as novel multi-target agents for treating cardiovascular pathology including MI. The present study demonstrates that overexpression of miR-145 protects against Ca2+ overload in cardiomyocytes under oxidative stress, in association with decreased CaMKIIδ expression.
Intracellular Ca2+ overload was initially considered an essential mechanism of
Acknowledgments
This research was supported by a Korea Science and Engineering Foundation grant funded by the Korean government (MEST) (2011-0019243, 2011-0019254), a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A120478), a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A085136), and Basic Science Research Program through the National Research Foundation of Korea (2011-0014595) funded by the Ministry of Education,
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These authors contributed equally to this work.