Programmed necrosis greatly contributes to the pathogenesis of cardiac disorders, especially severe myocardial damage such as I/R injury. Our present work identified that Syt-1 was an anti-necrosis and anti-I/R injury regulator. Syt1 inhibited I/R-induced necrotic cell death and I/R injury, and improved cardiac function. Syt1 was downregulated under oxidative stress, and overexpression of Syt1 suppressed H2O2-induced cardiomyocyte necrosis and mPTP opening. In further exploring the underlying mechanisms, we found that Syt1 interacted with Parkin and was required for Parkin to catalyze CypD ubiquitination. Syt1 regulated cardiomyocyte necrosis by targeting the Parkin-CypD-mPTP pathway. In addition, Syt1 expression was suppressed by miR-193b-3p. MiR-193b-3p mediated cardiomyocyte necrosis both in vivo and in vitro, and this regulatory effect was through targeting Syt1. Briefly, our work provides new insights into understanding the pathogenesis of I/R injury, and reveals a novel regulatory model of cardiomyocyte necrosis composed of miR-193b-3p, Syt1, Parkin, CypD and mPTP.
Syt1 was originally identified in the nervous system and functions in neurotransmitters release from presynaptic nerve terminals[16–21]. Further studies have demonstrated that Syt1 is expressed in some tumor tissues and tumor cells including thyroid cancer tissues, head and neck squamous cell carcinoma (HNSCC) tissues[39, 40]. Syt1 is also expressed in mouse islets tissues, insulinoma cell lines, mast cells from mucosal and connective tissues. In 2006, Syt1 was reported distributed in atrial cardiomyocytes from neonatal and adult rats[27]. In the present study, we investigated the expressions and functions of Syt1 in the left ventricle (LV) and cardiomyocyte. We found that Syt1 is highly expressed in the LV tissues of mouse hearts and H9C2 cells, and was significantly downregulated upon I/R or H2O2 challenge. The location of Syt1 in mitochondria was also identified. The wide distribution of Syt1 in cardiomyocytes and the significant expression change under oxidative stress suggests its potential role in pathogenesis of cardiac disorders. Our data revealed that Syt1 protected hearts against programmed necrosis and I/R injury. Enforced expression of Syt1 attenuated cardiac I/R injury, interstitial fibrosis, necrotic cell death and improved cardiac function.
A mass of reactive oxygen species (ROS) is generated in hearts with I/R, which directly injured the myocardium followed by cardiodepression. The burst release of ROS through prolonged mPTP opening results in dissipation of mitochondrial membrane potential, organelle swelling and rupture, mitochondrial dysfunction and cell death[8, 9, 41]. Syt1 has been reported to be associated with oxidative stress regulation in glioma, neuron, and diabetic encephalopathy, but direct evidence is lacking[28–30]. Here we demonstrated that Syt1 was abundantly distributed in the mitochondria of cardiomyocytes. Enforced expression of Syt1 significantly inhibited mPTP abnormal opening and mitochondria-dependent necrotic cell death in response to oxidative stress. Our data suggested Syt1 to be a cardiac antioxidant molecule. In addition to oxidative stress, mitochondrial Ca2+ overload is another key factor that drives cardiac cell death by triggering mPTP abnormal opening, which contributes to MI, myocardial I/R injury, obesity cardiomyopathy, and cardiac microvascular I/R injury[7, 42, 43]. ROS causes intracellular Ca2+ overload by enhancing extracellular Ca2+ influx, ultimately leading to Ca2+ homeostasis disruption[42]. Syt1 has been identified as a Ca2+ sensor[17]. Therefore, we hypothesized that Syt1 regulates mitochondrial Ca2+ hemostasis in cardiomyocytes probably via its Ca2+ sensor function, which needs to be further confirmed in the future.
Our previous work demonstrated that Parkin, an E3 ubiquitin-protein ligase, could translocate from cytoplasm to mitochondria, where it interacted with CypD and catalyzed CypD ubiquitination, thus suppressed both mPTP opening and cardiomyocyte necrosis under moderate oxidative stress, whereas Parkin was significantly downregulated under severe oxidative stress[15]. In this study, both Syt1 and Parkin were significantly decreased in the mitochondria of cardiomyocytes treated with high concentration of H2O2. Immunoprecipitation and Immunofluorescence results revealed a strong interaction between Syt1 and Parkin, and the interaction was disrupted by H2O2 or Syt1 deficiency. We further found that Parkin-catalyzed CypD ubiquitination was attenuated by knockdown of Syt1 and enhanced by overexpression of Syt1. Ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3) cooperatively catalyze the ubiquitination reaction. Beyond this conventional ubiquitination catalyzation, growing non-enzymatic ubiquitination regulatory factors have been identified, and these factors form ubiquitination or deubiquitination complex with ubiquitinase and substrates[44–46]. In the signaling pathway of cardiac hypertrophy, BAF55 suppresses ubiquitin-protein ligase WWP2-mediated PARP1 ubiquitination[44]. YTH N6-methyladenosine (m6A) RNA binding protein 1 (YTHDF1) translationally upregulated WWP1 and enhanced WWP1-catalyzed NLRP3 ubiquitination[45]. Abraxas brother 1 (ABRO1) acts as a scaffold protein to assemble substrate NLRP3 and ubiquitin-protein ligase BRCC3, and reduces the ubiquitination modification of NLRP3 by inactivating BRCC3[46]. Our present data showed that Syt1 interacted with Parkin and promoted Parkin-mediated CypD ubiquitination, suggesting that Syt1 is a member of the ubiquitination complex and functions as a ubiquitination regulatory factor. Regarding the underlying mechanisms, we proposed certain hypotheses. One is that Syt1 probably enhances Parkin translation, which could be predicted by the significant upregulation of Parkin protein level by overexpression of Syt1 (shown in Fig. 3a). Syt1 may enhance the enzyme activity of Parkin in catalyzing CypD ubiquitination. Another hypothesis is that Syt1 functions as a scaffold protein to enhance the interaction between Parkin and CypD. In what way does Syt1 affect Parkin-mediated CypD ubiquitination will be studied in detail in the future.
The upstream regulatory mechanisms of Syt1 remain an enigma so far. The present work identified that microRNA (miR)-193b-3p is a novel upstream regulator of Syt1. MicroRNAs, a class of single-stranded and highly-conserved small non-coding RNAs, suppress gene expression at post-transcriptional or translational levels, and is deeply implicated in cardiac development and disorders. Previous studies on the functions of miR-193b-3p were mainly focused on tumor[47, 48], inflammation[49, 50], and metabolism[36, 51]. Recent advances have revealed that miR-193b-3p participates in the regulation of pulmonary hypertension in heart failure[36], cardiac hypertrophy[37] and the apoptotic pathway in myocardial ischemia/reperfusion injury[38]. However, the roles of miR-193b-3p in the cardiomyocyte necrotic death have not been explored. Here we found that miR-193b-3p directly bound to the 3’ untranslated region (UTR) of Syt1 mRNA and downregulated the Syt1 protein level, and by this way miR-193b-3p promoted necrotic cell death and mPTP opening. We also demonstrated that miR-193b-3p regulated cardiomyocyte necrosis, interstitial fibrosis and cardiac function in mice with I/R.
In conclusion, this work demonstrates that Syt1 has a detrimental effect on mPTP opening and cardiomyocyte programmed necrosis, and thus exerts a cardioprotective effect in I/R injury. Mechanistically, Syt1 interacts with Parkin and promotes Parkin-mediated CypD ubiquitination. Syt1 expression is negatively regulated by miR-193b-3p. The study reveals a novel regulatory model of cardiomyocyte necrosis which is composed of miR-193b-3p, Syt1, Parkin, and CypD. These molecules may serve as potential therapeutic targets and modulating the model may become strategies to treat myocardial necrosis-associated heart diseases, including I/R injury, MI and heart failure.