(2015) ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation, Autophagy, 11:8, 1308-1325, DOI: 10.1080/15548627.2015 Keywords: ATG16L1, autophagy, cardiomyocyte, casein kinase 2, protein phosphatase 1 Abbreviations: ACTB, actinb; AMPK, AMP activated protein kinase; ATG, autophagy-related; BCL2, B-cell CLL/lymphoma 2; BECN1/Beclin 1, autophagy related; CD, Crohn disease; CSNK2, casein kinase 2; GFP, green fluorescent protein; GNB2L1/ RACK1, guanine nucleotide binding protein (G protein)bpolypeptide 2-like 1; GST, glutathione S-transferase; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; LAD, left anterior descending; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 b; mRFP, monomeric red fluorescent protein; MTORC1, mechanistic target of rapamycin complex 1; NRVCs, neonatal rat ventricular cardiomyocytes; OA, okadaic acid; PE, phosphatidylethanolamine; PPP1, protein phosphatase 1; PPP2, protein phosphatase 2; PVDF, polyvinylidenedifluoride; SNP, single nucleotide polymorphism; SUPT20H/p38IP, suppressor of Ty 20 homolog (S. cerevisiae); TBB, 4,5,6,7-tetrabromobenzotriazole; lPP, l protein phosphatase.Recent studies have shown that the phosphorylation and dephosphorylation of ULK1 and ATG13 are related to autophagy activity. Although ATG16L1 is absolutely required for autophagy induction by affecting the formation of autophagosomes, the post-translational modification of ATG16L1 remains elusive. Here, we explored the regulatory mechanism and role of ATG16L1 phosphorylation for autophagy induction in cardiomyocytes. We showed that ATG16L1 was a phosphoprotein, because phosphorylation of ATG16L1 was detected in rat cardiomyocytes during hypoxia/ reoxygenation (H/R). We not only demonstrated that CSNK2 (casein kinase 2) phosphorylated ATG16L1, but also identified the highly conserved Ser139 as the critical phosphorylation residue for CSNK2. We further established that ATG16L1 associated with the ATG12-ATG5 complex in a Ser139 phosphorylation-dependent manner. In agreement with this finding, CSNK2 inhibitor disrupted the ATG12-ATG5-ATG16L1 complex. Importantly, phosphorylation of ATG16L1 on Ser139 was responsible for H/R-induced autophagy in cardiomyocytes, which protects cardiomyocytes from apoptosis. Conversely, we determined that wild-type PPP1 (protein phosphatase 1), but not the inactive mutant, associated with ATG16L1 and antagonized CSNK2-mediated phosphorylation of ATG16L1. Interestingly, one RVxF consensus site for PPP1 binding in the C-terminal tail of ATG16L1 was identified; mutation of this site disrupted its association with ATG16L1. Notably, CSNK2 also associated with PPP1, but ATG16L1 depletion impaired the interaction between CSNK2 and PPP1. Collectively, these data identify ATG16L1 as a bona fide physiological CSNK2 and PPP1 substrate, which reveals a novel molecular link from CSNK2 to activation of the autophagy-specific ATG12-ATG5-ATG16L1 complex and autoph...

Background: Three-dimensional electroanatomic mapping systems have demonstrated a significant reduction in radiation exposure during radiofrequency catheter ablation procedures. We aimed to investigate the safety, feasibility and efficacy of a completely zero-fluoroscopy approach for catheter ablation of supraventricular tachycardia using the Ensite NavX navigation system compared with a conventional fluoroscopy approach. Methods: A multicenter prospective non-randomized registry study was performed in seven centers from January 2013 to February 2018. Consecutive patients referred for catheter ablation of supraventricular tachycardia were assigned either to a completely zero-fluoroscopic approach (ZF) or conventional fluoroscopy approach (CF) according to the operator's preference. Patients with atrial tachycardia were excluded. Results: Totally, 1020 patients were enrolled in ZF group; 2040 patients ablated by CF approach were selected for controls. There was no significant difference between the zero-fluoroscopy group and conventional fluoroscopy group as to procedure time (60.3 ± 20.3 vs. 59.7 ± 22.6 min, P = 0.90), immediate success rate of procedure (98.8% vs. 99.2%, P = 0.22), arrhythmia recurrence (0.4% vs. 0.5%, P = 0.85), total success rate of procedure (98.4% vs. 98.8%, P = 0.39) or complications (1.1% vs. 1.5%, P = 0.41). Compared with the conventional fluoroscopy approach, the zerofluoroscopy approach provided similar outcomes without compromising the safety or efficacy of the procedure. Conclusion: The completely zero-fluoroscopy approach demonstrated safety and efficacy comparable to a conventional fluoroscopy approach for catheter ablation of supraventricular tachycardia, and mitigated radiation exposure to both patients and operators.

Post-translational modifications of autophagy-related (ATG) genes are necessary to modulate their functions. However, ATG protein methylation and its physiological role have not yet been elucidated. The methylation of non-histone proteins by SETD7, a SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. Here we present evidence that the precise activity of ATG16L1 protein in hypoxia/reoxygenation (H/R)-treated cardiomyocytes is regulated by a balanced methylation and phosphorylation switch. We first show that H/R promotes autophagy and decreases SETD7 expression, whereas autophagy inhibition by 3-MA increases SETD7 level in cardiomyocytes, implying a tight correlation between autophagy and SETD7. Then we demonstrate that SETD7 methylates ATG16L1 at lysine 151 while KDM1A/LSD1 (lysine demethylase 1A) removes this methyl mark. Furthermore, we validate that this methylation at lysine 151 impairs the binding of ATG16L1 to the ATG12-ATG5 conjugate, leading to inhibition of autophagy and increased apoptosis in H/R-treated cardiomyocytes. However, the cardiomyocytes with shRNA-knocked down SETD7 or inhibition of SETD7 activity by a small molecule chemical, display increased autophagy and decreased apoptosis following H/R treatment. Additionally, methylation at lysine 151 inhibits phosphorylation of ATG16L1 at S139 by CSNK2 which was previously shown to be critical for autophagy maintenance, and vice versa. Together, our findings define a novel modification of ATG16L1 and highlight the importance of an ATG16L1 phosphorylation-methylation switch in determining the fate of H/R-treated cardiomyocytes.