Mechanism of FUNDC1 affacting apoptosis of H9c2 cardiomyocytes with high glucose injury by regulating mitochondrial fission

doi:10.11958/20220348

Abstract

Abstract:

Objective To investigate the mechanism of FUNDC1 affecting high glucose injured H9c2 cardiomyocyte apoptosis by regulating mitochondrial fission. Methods H9c2 cardiomyocytes were cultured in vitro, and the high glucose-induced injury model of cells was established. After cell transfection or AMPK activation by AICAR, the cells were divided into the control (CTRL) group, the high glucose injury (HG) group, the HG with transfected shRNA-NC (HG+shRNA-NC) group, the HG with transfected FUNDC1 shRNA (HG+shRNA-FUNDC1) group and the HG with AICAR (HG+AICAR) group. MTT method was used to detect the cell survival rate. The level of released LDH was measured with microplate reader. The structure of mitochondria was observed by laser confocal microscope. The proteins expression levels of mitochondrial dynamin-related protein1 (DRP1), fission protein 1 (FIS1), Bcl-2 associated X protein (Bax), B-lymphocytoma-2 (Bcl-2), cleaved cysteine-containing aspartate-spicific protease 3 (Cleaved Caspase-3), FUNDC1 and GAPDH were detected by Western blot assay. Results Compared with the CTRL group, the cell survival rate decreased, and the protein levels of DRP1-cyto and Bcl-2 were also decreased, the release of LDH and the protein expression levels of DRP1-mito, FIS1, Bax and Cleaved Caspase-3 increased in the HG group (P<0.05). Compared with the HG group, knocking down FUNDC1 increased the cell survival rate and the protein levels of DRP1-cyto and Bcl-2, decreased the release of LDH and the protein expression levels of DRP1-mito, FIS1, Bax and Cleaved Caspase-3 (P<0.05). There was no difference in each index between the HG group and the HG+shRNA-NC group (P>0.05). The mitochondria were mainly linear structure in the CTRL group, and the mitochondria were mainly pucta structure in the HG group and the HG+shRNA-NC group. Compared with the HG group, the mitochondrial linear structure increased and the puncta structure decreased in the HG+shRNA-FUNDC1 group. Compared with the CTRL group, the protein expression level of FUNDC1 increased in the HG group (P<0.05). Compared with the HG group, the protein level of FUNDC1 significantly decreased in the HG+AICAR group (P<0.05). Conclusion FUNDC1 affects H9c2 cardiomyocyte apoptosis induced by high glucose through regulating mitochondrial fission, and AMPK signaling pathway is involved in this process.

Key words: myocytes, cardiac, mitochondria, apoptosis, FUNDC1, signaling pathway

CLC Number:

R541

Figures/Tables 5

References 19

[1]	WU S, LU Q, DING Y, et al. Hyperglycemia-driven inhibition of AMP-activated protein kinase α2 induces diabetic cardiomyopathy by promoting mitochondria-associated endoplasmic reticulum membranes in vivo[J]. Circulation, 2019, 139(16):1913-1936. doi: 10.1161/CIRCULATIONAHA.118.033552.
[2]	ELORZA A A, SOFFIA J P. mtDNA heteroplasmy at the core of aging-associated heart failure. An integrative view of OXPHOS and mitochondrial life cycle in cardiac mitochondrial physiology[J]. Front Cell Dev Biol, 2021, 9:625020. doi: 10.3389/fcell.2021.625020.
[3]	TILOKANI L, NAGASHIMA S, PAUPE V, et al. Mitochondrial dynamics:Overview of molecular mechanisms[J]. Essays Biochem, 2018, 62(3):341-360. doi: 10.1042/EBC20170104.
[4]	LAHERA V, DE LAS HERAS N, LÓPEZ-FARRÉ A, et al. Role of mitochondrial dysfunction in hypertension and obesity[J]. Curr Hypertens Rep, 2017, 19(2):11. doi: 10.1007/s11906-017-0710-9.
[5]	DUAN C, KUANG L, XIANG X, et al. Drp1 regulates mitochondrial dysfunction and dysregulated metabolism in ischemic injury via Clec16a-,BAX-,and GSH- pathways[J]. Cell Death Dis, 2020, 11(4):251. doi: 10.1038/s41419-020-2461-9.
[6]	HAILESELASSIE B, MUKHERJEE R, JOSHI A U, et al. Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy[J]. J Mol Cell Cardiol, 2019, 130:160-169. doi: 10.1016/j.yjmcc.2019.04.006.
[7]	GILKERSON R, DE LA TORRE P, ST VALLIER S. Mitochondrial OMA1 and OPA1 as gatekeepers of organellar structure/function and cellular stress response[J]. Front Cell Dev Biol, 2021, 9:626117. doi: 10.3389/fcell.2021.626117.
[8]	LIU H, ZANG C, YUAN F, et al. The role of FUNDC1 in mitophagy, mitochondrial dynamics and human diseases[J]. Biochem Pharmacol, 2022, 197:114891. doi: 10.1016/j.bcp.2021.114891.
[9]	LIN K L, CHEN S D, LIN K J, et al. Quality Matters? The involvement of mitochondrial quality control in cardiovascular disease[J]. Front Cell Dev Biol, 2021, 9:636295. doi: 10.3389/fcell.2021.636295.
[10]	SHENOUDA S M, WIDLANSKY M E, CHEN K, et al. Altered mitochondrial dynamics contributes to endothelial dysfunction in diabetes mellitus[J]. Circulation, 2011, 124(4):444-453. doi: 10.1161/CIRCULATIONAHA.110.014506.
[11]	KIM D, SANKARAMOORTHY A, ROY S. Downregulation of Drp1 and Fis1 inhibits mitochondrial fission and prevents high glucose-induced apoptosis in retinal endothelial cells[J]. Cells, 2020, 9(7):1662. doi: 10.3390/cells9071662.
[12]	DUAN C, KUANG L, XIANG X, et al. Activated Drp1-mediated mitochondrial ROS influence the gut microbiome and intestinal barrier after hemorrhagic shock[J]. Aging(Albany NY), 2020, 12(2):1397-1416. doi: 10.18632/aging.102690.
[13]	PRUDENT J, ZUNINO R, SUGIURA A, et al. MAPL SUMOylation of Drp1 Stabilizes an ER/mitochondrial platform required for cell death[J]. Mol Cell, 2015, 59(6):941-955. doi: 10.1016/j.molcel.2015.08.001.
[14]	JOSHI A U, SAW N L, VOGEL H, et al. Inhibition of Drp1/Fis1 interaction slows progression of amyotrophic lateral sclerosis[J]. EMBO Mol Med, 2018, 10(3):e8166. doi: 10.15252/emmm.201708166.
[15]	IWASAWA R, MAHUL-MELLIER A L, DATLER C, et al. Fis1 and Bap31 bridge the mitochondria-ER interface to establish a platform for apoptosis induction[J]. EMBO J, 2011, 30(3):556-568. doi: 10.1038/emboj.2010.346.
[16]	WU S, LU Q, WANG Q, et al. Binding of FUN14 domain containing 1 with inositol 1,4,5-trisphosphate receptor in mitochondria-associated endoplasmic reticulum membranes maintains mitochondrial dynamics and function in hearts in vivo[J]. Circulation, 2017, 136(23):2248-2266. doi: 10.1161/CIRCULATIONAHA.117.030235.
[17]	CHEN M, CHEN Z, WANG Y, et al. Mitophagy receptor FUNDC1 regulates mitochondrial dynamics and mitophagy[J]. Autophagy, 2016, 12(4):689-702. doi: 10.1080/15548627.2016.1151580.
[18]	CAI Y, YANG E, YAO X, et al. FUNDC1-dependent mitophagy induced by tPA protects neurons against cerebral ischemia-reperfusion injury[J]. Redox Biol, 2021, 38:101792. doi: 10.1016/j.redox.2020.101792.
[19]	MAO S, TIAN S, LUO X, et al. Overexpression of PLK1 relieved the myocardial ischemia-reperfusion injury of rats through inducing the mitophagy and regulating the p-AMPK/FUNDC1 axis[J]. Bioengineered, 2021, 12(1):2676-2687. doi: 10.1080/21655979.2021.1938500.

组别	n	细胞存活率（%）	LDH释放量（A₄₉₀）
CTRL组	6	100.00±18.24	0.26±0.03
HG组	6	54.41±8.69^a	0.61±0.06^a
HG+shRNA-NC组	6	55.51±12.99	0.60±0.04
HG+shRNA-FUNDC1组	6	83.63±9.86^b	0.40±0.06^b
F		17.708^＊	71.294^＊

组别	n	细胞存活率（%）	LDH释放量（A₄₉₀）
CTRL组	6	100.00±18.24	0.26±0.03
HG组	6	54.41±8.69^a	0.61±0.06^a
HG+shRNA-NC组	6	55.51±12.99	0.60±0.04
HG+shRNA-FUNDC1组	6	83.63±9.86^b	0.40±0.06^b
F		17.708^＊	71.294^＊

组别		DRP1-mito	DRP1-cyto	FIS1
CTRL组		0.62±0.09	0.86±0.09	0.48±0.05
HG组		1.03±0.15^a	0.42±0.05^a	0.71±0.08^a
HG+shRNA-NC组		1.06±0.14	0.31±0.04	0.63±0.11
HG+shRNA-FUNDC1组		0.62±0.09^b	0.61±0.07^b	0.46±0.07^b
F		12.704^＊	38.953^＊	8.006^＊
组别	Bax	Bcl-2	Cleaved Caspase-3
CTRL组	0.38±0.05	0.84±0.05	0.72±0.09
HG组	1.10±0.14^a	0.60±0.03^a	1.00±0.09^a
HG+shRNA-NC组	1.18±0.15	0.59±0.06	0.94±0.11
HG+shRNA-FUNDC1组	0.48±0.06^b	0.79±0.08^b	0.75±0.09^b
F	42.624^＊	14.560^＊	5.891^＊

组别		DRP1-mito	DRP1-cyto	FIS1
CTRL组		0.62±0.09	0.86±0.09	0.48±0.05
HG组		1.03±0.15^a	0.42±0.05^a	0.71±0.08^a
HG+shRNA-NC组		1.06±0.14	0.31±0.04	0.63±0.11
HG+shRNA-FUNDC1组		0.62±0.09^b	0.61±0.07^b	0.46±0.07^b
F		12.704^＊	38.953^＊	8.006^＊
组别	Bax	Bcl-2	Cleaved Caspase-3
CTRL组	0.38±0.05	0.84±0.05	0.72±0.09
HG组	1.10±0.14^a	0.60±0.03^a	1.00±0.09^a
HG+shRNA-NC组	1.18±0.15	0.59±0.06	0.94±0.11
HG+shRNA-FUNDC1组	0.48±0.06^b	0.79±0.08^b	0.75±0.09^b
F	42.624^＊	14.560^＊	5.891^＊