title: Cardiac specific deletion of N-methyl-d-aspartate receptor 1 ameliorates mtMMP-9 mediated autophagy/mitophagy in hyperhomocysteinemia.
abstract: Autophagy is an important process in the pathogenesis of cardiovascular diseases; however, the proximal triggers for mitochondrial autophagy were unknown. The N-methyl-d-aspartate receptor 1 (NMDA-R1) is a receptor for homocysteine (Hcy) and plays a key role in cardiac dysfunction. Cardiac-specific deletion of NMDA-R1 has been shown to ameliorate Hcy-induced myocyte contractility. Hcy activates mitochondrial matrix metalloproteinase-9 (mtMMP-9) and induces translocation of connexin-43 (Cxn-43) to the mitochondria (mtCxn-43). We sought to show cardiac-specific deletion of NMDA-R1 mitigates Hcy-induced mtCxn-43 translocation, mtMMP-9-mediated mtCxn-43 degradation, leading to mitophagy, in part, by decreasing mitochondrial permeability (MPT). Cardiac-specific knockout (KO) of NAMDA-R1 was generated using the cre/lox approach. The myocyte mitochondria were isolated from wild type (WT), WT + Hcy (1.8 g of DL-Hcy/L in the drinking water for 6 weeks), NMDA-R1 KO + Hcy, and NR1(fl/fl)/Cre (NR1(fl/fl)) genetic control mice. Mitochondrial respiratory capacity and MPT were measured by fluorescence-dye methods. The mitochondrial superoxide and peroxinitrite levels were detected by confocal microscopy using Mito-SOX and dihydrorhodamine-123. The mtMMP-9 activity and expression were detected by zymography and RT-PCR analyses. The mtCxn-43 translocation was detected by confocal microscopy. The degradation of mtCxn-43 and LC3-I/II (a marker of autophagy) were detected by Western blot. These results suggested that Hcy enhanced intramitochondrial nitrosative stress in myocytes. There was a robust increase in mtMMP-9 activity. An increase in translocation and degradation of mtCxn-43 was also noted. These increases led to mitophagy. The effects were ameliorated by cardiac-specific deletion of NMDA-R1. We concluded that HHcy increased mitochondrial nitrosative stress, thereby activating mtMMP-9 and inciting the degradation of mtCxn-43. This led to mitophagy, in part, by activating NMDA-R1. The findings of this study will lead to therapeutic ramifications for mitigating cardiovascular diseases by inhibiting the mitochondrial mitophagy and NMDA-R1 receptor.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, University of Louisville, Louisville, KY 40292, USA.
authors.forename: Neetu
authors.initials: N
authors.lastname: Tyagi
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Vacek
authors.forename: Srikanth
authors.initials: S
authors.lastname: Givvimani
authors.forename: Utpal
authors.initials: U
authors.lastname: Sen
authors.forename: Suresh C
authors.initials: SC
authors.lastname: Tyagi
mesh_terms: D000818:Animals; D001343:Autophagy; D015153:Blotting, Western; D019069:Cell Respiration; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D005260:Female; D006710:Homocysteine / Q000494:pharmacology; D020138:Hyperhomocysteinemia / Q000378:metabolism* / Q000473:pathology; D019426:Integrases / Q000378:metabolism; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008929:Mitochondria, Heart / Q000378:metabolism* / Q000473:pathology; D033681:Mitochondrial Membrane Transport Proteins / Q000502:physiology; D000083162:Mitochondrial Permeability Transition Pore; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018384:Oxidative Stress; D010101:Oxygen Consumption; D030421:Peroxynitrous Acid / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018031:Connexin 43; D006710:Homocysteine; D019426:Integrases; D020780:Matrix Metalloproteinase 9; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D030421:Peroxynitrous Acid; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; C045073:Cre recombinase; C501830:Mmp9 protein, mouse