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.

Keywords::

• Mitochondrial MMP
• mitochondrial connexin-43 and microtubule-associated protein light chain 3
• nitrosative stress

Acknowledgments

A part of this study was supported by NIH grants: HL-71010, HL-74185, and HL-88012.

Declaration of interest

The authors report no conflicts of interest. The corresponding author are responsible for the content and writing of the paper.