Cardiac specific deletion ofN-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. KeywordsMitochondrial MMP; mitochondrial connexin-43 and microtubule-associated protein light chain 3; nitrosative stress

Oxidative Stress and Autophagy in Cardiovascular Homeostasis

“…One involves homocysteine-induced increases in mitochondrial calcium, which are activated by the homocysteine receptor N-methyl-daspartate receptor-1 (NAMDA-R1). Cardiomyocyte-specific knockout of NR1 (gene coding for NAMDA-R1) results in decreased NO production, inactivation of mitochondrial MMP-9, and improved cardiac function (77). A similar result was observed in a mouse model of pulmonary hypertension, where increased production of ROS, increased activity of MMPs, and increased autophagy were observed in the right ventricle (56).…”

“…Recent reports demonstrate that ROS can lead to the activation of matrix metalloproteinases (MMPs) in the heart, resulting in increased collagen deposition (78). Two mechanisms have been described (56,77). One involves homocysteine-induced increases in mitochondrial calcium, which are activated by the homocysteine receptor N-methyl-daspartate receptor-1 (NAMDA-R1).…”

Sustained Activation of N-Methyl-d-Aspartate Receptors in Podoctyes Leads to Oxidative Stress, Mobilization of Transient Receptor Potential Canonical 6 Channels, Nuclear Factor of Activated T Cells Activation, and Apoptotic Cell Death

“…It is now known that NMDA receptors are expressed in many different peripheral tissues (Patton et al, 1998;Parisi et al, 2009;Makhro et al, 2010;Mashkina et al, 2010;Tyagi et al, 2010), including podocytes (Rastaldi et al, 2006;Anderson et al, 2011), although their normal physiological functions in these tissues are not well understood. We have recently characterized the functional properties of NMDA receptors of podocytes (Anderson et al, 2011).…”

Cardiac N-methyl d-aspartate Receptors as a Pharmacological Target

“…Mitochondrial dysfunction was considered as one of the mechanisms of myocardial damage following NMDAR hyperactivation (45)(46)(47)(48)(49). Ca 2+ overload in cardiomyocytes was suggested to be involved in pathological response to the stimulation with high doses agonists of the NMDAR (47).…”