Xiao Y, Hu X, Rudolphi C F, Nollet E E, Nederlof R, Wang Q, Bakker D, Nikolaou Panagiota Efstathia, Knol J C, Haas R R Goeij-de, Henneman A A, Pham T V, Jimenez C R, Grootemaat A E, van der Wel N N, Girardin S E, Kaludercic N, van der Velden J, Onódi Z, Leszek P, Varga Z V, Ferdinandy P, Preckel B, Weber N C, Hollmann M W, Di Lisa F, Zuurbier C J
Laboratory of Experimental Intensive Care and Anesthesiology, Department of Anesthesiology, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.
Amsterdam Cardiovascular Sciences, Atherosclerosis & Ischemic Syndromes, Heart Failure & Arrhythmias, Amsterdam, The Netherlands.
Basic Res Cardiol. 2025 Jun 19. doi: 10.1007/s00395-025-01124-x.
NLRX1 is the only NOD-like innate immune receptor that localises to mitochondria. We previously demonstrated that NLRX1 deletion increased infarct size in isolated mouse hearts subjected to ischemia-reperfusion injury (IRI); however, underlying mechanisms are yet to be identified. Given the crucial role played by mitochondria in cardiac IRI, we here hypothesise that NLRX1 affects key mechanisms of cardiac IRI. Cardiac IRI was evaluated in isolated C57BL/6J (WT) and NLRX1 knock out (KO) mouse hearts. The following known modulators of IRI were explored in isolated hearts, isolated mitochondria; or permeabilised cardiac fibres: 1) mTOR/RISK/autophagy regulation, 2) AMPK and mitochondrial energy production, and 3) mitochondrial permeability transition pore (mPTP) opening. NLRX1 deletion increased IRI, and cardiac NLRX1 was decreased after IRI in mouse and pig hearts. NLRX1 ablation caused decreased mTOR and RISK pathway (Akt, ERK, and S6K) activation following IR, without affecting autophagy/inflammation/oxidative stress markers. The RISK activator Urocortin dissipated NLRX1 effects on mTOR, RISK pathway and IRI, indicating that increased cardiac IRI with NLRX1 deletion is, at least partly, due to impaired RISK activation. The energy sensor AMPK was activated in NLRX1 KO hearts, possibly due to slowed mitochondrial respiratory responses (impaired mitochondrial permeability) towards palmitoylcarnitine in permeabilised cardiac fibres. NLRX1 deletion completely abolished calcium-induced mPTP opening, and cyclosporine A (CsA) effects on mPTP, both before and after IR, and was associated with increased mitochondrial calcium content after IR. Mitochondrial sub-fractionation studies localised NLRX1 to the inner mitochondrial membrane. NLRX1 deletion associated with decreased phosphorylation of mitochondrial Got2, Cx43, Myl2, Ndufb7 and MICOS10. The mPTP inhibitor CsA abolished IRI differences between KO and WT hearts, suggesting that the permanent closure of mPTP due to NLRX1 deletion contributed to the increased IR sensitivity of NLRX1 KO hearts. This is the first demonstration that the mitochondrial NLRX1 is a novel factor required for mPTP opening and contributes to cardioprotection against acute IRI through RISK pathway activation and prevention of permanent mPTP closure.
NLRX1是唯一一种定位于线粒体的NOD样天然免疫受体。我们之前证明，在经历缺血再灌注损伤（IRI）的离体小鼠心脏中，NLRX1缺失会增加梗死面积；然而，其潜在机制尚待确定。鉴于线粒体在心脏IRI中发挥的关键作用，我们在此假设NLRX1影响心脏IRI的关键机制。在离体的C57BL/6J（野生型，WT）和NLRX1基因敲除（KO）小鼠心脏中评估心脏IRI。在离体心脏、离体线粒体或透化的心肌纤维中研究以下已知的IRI调节因子：1）mTOR/RISK/自噬调节，2）AMPK与线粒体能量产生，以及3）线粒体通透性转换孔（mPTP）开放。NLRX1缺失会增加IRI，在小鼠和猪心脏的IRI后，心脏NLRX1会减少。NLRX1缺失导致IR后mTOR和RISK通路（Akt、ERK和S6K）激活减少，而不影响自噬/炎症/氧化应激标志物。RISK激活剂尿皮质素消除了NLRX1对mTOR、RISK通路和IRI的影响，表明NLRX1缺失导致的心脏IRI增加至少部分是由于RISK激活受损。能量传感器AMPK在NLRX1基因敲除心脏中被激活，可能是由于透化的心肌纤维中对棕榈酰肉碱的线粒体呼吸反应减慢（线粒体通透性受损）。NLRX1缺失完全消除了钙诱导的mPTP开放以及环孢素A（CsA）对mPTP的影响，无论是在IR之前还是之后，并且与IR后线粒体钙含量增加有关。线粒体亚组分研究将NLRX1定位于线粒体内膜。NLRX1缺失与线粒体Got2、Cx43、Myl2、Ndufb7和MICOS10的磷酸化减少有关。mPTP抑制剂CsA消除了基因敲除心脏和野生型心脏之间的IRI差异，表明由于NLRX1缺失导致的mPTP永久关闭促成了NLRX1基因敲除心脏对IR敏感性的增加。这是首次证明线粒体NLRX1是mPTP开放所需的新因子，并通过RISK通路激活和防止mPTP永久关闭对急性IRI起到心脏保护作用。
