Pentacyclic triterpene oleanolic acid protects against cardiac aging through regulation of mitophagy and mitochondrial integrity

Advanced aging exhibits altered cardiac geometry and function involving mitochondrial anomaly. Natural compounds display promises in the regulation of cardiac homeostasis via governance of mitochondrial integrity in aging. This study examined the effect of oleanolic acid (OA), a natural pentacyclic triterpenoid with free radical scavenging and P450 cyclooxygenase-regulating properties, on cardiac aging and mechanisms involved with a focus on mitophagy. Young (4–5 month-old) and old (22–24 month-old) mice were treated with OA for 6 weeks prior to assessment of cardiac function, morphology, ultrastructure, mitochondrial integrity, cell death and autophagy. Our data revealed that OA treatment alleviated aging-induced changes in myocardial remodeling (increased heart weight, chamber size, cardiomyocyte area and interstitial fibrosis), contractile function and intracellular Ca²⁺ handling, apoptosis, necroptosis, inflammation, autophagy and mitophagy (LC3B, p62, TOM20 and FUNDC1 but not BNIP3 and Parkin). OA treatment rescued aging-induced anomalies in mitochondrial ultrastructure (loss of myofilament alignment, swollen mitochondria, increased circularity), mitochondrial biogenesis and O₂^? production without any notable effect at young age. Interestingly, OA-offered benefit against cardiomyocyte aging was nullified by deletion of the mitophagy receptor FUNDC1 using FUNDC1 knockout mice, denoting an obligatory role for FUNDC1 in OA-elicited preservation of mitophagy. OA reconciled aging-induced changes in E3 ligase MARCH5 but not FBXL2, and failed to affect aging-induced rises in IP3R3. Taken together, our data indicated a beneficial role for OA in attenuating cardiac remodeling and contractile dysfunction in aging through a FUNDC1-mediated mechanism.     

Cardiac-specific overexpression of catalase attenuates paraquat-induced myocardial geometric and contractile alteration: role of ER stress

Paraquat, a quaternary nitrogen herbicide, is a highly toxic pro-oxidant that causes multiorgan failure including that of the heart via generation of reactive oxygen species, although the underlying mechanism has not been well elucidated. This study examined the influence of cardiac-specific overexpression of catalase, an antioxidant detoxifying H(2)O(2), on paraquat-induced myocardial geometric and functional alterations, with a focus on ER stress. FVB and catalase transgenic mice were administered paraquat for 48h. Myocardial geometry, contractile function, apoptosis, and ER stress were evaluated using echocardiography, edge detection, caspase-3 activity, and immunoblotting. Our results revealed that paraquat treatment significantly enlarged left ventricular (LV) end diastolic and systolic diameters; increased LV mass and resting myocyte length; reduced fractional shortening, cardiomyocyte peak shortening, and maximal velocity of shortening/relengthening; and prolonged relengthening duration in the FVB group. Whereas the catalase transgene itself did not alter myocardial geometry and function, it mitigated or significantly attenuated paraquat-elicited myocardial geometric and functional changes. Paraquat promoted overt apoptosis and ER stress as evidenced by increased caspase-3 activity, apoptosis, and ER stress markers including Bax, Bcl-2, GADD153, calregulin, and phosphorylated JNK, IRE1α, and eIF2α; all were ablated by the catalase transgene. Paraquat-induced cardiomyocyte dysfunction was mitigated by the ER stress inhibitor tauroursodeoxycholic acid. Moreover, the JNK inhibitor SP600125 reversed paraquat-induced ER stress as evidenced by enhanced GADD153 and IRE1α phosphorylation. Taken together, these data revealed that catalase may rescue paraquat-induced myocardial geometric and functional alteration possibly by alleviating JNK-mediated ER stress.     

Inhibition of CYP2E1 attenuates myocardial dysfunction in a murine model of insulin resistance through NLRP3-mediated regulation of mitophagy

Insulin resistance leads to myocardial contractile dysfunction and deranged autophagy although the underlying mechanism or targeted therapeutic strategy is still lacking. This study was designed to examine the impact of inhibition of the cytochrome P450 2E1 (CYP2E1) enzyme on myocardial function and mitochondrial autophagy (mitophagy) in an Akt2 knockout model of insulin resistance. Adult wild-type (WT) and Akt2^?/? mice were treated with the CYP2E1 inhibitor diallyl sulfide (100?mg/kg/d, i.p.) for 4?weeks. Cardiac geometry and function were assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate autophagy, mitophagy, inducible NOS (iNOS), and the NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex. Akt2 deletion triggered insulin resistance, compromised cardiac contractile and intracellular Ca²⁺ property, mitochondrial ultrastructural damage, elevated O2^– production, as well as suppressed autophagy and mitophagy, accompanied with elevated levels of NLRP3 and iNOS, the effects of which were significantly attenuated or ablated by diallyl sulfide. In vitro studies revealed that the NLRP3 activator nigericin nullified diallyl sulfide-offered benefit against Akt2 knockout on cardiomyocyte mechanical function and mitophagy (using Western blot and colocalization of GFP-LC3 and MitoTracker Red). Moreover, inhibition of iNOS but not mitochondrial ROS production attenuated Akt2 deletion-induced activation of NLRP3, substantiating a role for iNOS-mediated NLRP3 in insulin resistance-induced changes in mitophagy and cardiac dysfunction. In conclusion, these data depict that insulin resistance through CYP2E1 may contribute to the pathogenesis of myopathic changes including myocardial contractile dysfunction, oxidative stress and mitochondrial injury, possibly through activation of iNOS and NLRP3 signaling.     

Deficiency in Beclin1 attenuates alcohol-induced cardiac dysfunction via inhibition of ferroptosis

BackgroundBinge drinking leads to compromised mitochondrial integrity and contractile function in the heart although little effective remedy is readily available. Given the possible derangement of autophagy in ethanol-induced cardiac anomalies, this study was designed to examine involvement of Beclin1 in acute ethanol-induced cardiac contractile dysfunction, in any, and the impact of Beclin1 haploinsufficiency on ethanol cardiotoxicity with a focus on autophagy-related ferroptosis.MethodsWT and Beclin1 haploinsufficiency (BECN^+/?) mice were challenged with ethanol for one week (2 g/kg, i.p. on day 1, 3 and 7) prior to assessment of cardiac injury markers (LDH, CK-MB), cardiac geometry, contractile and mitochondrial integrity, oxidative stress, lipid peroxidation, apoptosis and ferroptosis.ResultsEthanol exposure compromised cardiac geometry and contractile function accompanied with upregulated Beclin1 and autophagy, mitochondrial injury, oxidative stress, lipid peroxidation and apoptosis, and ferroptosis (GPx4, SLC7A11, NCOA4). Although Beclin1 deficiency did not affect cardiac function in the absence of ethanol challenge, it alleviated ethanol-induced changes in cardiac injury biomarkers, cardiomyocyte area, interstitial fibrosis, echocardiographic and cardiomyocyte mechanical properties along with mitochondrial integrity, oxidative stress, lipid peroxidation, apoptosis and ferroptosis. Ethanol challenge evoked pronounced ferroptosis (downregulated GPx4, SLC7A11 and elevated NCOA4, lipid peroxidation), the effect was alleviated by Beclin1 haploinsufficiency. Inhibition of ferroptosis using LIP-1 rescued ethanol-induced cardiac mechanical anomalies. In vitro study noted that ferroptosis induction using erastin abrogated Beclin1 haploinsufficiency-induced response against ethanol.ConclusionsIn sum, our data suggest that Beclin1 haploinsufficiency benefits acute ethanol challenge-induced myocardial remodeling and contractile dysfunction through ferroptosis-mediated manner.     

Mitochondrial E3 ligase MARCH5 regulates FUNDC1 to fine‐tune hypoxic mitophagy

Mitophagy is an essential process for mitochondrial quality control and turnover. It is activated by two distinct pathways, one dependent on ubiquitin and the other dependent on receptors including FUNDC1. It is not clear whether these pathways coordinate to mediate mitophagy in response to stresses, or how mitophagy receptors sense stress signals to activate mitophagy. We find that the mitochondrial E3 ligase MARCH5, but not Parkin, plays a role in regulating hypoxia‐induced mitophagy by ubiquitylating and degrading FUNDC1. MARCH5 directly interacts with FUNDC1 to mediate its ubiquitylation at lysine 119 for subsequent degradation. Degradation of FUNDC1 by MARCH5 expression desensitizes mitochondria to hypoxia‐induced mitophagy, whereas knockdown of endogenous MARCH5 significantly inhibits FUNDC1 degradation and enhances mitochondrial sensitivity toward mitophagy‐inducing stresses. Our findings reveal a feedback regulatory mechanism to control the protein levels of a mitochondrial receptor to fine‐tune mitochondrial quality.     

MARCH5-FUNDC1 axis fine-tunes hypoxia-induced mitophagy

Mitophagy is responsible for removal of damaged mitochondria and is therefore a fundamental process in mitochondrial quality control. Both ubiquitin-dependent and receptor-dependent pathways are considered to mediate mitophagy. These distinct mechanisms may be activated in response to distinct mitochondrial stresses. An intriguing question is whether and how crosstalk occurs between the distinct pathways to coordinate mitophagy. We have uncovered a striking piece of evidence to demonstrate that the mitophagy receptor FUNDC1 is a substrate of MARCH5, a mitochondrially localized E3 ubiquitin ligase. In response to hypoxia, MARCH5 degrades redundant FUNDC1 to fine-tune hypoxia-induced mitophagy, whereas ablation of MARCH5 leads to accumulation of FUNDC1 and an exaggerated mitophagic phenotype. Mechanistic studies demonstrate that hypoxic insult enhances the interaction of FUNDC1 with MARCH5, which ubiquitinates FUNDC1 at lysine 119 for subsequent degradation. MARCH5-based ubiquitination and degradation of FUNDC1 circumvents injudicious removal of cellular mitochondria. However, severe hypoxic stress leads to dephosphorylation of FUNDC1, increasing mitophagic flux.     

Mitophagy receptor FUNDC1 regulates mitochondrial dynamics and mitophagy

Mitochondrial fragmentation due to imbalanced fission and fusion of mitochondria is a prerequisite for mitophagy, however, the exact “coupling” of mitochondrial dynamics and mitophagy remains unclear. We have previously identified that FUNDC1 recruits MAP1LC3B/LC3B (LC3) through its LC3-interacting region (LIR) motif to initiate mitophagy in mammalian cells. Here, we show that FUNDC1 interacts with both DNM1L/DRP1 and OPA1 to coordinate mitochondrial fission or fusion and mitophagy. OPA1 interacted with FUNDC1 via its Lys70 (K70) residue, and mutation of K70 to Ala (A), but not to Arg (R), abolished the interaction and promoted mitochondrial fission and mitophagy. Mitochondrial stress such as selenite or FCCP treatment caused the disassembly of the FUNDC1-OPA1 complex while enhancing DNM1L recruitment to the mitochondria. Furthermore, we observed that dephosphorylation of FUNDC1 under stress conditions promotes the dissociation of FUNDC1 from OPA1 and association with DNM1L. Our data suggest that FUNDC1 regulates both mitochondrial fission or fusion and mitophagy and mediates the “coupling” across the double membrane for mitochondrial dynamics and quality control.     

Mitophagy receptor FUNDC1 regulates mitochondrial homeostasis and protects the heart from I/R injury

Mitophagy plays pivotal roles in the selective disposal of unwanted mitochondria, and accumulation of damaged mitochondria has been linked to aging-related diseases. However, definitive proof that mitophagy regulates mitochondrial quality in vivo is lacking. It is also largely unclear whether damaged mitochondria are the cause or just the consequence of these diseases. We previously showed that FUNDC1 is a mitophagy receptor that interacts with LC3 to mediate mitophagy in response to hypoxia in cultured cells. We established Fundc1 knockout mouse models and used genetic and biochemical approaches, including a synthetic peptide that blocks the FUNDC1-LC3 interaction, to demonstrate that mitophagy regulates both mitochondrial quantity and quality in vivo in response to hypoxia or hypoxic conditions caused by ischemia-reperfusion (I/R) heart injury. We found that hypoxic mitophagy regulates platelet activities. Furthermore, we found that hypoxic preconditioning induces FUNDC1-dependent mitophagy in platelets and reduces I/R-induced heart injury, suggesting a new strategy to protect cardiac function and fight cardiovascular diseases.     

FUNDC1 regulates mitochondrial dynamics at the ER–mitochondrial contact site under hypoxic conditions

In hypoxic cells, dysfunctional mitochondria are selectively removed by a specialized autophagic process called mitophagy. The ER–mitochondrial contact site (MAM) is essential for fission of mitochondria prior to engulfment, and the outer mitochondrial membrane protein FUNDC1 interacts with LC3 to recruit autophagosomes, but the mechanisms integrating these processes are poorly understood. Here, we describe a new pathway mediating mitochondrial fission and subsequent mitophagy under hypoxic conditions. FUNDC1 accumulates at the MAM by associating with the ER membrane protein calnexin. As mitophagy proceeds, FUNDC1/calnexin association attenuates and the exposed cytosolic loop of FUNDC1 interacts with DRP1 instead. DRP1 is thereby recruited to the MAM, and mitochondrial fission then occurs. Knockdown of FUNDC1, DRP1, or calnexin prevents fission and mitophagy under hypoxic conditions. Thus, FUNDC1 integrates mitochondrial fission and mitophagy at the interface of the MAM by working in concert with DRP1 and calnexin under hypoxic conditions in mammalian cells.     

ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy

Autophagy eliminates dysfunctional mitochondria in an intricate process known as mitophagy. ULK1 is critical for the induction of autophagy, but its substrate(s) and mechanism of action in mitophagy remain unclear. Here, we show that ULK1 is upregulated and translocates to fragmented mitochondria upon mitophagy induction by either hypoxia or mitochondrial uncouplers. At mitochondria, ULK1 interacts with FUNDC1, phosphorylating it at serine 17, which enhances FUNDC1 binding to LC3. A ULK1‐binding‐deficient mutant of FUNDC1 prevents ULK1 translocation to mitochondria and inhibits mitophagy. Finally, kinase‐active ULK1 and a phospho‐mimicking mutant of FUNDC1 rescue mitophagy in ULK1‐null cells. Thus, we conclude that FUNDC1 regulates ULK1 recruitment to damaged mitochondria, where FUNDC1 phosphorylation by ULK1 is crucial for mitophagy.     

Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells

Accumulating evidence has shown that dysfunctional mitochondria can be selectively removed by mitophagy. Dysregulation of mitophagy is implicated in the development of neurodegenerative disease and metabolic disorders. How individual mitochondria are recognized for removal and how this process is regulated remain poorly understood. Here we report that FUNDC1, an integral mitochondrial outer-membrane protein, is a receptor for hypoxia-induced mitophagy. FUNDC1 interacted with LC3 through its typical LC3-binding motif Y(18)xxL(21), and mutation of the LC3-interaction region impaired its interaction with LC3 and the subsequent induction of mitophagy. Knockdown of endogenous FUNDC1 significantly prevented hypoxia-induced mitophagy, which could be reversed by the expression of wild-type FUNDC1, but not LC3-interaction-deficient FUNDC1 mutants. Mechanistic studies further revealed that hypoxia induced dephosphorylation of FUNDC1 and enhanced its interaction with LC3 for selective mitophagy. Our findings thus offer insights into mitochondrial quality control in mammalian cells.     

MicroRNA-137 Is a Novel Hypoxia-responsive MicroRNA That Inhibits Mitophagy via Regulation of Two Mitophagy Receptors FUNDC1 and NIX

Mitophagy receptors mediate the selective recognition and targeting of damaged mitochondria by autophagosomes. The mechanism for the regulation of these receptors remains unknown. Here, we demonstrated that a novel hypoxia-responsive microRNA, microRNA-137 (miR-137), markedly inhibits mitochondrial degradation by autophagy without affecting global autophagy. miR-137 targets the expression of two mitophagy receptors NIX and FUNDC1. Impaired mitophagy in response to hypoxia caused by miR-137 is reversed by re-expression of FUNDC1 and NIX expression vectors lacking the miR-137 recognition sites at their 3′ UTR. Conversely, miR-137 also suppresses the mitophagy induced by fundc1 (CDS+3′UTR) but not fundc1 (CDS) overexpression. Finally, we found that miR-137 inhibits mitophagy by reducing the expression of the mitophagy receptor thereby leads to inadequate interaction between mitophagy receptor and LC3. Our results demonstrated the regulatory role of miRNA to mitophagy receptors and revealed a novel link between miR-137 and mitophagy.     

BNIP3L/NIX-mediated mitophagy protects against ischemic brain injury independent of PARK2

Cerebral ischemia induces massive mitochondrial damage. These damaged mitochondria are cleared, thus attenuating brain injury, by mitophagy. Here, we identified the involvement of BNIP3L/NIX in cerebral ischemia-reperfusion (I-R)-induced mitophagy. Bnip3l knockout (bnip3l^?/?) impaired mitophagy and aggravated cerebral I-R injury in mice, which can be rescued by BNIP3L overexpression. The rescuing effects of BNIP3L overexpression can be observed in park2^?/? mice, which showed mitophagy deficiency after I-R. Interestingly, bnip3l and park2 double-knockout mice showed a synergistic mitophagy deficiency with I-R treatment, which further highlighted the roles of BNIP3L-mediated mitophagy as being independent from PARK2. Further experiments indicated that phosphorylation of BNIP3L serine 81 is critical for BNIP3L-mediated mitophagy. Nonphosphorylatable mutant BNIP3L^S81A failed to counteract both mitophagy impairment and neuroprotective effects in bnip3l^?/? mice. Our findings offer insights into mitochondrial quality control in ischemic stroke and bring forth the concept that BNIP3L could be a potential therapeutic target for ischemic stroke, beyond its accepted role in reticulocyte maturation.     

NLRX1/FUNDC1/NIPSNAP1‐2 axis regulates mitophagy and alleviates intestinal ischaemia/reperfusion injury

ObjectivesMitophagy is considered to be a key mechanism in the pathogenesis of intestinal ischaemic reperfusion (IR) injury. NOD‐like receptor X1 (NLRX1) is located in the mitochondria and is highly expressed in the intestine, and is known to modulate ROS production, mitochondrial damage, autophagy and apoptosis. However, the function of NLRX1 in intestinal IR injury is unclear.Materials and methodsNLRX1 in rats with IR injury or in IEC‐6 cells with hypoxia reoxygenation (HR) injury were measured by Western blotting, real‐time PCR and immunohistochemistry. The function of NLRX1‐FUNDC1‐NIPSNAP1/NIPSNAP2 axis in mitochondrial homeostasis and cell apoptosis were assessed in vitro.ResultsNLRX1 is significantly downregulated following intestinal IR injury. In vivo studies showed that rats overexpressing NLRX1 exhibited resistance against intestinal IR injury and mitochondrial dysfunction. These beneficial effects of NLRX1 overexpression were dependent on mitophagy activation. Functional studies showed that HR injury reduced NLRX1 expression, which promoted phosphorylation of FUN14 domain‐containing 1 (FUNDC1). Based on immunoprecipitation studies, it was evident that phosphorylated FUNDC1 could not interact with the mitophagy signalling proteins NIPSNAP1 and NIPSNAP2 on the outer membrane of damaged mitochondria, which failed to launch the mitophagy process, resulting in the accumulation of damaged mitochondria and epithelial apoptosis.ConclusionsNLRX1 regulates mitophagy via FUNDC1‐NIPSNAP1/NIPSNAP2 signalling pathway. Thus, this study provides a potential target for the development of a therapeutic strategy for intestinal IR injury.     

Protective Effects of Chlorogenic Acid on Paraquat-induced Oxidative Stress in Rats

The protective effects of chlorogenic acid on paraquat-induced oxidative stress were examined in rats. The activities of erythrocytes and liver glutathione peroxidase, and of both liver catalase and glutathione reductase, which were increased by feeding paraquat, declined to the levels in the control rats by supplementing chlorogenic acid to the paraquat diet. The activity of superoxide dismutase was not changed by dietary paraquat or by supplementing chlorogenic acid to the paraquat diet. Paraquat in the diet markedly decreased the liver triacylglycerol and phospholipid concentrations, as well as the food intake and body weight gain, while chlorogenic acid protected against these decreases. These in vivo results and the in vitro superoxide anion scavenging activity of chlorogenic acid suggest that chlorogenic acid acted preventively against paraquat-induced oxidative stress.     

Receptor-mediated mitophagy in yeast and mammalian systems

Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease.     

Structural basis for the phosphorylation of FUNDC1 LIR as a molecular switch of mitophagy

Mitophagy is a fundamental process that determines mitochondrial quality and homeostasis. Several mitophagy receptors, including the newly identified FUNDC1, mediate selective removal of damaged or superfluous mitochondria through their specific interaction with LC3. However, the precise mechanism by which this interaction is regulated to initiate mitophagy is not understood. Here, we report the solution structure of LC3 in complex with a peptide containing the FUNDC1 LC3-interacting region (LIR) motif. The structure reveals a noncanonical LC3-LIR binding conformation, in which the third LIR residue (Val20) is also inserted into the hydrophobic pocket of LC3, together with the conserved residues Tyr18 and Leu21. This enables Tyr18 to be positioned near Asp19 of LC3, and thus phosphorylation of Tyr18 significantly weakens the binding affinity due to electrostatic repulsion. Functional analysis revealed that mitochondrial targeting of the LIR-containing cytosolic portion of FUNDC1 is necessary and sufficient to initiate mitophagy when Tyr18 is unphosphorylated, even in the absence of mitochondrial fragmentation. Thus, we demonstrated that phosphorylation of Tyr18 of FUNDC1 serves as a molecular switch for mitophagy. This may represent a novel target for therapeutic intervention.