A conventional immune regulator mitochondrial antiviral signaling protein blocks hepatic steatosis by maintaining mitochondrial homeostasis.

BACKGROUND AND AIMS: Although the prevalence of NAFLD has risen dramatically to 25% of the adult population worldwide, there are as yet no approved pharmacological interventions for the disease because of uncertainty about the underlying molecular mechanisms. It is known that mitochondrial dysfunction is an important factor in the development of NAFLD. Mitochondrial antiviral signaling protein (MAVS) is a critical signaling adaptor for host defenses against viral infection. However, the role of MAVS in mitochondrial metabolism during NAFLD progression remains largely unknown. APPROACH AND RESULTS: Based on expression analysis, we identified a marked down-regulation of MAVS in hepatocytes during NAFLD progression. By using MAVS global knockout and hepatocyte-specific MAVS knockout mice, we found that MAVS is protective against diet-induced NAFLD. MAVS deficiency induces extensive mitochondrial dysfunction during NAFLD pathogenesis, which was confirmed as impaired mitochondrial respiratory capacity and membrane potential. Metabolomics data also showed the extensive metabolic disorders after MAVS deletion. Mechanistically, MAVS interacts with the N-terminal stretch of voltage-dependent anion channel 2 (VDAC2), which is required for the ability of MAVS to influence mitochondrial function and hepatic steatosis. CONCLUSIONS: In hepatocytes, MAVS plays an important role in protecting against NAFLD by helping to regulate healthy mitochondrial function. These findings provide insights regarding the metabolic importance of conventional immune regulators and support the possibility that targeting MAVS may represent an avenue for treating NAFLD.

Deficiency of PHB complex impairs respiratory supercomplex formation and activates mitochondrial flashes.

Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins. PHBs form multimeric ring complexes acting as scaffolds in the inner mitochondrial membrane. Mitochondrial flashes (mitoflashes) are newly discovered mitochondrial signaling events that reflect electrical and chemical excitations of the organelle. Here, we investigate the possible roles of PHBs in the regulation of mitoflash signaling. Downregulation of PHBs increases mitoflash frequency by up to 5.4-fold due to elevated basal reactive oxygen species (ROS) production in the mitochondria. Mechanistically, PHB deficiency impairs the formation of mitochondrial respiratory supercomplexes (RSCs) without altering the abundance of individual respiratory complex subunits. These impairments induced by PHB deficiency are effectively rescued by co-expression of PHB1 and PHB2, indicating that the multimeric PHB complex acts as the functional unit. Furthermore, downregulating other RSC assembly factors, including SCAFI (also known as COX7A2L), RCF1a (HIGD1A), RCF1b (HIGD2A), UQCC3 and SLP2 (STOML2), all activate mitoflashes through elevating mitochondrial ROS production. Our findings identify the PHB complex as a new regulator of RSC formation and mitoflash signaling, and delineate a general relationship among RSC formation, basal ROS production and mitoflash biogenesis.

Protons Trigger Mitochondrial Flashes.

Emerging evidence indicates that mitochondrial flashes (mitoflashes) are highly conserved elemental mitochondrial signaling events. However, which signal controls their ignition and how they are integrated with other mitochondrial signals and functions remain elusive. In this study, we aimed to further delineate the signal components of the mitoflash and determine the mitoflash trigger mechanism. Using multiple biosensors and chemical probes as well as label-free autofluorescence, we found that the mitoflash reflects chemical and electrical excitation at the single-organelle level, comprising bursting superoxide production, oxidative redox shift, and matrix alkalinization as well as transient membrane depolarization. Both electroneutral H(+)/K(+) or H(+)/Na(+) antiport and matrix proton uncaging elicited immediate and robust mitoflash responses over a broad dynamic range in cardiomyocytes and HeLa cells. However, charge-uncompensated proton transport, which depolarizes mitochondria, caused the opposite effect, and steady matrix acidification mildly inhibited mitoflashes. Based on a numerical simulation, we estimated a mean proton lifetime of 1.42 ns and diffusion distance of 2.06 nm in the matrix. We conclude that nanodomain protons act as a novel, to our knowledge, trigger of mitoflashes in energized mitochondria. This finding suggests that mitoflash genesis is functionally and mechanistically integrated with mitochondrial energy metabolism.

Synergistic triggering of superoxide flashes by mitochondrial Ca2+ uniport and basal reactive oxygen species elevation.

Mitochondrial superoxide flashes reflect a quantal, bursting mode of reactive oxygen species (ROS) production that arises from stochastic, transient opening of the mitochondrial permeability transition pore (mPTP) in many types of cells and in living animals. However, the regulatory mechanisms and the exact nature of the flash-coupled mPTP remain poorly understood. Here we demonstrate a profound synergistic effect between mitochondrial Ca(2+) uniport and elevated basal ROS production in triggering superoxide flashes in intact cells. Hyperosmotic stress potently augmented the flash activity while simultaneously elevating mitochondrial Ca(2+) and ROS. Blocking mitochondrial Ca(2+) transport by knockdown of MICU1 or MCU, newly identified components of the mitochondrial Ca(2+) uniporter, or scavenging mitochondrial basal ROS markedly diminished the flash response. More importantly, whereas elevating Ca(2+) or ROS production alone was inefficacious in triggering the flashes, concurrent physiological Ca(2+) and ROS elevation served as the most powerful flash activator, increasing the flash incidence by an order of magnitude. Functionally, superoxide flashes in response to hyperosmotic stress participated in the activation of JNK and p38. Thus, physiological levels of mitochondrial Ca(2+) and ROS synergistically regulate stochastic mPTP opening and quantal ROS production in intact cells, marking the flash as a coincidence detector of mitochondrial Ca(2+) and ROS signals.

Superoxide flashes: elemental events of mitochondrial ROS signaling in the heart.

The role of mitochondrial reactive oxygen species (mitoROS) in cellular function remains obscure. By synthesizing recent data, we propose here that local dynamic mitoROS in the form of "superoxide flashes" serve as "signaling ROS" rather than "homeostatic ROS", distinguishable from basal mitoROS due to constitutive leakage of the electron transfer chain (ETC). Individual superoxide flashes are 10-s mitoROS bursts that are compartmentalized to a single mitochondrion or local mitochondrial networks. As a highly-conserved universal mitochondrial activity, it occurs in intact cells, in ex vivo beating hearts, and even in living animals. Unlike basal mitoROS, superoxide flashes are ignited by transient openings of a type of mitochondrial permeability transition pore (mPTP), and their incidence is richly regulated by an array of factors that converge on either the mPTP or ETC. Emerging evidence has shown that superoxide flashes decode dietary and metabolic status or exercise, gauge oxidative stress (e.g., during reoxygenation after hypoxia or anoxia), and constitute early mitochondrial signals that initiate oxidative stress-related apoptosis in a context-dependent manner. That they make only a miniscule contribution to global ROS attests to the high efficiency of local ROS signaling. However, the exact mechanisms underlying superoxide flash formation, regulation and function remain uncertain. Future investigation is warranted to uncover the cellular logic and molecular pathways of local dynamic mitoROS signaling in heart muscle cells and many other cell types.

Sophoricoside ameliorates cardiac hypertrophy by activating AMPK/mTORC1-mediated autophagy.

AIM: The study aims to evaluate protective effects of sophoricoside (Sop) on cardiac hypertrophy. Meanwhile, the potential and significance of Sop should be broadened and it should be considered as an attractive drug for the treatment of pathological cardiac hypertrophy and heart failure. METHODS: Using the phenylephrine (PE)-induced neonatal rat cardiomyocytes (NRCMs) enlargement model, the potent protection of Sop against cardiomyocytes enlargement was evaluated. The function of Sop was validated in mice received transverse aortic coarctation (TAC) or sham surgery. At 1 week after TAC surgery, mice were treated with Sop for the following 4 weeks, the hearts were harvested after echocardiography examination. RESULTS: Our study revealed that Sop significantly mitigated TAC-induced heart dysfunction, cardiomyocyte hypertrophy and cardiac fibrosis. Mechanistically, Sop treatment induced a remarkable activation of AMPK/mTORC1-autophagy cascade following sustained hypertrophic stimulation. Importantly, the protective effect of Sop was largely abolished by the AMPKα inhibitor Compound C, suggesting an AMPK activation-dependent manner of Sop function on suppressing pathological cardiac hypertrophy. CONCLUSION: Sop ameliorates cardiac hypertrophy by activating AMPK/mTORC1-mediated autophagy. Hence, Sop might be an attractive candidate for the treatment of pathological cardiac hypertrophy and heart failure.

Prohibitin 2 deficiency impairs cardiac fatty acid oxidation and causes heart failure.

Fatty acids are the most major substrate source for adult cardiac energy generation. Prohibitin 2 (PHB2), a highly conserved protein located in mitochondrial inner membrane, plays key roles in cellular energy metabolic homeostasis. However, its functions in regulating cardiac fatty acid metabolism have remained largely unknown. Our study demonstrates that cardiac-specific knockout of Phb2 leads to accumulation of lipid droplets and causes heart failure. Mechanistically, ablation of PHB2 impairs cardiac fatty acid oxidation (FAO) through downregulating carnitine palmitoyltransferase1b (CPT1b), a rate-limiting enzyme of cardiac mitochondrial FAO. Moreover, overexpression of CPT1b alleviates impaired FAO in PHB2-deficient cardiomyocytes. Thus, our study provides direct evidence for the link between PHB2 and cardiac fatty acid metabolism. Our study points out that PHB2 is a potential FAO regulator in cardiac mitochondrial inner membrane, as well as the connection between PHB2 and CPT1b and their relationships to cardiac pathology especially to cardiac fatty acid metabolic disorder.

Low-Dose Sorafenib Acts as a Mitochondrial Uncoupler and Ameliorates Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is becoming one of the leading causes of hepatocellular carcinoma (HCC). Sorafenib is the only first-line therapy for advanced HCC despite its serious adverse effects. Here, we report that at an equivalent of approximately one-tenth the clinical dose for HCC, sorafenib treatment effectively prevents the progression of NASH in both mice and monkeys without any observed significant adverse events. Mechanistically, sorafenib's benefit in NASH is independent of its canonical kinase targets in HCC, but involves the induction of mild mitochondrial uncoupling and subsequent activation of AMP-activated protein kinase (AMPK). Collectively, our findings demonstrate a previously unappreciated therapeutic effect and signaling mechanism of low-dose sorafenib treatment in NASH. We envision that this new therapeutic strategy for NASH has the potential to translate into a beneficial anti-NASH therapy with fewer adverse events than is observed in the drug's current use in HCC.

NDUFAB1 confers cardio-protection by enhancing mitochondrial bioenergetics through coordination of respiratory complex and supercomplex assembly.

The impairment of mitochondrial bioenergetics, often coupled with exaggerated reactive oxygen species (ROS) production, is a fundamental disease mechanism in organs with a high demand for energy, including the heart. Building a more robust and safer cellular powerhouse holds the promise for protecting these organs in stressful conditions. Here, we demonstrate that NADH:ubiquinone oxidoreductase subunit AB1 (NDUFAB1), also known as mitochondrial acyl carrier protein, acts as a powerful cardio-protector by conferring greater capacity and efficiency of mitochondrial energy metabolism. In particular, NDUFAB1 not only serves as a complex I subunit, but also coordinates the assembly of respiratory complexes I, II, and III, and supercomplexes, through regulating iron-sulfur biosynthesis and complex I subunit stability. Cardiac-specific deletion of Ndufab1 in mice caused defective bioenergetics and elevated ROS levels, leading to progressive dilated cardiomyopathy and eventual heart failure and sudden death. Overexpression of Ndufab1 effectively enhanced mitochondrial bioenergetics while limiting ROS production and protected the heart against ischemia-reperfusion injury. Together, our findings identify that NDUFAB1 is a crucial regulator of mitochondrial energy and ROS metabolism through coordinating the assembly of respiratory complexes and supercomplexes, and thus provide a potential therapeutic target for the prevention and treatment of heart failure.

Mitochondrial flashes regulate ATP homeostasis in the heart.

The maintenance of a constant ATP level ('set-point') is a vital homeostatic function shared by eukaryotic cells. In particular, mammalian myocardium exquisitely safeguards its ATP set-point despite 10-fold fluctuations in cardiac workload. However, the exact mechanisms underlying this regulation of ATP homeostasis remain elusive. Here we show mitochondrial flashes (mitoflashes), recently discovered dynamic activity of mitochondria, play an essential role for the auto-regulation of ATP set-point in the heart. Specifically, mitoflashes negatively regulate ATP production in isolated respiring mitochondria and, their activity waxes and wanes to counteract the ATP supply-demand imbalance caused by superfluous substrate and altered workload in cardiomyocytes. Moreover, manipulating mitoflash activity is sufficient to inversely shift the otherwise stable ATP set-point. Mechanistically, the Bcl-xL-regulated proton leakage through F1Fo-ATP synthase appears to mediate the coupling between mitoflash production and ATP set-point regulation. These findings indicate mitoflashes appear to constitute a digital auto-regulator for ATP homeostasis in the heart.

Identification of EFHD1 as a novel Ca(2+) sensor for mitoflash activation.

Mitochondrial flashes (mitoflashes) represent stochastic and discrete mitochondrial events that each comprises a burst of superoxide production accompanied by transient depolarization and matrix alkalinization in a respiratory mitochondrion. While mitochondrial Ca(2+) is shown to be an important regulator of mitoflash activity, little is known about its specific mechanism of action. Here we sought to determine possible molecular players that mediate the Ca(2+) regulation of mitoflashes by screening mitochondrial proteins containing the Ca(2+)-binding motifs. In silico analysis and targeted siRNA screening identified four mitoflash activators (MICU1, EFHD1, SLC25A23, SLC25A25) and one mitoflash inhibitor (LETM1) in terms of their ability to modulate mitoflash response to hyperosmotic stress. In particular, overexpression or down-regulation of EFHD1 enhanced or depressed mitoflash activation, respectively, under various conditions of mitochondrial Ca(2+) elevations. Yet, it did not alter mitochondrial Ca(2+) handling, mitochondrial respiration, or ROS-induced mitoflash production. Further, disruption of the two EF-hand motifs of EFHD1 abolished its potentiating effect on the mitoflash responses. These results indicate that EFHD1 functions as a novel mitochondrial Ca(2+) sensor underlying Ca(2+)-dependent activation of mitoflashes.

Remodeling of Mitochondrial Flashes in Muscular Development and Dystrophy in Zebrafish.

Mitochondrial flash (mitoflash) is a highly-conserved, universal, and physiological mitochondrial activity in isolated mitochondria, intact cells, and live organisms. Here we investigated developmental and disease-related remodeling of mitoflash activity in zebrafish skeletal muscles. In transgenic zebrafish expressing the mitoflash reporter cpYFP, in vivo imaging revealed that mitoflash frequency and unitary properties underwent multiphasic and muscle type-specific changes, accompanying mitochondrial morphogenesis from 2 to 14 dpf. In particular, short (S)-type mitoflashes predominated in early muscle formation, then S-, transitory (T)- and regular (R)-type mitoflashes coexisted during muscle maturation, followed by a switch to R-type mitoflashes in mature skeletal muscles. In early development of muscular dystrophy, we found accelerated S- to R-type mitoflash transition and reduced mitochondrial NAD(P)H amidst a remarkable cell-to-cell heterogeneity. This study not only unravels a profound functional and morphological remodeling of mitochondria in developing and diseased skeletal muscles, but also underscores mitoflashes as a useful reporter of mitochondrial function in milieu of live animals under physiological and pathophysiological conditions.

Regulation of superoxide flashes by transient and steady mitochondrial calcium elevations.

The mitochondria play essential roles in both intracellular calcium and reactive oxygen species signaling. As a newly discovered universal and fundamental mitochondrial phenomenon, superoxide flashes reflect transient bursts of superoxide production in the matrix of single mitochondria. Whether and how the superoxide flash activity is regulated by mitochondrial calcium remain largely unknown. Here we demonstrate that elevating mitochondrial calcium either by the calcium ionophore ionomycin or by increasing the bathing calcium in permeabilized HeLa cells increases superoxide flash incidence, and inhibition of the mitochondrial calcium uniporter activity abolishes the flash response. Quantitatively, the superoxide flash incidence is correlated to the steady-state mitochondrial calcium elevation with 1.7-fold increase per 1.0 ΔF/F 0 of Rhod-2 signal. In contrast, large mitochondrial calcium transients (e.g., peak ΔF/F 0 ∼ 2.8, duration ∼ 2 min) in the absence of steady-state elevations failed to alter the flash activity. These results indicate that physiological levels of sustained, but not transient, mitochondrial calcium elevation acts as a potent regulator of superoxide flashes, but its mechanism of action likely involves a multi-step, slow-onset process.