Philip G. Morgan

Mitochondrial Complex I Deficiency Increases Protein Acetylation and Accelerates Heart Failure

Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases, including heart failure, but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by the inactivation of the Ndufs4 gene, a protein critical for complex I assembly, in the mouse heart (cKO). Although complex I-supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD(+)/NADH ratio by complex I deficiency inhibited Sirt3 activity, leading to an increase in protein acetylation and sensitization of the permeability transition in mitochondria (mPTP). NAD(+) precursor supplementation to cKO mice partially normalized the NAD(+)/NADH ratio, protein acetylation, and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target.

Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans

Caenorhabditis elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear genes that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD(+)-dependent ketoacid oxidation occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations both among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease.

Effect of Anesthetics and a Convulsant on Normal and Mutant Caenorhabditis elegans

The authors have developed a method for studying the action of volatile anesthetics in Caenorhabditis elegans (C.e.), a free living nematode. C.e. appears to be a useful model for the study of the influence of genetics on susceptibility to anesthetics. This worm has a small, completely defined nervous system, easily manipulated genetics, and a large number of nervous system mutants. Under normal conditions C.e. moves almost constantly. When exposed to anesthetics there is an initial phase of increased locomotion, followed by uncoordinated motion that progresses to immobility. Motion returns quickly when the nematodes are removed from the anesthetic. The authors called loss of locomotion “anesthesia.” The ED50s of various anesthetics with C.e. are as follows: methoxyflurane 0.45%, chloroform 1.25%, halothane 2.7%, enflurane 4.2%, isoflurane 5.6%, fluroxene 9.9%. The authors also studied the action of a convulsant, flurothyl, on C.e. Flurothyl has anesthetizing properties in these animals with an ED50 of 8.1%. No convulsant activity was noted. However, mixtures of halothane and flurothyl were antagonistic in their effects, while halothane and enflurane were additive. Furthermore, the authors isolated a mutant strain (HS1) of C.e. that shows altered responses to several anesthetics and a convulsant. HS1 is uncoordinated when not exposed to anesthetics. Like the normal strain (N2) HS1 loses mobility when exposed to anesthetics. The ED50s for various anesthetics in HS1 were as follows: methoxyflurane 0.04%, chloroform 0.52%, halothane 0.85%, isoflurane 4.9%, enflurane 6.0%, fluroxene 10.9%. When compared with the normal C.e., HS1 exhibits a marked increase in sensitivity to methoxyflurane, chloroform, and halothane. No alterations in sensitivity to isoflurane or fluroxene were noted. HS1 exhibited decreased sensitivity to enflurane. HS1 reverts to normal motion when exposed to low concentrations of flurothyl and shows decreased sensitivity to the anesthetic effects of flurothyl with an ED50 of 11.8%. The slope of the curve of the log ED50 versus the log oil/gas partition coefficient is steeper in mutant than in normal nematodes.

Mitochondrial respiration and reactive oxygen species in mitochondrial aging mutants.

A powerful approach to understanding a complex process such as aging is to study the process in model organisms that are amenable to genetic dissection. Several mutant strains in different organisms have been identified that affect lifespan; data from these organisms indicate that mitochondrial function is a major factor affecting lifespan. Mutations which affect some aspect of mitochondrial function and affect lifespan have been isolated in yeast, nematodes, flies and mice. These results have revealed a general pattern that decreased metabolic rates are associated with increased lifespans. However, it is also clear that some strains with decreased metabolic rates have shortened lifespans. The emerging data is most consistent with the effects of reactive oxygen species also playing a major role in determining lifespan. Mitochondrial mutations are apparently capable of slowing metabolism with resulting increases or decreases in production of reactive oxygen species. In this review, we discuss the effects of mitochondrial mutations of lifespan with an emphasis on the role of reactive oxygen species.

Mitochondrial Complex I Function Modulates Volatile Anesthetic Sensitivity in C. elegans

Despite the widespread clinical use of volatile anesthetics, their mechanisms of action remain unknown [1-6]. An unbiased genetic screen in the nematode C. elegans for animals with altered volatile anesthetic sensitivity identified a mutant in a nuclear-encoded subunit of mitochondrial complex I [7,8]. This raised the question of whether mitochondrial dysfunction might be the primary mechanism by which volatile anesthetics act, rather than an untoward secondary effect [9,10]. We report here analysis of additional C. elegans mutations in orthologs of human genes that contribute to the formation of complex I, complex II, complex III, and coenzyme Q [11-14]. To further characterize the specific contribution of complex I, we generated four hypomorphic C. elegans mutants encoding different complex I subunits [15]. Our main finding is the identification of a clear correlation between complex I-dependent oxidative phosphorylation capacity and volatile anesthetic sensitivity. These extended data link a physiologic determinant of anesthetic action in a tractable animal model to similar clinical observations in children with mitochondrial myopathies [16]. This work is the first to specifically implicate complex I-dependent oxidative phosphorylation function as a primary mediator of volatile anesthetic effect.

INTERACTION OF GABA AND VOLATILE ANESTHETICS IN THE NEHATODE CAENORHABDITIS ELEGANS

The authors tested whether mutant strains of Caenorhabditis elegans with altered sensitivity to volatile anesthetics have altered responses to GABA or GABA-agonists. They determined the ED50s of the wild-type strain N2 and two mutant strains of C. elegans to a GABA-mimetic ivermectin (IVM) and to GABA. unc-79, a strain with increased sensitivity to halothane, was more sensitive than N2 to IVM and GABA. unc-9, a strain that suppresses the increased sensitivity of unc-79 to halothane, was less sensitive than N2 to IVM and GABA. The authors also tested whether doses of GABA or IVM and volatile anesthetics were additive in their effects on C. elegans. Halothane (2.1%) did not shift the ED50 of IVM, but was antagonistic to GABA. Enflurane (4%) was antagonistic to both IVM and GABA. However, ED50s of halothane and enflurane were unchanged in the presence of IVM (35 nM) or GABA (150 mM). The authors conclude that GABA by itself does not appear to mediate halothane or enflurane sensitivity in C. elegans.

Preclinical pharmacology of GW280430A (AV430A) in the rhesus monkey and in the cat: a comparison with mivacurium.

BackgroundNo replacement for succinylcholine is yet available. GW280430A (AV430A) is a representative of a new class of nondepolarizing neuromuscular blocking drugs called asymmetric mixed-onium chlorofumarates. It undergoes rapid degradation in plasma by chemical hydrolysis and inactivation by cysteine adduction, resulting in a very short duration of effect. The neuromuscular, cardiovascular, and autonomic pharmacology of GW280430A is compared herein with that of mivacurium. MethodsAdult male rhesus monkeys and adult male cats were anesthetized with nitrous oxide–oxygen–halothane and chloralose–pentobarbital, respectively. The neuromuscular blocking properties of GW280430A and mivacurium were compared at a stimulation rate of 0.15 Hz in the extensor digitorum of the foot (monkey) and the tibialis anterior (cat). Sympathetic responses were assayed in the cat in the nictitating membrane preparation, and vagal effects were evaluated in the cat via observation of bradycardic responses after stimulation of the cervical right vagus nerve. ResultsGW280430A and mivacurium were equipotent in the monkey (ED95 was 0.06 mg/kg in each case). GW280430A was half as potent as mivacurium in the cat. The total duration of action of GW280430A was less than half that of mivacurium in the monkey; recovery slopes were more than twice as rapid. The 25–75% recovery index of GW280430A did not vary significantly after various bolus doses or infusions, averaging 1.4–1.8 min in the monkey, significantly shorter than the same time interval (4.8–5.7 min) for mivacurium. Dose ratios for autonomic versus neuromuscular blocking properties in the cat were greater than 25 for both GW280430A and mivacurium. The ratio ED Hist:ED95 Neuromuscular Block in the monkey was significantly greater (approximately 53 vs. 13) for GW280430A, indicating approximately four times less relative prominence of the side effects of skin flushing and decrease of blood pressure, which are associated with release of histamine. ConclusionsThese experiments show a much shorter neuromuscular blocking effect and much-reduced side effects in the case of GW280430A vis-à-vis mivacurium. These results, together with the novel chemical degradation of GW280430A, suggest further evaluation in human subjects.

Mitochondrial respiration and reactive oxygen species in C. elegans

A powerful approach to understanding complex processes such as aging is to study longevity in organisms that are amenable to genetic dissection. The nematode Caenorhabditis elegans represents a superb model system in which to study the effects of mitochondrial function on longevity. Several mutant strains have been identified that indicate that mitochondrial function is a major factor affecting the organisms lifespan. Taken as a group, these mutant strains indicate that metabolic rate, per se, only affects longevity indirectly. Mutations causing lowered metabolic rate potential are capable of decreasing or increasing longevity.

Novel interactions between mitochondrial superoxide dismutases and the electron transport chain

The processes that control aging remain poorly understood. We have exploited mutants in the nematode, Caenorhabditis elegans, that compromise mitochondrial function and scavenging of reactive oxygen species (ROS) to understand their relation to lifespan. We discovered unanticipated roles and interactions of the mitochondrial superoxide dismutases (mtSODs): SOD‐2 and SOD‐3. Both SODs localize to mitochondrial supercomplex I:III:IV. Loss of SOD‐2 specifically (i) decreases the activities of complexes I and II, complexes III and IV remain normal; (ii) increases the lifespan of animals with a complex I defect, but not the lifespan of animals with a complex II defect, and kills an animal with a complex III defect; (iii) induces a presumed pro‐inflammatory response. Knockdown of a molecule that may be a pro‐inflammatory mediator very markedly extends lifespan and health of certain mitochondrial mutants. The relationship between the electron transport chain, ROS, and lifespan is complex, and defects in mitochondrial function have specific interactions with ROS scavenging mechanisms. We conclude that mtSODs are embedded within the supercomplex I:III:IV and stabilize or locally protect it from reactive oxygen species (ROS) damage. The results call for a change in the usual paradigm for the interaction of electron transport chain function, ROS release, scavenging, and compensatory responses.

Early Developmental Exposure to Volatile Anesthetics Causes Behavioral Defects in Caenorhabditis elegans

BACKGROUND:Mounting evidence from animal studies shows that anesthetic exposure in early life leads to apoptosis in the developing nervous system. This loss of neurons has functional consequences in adulthood. Clinical retrospective reviews have suggested that multiple anesthetic exposures in early childhood are associated with learning disabilities later in life as well. Despite much concern about this phenomenon, little is known about the mechanism by which anesthetics initiate neuronal cell death. Caenorhabditis elegans, a powerful genetic animal model, with precisely characterized neural development and cell death pathways, affords an excellent opportunity to study anesthetic-induced neurotoxicity. We hypothesized that exposing the nematode to volatile anesthetics early in life would induce neuron cell death, producing a behavioral defect that would be manifested in adulthood. METHODS:After synchronization and hatching, larval worms were exposed to volatile anesthetics at their 95% effective concentration for 4 hours. On day 4 of life, exposed and control worms were tested for their ability to sense and move to an attractant (i.e., to chemotax). We determined the rate of successful chemotaxis using a standardized chemotaxis index. RESULTS:Wild-type nematodes demonstrated striking deficits in chemotaxis indices after exposure to isoflurane (ISO) or sevoflurane (SEVO) in the first larval stage (chemotaxis index: untreated, 85 ± 2; ISO, 52 ± 2; SEVO, 47 ± 2; P < 0.05 for both exposures). The mitochondrial mutant gas-1 had a heightened effect from the anesthetic exposure (chemotaxis index: untreated, 71 ± 2; ISO, 29 ± 12; SEVO, 24 ± 13; P < 0.05 for both exposures). In contrast, animals unable to undergo apoptosis because of a mutation in the pathway that mediates programmed cell death (ced-3) retained their ability to sense and move toward an attractant (chemotaxis index: untreated, 76 ± 10; ISO, 73 ± 9; SEVO, 76 ± 10). Furthermore, we discovered that the window of greatest susceptibility to anesthetic neurotoxicity in nematodes occurs in the first larval stage after hatching (L1). This coincides with a period of neurogenesis in this model. All values are means ± SD. CONCLUSION:These data indicate that anesthetics affect neurobehavior in nematodes, extending the range of phyla in which early exposure to volatile anesthetics has been shown to cause functional neurological deficits. This implies that anesthetic-induced neurotoxicity occurs via an ancient underlying mechanism. C elegans is a tractable model organism with which to survey an entire genome for molecules that mediate the toxic effects of volatile anesthetics on the developing nervous system.