Impact of tafazzin deficiency on mitochondrial function
Barth syndrome (BTHS) is a mitochondrial myopathy resulting from mutations in the tafazzin (TAZ) gene encoding a phospholipid transacylase required for cardiolipin remodeling. Cardiolipin is a phospholipid of the inner mitochondrial membrane essential for the function of numerous mitochondrial proteins and processes. However, it is unclear exactly how tafazzin deficiency impacts cardiac mitochondrial metabolism. In studies funded by the Barth Syndrome Foundation, we addressed this question by utilizing Taz-shRNA “knockdown” (TazKD) mice, which exhibit defective cardiolipin remodeling and respiratory supercomplex instability characteristic of human BTHS, but normal cardiac function into adulthood. Results from these studies support links among cardiolipin abnormalities, respiratory supercomplex instability and mitochondrial oxidant production, and shed new light on the role of impaired CoA-dependent substrate oxidation as a primary metabolic consequence of tafazzin-deficiency in the mammalian heart. Recently, we published a detailed biochemical analyses of human BTHS heart samples that corroborate our findings in mice, which led to a new project funded by the Barth Syndrome Foundation to explore the benefits of a targeted nutritional supplement that bypasses the long-chain fatty oxidation pathway we believe to be defective in BTHS.
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Development of protocols and methodology for integrative assessment of mitochondrial function
High-resolution fluo-respirometry for integrative assessment of mitochondrial function
As an active member of the international Mitochondrial Physiology Network and Mitochondrial Physiology Society, our laboratory has engaged in several research collaborations and workshops with the Oroboros Instruments and other member laboratories to develop expertise in high-resolution respirometry (HRR) methodology and data interpretation. We were involved in the recent highly-collaborative publication that aimed to establish uniform standards for the evaluation of mitochondrial respiratory states and rates in this field, clarifying the theoretical and technical bases of these methods, and defining consistent nomenclature for their reporting and discussion. We continue to work with these groups to develop and apply new methodology and approaches to investigating mitochondrial function in biology and disease.
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- Mitochondrial Physiology
- Our lab also recently published a paper defining technical advantages and limitations of simultaneously measuring mitochondrial oxygen consumption and H2O2 release from muscle and cardiac samples, which we have applied to several published and ongoing studies in our laboratory.
Development of micro-metabolic multi-sensors
In collaboration with Dr. Thomas Chen in the Department of Electrical and Computer Engineering and Elaine Carnevale at the Equine Reproduction Lab, we have contributed to the development and testing of miniaturized metabolic microsensors capable of simultaneous monitoring oxygen consumption (mitochondrial respiration) and glycolytic flux (glucose uptake, lactate and H+ release) in single oocytes and early-stage embryos. Ongoing projects are using this technology to define the metabolic phenotype of equine and bovine oocytes and embryos during early development, and how this is influenced byt maternal age and diet or culture nutrient composition to modulate reproductive potential. Emerging collaboration with Drs. Randy Basaraba and Dan Reagan in the Department of Microbiology, Immunology and Pathology seek to utilize this technology to monitor metabolic cross-talk among immune cells, bacterial pathogens and cancer cells in hopes of developing metabolism-based therapies to combat infectious diseases and cancer.
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Mitochondrial adaptation to environmental stress
Extreme environmental conditions, such as heat, cold and high-altitude hypoxia, require integrated physiological adjustments to maintain organismal homeostasis. Mitochondria play a key role in these responses by managing cellular energy demands, redox balance and metabolic thermogenesis on both short-term and evolutionary time scales. Studies in our lab have investigated these processes through collaborations with scientists at CSU and around the world using our expertise in high-resolution fluoro-respirometry and integrative analysis of mitochondrial metabolism in dozens of biological species and sample types. A few of these projects are highlighted briefly below.
In collaboration with Rob Roach at the Altitude Research Center at the University of Colorado Anschutz Medical Campus, we participated in the multi-national 2012 AltitudeOmics expedition in Bolivia to study the cellular/molecular underpinnings of human acclimatization to high-altitude hypoxia. With the assistance of Hans Dreyer at the University of Oregon, we evaluated skeletal muscle mitochondrial function in 15 healthy volunteers in Eugene, OR (low altitude) and following 15 days at over 15,000 feet in a field laboratory atop Mount Chacaltaya in the Bolivian Andes. Results from these studies, published in the Journal of Biological Chemistry, indicated that mitochondria play a central role in muscle adaptation to high altitude by supporting higher resting phosphorylation potential and enhancing the efficiency of long-chain acylcarnitine oxidation. This enables redirection of glucose metabolism toward pathways that support cytosolic redox balance and help mitigate the effects of increased protein catabolism in hypoxia. Collectively, these studies illustrate how an integration of aerobic and anaerobic metabolism is required for physiological hypoxia adaptation in skeletal muscle, and highlight protein catabolism and allosteric regulation as unexpected orchestrators of metabolic remodeling in this context. These findings have important implications for the management of hypoxia-related diseases and other conditions associated with chronic catabolic stress. Current studies in our lab seek to better understand the role of sympatho-adrenal drive in high-altitude hypoxia adaption through collaboration with Ryan Maresh in the CSU Hypobaric Chamber Facility in the Department of Biomedical Sciences.
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Hibernating mammals such as the golden-mantled ground squirrel (Callospermophilus lateralis, GMGS) fast during the winter months in extended periods of reduced metabolic rate known as torpor In preparation for this remarkable feat, animals increase food intake in the summer and fall while simultaneously decreasing energy demands, leading to an approximate doubling of body mass primarily in the form of white adipose tissue before hibernation. During hibernation, animals rely primarily on the oxidation of these endogenous fat stores to fuel metabolic processes as internal body temperature decreases to near ambient temperatures as low as 5°C. Although significant effort has been devoted to understanding various aspects of hibernation physiology and metabolism, comparatively less is known about the metabolic adjustments that occur in preparation for the winter hibernation season. We hypothesized that mitochondria, the cellular sites of oxidative metabolism, undergo tissue-specific seasonal adjustments in carbohydrate and fatty acid utilization to facilitate or complement this remarkable phenotype. To address this, we performed high-resolution respirometry of mitochondria isolated from GMGS liver, heart, skeletal muscle, and brown adipose tissue (BAT) sampled during summer (active), fall (pre-hibernation), and winter (hibernation) seasons using multi-substrate titration protocols.
Studies revealed seasonal variations in mitochondrial oxidative phosphorylation capacity, substrate utilization, and coupling efficiency that reflected the distinct functions and metabolic demands of the tissues they support. A consistent finding across tissues was a greater influence of fatty acids (palmitoylcarnitine) on respiratory parameters during the pre-hibernation and hibernation seasons. In particular, fatty acids had a greater suppressive effect on pyruvate-supported oxidative phosphorylation in heart, muscle, and liver mitochondria and enhanced uncoupled respiration in BAT and muscle mitochondria in the colder seasons. Taken together, these studies indicate that mitochondria respond to seasonal variations in physical activity, temperature, and nutrient availability in a tissue-specific manner that complements circannual shifts in the bioenergetic and thermoregulatory demands of mammalian hibernators. Ongoing studies are comparing the metabolic interactions of seasonal cold exposure and hyperphagia in GMGS and laboratory mice to better understand the evolutionary basis of obesity and insulin resistance as both physiological and pathogenic responses to nutrient excess.
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Another major interest in our lab is the biochemical and genetic basis of mitochondrial plasticity on both organismal and evolutionary time scales. Comparative studies in mammals, ectotherms, insects, plants and fish species in collaboration with biologists at CSU and other institutions that have revealed several novel insights to how mitochondria adapt to physiological stress, a few of which are highlighted below:
In collaboration with Shane Kanatous in the Department of Biological Sciences, we evaluated the ontogeny of skeletal muscle mitochondrial function in Northern elephant seals (Mirounga angustirostris) – extreme, hypoxia-adapted endotherms that rely largely on aerobic metabolism during up to two-hour breath-hold dives in near freezing water temperatures. Results indicate that seal muscle maintains a high capacity for fatty acid oxidation despite a progressive decrease in total respiratory capacity as animals mature from pups to adults. This is explained by a progressive increase in phosphorylation control and fatty acid utilization over pyruvate in adult seals compared to humans and seal pups. The ontogeny of this phenotype suggests it is an adaptation of muscle to the prolonged breath-hold exercise and highly variable ambient temperatures experienced by mature elephant seals, rather than being genetically “programmed” at birth.
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In collaboration with Justin Havird at UT Austin and Dillion Chung at the University of British Colombia (presently at NHLBI), we have also evaluated the effects of geographic variations in aquatic insect and fish mitochondrial phenotype, with a particular interest in thermal adaptation and the role of membrane polyunsaturated fatty acid (PUFA) redistribution in this process. Ongoing and pending studies are targeting delta-6 desaturase, the rate-limiting enzyme in long-chain PUFA synthesis, as a potential regulator of membrane remodeling and mitochondrial plasticity during thermal acclimatization.
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Evolutionary bases of mitochondrial functional plasticity
Ongoing studies in collaboration with Rachel Mueller in the Department of Biological Sciences at CSU are investigating the comparative physiology of muscle and cardiac mitochondrial function across ectotherm species. We have a particular interest in elucidating the biological and evolutionary basis for variations in oxidative phosphorylation coupling efficiency as it relates to meeting variations in cellular bioenergetic and redox demands. Dr. Mueller’s lab has a particular interest in the cellular adaptations that facilitate wide variations in genome size in salamander species, which place unique demands on cellular physiology and architecture that have far-reaching implications for the basic understanding of cell biology and evolution.
We have also worked with Justin Havird at UT Austin and Dan Sloan in the Department of Biological Sciences to develop new high-resolution respirometry protocols for comparative evaluation of mitochondrial function in plant species, with a particular interest in the functional consequences of evolutionary variations in the expression of mitochondrial- vs. nuclear DNA-encoded subunits of electron transfer complexes.
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Basic and applied aspects of bacterial bioenergetics
The widely accepted endosymbiont hypothesis states that mitochondria evolved from an ancient bacterial progenitor that was engulfed by a host cell, and both lived on in an endosymbiotic relationship to form the first eukaryotic cells from which most complex life has evolved. Indeed, modern bacteria utilize many of the same biochemical pathways and enzyme complexes to generate energy as mitochondria. However, they have also evolved an impressive array of alternative mechanisms to support oxidative phosphorylation under a wide range of environmental conditions. In collaboration with faculty and the Departments of Chemistry and Microbiology, Immunology and Pathology, our lab uses high-resolution fluo-respirometry to investigate the impacts of bactericidal drugs and evolutionary diversity on bacterial bioenergetics. A current project recently funded by the National Science Foundation in collaboration with Professors Debbie Crans and Dean Crick aims to elucidate to functional consequence of variations in menaquinone structure on bacterial electron transfer and the efficiency of oxidative metabolism.
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