There is accumulating evidence that mitochondrial respiratory malfunction is associated with aging-associated complex diseases. However, progress in our understanding of these diseases has been hampered by the sensitivity and throughput of systems employed to quantify dysfunction and inherent limitations of the biological systems studied. In this review, we describe and contrast two methodologies that have been developed for measuring mitochondrial function to address the need for improved sensitivity and increased throughput. We then consider the utility of each methodology in studying three biological systems: isolated mitochondria, cultured cells, and cell fibers and tissues. Finally, we discuss the application of each methodology in the study of mitochondrial dysfunction in Alzheimer’s disease, type 2 diabetes mellitus, and aging-associated autophagy impairment and mitochondrial malfunction. We conclude that the methodologies are complementary, and researchers may need to examine multiple biological systems to unravel complex diseases of aging.

Changes in environmental temperature can pose considerable challenges to animals and shifts in thermal habitat have been shown to be a major force driving species’ adaptation. These adaptations have been the focus of major research efforts to determine the physiological or metabolic constraints related to temperature and to reveal the phenotypic characters that can or should adjust. Considering the current consensus on climate change, the focus of research will likely shift to questioning whether ectothermic organisms will be able to survive future modifications of their thermal niches. Organisms can adjust to temperature changes through physiological plasticity (e.g., acclimation), genetic adaptation, or via dispersal to more suitable thermal habitats. Thus, it is important to understand what genetic and phenotypic attributes—at the individual, population, and species levels—could improve survival success. These issues are particularly important for ectotherms, which are in thermal equilibrium with the surrounding environment. To start addressing these queries, we should consider what physiological or metabolic functions are responsible for the impact of temperature on organisms. Some recent developments indicate that mitochondria are key metabolic structures determining the thermal range that an organism can tolerate. The catalytic capacity of mitochondria is highly sensitive to thermal variation and therefore should partly dictate the temperature dependence of biological functions. Mitochondria contain a complex network of different enzymatic reaction pathways that interact synergistically. The precise regulation of both adenosine triphosphate (ATP) and reactive oxygen species (ROS) production depends on the integration of different enzymes and pathways. Here, we examine the temperature dependence of different parts of mitochondrial pathways and evaluate the evolutionary challenges that need to be overcome to ensure mitochondrial adaptations to new thermal environments.

The oxidative phosphorylation (OxPhos) pathway is responsible for most aerobic ATP production and is the only pathway with both nuclear and mitochondrial encoded proteins. The importance of the interactions between these two genomes has recently received more attention because of their potential evolutionary effects and how they may affect human health and disease. In many different organisms, healthy nuclear and mitochondrial genome hybrids between species or among distant populations within a species affect fitness and OxPhos functions. However, what is less understood is whether these interactions impact individuals within a single natural population. The significance of this impact depends on the strength of selection for mito-nuclear interactions. We examined whether mito-nuclear interactions alter allele frequencies for ~11,000 nuclear SNPs within a single, natural Fundulus heteroclitus population containing two divergent mitochondrial haplotypes (mt-haplotypes). Between the two mt-haplotypes, there are significant nuclear allele frequency differences for 349 SNPs with a p-value of 1% (236 with 10% FDR). Unlike the rest of the genome, these 349 outlier SNPs form two groups associated with each mt-haplotype, with a minority of individuals having mixed ancestry. We use this mixed ancestry in combination with mt-haplotype as a polygenic factor to explain a significant fraction of the individual OxPhos variation. These data suggest that mito-nuclear interactions affect cardiac OxPhos function. The 349 outlier SNPs occur in genes involved in regulating metabolic processes but are not directly associated with the 79 nuclear OxPhos proteins. Therefore, we postulate that the evolution of mito-nuclear interactions affects OxPhos function by acting upstream of OxPhos. Maintaining two distinct mitochondrial haplotypes with functionally important nucleotide variations in a single population is difficult. If interactions with the mitochondria affect the nuclear genome such that some alleles are more advantageous when they occur with a specific mitochondrial genome, then there is an additional genetic burden that also should favor a single mitochondrion. Yet in a single, well mixed Fundulus heteroclitus (teleost fish) population where individuals appear to freely interbreed, there are two mitochondria with five amino acid substitutions between them. We investigate this population and show that individuals with different mitochondria have several hundred loci with extreme allele frequency biases. These differences are not due to neutral or demographic processes and affect cardiac energetics. We conclude that the allele frequency differences are most likely evolving by natural selection and affecting ATP production and energy homeostasis.