Mitonuclear Imbalance Research Articles

Abstract 533: Doxycycline Fuels Mitochondrial Dysfunction In Aortic Smooth Muscle Cells

Introduction: The antibiotic doxycycline is known to inhibit inflammation, and was therefore considered as a therapeutic to prevent abdominal aortic aneurysm (AAA) growth. However, in two clinical trials, doxycycline-treated AAA patients did not experience reduced aneurysm growth. Doxycycline was previously shown to induce mitonuclear imbalance and reduce mitochondrial function. Objective: To determine that doxycycline would also impair mitochondrial function in the aorta and aortic smooth muscle cells (SMCs), giving rise to SMC phenotype switching. Results: Oral doxycycline intake induced mitonuclear imbalance in the murine aorta, human aortic aneurysm tissue and in human aortic SMCs. Consequently, doxycycline reduced mitochondrial activity and respiration in aortic SMCs. Transcription factor krüppel-like factor 4 (KLF4), a player in mitochondrial homeostasis, is upregulated after doxycycline treatment. Moreover, the integrated stress response effector, activating transcription factor 4 (ATF4), is enhanced to restore cellular homeostasis. These changes result in diminished expression of typical contractile SMC markers. Treatment with the drug Elamipretide (SS-31) improves mitochondrial markers and cellular stress management. Conclusion: Doxycycline leads to mitochondrial dysfunction and ER stress in aortic SMCs. These changes may contribute to aortic aneurysm growth by impairing SMC function. Elamipretide offers rescue from some of the harmful effects of doxycycline by improving mitochondrial function.

Exercise alters the mitochondrial proteostasis and induces the mitonuclear imbalance and UPRmt in the hypothalamus of mice

The maintenance of mitochondrial activity in hypothalamic neurons is determinant to the control of energy homeostasis in mammals. Disturbs in the mitochondrial proteostasis can trigger the mitonuclear imbalance and mitochondrial unfolded protein response (UPRmt) to guarantee the mitochondrial integrity and function. However, the role of mitonuclear imbalance and UPRmt in hypothalamic cells are unclear. Combining the transcriptomic analyses from BXD mice database and in vivo experiments, we demonstrated that physical training alters the mitochondrial proteostasis in the hypothalamus of C57BL/6J mice. This physical training elicited the mitonuclear protein imbalance, increasing the mtCO-1/Atp5a ratio, which was accompanied by high levels of UPRmt markers in the hypothalamus. Also, physical training increased the maximum mitochondrial respiratory capacity in the brain. Interestingly, the transcriptomic analysis across several strains of the isogenic BXD mice revealed that hypothalamic mitochondrial DNA-encoded genes were negatively correlated with body weight and several genes related to the orexigenic response. As expected, physical training reduced body weight and food intake. Interestingly, we found an abundance of mt-CO1, a mitochondrial DNA-encoded protein, in NPY-producing neurons in the lateral hypothalamus nucleus of exercised mice. Collectively, our data demonstrated that physical training altered the mitochondrial proteostasis and induced the mitonuclear protein imbalance and UPRmt in hypothalamic cells.

High-intensity exercise training induces mitonuclear imbalance and activates the mitochondrial unfolded protein response in the skeletal muscle of aged mice.

The impairment of mitochondrial metabolism is a hallmark of aging. Mitonuclear imbalance and the mitochondrial unfolded protein response (UPRmt) are two conserved mitochondrial mechanisms that play critical roles in ensuring mitochondrial proteostasis and function. Here, we combined bioinformatics, physiological, and molecular analyses to examine the role of mitonuclear imbalance and UPRmt in the skeletal muscle of aged rodents and humans. The analysis of transcripts from the skeletal muscle of aged humans (60-70years old) revealed that individuals with higher levels of UPRmt-related genes displayed a consistent increase in several mitochondrial-related genes, including the OXPHOS-associated genes. Interestingly, high-intensity interval training (HIIT) was effective in stimulating the mitonuclear imbalance and UPRmt in the skeletal muscle of aged mice. Furthermore, these results were accompanied by higher levels of several mitochondrial markers and improvements in physiological parameters and physical performance. These data indicate that the maintenance or stimulation of the mitonuclear imbalance and UPRmt in the skeletal muscle could ensure mitochondrial proteostasis during aging, revealing new insights into targeting mitochondrial metabolism by using physical exercise.

Loss of high-temperature requirement protein A2 protease activity induces mitonuclear imbalance via differential regulation of mitochondrial biogenesis in sarcopenia.

Cellular homeostasis requires tight coordination between nucleus and mitochondria, organelles that each possesses their own genomes. Disrupted mitonuclear communication has been found to be implicated in many aging processes. However, little is known about mitonuclear signaling regulator in sarcopenia which is a major contributor to the risk of poor health-related quality of life, disability, and premature death in older people. High-temperature requirement protein A2 (HtrA2/Omi) is a mitochondrial protease and plays an important role in mitochondrial proteostasis. HtrA2mnd2(-/-) mice harboring protease-deficient HtrA2/Omi Ser276Cys missense mutants exhibit premature aging phenotype. Additionally, HtrA2/Omi has been established as a signaling regulator in nervous system and tumors. We therefore asked whether HtrA2/Omi participates in mitonuclear signaling regulation in muscle degeneration. Using motor functional, histological, and molecular biological methods, we characterized the phenotype of HtrA2mnd2(-/-) muscle. Furthermore, we isolated the gastrocnemius muscle of HtrA2mnd2(-/-) mice and determined expression of genes in mitochondrial unfolded protein response (UPRmt ), mitohormesis, electron transport chain (ETC), and mitochondrial biogenesis. Here, we showed that HtrA2/Omi protease deficiency induced denervation-independent skeletal muscle degeneration with sarcopenia phenotypes. Despite mitochondrial hypofunction, upregulation of UPRmt and mitohormesis-related genes and elevated total reactive oxygen species (ROS) production were not observed in HtrA2mnd2(-/-) mice, contrary to previous assumptions that loss of protease activity of HtrA2/Omi would lead to mitochondrial dysfunction as a result of proteostasis disturbance and ROS burst. Instead, we showed that HtrA2/Omi protease deficiency results in different changes between the expression of nuclear DNA- and mitochondrial DNA-encoded ETC subunits, which is in consistent with their transcription factors, nuclear respiratory factors 1 and 2, and coactivator peroxisome proliferator-activated receptor γ coactivator 1α. These results reveal that loss of HtrA2/Omi protease activity induces mitonuclear imbalance via differential regulation of mitochondrial biogenesis in sarcopenia. The novel mechanistic insights may be of importance in developing new therapeutic strategies for sarcopenia.

Aerobic Exercise Training Induces the Mitonuclear Imbalance and UPRmt in the Skeletal Muscle of Aged Mice.

The impairment of the mitochondrial functions is a hallmark of aging. During aging, there is a downregulation of two mechanisms strictly associated with mitochondrial integrity, including the mitonuclear imbalance (eg, imbalance in mitochondrial- versus nuclear-encoded mitochondrial proteins) and the mitochondrial unfolded protein response (UPRmt). Here, we evaluated the effects of aerobic exercise in the mitonuclear imbalance and UPRmt markers in the skeletal muscle of old mice. We combined the physiological tests, molecular and bioinformatic analyzes to evaluate the effects of 4 weeks of aerobic exercise training on mitonuclear imbalance and UPRmt markers in the skeletal muscle of young (2 months) and aged (24 months) C57BL/6J mice. Initially, we found that aging reduced several mitochondrial genes in the gastrocnemius muscle, and it was accompanied by the low levels of UPRmt markers, including Yme1l1 and Clpp mRNA. As expected, physical training improved the whole-body metabolism and physical performance of aged mice. The aerobic exercise increased key proteins involved in the mitochondrial biogenesis/functions (VDAC and SIRT1) along with mitochondrial-encoded genes (mtNd1, mtCytB, and mtD-Loop) in the skeletal muscle of old mice. Interestingly, aerobic exercise induced the mitonuclear imbalance, increasing MTCO1/ATP5a ratio and UPRmt markers in the skeletal muscle, including HSP60, Lonp1, and Yme1L1 protein levels in the gastrocnemius muscle of aged mice. These data demonstrate that aerobic exercise training induced mitonuclear imbalance and UPRmt in the skeletal muscle during aging. These phenomena could be involved in the improvement of the mitochondrial metabolism and oxidative capacity in aged individuals.

Mitochondrial haplotypes affect metabolic phenotypes in the Drosophila Genetic Reference Panel.

The nature and extent of mitochondrial DNA variation in a population and how it affects traits is poorly understood. Here we resequence the mitochondrial genomes of 169 Drosophila Genetic Reference Panel lines, identifying 231 variants that stratify along 12 mitochondrial haplotypes. We identify 1,845 cases of mitonuclear allelic imbalances, thus implying that mitochondrial haplotypes are reflected in the nuclear genome. However, no major fitness effects are associated with mitonuclear imbalance, suggesting that such imbalances reflect population structure at the mitochondrial level rather than genomic incompatibilities. Although mitochondrial haplotypes have no direct impact on mitochondrial respiration, some haplotypes are associated with stress- and metabolism-related phenotypes, including food intake in males. Finally, through reciprocal swapping of mitochondrial genomes, we demonstrate that a mitochondrial haplotype associated with high food intake can rescue a low food intake phenotype. Together, our findings provide new insight into population structure at the mitochondrial level and point to the importance of incorporating mitochondrial haplotypes in genotype-phenotype relationship studies.

Effect of Tim23 knockdown in vivo on mitochondrial protein import and retrograde signaling to the UPRmt in muscle.

The mitochondrial unfolded protein response (UPRmt) is a protein quality control mechanism that strives to achieve proteostasis in the face of misfolded proteins. Because of the reliance of mitochondria on both the nuclear and mitochondrial genomes, a perturbation of the coordination of these genomes results in a mitonuclear imbalance in which holoenzymes are unable to assume mature stoichiometry and thereby activates the UPRmt. Thus, we sought to perturb this genomic coordination by using a systemic antisense oligonucleotide (in vivo morpholino) targeted to translocase of the inner membrane channel subunit 23 (Tim23), the major channel of the inner membrane. This resulted in a 40% reduction in Tim23 protein content, a 32% decrease in matrix-destined protein import, and a trend to elevate reactive oxygen species (ROS) emission under maximal respiration conditions. This import defect activated the C/EBP homologous protein (CHOP) branch of the UPRmt, as evident from increases in caseinolytic mitochondrial matrix peptidase proteolytic subunit (ClpP) and chaperonin 10 (cpn10) but not the activating transcription factor 5 (ATF5) arm. Thus, in the face of proteotoxic stress, CHOP and ATF5 could be activated independently to regain proteostasis. Our second aim was to investigate the role of proteolytically derived peptides in mediating retrograde signaling. Peptides released from the mitochondrion following basal proteolysis were isolated and incubated with import reactions. Dose- and time-dependent effect of peptides on protein import was observed. Our data suggest that mitochondrial proteolytic byproducts exert an inhibitory effect on protein import, possibly to reduce excessive protein import as a potential negative feedback mechanism. The inhibition of import into the organelle also serves a retrograde function, possibly via ROS emission, to modify nuclear gene expression and ultimately improve folding capacity.

Mitochondrial haplotypes influence metabolic traits across bovine inter- and intra-species cybrids

In bovine species, mitochondrial DNA polymorphisms and their correlation to productive or reproductive performances have been widely reported across breeds and individuals. However, experimental evidence of this correlation has never been provided. In order to identify differences among bovine mtDNA haplotypes, transmitochondrial cybrids were generated, with the nucleus from MAC-T cell line, derived from a Holstein dairy cow (Bos taurus) and mitochondria from either primary cell line derived from a domestic Chinese native beef Luxi cattle breed or central Asian domestic yak (Bos grunniens). Yak primary cells illustrated a stronger metabolic capacity than that of Luxi. However, all yak cybrid parameters illustrated a drop in relative yak mtDNA compared to Luxi mtDNA, in line with a mitonuclear imbalance in yak interspecies cybrid. Luxi has 250 divergent variations relative to the mitogenome of Holsteins. In cybrids there were generally higher rates of oxygen consumption (OCR) and extracellular acidification (ECAR), and lower mRNA expression levels of nuclear-encoded mitochondrial genes, potentially reflecting active energy metabolism and cellular stress resistance. The results demonstrate that functional differences exist between bovine cybrid cells. While cybrid viability was similar between Holstein and Luxi breeds, the mitonuclear mismatch caused a marked metabolic dysfunction in cattle:yak cybrid species.