Absolute quantification of plasma mitochondrial DNA by droplet digital PCR marks COVID-19 severity over time during intensive care unit admissions.

Increased plasma mitochondrial DNA concentrations are associated with poor outcomes in multiple critical illnesses, including COVID-19. However, current methods of cell-free mitochondrial DNA quantification in plasma are time-consuming and lack reproducibility. Here, we used next-generation sequencing to characterize the size and genome location of circulating mitochondrial DNA in critically ill subjects with COVID-19 to develop a facile and optimal method of quantification by droplet digital PCR. Sequencing revealed a large percentage of small mitochondrial DNA fragments in plasma with wide variability in coverage by genome location. We identified probes for the mitochondrial DNA genes, cytochrome B and NADH dehydrogenase 1, in regions of relatively high coverage that target small sequences potentially missed by other methods. Serial assessments of absolute mitochondrial DNA concentrations were then determined in plasma from 20 critically ill subjects with COVID-19 without a DNA isolation step. Mitochondrial DNA concentrations on the day of enrollment were increased significantly in patients with moderate or severe acute respiratory distress syndrome (ARDS) compared with those with no or mild ARDS. Comparisons of mitochondrial DNA concentrations over time between patients with no/mild ARDS who survived, patients with moderate/severe ARDS who survived, and nonsurvivors showed the highest concentrations in patients with more severe disease. Absolute mitochondrial DNA quantification by droplet digital PCR is time-efficient and reproducible; thus, we provide a valuable tool and rationale for future studies evaluating mitochondrial DNA as a real-time biomarker to guide clinical decision-making in critically ill subjects with COVID-19.

Mcl-1-mediated mitochondrial fission protects against stress but impairs cardiac adaptation to exercise.

Myeloid cell leukemia-1 (Mcl-1) is a structurally and functionally unique anti-apoptotic Bcl-2 protein. While elevated levels of Mcl-1 contribute to tumor cell survival and drug resistance, loss of Mcl-1 in cardiac myocytes leads to rapid mitochondrial dysfunction and heart failure development. Although Mcl-1 is an anti-apoptotic protein, previous studies indicate that its functions extend beyond regulating apoptosis. Mcl-1 is localized to both the mitochondrial outer membrane and matrix. Here, we have identified that Mcl-1 in the outer mitochondrial membrane mediates mitochondrial fission, which is independent of its anti-apoptotic function. We demonstrate that Mcl-1 interacts with Drp1 to promote mitochondrial fission in response to various challenges known to perturb mitochondria morphology. Induction of fission by Mcl-1 reduces nutrient deprivation-induced cell death and the protection is independent of its BH3 domain. Finally, cardiac-specific overexpression of Mcl-1OM, but not Mcl-1Matrix, contributes to a shift in the balance towards fission and leads to reduced exercise capacity, suggesting that a pre-existing fragmented mitochondrial network leads to decreased ability to adapt to an acute increase in workload and energy demand. Overall, these findings highlight the importance of Mcl-1 in maintaining mitochondrial health in cells.

Frequency and Complexity of De Novo Structural Mutation in Autism.

Genetic studies of autism spectrum disorder (ASD) have established that de novo duplications and deletions contribute to risk. However, ascertainment of structural variants (SVs) has been restricted by the coarse resolution of current approaches. By applying a custom pipeline for SV discovery, genotyping, and de novo assembly to genome sequencing of 235 subjects (71 affected individuals, 26 healthy siblings, and their parents), we compiled an atlas of 29,719 SV loci (5,213/genome), comprising 11 different classes. We found a high diversity of de novo mutations, the majority of which were undetectable by previous methods. In addition, we observed complex mutation clusters where combinations of de novo SVs, nucleotide substitutions, and indels occurred as a single event. We estimate a high rate of structural mutation in humans (20%) and propose that genetic risk for ASD is attributable to an elevated frequency of gene-disrupting de novo SVs, but not an elevated rate of genome rearrangement.

Mending a broken heart: the role of mitophagy in cardioprotection.

The heart is highly energy dependent with most of its energy provided by mitochondrial oxidative phosphorylation. Mitochondria also play a role in many other essential cellular processes including metabolite synthesis and calcium storage. Therefore, maintaining a functional population of mitochondria is critical for cardiac function. Efficient degradation and replacement of dysfunctional mitochondria ensures cell survival, particularly in terminally differentiated cells such as cardiac myocytes. Mitochondria are eliminated via mitochondrial autophagy or mitophagy. In the heart, mitophagy is an essential housekeeping process and required for cardiac homeostasis. Reduced autophagy and accumulation of impaired mitochondria have been linked to progression of heart failure and aging. In this review, we discuss the pathways that regulate mitophagy in cells and highlight the cardioprotective role of mitophagy in response to stress and aging. We also discuss the therapeutic potential of targeting mitophagy and directions for future investigation.

DRD4 genotype predicts longevity in mouse and human.

Longevity is influenced by genetic and environmental factors. The brain's dopamine system may be particularly relevant, since it modulates traits (e.g., sensitivity to reward, incentive motivation, sustained effort) that impact behavioral responses to the environment. In particular, the dopamine D4 receptor (DRD4) has been shown to moderate the impact of environments on behavior and health. We tested the hypothesis that the DRD4 gene influences longevity and that its impact is mediated through environmental effects. Surviving participants of a 30-year-old population-based health survey (N = 310; age range, 90-109 years; the 90+ Study) were genotyped/resequenced at the DRD4 gene and compared with a European ancestry-matched younger population (N = 2902; age range, 7-45 years). We found that the oldest-old population had a 66% increase in individuals carrying the DRD4 7R allele relative to the younger sample (p = 3.5 × 10(-9)), and that this genotype was strongly correlated with increased levels of physical activity. Consistent with these results, DRD4 knock-out mice, when compared with wild-type and heterozygous mice, displayed a 7-9.7% decrease in lifespan, reduced spontaneous locomotor activity, and no lifespan increase when reared in an enriched environment. These results support the hypothesis that DRD4 gene variants contribute to longevity in humans and in mice, and suggest that this effect is mediated by shaping behavioral responses to the environment.

Mcl-1 Differentially Regulates Autophagy in Response to Changes in Energy Status and Mitochondrial Damage.

Myeloid cell leukemia-1 (Mcl-1) is a unique antiapoptotic Bcl-2 member that is critical for mitochondrial homeostasis. Recent studies have demonstrated that Mcl-1's functions extend beyond its traditional role in preventing apoptotic cell death. Specifically, data suggest that Mcl-1 plays a regulatory role in autophagy, an essential degradation pathway involved in recycling and eliminating dysfunctional organelles. Here, we investigated whether Mcl-1 regulates autophagy in the heart. We found that cardiac-specific overexpression of Mcl-1 had little effect on baseline autophagic activity but strongly suppressed starvation-induced autophagy. In contrast, Mcl-1 did not inhibit activation of autophagy during myocardial infarction or mitochondrial depolarization. Instead, overexpression of Mcl-1 increased the clearance of depolarized mitochondria by mitophagy independent of Parkin. The increase in mitophagy was partially mediated via Mcl-1's LC3-interacting regions and mutation of these sites significantly reduced Mcl-1-mediated mitochondrial clearance. We also found that Mcl-1 interacted with the mitophagy receptor Bnip3 and that the interaction was increased in response to mitochondrial stress. Overall, these findings suggest that Mcl-1 suppresses nonselective autophagy during nutrient limiting conditions, whereas it enhances selective autophagy of dysfunctional mitochondria by functioning as a mitophagy receptor.

Aging is associated with a decline in Atg9b-mediated autophagosome formation and appearance of enlarged mitochondria in the heart.

Advancing age is a major risk factor for developing heart disease, and the biological processes contributing to aging are currently under intense investigation. Autophagy is an important cellular quality control mechanism that is reduced in tissues with age but the molecular mechanisms underlying the age-associated defects in autophagy remain poorly characterized. Here, we have investigated how the autophagic process is altered in aged mouse hearts. We report that autophagic activity is reduced in aged hearts due to a reduction in autophagosome formation. Gene expression profile analysis to evaluate changes in autophagy regulators uncovered a reduction in Atg9b transcript and protein levels. Atg9 proteins are critical in delivering membrane to the growing autophagosome, and siRNA knockdown of Atg9b in cells confirmed a reduction in autophagosome formation. Autophagy is also the main pathway involved in eliminating dysfunctional mitochondria via a process known as mitophagy. The E3 ubiquitin ligase Parkin plays a key role in labeling mitochondria for mitophagy. We also found increased levels of Parkin-positive mitochondria in the aged hearts, an indication that they have been labeled for mitophagy. In contrast, Nrf1, a major transcriptional regulator of mitochondrial biogenesis, was significantly reduced in aged hearts. Additionally, our data showed reduced Drp1-mediated mitochondrial fission and formation of enlarged mitochondria in the aged heart. Overall, our findings suggest that cardiac aging is associated with reduced autophagosome number, decreased mitochondrial turnover, and formation of megamitochondria.

PINK1 Is Dispensable for Mitochondrial Recruitment of Parkin and Activation of Mitophagy in Cardiac Myocytes.

Myocyte function and survival relies on the maintenance of a healthy population of mitochondria. The PINK1/Parkin pathway plays an important role in clearing defective mitochondria via autophagy in cells. However, how the PINK1/Parkin pathway regulates mitochondrial quality control and whether it coordinates with other mitophagy pathways are still unclear. Therefore, the objective of this study was to investigate the effect of PINK1-deficiency on mitochondrial quality control in myocytes. Using PINK1-deficient (PINK1-/-) mice, we found that Parkin is recruited to damaged cardiac mitochondria in hearts after treatment with the mitochondrial uncoupler FCCP or after a myocardial infarction even in the absence of PINK1. Parkin recruitment to depolarized mitochondria correlates with increased ubiquitination of mitochondrial proteins and activation of mitophagy in PINK1-/- myocytes. In addition, induction of mitophagy by the atypical BH3-only protein BNIP3 is unaffected by lack of PINK1. Overall, these data suggest that Parkin recruitment to depolarized cardiac mitochondria and subsequent activation of mitophagy is independent of PINK1. Moreover, alternative mechanisms of Parkin activation and pathways of mitophagy remain functional in PINK1-/- myocytes and could compensate for the PINK1 deficiency.