Biomarkers of mitochondrial stress and DNA damage during pediatric catheter-directed neuroangiography - a prospective single-center study.

BACKGROUND: Neuroangiography represents a critical diagnostic and therapeutic imaging modality whose associated radiation may be of concern in children. The availability of in vivo radiation damage markers would represent a key advancement for understanding radiation effects and aid in the development of radioprotective strategies. OBJECTIVE: Determine if biomarkers of cellular damage can be detected in the peripheral blood mononuclear cells (PBMC) of children undergoing neuroangiography. MATERIALS AND METHODS: Prospective single-site study of 27 children. Blood collected pre and post neuroangiography, from which PBMC were isolated and assayed for biomarkers of mitochondrial stress (mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitochondrial DNA (mtDNA)) and DNA damage (γH2AX). Dose response of biomarkers vs. radiation dose was analyzed using linear regressions. The cohort was divided into higher (HD) and lower dose (LD) groups and analyzed using linear mixed models and compared using Welch's t-tests. RESULTS: No biomarker exhibited a dose-dependent response following radiation (γH2AX: R2 = 0.0012, P = 0.86; MMP: R2 = 0.016, P = 0.53; mtDNA: R2 = 0.10, P = 0.11; ROS: R2 = 0.0023, P = 0.81). Groupwise comparisons showed no significant differences in γH2AX or ROS after radiation (γH2AX: LD: 0.6 ± 6.0, P = 0.92; HD: -7.5 ± 6.3 AU, P = 0.24; ROS: LD: 1.3 ± 2.8, P = 0.64; HD: -3.6 ± 3.0 AU, P = 0.24). Significant changes were observed to mitochondrial markers MMP (-53.7 ± 14.7 AU, P = 0.0014) and mtDNA (-1.1 ± 0.4 AU, P = 0.0092) for HD, but not the LD group (MMP: 26.1 ± 14.7 AU, P = 0.090; mtDNA: 0.2 ± 0.4, P = 0.65). CONCLUSIONS: Biomarkers of mitochondrial stress in PBMC were identified during pediatric neuroangiography and warrant further investigation for radiation biodosimetry. However, isolating radiation-specific effects from those of procedural stress and general anesthesia requires further investigation.

DNM1L Variant Alters Baseline Mitochondrial Function and Response to Stress in a Patient with Severe Neurological Dysfunction.

Mitochondria play vital roles in brain development and neuronal activity, and mitochondrial dynamics (fission and fusion) maintain organelle function through the removal of damaged components. Dynamin-like protein-1 (DRP-1), encoded by DNM1L, is an evolutionarily conserved GTPase that mediates mitochondrial fission by surrounding the scission site in concentric ring-like structures via self-oligomerization, followed by GTPase-dependant constriction. Here, we describe the clinical characteristics and cellular phenotype of a patient with severe neurological dysfunction, possessing a homozygous DNM1L variant c.305C>T (p.T115M) in the GTPase domain. For comparative analysis, we also describe a previously identified heterozygous variant demonstrating a rapidly fatal neurocognitive phenotype (c.261dup/c.385:386del, p.W88M*9/E129K*6). Using patient-generated fibroblasts, we demonstrated both DNM1L variants undergo adverse alterations to mitochondrial structure and function, including impaired mitochondrial fission, reduced membrane potential, and lower oxidative capacity including an increased cellular level of reactive oxygen species (ROS) and dsDNA breaks. Mutation of DNM1L was also associated with impaired responses to oxidative stress, as treatment with hydrogen peroxide dramatically increased cellular ROS, with minimal exacerbation of already impaired mitochondrial function. Taken together, our observations indicate that homozygous p.T115M variant of DNM1L produces a neurological and neurodevelopmental phenotype, consistent with impaired mitochondrial architecture and function, through a diminished ability to oligomerize, which was most prevalent under oxidative stress.

2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca(2+) sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of cross-bridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM.

The importance of size and disorder in the cryoprotective effects of dehydrins.

Dehydrins protect plant proteins and membranes from damage during drought and cold. Vitis riparia K2 is a 48-residue protein that can protect lactate dehydrogenase from freeze-thaw damage by preventing the aggregation and denaturation of the enzyme. To further elucidate its mechanism, we used a series of V. riparia K2 concatemers (K4, K6, K8, and K10) and natural dehydrins (V. riparia YSK2, 60 kilodalton peach dehydrin [PCA60], barley dehydrin5 [Dhn5], Thellungiella salsuginea dehydrin2 [TsDHN-2], and Opuntia streptacantha dehydrin1 [OpsDHN-1]) to test the effect of the number of K-segments and dehydrin size on their ability to protect lactate dehydrogenase from freeze-thaw damage. The results show that the larger the hydrodynamic radius of the dehydrin, the more effective the cryoprotection. A similar trend is observed with polyethylene glycol, which would suggest that the protection is simply a nonspecific volume exclusion effect that can be manifested by any protein. However, structured proteins of a similar range of sizes did not show the same pattern and level of cryoprotection. Our results suggest that with respect to enzyme protection, dehydrins function primarily as molecular shields and that their intrinsic disorder is required for them to be an effective cryoprotectant. Lastly, we show that the cryoprotection by a dehydrin is not due to any antifreeze protein-like activity, as has been reported previously.