Nemaline myopathy (NM) is a genetically and clinically heterogeneous disease that is diagnosed on the basis of the presence of nemaline rods on skeletal muscle biopsy. Although NM has typically been classified by causative genes, disease severity or prognosis cannot be predicted. The common pathologic end point of nemaline rods (despite diverse genetic causes) and an unexplained range of muscle weakness suggest that shared secondary processes contribute to the pathogenesis of NM. We speculated that these processes could be identified through a proteomewide interrogation using a mouse model of severe NM in combination with pathway validation and structural/functional analyses. A proteomic analysis was performed using skeletal muscle tissue from the Neb conditional knockout mouse model compared with its wildtype counterpart to identify pathophysiologically relevant biological processes that might impact disease severity or provide new treatment targets. A differential expression analysis and Ingenuity Pathway Core Analysis predicted perturbations in several cellular processes, including mitochondrial dysfunction and changes in energetic metabolism and stressrelated pathways. Subsequent structural and functional studies demonstrated abnormal mitochondrial distribution, decreased mitochondrial respiratory function, an increase in mitochondrial transmembrane potential, and extremely low ATP content in Neb conditional knockout muscles relative to wild type. Overall, the findings of these studies support a role for severe mitochondrial dysfunction as a novel contributor to muscle weakness in NM. (Am J Pathol 2023, 193: 1528e1547; https://doi.org/10.1016/ j.ajpath.2023.06.009)

ACTA1 encodes skeletal muscle-specific a-actin, which polymerizes to form the thin filament of the sarco-mere. Mutations in ACTA1 are responsible for approximately 30% of nemaline myopathy (NM) cases. Previous studies of weakness in NM have focused on muscle structure and contractility, butgenetic issues alone do not explain the phenotypic heterogeneity observed in patients with NM or NM mouse models. To identify additional biological processes related to NM phenotypic severity, proteomic analysis was performed using muscle protein isolates from wild-type mice in comparison to moderately affected knock-in (KI) Acta1H40Y and the minimally affected transgenic (Tg) ACTA1D286G NM mice. This analysis revealed abnormalities in mitochondrial function and stress-related pathways in both mouse models, supporting an in-depth assessment of mitochondrial biology. Interestingly, evaluating each model in comparison to its wild-type counterpart identified different degrees of mitochondrial abnormality that correlated well with the phenotypic severity of the mouse model. Muscle histology, mitochondrial respiration, electron transport chain function, and mitochondrial transmembrane potential were all normal or minimally affected in the TgACTA1D286G mouse model. In contrast, the more severely affected KI.Acta1H40Y mice displayed significant abnormalities in relation to muscle histology, mitochondrial respirometry, ATP, ADP, and phosphate content, and mitochondrial transmembrane potential. These findings suggest that abnormal energy metabolism is related to symptomatic severity in NM and may constitute a contributor to phenotypic variability and a novel treatment target.

Mitochondria play a key role in cytosolic Ca2+ regulation and buffering, with Ca2+ uptake mainly via the inner membrane mitochondrial Ca2+ uniporter (MCU), and efflux of mitochondrial Ca2+ largely via the Na+/Ca2+ exchanger (mNCE). Previous reports have shown brain synaptic and non‐synaptic mitochondria display marked differences in Ca2+ uptake before permeabilization of the mitochondrial membranes, the so‐called permeable transition pore (mPTP). However, the kinetics of mitochondrial Ca2+ handling and its implications for Ca2+ buffering after boluses of CaCl2 have not been reported. In this study, we aimed to define the kinetics of Ca2+ handling in synaptic and non‐synaptic mitochondria isolated from rat brains via discontinuous percoll centrifugation followed by differential centrifugation. Respiratory control index (RCI), and subsequent changes in calcium retention capacity (CRC) and membrane potential (ΔΨm), were assessed in isolated mitochondria. CRC and ΔΨm were evaluated with Fura 4F penta‐K salt and TMRM dyes, respectively, using fluorescence spectrophotometry (Photon Technology). Mitochondria were energized with glutamate and malate, and CRC and ΔΨm were assessed during state 2 respiration. To investigate the potential role of the mNCE in Ca2+ handling, the NCE blocker CGP37157 (CGP) was used. In addition, experiments were conducted to determine the expression levels of the MCU and mNCE in the two mitochondria pools using western blot. Mitochondrial RCI was higher in the synaptic group compared to the non‐synaptic group, which is consistent with past results. Ca2+ uptake and retention without CGP were lower in synaptic mitochondria compared to non‐synaptic mitochondria. Addition of CGP markedly enhanced CRC in the synaptic mitochondria to levels found in non‐synaptic mitochondria without CGP. In contrast, the CGP induced changes in CRC were not observed in the non‐synaptic mitochondria. Membrane potential depolarization occurred in both synaptic and non‐synaptic mitochondria as matrix free Ca2+ accumulated, reflecting the decrease in Ca2+ sequestration or Ca2+ efflux in the non‐synaptic and synaptic mitochondria, respectively. MCU expression was higher in the non‐synaptic mitochondria compared to the synaptic mitochondria, whereas mNCE expression was not different. The increased Ca2+ uptake and retention in the presence of CGP in the synaptic mitochondria implies that mNCE is active in synaptic mitochondria, which leads to faster extrusion of added Ca2+ before it is sequestered. The lack of change in Ca2+ kinetics in non‐synaptic mitochondria suggests that NCE activity is lower. The ΔΨm and CRC results likely indicate that synaptic mitochondria can buffer Ca2+ like that in non‐synaptic mitochondria, but only do so when the mNCE is blocked. We propose that synaptic mitochondria may have increased mNCE activity to regulate its matrix free Ca2+, while non‐synaptic mitochondria depend mostly on buffering of the free Ca2+; the implications for these differential responses may be related to the physiological role of these two populations of mitochondria.Support or Funding InformationFunding: NIH T35 HL072483 and MCW‐AHWThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The adenine nucleotide translocase (ANT) is the most abundant protein in the inner mitochondrial membrane and the main transporter for ATP and ADP exchange across the membrane. Ischemia reperfusion (IR) injury causes mitochondrial accumulation of reactive O2 and N2 species (ROS/RNS), which can induce irreversible and deleterious tyrosine nitration (NTyr) of protein. In this study, we investigated IR‐induced NTyr of ANT and its impact on cell damage during oxidative stress. Isolated guinea pig hearts were subjected to 35 min ischemia and 20 min reperfusion or no‐IR (control). At the end of reperfusion, ANT was purified from IR or control heart mitochondria and subjected to Western blotting with antibodies to ANT and nitro‐Tyr. We found that IR induced NTyr of ANT. Mass spectrometry analysis identified ANT tyrosines (Y) residues Y81, Y191 and Y195 were nitrated when mitochondria were treated with 100 µM peroxynitrite, an oxidant. To determine the contribution of nitration of ANT Y81, Y191 and Y195 on cellular injury induced by oxidative stress, a single mutant Y81F (phenyalanine) and a double mutant Y192F and Y195F were constructed. After transfection of these mutants into a cardiomyocyte cell line, HL‐1, we found that overexpression of both the single and double mutants of ANT displayed less lactic acid dehydrogenase release induced by hypoxia and reoxygeneration compared to the wild type ANT. Moreover, the association of Voltage‐dependent anion channels with ANT was reduced after IR. In summary, we have identified specific NTyr residues in ANT following oxidative stress, and demonstrated that preventing nitration of these sites attenuated cell oxidative stress injury.

Small and big conductance Ca2+‐sensitive K+ channel (SKCa, BKCa) agonists have been shown to provide preconditioning protection against cardiac ischemia reperfusion (IR) injury. We tested if activation or block of SKCa vs. BKCa channels, initiated before ischemia through initial reperfusion, differently reduce or worsen cardiac global IR injury in isolated rodent hearts, and if these drugs are effective if given IV in vivo before and during regional (LAD) coronary artery occlusion. We found that activating SKCa or BKCa channels by DCEB or NS1619 reduced infarct size (IS) after 35 min of ischemia by 26 and 39% in vivo, and by 56 and 62% in vitro vs. IR alone respectively; BKCa and SKCa blockers paxilline+NS8593, respectively, increased IS by 17% in vivo and by 30% in vitro vs. IR alone. In vitro the two agonists also increased LVP by 90 and 88% and the blockers reduced it by 45% vs. IR alone. Activation of SKCa and BKCa channels improved the respiratory control index (state3/4), increased Ca2+ retention capacity, and restored membrane repolarization in mitochondria isolated after IR. Our results show similar effects of these drugs when given in vivo or in vitro indicating their bioavailability in the heart when given IV. Since each of SKCa ‐ BKCa agonists/antagonists exert effects on isolated cardiac mitochondria, our data also suggest that activation of KCa channels endogenously protects against cardiac injury in part by improving mitochondrial function.Grant Funding Source: R01 HL 089514