Marin Mathieu, Natalia

In human airway smooth muscle (hASM), mitochondrial volume density is greater in asthmatic patients compared with normal controls. There is also an increase in mitochondrial fragmentation in hASM of moderate asthmatics associated with an increase in dynamin-related protein 1 (Drp1) and a decrease in mitofusin 2 (Mfn2) expression, mitochondrial fission, and fusion proteins, respectively. Proinflammatory cytokines such TNFα contribute to hASM hyperreactivity and cell proliferation associated with asthma. However, the involvement of proinflammatory cytokines in mitochondrial remodeling is not clearly established. In nonasthmatic hASM cells, mitochondria were labeled using MitoTracker Red and imaged in three dimensions using a confocal microscope. After 24-h TNFα exposure, mitochondria in hASM cells were more fragmented, evidenced by decreased form factor and aspect ratio and increased sphericity. Associated with increased mitochondrial fragmentation, Drp1 expression increased while Mfn2 expression was reduced. TNFα also increased mitochondrial biogenesis in hASM cells reflected by increased peroxisome proliferator-activated receptor-γ coactivator 1α expression and increased mitochondrial DNA copy number. Associated with mitochondrial biogenesis, TNFα exposure also increased mitochondrial volume density and porin expression, resulting in an increase in maximum O 2 consumption rate. However, when normalized for mitochondrial volume density, O 2 consumption rate per mitochondrion was reduced by TNFα exposure. Associated with mitochondrial fragmentation and biogenesis, TNFα also increased hASM cell proliferation, an effect mimicked by siRNA knockdown of Mfn2 expression and mitigated by Mfn2 overexpression. The results of this study support our hypothesis that in hASM cells exposed to TNFα mitochondria are more fragmented, with an increase in mitochondrial biogenesis and mitochondrial volume density resulting in reduced O 2 consumption rate per mitochondrion.

In aged rats, diaphragm muscle (DIAm) reduced specific force and fiber cross-sectional area, sarcopenia, is selective for vulnerable type IIx and/or IIb DIAm fibers, with type I and IIa fibers being resilient. In humans, the oxidative capacity [as measured by maximum succinate dehydrogenase (SDH max ) activity] of fast-type muscle is reduced with aging, with slow-type muscle being unaffected. We hypothesized that in aged Fischer rat DIAm exhibiting sarcopenia, reduced SDH max activity would occur in type IIx and/or IIb fibers. Rats obtained from the NIA colony (6, 18, and 24 mo old) were euthanized, and ~2-mm-wide DIAm strips were obtained. For SDH max and fiber type assessments, DIAm strips were stretched (approximately optimal length), fresh frozen in isopentane, and sectioned on a cryostat at 6 μm. SDH max , quantified by intensity of nitroblue tetrazolium diformazan precipitation, was assessed in a fiber type-specific manner by comparing serial sections labeled with myosin heavy chain (MyHC) antibodies differentiating type I (MyHC Slow ), IIa (MyHC 2A ), and IIx and/or IIb fibers. Isometric DIAm force and fatigue were assessed in DIAm strips by muscle stimulation with supramaximal pulses at a variety of frequencies (5–100 Hz) delivered in 1-s trains. By 24 mo, DIAm sarcopenia was apparent and SDH max in type IIx and/or IIb fibers activity was reduced ~35% compared with 6-mo-old control DIAm. These results underscore the remarkable fiber type selectivity of type IIx and/or IIb fibers to age-associated perturbations and suggest that reduced mitochondrial oxidative capacity is associated with DIAm sarcopenia. NEW & NOTEWORTHY We examined the oxidative capacity as measured by maximum succinate dehydrogenase activity in older (18 or 24 mo old) Fischer 344 rat diaphragm muscle (DIAm) compared with young rats (6 mo old). In 24-mo-old rats, SDH activity was reduced in type IIx/b DIAm fibers. These SDH changes were concomitant with sarcopenia (reduced specific force and atrophy of type IIx/b DIAm fibers) at 24 mo old. At 18 mo old, there was no change in SDH activity and no evidence of sarcopenia.