Darmyn Ritchie

Mitochondrial structure and function are disrupted by standard isolation methods.

Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca2+ handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca2+ challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H2O2 production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented.

Mitochondrial functional impairment with aging is exaggerated in isolated mitochondria compared to permeabilized myofibers

Mitochondria regulate cellular bioenergetics and apoptosis and have been implicated in aging. However, it remains unclear whether age‐related loss of muscle mass, known as sarcopenia, is associated with abnormal mitochondrial function. Two technically different approaches have mainly been used to measure mitochondrial function: isolated mitochondria and permeabilized myofiber bundles, but the reliability of these measures in the context of sarcopenia has not been systematically assessed before. A key difference between these approaches is that contrary to isolated mitochondria, permeabilized bundles contain the totality of fiber mitochondria where normal mitochondrial morphology and intracellular interactions are preserved. Using the gastrocnemius muscle from young adult and senescent rats, we show marked effects of aging on three primary indices of mitochondrial function (respiration, H2O2 emission, sensitivity of permeability transition pore to Ca2+) when measured in isolated mitochondria, but to a much lesser degree when measured in permeabilized bundles. Our results clearly demonstrate that mitochondrial isolation procedures typically employed to study aged muscles expose functional impairments not seen in situ. We conclude that aging is associated with more modest changes in mitochondrial function in sarcopenic muscle than suggested previously from isolated organelle studies.

Alterations in intrinsic mitochondrial function with aging are fiber type‐specific and do not explain differential atrophy between muscles

To determine whether mitochondrial dysfunction is causally related to muscle atrophy with aging, we examined respiratory capacity, H2O2 emission, and function of the mitochondrial permeability transition pore (mPTP) in permeabilized myofibers prepared from four rat muscles that span a range of fiber type and degree of age‐related atrophy. Muscle atrophy with aging was greatest in fast‐twitch gastrocnemius (Gas) muscle (−38%), intermediate in both the fast‐twitch extensor digitorum longus (EDL) and slow‐twitch soleus (Sol) muscles (−21%), and non‐existent in adductor longus (AL) muscle (+47%). In contrast, indices of mitochondrial dysfunction did not correspond to this differential degree of atrophy. Specifically, despite higher protein expression for oxidative phosphorylation (oxphos) system in fast Gas and EDL, state III respiratory capacity per myofiber wet weight was unchanged with aging, whereas the slow Sol showed proportional decreases in oxphos protein, citrate synthase activity, and state III respiration. Free radical leak (H2O2 emission per O2 flux) under state III respiration was higher with aging in the fast Gas, whereas state II free radical leak was higher in the slow AL. Only the fast muscles had impaired mPTP function with aging, with lower mitochondrial calcium retention capacity in EDL and shorter time to mPTP opening in Gas and EDL. Collectively, our results underscore that the age‐related changes in muscle mitochondrial function depend largely upon fiber type and are unrelated to the severity of muscle atrophy, suggesting that intrinsic changes in mitochondrial function are unlikely to be causally involved in aging muscle atrophy.

Adaptations in Capillarization and Citrate Synthase Activity in Response to Endurance Training in Older and Young Men

The time-course of adaptation in cardiorespiratory fitness, measures of capillarization, and citrate synthase (CS) activity were examined in seven older (O; 69 ± 7 years) and seven young (Y; 22 ± 1 years) men pre-, mid-, and posttraining during a 12-week endurance training program. Training was performed on a cycle ergometer three times per week for 45 minutes at ~70% of maximal VO(2) (VO(2max)). VO(2max) and maximal cardiac output increased similarly from pre- to posttraining in O and Y (p < .05), and maximal a-vO(2diff) was greater (p < .05) posttraining in O and Y. CS was elevated at mid- and posttraining compared with pretraining in both O and Y (p < .05). Indices of capillarization increased 30%-40% in O and 20%-30% in Y and were elevated at posttraining compared with pre- and midtraining in both groups (p < .05). This study showed that both O and Y undertaking similar endurance training displayed capillary angiogenesis and improved mitochondrial respiratory capacity.

Mitochondrial Function in Permeabilized Cardiomyocytes Is Largely Preserved in the Senescent Rat Myocardium

The aging heart is characterized by a progressive decline in contractile function and diastolic relaxation. Amongst the factors implicated in these changes is a progressive replacement fibrosis secondary to cardiomyoctye death, oxidative damage, and energetic deficit, each of which may be secondary to impaired mitochondrial function. Here, we performed an in-depth examination of mitochondrial function in saponin-permeabilized cardiomyocyte bundles, a preparation where all mitochondria are represented and their structure intact, from young adult (YA) and senescent (SEN) rats (n = 8 per group). When accounting for increased fibrosis (+19%, P<0.01) and proportional decrease in citrate synthase activity in the SEN myocardium (−23%, P<0.05), mitochondrial respiration and reactive oxygen species (H2O2) emission across a range of energized states was similar between age groups. Accordingly, the abundance of electron transport chain proteins was also unchanged. Likewise, except for CuZnSOD (−37%, P<0.05), the activity of antioxidant enzymes was unaltered with aging. Although time to mitochondrial permeability transition pore (mPTP) opening was decreased (−25%, P<0.05) in the SEN heart, suggesting sensitization to apoptotic stimuli, this was not associated with a difference in apoptotic index measured by ELISA. Collectively, our results suggest that the function of existing cardiac ventricular mitochondria is relatively preserved in SEN rat heart when measured in permeabilized cells.