Abstract
Compromised mitochondrial respiratory function is associated with advancing age. Damage due to an increase in reactive oxygen species (ROS) with age is thought to contribute to the mitochondrial deficits. The coenzyme nicotinamide adenine dinucleotide in its reduced (NADH) and oxidized (NAD+) forms plays an essential role in the cyclic sequence of reactions that result in the regeneration of ATP by oxidative phosphorylation in mitochondria. Monitoring mitochondrial NADH/NAD+ redox status during recovery from an episode of high energy demand thus allows assessment of mitochondrial function. NADH fluoresces when excited with ultraviolet light in the UV-A band and NAD+ does not, allowing NADH/NAD+ to be monitored in real time using fluorescence microscopy. Our goal was to assess mitochondrial function by monitoring the NADH fluorescence response following a brief period of high energy demand in muscle from adult and old wild-type mice. This was accomplished by isolating whole lumbrical muscles from the hind paws of 7- and 28-month-old mice and making simultaneous measurements of force and NADH fluorescence responses during and after a 5 s maximum isometric contraction. All muscles exhibited fluorescence oscillations that were qualitatively similar and consisted of a brief transient increase followed by a longer transient period of reduced fluorescence and, finally, an increase that included an overshoot before recovering to resting level. Compared with the adult mice, muscles from the 28 mo mice exhibited a delayed peak during the first fluorescence transient and an attenuated recovery following the second transient. These findings indicate an impaired mitochondrial capacity to maintain NADH/NAD+ redox homeostasis during contractile activity in skeletal muscles of old mice.
Highlights
Mitochondria are the main source of the adenosine triphosphate (ATP) required to support skeletal muscle contractile function
Because the fluorescence captured during whole cell or tissue UV excitation is dominated by mitochondrial NADH (Mayevsky and Rogatsky, 2007), tracking the fluorescence response of skeletal muscle following a maximum tetanic contraction provides a view of mitochondrial function and mitochondrial nicotinamide adenine dinucleotide (NAD) redox status during recovery from an episode of high energy demand
Based upon these preliminary experiments, a 5 s tetanic contraction was chosen as the test condition for the remainder of the study as it resulted in a robust, characteristic fluorescence response without the large decline in force observed during longer contractions
Summary
Mitochondria are the main source of the adenosine triphosphate (ATP) required to support skeletal muscle contractile function. The mitochondrial processes that result in the regeneration of ATP give rise to reactive oxygen species (ROS). The coenzyme nicotinamide adenine dinucleotide (NAD) in its reduced (NADH) and oxidized (NAD+) forms plays an essential role in the cyclic sequence of reactions that result in the regeneration of ATP by oxidative phosphorylation in mitochondria. Because the fluorescence captured during whole cell or tissue UV excitation is dominated by mitochondrial NADH (Mayevsky and Rogatsky, 2007), tracking the fluorescence response of skeletal muscle following a maximum tetanic contraction provides a view of mitochondrial function and mitochondrial NAD redox status during recovery from an episode of high energy demand. Since the mitochondrial NAD redox state reflects the global mitochondrial redox state (Mayevsky and Rogatsky, 2007; Zorov et al, 2014) which, in turn, is an indicator of the probability of ROS formation (Zorov et al, 2014), the fluorescence response serves as a continuous, real-time signal that predicts the development of oxidative stress
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