Gary Diffee

Exercise training attenuates aging-associated mitochondrial dysfunction in rat skeletal muscle: Role of PGC-1α

Aged skeletal muscle demonstrates declines in muscle mass and deterioration of mitochondrial content and function. Peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) plays an important role in promoting muscle mitochondrial biogenesis in response to exercise training, but its role in senescent muscle is not clear. In the present study we hypothesize that a downregulation of the PGC-1α signaling pathway contributes to mitochondrial deterioration in aged muscle whereas endurance training ameliorates the deficits. Three groups of Fischer 344/BNF1 rats were used: young, sedentary (Y, 4 months); old, sedentary (O, 22 months); and old trained (OT, 22 months), subjected to treadmill running at 17.5 m/min, 10% grade for 45 min/day, 5 days/week for 12-weeks. PGC-1α mRNA and nuclear PGC-1α protein content in the soleus muscle were both decreased in O vs. Y rats, whereas OT rats showed a 2.3 and 1.8-fold higher PGC-1α content than O and Y rats, respectively (P<0.01). Mitochondrial transcription factor A (Tfam), cytochrome c (Cyt c) and mitochondrial (mt) DNA contents were significantly decreased in O vs. Y rats, but elevated by 2.2 (P<0.01), 1.4 (P<0.05) and 2.4-fold (P<0.01), respectively, in OT vs. O rats. In addition, Tfam and mtDNA showed 1.6 and 1.8-fold (P<0.01) higher levels, respectively, in OT vs. Y rats. These adaptations were accompanied by significant increases in the expression of the phosphorylated form of AMP-activated kinase (AMPK) (P<0.01), p38 mitogen-activated kinase (MAPK) (P<0.05) and silent mating type information regulator 2 homolog 1 (SIRT1) (P<0.01) in OT rats. Furthermore, OT rats showed great levels of phosphorylation in cAMP responsive element binding protein (p-CREB) and DNA binding compared to O and Y rats. These data indicate that endurance training can attenuate aging-associated decline in mitochondrial protein synthesis in skeletal muscle partly due to upregulation of PGC-1α signaling.

Phosphorylation of myosin regulatory light chain eliminates force-dependent changes in relaxation rates in skeletal muscle.

The rate of relaxation from steady-state force in rabbit psoas fiber bundles was examined before and after phosphorylation of myosin regulatory light chain (RLC). Relaxation was initiated using diazo-2, a photolabile Ca2+ chelator that has low Ca2+ binding affinity (K(Ca) = 4.5 x 10(5) M(-1)) before photolysis and high affinity (K(Ca) = 1.3 x 10(7) M(-1)) after photolysis. Before phosphorylating RLC, the half-times for relaxation initiated from 0.27 +/- 0.02, 0.51 +/- 0.03, and 0.61 +/- 0.03 Po were 90 +/- 6, 140 +/- 6, and 182 +/- 9 ms, respectively. After phosphorylation of RLC, the half-times for relaxation from 0.36 +/- 0.03 Po, 0.59 +/- 0.03 Po, and 0.65 +/- 0.02 Po were 197 +/- 35 ms, 184 +/- 35 ms, and 179 +/- 22 ms. This slowing of relaxation rates from steady-state forces less than 0.50 Po was also observed when bundles of fibers were bathed with N-ethylmaleimide-modified myosin S-1, a strongly binding cross-bridge derivative of S1. These results suggest that phosphorylation of RLC slows relaxation, most likely by slowing the apparent rate of transition of cross-bridges from strongly bound (force-generating) to weakly bound (non-force-generating) states, and reduces or eliminates Ca2+ and cross-bridge activation-dependent changes in relaxation rates.

Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers

To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers.

Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.

We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.

Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction

Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia.

Effect of Aging on Power Output Properties in Rat Skinned Cardiac Myocytes

Aging is generally associated with a decline in several indices of cardiac function. The cellular mechanisms for this decline are not completely understood. The ability of the myocardium to perform external work (power output) is a critical aspect of ventricular function. The purpose of this study was to determine the effect of aging on loaded shortening and power output properties. We measured force-velocity properties in permeabilized (skinned) myocytes from the hearts of 9-, 24-, and 33-month-old male Fisher 344 × Brown Norway F1 hybrid rats (F344BN) during loaded contractions using a force-clamp technique. Power output was calculated by multiplying force and shortening velocity values. We found that peak power output normalized to maximal force was significantly decreased by 18% and 31% in myocytes from 24- and 33-month-old group, respectively, compared with 9-month group (p < .05). These results suggest that aging is associated with a significant decrease in the ability of the myocardium to do work.

Moderate Intensity, but Not High Intensity, Treadmill Exercise Training Alters Power Output Properties in Myocardium From Aged Rats

Aging is characterized by a progressive decline in cardiac function, but endurance exercise training has been shown to retard a number of deleterious effects of aging. However, underlying mechanisms by which exercise training improves age-related decrements in myocardial contractile function are not well understood. The purpose of this study was to determine the effects of exercise training on power output properties in permeablized (skinned) myocytes of old rats. Thirty-month-old rats were divided into sedentary control (C) and groups undergoing 11 weeks of treadmill exercise training at moderate intensity (MI) and at high intensity (HI). Peak power output normalized to maximal force was significantly increased in MI but not in HI compared to C with significant increases in atrial myosin light chain 1 in ventricle. These results suggest that MI exercise training is beneficial as a significant increase was seen in the ability of the myocardium to do work, but this effect was not seen with HI training.

Mitochondrial and Metabolic Gene Expression in the Aged Rat Heart.

Aging is associated with a decline in cardiac function. Exercise intervention has been suggested as a way to improve this decrement. Age-related decline in cardiac function is associated with decreases in fatty acid oxidation, mitochondrial function, and AMP-activated protein kinase (AMPK) activity. The molecular mechanisms involved with age-related changes in mitochondrial function and substrate metabolism are poorly understood. We determined gene expression differences in hearts of Young (6 mo), Old (33 mo), and old exercise trained (Old + EXE) (34 mo) FBN rats, using Qiagen PCR arrays for Glucose, Fatty acid, and Mitochondrial metabolism. Old rats demonstrated decreased (p < 0.05) expression for key genes in fatty acid oxidation, mitochondrial function, and AMPK signaling. There were no differences in the expression of genes involved in glucose metabolism with age. These gene expression changes occurred prior to altered protein translation as we found no differences in the protein content of peroxisome proliferator activated receptor gamma, coactivators 1 alpha (PGC-1α), peroxisome proliferator activated receptor alpha (PPARα), and AMPKα2 between young and old hearts. Four months of exercise training did not attenuate the decline in the gene expression in aged hearts. Despite this lack of change in gene expression, exercise-trained rats demonstrated increased exercise capacity compared to their sedentary counterparts. Taken together, our results show that differential expression of genes associated with fatty acid metabolism, AMPK signaling and mitochondrial function decrease in the aging heart which may play a role in age-related declines in fatty acid oxidation, AMPK activity, and mitochondrial function in the heart.

Challenges in Exercise Physiology Research and Education

Similar to other subdisciplines in kinesiology, exercise physiology (EP) as a field is facing challenges in both research (creation and dissemination of new knowledge) and education (classroom instruction and student mentoring). In the current communication, we will learn from the history, analyze the current status of the field, and provide some perspectives based primarily on our knowledge and experience working as faculty members at the University of Wisconsin–Madison.

Linking metabolic and contractile dysfunction in aged cardiac myocytes

Aging is associated with declining cardiac contractile function as well as changes in metabolism and mitochondrial function. The relationship between age‐related changes in cardiac metabolism and declining cardiac contractile function has not been determined. In order to define the role energetics play in changes in contractile function, we measured mitochondrial NADH, [NADH]m, during continuous contractions of isolated left ventricular myocytes from young (Y) and old (O) FBN rats. Second, we explored the role of metabolic disruption with rotenone and increased workload with isoproterenol (ISO) had on age‐related changes in myocytes shortening. Single, intact myocytes were stimulated for 10 min of continuous contraction at either 2 Hz or 4 Hz while being perfused with Ringers solution. Properties of shortening (peak shortening and rate of shortening) were measured at the onset (T0) and after 10 min (T10) of continuous contraction, and the decline in shortening over time (T10/T0) was determined. Although young and old myocytes had similar contractile function under resting conditions, old myocytes demonstrated decrements in [NADH]m during continuous stimulation, while young myocytes maintained constant [NADH]m over this time. In addition, old myocytes exhibited impaired contractile function to a workload (ISO) and metabolic (rotenone) stress compared to young myocytes. Taken together, these results demonstrated that old myocytes are susceptible to stress‐induced contractile dysfunction which may be related to altered cellular energetics.