Susan H. McKiernan

Different lots of bovine serum albumin inhibit or stimulate in vitro development of hamster embryos

Dear Editor: In recent years, much emphasis has been placed on eliminating serum from culture media for somatic cells and preimplantation embryos, in part because of the high cost and inconsistency of this product, and in part to examine effects of specific growth factors and hormones without experiments being compromised by serum factors (3). In many studies, serum has been replaced with bovine serum albumin (BSA) (8,14). However, serum albumin, in general, binds biological substances such as fatty acids, steroids and many trace metals, provides an accessible protein reserve, serves as a colloid osmotic regulator and acts as a transport protein (1). Thus, BSA is not an inert constituent of culture media and may provide low molecular weight growth-promoting factors (9) as well as chelate heavy metals and other potentially toxic contaminants (4,7,10). Considerable progress has been made in supporting development of hamster preimplantation embryos in vitro using a simple proteinfree, chemically defined medium generically designated Hamster Embryo Culture Medium (HECM), from 0% 2-cell embryo development a few years ago (12) to approximately 50% blastocysts currently (11). However, this culture medium may lack important factors found in vivo that could improve development; accordingly, we examined the effect of incorporating bovine serum albumin into the medium. We tested the effect of four Fraction V BSA preparations on the development of hamster 2-cell embryos in vitro. Hamster 2-cell embryos, collected 32 h post egg-activation from PMSG-stimulated females mated to fertile males (13), were cultured in 100 #1 drops of HECM1 (13) containing 0.1 mg/ml polyvinyl alcohol (PVA) under a silicone oil overlay in an atmosphere of 10% CO2:10% 02:80% N 2 at 37.5 ° C for 72 h (11). The experiment consisted of five treatments, arranged as five drops of media in a 60 mm petri dish. The control treatment contained no BSA, while the four experimental treatments each contained a different batch of commercial Fraction V BSA at 3 mg/ml. BSA-1, BSA-2 and BSA-3 were obtained from Sigma Chemical Co., all purchased under catalog number A-9647, with lot numbers 41-F-0593, 106-F-0580 and 26-F-0202, respectively. BSA-4 was obtained from Armour Pharmaceuticals: catalog number 0140-00, lot number B-10012. The experiment was repeated four times. Embryo development, expressed as a percentage of the total number of embryos in each treatment, was determined at 48 h. The number of embryos that developed to the hatching blastocyst stage was recorded at 72 h. Transformed data were subjected to a 2-way ANOVA. F values were determined, and when they were significant, levels of treatments were compared using Newman-Keuls multiple range test. The four lots of BSA were examined using one-dimensional polyacrylamide gel electrophoresis. In the control treatment, containing no BSA, 49% of the 2-cell embryos cultured developed to the blastocyst stage; 14% were

Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys.

Mitochondrial DNA (mtDNA) deletion mutations co‐localize with electron transport system (ETS) abnormalities in rhesus monkey skeletal muscle fibers. Using laser capture microdissection in conjunction with PCR and DNA sequence analysis, mitochondrial genomes from single sections of ETS abnormal fibers were characterized. All ETS abnormal fibers contained mtDNA deletion mutations. Deletions were large, removing 20–78% of the genome, with some to nearly all of the functional genes lost. In one‐third of the deleted genomes, the light strand origin was deleted, whereas the heavy strand origin of replication was conserved in all fibers. A majority (27/39) of the deletion mutations had direct repeat sequences at their breakpoints and most (36/39) had one breakpoint within or in close proximity to the cytochrome b gene. Several pieces of evidence support the clonality of the mtDNA deletion mutation within an ETS abnormal region of a fiber: (a) only single, smaller than wild‐type, PCR products were obtained from each ETS abnormal region; (b) the amplification of mtDNA from two regions of the same ETS abnormal fiber identified identical deletion mutations, and (c) a polymorphism was observed at nucleotide position 16103 (A and G) in the wild‐type mtDNA of one animal (sequence analysis of an ETS abnormal region revealed that mtDNA deletion mutations contained only A or G at this position). Species‐specific differences in the regions of the genomes lost as well as the presence of direct repeat sequences at the breakpoints suggest mechanistic differences in deletion mutation formation between rodents and primates.

Regulation of Hamster Embryo Development In Vitro by Amino Acids

Considerable attention has been directed towards determining carbohydrate energy substrates for supporting in vitro development of pre-implantation embryos, mostly in studies with mice (1,2). In contrast, there has been little interest in examining amino acid requirements, doubtless because no regulatory role for amino acids was found in studies with mouse embryos. Although glycine as the sole fixed-nitrogen source was able to support 8-cell mouse embryo development (3), later studies showed that a fixed-nitrogen source was not essential for development of 2-cell mouse embryos (4).

Early-onset calorie restriction conserves fiber number in aging rat skeletal muscle

The purpose of this work was to determine the effect of early‐onset calorie restriction on sarcopenia in the aging rat. Ad libitum (AL) fed animals were examined at 5, 18, 21, and 36 months of age. Calorie‐restricted (CR) rats, 40% restricted since 4 months of age, were examined at 21 and 36 months of age. By 36 months, vastus lateralis, rectus femoris and soleus muscles, from AL‐fed rats, had significant muscle mass and fiber loss, and reduced muscle cross‐sectional area. Mean fiber diameter decreased with age in the vastus lateralis and rectus femoris but not the soleus of AL‐fed rats. The number of Type I fibers significantly increased in the vastus lateralis with age. Calorie restriction did not prevent muscle mass loss with age; however, it significantly reduced muscle mass loss between 21 and 36 months of age compared with age‐ matched AL cohorts. Calorie restriction prevented fiber loss with age, and this conservation of fiber number reduced muscle mass loss with age.

Mitochondrial DNA Deletion Mutations and Sarcopenia

This manuscript summarizes our studies on mitochondrial DNA and enzymatic abnormalities that accumulate, with age, in skeletal muscle. Specific quadricep muscles, rectus femoris in the rat and vastus lateralis in the rhesus monkey, were used in these studies. These muscles exhibit considerable sarcopenia, the loss of muscle mass with age. The focal accumulation of mtDNA deletion mutations and enzymatic abnormalities in aged skeletal muscle necessitates a histologic approach in which every muscle fiber is examined for electron transport system (ETS) enzyme activity along its length. These studies demonstrate that ETS abnormalities accumulate to high levels within small regions of aged muscle fibers. Concomitant with the ETS abnormalities, we observe intrafiber atrophy and, in many cases, fiber breakage. Laser capture microdissection facilitates analysis of individual fibers from histologic sections and demonstrates a tight association between mtDNA deletion mutations and the ETS abnormalities. On the basis of these results, we propose a molecular basis for skeletal muscle fiber loss with age, a process beginning with the mtDNA deletion event and culminating with muscle fiber breakage and loss.

Calorie restriction limits the generation but not the progression of mitochondrial abnormalities in aging skeletal muscle.

The effect of early‐onset calorie restriction and aging on the accumulation of electron transport system (ETS) abnormalities was studied in rat skeletal muscle. Rectus femoris and vastus lateralis muscle fibers were analyzed for cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) enzyme activities. Fibers displaying COX negative and SDH hyper reactive (COX–/SDH++) phenotype were followed through 1000–2000 micrometers to determine the frequency and length of these abnormalities as well as the physiological impact on fiber structure. Calorie restricted rats had fewer ETS abnormal muscle fibers. The mean length of ETS abnormal regions in ad libitum rat muscle fibers was similar to calorie restricted rat muscles. ETS abnormal fibers from both diet groups exhibited intra‐fiber atrophy. A negative correlation between ETS abnormality length and fiber cross‐sectional area (CSA) ratio was observed in both ad libitum and calorie‐ restricted rats. Although calorie restriction reduced the number of ETS abnormalities, it did not affect the length or associated fiber atrophy of ETS abnormal regions once the abnormality was established. Thus, calorie restriction affects the onset but not the progression of electron transport system abnormalities, thereby, limiting a process that ultimately results in fiber breakage and fiber loss.

Muscle mass loss in Rhesus monkeys: age of onset.

Sarcopenia, the decline in skeletal muscle mass and function with age, contributes to increased frailty and decreased functional performance in the aging human population. The negative health consequences of muscle mass loss emphasize the need for development of a nonhuman primate model for the prevention or attenuation of sarcopenia. The age of onset for muscle mass loss in Rhesus macaques was determined using three datasets; (i) dual-energy X-ray absorptiometry (DXA) data from a cross-sectional study of 90 adult Rhesus monkeys; (ii) lean tissue mass and estimated skeletal muscle mass (ESM) from 727 DXA scans taken in 38 monkeys in a long-term, longitudinal aging study; and, (iii) quadriceps weights taken at necropsy from 13 male and 28 female Rhesus monkeys. These data indicate that both male and female Rhesus monkeys develop sarcopenia with age. The onset of sarcopenia is 14.1 +/- 2.8 years in females and 15.8 +/- 2.5 years in males. Muscle loss reaches 20% in males by 23.2 years of age and in females by 24.5 years of age. Furthermore, our data indicate percentage declines in ESM similar to those seen in humans with advancing age. These data support the suitability of the Rhesus monkey as a primate sarcopenia model.

Cellular adaptation contributes to calorie restriction-induced preservation of skeletal muscle in aged rhesus monkeys

We have previously shown that a 30% reduced calorie intake diet delayed the onset of muscle mass loss in adult monkeys between ~16 and ~22 years of age and prevented multiple cellular phenotypes of aging. In the present study we show the impact of long term (~17 years) calorie restriction (CR) on muscle aging in very old monkeys (27-33 yrs) compared to age-matched Control monkeys fed ad libitum, and describe these data in the context of the whole longitudinal study. Muscle mass was preserved in very old calorie restricted (CR) monkeys compared to age-matched Controls. Immunohistochemical analysis revealed an age-associated increase in the proportion of Type I fibers in the VL from Control animals that was prevented with CR. The cross sectional area (CSA) of Type II fibers was reduced in old CR animals compared to earlier time points (16-22 years of age); however, the total loss in CSA was only 15% in CR animals compared to 36% in old Controls at ~27 years of age. Atrophy was not detected in Type I fibers from either group. Notably, Type I fiber CSA was ~1.6 fold greater in VL from CR animals compared to Control animals at ~27 years of age. The frequency of VL muscle fibers with defects in mitochondrial electron transport system enzymes (ETS(ab)), the absence of cytochrome c oxidase and hyper-reactive succinate dehydrogenase, were identical between Control and CR. We describe changes in ETS(ab) fiber CSA and determined that CR fibers respond differently to the challenge of mitochondrial deficiency. Fiber counts of intact rectus femoris muscles revealed that muscle fiber density was preserved in old CR animals. We suggest that muscle fibers from CR animals are better poised to endure and adapt to changes in muscle mass than those of Control animals.

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.

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.