Mika Oe

Differential expression of the skeletal muscle proteome in grazed cattle.

The objective of this study was to investigate the differences in the muscle proteome of grass-fed and grain-fed cattle. Eight Japanese Black Cattle 10 mo of age were separated randomly into 2 groups: 1) grazing (grass-fed) and 2) concentrate (grain-fed) groups. All cattle were first housed individually in a stall barn and fed a combination of concentrate ad libitum and Italian ryegrass hay until 21 mo of age. After this control period, the 4 grass-fed cattle were placed on outdoor pasture, whereas the other 4 grain-fed cattle continued on the concentrate diet. The cattle were slaughtered at 27 mo of age, and tissues from the semitendinosus muscle were obtained for use in proteome analysis. Differential expression of muscle proteins in the 2 groups was carried out using 2-dimensional gel electrophoresis (2DE) and Western blot analyses, with subsequent mass spectrometry. Approximately 200 individual protein spots were detected and compared in each group using 2DE, of which 20 and 9 spots, respectively, showed differences in the spot intensity for the sarcoplasmic fraction and myofibrillar fraction. In the grazing group, the relative intensity of spots was significantly greater for adenylate kinase 1 and myoglobin in the sarcoplasmic fraction, and for slow-twitch myosin light chain 2 in the myofibrillar fraction (P < 0.05), than the concentrate group. The relative spot intensity of several glycolytic enzymes was significantly greater in the grazing group, such as beta-enolase 3, fructose-1,6-bisphosphate aldolase A, triosephosphate isomerase, and heat shock 27 kDa protein (P < 0.05). Moreover, significantly greater slow twitch of troponin T, troponin I, and myosin heavy chain of semitendinosus muscle was detected in the grazing group than in the concentrate group using Western blot analysis (P < 0.05). Several previous reports have described that the slow-twitch muscle contents affect elements of nutrition, flavor, and food texture of meat. This study revealed muscle fiber type conversion to slow-twitch tissues from fast-twitch tissues occurring with change in the energy metabolic enzyme when cattle were grazed in the latter fattening period. Although analyses of the influence on elements of nutrition, flavor, and food texture were not done for this study, these results show that slow-twitch converted muscle resulting from the grazing of cattle might modify several meat characteristics.

Profiling of differentially expressed microRNA and the bioinformatic target gene analyses in bovine fast- and slow-type muscles by massively parallel sequencing.

MicroRNA (miRNA) are highly conserved, noncoding small RNA involved in post-transcriptional gene regulation in a variety of biological processes. To elucidate roles of miRNA in bovine muscle type specification and maintenance, we sought to determine differentially expressed miRNA between semitendinosus (STD) and masseter (MS) muscles from 3 Japanese black cattle by massively parallel sequencing. Differential gene expression of myosin heavy chain (MyHC) isoforms confirmed that STD and MS were MyHC-2x- and MyHC-1-abundant muscles, respectively. In total, 192 known miRNA and 20 potential new bovine miRNA were obtained from the sequencing. The differentially expressed miRNA with more than 2-fold difference in each muscle were identified. In particular, miR-196a and miR-885 were exclusively expressed in STD muscle, which was validated by quantitative reverse transcription-PCR (P=0.045 and P<0.001, respectively), whereas a slow type-directing miR-208b was highly expressed in MS compared with STD (false discovery rate<0.05). In addition, 16 potential novel miRNA were mapped and confirmed for their precursor structures by computational analyses. The results of functional annotation combined with in silico target analysis showed that the predicted target genes of miR-196a/b and miR-885 enriched gene ontology (GO) terms related to skeletal system development and regulation of transcription, respectively. Moreover, GO terms enriched from predicted targets miRNA suggested that STD-abundant- and MS-abundant-miRNA were associated with embryonic body planning and organ/tissue pattern formation, respectively. The present results revealed that the differentially expressed miRNA between the STD and MS muscles may play key roles to determine muscle type-specific tissue formation and maintenance in cattle thorough attenuating putative target genes involved in different developmental events.

Postmortem changes in bovine troponin T isoforms on two-dimensional electrophoretic gel analyzed using mass spectrometry and western blotting: The limited fragmentation into basic polypeptides

To comprehend postmortem changes in troponin T (TnT), whole beef proteins were developed on a two-dimensional electrophoretic gel. Multiple TnT-related spots were identified by both western blotting and MALDI-TOF MS utilizing bovine TnT isoform mRNA sequences. More than 10 TnT fast-type isoform spots (pI 5.7-9.6<) and the two slow-type isoform spots (pI 5.6-5.7) were observed at slaughter. All the isoforms were degraded exclusively into basic spots (pI 9.6<) at day 14 postmortem. Some TnT-related phosphorylated spots present at slaughter had disappeared by day 14, suggesting that the phosphorylated N-terminal region was cut off during beef aging. The intact isoforms and the fragments were identified by the MS with sequence coverage of 20.8-62.7%, and two of the fragments included the cutting site peptide of a conventional 30kDa or of a slow TnT-derived fragment. These results revealed that all of the TnT isoforms are cut exclusively in the glutamic acid-rich amino-terminal region during postmortem aging.

Difference in postmortem degradation pattern among troponin T isoforms expressed in bovine longissimus, diaphragm, and masseter muscles.

The postmortem degradation of troponin T (TnT) in bovine longissimus (LT), diaphragm (DP), and masseter (MS) was analyzed. A 28.3kDa (conventional 30kDa) fragment of fast-type TnT isoforms showed the highest content in both LT and DP, where a 35.4kDa isoform had the highest expression among the other fast isoforms. Meanwhile, a 26.0kDa fragment was found to be the most highly produced among the fast TnT fragments in MS, where the expression of 36.5 and 32.8kDa isoforms was higher than that of 35.4 and 34.8kDa isoforms. Thus, the compositions of both the intact TnT isoform proteins and the postmortem fragments differed among the muscles examined, indicating that each TnT isoform degrades into a specific fragment in each muscle. Among the muscles, the LT muscle showed a high extent of TnT degradation and the highest expression of fast TnT isoforms containing a taste-related peptide sequence.

Muscle type specific expression of tropomyosin isoforms in bovine skeletal muscles

Nucleotide sequences encoding an entire coding region for bovine tropomyosin (TPM) isoforms were determined. Three TPM isoforms, TPM1, TPM2 and TPM3, were expressed in bovine skeletal muscles, and exhibited a 93.3%, 99.6% and 100% amino acid homology to the human sequence, respectively. Based on the sequences, the composition of TPM isoforms was analyzed on cDNA and protein levels from five physiologically different muscles (masseter, diaphragm, psoas major, longissimus thoracis and semitendinosus) using RT-PCR and proteome analyses. Although the content of TPM2 was constantly about 50% of the total TPM in all muscles, the contents of TPM1 and TPM3 were different in muscles according to their function in muscle contraction. In masseter, the content of TPM3 cDNA was about 50% and higher than that of other muscles. In longissimus thoracis and semitendinosus, the contents of TPM1 cDNA were 29.6% and 31.7%, respectively, which were comparatively higher than that of other muscles. The result suggests that the TPM dimer consists of the TPM2 subunit regularly and TPM1 or TPM3 depending on whether the muscle is fast or slow type, respectively.

Effect of phase limited inhibition of MyoD expression on the terminal differentiation of bovine myoblasts: No alteration of Myf5 or myogenin expression

To investigate the roles played by MyoD in the terminal differentiation of satellite cell‐derived myoblasts, the effect of antisense inhibition of MyoD expression was examined in bovine adult myoblast culture, in which inhibition treatment was limited to the terminal differentiation phase. MyoD antisense oligonucleotide DNA (AS‐mD) suppressed the formation of multinucleated myotubes in the cell culture. Myotube formation was suppressed even when AS‐mD treatment was limited to the period preceding the onset of myotube formation. Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis revealed that treatment with AS‐mD suppressed the expression of myosin heavy chain embryonic isoform and troponin T isoforms at 4 days after the induction of differentiation. AS‐mD also suppressed the expression of MRF4, but did not alter the expression of either Myf5 or myogenin, in contrast to previous results using mouse cells possessing MyoD(–/–) genetic background. These findings suggest that MyoD controls myogenesis but not Myf5 or myogenin mRNA expression during the terminal differentiation phase. Furthermore, among the α4, α5, α6, and α7 integrins, α4, α5, and α7 integrin expression was suppressed by AS‐mD treatment, in parallel with the suppression of myotube formation, which suggests that MyoD controls myotube formation by regulating the expression of α4, α5, and α7 integrins.

The importance of subfragment 2 and C-terminus of myosin heavy chain for thick filament assembly in skeletal muscle cells.

In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 (S2) + light meromyosin (LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C-terminus on thick filament assembly. C-terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C-terminus of LMM.

A non-destructive method to monitor changes in a troponin T peptide in beef drip with a monoclonal antibody.

To simplify the monitoring of postmortem beef aging, we established a system to detect a troponin T (TnT) peptide fragment in bovine muscle drip (natural exudates) with an original monoclonal antibody. The antibody was raised against a synthetic peptide APPPPAEVPEVHEEVH corresponding to the N-terminal region of bovine fast-type TnT. In a competitive enzyme-linked immunosorbent assay (ELISA), our antibody detected the standard peptide dose-dependently. According to the monitoring examination with a competitive ELISA during 22days postmortem, the concentration of the peptide in both the drip and trichloroacetic acid extracts from the longissimus muscle (n=4) significantly increased in parallel, up to 10nmol/ml and 16.4nmol/g at day 14 postmortem, respectively. These events were accompanied by an increase in the conventional 30kDa fragment in western blot analysis and a decrease in the Warner-Bratzler shear force value of the beef from 5.0 to 2.4N/cm(2). The peptide detection system using drips with the antibody has advantages applicable to a non-destructive, simple, quick, and on-site monitoring method, such as immunochromatography.

Comparison of liver mitochondrial proteins derived from newborn cloned calves and from cloned adult cattle by two-dimensional differential gel electrophoresis.

Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for such a developmental deficiency. However, detailed knowledge of protein expression during somatic cell nuclear transfer (SCNT) in cattle is lacking. In the present study, variations in mitochondrial protein levels between SCNT‐derived and control cattle, and from calves derived by artificial insemination were investigated. Mitochondrial fractions were prepared from frozen liver samples and subjected to two‐dimensional (2‐D) fluorescence differential gel electrophoresis (DIGE) using CyDye™ dyes. Protein expression changes were confirmed with a volume ratio greater than 2.0 (P < 0.05). 2D‐DIGE analysis revealed differential expression of three proteins for SCNT cattle (n = 4) and seven proteins for SCNT calves (n = 6) compared to controls (P < 0.05). Different protein patterning was observed among SCNT animals even if animals were generated from the same donor cell source. No differences were detected in two of the SCNT cattle. Moreover, there was no novel protein identified in any of the SCNT cattle or calves. In conclusion, variation in mitochondrial protein expression concentrations was observed in non‐viable, neonatal SCNT calves and among SCNT individuals. This result implicates mitochondrial‐related gene expression in early developmental loss of SCNT embryos. Comparative proteomic analysis represents an important tool for further studies on SCNT animals. Mol. Reprod. Dev. 78:263–273, 2011.

Dynamics of protein secretion during adipocyte differentiation

The major functions of adipocytes include both lipid storage and the production of secretory factors. However, the type of proteins released from mouse 3T3‐L1 cells during adipocyte differentiation remains poorly understood. We examined the dynamics of secreted proteins during adipocyte differentiation using mass spectrometry (MS) combined with an iTRAQ® labeling method that enables the simultaneous analysis of relative protein expression levels. A total of 215 proteins were identified and quantified from approximately 10 000 MS/MS spectra. Of these, approximately 38% were categorized as secreted proteins based on gene ontology classification. Adipokine secretion levels were increased with the progression of differentiation. By contrast, levels of fibril collagen components, such as subunits of type I and III collagens, were decreased during differentiation. Basement membrane components attained their peak levels at day 4 when small lipid droplets accumulated in differentiated 3T3‐L1 cells. Simultaneously, peak levels of collagen microfibril components that comprise type V and VI collagen subunits were also observed. Our data demonstrated that extracellular matrix components were predominantly released during the early and middle stages of adipocyte differentiation, with a subsequent increase in the secretion of adipokines. This suggests that 3T3‐L1 cells secrete adipokines after their ECM is constructed during adipocyte differentiation.