The effect of postmortem pH decline rate on mitochondrial apoptosis and tenderness of bovine skeletal muscle during aging

This study aimed to investigate the effect of postmortem pH decline rate on mitochondrial dysfunction-induced apoptosis and bovine muscle tenderness during aging. Protein denaturation, reactive oxygen species (ROS) levels, mitochondrial apoptosis factors, and shear force were assessed in bovine muscles with different pH decline rates. The results showed that compared to the slow group, fast pH decline in bovine muscle significantly increased sarcoplasmic protein denaturation at 6–12 h and mitochondrial ROS levels at 6–24 h (P<0.05), and in turn significantly accentuated mitochondrial dysfunction and cytochrome c oxidation levels (P<0.05), resulting in caspase-3 activation, apoptosis, and reduced muscle shear force (P<0.05). These results demonstrated that the fast postmortem pH decline enhanced mitochondrial apoptosis and bovine muscle tenderization by inducing ROS accumulation during aging.

The circular RNA circCPE regulates myoblast development by sponging miR-138

Background Skeletal muscle development, a long-term and complex process, is controlled by a set of the myogenic genes. Circular RNAs (circRNAs), a class of noncoding RNA, have been shown to regulate various biological processes. Recent studies indicate circRNAs may be involved in myogenesis, but the role and regulatory mechanism of circRNAs in myogenesis is largely unknown. In the present study, circCPE was firstly found to promote the bovine myoblast proliferation and inhibit cell apoptosis and differentiation by influencing the expression of FOXC1 in a miR138-mediated manner. And in vivo experiments revealed that overexpression of circCPE attenuates skeletal muscle regeneration. Results We identified a novel circular RNA circCPE by analyzing circRNAs sequencing data of bovine muscle tissue. Sequencing verification, RNase R treatment and Actinomycin D treatment confirmed the circular nature of circCPE in bovine muscle. Functional assays showed that overexpression of circCPE could inhibit bovine myoblast apoptosis and differentiation, as well as facilitate cell proliferation. Moreover, in vivo experiments revealed that overexpression of circCPE attenuates skeletal muscle regeneration. In consideration of circRNA action as miRNAs sponge, we found that circCPE harbors miR-138 binding sites and absorbed miR-138. Mechanistically, the rescue experiments showed that the overexpression of circCPE can counteract the inhibitory effect of miR-138 on the cell proliferation and the accelerated effects on the differentiation and apoptosis. Subsequently, we found that circCPE sequester the inhibitory effect of miR-138 on FOXC1 so as to involve in myogenesis. Conclusions Collectively, we constructed a novel circCPE/miR-138/FOXC1 regulatory network in bovine myogenesis, which further provide stronger evidence that circRNA involved in muscle development acting as miRNA sponge.

MSTN mutant promoted bovine muscle satellite cell proliferation by regulating the binding of SMAD2/SMAD3 with CDKN1C

Background: Myostatin (MSTN), also known as growth/differentiation factor 8, mostly expressed in skeletal muscle and plays negative roles in regulation of muscle development. Previous studies had proved that MSTN have important effect on cell proliferation. Therefore we aimed to investigate the mechanism of MSTN in regulating the proliferation of bovine muscle satellite cells (MSCs).Methods: Bovine MSCs of MSTN mutant (MT) and wild type (WT) were obtained, we detected the cell proliferation and cell cycle by EdU proliferation assay and Flow cytometry. Then we detected the expression of genes associated with cell cycle by Real-time PCR and Western blotting . RNA-seq and Chromatin immunoprecipitation (ChIP)assay were performed to research the mechanism of MSTN in regulating the cell proliferation. Results: In this study, we found that MSTN mutant promoted the proliferation of MSCs. The expression of CyclinA, CyclinD and CyclinE were all increased after MSTN mutant, while the expression of CDKN1C (P57), CDKN2A, CDKN2C and CDKN2D were down-regulated, which were consistent with the promotion of cell proliferation. Among these genes, CDKN1C(P57) down-regulated most significantly. RNA-seq results showed that MSTN mutant affected the SMAD binding, so we performed ChIP-qPCR and demonstrated that the SMAD2/SMAD3 transcription factor combined with the promoter of CDKN1C thus to increase the expression of CDKN1C, this demonstrating that MSTN regulated the expression of CDKN1C through SMAD2/SMAD3 complex. Finally, overexpression of SMAD3 in wild type cells increased the expression of CDKN1C, further suggested that SMAD3 regulated the expression of CDKN1C. Conclusion: MSTN mutant down-regulated the expression of SMAD2/SMAD3, then reduced the promotion of SMAD2/SMAD3 to the expression of CDKN1C, thus to inhibit the expression of CDKN1C, then promoting the cell cycle.

Feasibility of noninvasive thermometry in hyperthermia regime using harmonics generated by nonlinear ultrasound wave propagation

Hyperthermia is a cancer treatment modality that could be delivered as a stand-alone treatment or in conjunction with chemotherapy or radiation therapy. Noninvasive and real-time temperature monitoring of the heated tissue improves the efficacy and safety of the treatment. Ultrasound-based thermometry requires a temperature-sensitive acoustic parameter that can be used to estimate the temperature by tracking changes in that parameter during heating. This dissertation describes the experiments and simulations performed to obtain the temperature dependence of acoustic harmonics generated by nonlinear ultrasound propagation in several media including: water, an attenuating tissue-mimicking liquid, ex vivo bovine muscle tissues, and tissue-mimicking gel phantoms. The mechanisms of action of harmonic generation in water and in the attenuating liquid, made by a mixture of 90% glycerol and 10% water (by volume), as a function of temperature at various frequencies have been investigated using a temperature dependent Khokhlov–Zabolotskaya–Kuznetsov (KZK) nonlinear acoustic wave propagation model. The simulation results were compared with and validated by measurements. In water, the harmonic amplitudes decrease with increasing the temperature at low frequencies (1 and 3.3 MHz), while the opposite temperature dependence was observed at higher frequencies (13 and 20 MHz). The harmonic generation significantly increased with temperature in the tissue-mimicking liquid at both frequencies of 5 and 13 MHz. The temperature dependence of harmonics in tissue-mimicking gel phantoms and ex vivo bovine muscle tissues were measured using a commercial high-frequency ultrasound imaging system, and a new noninvasive ultrasound-based thermometry has been developed that is based on the backscattered energy of the harmonics. The sensitivity of this new thermometry technique to medium’s motion was studied and compared with the conventional echo-shift thermometry technique. Based on this study, it is suggested that noninvasive temperature estimation is feasible using acoustic harmonics with lower sensitivity to motion artifacts compared to the conventional echo-shift technique.

Feasibility of noninvasive thermometry in hyperthermia regime using harmonics generated by nonlinear ultrasound wave propagation

Hyperthermia is a cancer treatment modality that could be delivered as a stand-alone treatment or in conjunction with chemotherapy or radiation therapy. Noninvasive and real-time temperature monitoring of the heated tissue improves the efficacy and safety of the treatment. Ultrasound-based thermometry requires a temperature-sensitive acoustic parameter that can be used to estimate the temperature by tracking changes in that parameter during heating. This dissertation describes the experiments and simulations performed to obtain the temperature dependence of acoustic harmonics generated by nonlinear ultrasound propagation in several media including: water, an attenuating tissue-mimicking liquid, ex vivo bovine muscle tissues, and tissue-mimicking gel phantoms. The mechanisms of action of harmonic generation in water and in the attenuating liquid, made by a mixture of 90% glycerol and 10% water (by volume), as a function of temperature at various frequencies have been investigated using a temperature dependent Khokhlov–Zabolotskaya–Kuznetsov (KZK) nonlinear acoustic wave propagation model. The simulation results were compared with and validated by measurements. In water, the harmonic amplitudes decrease with increasing the temperature at low frequencies (1 and 3.3 MHz), while the opposite temperature dependence was observed at higher frequencies (13 and 20 MHz). The harmonic generation significantly increased with temperature in the tissue-mimicking liquid at both frequencies of 5 and 13 MHz. The temperature dependence of harmonics in tissue-mimicking gel phantoms and ex vivo bovine muscle tissues were measured using a commercial high-frequency ultrasound imaging system, and a new noninvasive ultrasound-based thermometry has been developed that is based on the backscattered energy of the harmonics. The sensitivity of this new thermometry technique to medium’s motion was studied and compared with the conventional echo-shift thermometry technique. Based on this study, it is suggested that noninvasive temperature estimation is feasible using acoustic harmonics with lower sensitivity to motion artifacts compared to the conventional echo-shift technique.

Application of shearwave™ elastography to evaluate heat-induced changes in the young's modulus of fresh bovine muscle: A preliminary study

PurposeThe purpose of this study was to investigate the variation in the Young’s modulus (E) of bovine muscle samples as a function of temperature change generated by therapeutic ultrasound using Shearwave™ Elastography.MethodsInitially, the bovine muscle was heated via therapeutic ultrasound with a frequency of 3 MHz, nominal intensity of 2 W·cm-2, and application time of 2 min. Immediately following cessation of therapeutic irradiation, an E image was recorded and the stiffness was measured in circular area positioned at six depths (from 0.4 to 2.9 cm) in the center of the region of interest. Next, an E image was recorded every minute for the first 5 min. Over the next 30 min, an image was recorded every 5 min. Finally, an image was acquired 60 min after cessation of therapeutic irradiation. In the second test, the same experimental procedure was performed 60 min later with the physiotherapy equipment configured with a 10-min application time. Finally, during the ultrasonic irradiation of a new bovine muscle sample, the physiotherapeutic transducer was applied in a circular motion and with an angular velocity of 3.6 ± 0.3 rad·s-1.ResultsIn the first test, the bovine muscle E decreased from 212.2 ± 32.8 to 80.1 ± 13.8 kPa at 0.4 cm-depth, as the temperature increased from 18.2 to 44.9 °C. This effect was reversed when the temperature decreased. In the second test, denaturation and cell death occurred, so an artifact appeared in the elastographic image and the Shearwave™ Elastography did not capture the E from the depth of 1.9 cm.ConclusionWe confirmed that it is possible to use Shearwave™ Elastography to investigate heat-induced changes in the elastic modulus of biological tissue.