BackgroundOutflow tract (OFT) septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. This study, therefore, aimed to investigate the septation of the aortic sac and the OFT in the early embryonic human heart.

MethodsSerial sections of 27 human embryonic hearts from Carnegie stage (CS) 10 to CS19 were immunohistochemically stained with antibodies against α-smooth muscle actin (α-SMA) and myosin heavy chain.

ResultsAt CS10-CS11, the OFT wall was an exclusively myocardial structure that was continuous with the aortic sac at the margin of the pericardial cavity. From CS13 onward, the OFT was divided into nonmyocardial and myocardial portions. The cushion formed gradually, and its distal border with the OFT myocardium was consistently maintained. The aortic sac between the fourth and sixth aortic arch arteries was degenerated. At CS16, the α-SMA-positive aortopulmonary septum formed and fused with the two OFT cushions, thus septating the nonmyocardial portion of the OFT into two arteries. At this stage, the cushions were not fused. At CS19, the bilateral cushions were fused to septate the myocardial portion of the OFT.

ConclusionsData suggest that the OFT cushion is formed before the aortopulmonary septum is formed. Thus, the OFT cushion is not derived from the aortopulmonary septum. In addition, the nonmyocardial part of the OFT is septated into the aorta and pulmonary trunk by the aortopulmonary septum, while the main part of the cushion fuses and septates the myocardial portion of the OFT.

Actins ; metabolism ; Alkaline Phosphatase ; metabolism ; Aorta ; embryology ; Heart ; embryology ; Heart Valves ; embryology ; Humans ; Immunohistochemistry ; Myosin Heavy Chains ; metabolism

AIMTo detect effect of the different frequency of chronic electrical stimulation (CES) on myofibrillar isoform, myosin heavy chain (MHC) and metabolic enzyme activities.

METHODSThe histochemical method and SDS-polyacrylamide gel electrophoresis were respectively employed.

RESULTS(1)There were a significant increase in I myo-fibrillar isoform and I MHC isoform and decrease in II B myofibrillar isoform and II B MHC isoforms in the chronic low frequency electrical stimulation (CLFES) 10 Hz and 20 Hz groups, but opposite results were found in the chronic high frequency electrical stimulation (CHFES) 50 Hz and 100 Hz groups. (2) There were a significant increase in the aerobic-oxidative enzyme activities and capacity, and a concomitant significant drop in glycolysis enzyme activities in CLFES groups, but opposite results were found in CHFES 50 Hz and 100 Hz groups.

CONCLUSIONIt was suggested that there was a significant dependent relation between chronic electrical stimulation frequency and myofibrilla isoforms, myosin heavy chain (MHC) and metabolic enzyme activities.

Adaptation, Physiological ; Animals ; Diaphragm ; enzymology ; metabolism ; physiology ; Electric Stimulation ; Muscle Contraction ; Myosin Heavy Chains ; metabolism ; Nonmuscle Myosin Type IIB ; metabolism ; Protein Isoforms ; Rabbits

OBJECTIVE: To investigate the expression of myosin heavy chain (MHC) in normal laryngeal muscle and the difference between the adductor and abductor. METHOD: Seven patients with total laryngectomy were enrolled in this study. The adductor muscles were acquired from the lateral cricoarytenoid (LCA) muscle and the abductor muscles were acquired from the posterior cricoarytenoid (PCA) muscle. The expression of myosin heavy chain were detected with fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining respectively. RESULT: (1) MHC-II b was expressed in laryngeal muscles at mRNA levels, and not expressed at the protein level; (2) At both mRNA level and protein level, the expression of MHC-I was higher in the PCA muscles than in the LCA muscles while MHC-II level was higher in the LCA muscles than in the PCA muscles. CONCLUSION (1) MHC-II b protein was not expressed in human laryngeal muscles; (2) Phenotypic differences were significant in laryngeal adductor and abductor muscles based on their different functions. PCA contained larger percentage of MHC-I fibers, while LCA contained more MHC-II fibers.
Aged ; Female ; Humans ; Laryngeal Muscles ; chemistry ; metabolism ; Male ; Middle Aged ; Myosin Heavy Chains ; genetics ; metabolism ; Phenotype ; Protein Isoforms

OBJECTIVE: To investigate the expression of Myosin VI and Disabled-2 (Dab2) in the cochlea of mice at different ages. METHOD: Forty KM mice were divided into four groups according to age, named as postnatal 2 week (P2w), P5w, P9w, P16month. The localization of protein in the basilar membrane of mice cochlea was detected by immunofluorescence staining and laser scanning confocal microscope (LSCM). The mRNA expression level of protein in cochlear at different ages was evaluated by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Statistical analysis was performed by the SPSS18.0 software. RESULT: Myosin VI and Disabled-2 protein mainly expressed at the apical cytoplasm of hair cells. As for the inner hair cell, Dab2 labeling was abundant especially at the cuticular plate and nearby. Comparing four immunofluorescence staining images of Myosin VI, we found the fluorescence intensity of P2w and P16m were weaker than that of P5w and P9w. After setting P9w as the control group, qRT-PCR revealed that the mRNA expression of MyosinVI and Dab2 in P2w was less than that in the control group (P < 0.01), while no significant difference was found between P5w and the control group, nor between P16m and the control group (P > 0.05). CONCLUSION Myosin VI and Dab2, two proteins which regulated the clathrin-mediated endocytosis, expressed at hair cells of mice cochlea. In the inner hair cell, this process of endocytosis may be more efficient at the cuticular plate and nearby. The expression level of protein may change in different ages, and this probably leads to a difference of CME, it also may cause a defect of inner hair cells function.
Adaptor Proteins, Vesicular Transport ; metabolism ; Aging ; Animals ; Cochlea ; metabolism ; Endocytosis ; Hair Cells, Auditory ; metabolism ; Hair Cells, Auditory, Inner ; metabolism ; Mice ; Microscopy, Confocal ; Myosin Heavy Chains ; metabolism

OBJECTIVETo explore the effect of Salvianolate on myosin heavy chain (MHC) in cardiomyocytes of congestive heart failure (CHF) rats.

METHODSSixty male SD rats were divided into 6 groups according to random digit table, i.e., the normal control group (NCG), the model group, the Captopril group (CAG), the low dose Salvianolate group (LSG), the high dose Salvianolate group (HSG), the Captopril and high dose Salvianolate group (CSG), 10 in each group. CHF rat model was established with peritoneal injection of adriamycin in all rats except those in the NCG. Equal volume of normal saline was peritoneally injected to rats in the NCG, once per week for 6 successive weeks. Corresponding medication was started from the 5th week of injecting adriamycin. Rats in the CAG were administered with Captopril solution at the daily dose of 10 mg/kg by gastrogavage. Rats in the LSG and the HSG were administered with Salvianolate solution at the daily dose of 24.219 mg/kg and 48.438 mg/kg respectively by gastrogavage. Salvianolate was dissolved in 2 mL 5% glucose solution and administered by peritoneal injection. Rats in the CSG were peritoneally injected with high dose Salvianolate solution and administered with Captopril solution by gastrogavage. Two mL normal saline was peritoneally injected to rats in the model group, once per day for 8 successive weeks. Eight weeks later, the cardiac function and myocardial hypertrophy indices were detected by biological signal collecting and processing system. mRNA expression levels of alpha-MHC and beta-MHC in cardiac muscle were detected by fluorescence quantitative PCR. Expressions of protein kinase C (PKC) in cardiac muscle were detected by Western blot.

RESULTSCompared with the normal control group, heart mass index (HMI) and left ventricular mass index (LVMI) obviously increased in the model group (P < 0.01). Compared with the model group, HMI and LVMI decreased in HSG, CAG, and CSG groups (P < 0.05, P < 0.01). It was more obviously lowered in the CSG group than in the CAG group (P < 0.05). Compared with the NCG, the mRNA expression level of alpha-MHC in cardiac muscle decreased, the mRNA expression level of p-MHC and the expression of PKC in cardiac muscle increased in the model group (P < 0.01). Compared with the model group, the mRNA expression level of alpha-MHC in cardiac muscle was increased, and the mRNA expression level of beta-MHC and the expression of PKC in cardiac muscle were decreased in HSG, CAG, and CSG groups (P < 0.05, P < 0.01). There was statistical difference between the CSG group and the CAG group (P < 0.05).

CONCLUSIONSSalvianolate could up-regulate the mRNA expression level of alpha-MHC, and down-regulate the mRNA expression level of beta-MHC in cardiac muscle. Its mechanism might be related to decreasing the expression of PKC.

Animals ; Captopril ; Doxorubicin ; Drugs, Chinese Herbal ; Heart Failure ; metabolism ; Male ; Myocardium ; Myocytes, Cardiac ; drug effects ; metabolism ; Myosin Heavy Chains ; metabolism ; Plant Extracts ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the role of miRNA-199a on cardiac hypertrophy.

METHODS(1) Male Sprague-Dawley rats were subjected to pressure overload induced by abdominal aortic constriction (AAC, n = 6) and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the change of microRNAs (miRNAs). (2) Neonatal rat ventricular myocytes were isolated from 2-day old Sprague-Dawley rats. The myocytes were divided into two groups: adenovirus miRNA-199a (Ad-miRNA-199a) or adenovirus vector (Ad-vector). They were transfected in cardiomyocytes for 48 h using Lipofectamine 2000. qRT-PCR was used to detect the change of myocardial hypertrophy markers α-myosin heavy chain (αMHC, myh6), β-myosin heavy chain (βMHC, myh7) and atrial natriuretic peptide (ANP, Nppa). Software Axio Vision was used to detect the change of cardiomyocytes surface areas. (3) Neonatal rat ventricular myocytes were divided into two groups: antisense oligonucleotide-miRNA-199a (As-miRNA-199a) and scramble oligonucleotides (As-ctl). They were transfected to cardiomyocytes respectively for 48 h. qRT-PCR was used to detect the change of miRNA-199a. (4) Neonatal rat ventricular myocytes were divided into four groups: A: control (ctl), B: phenylephrine (PE), C: PE + As-ctl, D: PE + As-miRNA-199a. qRT-PCR was used to detect the change of myh6, myh7 and Nppa. Software Axio Vision was used to detect the change of cardiomyocytes surface areas.

RESULTS(1) qRT-PCR results showed that miRNA-1, miRNA-133, miRNA-181a and miRNA-499 were significantly decreased, while the miRNA-199a was significantly increased at 1 week post AAC hearts compared with the sham group. (2) qRT-PCR results showed that miRNA-199a and myh7 were increased and myh6 was decreased significantly in Ad-miRNA-199a group compared with Ad-vector group. The cardiomyocytes surface area was increased in Ad-miRNA-199a group detected by immunofluorescence. (3) qRT-PCR results showed that miRNA-199a was significantly decreased in As-miRNA-199a group compared with Ad-vector group. (4) The Nppa and myh7 were significantly increased and myh6 was decreased in cardiomyocytes stimulated by PE for 48 h. The cardiomyocytes surface area determined by immunofluorescence was increased in PE + As-miRNA-199a groups compared with PE + As-ctl groups.

CONCLUSIONmiRNA-199a may play a regulatory role in cardiac hypertrophy.

Animals ; Animals, Newborn ; Atrial Natriuretic Factor ; metabolism ; Cardiomegaly ; metabolism ; pathology ; Male ; MicroRNAs ; Myosin Heavy Chains ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

Activities of 58 miRNAs for BHK21, HEK293, and Vero cell lines were screened using the high-throughput miRNA activity profiling method. miR-206 activity was found specifically high in BHK21. Considering miR-206 was recognized as a muscle-specific miRNA, we further detected the expression and activity level of miR-206 in BHK21 cells, with myoblast cells C2C12 as positive control and HEK293 cells as negative control. Then, we induced BHK21 by culturing with medium containing 2% horse serum (HS) and tested expression level of slow skeletal myosin heavy chain (MHC), activity and expression levels of miR-206, and expression level of Connexin43 (Cx43) which was reported negatively regulated by miR-206. Results demonstrated that activity and expression levels of miR-206 were both higher in BHK21 cells than in C2C12 cells. After induction of HS, MHC expression level was increased in BHK21 cells. The activity and expression levels of miR-206 were further enhanced. The Cx43 expression level was decreased. These results suggested that BHK21 had the characters of myoblast cells. In conclusion, we firstly discovered that miR-206 activity was specifically high in BHK21 cells, suggesting that BHK21 cells were derived from interstitial cells other than parenchyma cells of kidney from miRNA point of view. Our study also indicated that BHK21 cells were able to be used as models in vitro for research of miR-206 function.
Animals ; Cell Line ; Cercopithecus aethiops ; Connexin 43 ; metabolism ; Cricetinae ; Gene Expression Regulation ; HEK293 Cells ; Humans ; MicroRNAs ; genetics ; Myosin Heavy Chains ; metabolism ; Vero Cells

OBJECTIVETo investigate the influence of mechanical stretch on the expression of myosin heavy chain (MHC) mRNA in cardiomyocyte subjected to ischemia and hypoxia.

METHODSMechanical stretch model of in vitro cultured cardiomyocyte was established for the study. The cells were processed by non-sugar hypoxic stimuli to simulate postburn ischemic and/or hypoxic injuries. The cells were then divided into normal control (N), 10% stretch (S), ischemic and hypoxic culture (IHC), 10% stretch with ischemia and hypoxia (SIHC) groups. The changes in MHC mRNA expression were observed at 1, 3, 6 and 12 post treatment hours (PTHs) by RT-PCR and were statistically analyzed with gel image analysis software.

RESULTSThe expression of both alpha and beta MHC mRNA increased in 10% stretch group, especially of beta MHC mRNA (P < 0.01). The transformation of alpha MHC mRNA to beta MHC mRNA was accelerated in IHC group, and alpha MHC mRNA expression was decreased at 12 PTH. The expression of beta MHC mRNA was increased after ischemia and hypoxia, peaked at 6 PTH, and decreased thereafter (P < 0.05). The transformation of alpha MHC mRNA to beta MHC mRNA was more obvious in SIHC group, and which was intensified along with the elapse of stimulation time. The expression of both alpha and beta MHC mRNA were down-regulated at 12 PTH (P < 0.05).

CONCLUSIONDown regulation of MHC mRNA expression by ischemia and hypoxia could be aggravated by mechanical stretch, indicating that mechanical stretch might be a possible cause for cardiac dysfunction.

Animals ; Cell Hypoxia ; Cells, Cultured ; Myocardial Ischemia ; metabolism ; Myocytes, Cardiac ; metabolism ; Myosin Heavy Chains ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo isolate human adipose tissue-derived stromal cells and study the potential of osteogenic differentiation after inductive culture.

METHODSLiposuction human adipose tissues were minced and digested with collagenase type I. The obtained stromal cells were plated in BGJb medium as primary culture for ten days. The second passage cells were harvested and plated in DMEM/F12 medium supplemented with 10% FBS, 5% horse serum and 50 micromol/L hydrocortisone for myogenic induction culture. The cell-anchored slips were removed and fixed in 4% formaldehydam polymerisatum. Toluidine blue, Mallory's phosphotungstic hematoxylin staining and monoclonal antibody to human skeletal muscle myosin heavy chain immunocytochemical methods were used to assay the differentiation of cells.

RESULTSIt was observed that the size and shape of induced cells were much different from those of non-induced cells. Toluidine blue, Mallory's phosphotungstic hematoxylin staining demonstrated there were many basophilic striations within cytoplasm and multinucleated myotubes were formed. Immunocytochemical stain indicated that characterastic skeletal myosin heavy chain was positive in myogenic induced cells.

CONCLUSIONIt seems that human adipose tissue represents an abundant reservoir of adult stem cells that have multi-germline potential to differentiate into myoblasts. Adipose tissue derived stromal cells will be another alternative source for cell-based tissue engineering in skeletal muscle reconstruction.

Adipose Tissue ; cytology ; Adult Stem Cells ; cytology ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Culture Media ; Humans ; Myoblasts ; cytology ; Myosin Heavy Chains ; metabolism ; Stromal Cells ; cytology

OBJECTIVE: To investigate the effects of hydrogen sulfide (HS) on the negatively regulation of cardiomyocyte hypertrophy and the relationship between the effect of HS with miRNA-133a-mediated Ca/calcineurin/NFATc4 signal pathway. METHODS: Cardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (Leica). The expression of brain natriuretic peptide(BNP), β-myosin heavy chain(β-MHC), cystathionase (CSE), miRNA-133a, calcineurin (CaN) were detected by qRT-PCR. The protein expressions of CaN、nuclear factors of activated T cells (NFATc4) were detected by Western blot. The concentration of HS in the cardiomyocyte was detected by Elisa. The concentration of intracellular calcium was measured by calcium imaging using confocal microscope. The nuclear translocation of NFATc4 was checked by immuno-fluorescence cell staining technique. RESULTS: ①The level of system of CSE/HS and expression of miRNA-133a were significantly reduced in cardiomyocyte hypertrophy. Pretreatment with NaHS increased the concentration of HS and the expression of miRNA-133a mRNA in cardiomyocytes, and suppressed cardiomyocyte hypertrophy. ②The concentration of intracellular calcium, the expression of CaN and nulear protein NFATc4 were significantly increased, and the nuclear translocation of NFATc4 were obviously enhanced in cardiomyocyte hypertrophy. NaHS pretreatment markedly inhibited these effects of ISO induced cardiomyocyte hypertrophy. ③Application of antagomir-133a reversed the inhibitory effects of NaHS on cardiomyocyte hypertrophy, and increased the influx of intracellular calcium, and elevated the expression of CaN and nuclear protein NFATc4, and enhanced the nuclear translocation of NFATc4. CONCLUSIONS HS can negatively regulate cardiomyocyte hypertrophy. The effects might be associated with HS increasing expression of miRNA-133a and inhibiting inactivation of Ca/calcineurin/NFATc4 signal pathway.
Animals ; Calcineurin ; metabolism ; Cardiomegaly ; chemically induced ; metabolism ; Cells, Cultured ; Cystathionine gamma-Lyase ; metabolism ; Hydrogen Sulfide ; metabolism ; MicroRNAs ; metabolism ; Myocytes, Cardiac ; metabolism ; Myosin Heavy Chains ; metabolism ; NFATC Transcription Factors ; metabolism ; Natriuretic Peptide, Brain ; metabolism ; Nerve Tissue Proteins ; metabolism ; Rats ; Signal Transduction