In vitro and in vivo characteristics of connexin 43-modified human skeletal myoblasts as candidates for prospective stem cell therapy for the failing heart

Background

Previously, connexin 43-modified skeletal myoblasts (MbCx) were shown to reduce the pro-arrhythmic effect during the regeneration of heart tissue in an animal model of infarction. To increase the relevance to clinical implementation, in this study, we introduced connexin 43 into human myoblasts using a highly safe non-viral vector and demonstrated that their transplantation had a positive effect on the function of the injured heart.

Methods and results

Myoblasts were efficiently transfected with a pCiNeo-GJA1 plasmid (65.72%). qPCR analysis revealed over 32-fold higher expression of the connexin 43 gene in the MbCx cell population compared to ‘native’ controls. The susceptibility of the myoblasts to oxidative stress conditions (p < 0.001) and the fusion index (p < 0.01) were increased in the MbCx cells. Additionally, we observed changes in the MYOG and MYH2 gene expression levels in the GJA1-modified myoblasts. Finally, we observed a significant improvement in the post-infarction echocardiographic parameters after intervention using MbCx cells compared with non-transfected myoblasts (MbWt) and the control (0.9% NaCl), wherein a significant decrease in the left ventricular area change in the short axis (SAX AC%) was observed at the two-month follow-up (p < 0.05 and p < 0.01, respectively).

Conclusions

We demonstrated the positive effect of connexin 43 overexpression on the biology and function of human skeletal myoblasts in the context of their potential clinical applications. Our preclinical studies using a mouse infarction model indicated the positive effect of MbCx implantation on the function of the injured heart.     

七氟醚预处理对大鼠心肌缺血再灌注时缝隙连接蛋白43的影响

目的 评价七氟醚预处理对大鼠心肌缺血再灌注时缝隙连接蛋白43的影响.方法 健康成年雄性Wistar大鼠,体重220 ～ 250 g,采用改良Langendorff灌注装置制备离体心脏灌注模型.取离体心脏模型16个,采用随机数字表法,将其随机分为2组(n=8):缺血再灌注组(I/R组)和七氟醚预处理组(SP组).平衡灌注30 min时,I/R组继续灌注K-H液20 min;SP组将2.4％七氟醚持续吹入K-H液中,用预充2.4％七氟醚的K-H液持续灌注15 min,然后用K-H液洗脱5 min.之后两组均行全心缺血40 min,再灌注60 min.分别于平衡灌注末、缺血前即刻、再灌注30和60 min时,记录HR、左室发展压(LVDP)、左心室内压最大上升速率(+dp/dtmax)及左心室内压最大下降速率(- dp/dtmax).于再灌注60 min时,取部分心尖组织,观察心肌病理学改变;取左室部分心肌,观察缝隙连接蛋白43的分布情况,测定缝隙连接蛋白43的表达.结果 与平衡灌注末比较,I/R组和SP组缺血前即刻+ dp/dtmax和- dp/dtmax降低,再灌注30和60 min时HR、LVDP、+dp/dtmax和- dp/dtmax降低(P＜0.01);与缺血前即刻比较,I/R组和SP组再灌注30和60 min时HR、LVDP、+dp/dtmax和- dp/dtmax降低(P ＜0.05或0.01);与I/R组比较,SP组缺血前即刻HR、LVDP、+dp/dtmax和- dp/dtmax降低,再灌注30和60 min时HR、LVDP、+dp/dtmax和- dp/dtmax升高(P＜0.01),病理学损伤减轻.缝隙连接蛋白43 I/R组分布不均,闰盘处少见;SP组分布规律呈条带状,主要位于闰盘处.两组缝隙连接蛋白43表达比较差异无统计学意义(P＞0.05).结论 七氟醚预处理减轻大鼠心肌缺血再灌注损伤的机制可能与抑制缝隙连接蛋白43的再分布有关,而与缝隙连接蛋白43的表达无关.     

Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation

Connexin 43 (Cx43) mediates osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/osteocytes or only in osteocytes (Cx43(ΔOt) mice) exhibit increased osteocyte apoptosis, endocortical resorption, and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated. Anatomical mapping of apoptotic osteocytes, osteocytic protein expression, and resorption and formation suggests that Cx43 controls osteoclast and osteoblast activity by regulating osteoprotegerin and sclerostin levels, respectively, in osteocytes located in specific areas of the cortex. Whereas empty lacunae and living osteocytes lacking osteoprotegerin were distributed throughout cortical bone in Cx43(ΔOt) mice, apoptotic osteocytes were preferentially located in areas containing osteoclasts, suggesting that osteoclast recruitment requires active signaling from dying osteocytes. Furthermore, Cx43 deletion in cultured osteocytic cells resulted in increased apoptosis and decreased osteoprotegerin expression. Thus, Cx43 is essential in a cell-autonomous fashion in vivo and in vitro for osteocyte survival and for controlling the expression of osteocytic genes that affect osteoclast and osteoblast function.     

TGF-β1对大鼠睾丸Leydig细胞间连接蛋白43介导缝隙连接通讯功能的影响

目的:观察转化生长因子TGF-β1对成年大鼠睾丸Leydig细胞间连接蛋白Cx43表达以及由Cx43介导的缝隙连接细胞间通讯(GJIC)功能的影响,旨在探讨TGF-β1对Leydig细胞的影响是否与其改变细胞间GJIC功能相关。方法:将原代培养纯化的Leydig细胞分为6组:实验组分别以1、2、5、10 ng/ml的TGF-β1处理细胞20 h,空白对照组以含10%胎牛血清的DMEM/F12培养液处理细胞,部分实验中以GJIC抑制剂甘珀酸(Carbenox-olone)处理细胞作为阳性对照组。采用免疫荧光法和Western印迹观察Cx43在细胞中的定位和表达变化,用荧光漂白恢复实验(FRAP)检测细胞间GJIC功能的改变。结果:Cx43表达呈斑点状散在分布于Leydig细胞的胞质和胞膜中,TGF-β1处理20 h后,Cx43在胞质中的表达强度随着TGF-β1浓度的增加较空白对照组明显增强,而其在胞膜中的表达则无明显改变。Western印迹结果显示磷酸化状态的Cx43随着TGF-β1浓度增加表现出较空白对照明显增强的趋势(P<0.05),而非磷酸化状态则无显著差异。FRAP结果显示经5 ng/ml TGF-β1作用20 h后细胞内荧光强度较空白对照明显减弱,具有显著性差异(P<0.01),且其平均荧光恢复率仅为(43.58±1.87)%。结论:TGF-β1可显著下调Leydig细胞间的GJIC功能,这种抑制作用可能通过增加其连接蛋白Cx43在细胞质中的表达,提高其磷酸化水平来实现。     

缝隙连接蛋白43在受照人血管内皮细胞中的改变及其对受照细胞刚性的作用

目的 研究X射线对人脐静脉内皮细胞（HUVEC）中缝隙连接蛋白43（Cx43）的表达、分布和细胞刚性的影响,初步探讨Cx43对受照细胞刚性的调控作用。方法 采用Western blot方法检测10 Gy X射线照射后不同时间（0、6、12、24和48 h）和不同剂量X射线（0、2.5、5、10和20 Gy）照射后12 h HUVEC细胞中Cx43表达水平的改变,以及不同剂量（0、5和10 Gy） X射线照射后不同时间（3、6、24和48 h） Cx43 3个磷酸化位点（Ser279/282、Ser368和Tyr265）磷酸化水平的变化。采用细胞免疫荧光方法检测Cx43蛋白在受照HUVEC细胞中的分布变化。采用原子力显微镜检测受照细胞在探针压入不同深度（50、100和200 nm）时杨氏模量（细胞刚性）的变化,以及Cx43过表达对受照细胞杨氏模量（细胞刚性）的影响。结果 10 Gy X射线照射后6、12、24、48 h,HUVEC细胞中Cx43表达量降低（t=3.262、3.708、3.686、6.825,P<0.05）,且在2.5、5、10和20 Gy照射后24 h Cx43表达水平的降低与受照剂量呈现剂量依赖性（t=3.034、10.720、13.130、13.650,P<0.05）。5、10和20 Gy X射线照射后24 h,HUVEC细胞中Cx43分布由细胞间隙转移入核及核周,照射后24和48 h,Cx43的Ser368位点磷酸化水平升高并且随剂量增加而增加。10 Gy X射线照射HUVEC细胞后24 h,照射组与对照组相比,在探针压入100和200 nm时,杨氏模量均明显降低（t=3.362、5.122,P<0.05）;过表达Cx43的受照组与空载体受照组相比,在探针压入100和200 nm时,杨氏模量增高（t=2.674、4.398,P<0.05）。结论 X射线照射可导致HUVEC细胞内Cx43 Ser368位点磷酸化,促进Cx43降解和分布变化,降低细胞刚性。提高Cx43的表达水平,有助于受照细胞刚性的恢复,提示Cx43可能是调控X射线致血管内皮细胞损伤的作用靶点。     

In Vivo and In Vitro Expression of Connexin 43 in Human Teeth

Gap junctions are composed of transmembrane proteins belonging to the connexin family. These proteins permit the exchange of small regulatory molecules directly between cells for the control of growth, development and differentiation. Although the presence of gap junctions in teeth has been already evidenced, the involved connexins have not yet been identified in human species. Here, we examined the distribution of connexin 43 (Cx43) in embryonic and permanent intact and carious human teeth. During tooth development, Cx43 localized both in epithelial and mesenchymal dental cells, correlated with cytodifferentiation gradients. In adult intact teeth, Cx43 was distributed in odontoblast processes. While Cx43 expression was downregulated in mature intact teeth, Cx43 appeared to be upregulated in odontoblasts facing carious lesions. In cultured pulp cells, Cx43 expression was related to the formation of mineralized nodules. These results indicate that Cx43 expression is developmentally regulated in human dental tissues, and suggest that Cx43 may participate in the processes of dentin formation and pathology.     

Drug development in dementia

Dementia is a progressive, irreversible decline in cognition that, by definition, impacts on a patient's pre-existing level of functioning. The clinical syndrome of dementia has several aetiologies of which Alzheimer's disease (AD) is the most common. Drug development in AD is based on evolving pathophysiological theory. Disease modifying approaches include the targeting of amyloid processing, aggregation of tau, insulin signalling, neuroinflammation and neurotransmitter dysfunction, with efforts thus far yielding abandoned hopes and ongoing promise. Reflecting its dominance on the pathophysiological stage the amyloid cascade is central to many of the emerging drug therapies. The long preclinical phase of the disease requires robust biomarker means of identifying those at risk if timely intervention is to be possible.     

Human Coronavirus OC43 Interacts with Major Histocompatibility Complex Class I Molecules at the Cell Surface to Establish Infection

Human coronaviruses have been associated with common colds, diarrhea and enterocolitis, and have been implicated in multiple sclerosis. HLA class I molecules may play a critical role as receptor for OC43 because monoclonal antibody (mAb)W6/32 to HLA-A, -B and -C specificities completely blocks infectivity in human rhabdomyosarcoma (RD) cells. The role of HLA class 1 antigen as the virus receptor was examined using HLA-A3.1 stably transfected human plasma cells and untransfected HMY.C1R cells which do not express HLA-A and -B molecules. When the cells (5x10⁶) were infected at a multiplicity of one, the HLA.A3 transfected cells produced 10⁸ PFU of virus whereas no replication occurred in the HMY.C1R cells mAb W6/32 reduced the virus yield by 99.9% Cell membranes from HMY.C1R, HMY.A3 cells and chicken erythrocytes were biotinylated as live cells. Immunoprecipitation with polyclonal antiviral antibody to detect binding of biotinylated cell membranes to virus revealed that biotinylated HMY.A3 membranes co-precipitated with virus-antibody complexes when the immunoprecipitates were electrophoresed on SDS-PAGE gel, electroblotted and stained with Avidin-horseradish peroxidase. The results provide direct evidence that OC43 virus can recognize HLA class I as receptor on the cell surface.     

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

The spliceosome is a single-turnover enzyme that needs to be dismantled after catalysis to both release the mRNA and recycle small nuclear ribonucleoproteins (snRNPs) for subsequent rounds of pre-mRNA splicing. The RNP remodeling events occurring during spliceosome disassembly are poorly understood, and the composition of the released snRNPs are only roughly known. Using purified components in vitro, we generated post-catalytic spliceosomes that can be dissociated into mRNA and the intron-lariat spliceosome (ILS) by addition of the RNA helicase Prp22 plus ATP and without requiring the step 2 proteins Slu7 and Prp18. Incubation of the isolated ILS with the RNA helicase Prp43 plus Ntr1/Ntr2 and ATP generates defined spliceosomal dissociation products: the intron-lariat, U6 snRNA, a 20–25S U2 snRNP containing SF3a/b, an 18S U5 snRNP, and the “nineteen complex” associated with both the released U2 snRNP and intron-lariat RNA. Our system reproduces the entire ordered disassembly phase of the spliceosome with purified components, which defines the minimum set of agents required for this process. It enabled us to characterize the proteins of the ILS by mass spectrometry and identify the ATPase action of Prp43 as necessary and sufficient for dissociation of the ILS without the involvement of Brr2 ATPase.     

A unique mouse model for investigating the properties of amyotrophic lateral sclerosis-associated protein TDP-43, by in utero electroporation

TDP-43 is a discriminative protein that is found as intracellular aggregations in the neurons of the cerebral cortex and spinal cord of patients with amyotrophic lateral sclerosis (ALS); however, the mechanisms of neuron loss and its relation to the aggregations are still unclear. In this study, we generated a useful model to produce TDP-43 aggregations in the motor cortex using in utero electroporation on mouse embryos. The plasmids used were full-length TDP-43 and C-terminal fragments of TDP-43 (wild-type or M337V mutant) tagged with GFP. For the full-length TDP-43, both wild-type and mutant, electroporated TDP-43 localized mostly in the nucleus, and though aggregations were detected in embryonic brains, they were very rarely observed at P7 and P21. In contrast, TDP-43 aggregations were generated in the brains electroporated with the C-terminal TDP-43 fragments as previously reported in in vitro experiments. TDP-43 protein was distributed diffusely—not only in the nucleus, but also in the cytoplasm—and the inclusion bodies were ubiquitinated and included phosphorylated TDP-43, which reflects the human pathology of ALS. This model using in utero electroporation of pathogenic genes into the brain of the mouse will likely become a useful model for studying ALS and also for evaluation of agents for therapeutic purpose, and may be applicable to other neurodegenerative diseases, as well.