Primary structure and developmental acidic to basic transition of 13 alternatively spliced mouse fast skeletal muscle troponin T isoforms

Large samples of original cDNAs encoding neonatal and adult mouse fast skeletal muscle troponin T (fTnT) have been isolated and characterized. The results demonstrate expression relationships of 8 alternatively spliced exons of the fTnT gene and reveal the primary structure of as many as 13 fTnT isoforms that diverge into acidic and basic classes due to differential mRNA splicing in the N-terminal variable region. In the C-terminal variable region encoded by the mutually exclusive exons 16 and 17, the splicing pathway and structure of exon 16 appears to be adult fTnT-specific, suggesting an adaptation to the functional demands of mature fast skeletal muscle. The cloned cDNAs were expressed in E. coli as standards to identify a high M_r to low M_r, acidic to basic fTnT isoform transition in postnatal developing skeletal muscles. Different from the developmental cardiac TnT switch generated by alternative splicing of a single exon, the fTnT isoform transition is an additive effect of alternative splicing of multiple N-terminal-coding exons, especially exons 4, 8 and fetal that are expressed at higher frequencies in the neonatal than in the adult muscle. The developmental fTnT isoform primary structure transition in both N- and C-terminal variable regions suggest a physiological importance of the apparently complex TnT isoform expression.     

Troponin T3 expression in skeletal and smooth muscle is required for growth and postnatal survival: Characterization of Tnnt3tm2a(KOMP)Wtsi mice

The troponin complex, which consists of three regulatory proteins (troponin C, troponin I, and troponin T), is known to regulate muscle contraction in skeletal and cardiac muscle, but its role in smooth muscle remains controversial. Troponin T3 (TnnT3) is a fast skeletal muscle troponin believed to be expressed only in skeletal muscle cells. To determine the in vivo function and tissue‐specific expression of Tnnt3, we obtained the heterozygous Tnnt3+/flox/lacZ mice from Knockout Mouse Project (KOMP) Repository. Tnnt3^lacZ/+ mice are smaller than their WT littermates throughout development but do not display any gross phenotypes. Tnnt3^lacZ/lacZ embryos are smaller than heterozygotes and die shortly after birth. Histology revealed hemorrhagic tissue in Tnnt3^lacZ/lacZ liver and kidney, which was not present in Tnnt3^lacZ/+ or WT, but no other gross tissue abnormalities. X‐gal staining for Tnnt3 promoter‐driven lacZ transgene expression revealed positive staining in skeletal muscle and diaphragm and smooth muscle cells located in the aorta, bladder, and bronchus. Collectively, these findings suggest that troponins are expressed in smooth muscle and are required for normal growth and breathing for postnatal survival. Moreover, future studies with this mouse model can explore TnnT3 function in adult muscle function using the conditional‐inducible gene deletion approach genesis 51:667–675. © 2013 Wiley Periodicals, Inc.     

Affinity-chromatographic isolation and some properties of troponin C from different muscle types.

1. The formation of a complex between troponin I and troponin C that is stable in 6M-urea and dependent on Ca2+ was demonstrated in extracts of vertebrate striated and smooth muscles. 2. A method using troponin I coupled to Sepharose is described for the rapid isolation of troponin C from striated and smooth muscles of vertebrates. 3. Troponin C of rabbit cardiac muscle differs significantly in amino acid composition from troponin C of skeletal muscle. The primary structures of troponin C of red and white skeletal muscle are very similar. 4. The troponin C-like protein isolated from rabbit uterus muscle has a slightly different amino acid composition, but possess many similar properties to the forms of troponin C isolated from other muscle types. 5. The electrophoretic mobilities of the I-troponin C complexes formed from components isolated from different muscle types are determined by the troponin I component.     

Heterogeneity of desmin, the muscle-type intermediate filament protein, in blood vessels and astrocytes

Monoclonal antibodies were isolated from mice immunized with chicken gizzard desmin. Antibodies reacting with desmin on immunoblots and selectively decorating chicken and rat intestinal smooth muscle as well as the Z-line in striated muscle, were selected for this study. Based on their staining pattern on cryostat sections of chicken and rat cerebellum, spleen, kidney, aorta and femoral artery, monoclonal supernatants could be divided in three groups: (i) antibodies decorating astrocytes and vascular smooth muscle; (ii) antibodies decorating only vascular smooth muscle; (iii) antibodies decorating only astrocytes. Antibodies in group (i) and (iii) also stained GFA-negative Bergmann glia in chicken cerebellum. It is proposed that desmin may vary depending on the histological localization.     

小鼠主动脉血管平滑肌原代细胞的分离培养及鉴定

目的:血管平滑肌细胞在人类心血管疾病中具有重要的作用,而作为重要的遗传学研究模式生物的小鼠血管平滑肌材料有限,因此建立一种简单高效的小鼠血管平滑肌原代细胞分离培养方法很重要。方法:分离小鼠主动脉中膜层,胶原酶消化法获得原代平滑肌细胞,免疫荧光方法检测细胞的纯度和分化状态;分离平滑肌细胞特异的报告小鼠的平滑肌细胞,LacZ染色鉴定。结果:用该方法分离的原代平滑肌细胞生长迅速,3d后即可达5×106个。免疫荧光显示,细胞传至第3代后纯度在98%以上,细胞传至8代分化状态没有改变。LacZ染色鉴定报告小鼠分离的3代平滑肌细胞98%以上显示特异的蓝染。两种实验证明,应用此方法分离原代平滑肌细胞可以满足平滑肌体外功能实验的需求。结论:与传统的组织块培养法相比,该方法操作简便、经济,可以获得更多高纯度的血管平滑肌细胞。     

Polycystin-2 interacts with troponin I,an angiogenesis inhibitor

Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in 10-15% of autosomal dominant polycystic kidney disease (ADPKD) patients. PC2 is a Ca(2+)-permeable nonselective cation channel and is present in kidney and many other organs. Likewise, PKD2-mutated patients and mice exhibit extrarenal abnormalities. In comparison with cysts in the kidney, liver, and pancreas, abnormalities in the heart, brain, and vascular vessels are less understood. In particular, roles of PC2 in muscle and endothelia remain largely unknown. In the present study, using a yeast two-hybrid screening, we discovered that the PC2 carboxyl terminal domain (D682-V968) interacts with the cardiac troponin I, an important regulatory component of the actin microfilament in cardiac muscle cells. This interaction was demonstrated by GST pull-down and microtiter binding assays. Dose-dependent binding between PC2 and troponin I followed a Michaelis-Menten relationship, indicating a 1:1 binding stoichiometry. The interacting domains were located to the R872-H927 segment of PC2 and the M1-V107 and K106-L158 segments of troponin I. Co-immunoprecipitation experiments demonstrated that the cardiac and two skeletal isoforms of troponin I were all associated with PC2, when coexpressed in mouse fibroblast NIH 3T3 cells and Xenopus oocytes. Furthermore, reciprocal co-immunoprecipitation verified the interaction between the native polycystin-2 and troponin I in human adult heart tissues. This study thus provides new evidence for a direct attachment of PC2 to the actin microfilament network, in addition to the recently identified association between PC2 and trypomyosin-1. Troponin I functions as an inhibitory subunit of the troponin complex for calcium-dependent regulation of muscle contraction and as an inhibitor of angiogenesis seen in ADPKD. It is possible that altered interaction due to pathogenic polycystin-1 or -2 mutations can account for angiogenesis in ADPKD and may be corrected to some extent by exogenous troponin I.     

Noxa1 is a central component of the smooth muscle NADPH oxidase in mice

NADPH oxidase is the most important source of oxygen-derived radicals (ROS) in the vascular wall. In vascular smooth muscle cells (VSMC), NADPH oxidase is characterized by the expression of the membrane subunit Nox1, which is activated by cytoplasmic proteins binding to its activation domain. We set out to identify the cytoplasmic protein involved in NADPH oxidase activation in mouse VSMC. Western blot analysis revealed that human endothelial cells and leukocytes but not VSMC from the aorta of the rat and the mouse express the classic NADPH oxidase activator p67phox. In mouse VSMC, however, the p67phox homologue Noxa1 was detected. Using antibodies generated against mouse Noxa1, the protein was observed in the cytosolic fraction of mouse VSMC with a molecular weight of about 51 kDa. Immunohistochemistry revealed that Noxa1 is expressed in the smooth muscle layer but not in endothelium or the adventitia of the mouse carotid artery. Fluorescent fusion proteins of Noxa1 were observed to be expressed in the cytoplasm of VSMC and coexpression of the NADPH oxidase organizer Noxo1 targeted the complex to membrane. An antisense plasmid of Noxa1 attenuated the endogenous Noxa1 protein expression in VSMC. This plasmid attenuated the ROS formation in mouse VSMC as detected using L012 chemiluminescence and prevented the agonist-induced ROS production in response to basic fibroblast growth factor and epidermal growth factor. In conclusion, these data indicate that Noxa1 replaces p67phox in VSMC and plays a central role in the activation of the NADPH oxidase in the vascular wall.     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

PDGF-BB enhances expression of,and reduces adhesion to,laminin-5 in vascular smooth muscle cells

The laminin family of extracellular matrix (ECM) proteins plays crucial roles in regulating cellular growth, migration, and differentiation. We report here that laminin-5 is expressed in the tunica media of the rat aorta and pulmonary arteries. Using indirect immunofluorescence microscopy, Western blots, and RT-PCR analysis, we found that primary cultures of rat arterial smooth muscle cells express laminin-5 and deposit it into their insoluble ECM. These cells also attach strongly to laminin-5 via beta1 integrin receptors in 30 min adhesion assays. Laminin-5 expression in these cells is upregulated by growth factors in vitro and platelet-derived growth factor (PDGF-BB) stimulation reduces adhesion to laminin-5. As laminin-5 promotes enhanced migration of other cell types, the production of and adhesion to laminin-5 by vascular smooth muscle cells may play a role in the pathological growth and migration of these cells associated with restenosis following vascular injury.     

Identification of a novel smooth muscle associated protein,smap2, upregulated during neointima formation in a rat carotid endarterectomy model

Proliferation of aortic smooth muscle cells is an important event in vascular lesion formation. To identify new genes that are involved in neointima formation, we constructed an aortic 3'-directed cDNA library. The novel cDNA of a gene designated smooth muscle associated protein 2 (smap2) was isolated. The full-length cDNA of smap2 is 2914 base pairs long and contains an open reading frame of 1338 base pairs. Dot blot analysis revealed that smap2 was expressed particularly in aorta. The deduced amino acid sequence of smap2 contains two thyroglobulin type-1 domains, two EF-hand calcium-binding domains and putative signal peptide. Furthermore, we demonstrated that smap2 mRNA was upregulated during neointima formation in a rat carotid endarterectomy model. These findings suggest that smap2 might be involved in the progression of atherosclerosis in aorta.     

Human cardiac troponin complex. Structure and functions

Troponin complex is a component of skeletal and cardiac muscle thin filaments. It consists of three subunits — troponin I, T, and C, and it plays a crucial role in muscle activity, connecting changes in intracellular Ca²⁺ concentration with generation of contraction. In spite of more than 40 years of studies, many aspects of troponin functioning are still not completely understood, and several models describing the mechanism of muscle contraction exist. Being a key factor in the regulation of cardiac muscle contraction, troponin complex is utilized in medicine as a target for some cardiotonic drugs used in the treatment of heart failure. A number of mutations in troponin subunits are associated with development of different types of cardiomyopathy. Moreover, for the last 25 years cardiac isoforms of troponin I and T have been widely used for immunochemical diagnostics of pathologies associated with cardiomyocyte death (myocardial infarction, myocardial trauma, and others). This review summarizes the existing evidence on the structure and function of troponin complex subunits, their role in the regulation of cardiac muscle contraction, and their clinical applications.     

Apoptosis repressor with caspase recruitment domain is dramatically reduced in cardiac, skeletal, and vascular smooth muscle during hypertension

Apoptosis repressor with caspase recruitment domain (ARC) is a unique anti-apoptotic protein with a distinct tissue distribution. In addition, unlike most anti-apoptotic proteins which act on one pathway, ARC can inhibit apoptosis mediated by both the death-receptor and mitochondrial signaling pathways. In this study, we confirm previous reports showing high levels of ARC protein in rat heart and skeletal muscle, but demonstrate for the first time that ARC is also expressed in rat aorta. Immunoblot analysis on endothelium-denuded aorta as well as immunohistochemical analysis on intact aorta demonstrated that ARC was highly expressed in smooth muscle. Immunoblot analysis also found that ARC protein was severely downregulated in skeletal muscle (−82%; P < 0.001), heart (−80%; P < 0.001), and aorta (−71%; P < 0.001) of spontaneously hypertensive rats (SHR) compared to normotensive Wistar-Kyoto (WKY) rats. Decreased ARC levels were also confirmed in tissues of hypertensive animals by immunohistochemical analysis. Collectively, this data suggests that ARC protein is expressed in vascular smooth muscle and is significantly reduced in several target tissues during hypertension.     

Blood pressure regulates the activity and function of the Na-K-2Cl cotransporter in vascular smooth muscle

The Na-K-2Cl cotransporter (NKCC1) is one of several transporters that have been linked to hypertension, and its inhibition reduces vascular smooth muscle tone and blood pressure. NKCC1 in the rat aorta is stimulated by vasoconstrictors and inhibited by nitrovasodilators, and this is linked to the contractile state of the smooth muscle. To determine whether blood pressure also regulates NKCC1, we examined the acute effect of hypertension on NKCC1 in rats after aortic coarctation. In the hypertensive aorta (28-mmHg rise in mean blood pressure), an increase in NKCC1 activity (measured as bumetanide-sensitive (86)Rb efflux) was apparent by 16 h and reached a plateau of 62% greater than control at 48 h. In contrast, there was a slight decrease in NKCC1 activity in the hypotensive aorta (21% decrease in mean blood pressure). Measurement of NKCC1 mRNA by real-time PCR revealed a fivefold increase in the hypertensive aorta compared with the hypotensive aorta or sham aorta. The inhibition by bumetanide of isometric force response to phenylephrine was significantly greater in the hypertensive aorta than in the control aorta or hypotensive aorta. We conclude that NKCC1 in rat aortic smooth muscle is regulated by blood pressure, most likely through changes in transporter abundance. This upregulation of NKCC1 is associated with a greater contribution to force generation in the hypertensive aorta. This is the first demonstration that NKCC1 in vascular smooth muscle is regulated by blood pressure and indicates that this transporter is important in the acute response of vascular smooth muscle to hypertension.     

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-α-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.     

Isolation of a morphologically and functionally distinct smooth muscle cell type from the intimal aspect of the normal rat aorta. Evidence for smooth muscle cell heterogeneity

Summary Recent studies indicate that the neointima of injured rat arteries is composed of a subpopulation of smooth muscle cells (SMCs) distinct from medial smooth muscle cells. However, SMC diversity in normal adult aorta has remained elusive. This study characterizes two morphologically and functionally distinct SMC types isolated from different anatomic regions of the normal rat aorta. Rat aortic medial smooth muscle cells (MSMCs) were isolated from the media after removal of the intimal and adventitial cells. Rat aortic intimal smooth muscle cells (ISMCs) were isolated from the intimal aspect of everted rat aortas. The two cell types were characterized morphologically and immunohistochemically and were compared for their capacity to contract collagen gels in response to endothelin-1. MSMCs were spindle-shaped and grew in hills and valleys showing features previously described for vascular SMCs. Conversely, ISMCs displayed a polygonal and epithelioid shape, grew mainly as a monolayer, and had a higher proliferative rate. Both cell types expressed alpha-smooth muscle actin and were negative for Factor VIII-RAg. ISMCs produced large amounts of a laminin and type IV collagen-rich extracellular matrix which had a characteristic pericellular distribution. ISMCs, but not MSMCs, rapidly contracted collagen gels in response to endothelin-1. This study indicates that the normal rat aorta contains two types of SMCs located in anatomically distinct regions of the vessel wall. Because of their functional characteristics, the SMCs isolated from the intimal aspect of the aorta may play an important role in physiologic as well as pathologic conditions.