胰岛素及cAMP对培养人动脉平滑肌细胞HDL受体功能的影响

对胰岛素ｃＡＭＰ对培养人动脉平滑肌细胞（ＳＭＣ）ＨＤＬ受体功能的影响进行了研究,结果发现：胰岛素使ＳＭＣＨＤＬ受体的结合容量Ｂｍａｘ即受体数目显著下降,而对ＳＭＣＨＤＬ受体的Ｋｄ值亲和力无影响;ｃＡＭｐ则ＳＭＣＨＤＬ受体亲和力增加,而对受体数目无影响。     

Myofilament incorporation determines the stoichiometry of troponin I in transgenic expression and the rescue of a null mutation

The highly organized contractile machinery in skeletal and cardiac muscles requires an assembly of myofilament proteins with stringent stoichiometry. To understand the maintenance of myofilament protein stoichiometry under dynamic protein synthesis and catabolism in muscle cells, we investigated the equilibrium of troponin I (TnI) in mouse cardiac muscle during developmental isoform switching and in under- and over-expression models. Compared with the course of developmental TnI isoform switching in normal hearts, the postnatal presence of slow skeletal muscle TnI lasted significantly longer in the hearts of cardiac TnI (cTnI) knockout (cTnI-KO) mice, in which the diminished synthesis was compensated by prolonging the life of myofilamental TnI. Transgenic postnatal expression of an N-terminal truncated cTnI (cTnI-ND) using α-myosin heavy chain promoter effectively rescued the lethality of cTnI-KO mice and shortened the postnatal presence of slow TnI in cardiac muscle. cTnI-KO mice rescued with different levels of cTnI-ND over-expression exhibited similar levels of myocardial TnI comparable to that in wild type hearts, demonstrating that excessive synthesis would not increase TnI stoichiometry in the myofilaments. Consistently, haploid under-expression of cTnI in heterozygote cTnI-KO mice was sufficient to sustain the normal level of myocardial cTnI, indicating that cTnI is synthesized in excess in wild type cardiomyocytes. Altogether, these observations suggest that under wide ranges of protein synthesis and turnover, myofilament incorporation determines the stoichiometry of troponin subunits in muscle cells.     

Removal of the Cardiac Troponin I N-terminal Extension Improves Cardiac Function in Aged Mice

The cardiac troponin I (cTnI) isoform contains a unique N-terminal extension that functions to modulate activation of cardiac myofilaments. During cardiac remodeling restricted proteolysis of cTnI removes this cardiac specific N-terminal modulatory extension to alter myofilament regulation. We have demonstrated expression of the N-terminal-deleted cTnI (cTnI-ND) in the heart decreased the development of the cardiomyopathy like phenotype in a β-adrenergic-deficient transgenic mouse model. To investigate the potential beneficial effects of cTnI-ND on the development of naturally occurring cardiac dysfunction, we measured the hemodynamic and biochemical effects of cTnI-ND transgenic expression in the aged heart. Echocardiographic measurements demonstrate cTnI-ND transgenic mice exhibit increased systolic and diastolic functions at 16 months of age compared with age-matched controls. This improvement likely results from decreased Ca²⁺ sensitivity and increased cross-bridge kinetics as observed in skinned papillary bundles from young transgenic mice prior to the effects of aging. Hearts of cTnI-ND transgenic mice further exhibited decreased β myosin heavy chain expression compared to age matched non-transgenic mice as well as altered cTnI phosphorylation. Finally, we demonstrated cTnI-ND expressed in the heart is not phosphorylated indicating the cTnI N-terminal is necessary for the higher level phosphorylation of cTnI. Taken together, our data suggest the regulated proteolysis of cTnI during cardiac stress to remove the unique cardiac N-terminal extension functions to improve cardiac contractility at the myofilament level and improve overall cardiac function.     

SELDI-TOF MS analysis of the Cardiac Troponin I forms present in plasma from patients with myocardial infarction

The troponin (Tn) complex is composed of troponin T, troponin C and troponin I. The cardiac isoform of TnI (cTnI) is modified and released in blood of patients with cardiovascular diseases as a heterogeneous mixture of free, complexed and posttranslationally modified forms. With the aim to determine later, whether specific forms of cTnI could be associated with the different pathologies leading to cTnI release, the cTnI forms present in the plasma from 64 patients with acute myocardial infarction (AMI) have been analysed by SELDI-TOF MS using anti-TnI mAbs coupled to PS20 ProteinChips arrays. Upfront immunoaffinity enrichment using anti-cTnI 19C7 mAb allowed us to detect cTnI and bis-phosphorylated cTnI in 11/12 and 9/12 analyses respectively, as well as truncated cTnI in plasma with concentration of cTnI as low as 8 ng/mL. Cardiac troponin C (cTnC) and covalent TnIC complex were also found in pools of plasma with higher concentrations of cTnI. MAb 19C7-affinity SELDI-TOF MS analysis performed after immunopurification of one pool of AMI plasma with anti-free cTnI, anti-cTnC, and anti-phosphorylated cTnI mAbs indicated that intact and bis-phosphorylated cTnI were mostly under the free form. Besides, a 18 718 m/z peak could correspond to a truncated phosphorylated form initially complexed with cTnC.     

肌钙蛋白I2辅助活化多种核受体的反式作用

为深入研究乳腺癌中孤儿受体ERRα1参与基因表达调控的详细机理,特别是核受体辅激活蛋白在其中的作用,以ERRα1的LBD为诱饵,用酵母双杂交系统筛选人乳腺组织cDNA文库得到了与其有明显相互作用的快速骨骼肌型肌钙蛋白I(TNNI2).应用酵母双杂交技术研究表明,TNNI2与多种核受体存在相互作用,且这种作用依赖于功能性的核受体AF2结构域.在哺乳细胞瞬时共转染实验中,TNNI2显示了对多种核受体反式激活功能的辅助活化作用.研究证明,TNNI2与许多辅激活蛋白类似,以配体依赖(对类固醇激素受体而言)或非依赖(对孤儿受体而言)的方式与核受体功能性AF2结构域相互作用,并增强多种核受体介导的反式作用.     

Mutual Rescues between Two Dominant Negative Mutations in Cardiac Troponin I and Cardiac Troponin T

Troponin T (TnT) and troponin I (TnI) are two evolutionarily and functionally linked subunits of the troponin complex that regulates striated muscle contraction. We previously reported a single amino acid substitution in the highly conserved TnT-binding helix of cardiac TnI (cTnI) in wild turkey hearts in concurrence with an abnormally spliced myopathic cardiac TnT (cTnT) (Biesiadecki, B. J., Schneider, K. L., Yu, Z. B., Chong, S. M., and Jin, J. P. (2004) J. Biol. Chem. 279, 13825–13832). To investigate the functional effect of this cTnI mutation and its potential value in compensating for the cTnT abnormality, we developed transgenic mice expressing the mutant cTnI (K118C) in the heart with or without the deletion of the endogenous cTnI gene to mimic the homozygote and heterozygote of wild turkeys. Double and triple transgenic mice were created by crossing the cTnI-K118C lines with transgenic mice overexpressing the myopathic cTnT (exon 7 deletion). Functional studies of ex vivo working hearts found that cTnI-K118C alone had a dominantly negative effect on diastolic function and blunted the inotropic responses of cardiac muscle to β-adrenergic stimuli without abolishing the protein kinase A-dependent phosphorylation of cTnI. When co-expressed with the cTnT mutation, cTnI-K118C corrected the significant depression of systolic function caused by cTnT exon 7 deletion, and the co-existence of exon 7-deleted cTnT minimized the diastolic abnormality of cTnI-K118C. Characterization of this naturally selected pair of mutually rescuing mutations demonstrated that TnI-TnT interaction is a critical link in the Ca²⁺ signaling and β-adrenergic regulation in cardiac muscle, suggesting a potential target for the treatment of troponin cardiomyopathies and heart failure.     

Troponin I: Inhibitor or facilitator

TN-I occurs as a homologous group of proteins which form part of the regulatory system of vertebrate and invertebrate striated muscle. These proteins are present in vertebrate muscle as isoforms, Mr 21000-24000, that are specific for the muscle type and under individual genetic control. TN-I occupies a central position in the chain of events starting with the binding of calcium to troponin C and ending with activation of the Ca2+ stimulated MgATPase of the actomyosin filament in muscle. The ability of TN-I to inhibit the MgATPase of actomyosin in a manner that is accentuated by tropomyosin is fundamental to its role but the molecular mechanism involved is not yet completely understood. For the actomyosin ATPase to be regulated the interaction of TN-I with actin, TN-C and TN-T must undergo changes as the calcium concentration in the muscle cell rises, which result in the loss of its inhibitory activity. A variety of techniques have enabled the sites of interaction to be defined in terms of regions of the polypeptide chain that must be intact to preserve the biological properties of TN-I. There is also evidence for conformational changes that occur when the complex with TN-C binds calcium. Nevertheless a detailed high resolution structure of the troponin complex and its relation to actin/tropomyosin is not yet available. TN-I induces changes in those proteins with which it interacts, that are essential for their function. In the special case of cardiac TN-I its effect on the calcium binding properties of TN-C is modulated by phosphorylation. It has yet to be determined whether TN-I acts directly as an inhibitor or indirectly by interacting with associated proteins to facilitate their role in the regulatory system.     

亲和层析法分离人心脏特异性肌钙蛋白Ⅰ(CSTnⅠ)

在Ca~(2+)存在下,肌钙蛋白C(TnC)和肌钙蛋白Ⅰ(TnⅠ)可于高浓度的尿素溶液中形成稳定的复合体,而该复合体又能被EGTA解离。并且TnC和TnⅠ的Ca~(2+)依赖性结合反应没有种间或者肌肉类型间的特异性。将提纯的兔快肌TnC和活化的琼脂糖凝胶进行偶联,制备TnC-Sepharose 4B亲和层析柱,用来从人心肌中直接提纯心脏特异性肌钙蛋白Ⅰ(CSTnⅠ)。SDS-PAGE测得其分子量为29kD左右。对其氨基酸组成亦进行了分析。     

PKA-mediated protein phosphorylation protects ezrin from calpain I cleavage

Ezrin is localized to the apical membrane of parietal cells and couples the cAMP-dependent protein kinase (PKA) activation cascade to the regulated HCl secretion in gastric parietal cells. Our recent studies demonstrate the functional relevance of PKA-mediated phosphorylation of ezrin in parietal cell secretion [R. Zhou, X. Cao, C. Watson, Y. Miao, Z. Guo, J.G. Forte, X. Yao, Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation, J. Biol. Chem. 278 (2003) 35651]. Here we show that activation of PKA protects ezrin from calpain I-mediated proteolysis without alteration of calpain I activation and fodrin breakdown. To determine whether phosphorylation of Ser66 by PKA affects the insensitivity to the calpain I-mediated cleavage, recombinant proteins of ezrin, both wild type and S66A/D mutants, were incubated with the purified calpain I. Indeed, phosphorylation-like S66D mutant ezrin is resistant to calpain I-mediated proteolysis while wild type and S66A mutant were sensitive. In fact, expression of phosphorylation-like S66D, but not S66A, mutant in parietal cells confers its resistance to calpain I-mediated proteolysis. Taken together, these results indicate that phosphorylation of ezrin by PKA modulates its sensitivity to calpain I cleavage.     

心肌肌钙蛋白T基因突变在心肌病发病机制中的研究进展

肥厚型和扩张型心肌病中,基因缺陷分别占发病的50％和35％,其病理生理机制,主要包括肌小节蛋白基因突变引起的收缩力产生缺陷,细胞骨架蛋白基因突变引起的收缩力传递缺陷等。心肌肌钙蛋白T将肌钙蛋白C和肌钙蛋白I连接到肌动蛋白和原肌球蛋白上,在心肌细胞收缩和舒张过程中发挥重要作用。在肥厚型和扩张型心肌病中发现了多种心肌肌钙蛋白T的基因突变,围绕心肌肌钙蛋白T的研究有助于阐明心肌病的发病机制。本文总结了心肌肌钙蛋白T基因突变在心肌病发病机制中的研究情况。     

Troponin subunits contribute to altered myosin ATPase activity in diabetic cardiomyopathy

Our group has documented that myocardial performance is impaired in the hearts of chronically diabetic rats and rabbits. Abnormalities in the contractile proteins and regulatory proteins may be responsible for the mechanical defects in the streptozotocin (STZ)-diabetic hearts. Previously, the major focus of our research on contractile proteins in abnormal states has concentrated on myosin ATPase and its isoenzymes. Our present study is based on the overall hypothesis that regulatory proteins, in addition to contractile protein, myosin contribute to altered cardiac contractile performance in the rat model of diabetic cardiomyopathy. The purpose of our research was to define the role of cardiac regulatory proteins (troponin-tropomyosin) in the regulation of actomyosin system in diabetic cardiomyopathy.For baseline data, myofibrillar ATPase studies were conducted in the myofibrils from control and diabetic rats. To focus on the regulatory proteins (troponin and tropomyosin), individual proteins of the cardiac system were reconstituted under controlled conditions. By this approach, myosin plus actin and troponin-tropomyosin from the normal and diabetic animals could be studied enzymatically. The proteins were isolated from the cardiac muscle of control and STZ-diabetic (4 weeks) rats. Sodium dodecyl sulfate gel electrophoretic patterns demonstrate differences in the cardiac TnT and TnI regions of diabetic animals suggesting the different amounts of TnT and/or TnI or possibly different cardiac isozymes in the regulatory protein complex. Myofibrils probed with a monoclonal antibody TnI-1 (specific for adult cardiac TnI) show a downregulation of cardiac TnI in diabetics when compared to its controls. Enzymatic data confirm a diminished calcium sensitivity in the regulation of the cardiac actomyosin system when regulatory protein(s) complex was recombined from diabetic hearts. Actomyosin ATPase activity in the hearts of diabetic animals was partially reversed when myosin from diabetic rats was regulated with the regulatory protein complex isolated from control hearts. To our knowledge, this is the first study which demonstrates that the regulatory proteins from normal hearts can upregulate cardiac myosin isolated from a pathologic rat model of diabetes. This diminished calcium sensitivity along with shifts in cardiac myosin heavy chain (V1V3) may be partially responsible for the impaired cardiac function in the hearts of chronic diabetic rats. (Mol Cell Biochem151: 165–172, 1995)     

肺炎合并心力衰竭患者血浆BNP,cTn1的变化及其临床意义

目的:探讨血浆脑钠肽(BNP)和心肌肌钙蛋白(cTn1)在肺炎合并心力衰竭患者血浆脑钠肽(BNP)和心肌肌钙蛋白(cTn1)的变化情况及、肺炎未合并心衰患者及健康对照组中的不同表达,探讨血浆脑钠肽(BNP)和心肌肌钙蛋白(cTn1)与疾病变化的关系及在肺炎合并心力衰竭中的临床诊断的意义.方法:回顾性分析我院自2010年1月至2012年1月收治的42例肺炎合并心衰患者,同期收治的肺炎末合并心衰患者34例为阳性对照组,以及同期在门诊进行体检的30例健康患者为阴性对照组.在入院后24h之内评估心脏功能并检测血浆BNP及cTn1水平变化,以及心衰合并肺炎患者入院24h急性期及心衰恢复期BNP和cTn1水平的变化,比较BNP和cTn1在的不同表达.结果:心衰组、阳性对照组、阴性对照组患者BNP(378.14,142.53,0.74±0.15)和cTn1 (0.84,0.32,0.18)比较,在统计学上具有显著性意义(H=140.67,H=30.14,P＜0.001).心衰急性期与心衰恢复期BNP(378.14,140.32)和cTn1(0.84,0.04)水平比较,在统计学上具有显著性差异(t=2.044,t=2.051,P＜ 0.05).结论:BNP与cTn1在肺炎合并心衰患者为高表达,显著高于肺炎未合并心衰患者,合并心衰与未合并心衰组的BNP与cTn1水平也显著高于健康对照组,表明BNP与cTn1的表达与病情呈正相关.且在肺炎合并心衰急性期的表达高于恢复期,表明BNP、cTnⅠ水平变化可为诊断患者病情严重程度及肺炎合并心衰为急性期或慢性提高依据,可以为早期心功能衰竭提高临床参考.     

Isoform-specific downregulation of peroxiredoxin in human failing myocardium

Peroxiredoxins (Prx) are a family of antioxidant thioredoxin or glutathione dependent peroxidases. The major functions of Prx comprise modulation of signalling cascades that apply hydrogen peroxide (H(2)O(2)) and cellular protection against oxidative stress. Nothing is known about Prx isoforms in human myocardium. We investigated the protein expression of Prx isoforms 1-6 in human non-failing (NF, donor hearts, n=6, male, age: 53.3+/-2.1 years) and failing myocardium (DCM, orthotopic heart transplantation, dilated cardiomyopathy, n=15, male, 57.0+/-1.7 years). In addition, we performed immunohistochemical stainings and measured Prx 4 mRNA expression levels (RNAse protection assay). The protein expression of Prx 1-2 was similar in NF and DCM. The protein expression of Prx 3-6 and the mRNA-expression of Prx 4 were decreased in DCM. Immunohistochemical analyses provided evidence that all Prx isoforms are present in cardiomyocytes and endothelial cells. Whereas Prx 1-5 staining was more pronounced in endothelial cells, Prx6 staining was more evident in cardiomyocytes. This study provides evidence that Prx are differentially regulated in DCM. The selective downregulation of peroxiredoxin 3-6 isoforms may point towards a subcellular specific dysregulation of the antioxidative defence during the development of DCM.     

Augmented phosphorylation of cardiac troponin I in hypertensive heart failure

An altered cardiac myofilament response to activating Ca(2+) is a hallmark of human heart failure. Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility and Ca(2+) sensitivity of cardiac muscle. cTnI can be phosphorylated by protein kinase A (PKA) at Ser(22/23) and protein kinase C (PKC) at Ser(22/23), Ser(42/44), and Thr(143). Whereas the functional significance of Ser(22/23) phosphorylation is well understood, the role of other cTnI phosphorylation sites in the regulation of cardiac contractility remains a topic of intense debate, in part, due to the lack of evidence of in vivo phosphorylation. In this study, we utilized top-down high resolution mass spectrometry (MS) combined with immunoaffinity chromatography to determine quantitatively the cTnI phosphorylation changes in spontaneously hypertensive rat (SHR) model of hypertensive heart disease and failure. Our data indicate that cTnI is hyperphosphorylated in the failing SHR myocardium compared with age-matched normotensive Wistar-Kyoto rats. The top-down electron capture dissociation MS unambiguously localized augmented phosphorylation sites to Ser(22/23) and Ser(42/44) in SHR. Enhanced Ser(22/23) phosphorylation was verified by immunoblotting with phospho-specific antibodies. Immunoblot analysis also revealed up-regulation of PKC-α and -δ, decreased PKCε, but no changes in PKA or PKC-β levels in the SHR myocardium. This provides direct evidence of in vivo phosphorylation of cTnI-Ser(42/44) (PKC-specific) sites in an animal model of hypertensive heart failure, supporting the hypothesis that PKC phosphorylation of cTnI may be maladaptive and potentially associated with cardiac dysfunction.     

Functional Effects of a Restrictive-Cardiomyopathy-Linked Cardiac Troponin I Mutation (R145W) in Transgenic Mice

The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an ∼ 13-16% increase in maximal Ca²⁺-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate (g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca²⁺ sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca²⁺] transients and increased time to peak force. Analysis of force and Ca²⁺ transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca²⁺ transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca²⁺ transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype.     

Calcium/calmodulin-dependent protein kinase I inhibits neuronal nitric-oxide synthase activity through serine 741 phosphorylation

We demonstrate here that neuronal nitric-oxide synthase (nNOS) is phosphorylated and inhibited by a constitutively active form of Ca²⁺/calmodulin (CaM)-dependent protein kinase I (CaM-K I1-293). Substitution of Ser⁷⁴¹ to Ala in nNOS blocked the phosphorylation and the inhibitory effect. Mimicking phosphorylation at Ser⁷⁴¹ by Ser to Asp mutation resulted in decreased binding of and activation by CaM, since the mutation was within the CaM-binding domain. CaM-K I1-293 gave phosphorylation of nNOS at Ser⁷⁴¹ in transfected cells, resulting in 60–70% inhibition of nNOS activity. Wild-type CaM-K I also did phosphorylate nNOS at Ser⁷⁴¹ in transfected cells, but either CaM-K II or CaM-K IV did not. These results raise the possibility of a novel cross-talk between nNOS and CaM-K I through the phosphorylation of Ser⁷⁴¹ on nNOS.     

Cardiac troponin I for predicting right ventricular dysfunction and intermediate risk in patients with normotensive pulmonary embolism

Background

Right ventricular dysfunction (RVD) and cardiac troponin I (cTnI) are important tools for risk stratification in pulmonary embolism (PE). We investigate the association of RVD and cTnI in normotensive PE patients and calculate a cTnI cut-off level for predicting RVD and submassive PE.

Methods

Clinical, laboratory, radiological and echocardiagraphic data were analysed. Patients were categorised into groups with or without RVD and compared focussing on cTnI. Effectiveness of cTnI for predicting RVD and submassive PE was tested.

Results

One hundred twenty-nine normotensive PE patients, 71 with and 58 without RVD, were included. Patients with RVD were older (75.0 years (61.3/81.0) vs. 66.0 years (57.7/75.1), P = 0.019). cTnI (0.06 ng/ml (0.02/0.23) vs. 0.01 ng/ml (0.00/0.03), P < 0.0001) and D-dimer values (2.00 mg/l (1.08/4.05) vs. 1.23 mg/l (0.76/2.26), P = 0.016) were higher in PE with RVD. cTnI was associated with RVD (OR 3.95; 95 % CI 1.95–8.02, p = 0.00014). AUC for cTnI diagnosing RVD was 0.79, and for submassive PE0.87. Cut-off values for cTnI predicting RVD and submassive PE were 0.01 ng/ml, with a negative predictive value of 73 %. cTnI was positively correlated with age, D-dimer and creatinine.

Conclusions

In normotensive PE patients, cTnI is helpful for risk stratification and excluding RVD. cTnI elevation is correlated with increasing age and reduced kidney function.     

Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca²⁺ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca²⁺-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca²⁺ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca²⁺ content. This abnormal Ca²⁺ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na⁺-Ca²⁺ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.     

心力衰竭超声指数联合血清肌钙蛋白I、脑钠肽对老年舒张性心力衰竭患者不良心血管事件的预测价值分析

摘要 目的：研究心力衰竭超声指数（HFEI）联合血清肌钙蛋白I（cTnI）、脑钠肽（BNP）对老年舒张性心力衰竭患者不良心血管事件（MACE）的预测价值。方法：将医院从2018年1月～2019年12月期间收治的80例老年舒张性心力衰竭患者纳入研究。将其按照随访1年结局的差异分作研究组（发生MACE）35例和对照组（未发生MACE）45例。检测并比较两组HFEI以及血清cTnI、BNP水平,同时分析两组基线资料以及心功能指标水平的差异。通过多因素Logistic回归分析明确MACE的相关影响因素,并以受试者工作特征（ROC）曲线分析HFEI以及血清cTnI、BNP联合预测MACE的效能。结果：研究组HFEI以及血清cTnI、BNP水平均高于对照组（均P＜0.05）。研究组年龄和左室舒张末期内径（LVEDd均高于对照组,而左室射血分数（LVEF）、二尖瓣血流频谱E峰和A峰比值（E/A）比值均低于对照组（均P＜0.05）。经多因素Logistic回归分析可得：HFEI以及血清cTnI、BNP水平均是老年舒张性心力衰竭患者MACE的独立危险因素（均OR＞1,P＜0.05）。经ROC曲线分析可得：HFEI以及血清cTnI、BNP联合预测MACE的曲线下面积、灵敏度、特异度、约登指数均高于上述三项指标单独检测。结论：HFEI以及血清cTnI、BNP在预测老年舒张性心力衰竭患者MACE方面的价值较高,值得临床推广应用。     

Immunohistochemical Differentiation of Fiber Types in Human Skeletal Muscle Using Monoclonal Antibodies to Slow and Fast Isoforms of Troponin I Subunit

The cDNA sequence of troponin I (TnI), one of the subunits of the skeletal muscle regulatory protein, differs between slow-twitch muscle and fast-twitch muscle. We prepared monoclonal antibodies td the slow and fast isoforms of human TnI for the purpose of differentiating muscle fiber types in human neuromuscular disorders. Slow TnI antibody was labeled with tetramethylrhodamine isothiocyanate while fast TnI antibody was labeled with fluorescein isothiocyanate; then these two antibodies were mixed. This mixture was then used to stain biopsied muscle from patients with neuromuscular disorders. It was possible to differentiate muscle fibers into slow, fast and intermediate fibers having various contents of slow and fast TnI. In tissue composed of small muscle fibers, this method facilitated differentiation of types of muscle fibers by allowing staining of only a single section. The usefulness of our technique using slow and fast TnI antibodies is discussed in comparison with ATPase staining. Because our staining method can distinguish slow and fast fiber components, it is useful for clinical application.