Molecular cloning of rat cardiac troponin I and analysis of troponin I isoform expression in developing rat heart

We have isolated and sequenced a cDNA encoding rat cardiac troponin I. The predicted amino acid sequence was highly identical with previously reported chemically derived amino acid sequences for rabbit and bovine cardiac troponin I. Clones for slow skeletal muscle troponin I were also obtained from neonatal rat cardiac ventricle by the polymerase chain reaction. The nucleotide sequences of these clones were determined to be more than 99% identical with a previously reported rat slow skeletal troponin I cDNA [Koppe et al. (1989) J. Biol. Chem. 264, 14327-14333]. The troponin I clones hybridized to RNA from the appropriate muscle from adult animals. However, RNA from fetal and neonatal rat heart also hybridized with the slow skeletal troponin I cDNA, demonstrating its expression in fetal and neonatal rat heart. Slow skeletal troponin I steady-state mRNA levels decreased with increasing age, but cardiac troponin I mRNA levels increased through fetal and early neonatal cardiac development. Thus, during fetal and neonatal development, slow skeletal and cardiac troponin I isoforms are coexpressed in the rat heart and regulated in opposite directions. The degree of primary sequence differences in these isoforms, especially at phosphorylation sites, may result in important functional differences in the neonatal myocardium.     

Troponin T switching in the developing rat heart

A monoclonal antibody specific for cardiac troponin T has been used to investigate troponin changes during development in the rat heart. Specificity of the antibody was determined by immunoblot analysis with purified bovine cardiac troponin. In the rat heart, immunoblot analysis shows that anticardiac troponin T reacts with a 42.5-kDa band in fetal ventricles and with a 41-kDa band in adult ventricles. The faster migrating troponin T is present in traces in the fetal heart and increases markedly during the first 2 weeks after birth, concomitantly with the progressive decrease of the slower migrating form that is no longer detectable in the adult. The pattern of reactivity of the monoclonal antibody is not modified by alkaline phosphatase pretreatment, suggesting that the antibody is not specific for a phosphorylated epitope. Conditions known to affect cardiac myosin composition, such as hypothyroidism and hypertrophy secondary to systemic hypertension, do not change the troponin T isoform profile of adult rat ventricles. The expression and accumulation of the adult isoforms of troponin T are not suppressed by propylthiouracil treatment of pregnant and nursing rats.     

Troponin I switching in the developing heart

Monoclonal antibodies identify two distinct isoforms of troponin I in rat cardiac muscle, one predominant in the embryonic and fetal heart and one predominant in the adult heart. The two isoforms can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with apparent molecular weights of 27,000 and 31,500, respectively. The adult isoform is specifically recognized by a monoclonal antibody that is unreactive with the embryonic variant, while two other monoclonal antibodies recognize both isoforms. A monoclonal antibody to cardiac troponin T was used to isolate by affinity chromatography the troponin complex from adult and neonatal rat heart. Affinity purified troponin from neonatal heart was found to contain both the embryonic and adult isoforms of troponin I. Comparative immunoblotting analysis with different muscle tissues shows that embryonic troponin I is identical with respect to electrophoretic mobility and pattern of immunoreactivity to the major troponin I isoform found in adult slow skeletal muscle. Troponin I switching may be implicated in developmental changes involving Ca2+ and pH sensitivity of the contractile system and response to beta-adrenergic stimulation.     

A cardiac troponin T epitope conserved across phyla.

Troponin T is a thin filament protein that is important in regulating striated muscle contraction. We have raised a monoclonal antibody against rabbit cardiac troponin T, monoclonal (mAb) 13-11, that recognizes its epitope in cardiac troponin T isoforms from fish, bird, and mammal but not from frog. The number of these isoforms expressed in cardiac muscle varies among species and during development. Cardiac troponin T isoforms were not found in adult skeletal muscle, while they were expressed transiently in immature skeletal muscle. We have mapped the epitope recognized by mAb 13-11 using rabbit cardiac troponin T isoforms. Analysis of stepwise cyanogen bromide digestion, which allowed association of the epitope to regions spanning methionine residues, coupled with immunoactivity of synthetic peptides, corresponding to sequences containing methionine residues, indicated that mAb 13-11 recognized its epitope in a 17-residue sequence containing the methionine at position 68, SKPKPRPFMPNLVPPKI. Comparison of skeletal and cardiac troponin T sequences suggested that the epitope was contained within the sequence FMPNLVPPKI. Synthetic peptides PFMPNLVPPKI and FMPNLVPPKI were recognized by mAb 13-11 on slot-blots. Enzyme-linked immunosorbent assay demonstrated mAb 13-11 recognized, in order of descending affinity, the 17-, 11-, and 10-residue sequence. Preabsorption of mAb 13-11 with each of these sequences blocked the recognition of the 17-residue peptide by mAb 13-11. The domain, PFMPNLVPPKI is encoded by the 5' region of the cardiac gene exon 10 and is present in hearts across a broad range of phyla. These findings suggest that this cardiac troponin T-specific sequence confers onto myofilaments structural and functional properties unique to the heart.     

牛心肌钙蛋白T的纯化

以小牛心肌为原料,用匀浆提取、热处理、硫酸铵沉淀、ＤＥＡＥ—纤维素柱层析纯化了牛心肌钙蛋白Ｔ（ｃＴｎＴ）。纯化的蛋白在ＳＤＳ—聚丙烯酰胺凝胶电泳上为一条带,分子量为３７,０００道尔顿。用ＴｒｏｐｏｎｉｎＴ快速半定量试纸条测定该蛋白,呈现强阳性反应。这说明我们纯化的蛋白为牛心肌钙蛋白Ｔ。     

Immunological characterization of the mitochondrial 2-oxoglutarate carrier from liver and heart. Organ specificity

Antibodies have been prepared against the 2-oxoglutarate transport proteins purified from bovine heart and rat liver mitochondria. The anti-heart antiserum cross-reacts with the 2-oxoglutarate carrier (OGC) from beef, pig, rat and rabbit heart, but not with the OGC from liver of the same animals. Conversely, the anti-liver antiserum recognizes the carrier protein from liver of all species tested but not from heart. Immunoinactivation of oxoglutarate transport activity by the antibodies is also tissue specific. Peptide maps of purified OGC show structural differences between the carrier from heart and liver of the same animal species. These results indicate the existence of isoforms of the OGC in heart and liver.     

Toad Heart Utilizes Exclusively Slow Skeletal Muscle Troponin T: AN EVOLUTIONARY ADAPTATION WITH POTENTIAL FUNCTIONAL BENEFITS

The three isoforms of vertebrate troponin T (TnT) are normally expressed in a muscle type-specific manner. Here we report an exception that the cardiac muscle of toad (Bufo) expresses exclusively slow skeletal muscle TnT (ssTnT) together with cardiac forms of troponin I and myosin as determined using immunoblotting, cDNA cloning, and/or LC-MS/MS. Using RT-PCR and 3'- and 5'-rapid amplification of cDNA ends on toad cardiac mRNA, we cloned full-length cDNAs encoding two alternatively spliced variants of ssTnT. Expression of the cloned cDNAs in Escherichia coli confirmed that the toad cardiac muscle expresses solely ssTnT, predominantly the low molecular weight variant with the exon 5-encoded NH(2)-terminal segment spliced out. Functional studies were performed in ex vivo working toad hearts and compared with the frog (Rana) hearts. The results showed that toad hearts had higher contractile and relaxation velocities and were able to work against a significantly higher afterload than that of frog hearts. Therefore, the unique evolutionary adaptation of utilizing exclusively ssTnT in toad cardiac muscle corresponded to a fitness value from improving systolic function of the heart. The data demonstrated a physiological importance of the functional diversity of TnT isoforms. The structure-function relationship of TnT may be explored for the development of new treatment of heart failure.     

Structure-function relationships in cardiac troponin T

Regions of rabbit and bovine cardiac troponin T that are involved in binding tropomyosin, troponin C and troponin I have been identified. Two sites of contact for tropomyosin have been located, situated between residues 92-178 and 180-284 of troponin T. A cardiac-specific binding site for troponin I has been identified between residues 1-68 of cardiac troponin T, within a region of the protein that has previously been shown to be encoded by a series of exons that are expressed in a tissue-specific and developmentally regulated manner. The binding site for troponin C is located between residues 180-284 of cardiac troponin T. When isolated from fresh bovine hearts, cardiac troponin T contained 0.21 +/- 0.11 mol phosphate per mol; incubation with phosphorylase kinase increased the phosphate content to approx. 1 mol phosphate per mol. One site of phosphorylation was identified as serine-1; a second site of phosphorylation was located within peptide CB3 (residues 93-178) and has been tentatively identified as serine-176. Addition of troponin C to cardiac troponin T does not inhibit the phosphorylation of this latter protein that is catalysed by phosphorylase b kinase.     

Role of troponin I isoform switching in determining the pH sensitivity of Ca(2+) regulation in developing rabbit cardiac muscle

Skinned muscle fibers prepared from fetal rabbit heart (28 days of gestation) showed a marked resistance to acidic pH in the Ca(2+) regulation of force generation, compared to the fibers prepared from adult heart. SDS-PAGE and immunoblot analysis showed that the slow skeletal troponin I was predominantly expressed in the fetal cardiac muscle, while the cardiac isoform was predominantly expressed in the adult cardiac muscle. Direct exchange of purified slow skeletal and cardiac troponin I isoforms into these skinned muscle fibers revealed that cardiac troponin I made the Ca(2+) regulation of contraction sensitive to acidic pH just as in the adult fibers, whereas slow skeletal troponin I made the Ca(2+) regulation of contraction resistant to acidic pH just as in the fetal fibers. These results demonstrate that the troponin I isoform switching accounts fully for the change in the pH dependence of Ca(2+) regulation of contraction in developmental cardiac muscle.     

Bovine cardiac troponin T: amino acid sequences of the two isoforms

Troponin T (TnT) is the tropomyosin-binding subunit of troponin, the thin filament regulatory complex that confers calcium sensitivity to striated muscle contraction and actomyosin ATPase activity. Bovine cardiac muscle contains two isoforms (TnT-1 and TnT-2) of TnT that differ in sequence near their amino termini. Thin filaments containing TnT-2 require less calcium to activate the MgATPase rate of myosin than do thin filaments containing TnT-1. Using whole troponin T purified from adult bovine cardiac muscle, we have determined the complete amino acid sequence of the larger, more abundant isoform TnT-1. We confirmed that sequence differences between TnT-1 and TnT-2 are confined to the amino-terminal regions and found that TnT-1 makes up approximately 75% of the total troponin T isolated. Partial sequencing of the separated isoforms showed that the difference between them is due solely to residues 15-19 (Glu-Ala-Ala-Glu-Glu) of TnT-1 being absent from TnT-2. The deleted segment may correspond to the product of exon 4 of the chicken cardiac TnT gene [Cooper, T.A., & Ordahl, C.P. (1985) J. Biol. Chem. 260, 11140-11148]. Exon 5, which is developmentally regulated in the chicken, is not expressed in either TnT-1 or TnT-2. TnT-1 contains 284 amino acid residues and has a Mr of 33,808, while TnT-2 contains 279 amino acid residues and has a Mr of 33,279. Bovine cardiac TnT contains the only known thiol group in any isolated TnT (Cys-39 of TnT-1, Cys-34 of TnT-2). Comparison of bovine, rabbit, and chicken cardiac TnT sequences shows near identity of the amino-terminal 13 amino acid residues (exons 2 and 3 of the chicken cardiac gene), many differences in the following 60 residues (exons 4-8), and great similarity in the C-terminal 230 residues (exons 9-18).     

Rat liver mitochondria contain two immunologically distinct dihydrolipoamide dehydrogenases

We have raised antisera against dihydrolipoamide dehydrogenase. One antigen was isolated from purified bovine kidney pyruvate dehydrogenase complex (PDC). The other antigen was a commercial preparation of porcine heart dihydrolipoamide dehydrogenase (E3) which did not first involve purification of the alpha-keto acid dehydrogenase complex(es). Both antibody preparations cross-reacted with the E3 components of PDC, alpha-ketoglutarate dehydrogenase complex, and branched-chain keto acid dehydrogenase complex. This demonstrates the immunological identity of the E3 components. These sera totally precipitated E3 activity from the purified complexes, from purified preparations of E3, and from extracts of rat heart and kidney mitochondria. The two sera vary in their reaction with rat liver mitochondrial extracts: the anti PDC-E3 serum left residual E3 activity (approximately 50% of the original) that was precipitable by the anti-E3 anti-serum. This indicates that liver contains two immunologically distinct forms of E3. Metabolic assays measuring the differential effects of the two sera on the glycine decarboxylation reaction suggest that the form which is immunologically nonreactive with the anti-PDC-E3 serum could represent the E3 involved in the glycine cleavage system.     

Replacement of three troponin components with cardiac troponin components within single glycerinated skeletal muscle fibers.

The tension of single glycerinated rabbit skeletal muscle fiber was desensitized to a Ca(2+)-concentration after treatment with an excessive amount of bovine cardiac troponin T and reached a level of about 70% of the maximum tension of the untreated fiber. A SDS-gel electrophoretic examination indicated that troponin C.I.T complex in the fiber was replaced with the added cardiac troponin T. The Ca(2+)-sensitivity of the tension of the troponin T-treated fiber was then recovered by the addition of bovine cardiac troponins I and C. The rabbit skeletal muscle fiber thus hybridized with bovine cardiac troponin C.I.T showed the same cooperativity of Ca(2+)-activation as the cardiac muscle.     

Polyclonal Antisera to the Individual Neurofilament Triplet Protdins: A Characterization Using ELISA and Immunoblotting

In this article, the preparation and characterization of polyclonal rabbit antisera against the individual polypeptides of bovine neurofilament (68, 150, and 200 kilodaltons) is described. Selected antisera against the 68- and 150-kilodalton neurofilament polypeptides were specific for the corresponding antigen in homogenates of bovine, rat, and human brain as judged by immunoblots. The antisera against the 200-kilodalton neurofilament polypeptide cross-reacted to some extent with the 150-kilodalton neurofilament polypeptide, especially with the human antigen. The most specific antisera were used to develop an enzyme-linked immunosorbent assay (ELISA), and the cross-reactivities between the antisera and the different bovine and rat neurofilament polypeptides were determined. Contrary to the results in the immunoblots, the antiserum against the 200-kilodalton neurofilament polypeptide was subunit-specific, as was the 150-kilodalton antiserum. The 68-kilodalton antiserum displayed a minute cross-reactivity against bovine 150- and 200-kilodalton neurofilaments, but it cross-reacted somewhat more with the rat 150- and 200-kilodalton antigens. Even so, the subunit specificity of the antisera is high enough to enable the development of a quantitative ELISA for determination of the individual bovine or rat neurofilament polypeptides in a mixture. This study is the necessary preparation for such an assay.     

Immunological characterization of bovine lysyl oxidase

Antibodies to homogeneously purified bovine aortic lysyl oxidase were prepared in chickens. The chicken anti-lysyl oxidase antiserum effectively inhibited bovine aortic lysyl oxidase activity. Non-immune antiserum from chickens, goats and humans was found to enhance bovine aortic lysyl oxidase activity, while non-immune rabbit serum inhibited enzyme activity. A competitive ELISA was developed to monitor immunoreactive lysyl oxidase during purification. Chromatography of bovine lysyl oxidase on Sephacryl S-200, the final step in purification, revealed two peaks of immunoreactive lysyl oxidase. The large molecular weight peak appears to contain inactive multimeric forms of the enzyme.     

Cardiac troponin T isoforms affect the Ca2+ sensitivity and inhibition of force development. Insights into the role of troponin T isoforms in the heart

At least four isoforms of troponin T (TnT) exist in the human heart, and they are expressed in a developmentally regulated manner. To determine whether the different N-terminal isoforms are functionally distinct with respect to structure, Ca(2+) sensitivity, and inhibition of force development, the four known human cardiac troponin T isoforms, TnT1 (all exons present), TnT2 (missing exon 4), TnT3 (missing exon 5), and TnT4 (missing exons 4 and 5), were expressed, purified, and utilized in skinned fiber studies and in reconstituted actomyosin ATPase assays. TnT3, the adult isoform, had a slightly higher alpha-helical content than the other three isoforms. The variable region in the N terminus of cardiac TnT was found to contribute to the determination of the Ca(2+) sensitivity of force development in a charge-dependent manner; the greater the charge the higher the Ca(2+) sensitivity, and this was primarily because of exon 5. These studies also demonstrated that removal of either exon 4 or exon 5 from TnT increased the cooperativity of the pCa force relationship. Troponin complexes reconstituted with the four TnT isoforms all yielded the same maximal actin-tropomyosin-activated myosin ATPase activity. However, troponin complexes containing either TnT1 or TnT2 (both containing exon 5) had a reduced ability to inhibit this ATPase activity when compared with wild type troponin (which contains TnT3). Interestingly, fibers containing these isoforms also showed less relaxation suggesting that exon 5 of cardiac TnT affects the ability of Tn to inhibit force development and ATPase activity. These results suggest that the different N-terminal TnT isoforms would produce different functional properties in the heart that would directly affect myocardial contraction.     

Purification and characterization of hormone-regulated isoforms of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovaries

The regulatory subunit (R-II) of cAMP-dependent protein kinase type II is induced in rat ovarian granulosa cells by the synergistic actions of estradiol and follicle-stimulating hormone. The R-II from rat ovaries was compared with R-II from rat heart, rat brain, bovine heart, and bovine brain using immunological methods, 8-N3[32P]cAMP photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three isoforms of R-II were identified in rat ovarian cell extract (R-II54 Mr = 54,000, R-II52 Mr = 52,000, R-II51 Mr = 51,000), two isoforms of R-II in rat brain cell extract (Mr = 54,000, Mr = 52,000), and one isoform of R-II in rat heart cell extract (Mr = 54,000). Rat ovarian R-II54, heart R-II, and brain R-II (Mr = 54,000) were recognized by antiserum against rat heart R-II, whereas rat ovarian R-II52/R-II51 and rat brain R-II (Mr = 52,000) were not. In contrast, an antiserum raised against bovine heart R-II recognized all three isoforms of ovarian R-II as well as the lower molecular weight form of rat brain R-II. Ovarian types R-II52 and R-II51 but not R-II54 were increased selectively in granulosa cells by estradiol and follicle-stimulating hormone. In addition: 1) ovarian R-II52/51 subunits were purified to homogeneity and shown to recombine with C subunit from bovine heart to form a cAMP-dependent protein kinase; 2) pure R-II52/51 were not interconvertible to a higher molecular weight form by C subunit-dependent phosphorylation; 3) pure rat heart R-II (Mr = 54,000) and ovarian R-II52/51 exhibited distinct differences based on one- and two-dimensional peptide mapping; and 4) by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis pure R-II52/51 were resolved as three (rather than two) isoelectric variants which were clearly different from pure rat heart R-II54. Thus, the hormone-regulated form of R-II in rat ovarian granulosa cells appears to represent a gene product distinct from R-II54 in rat heart.     

Cross-reactivity of metallothioneins from different origins with rabbit anti-rat hepatic metallothione in antibody.

Rat hepatic Cd-metallothionein was purified and isolated into its two components, metallothionein 1 and 2, by disc electrophoresis. Antibodies to metallothionein 2 were generated in rabbits. The antiserum reacted with the protein and formed a single precipitin band on a double diffusion plate. By ammonium sulfate precipitations, it was found that the antiserum cross-reacted with rat hepatic metallothionein 1. Cross-reactivity of the antiserum was also observed for components of rat renal Cd-metallothionein, rabbit hepatic Cd-metallothionein and human renal metallothionein.     

Proteomic changes in the heart of diet-induced pre-diabetic mice

The development of type 2 diabetes (T2D) is strongly associated with obesity. In humans, T2D increases the risk for end organ complications. Among these, heart disease has been ranked as the leading cause of death. We used a proteomic methodology to test the hypothesis that a pre-diabetic state generated by high-fat diet leads to changes in proteins related to heart function and structure. Over 300 protein spots were resolved by two-dimensional gel electrophoresis (2-DE). Fifteen protein spots were found to be altered (7 decreased and 8 increased) in pre-diabetic hearts. The protein spots were then identified by mass spectrometry and immunoblots. Among the decreased proteins, 3 are involved in heart structure (one isoform of desmin, troponin T2 and α-cardiac actin), 3 are involved in energy metabolism (mitochondrial ATP synthase β subunit, adenylate kinase and creatine kinase) and one is a component of the citric acid cycle (isocitrate dehydrogenase 3). In contrast, proteins involved in fatty acid oxidation (two isoforms of peroxisomal enoyl-CoA hydratase) and the citric acid cycle (three isoforms of malate dehydrogenase) were increased in pre-diabetic hearts. The results suggest that changes in the levels of several heart proteins may have implications in the development of the cardiac phenotype associated to T2D.     

Immunofluorescent localization of 100K coated vesicle proteins

A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.     

Co-expression of skeletal and cardiac troponin T decreases mouse cardiac function

In contrast to skeletal muscles that simultaneously express multiple troponin T (TnT) isoforms, normal adult human cardiac muscle contains a single isoform of cardiac TnT. To understand the significance of myocardial TnT homogeneity, we examined the effect of TnT heterogeneity on heart function. Transgenic mouse hearts overexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT was investigated in vivo and ex vivo as an experimental system of concurrent presence of two classes of TnT in the adult cardiac muscle. This model of myocardial TnT heterogeneity produced pathogenic phenotypes: echocardiograph imaging detected age-progressive reductions of cardiac function; in vivo left ventricular pressure analysis showed decreased myocardial contractility; ex vivo analysis of isolated working heart preparations confirmed an intrinsic decrease of cardiac function in the absence of neurohumoral influence. The transgenic mice also showed chronic myocardial hypertrophy and degeneration. The dominantly negative effects of introducing a fast TnT into the cardiac thin filaments to produce two classes of Ca(2+) regulatory units in the adult myocardium suggest that TnT heterogeneity decreases contractile function by disrupting the synchronized action during ventricular contraction that is normally activated as an electrophysiological syncytium.