Cooperative Interaction between Developmentally Regulated Troponin T and Tropomyosin Isoforms in the Absence of F-actin

119

137

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 ␣-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.

Section: Discussionmentioning

confidence: 99%

Cardiac Troponin T Isoforms Affect the Ca2+Sensitivity and Inhibition of Force Development

Gomes¹,

Guzman

Zhao

et al. 2002

119

137

“…All cTnT isoforms eluted in the same fractions suggesting that the affinities of the cTnT isoforms for cTnC are not significantly different from each other (data not shown). A previous report (38) has shown that the higher molecular weight acidic fast twitch skeletal TnT (fsTnT) bound to ␣/␤-Tm with a greater affinity than the lower molecular weight basic fsTnT (adult isoform). TnT is involved in activating the actomyosin ATPase to levels greater than that of actomyosin-Tm alone.…”

Section: ϫ015) Than Intact Fibers Similar To What Has Been Reported mentioning

confidence: 99%

Cardiac Troponin T Isoforms Affect the Ca2+ Sensitivity of Force Development in the Presence of Slow Skeletal Troponin I

Gomes¹,

Venkatraman

Davis

et al. 2004

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca 2؉ sensitivity of force development compared with cTnT3-and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca 2؉ was released from site II of cTnC in the cTnI⅐cTnC complex (122/s) was 12.5-fold faster than for the ssTnI⅐cTnC complex (9.8/s). Addition of cTnT3 to the cTnI⅐cTnC complex resulted in a 3.6-fold decrease in the Ca 2؉ dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI⅐cTnC complex resulted in a 1.9-fold increase in the Ca 2؉ dissociation rate from site II of cTnC. The rate at which Ca 2؉ dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca 2؉ dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.

“…The purification protocols for TnC, TnI, TnT, and ␣/␤-Tm from chicken skeletal muscle have been described previously, and the purity of these preparations have been demonstrated (17,18). Concentrations for all protein preparations were determined by the Nanoorange protein quantitation kit (Molecular Probes, Eugene, OR) with the exception of TnC, which was determined by absorption, assuming an extinction coefficient of 0.132 at 259 nm for a 1-mg/ml solution.…”

Section: Reverse Transcription (Rt)-pcr Cloning Of Mouse Nebulin Andmentioning

confidence: 99%

“…Hybridomas were selected by HAT (0.1 mM hypoxanthine, 0.4 M aminopterin, and 16 M thymidine) media containing 20% fetal bovine serum and screened by indirect enzyme-linked immunosorbent assay against coated CN5 using horseradish peroxidase-labeled goat anti-mouse total immunoglobulin (Sigma) as the second antibody. The positive hybridoma culture supernatants were verified by Western blotting against the CN5 protein using alkaline phosphatase-labeled goat anti-mouse total immunoglobulin second antibody (Sigma) as described previously (17). The anti-CN5 antibodysecreting hybridomas were subcloned three times by limiting the dilution using young BALB/c mouse spleen cells as a feeder to establish stable cell lines.…”

Section: Reverse Transcription (Rt)-pcr Cloning Of Mouse Nebulin Andmentioning

confidence: 99%

See 1 more Smart Citation

Interactions between Nebulin-like Motifs and Thin Filament Regulatory Proteins

Ogut

Hossain

Jin

2003

Self Cite

Nebulin (600 -900 kDa) and nebulette (107-109 kDa) are two homologous thin filament-associated proteins in skeletal and cardiac muscles, respectively. Both proteins are capped with a unique region at the amino terminus as well as a serine-rich linker domain and SH3 domains at the COOH terminus. Their significant size difference is attributed to the length of the central region wherein both proteins are primarily composed of ϳ35 amino acid repeats termed nebulin-like repeats or motifs. These motifs are marked by a conserved SXXXY sequence and high affinity binding to F-actin. To further characterize the effects that nebulin-like proteins may have on the striated muscle thin filament, we have cloned, expressed, and purified a five-motif chicken nebulette fragment and tested its interaction with the thin filament regulatory proteins. Both tropomyosin and troponin T individually bound the nebulette fragment, although the affinity of this interaction was significantly increased when tropomyosin-troponin T was tested as a binary complex. The addition of troponin I to the tropomyosin-troponin T complex decreased the binding to the nebulette fragment, indicating an involvement of the conserved T2 region of troponin T in this interaction. F-actin cosedimentation demonstrated that the nebulette fragment was able to significantly increase the affinity of the tropomyosin-troponin assembly for F-actin. The relationships provide a means for nebulin-like motifs to participate in the allosteric regulation of striated muscle contraction.Nebulin (600 -900 kDa) and nebulette (107-109 kDa) are two homologous thin filament-associated proteins in skeletal and cardiac muscles, respectively (1, 2). cDNA sequences of nebulin and nebulette have shown that both proteins are primarily composed of ϳ35 amino acid repeats termed nebulin-like repeats or motifs, each marked by a conserved SXXXY sequence (2, 3). Both nebulin and nebulette are capped by a unique region at the amino terminus in addition to serine-rich linker and SH3 domains at the carboxyl terminus (2-6). Immunoelectron microscopy has demonstrated that a single nebulin molecule extends from the Z-line (COOH terminus of the protein) to the pointed ends of the thin filament at its NH 2 terminus (7).Nebulette is similarly anchored to the Z-line through its COOH terminus, but because of its relatively smaller size, it does not extend as far as the pointed ends of the actin filament. Experiments using in vitro systems have formed a consensus that nebulin-like motifs from both nebulin and nebulette associate with F-actin (4, 6, 8). Additional experiments have extended this observation to demonstrate that nebulin-like motifs may interact directly with tropomyosin (Tm) 1 and troponin (Tn) (4) along the striated muscle thin filament.At the Z-line, the nebulin/nebulette interactions are probably mediated by contributions from the linker domain as well as interactions of the SH3 domains with ␣-actinin (6, 9). However, results from Ojima and coworkers (10) suggest that neither the serine-rich...