Ca 2+ activation of myofilaments from transgenic mouse hearts expressing R92Q mutant cardiac troponin T

The effects of Troponin T (TnT) mutants R141W and ⌬K210, the only two currently known mutations in TnT that cause dilated cardiomyopathy(DCM) independent of familial hypertrophic cardiomyopathy (FHC), and TnT-K273E, a mutation that leads to a progression from FHC to DCM, were investigated. Studies on the Ca 2؉ sensitivity of force development in porcine cardiac fibers demonstrated that TnT-⌬K210 caused a significant decrease in Ca 2؉ sensitivity, whereas the TnT-R141W did not result in any change in Ca 2؉ sensitivity when compared with human cardiac wild-type TnT (HCWTnT). TnT-⌬K210 also caused a decrease in maximal force when compared with HCWTnT and TnT-R141W. In addition, the TnT-⌬K210 mutant decreased maximal ATPase activity in the presence of Ca 2؉ . However, the TnT-K273E mutation caused a significant increase in Ca 2؉ sensitivity but behaved similarly to HCWTnT in actomyosin activation assays. Inhibition of ATPase activity in reconstituted actin-activated myosin ATPase assays was similar for all three TnT mutants and HCWTnT. Additionally, circular dichroism studies suggest that the secondary structure of all three TnT mutants was similar to that of the HCWTnT. These results suggest that a rightward shift in Ca 2؉ sensitivity is not the only determinant for the phenotype of DCM.

Section: Discussionmentioning

confidence: 99%

Different Functional Properties of Troponin T Mutants That Cause Dilated Cardiomyopathy

Venkatraman

Harada

Gomes

et al. 2003

“…Quantification of cTn Exchange by Western Blot-Recombinant TNT in the present study included an NH 2 terminus myc tag to allow for quantification of the amount of cTn exchange by Western blotting techniques (17,18). Previously, our group has demonstrated that the presence of this myc tag does not affect myofilament function (17).…”

Section: Methodsmentioning

confidence: 99%

“…The expression and purification of the recombinant human cardiac TNT containing an NH 2 -terminal myc tag was carried out as previously described with slight modification of the purification protocol (17,18). Reconstitution and purification of intact Tn complex was carried out by sequential dialysis to remove urea and decrease salt of an equimolar amount of purified cTn components and purification using Resource-Q (1 ml; Amersham Biosciences) (17,18).…”

Section: Methodsmentioning

confidence: 99%

Increased Cross-bridge Cycling Kinetics after Exchange of C-terminal Truncated Troponin I in Skinned Rat Cardiac Muscle

Tachampa

Kobayashi

Wang

et al. 2008

Self Cite

The precise mechanism of cardiac troponin I (cTnI) proteolysis in myocardial stunning is not fully understood. Accordingly, we determined the effect of cTnI C terminus truncation on chemo-mechanical transduction in isolated skinned rat trabeculae. Recombinant troponin complex (cTn), containing either mouse cTnI-(1-193) or human cTnI-(1-192) was exchanged into skinned cardiac trabeculae; Western blot analysis confirmed that 60 -70% of the endogenous cTn was replaced by recombinant Tn. Incorporation of truncated cTnI induced significant reductions (ϳ50%) in maximum force and cooperative activation as well as increases (ϳ50%) in myofilament Ca 2؉ sensitivity and tension cost. Similar results were obtained with either mouse or human truncated cTn. Presence of truncated cTnI increased maximum actin-activated S1 ATPase activity as well as its Ca 2؉ sensitivity in vitro. Partial exchange (50%) for truncated cTnI resulted in similar reductions in maximum force and cooperativity; tension cost was increased in proportion to truncated cTnI content. In vitro, to determine the molecular mechanism responsible for the enhanced myofilament Ca 2؉ sensitivity, we measured Ca 2؉binding to cTn as reported using a fluorescent probe. Incorporation of truncated cTnI did not affect Ca 2؉ binding affinity to cTn alone. However, when cTn was incorporated into thin filaments, cTnI truncation induced a significant increase in Ca 2؉ binding affinity to cTn. We conclude that cTnI truncation induces depressed myofilament function. Decreased cardiac function after ischemia/reperfusion injury may directly result, in part, from proteolytic degradation of cTnI, resulting in alterations in cross-bridge cycling kinetics.In experiments described here, we investigated the functional significance of losing the C terminus region of cardiac TnI as found in myocardial stunning. Myocardial stunning is a form of postischemic dysfunction that persists after restoration of normal coronary flow in the absence of irreversible damage. It has become increasingly evident that myocardial stunning may contribute significantly to the morbidity associated with coronary artery disease (1). As such, elucidation of molecular mechanisms underlying myocardial stunning is critical to aid in the development of novel therapeutic strategies. Proteolytic degradation of cardiac cTn I (cTnI) 2 has emerged has a potential cellular mechanism underlying the depressed contractile function seen in myocardial stunning, possibly as the result of Ca 2ϩ activation of the protease calpain-I upon reperfusion (1) and/or increased mechanical load on the heart (2). However, these findings are species-dependent (i.e. no degradation of cTnI was observed in larger animal models of myocardial stunning, such as the pig (3) or dog (4)). In addition, although application of activated calpain-I to rodent skinned myocardium results in depressed myofilament function (5, 6), this effect is reported to be correlated with the degradation of cTnI in some (5) but not all studies (6). Importantly, cTnI degra...

“…Western Blot Analysis of Recombinant Troponin ExchangeThe Western blot method to measure the extent of recombinant troponin exchange has been previously reported (40). Briefly, isolated skinned myocytes were exchanged overnight with recombinant troponin and after twice resuspending in relaxing solution myocytes were solubilized in 8 M Tris-urea denaturing buffer, and Laemmli buffer was added.…”

Section: Methodsmentioning

confidence: 99%

“…The exchange efficiency was calculated as the percentage ratio of exogenous myc-cTnT (top band) relative to total cTnT (top ϩ bottom band), which was quantified using the ImageJ gel densitometry analysis toolkit. The presence of the myc-tag on cTnT has been shown to not affect the cardiac force- [Ca 2ϩ ] relationship (40). Isometric Force Measurement-Permeabilized (skinned) myocytes in relaxing solution were placed in a 0.1% BSA-coated 35-mm culture dish on the stage of an inverted microscope (Leica DMIRB).…”

Section: Methodsmentioning

confidence: 99%

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation

Kumar

Govindan

Zhang

et al. 2015

Self Cite

Background: Myofilament length-dependent activation (LDA) is modulated by phosphorylation. Results: Myosin binding protein C (MyBP-C) constitutes the dominant molecular mechanism underlying phosphorylation, with a minor and independent contribution of cardiac troponin-I (cTnI). Conclusion: MyBP-C, and to a lesser extent cTnI, both contribute to enhance LDA. Significance: Novel insights into the molecular basis LDA and its regulation by phosphorylation.