Lys184 deletion in troponin I impairs relaxation kinetics and induces hypercontractility in murine cardiac myofibrils.

AIMS: To understand the functional consequences of the Lys184 deletion in murine cardiac troponin I (mcTnI(DeltaK184)), we have studied the primary effects of this mutation linked to familial hypertrophic cardiomyopathy (FHC) at the sarcomeric level. METHODS AND RESULTS: Ca(2+) sensitivity and kinetics of force development and relaxation were investigated in cardiac myofibrils from transgenic mice expressing mcTnI(DeltaK184), as a model which co-segregates with FHC. Ca(2+)-dependent conformational changes (switch-on/off) of the fluorescence-labelled human troponin complex, containing either wild-type hcTnI or mutant hcTnI(DeltaK183), were investigated in myofibrils prepared from the guinea pig left ventricle. Ca(2+) sensitivity and maximum Ca(2+)-activated and passive forces were significantly enhanced and cooperativity was reduced in mutant myofibrils. At partial Ca(2+) activation, mutant but not wild-type myofibrils displayed spontaneous oscillatory contraction of sarcomeres. Both conformational switch-off rates of the incorporated troponin complex and the myofibrillar relaxation kinetics were slowed down by the mutation. Impaired relaxation kinetics and increased force at low [Ca(2+)] were reversed by 2,3-butanedione monoxime (BDM), which traps cross-bridges in non-force-generating states. CONCLUSION: We conclude that these changes are not due to alterations of the intrinsic cross-bridge kinetics. The molecular mechanism of sarcomeric diastolic dysfunction in this FHC model is based on the impaired regulatory switch-off kinetics of cTnI, which induces incomplete inhibition of force-generating cross-bridges at low [Ca(2+)] and thereby slows down relaxation of sarcomeres. Ca(2+) sensitization and impairment of the relaxation of sarcomeres induced by this mutation may underlie the enhanced systolic function and diastolic dysfunction at the sarcomeric level.

Mn2+-dependent protein phosphatase 1 enhances protein kinase A-induced Ca2+ desensitisation in skinned murine myocardium.

OBJECTIVE: Phosphorylation of proteins in cardiac myofilaments is a major determinant in the regulation of the Ca(2+) sensitivity of contraction. Whereas most reports have focused on effects of phosphorylation, little is known about reverse effects of dephosphorylation in skinned myocardium. Here we studied the effect of the Mn(2+)-dependent catalytic subunit of protein phosphatase 1 (PP1c-alpha) on the Ca(2+) regulation of contraction. In particular, we tested the hypothesis that phosphorylation persists after the skinning procedure and thereby attenuates protein kinase A (PKA)-induced Ca(2+) desensitisation. METHODS: Effects of Mn(2+) and Mn(2+)-PP1c on the Ca(2+) sensitivity of contraction (pCa(50)) were investigated in triton-skinned cardiac fibres from mice and compared with those of PKA treatment. Phosphorylation of proteins was monitored by autoradiography. RESULTS: PKA treatment significantly decreased the pCa(50) by 0.04 pCa units. In contrast, treatment with PP1c or Mn(2+)-containing PP1c buffer significantly increased the pCa(50) by 0.26 units or 0.09 units, respectively. These Ca(2+) sensitisations were completely reversed by subsequent PKA treatment. Replacement of the endogenous cardiac troponin I (cTnI) in fibres with the phospho-mimicking mutant human cTnI(S22/23D) abolished the PP1c-induced Ca(2+) sensitisation. PP1c removed (32)P which had been incorporated into cTnI and cardiac myosin binding protein C by PKA treatment. PKA incorporated twofold more (32)P into cTnI in fibres pre-treated with PP1c. CONCLUSIONS: Mn(2+)-dependent PP1c increases the Ca(2+) sensitivity of contraction of skinned cardiac fibres. This can be ascribed to dephosphorylation of PKA-dependent phosphorylation sites. Hence PKA-dependent phosphorylation of sarcomeric proteins persists to a functionally relevant degree after the skinning procedure.

Kinetic mechanism of the Ca2+-dependent switch-on and switch-off of cardiac troponin in myofibrils.

The kinetics of Ca(2+)-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca(2+)] or the Ca(2+)-buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca(2+)] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s(-1) and approximately 330 s(-1), respectively. At low [Ca(2+)] (pCa 6.6), the slower rate was reduced to approximately 25 s(-1), but was still approximately 50-fold higher than the rate constant of Ca(2+)-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca(2+)] from pCa 5.0-7.9 induced a fluorescence decay with a rate constant of 39 s(-1), which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca(2+)-binding (k(B) approximately 120 microM(-1) s(-1)) and dissociation (k(D) approximately 550 s(-1)); and 2), slower switch-on (k(on) = 390s(-1)) and switch-off (k(off) = 36s(-1)) kinetics. At high [Ca(2+)], approximately 90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation.

Cardiac troponin I but not cardiac troponin T induces severe autoimmune inflammation in the myocardium.

BACKGROUND: Cardiac troponins in blood are the most preferred markers of myocardial damage. The fact that they are normally not found in the circulation provides a high level of clinical sensitivity and specificity even when cardiac lesions are small. After myocardial injury, the troponins enter the circulation, where they can be used for diagnosis of acute coronary syndromes. Thus, the cardiac troponins are paramount for disease classification and risk stratification. However, little is known about the long-term effects of the released troponins on cardiac function. METHODS AND RESULTS: In this study we prepared recombinant murine cardiac troponin I (mc-TnI) and murine cardiac troponin T and used them to immunize mice. We report that A/J mice immunized with mc-TnI developed severe inflammation of the myocardium with increased expression of inflammatory chemokines RANTES (regulated on activation normal T cell expressed and secreted), monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, T-cell activation gene 3, and eotaxin and chemokine receptors CCR1, CCR2, and CCR5. The inflammation was followed by cardiomegaly, fibrosis, reduced fractional shortening, and 30% mortality over 270 days. In contrast, mice immunized with murine cardiac troponin T or with the control buffer showed little or no inflammation and no death. Furthermore, we demonstrate that mice preimmunized with mc-TnI before left anterior descending coronary artery ligation showed greater infarct size, more fibrosis, higher inflammation score, and reduced fractional shortening. CONCLUSIONS: Overall, our results show for the first time that provocation of an autoimmune response to mc-TnI induces severe inflammation in the myocardium followed by fibrosis and heart failure with increased mortality in mice.

Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities.

The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.

Isolation and characterization of bifunctional Escherichia coli TatA mutant proteins that allow efficient tat-dependent protein translocation in the absence of TatB.

In Escherichia coli, the Tat system promotes the membrane translocation of a subset of exported proteins across the cytoplasmic membrane. Four genes (tatA, tatB, tatC, and tatE) have been identified that encode the components of the E. coli Tat translocation apparatus. Whereas TatA and TatE can functionally substitute for each other, the TatB and the TatC proteins have been shown to perform distinct functions. In contrast to Tat systems of the ABC(E) type found in E. coli and many other bacteria, some microorganisms possess a TatAC-type translocase that consists of TatA and TatC only, suggesting that, in these systems, TatB is not required or that one of the remaining components (TatA or TatC) additionally takes over the TatB function. We have addressed the molecular basis for the difference in subunit composition between TatABC(E) and TatAC-type systems by using a genetic approach. A plasmid-encoded E. coli minimal Tat translocase consisting solely of TatA and TatC was shown to mediate a low level translocation of a sensitive Tat-dependent reporter protein. Suppressor mutations in the minimal Tat translocase were isolated that compensate for the absence of TatB and that showed substantial increases in translocation activities. All of the mutations mapped to the extreme amino-terminal domain of TatA. No mutations affecting TatC were identified. These results suggest that in TatAC-type systems, the TatA protein represents a bifunctional component fulfilling both the TatA and TatB functions. Furthermore, our results indicate that the structure of the amino-terminal domain of TatA is decisive for whether or not TatB is required.

Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane.

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.