Mechanical properties of sarcomeres during cardiac myofibrillar relaxation: stretch-induced cross-bridge detachment contributes to early diastolic filling.

Sudden Ca2+ removal from isometrically contracting cardiac myofibrils induces a biphasic relaxation: first a slow, linear force decline during which sarcomeres remain isometric and then a rapid, exponential decay originating from sequential lengthening, i.e., successive mechanical relaxation, of individual sarcomeres (Stehle et al. 2002; Biophys J 83:2152-2162). Step-stretches were applied to the myofibrils, in order to study the mechanical properties of sarcomeres during this dynamic relaxation process. Stretch applied soon (approximately 10 ms) after Ca2+ removal accelerated the initiation of the rapid, exponential force decay and of the sequential sarcomere lengthening. After the stretch, a short, transient period (approximately 24 ms) remained, during which time force was enhanced and sarcomeres were homogenously elongated by the stretch. This period was similar to the duration of the switching-off of troponin C in myofibrils, as measured by stopped-flow. In contrast, when the stretch was applied during the rapid, exponential relaxation phase, force quickly decayed after stretch, back to the force level of isometric controls or even lower. Smaller stretches lengthened only those sarcomeres that were located at the wave front of the sequential sarcomere relaxation. The more the stretch-size was increased, the more of the contracting sarcomeres became lengthened by the stretch; those sarcomeres that were relaxed prior to stretch were barely elongated. These results indicate that the stretch accelerates myofibrillar relaxation by forcing the cross-bridges in contracting sarcomeres to detach. Subsequent rapid cross-bridge reattachment occurs during a short period after Ca2+ removal until troponin C is switched off. However, this switch off occurs approximately 5 times too fast to directly rate-limit the force relaxation under the isometric condition. After troponin C is switched off, stretching induces cross-bridge detachment without subsequent reattachment, and force rapidly decays below the isometric level. This may explain the rapid distention of the ventricular myocardium during early diastolic filling.

Effects of the mutation R145G in human cardiac troponin I on the kinetics of the contraction-relaxation cycle in isolated cardiac myofibrils.

Familial hypertrophic cardiomyopathy (FHC) has been linked to mutations in sarcomeric proteins such as human cardiac troponin I (hcTnI). To elucidate the functional consequences of the mutation hcTnI(R145G) on crossbridge kinetics, force kinetics were analysed in murine cardiac myofibrils carrying either the mutant or the wild-type protein. The mutation was introduced into the myofibrils in two different ways: in the first approach, the endogenous Tn was replaced by incubation of the myofibrils with an excess of reconstituted recombinant hcTn containing either hcTnI(WT) or hcTnI(R145G). Alternatively, myofibrils were isolated either from non-transgenic or transgenic mice expressing the corresponding mcTnI(R146G) mutation. In myofibrils from both models, the mutation leads to a significant upward shift of the passive force-sarcomere length relation determined at pCa 7.5. Addition of 5 mm BDM (2,3-butandione-2-monoxime), an inhibitor of actomyosin ATPase partially reverses this shift, suggesting that the mutation impairs the normal function of cTnI to fully inhibit formation of force-generating crossbridges in the absence of Ca(2)(+). Maximum force development (F(max)) is significantly decreased by the mutation only in myofibrils exchanged with hcTnI(R145G) in vitro. Ca(2)(+) sensitivity of force development was reduced by the mutation in myofibrils from transgenic mice but not in exchanged myofibrils. In both models the rate constant of force development k(ACT) is reduced at maximal [Ca(2)(+)] but not at low [Ca(2)(+)] where it is rather increased. Force relaxation is significantly prolonged due to a reduction of the relaxation rate constant k(REL). We therefore assume that the impairment in the regulatory function of TnI by the mutation leads to modulations in crossbridge kinetics that significantly alter the dynamics of myofibrillar contraction and relaxation.

Specificity of signal peptide recognition in tat-dependent bacterial protein translocation.

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.

The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding.

The periplasmic, NADP-containing glucose-fructose oxidoreductase of the gram-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose-fructose oxidoreductase into the periplasm uses a Sec-independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:beta-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glucose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.