Two cardiomyopathy-causing mutations, E244D and K247R, in human cardiac troponin T (TnT) are located in the coiled-coil region of the Tn-core domain. To elucidate effects of mutations in this region on the regulatory function of Tn, we measured Ca(2+)-dependent ATPase activity of myofibrils containing various mutants of TnT at these residues. The results confirmed that the mutant E244D increases the maximum ATPase activity without changing the Ca(2+)-sensitivity. The mutant K247R was shown for the first time to have the effect similar to the mutant E244D. Furthermore, various TnT mutants (E244D, E244M, E244A, E244K, K247R, K247E, and K247A) showed various effects on the maximum ATPase activity while the Ca(2+)-sensitivity was unchanged. Molecular dynamics simulations of the Tn-core containing these TnT mutants suggested that the hydrogen-bond network formed by the side chains of neighboring residues around residues 244 and 247 is important for Tn to function properly.

Extensive efforts have been devoted to study A-type lamins because mutations in their gene, LMNA in humans, are associated with a number of diseases. The mouse germline mutations in the A-type lamins (encoded by Lmna) exhibit postnatal lethality at either 4-8 postnatal (P) weeks or P16-18 days, depending on the deletion alleles. These mice exhibit defects in several tissues including hearts and skeletal muscles. Despite numerous studies, how the germline mutation of Lmna, which is expressed in many postnatal tissues, affects only selected tissues remains poorly understood. Addressing the tissue specific functions of Lmna requires the generation and careful characterization of conditional Lmna null alleles. Here we report the creation of a conditional Lmna knockout allele in mice by introducing loxP sites flanking the second exon of Lmna. The Lmna(flox/flox) mice are phenotypically normal and fertile. We show that Lmna homozygous mutants (Lmna(Δ/Δ)) generated by germline Cre expression display postnatal lethality at P16-18 days with defects similar to a recently reported germline Lmna knockout mouse that exhibits the earliest lethality compared to other germline knockout alleles. This conditional knockout mouse strain should serve as an important genetic tool to study the tissue specific roles of Lmna, which would contribute toward the understanding of various human diseases associated with A-type lamins.

During heartbeat, the repeated contractions of myocardium are induced by the oscillation of intracellular Ca(2+) concentration. On the other hand, when intermediately activated at a certain Ca(2+) concentration, cardiac myofibrils exhibit the spontaneous sarcomeric oscillation (Ca-SPOC) under steady ionic conditions. In the present study, we found that Ca-SPOC occurred over a wide range of Ca(2+) concentrations, including physiological contractile conditions, in skinned myocardium prepared from various animal species (rat, rabbit, pig, and cow). The period of sarcomeric oscillation fell within the same range as the period of heartbeat of each animal species. On the basis of these results we propose that the intrinsic auto-oscillatory property of sarcomeres (myofibrils) significantly contributes to myocardial beating in vivo.

Stress fibers (SFs) composed of nonmuscle actin and myosin II play critical roles in various cellular functions such as structural remodeling in response to changes in cell stress or strain. Previous studies report that SFs rapidly disassemble upon loss of tension caused by reduced myosin activity or sudden cell shortening, but the mechanism is unclear. Here, we showed that Rho-kinase inhibition with Y-27632 led to detachment of intact actin filaments from the SFs rather than depolymerization. Loss of tension may allow SFs to shorten via MgATP-driven cross-bridge cycling, thus we investigated the effects of MgATP concentration on SF shortening and stability. We performed the experiments using extracted SFs to allow control over MgATP concentration. SF contraction and disassembly rates each increased with increasing MgATP concentration. SFs transitioned from conventional SF shortening to rapid disassembly as MgATP concentration increases from 2 to 5mM, which is within the physiological range of intracellular MgATP concentrations. Thus, we submit that SFs in intact cells are inherently on the verge of disassembly, which is likely due to the small number of actomyosin cross-bridges in SFs compared to those found in relatively stable myofibrils. Given that recent studies have revealed that loss of resistive force against myosin II could lower the fraction of the MgATPase cycle time that the myosin head is attached to actin (i.e., the duty ratio), binding of cytoplasmic levels of MgATP to myosin II may be sufficient to cause the disassembly of unloaded SFs. The present study thus describes a putative mechanism for rapid SF disassembly caused by decreased myosin activity or sudden cell shortening.