Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaMKII) play important roles in the development of heart failure. In this study, we evaluated the effects of CaM on mitochondrial membrane potential (DeltaPsi(m)), permeability transition pore (mPTP) and the production of reactive oxygen species (ROS) in permeabilized myocytes; our findings are as follows. (1) CaM depolarized DeltaPsi(m) dose-dependently, but this was prevented by an inhibitor of CaM (W-7) or CaMKII (autocamtide 2-related inhibitory peptide (AIP)). (2) CaM accelerated calcein leakage from mitochondria, indicating the opening of mPTP, however this was prevented by AIP. (3) Cyclosporin A (an inhibitor of the mPTP) inhibited both CaM-induced DeltaPsi(m) depolarization and calcein leakage. (4) CaM increased mitochondrial ROS, which was related to DeltaPsi(m) depolarization and the opening of mPTP. (5) Chelating of cytosolic Ca(2+) by BAPTA, the depletion of SR Ca(2+) by thapsigargin (an inhibitor of SERCA) and the inhibition of mitochondrial Ca(2+) uniporter by Ru360 attenuated the effects of CaM on mitochondrial function. (6) CaM accelerated Ca(2+) extrusion from mitochondria. We conclude that CaM/CaMKII depolarized DeltaPsi(m) and opened mPTP by increasing ROS production, and these effects were strictly regulated by the local increase in cytosolic Ca(2+) concentration, initiated by Ca(2+) releases from the SR. In addition, CaM was involved in the regulation of mitochondrial Ca(2+) homeostasis.

Although mitochondrial oxidative catabolism of fatty acid (FA) is a major energy source for the adult mammalian heart, cardiac lipotoxity resulting from elevated serum FA and enhanced FA use has been implicated in the pathogenesis of heart failure. To investigate the effects of intermediates of FA metabolism [palmitoyl-l-carnitine (Pal-car) and palmitoyl-CoA (Pal-CoA)] on mitochondrial function, we measured membrane potential (DeltaPsi(m)), opening of the mitochondrial permeability transition pore (mPTP), and the production of ROS in saponin-treated rat ventricular myocytes with a laser scanning confocal microscope. Our results revealed that 1) lower concentrations of Pal-car (1 and 5 muM) caused a slight hyperpolarization of DeltaPsi(m) [tetramethylrhodamine ethyl ester (TMRE) intensity increased to 115.5 +/- 5.4% and 110.7 +/- 1.6% of baseline, respectively, P < 0.05] but did not open the mPTP, 2) a higher concentration of Pal-car (10 microM) depolarized DeltaPsi(m) (TMRE intensity decreased to 61.9 +/- 12.2% of baseline, P < 0.01) and opened the mPTP (calcein intensity decreased to 70.7 +/- 2.8% of baseline, P < 0.01), 3) Pal-CoA depolarized DeltaPsi(m) without opening the mPTP, and 4) only the higher concentration of Pal-car (10 muM) increased ROS generation (2',7'-dichlorofluorescein diacetate intensity increased to 3.4 +/- 0.3-fold of baseline). We concluded that excessive exogenous intermediates of long-chain saturated FA may disturb mitochondrial function in different ways between Pal-car and Pal-CoA. The distinct mechanisms of the deteriorating effects of long-chain FA on mitochondrial function are important for our understanding of the development of cardiac diseases in systemic metabolic disorders.

In this paper, we discuss the conformational properties of the hydroxymethyl group of β-glucopyranose in aqueous solution and its reorientation mechanism. First, using the values for the hydroxymethyl torsion (O5−C5−C6−O6) angle obtained by our ab initio simulations, we reestimate the experimental ratio of the hydroxymethyl rotamer populations. The reestimated ratio is found to be in agreement with those previously reported in several computational studies, which probably partly explains the discrepancies between theoretical and experimental studies that have been discussed in the literature. Second, our time-frequency analysis on a reorientation in the hydroxymethyl group in an ab initio molecular dynamics trajectory suggests that, before the reorientation, the O6−H6 stretching mode is vibrationally coupled with a proton-accepting first-hydration-shell water molecule, whereas the C6−O6 stretching mode is vibrationally coupled with a proton-donating one. The amount of the total vibrational energy induced by these vibrational couplings is estimated to be comparable to typical values for the potential barriers between hydroxymethyl rotamers. To elucidate the vibrational couplings, we investigate the hydrogen-bonding properties around the hydroxymethyl group during the pretransition period. The implications, validity, and limitation of a possible reorientation mechanism based on these findings are also discussed.

Aldosterone has non-genomic effects that express within minutes and modulate intracellular ion milieu and cellular function. However, it is still undefined whether aldosterone actually alters intracellular ion concentrations or cellular contractility. To clarify the non-genomic effects of aldosterone, we measured [Na+]i, Ca2+ transient (CaT), and cell volume in dye-loaded rat ventricular myocytes, and we also evaluated myocardial contractility. We found the following: (i) aldosterone increased [Na+]i at the concentrations of 100 nmol/L to 10 micromol/L; (ii) aldosterone (up to 10 micromol/L) did not alter CaT and cell shortening in isolated myocytes, developed tension in papillary muscles, or left ventricular developed pressure in Langendorff-perfused hearts; (iii) aldosterone (100 nmol/L) increased the cell volume from 47.5 +/- 3.6 pL to 49.8 +/- 3.7 pL (n=8, p<0.05); (iv) both the increases in [Na+]i and cell volume were blocked by a Na+-K+-2Cl- co-transporter (NKCCl) inhibitor, bumetanide, or by a Na+/H+ exchange (NHE) inhibitor, 5-(N-ethyl-N-isopropyl) amiloride; and (v) spironolactone by itself increased in [Na+]i and cell volume. In conclusion, aldosterone rapidly increased [Na+]i and cell volume via NKCC1 and NHE, whereas there were no changes in CaT or myocardial contractility. Hence the non-genomic effects of aldosterone may be related to cell swelling rather than the increase in contractility.

For the purpose of investigating the structure of solutions of muscle protein F–actin and the effect of external force on this structure, simultaneous measurements of rigidity, viscosity, and birefringence were made under rotation and oscillation at very low rates of shear, and as a result a consistent picture of F–actin solutions has been obtained. The F–actin solutions of various concentrations are made by adding a small amount of magnesium ions to a salt‐free G–actin solution. Rigidity, viscosity, and degree of flow birefringence all increase in proportion to the F–actin concentration. Even at very low concentrations, F–actin solutions have a large rigidity. This rigidity is found to be due to a network structure of semiflexible F–actin filaments composed of very many G–actin molecules. The linkage‐to‐linkage length and the flexibility of filaments are estimated from experimental data. Rotation at very low rate of shear causes rigidity and viscosity to decrease rapidly. All results are reasonably explained by assuming that the filaments are elongated and then linkages are detached by shear of rotation. With a decrease in the number of linkages, F–actin filaments are completely oriented. Once attained as a result of shear, the orientation decreases very slowly on standing and gives an anomalous decay curve, probably as a result of setting of the network structure of oriented filaments. At high concentrations of F–actin, a spontaneous orientation occurs. Hysteresis phenomena of F–actin solutions observed under various mechanical treatments are all attributable to formation and detachment of linkages between semiflexible F–actin filaments.