Ruth A. Altschuld

Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes

Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium derivative and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chemical structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc.115, 11799–11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+uptake into mitochondria in vitro or in intact cells has not been determined. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nm) than ruthenium red (IC50 = 6.85 nm) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (K d = 0.34 nm, B max = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nm, indicating that saturation of a specific binding site is responsible for the inhibition of Ca2+uptake. Ru360, as high as 10 μm, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 μm) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells.

Metabolic Compartmentalization in Neonatal Swine Myocytes

The transport of metabolites across the mitochondrial membrane is regulated by specific exchange and shuttle systems that are often dependent on the mitochondrial membrane potential. Thus, metabolite concentrations in the cytosol and mitochondrial compartments are largely determined by the energy state of the cardiac muscle cell. The purpose of this study was to investigate metabolic compartmentalization in ventricular myocytes isolated from newborn(<24 h) swine hearts. Furthermore, the effect of respiratory inhibition on these distribution patterns was examined. Freshly isolated cells contained 33 nmol of ATP and 37 nmol of total adenine nucleotides (AN) per mg of myocyte protein. Rapid digitonin fractionation indicated that 95% of ATP and 86% of AN were cytosolic, whereas >50% of the pyridine nucleotides were mitochondrial. With 11 mM added glucose, myocytes treated with the respiratory inhibitor, rotenone, maintained ATP at 88% of that of aerobic myocytes, but phosphocreatine declined by 50% over 30 min. Rotenone treatment caused the mitochondrial NAD/NADH ratio to decline from 1.2 to 0.06, whereas the cytosolic pyridine nucleotides remained >90% oxidized. Total adenine and pyridine nucleotide content and their compartmentalization were unaffected by respiratory inhibition. Comparisons of metabolite content and respiratory activity between isolated piglet mitochondria and the mitochondrial compartment of piglet myocytes indicated that mitochondria account for ≈30% of total myocyte protein. A similar value (29%) was obtained for the aqueous volume fraction of the in situ mitochondrial matrix using the 4000Mr 14C-labeled polyethylene glycol-impermeable3 H2O spaces of intact and lysed myocytes. These results are comparable to literature values for myocardium from other species and age groups.

FK506 alters sarcoplasmic reticulum calcium release in neonatal piglet cardiac myocytes.

Tacrolimus (FK506) is a potent immunosuppressive drug that, when complexed to a family of immunophilin proteins known as FK binding proteins, inhibits calcineurin in T lymphocytes. Although i.v. use of FK506 in pediatric transplant recipients has been linked to development of cardiomyopathies, its mechanism of cardiotoxicity has not been examined in a neonatal animal model. In our study the effects of FK506 were investigated in cardiac myocytes isolated from newborn piglets. The peak amplitude of electrically triggered fura-2 Ca2+ transients was increased in FK506-treated myocytes, but Ca2+ transient duration and baseline fura-2 Ca2+ ratios were not altered. 45Ca2+ uptake by digitonin-lysed piglet cells decreased at pCa ≤ 6.0, indicating that sarcoplasmic reticulum efflux channels were leaky. The results suggest that elevated release of sarcoplasmic reticulum Ca2+ during systole contributes to cardiotoxic effects observed in children.