Regulation of Mitochondrial Ca2+ Uptake by Mitochondrial Ryanodine Receptor in Control and Malignant Hyperthermia Mouse Heart

Malignant hyperthermia (MH) is a severe hypermetabolic response triggered by exposure to volatile anesthetics and caused by mutations in the genome of ryanodine receptor subtype 1 (RYR1) in the skeletal muscle. The presence of mitochondrial ryanodine receptor (mRYR) in heart, which behaves immunologically and pharmacologically like RyR1, raises the question of its primary cardiac pathological role in MH.To address this, we used genetically modified micecontaining RyR1 with a single point mutation at the position 522 (tyrosine versus serine; YS mice, obtained from Dr. Susan Hamilton, Baylor College of Medicine) and wild type (WT) mice. Ca2+ uptake of isolated heart mitochondria from both animal groups was measured (N=3) using ArsenazoIII. Our data show, that in 1 year old YS mice, the total free Ca2+ load tolerated before full mitochondrial permeability transition pore (mPTP) opening was lower (275.22±42.4 nmol free Ca2+/ mg protein) compared to WT (474.77±65.7 nmol free Ca2+/ mg protein). The time constant (T), calculated from the velocity of Ca2+ uptake during the first 2 minutes after applying a 5μM Ca2+ pulse was not significantly different in YS than in WT. However, significant differences were observed using RYR1 specific inhibitors (10μM dantrolene and 10μM azumolene). In presence of these inhibitors, WT showed a prolonged T (383.09±41.65sec with dantrolene and 305.75±38.52sec with azumolene) suggesting that mRYR was inhibited. Surprisingly, YS showed less inhibition due to the difficulty of blocking leaky channel (265.55±26.07sec and 202.56±31.45sec respectively).Measuring the membrane potential with TMRE showed that baseline fluorescence of YS mitochondria was significantly lower, indicating that these mitochondria are depolarized (1.61±0.02a.u. versus 1.93±0.02a.u. in WT, P<0.05).Depolarized mitochondrial membranes due to leaky mRYR suggest a primary role of this protein in heart pathology in MH.