Fig. 6. Fission of mitochondrial reticulum coupled with changes in mitochondrial membrane potential in human skeletal muscle myoblasts. Human skeletal muscle myoblasts were grown with or without bupivacaine (24 h) and with or without recombinant human erythropoietin (1 U/ml, 8 h before bupivacaine treatment). Normal mitochondrial reticulum visualized by fluorescence microscopy and mitochondrial membrane potential obtained by spectrofluorometry are shown in  A and  E . Control (  A ), concentrations of 1 μm (  B ), 1 mm (  C ), and 5 mm bupivacaine (  D ) led to alterations in the mitochondrial network morphology, coupled with the loss of mitochondrial membrane potential (ΔΨ) (  F –  H , respectively). The images in  E –  H and  M –  P are pseudocolor, merged images using the two emission wavelengths monitored for JC-1 quantitation. Recombinant human erythropoietin alone induced no changes in either mitochondria network morphology (  I ) or ΔΨ (  M ). Recombinant human erythropoietin pretreatment prevented fission of the mitochondrial reticulum for low concentrations of bupivacaine (1 μm–1 mm,  J –  K ) but not for the 5 mm concentration (  L ). Parallel effects were observed with regard to ΔΨ (  N –  P , respectively).  Scale bar , 80 μm.