Figure 1.
Morphological and biochemical characteristics of two mitochondrial populations in heart muscle.
A, Electron micrographs of subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria within the heart cell (In Situ) and after isolation (Isolated). Magnification: x10, 000. B, Expression of mitochondria-specific marker proteins in SSM and IFM. Top panel displays Western blots of citrate synthase (CS) and adenine nucleotide transporter 1 (ANT1). Bottom panel displays bar graphs of the activity of CS (SSM, open bars; IFM, filled bars).
Figure 2.
Ca2+ handling and oxidative phosphorylation capacity of subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria.
A, Typical tracing of Ca2+ loading in SSM (left panel) and IFM (right panel). Mitochondria were loaded with consecutive Ca2+ pulses (arrows), and each pulse delivers 50 nmol of Ca2+. B, Average Ca2+-accumulating capacity of SSM (open bar) and IFM (filled bar), n = 6, p<0.05. C, Typical tracing of ADP-induced changes in oxygen consumption (top) and changes in membrane potential from baseline (bottom) of SSM and IFM. D, Average ADP-stimulated (State 3) respiration of SSM and IFM (n = 6, p<0.05). E, The rate of ATP production in SSM (left bars) and IFM (right bars) before (B/W bars) and after (gray bars) loading with 150 nmol Ca2+/mg protein, n = 3, p<0.05. Inset: Alternative monitoring of ATP production in mitochondria using coupled enzymes. Asterisks signify statistical differences with p<0.05.
Figure 3.
Effect of diazoxide on the membrane potential and Ca2+ handling in subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria.
A, Depolarizing effect of diazoxide (100 µM) in SSM (open bar) and IFM (filled bar), n = 6, p<0.05. B, Mitochondrial Ca2+ uptake in SSM and IFM in the absence (-DZ) and presence (+DZ) of diazoxide (100 µM). C, Inhibition of the rate of Ca2+ uptake in SSM (open bar) and IFM (filled bar), n = 6, p<0.05. D, Diazoxide (100 µM) mediated Ca2+ release from preloaded SSM and IFM. E, Average rate of diazoxide-mediated Ca2+ release from mitochondria, n = 3, p<0.05. F and G, Dose-dependent effect of diazoxide on Ca2+ uptake (F) and Ca2+ release (G) in SSM (triangles) and IFM (circles), n = 6.
Figure 4.
Diazoxide-mediated recovery of Ca2+-inhibited ATP synthesis.
A, Dose-dependent effect of diazoxide on restoration of Ca2+-inhibited State 3 respiration in subsarcolemmal (SSM) (triangles) and interfibrillar (IFM) (circles) mitochondria. B, Effect of diazoxide on Ca2+-inhibited State 3 respiration in SSM and IFM preloaded with (0–150 nmol Ca2+/mg protein). C, Bar graphs of diazoxide (100 µM)-mediated recovery of Ca2+-inhibited State 3 respiration in SSM and IFM loaded with 150 (open bars) and 30 nmol Ca2+/mg protein (hatched bars), n = 3. Asterisks signify statistical differences with p<0.05.
Figure 5.
Schematic illustration of preferential targeting of subsarcolemmal mitochondria (SSM) by diazoxide.
A, Before ischemic insult (Normoxia), SSM and interfibrillar mitochondria (IFM) produce sufficient ATP to feed ionic pumps and maintain ionic homeostasis of cell. During ischemic period and decreased supply of substrates and oxygen, the mitochondrial ATP formation decreases and disables ionic pumps, resulting in increased cytosolic Ca2+. B, At reperfusion (Reperfusion), restored supply of substrates and oxygen reenergizes the SSM, resulting in excessive Ca2+ uptake (from high Ca2+ cytosol) and inhibition of ATP production. C, In the presence of diazoxide during reperfusion (Reperfusion + DZ) Ca2+ uptake into SSM will be decreased, thus preserving mitochondrial ability to produce ATP, which is required for the activity of ionic pumps and restoration of normal cellular homeostasis.