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Supplementary material
Explanation of the substrates and inhibitors used in Figure 2c.
H9c2 myoblasts were permeabilized with digitoninbefore the determination of the activities of the enzyme complexes of the electron transport chain (ETC). After permeabilization, the following experiments were performed.
Activity of complex I+III.
1. Glutamate and malateare transported into the mitochondria via the aspartate shuttle. Glutamate is converted to α-ketoglutarate, a reaction yielding NADH which is the substrate of complex I. Malate is converted in two steps to aspartate, thereby facilitating the metabolism and transport of glutamate.
2.ADP is converted to ATP by the F0F1ATPase, a reaction reducing the proton gradient across the inner mitochondrial membrane and thereby stimulating the activity of the ETC.
3. Oligomycin inhibits the F0F1ATPase. Oxygen consumed in the presence of oligomycin is independent of the production of ATP and therefore originates from uncoupled oxidative phosphorylation.
4. FCCP(carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) is an artificial uncoupler of oxidative phosphorylation. In the presence of FCCP, the activity of the ETC is maximal.
5. Cytochrome c must cross the outer mitochondrial membrane to serve as an electron carrier. If oxygen consumption is not significantly increased after addition of cytochrome c, the mitochondria are considered to be intact.
6. Rotenone is a specific inhibitor of complex I. Residual activity reflects the presence of compounds yielding for instance FADH2 when they are metabolized (e.g. succinate and fatty acids). FADH2 enters the ETC at complex II.
7. Duroquinol is an artificial substrate for complex III. Under the conditions of the experiment, with FCCP present, the activity of complex III can be expected to be maximal.
8. Antimycin A is a specific inhibitor of complex III.
Activity of complex II+IV.
1.Succinate is transported actively into the mitochondria and converted to fumarate by succinate dehydrogenase. This reaction yields FADH2, which is a substrate of complex II.
2.Rotenone is a specific inhibitor of complex I. Since complex I and complex II feed electrons parallel into complex III and compounds yielding NADH (the substrate of complex I) are abundant, it is important to inhibit complex I when complex II is determined.
3.ADP is converted to ATP by the F0F1ATPase, a reaction reducing the proton gradient across the inner mitochondrial membrane and thereby stimulating the activity of the ETC.
4.Oligomycin inhibits the F0F1ATPase. Oxygen consumed in the presence of oligomycin is independent of the production of ATP and therefore originates from uncoupled oxidative phosphorylation.
5.FCCP is an artificial uncoupler of oxidative phosphorylation. In the presence of FCCP, the activity of the ETC is maximal.
6.Cytochrome c must cross the outer mitochondrial membrane to serve as an electron carrier. If oxygen consumption is not significantly increased after addition of cytochrome c, the mitochondria are considered to be intact.
7.Antimycin A is a specific inhibitor of complex III.
8.Ascorbate and TMPD(N,N,N',N'-tetramethyl-p-phenylenediaminedihydrochloride) are artificial substrates of complex IV. Under the conditions of the experiment, with FCCP present, the activity of complex IV can be expected to be maximal.
Explanation of the substrates and inhibitors used in Figure 5b.
Muscle bundles were isolated from cardiac tissue and permeabilized using saponin. The function of the enzyme complexes of the electron transport chain of the mitochondria in these muscle bundles was assessed similar to the mitochondria in H9c2 myoblasts.
The sequence of the addition of substrates and inhibitors was as follows: 1. glutamate and malate, 2. ADP, 3. rotenone, 4. succinate, 5. antimycin A, 6. TMPD and ascorbate, 7. cytochrome c.
The function of the substrates and inhibitors used is described in the explanation for Figure 2b.