Heme-Induced Oxidation of Cysteine Groups of Myofilament Proteins Leads to Contractile Dysfunction of Permeabilized Human Skeletal Muscle Fibres

Figures

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  Figure 1. Functional effects of heme on human skeletal muscle fibres. Typical photomicrographs of permeabilized human skeletal muscle fibres. Muscle preparations were incubated in the presence of reaction buffer (A) or 20 µM heme (B) for 20 min at room temperature (~25 °C). The normal cross striation pattern is clear in the control. Contractile parameters such as Ca2+-dependent (active) force (C) and Ca2+-independent (passive) force (D) were determined before and after exposures to the buffer alone, vehicle of heme solvent (NaOH) or heme/NaOH (20 µM). Active force was also plotted as a function of the applied Ca2+ concentration. Active force was either normalized to the maximal contractile force before treatments (E) or to the maximal contractile force recorded at the highest Ca2+ concentration after treatments (F). In this latter case the pCa50 (i.e., the value representing the Ca2+ sensitivity of force production) is also shown in the insert. Bars and symbols show means ± S.E.M. and statistical significance is indicated by asterisks (*) when p < 0.05. The number of observations was n = 5 for all groups.

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  Figure 2. Heme-induced SH-group oxidation. (A) Skeletal muscle fibres were isolated and permeabilized as described in the Methods section. About 25 µg of the skinned muscle fibres were exposed to heme (300 µM), DTT (50 mM) or DTDP (2.5 mM) as indicated in (A). Then cysteinyl SH groups were labelled by biotin and the labelled proteins were analyzed by SDS-PAGE. Biotinylated proteins were visualized by streptavidin and shown in (A). The apparent positions of molecular weight standards are shown on the left. The position of some proteins was tested on the same membranes by antibodies specific for myosin heavy chain (MHC), myomesin-2, myosin-binding protein C (MyBPC) or α-actinin. Results of typical western immunoblots are shown on the right side of (A). Results of the densitometric analyses of the bands are given in (B–E). The apparent molecular weights and the identity of the co-migrating proteins are indicated in the headers of the graphs. Treatments are the same as described in (A). Data are from three independent assays and were normalized to the control. Bars show means ± S.E.M. and statistical significance is shown by asterisks (*) when p < 0.05.

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  Figure 3. Heme-induced SH group oxidation in large myofibrillar proteins. Human skeletal muscle fibres were treated as described in Figure 2. Proteins were solubilized and subjected to agarose strengthened SDS-polyacrylamide gels. Proteins were stained by Sypro-Ruby (total protein), and biotinylation was tested by streptavidin (A,C). Representative gel pictures (A,C) and values from the densitometric analyses are shown on bar graphs (B,D). The apparent molecular weights of the bands and the identified proteins co-migrating with these bands are indicated in the headers. Data represent the results of three independent experiments and were normalized to the control. Bars show means ± S.E.M. and statistical significance is shown by asterisks (*) when p < 0.05.

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  Figure 4. Prevention of heme evoked deterioration by heme binding proteins or heme “scavengers”. Human skeletal muscle fibres were treated with heme as described in Figure 1. In addition, heme-protein complexes were pre-treated with heme-hemopexin (Hpx) and heme-α-1-microglobulin (A1M) complexes as shown. (A) shows original force recordings. Active (B) and passive (C) force values are normalized to the control and are shown in bar graphs. Biotinylation was performed as described in Figure 2. Representative blots are shown in (D,F), while the results of the densitometric analyses are shown in bar graphs (E,G). Data represent the results of three independents experiments and were normalized to the control. Bars show means ± S.E.M. and statistical significance is shown by asterisks (*) when p < 0.05.

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  Figure 5. Sulfenylation of skeletal muscle proteins with low molecular weights by heme. Human skeletal muscle fibres were isolated and treated as described in Figure 2. Proteins were subjected to SDS-PAGE and plotted onto nitrocellulose membranes. Proteins were visualized by the non-specific Sypro Ruby dye and the same membranes were subsequently processed with sulfenyl specific labelling. (A) shows representative membranes. Bar graphs (B–E) show the results of densitometric analyses. The headers of the graphs indicate the molecular weights of the evaluated bands and the identified proteins. Data represent three independents experiments and were normalized to the control. Bars show means ± S.E.M. and statistical significance is shown by asterisks (*) when p < 0.05.

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  Figure 6. Sulfenylation of skeletal muscle proteins with high molecular weights by heme. Human skeletal muscle fibres were isolated as described in Figure 2. Proteins were subjected to agarose strengthened SDS-PAGE and transferred to nitrocellulose membranes. Membranes were stained for protein by Sypro Ruby and sulfenylation was detected by western immunoblot (labelled by ECL-BP1). Representative membranes are shown in panel (A,C). Results of densitometric evaluations are shown in panel (B,D). Data illustrate the results of three independents experiments and were normalized to the control. Bars show means ± S.E.M. and statistical significance is shown by asterisks (*) when p < 0.05.