H2O2 alters rat cardiac sarcomere function and protein phosphorylation through redox signaling

Abstract

ROS, such as H(2)O(2), are a component of pathological conditions in many organ systems and have been reported to be elevated in cardiac pathophysiology. The experiments presented here test the hypothesis that H(2)O(2) induces alterations in cardiac myofilament function by the posttranslational modification of sarcomeric proteins indirectly through PKC signaling. In vitro assessment of actomyosin Mg(2+)-ATPase activity of myofibrillar fractions showed blunted relative ATP consumption in the relaxed state (pCa 8.0) in response to treatment with 0.5 mM H(2)O(2) before myofilament isolation. The effect was attributable to downstream "redox signaling," inasmuch as the direct application of H(2)O(2) to isolated myofibrils did not alter Mg(2+)-ATPase activity. Ca(2+)-ATPase activity, which was used as a measure of myofibrillar myosin function, was unaffected by H(2)O(2). Functional experiments using rat cardiac trabeculae treated with 0.5 or 5 mM H(2)O(2) followed by detergent extraction of membranes demonstrated increased Ca(2+) sensitivity of force production, a faster rate of force redevelopment, and (for 5 mM) decreased maximum tension. Biochemical analysis of myocardial samples treated with 0.5 mM H(2)O(2) demonstrated increased phosphorylation of two sarcomeric proteins: cardiac troponin I and myosin-binding protein-C. These changes were eliminated by a general PKC inhibitor. However, H(2)O(2) and the general PKC activator PMA induced different phosphorylation patterns in cardiomyocytes in which PKC-delta was elevated by viral infection. These data provide evidence that PKC-dependent redox signaling affects the function of cardiac myofilaments and indicate modification of specific proteins through this signaling mechanism.

Fig. 1.

Data showing in vitro actomyosin Mg²⁺-ATPase of myofibrillar fractions at relaxing conditions relative to activating conditions after treatment with H₂O₂. Myofilament protein samples were extracted from rat ventricular tissue after treatment with PBS (control) or 0.5 mM H₂O₂ for 10 min. Measurements of myofibrillar ATP hydrolysis are expressed as percentages of activation under relaxing conditions (pCa 8.0; control: 28.1 ± 3.6 nmol phosphate·mg protein⁻¹·min⁻¹ and H₂O₂: 23.6 ± 3.0 nmol phosphate·mg protein⁻¹·min⁻¹) relative to maximally activating conditions (pCa 4.5; control: 120 ± 12 nmol phosphate·mg protein⁻¹·min⁻¹ and H₂O₂: 131 ± 12 nmol phosphate·mg protein⁻¹·min⁻¹). Values are means ± SE; n = 10. *Statistically significant difference from the untreated sample (P < 0.05).

Fig. 2.

Data showing actomyosin Mg²⁺-ATPase of myofibrillar fractions after “direct” treatment with H₂O₂ (H₂O₂ added after the extraction of the myofibrillar fraction). After the isolation of the myofilament fraction by detergent extraction, homogenization buffer (control) or 5 mM H₂O₂ was added for 10 min to directly oxidize the protein samples. Measurements of myofibrillar ATP hydrolysis are expressed as percentages of activation under relaxing conditions (pCa ∼ 7.2; control: 30.4 ± 8.4 nmol phosphate·mg protein⁻¹·min⁻¹ and H₂O₂: 41.0 ± 9.0 nmol phosphate·mg protein⁻¹·min⁻¹) relative to maximally activating conditions (pCa < 4.5; control: 178 ± 26 nmol phosphate·mg protein⁻¹·min⁻¹ and H₂O₂: 223 ± 30 nmol phosphate·mg protein⁻¹·min⁻¹). Values are means ± SE; n = 7.

Fig. 3.

Data showing myosin Ca²⁺-activated ATPase of myofibrillar fractions after treatment with H₂O₂. Myofilament protein samples from tissue treated “indirectly” (treatment before homogenization and detergent extraction) with 0.5 mM H₂O₂ for 10 min were compared with PBS-treated control samples in an assay of ATP hydrolysis by myofibrillar myosin as described in materials and methods. Values are means ± SE; n = 7.

Fig. 4.

Tension-pCa and ATPase-pCa relationships from rat cardiac trabeculae with detergent extractraction after H₂O₂ treatment. Trabeculae from rat hearts were incubated with H₂O₂ or PBS (control) for 10 min before detergent extraction and measurements of tension generation and ATPase activity. A: averaged tension measurements at varying pCa values (in mN/mm²). B: averaged ATPase measurements at varying pCa values (in pmol ATP hydrolyzed·s⁻¹·mm⁻³). Values are means ± SE; n = 6 control trabeculae and 4 H₂O₂ (5 mM)-treated trabeculae. See Table 1 for summary data.

Fig. 5.

Stained SDS-PAGE gels showing levels of myofilament protein phosphorylation after treatment with H₂O₂, the PKC agonist PMA, and inhibitors. A: phosphorylation levels of proteins from isolated rat cardiomyocytes were compared after treatment with agonists and inhibitors (as specified) using SDS-PAGE separation (15 μg total protein/lane) and ProQ Diamond phosphoprotein stain. B: the gel from A was stained with SYPRO ruby to normalize for loading. Concentrations were as follows: H₂O₂, 0.5 mM; PMA, 0.1 mM; GF-109203X, 5 μM; and genistein, 10 μM. cTnI, cardiac troponin I; MyBP-C, myosin-binding protein-C/C-protein. The dark vertical lines indicate that irrelevant lanes were cropped during the preparation of this article. n = 6.

Fig. 6.

Histograms showing percent increases in myofilament protein phosphorylation after treatment with H₂O₂, the PKC agonist PMA, and inhibitors. The histograms compare relative levels of phosphorylation of the myofilament proteins cTnI and MyBP-C, indicating differential agonist-induced, PKC-dependent phosphorylation. Numbers represent percent increases compared with the PBS-treated control sample. P, PMA; GF, GF-109203X; Gen, genistein; H, H₂O₂. *Statistically significant difference compared with the untreated sample (P < 0.05); †statistically significant difference compared with the antagonist-free sample with the same agonist (PMA or H₂O₂) (P < 0.05); ‡statistically significant difference compared with the PMA-treated sample (P < 0.05). Values are means ± SE; n = 6.

Fig. 7.

Representative image showing two-dimensional difference in-gel electrophoresis analysis of protein differences between PMA- and H₂O₂-treated myocyte samples. The myocyte samples shown here overexpressed PKC-δ as described in materials and methods. Proteins extracted from PMA-treated samples were labeled with Cy3 (green) and H₂O₂-treated samples with Cy5 (red). Proteins were labeled separately, mixed equally, and separated by charge (horizontal) and size (vertical) by isoelectric focusing and electrophoresis, respectively. A: superimposed Cy3/Cy5 image. Boxes highlight protein species identified by molecular weight (MW)/isoelectric point (pI) as MyBP-C and cTnI. B and C: boxed areas enlarged for closer examination. See text for details.