Myocardial infarction in mice alters sarcomeric function via post-translational protein modification

Abstract

Myocardial physiology in the aftermath of myocardial infarction (MI) before remodeling is an under-explored area of investigation. Here, we describe the effects of MI on the cardiac sarcomere with focus on the possible contributions of reactive oxygen species. We surgically induced MI in 6-7-month-old female CD1 mice by ligation of the left anterior descending coronary artery. Data were collected 3-4 days after MI or sham (SH) surgery. MI hearts demonstrated ventricular dilatation and systolic dysfunction upon echo cardiographic analysis. Sub-maximum Ca-activated tension in detergent-extracted fiber bundles from papillary muscles increased significantly in the preparations from MI hearts. Ca(2+) sensitivity increased after MI, whereas cooperativity of activation decreased. To assess myosin enzymatic integrity we measured splitting of Ca-ATP in myofibrillar preparations, which demonstrated a decline in Ca-ATPase activity of myofilament myosin. Biochemical analysis demonstrated post-translational modification of sarcomeric proteins. Phosphorylation of cardiac troponin I and myosin light chain 2 was reduced after MI in papillary samples, as measured using a phospho-specific stain. Tropomyosin was oxidized after MI, forming disulfide products detectable by diagonal non-reducing-reducing SDS-PAGE. Our analysis of myocardial protein oxidation post-MI also demonstrated increased S-glutathionylation. We functionally linked protein oxidation with sarcomere function by treating skinned fibers with the sulfhydryl reducing agent dithiothreitol, which reduced Ca(2+) sensitivity in MI, but not SH, samples. Our data indicate important structural and functional alterations to the cardiac sarcomere after MI, and the contribution of protein oxidation to this process.

Fig. 1. Representative examples of echo cardiographic images and Evans Blue stain of SH and MI hearts

A. M-mode echo cardiographic images from sham-operated (SH) and post-myocardial infarction (MI) hearts were obtained at the mid-papillary level of the left ventricle. Analysis was performed 3 days post-surgery. Measurements showed significant left ventricular contractile function decline in post-MI animals. Line segments mark LV end-diastolic diameter (LVIDd) and end-systolic diameter (LVIDs). n = 4 SH, 6 MI B. Hearts were stained with Evans blue dye and triphenyltetrazolium chloride (TTZ). Sections (1 mm thick) from SH and MI hearts are displayed sequentially (from base to apex going left to right). The “remote” zones that continued to receive blood flow after MI are stained blue. Red tissue represents viable tissue within the ischemic zone. White (unstained) tissue is irreversibly infarcted. Arrows indicate the left ventricular papillary muscles.

Fig. 2. Plot of the tension-pCa relation of papillary muscle “skinned fibers”

A. Data represent averaged tension at varying pCa values for detergent-extracted fiber bundles from sham (SH) and non-infarcted left ventricular papillary muscles from hearts stressed by myocardial infarction (MI). Inset indicates per cent increase in tension in MI hearts over controls at sub-maximal pCa values. B. Data represent tensions normalized to maxium tension. MI fibers showed an increase in Ca⁺⁺ sensitivity and a decrease in cooperativity of activation. n = 16 SH, 14 MI See Table 1 for data summary and analysis.

Fig 3. Histogram showing in vitro ATPase activity in ventricular protein samples

Myofibrillar protein extracts from SH (sham) and MI (infarcted) hearts were homogenized and assayed for myosin ATPase activity using a colorimetric assay for free PO₄ generation. Two reaction solutions were used to measure myofilament myosin Ca-ATPase and K-ATPase activities Units given in nmol PO₄/mg protein/min n = 20 (Ca-ATPase), 12 (K-ATPase) * statistically significant difference between SH and MI

Fig 4. Gel and histogram showing net phosphorylation status of cTnI in papillary samples

A. Myofilament proteins from skinned fibers dissected from the viable portions of detergent-extracted papillary muscles from SH and MI hearts were separated by SDS-PAGE. Levels of phosphorylation were determined by ProQ Diamond Phosphoprotein Stain, and the gel was stained with Coomassie Blue to visualize total protein loading. The leftmost lane is a molecular mass standard (DualColor; BioRad) with bands corresponding to the indicated masses in kD; sample lanes are labeled as MI (M) or SH (S). A duplicate molecular mass standard has been cropped from the right side of each image. B. Histogram depicting the data from A. Values for protein phosphorylation in SH (white) and MI (gray) samples represent arbitrary units defined by ratios of protein phosphorylation normalized for abundance of actin. Sarcomeric proteins listed are myosin binding protein C (MyBP-C), desmin, cardiac troponin T (cTnT), tropomyosin (Tm), cardiac troponin I (cTnI), and myosin light chain 2 (MLC2). cTnI and MLC2 phosphorylation was ~20% lower in MI samples. n = 5 * statistically significant difference between SH and MI

Fig 5. Representative non-reducing/reducing “diagonal gel” showing Tm oxidation

A. Myofilament fraction homogenates from ventricular tissue were separated by SDS-PAGE under non-reducing and reducing conditions, and stained with Coomassie Blue to visualize the proteins. Reduction-sensitive putative disulfide products appear to the left of the diagonal in each protein sample. The circled spot represents an oxidized product containing Tm. Tm oxidation was higher in the MI sample in 7 out of 8 pairings from sarcomeric fractions of whole-ventricles and in 7 out of 9 pairings from viable papillary skinned fiber samples. B. Putative oxidized Tm products from the experiment series shown in A. were subjected to LC-MS/MS analysis. Peptides were analyzed on an LTQ-FT-ICR instrument equipped with a capillary column and nanospray source. α-tropomyosin was identified with high confidence (100% protein probability) in an analysis of 13 unique peptides, 18 unique spectra, and 39 total spectra; 99/284 amino acids were represented in the analysis for 35% sequence coverage of the protein. Shown above the spectra is the sequence coverage of Tm with peptides in yellow. MS/MS spectra of doubly charged m/z 657.89 ion, corresponding to Tm peptide 168–178 (KLVIIESDLER), are shown in the lower portion. Tm was the only protein confidently identified in 4 out of 4 MI samples.

Fig 6. Western blot and histogram depicting protein glutathionylation

A. Western blot (left) and Ponceau stain (right) comparing SHAM (S), MI (M), positive control (+; reduced glutathione and diamide), and negative control (−; DTT) samples. Myofilament fraction homogenates prepared with NEM were subjected to SDS-PAGE/Western analysis and probed with an anti-glutathione antibody. Membranes were stained with Ponceau S to normalize for total protein loading. B. Histogram representation of the results of A. Glutathionylation of a high molecular weight protein most likely titin is higher after MI. n = 6 SH, 4 MI * statistically significant difference between SH and MI

Fig 7. Before-after plots showing the effect of a reducing agent on Ca⁺⁺ sensitivity in papillary “skinned fibers”

Tension-pCa relations were obtained for non-infarcted fiber bundles from detergent-extracted papillary muscles over two contraction series. Prior to the second set of measurements, each fiber was treated with 0.1 M DTT for 10 min. DTT treatment did not significantly alter Ca⁺⁺ sensitivity in SH fibers, but reduced Ca⁺⁺ sensitivity in MI samples. Values represent paired measurements of non-reduced and reduced pCa₅₀ value for each fiber. n = 7 SH, 9 MI SH p N.S., MI p < 0.005 Unpaired sample means ± SEM are SH: 5.82 ± 0.02 before, 5.82 ± 0.02 after; MI: 5.87 ± 0.03 before, 5.84 ± 0.02 after. ΔpCa₅₀ means are SH 0.01 ± 0.01, MI 0.03 ± 0.01, p < 0.05. Samples whose ΔpCa₅₀ deviated by > 2 S.D. were excluded from the analysis.