Alterations in ACE and ACE2 Activities and Cardiomyocyte Signaling Underlie Improved Myocardial Function in a Rat Model of Repeated Remote Ischemic Conditioning

Figures

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  Figure 1. Scheme of the experimental protocol. The left anterior descending coronary artery (LAD) was ligated to induce myocardial infarction (MI group, n = 6) in 12-week-old male rats. rRIC was performed by 3 cycles of 5-min-long unilateral hind limb ischemia and 5 min of reperfusion once a day for 2 weeks, 4 weeks after LAD ligation in a separate group of animals (MI + rRIC group; n = 6). Sham operated animals (Sham group, n = 4) served as controls. Myocardial tissues were collected at 18 weeks for functional and biochemical measurements.

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  Figure 2. Histological analysis and functional characteristics of the infarcted hearts. Representative Masson’s trichrome-stained cross-sections are shown from the Sham, MI, and MI + rRIC groups (A). Red staining identifies viable myocardial tissues without scar formation in the inferior region (non-infarcted area) of the left ventricle (LV), while blue staining shows scar formation in the anterior region (infarcted area) of LV following MI. Quantitative aspects of myocardial infarct sizes are given in a bar graph (B). Time courses of systolic LV anterior wall thickness and LV dimensions at baseline, 4 or 6 weeks after MI induction as determined by echocardiography. Panels (C–E) illustrate systolic LV anterior wall thickness, LV end-systolic diameter (LVESD), and LV end-diastolic diameter (LVEDD) in the Sham, MI and MI + rRIC groups. Heart rate, LV peak systolic pressure (LVSP), maximal rate of rise of LV pressure ( + dP/dtmax), and LV end-diastolic pressure (LVEDP) at six weeks after MI induction are also given for the three experimental groups panels (F–I). Values are given as mean ± SEM; n = 4–6 animals/group. * p < 0.05.

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  Figure 3. Changes in LV ejection fraction (LVEF) upon rRIC. Representative echocardiographic images during the determinations of LVEF (A). LVEF at baseline, 4 weeks and 6 weeks after MI induction (B). LVEF of individual rats in the MI group (C) and in the MI + rRIC group (D) at baseline, 4 and 6 weeks after MI induction. Values are given as mean ± SEM; n = 4–6 animals/group. * p < 0.05.

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  Figure 4. Myocardial ACE and ACE2 activities in cardiac tissue samples 6 weeks after MI induction. ACE (A,B) and ACE2 activities (C,D) were determined in anterior (A,C) and inferior (B,D) cardiac tissue samples from all experimental groups. Values are given as mean ± SEM; n = 4–6 rats/group. * p < 0.05.

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  Figure 5. Ca2+-sensitivity of force production (pCa50) in isolated, permeabilized cardiomyocytes of the anterior (A,C,E) and inferior LV areas (B,D,F). Absolute values of activate force (Factive) production in isolated myocyte-sized preparations at different Ca2+ concentrations in the anterior (A) and inferior (B) areas from the Sham, MI, and MI + rRIC groups. Sarcomere length was adjusted to 2.3 µm. Factive values at submaximal Ca2+ concentrations (pCa 5.4–7.0) were expressed relative to Fmax (pCa 4.75) to determine normalized pCa-force relationships (C,D). Bar graphs (E,F) highlight the midpoints of normalized pCa–force relationships. Data are expressed as mean ± SEM, * p < 0.05; n = 8 from at least four different hearts.

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  Figure 6. PKA- and PKC-specific phosphorylation of cTnI in the anterior (A,C) and inferior (B,D) LV regions. Cardiac troponin-I (cTnI) phosphorylation levels at the PKA-specific serine-22/23 (Ser-22/23-P) (A,B) and PKC-specific threonine-144 (Thr-144-P) (C,D) residues were screened by Western immunoblotting in Sham, MI, and MI + rRIC groups of anterior and inferior LV areas. The phosphorylation sites of cTnI were labelled with specific antibodies. Total protein amounts were assessed by a super sensitive blot stain. Bars represent means ± SEM (normalized to the mean of the Sham group as control), * p < 0.05, n = 7–10 independent determinations from at least 4–5 different hearts.

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  Figure 7. cMyBP-C phosphorylation in cardiomyocytes in the anterior (A) and inferior (B) LV regions. Total phosphorylation levels of cMyBP-C were investigated with Pro-Q® Diamond phosphoprotein staining. The upper bands illustrate the phosphorylation status of cMyBP-C and the lower bands indicate the total protein amounts for each representative membrane (n = 5–13 independent determinations from at least 4–5 different heart samples). Values are given as mean ± SEM, * p < 0.05 vs. control (Sham) groups.

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  Figure 8. Titin-based passive tension and titin protein phosphorylation in the anterior (A,C) and inferior (B,D) LV regions. Titin-dependent passive tension (Fpassive) in permeabilized cardiomyocytes was determined at a sarcomere length of 2.3 μm (A,B) (n = 8–10 cardiomyocytes/groups). Total phosphorylation status of the N2B titin isoform was assessed by Pro-Q® Diamond phosphoprotein staining. Total titin amount was visualized by Coomassie-blue staining (C,D). Protein homogenates were collected from four control and six infarcted hearts and assays were repeated four to six times. Data are given as mean ± SEM.