Myocardial overexpression of protein phosphatase 2A-B56α improves resistance against ischemia-reperfusion injury

Figures

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  Fig. 1. Improved left ventricular pressure development in TG after ischemia. Isolated, Langendorff-perfused hearts of WT and TG mice were equilibrated (B=basal) and then subjected to a 20-min global no-flow ischemia that was followed by a 40-min reperfusion period. The maximum left ventricular (LV) pressure (A), the maximum rate of LV pressure development (B) and the maximum rate of LV pressure decline (C) were constantly monitored in the absence and presence of 3 nM okadaic acid (±OA) (*P < 0.05 vs. WT; two-way ANOVA RM with Holm-Sidak's posttest; N = 5 hearts each group).

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  Fig. 2. Attenuated ischemic contracture in TG hearts. Isolated, Langendorff-perfused hearts of both genotypes were equilibrated and then subjected to a 20-min global non-flow ischemia that was followed by a 40-min reperfusion. The maximum diastolic left ventricular pressure was measured in the absence (A) and presence (B) of 3 nM okadaic acid (+OA) (N = 5 hearts each group). The time of ischemic contracture (C) was detected when the diastolic pressure reached 30 mm Hg (red dotted line) (*P < 0.05 vs. non-treated WT; unpaired Student's t-test; N = 5–12 hearts).

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  Fig. 3. Reduced phosphorylation of cardiac regulatory proteins in TG hearts in late reperfusion. Isolated, Langendorff-perfused hearts of WT and TG mice were equilibrated and then subjected to a 20-min global non-flow ischemia that was followed by reperfusion. Hearts were analyzed for relative protein phosphorylation 40 min after reperfusion in the absence (non-treated) and presence (+OA) of 3 nM okadaic acid. The following phosphoproteins were detected: MyBP-C, myosin-binding protein C (A), MLC2, myosin light chain type 2 (B), TnI, troponin inhibitor (C), RyR2, ryanodine receptor type 2 (D). The levels of phosphoproteins were related to the expression of total proteins. Shown are representative immunoblots of non-phosphorylated and phosphorylated proteins (*P < 0.05 vs. WT; unpaired Student's t-test; N = 5–6 hearts). Protein phosphatase activity was measured in homogenates of hearts sampled under basal conditions, 5 min and 40 min after reperfusion. 3 nM okadaic acid was used to discriminate between type 2A (E) and type 1 (F) phosphatase activities (*P < 0.05 vs. WT; one-way ANOVA with Student-Newman-Keuls posttest; N = 5 hearts each group). More original examples of immunoblots are…

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  Fig. 4. Enhanced phosphorylation of ERK1 in TG hearts in early reperfusion. Isolated, Langendorff-perfused hearts were subjected to global ischemia and subsequent reperfusion. Thereafter, the expression of RISK phosphoproteins was determined under basal conditions and after early (5 min) and late (40 min) phases of reperfusion in heart homogenates. The summarized data are shown for relative phosphorylation of Akt (A), ERK1 (B) and ERK2 (C). The level of phosphorylation was related to the expression of the total protein. Representative immunoblots are depicted on the right-hand site (*P < 0.05 vs. WT; one-way ANOVA with Student-Newman-Keuls posttest; N = 4–7 hearts). More original examples of immunoblots are provided in Supplemental Fig. 2. Enhanced phosphorylation of ERK1 in TG hearts in early reperfusion. Isolated, Langendorff-perfused hearts were subjected to global ischemia and subsequent reperfusion. Thereafter, the expression of RISK phosphoproteins was determined under basal conditions and after early (5 min) and late (40 min) phases of reperfusion in heart homogenates. The summarized data are shown for relative phosphorylation of Akt (A), ERK1 (B) and ERK2 (C). The level of…

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  Fig. 5. Delayed development of acidosis in TG cardiomyocytes during ischemia. (A) Shown are representative images of isolated WT cardiomyocytes labeled with pHrodo Red (A). Cells were gassed with carbogen (5 % CO2 and 95 % O2) and the basal fluorescence signal was detected. Thereafter, cardiomyocytes were incubated with 20 % CO2. A decrease in pH results in higher fluorescence intensities. The summarized data of percent increase of maximal fluorescence signal after incubation with 20 % CO2 are given (B). The time to 50 % increase in F1/F0 signal was determined in both genotypes (C) (*P < 0.05 vs. WT; unpaired Student's t-test; n = 25–27 cardiomyocytes; N = 3 hearts each group). Mitochondrial preparations were obtained from isolated, Langendorff-perfused hearts subjected to a standard I/R protocol. The mitochondrial expression of B56α was measured under basal conditions, after a 30-min period of global ischemia and after 30 min of reperfusion. The summarized results are given (D, left panel) (*P < 0.05 vs. basal; one-way ANOVA with Student-Newman-Keuls posttest; N = 3 hearts each group). Representative immunoblots of B56α and the voltage-dependent anion channel, VDAC, which served as a…

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  Fig. 6. Reduced fibrosis in heart sections of TG mice after coronary banding. Myocardial infarction was induced by 120 min ligation of the left anterior descending artery. Mice were killed 3 weeks after ligation by whole-body perfusion and fixation with paraformaldehyde (4 %). Hearts were excised, embedded in paraffin and then cut into 5-μm sections. Subsequently, sections were deparaffinized, rehydrated, and subjected to Heidenhain's AZAN trichrome staining which colorizes connective tissue such as the infarct scar deep blue, in contrast to the red cardiomyocytes of the remote heart tissue appearing brick-red. Representative sections of the same transverse heart level are shown for both genotypes (A). The amount of fibrotic area in percent of the whole heart section area was determined (B) (*P < 0.05 vs. WT; unpaired Student's t-test; N = 8 hearts each group).

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  Fig. 7. Identification of potential PP2A-B56α targets under I/R conditions. Samples were taken from isolated Langendorff-perfused hearts of WT and TG mice after 40 min of reperfusion. After 2D-gel electrophoresis and ProQ Diamond staining, differentially regulated spots (showing a WT/TG ratio of > ± 1.5, P ≤ 0.05) where excised and analyzed by mass spectrometry (A). The individual phosphorylation levels of up- and downregulated proteins are represented by a heat map (B). A larger detailed image of the 2D gel with circled spots is shown in Supplemental Fig. 3. More details on the mass spectrometry analysis and Mascot engine search results are provided in Supplemental Table 1. Identification of potential PP2A-B56α targets under I/R conditions. Samples were taken from isolated Langendorff-perfused hearts of WT and TG mice after 40 min of reperfusion. After 2D-gel electrophoresis and ProQ Diamond staining, differentially regulated spots (showing a WT/TG ratio of > ± 1.5, P ≤ 0.05) where excised and analyzed by mass spectrometry (A). The individual phosphorylation levels of up- and downregulated proteins are represented by a heat map (B). A larger detailed image of the 2D gel with circled…

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  Fig. 8. Hypothesis of preservation of cardiac function in PP2A-B56α-overexpressing mice. During ischemia (-O2) overexpression of PP2A-B56α prevents cardiac stiffness probably by an inhibition of myocellular Ca2+ overload and mPTP opening. The prolonged acidification in TG cardiomyocytes may contribute to this process leading to a decreased fibrosis in transiently LAD-ligated mice. During early reperfusion (+O2) the B56α-mediated attenuation of PP2A activity might activate ERK1 resulting in inhibition of mPTP opening and cardioprotection. The translocation of B56α into mitochondria might help to inhibit PP2A, which suppresses the release of cytochrome c. At the late phase of reperfusion, higher PP2A activity was restored, which was paralleled by an increased contractility and lower okadaic acid-sensitive phosphorylation levels of MyBP-C and the RyR2.