A Sarcoplasmic Reticulum Localized Protein Phosphatase Regulates Phospholamban Phosphorylation and Promotes Ischemia Reperfusion Injury in the Heart

Figures

• 

  Figure 1. PP2Ce is an ER Membrane-Targeted Protein Phosphatase and Detected in Adult Mouse Hearts (A) Schematic description of protein phosphatase 2C on endoplasmic reticulum (ER) membrane (PP2Ce) protein with signal peptide sequence (aa20-40), transmembrane domain, and conserved PP2C phosphatase activity domain highlighted. (B) Northern-blot analysis of PP2Ce messenger ribonucleic acid (mRNA) expression in different tissues from adult mice with significant expression detected in in the brain, heart, and diaphragm. (C) Immunohistochemistry results displayed PP2Ce (green) was colocalized with sarcolemma-endoplasmic reticulum calcium ATPase (SERCA) 2a (red) in adult rabbit ventricular cardiomyocytes as supported in merged image and line-scanning intensity analysis. (D) Relative expression of PP2Ce mRNA detected by quantitative real-time polymerase chain reaction in heart samples from ischemic cardiomyopathy (ICM) (n = 3) and dilated cardiomyopathy (DCM) (n = 3) compared with non-heart failure hearts (normal, n = 3). *p < 0.05 versus normal.

• 

  Figure 2. PP2Ce Expression on ER Stress Signaling Pathway in NRVM (A) Representative immunoblot of ER stress regulators as indicated in NRVM expressing GFP and PP2Ce in response to 50 μmol/l H2O2 treatment at specific time points as indicated. The 5 μg/ml tunicamycin-treated NRVM was used as a positive control. (B) Quantification of immunoblot signals with independent experimental replicate number labeled in each column. **p < 0.01. ER = endoplasmic reticulum; GFP = green fluorescent protein; NRVM = neonatal rat ventricular myocyte; PP2Ce = protein phosphatase 2C on ER membrane.

• 

  Figure 3. PP2Ce is a Specific Phosphatase for PLN Thr-17 Phosphorylation Site (A) Representative immunoblot from NRVM-expressing GFP or PP2Ce were treated with 1 μmol/l isoproterenol for 0.5 and 2 h as indicated. (B) Quantification of immunoblot signals with independent experimental replicate number labeled in each corresponding column. (C) Western blot for NRVM coexpressing active CaMKII (CaMKII-CA) and increasing amount of PP2Ce as indicated. *p < 0.05; **p < 0.01 versus untreated; and #p < 0.05; between PP2Ce and GFP at each time point. PLN = phospholamban; other abbreviations as in Figure 2.

• 

  Figure 4. Specificity of PP2Ce Activity to PLN Thr-17 Phosphorylation (A) Immunoblot of PP2Ce, GFP, and PLN phosphorylation (Ser-16 and Thr-17) in NRVM expressing GFP or PP2Ce wild-type or PP2Ce phosphatase dead mutant (D320A) following 1 μmol/l isoproterenol treatment at different time points as indicated. (B) ISO-induced PP2Ce expression and PLN phosphorylation in NRVM-expressing PP2Ce truncation mutant (⊿1–57), which has lost ER targeting capacity 28, 29. (C) PP2Ce–GST fusion protein was incubated with PLN isolated from NRVM with or without ISO treatment, followed by immunoblot for total and phosphorylation levels of PLN at Ser-16 and Thr-17. (D) Coimmunoprecipitation of PP2Ce and PLN in mouse heart using PP2Ce TG and PP2Ce KO as positive and negative control, respectively. ISO = isoproterenol; TG = transgenic; other abbreviations as in Figures 2 and 3.

• 

  Figure 5. ISO-Mediated Regulation of PP2Ce Protein in Cardiomyocytes (A) PP2Ce and PP2Cα expression in NRVM following ISO treatment. (B) PP2Ce expression in NRVM treated with ISO (1 μmol/l) with or without cotreatment of proteasome inhibitor MG132 (10 μmol/l) or lysosomal inhibitor BAF (50 nmol/l) as indicated. (C) Quantification of PP2Ce expression in NRVM treated with ISO with or without additional treatment of cycloheximide (CHX), MG132, or BAF as indicated. (D) PP2Ce expression in NRVM-treated ISO or forskolin (FSK) at 0.1, 1, and 10 μmol/l concentrations as indicated. (E) PP2Ce expression in NRVM treated with FSK (1 μmol/l) with or without PKA inhibitor H89 (1 μmol/l) as indicated. n indicates the number of total experimental replicates. *p < 0.05; **p < 0.01. Abbreviations as in Figures 2, 3, and 4.

• 

  Figure 6. Functional Effect of PP2Ce Expression on SR Calcium Homeostasis in Adult Rat Cardiomyocytes (A) Representative images of calcium transients from control rat myocytes at basal and following ISO (0.1 μmol/l) treatment. (B) Representative recording of calcium signal (F/F0) at basal and post-ISO treatment in LacZ-expressing rat adult cardiomyocytes. (C) Representative recording of calcium signal at basal and post-ISO treatment in PP2Ce-expressing rat adult cardiomyocytes. (D) Summary data of changes in calcium amplitude and decay time in adult cardiomyocytes expressing LacZ (n = 5) versus PP2Ce (n = 3) between basal and post-ISO treatment. *p < 0.05; **p < 0.01 LacZ versus PP2Ce, respectively. ⊿ = the difference between post- and pre-ISO treatment; other abbreviations as in Figures 2, 3, and 4.

• 

  Figure 7. Effect of PP2Ce on Calcium Homeostasis in Intact Mouse Heart (A) Schematic drawing of PP2Ce-transgenic mouse with tissue-specific expression of PP2Ce in heart. (B) PP2Ce protein expression in mouse hearts from 4 genotypes carrying either none, single, or double transgenes of αMHC-tTA expressing tet-off suppressor (tTA) and αHMC-TetO-PP2Ce (iPP2Ce). PP2Ce expression was detected only from double transgenic hearts in the absence of tetracycline. PC is the positive control from NRVM-expressing adv-PP2Ce. (C) Representative immunoblot analysis. (D) Quantification of key proteins involved in calcium homeostasis regulation in control and PP2Ce transgenic (TG) hearts. Number of replicates is labeled in each column. (E) Calcium transient peak amplitudes (F/F0), time to peak and decay measured from isolated cardiomyocytes of control (n = 8) and PP2Ce TG (n = 8) hearts. *p < 0.05; **p < 0.01. Abbreviations as in Figures 2, 3, and 4.

• 

  Figure 8. PP2Ce Expression Impairs ISO Stimulated Enhancement of Contractility (A) Representative left ventricle pressure (LVP) measured from isolated hearts of wildtype or PP2Ce TG mice at basal or perfused by 1 μmol/l of ISO. (B) Hemodynamic parameters from control (n = 9) and PP2Ce TG (n = 6) hearts at basal or treated by 10 nmol/l ISO. *p < 0.05; **p < 0.01 between basal and I/R within control or PP2Ce TG groups. #p < 0.05 between corresponding control and PP2Ce TG. dP/dt max = maximal systolic pressure derivative; dP/dt min = maximal diastolic pressure derivative; HR = heart rate; I/R = ischemia/reperfusion; LVDP = left ventricle developed pressure (Pmax − Pmin); Pmax = systolic pressure; Pmin = diastolic pressure; Tau = relaxation index; other abbreviations as in Figures 2, 3, and 4.

• 

  Figure 9. PP2Ce Expression Impairs Function Recovery Following Ischemia/Reperfusion Injury (A) Left ventricle dP/dt max during 15-min global ischemia followed by 60-min reperfusion in isolated hearts from nontransgenic control subjects (control, n = 9) and PP2Ce TG (TG, n = 6) mice. Left ventricle function parameters measured as (B) left ventricular developed pressure (LVDP), (C) systolic pressure increase (dP/dt max), (D) diastolic pressure decrease (dP/dt min), and (E) relaxation time index (Tau) in nontransgenic control subjects (control, n = 9) and PP2Ce TG hearts (n = 6) at basal and 45 min after reperfusion. **p < 0.01 between control and PP2Ce TG groups. #p < 0.05; ##p < 0.01 between corresponding control and PP2Ce TG groups at each time point.

• 

  Figure 10. Induced PP2Ce Expression Promotes Cardiomyocyte Death in Response to I/R Injury (A) Representative TTC staining cross section images from 1 PP2Ce TG and 1 control heart following 15 min of ischemia and 120 min of reperfusion (I/R). (B) Infarct sizes of control (n = 5) and PP2Ce TG (n = 6) hearts as measured from (A). (C) Apoptotic index measured by TUNEL staining in control (n = 4) and PP2Ce TG (n = 4) hearts. (D) Representative immunoblot for level of activated caspase 9 in control (n = 10) and PP2Ce TG (n = 8) hearts following the same I/R protocol. (E) Quantification of level of cleaved caspase 9 signal from immunoblots. *p < 0.05 between control and PP2Ce TG groups. **p < 0.01 between control and PP2Ce TG groups. Abbreviations as in Figures 2, 3, and 4.

• 

  Figure 11. PP2Ce Expression Promotes Myocytes Death and Perturbs Prosurvival Signaling Following Oxidative Injury (A) NRVM viability measured by MTT assay in NRVM expressing GFP or PP2Ce following H2O2 treatment for 16 h at different doses as indicated. Replicate number is labeled in each corresponding column. (B) NRVM viability expressing GFP (n = 8) or PP2Ce (n = 8) under untreated or following 24 h of treatment of 2 μmol/l ionomycin. (C) Immunoblot analysis of intracellular signaling molecules as indicated in NRVM-expressing GFP or PP2Ce with 50 μmol/l H2O2 treatment at different time points as indicated. Tunicamycin (5 μg/ml for 4 h) was used as a control. (D) Quantification of signaling molecules as indicated with independent experimental replicate number labeled in each column. **p < 0.01 versus untreated. ##p < 0.01 between GFP and PP2Ce groups at same doses. Abbreviations as in Figures 2, 3, and 4.

• 

  Figure 12. Illustration of PP2Ce Function in Cardiac Myocytes Abbreviations as in Figures 2 and 4.