Phosphorylation and translocation of heat shock protein 27 and αB-crystallin in human myocardium after cardioplegia and cardiopulmonary bypass Richard.

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Phosphorylation and translocation of heat shock protein 27 and αB-crystallin in human myocardium after cardioplegia and cardiopulmonary bypass Richard T. Clements, PhD, Neel R. Sodha, MD, Jun Feng, MD, PhD, Shigetoshi Mieno, MD, PhD, Munir Boodhwani, MD, Basel Ramlawi, MD, Cesario Bianchi, MD, PhD, Frank W. Sellke, MD The Journal of Thoracic and Cardiovascular Surgery Volume 134, Issue 6, Pages 1461-1470.e3 (December 2007) DOI: 10.1016/j.jtcvs.2007.06.026 Copyright © 2007 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 p38 MAPK is phosphorylated after cardioplegic arrest. A, Representative blots of phospho-p38MAPK and total p38-MAPK pre-CP/CPB (Pre) and post-CP/CPB (Post) in atria and skeletal muscle. B, Quantitative densitometry of the data shown in A. Phosphorylation data normalized to the level of total p38-MAPK and presented as the mean fold increase ± standard error of the mean (n = 6). *P < .05 versus the respective pre-CP/CPB group. MAPK, Mitogen-activated protein kinase; CP/CPB, cardioplegia/cardiopulmonary bypass. The Journal of Thoracic and Cardiovascular Surgery 2007 134, 1461-1470.e3DOI: (10.1016/j.jtcvs.2007.06.026) Copyright © 2007 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 CP/CPB increases phosphorylation of HSP27 in cardiac tissue. A, Representative blots of total HSP27 and HSP27 phosphorylated on ser15, ser78, and ser82 in atrial and skeletal muscle pre-CP/CPB (Pre) and post-CP/CPB (Post). B-D, Densitometry values of phospho-HSP27 normalized to total HSP27 in atrial and skeletal muscle tissue pre- and post-CP/CPB: pser15-HSP27 (B), pser78-HSP27 (C), and pser82-HSP27 (D), minimum n = 6 for each residue. Data presented as the mean ± standard error of the mean in all graphs. *P < .05 versus the respective pre-CP/CPB group. #P < .05 versus the atrial pre-CP/CPB value. pser, Phosphorylated serine; HSP, heat shock protein; CP/CPB, cardioplegia/cardiopulmonary bypass. The Journal of Thoracic and Cardiovascular Surgery 2007 134, 1461-1470.e3DOI: (10.1016/j.jtcvs.2007.06.026) Copyright © 2007 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 CP/CPB increases phosphorylation of cryAB in cardiac tissue. A, Representative blots of total cryAB and cryAB phosphorylated on ser19, ser45, and ser59 in atria and skeletal muscle pre- and post-CP/CPB. B-D, Densitometry values of phospho-cryAB normalized to total cryAB in atrial and skeletal muscle tissue pre- and post-CP/CPB: pser19-cryAB (B), pser45-cryAB (C), and pser59-cryAB (D), minimum n = 6 for each residue. Data presented as the mean ± standard error of the mean in all graphs. *P < .05 versus the respective pre-CP/CPB group. #P < .05 versus the atrial pre-CP/CPB value. pser, Phosphorylated serine; cryAB, αB-crystallin; CP/CPB, cardioplegia/cardiopulmonary bypass. The Journal of Thoracic and Cardiovascular Surgery 2007 134, 1461-1470.e3DOI: (10.1016/j.jtcvs.2007.06.026) Copyright © 2007 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 CP/CPB-induces sarcomeric translocation of HSP27. Human atrial sections pre- (A, C, E, G) and post-CP/CPB (B, B’, D, D’, F, F’, H, H’, I-L) were labeled with antibodies to HSP27 (red: A, B, B’, I), pser15-HSP27 (red: C, D, D’, J), pser78-HSP27 (red: E, F, F’, K), or pser82-HSP27 (red: G, H, H’, L). Sections were co-labeled with the atrial myocyte marker MLC-2a (green) and nuclei stain ToPro-3 (Invitrogen, San Diego, Calif) (blue) (B’, D’, F’, H’, I-L). Intercalated disc localization (arrows); nuclear localization (arrowheads). Representative micrographs are shown, minimum n = 3. Bar (H’) = 20 μm (A-H’). Bar (L) = 10 μm (I-L). pSer, Phosphorylated serine; HSP, heat shock protein; CP/CPB, cardioplegia/cardiopulmonary bypass. The Journal of Thoracic and Cardiovascular Surgery 2007 134, 1461-1470.e3DOI: (10.1016/j.jtcvs.2007.06.026) Copyright © 2007 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 CP/CPB induces sarcomeric translocation of cryAB. Human atrial sections pre- (A, C, E, G) and post-CP/CPB (B, B’, D, D’, F, F’, H, H’, I-L) were labeled with antibodies to cryAB (red: A, B, B’, I), pser19-cryAB (red: C, D, D’, J), pser45-cryAB (red: E, F, F’, K), or pser59-cryAB (red: G, H, H’, L). Sections were co-labeled with the atrial myocyte marker MLC-2a (green) and the nuclei stain ToPro-3 (blue) (B’, D’, F’, H’, I-L). Intercalated disc localization (arrows) and nuclear localization (arrowheads). Representative micrographs are shown, minimum n = 3. Bar (H’) = 20 μm (A-H’). Bar (L) = 10 μm (I-L). cryAB, αB-crystallin; pSer, phosphorylated serine; CP/CPB, cardioplegia/cardiopulmonary bypass. The Journal of Thoracic and Cardiovascular Surgery 2007 134, 1461-1470.e3DOI: (10.1016/j.jtcvs.2007.06.026) Copyright © 2007 The American Association for Thoracic Surgery Terms and Conditions

Figure 6 Cardioplegic arrest induces cytoskeletal translocation of HSP27 and cryAB. Human atrial samples were fractionated into Triton-soluble and insoluble tissue lysates. Phosphorylation of HSP-27 (A) and cryAB (B) (upper) post-CP/CPB was associated with movement of a significant fraction of total HSP27 and cryAB to the Triton X-100–insoluble protein pool (lower). (C) There were no CP/CPB-induced solubility changes of the highly related myotonic dystrophy protein kinase binding protein. Representative blots shown from 1 patient, total n = 4. S, Soluble; I, insoluble; pSer, phosphorylated serine; HSP, heat shock protein; MKBP, myotonic dystrophy protein kinase binding protein. The Journal of Thoracic and Cardiovascular Surgery 2007 134, 1461-1470.e3DOI: (10.1016/j.jtcvs.2007.06.026) Copyright © 2007 The American Association for Thoracic Surgery Terms and Conditions

Figure E1 HSP27 and cryAB colocalize and interact pre- and post-CP/CPB. Human atrial sections pre- (A-C) and post-CP/CPB (D-I) were labeled with antibodies to cryAB (green: A, C, D, F, G, I) or HSP27 (red: B, C, E, F, H, I) and the nuclei stain ToPro-3 (blue) (C, F, H). Representative micrographs are shown (n = 3). Bar (F) = 20 μm (A-F). Bar (G) = 10 μM (G-I). J, Human atrial radioimmunoprecipitation assay lysate pre- (lanes 3, 5, 7, and 9) and post-CP/CPB (lanes 1, 2, 4, 6, 8, and 10) was immunoprecipitated for HSP27 (lanes 5 and 6, IP fraction; lanes 3 and 4, post-IP radioimmunoprecipitation assay fraction), cryAB (lanes 9 and 10, IP; lanes 7 and 8, post-IP radioimmunoprecipitation assay fraction), or nonspecific mouse immunoglobulin-G (lane 2, IP fraction; lane 1, post-IP fraction, post-CP/CPB only). Duplicate gels were inmmunoblotted for HSP27 (upper) and cryAB (lower). Blots are representative of 3 independent experiments. cryAB, αB-crystallin; HSP, heat shock protein; CP/CPB, cardioplegia/cardiopulmonary bypass; Ig, immunoglobulin; IP, immunoprecipitation. The Journal of Thoracic and Cardiovascular Surgery 2007 134, 1461-1470.e3DOI: (10.1016/j.jtcvs.2007.06.026) Copyright © 2007 The American Association for Thoracic Surgery Terms and Conditions

Figure E2 Increased phosphorylation of HSP27 and cryAB correlates with depressed post-surgery cardiac function. Fold increase phosphorylation of HSP27 and cryAB (phospho-ser82 (A, C, E) and ser59 (B, D, F) pre- and post-CP/CPB were plotted against cardiac index at ICU admittance (A, B), the 4-hour postsurgery ICU cardiac index (C, D), and percent change presurgery versus postsurgery EF (E, F) (n = 12 for A-D; n = 14 for E and F). pser, Phosphorylated serine; HSP, heat shock protein. The Journal of Thoracic and Cardiovascular Surgery 2007 134, 1461-1470.e3DOI: (10.1016/j.jtcvs.2007.06.026) Copyright © 2007 The American Association for Thoracic Surgery Terms and Conditions