Identification of Protein Disulfide Isomerase as a Cardiomyocyte Survival Factor in Ischemic Cardiomyopathy
Anna Severino, PhD*,
Mara Campioni, BSc ,
Stefania Straino, BSc ,
Fadi N. Salloum, PhD ,
Nina Schmidt, BSc||,
Ulrike Herbrand, BSc||,
Stilla Frede, BSc||,**,
Gabriele Toietta, PhD,
Giuliana Di Rocco, PhD¶,
Rossana Bussani, MD#,
Furio Silvestri, MD#,
Maddalena Piro, MD*,
Giovanna Liuzzo, MD*,
Luigi M. Biasucci, MD*,
Pasquale Mellone, MD,
Florinda Feroce, MD,
Maurizio Capogrossi, MD,
Feliciano Baldi, MD,
Joachim Fandrey, PhD||,**,
Michael Ehrmann, PhD||,
Filippo Crea, MD*,
Antonio Abbate, MD and
Alfonso Baldi, MD,*
* Institute of Cardiology, Catholic University, Rome, Italy
Istituto Dermopatico dell’Immacolata, Rome, Italy
Department of Biochemistry, Pathology Section, Second University of Naples, Naples, Italy
¶ Laboratory of Vascular Biology and Gene Therapy, Centro Cardiologico Fondazione Monzino, Milan, Italy
# Department of Pathology, University of Trieste, Trieste, Italy
VCU Pauley Heart Center, Virginia Commonwealth University, Richmond, Virginia
|| Centre for Medical Biotechnology, University of Duisburg-Essen, Essen, Germany
** Institute of Physiology, University of Duisburg-Essen, Essen, Germany.
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Figure 1 Analysis of PDI Expression on Autoptic Human Infarcted Hearts
(A) Semiquantitative reverse transcriptase polymerase chain reaction showing higher expression of protein disulfide isomerase (PDI) transcript in the ribonucleic acid (RNA) extracted close to the infarcted area, with respect to the RNA extracted in the area remote from the infarct. The PDI expression in RNA extracted from a nonischemic heart as well as a reaction made with no RNA were used as controls. (B) Immunohistochemical expression of PDI protein in the infarcted area (original magnification x40). (C) Immunohistochemical expression of PDI in the area remote from the infarct (original magnification x40). (D) Double staining for PDI protein and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) in the infarcted area: only a few cardiomyocytes expressing high levels of PDI were TUNEL positive, as indicated by an asterisk (original magnification x40). (E) Correlation between PDI expression and apoptotic rate in the peri-infarct region. (F) Correlation between PDI expression and congestive heart failure (CHF) signs. (G) Correlation between PDI expression and biventricular enlargement.
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Figure 2 PDI Over-Expression Protects HL1 Cardiomyocytes From Apoptosis Under Hypoxic Conditions
(A) Endogenous protein disulfide isomerase (PDI) expression is induced under hypoxic conditions in mouse cardiomyocytes. HL1 cells were cultured under hypoxia conditions for 24 h and 48 h. The PDI expression was analyzed by Western blotting. (B) Schematic representation of PDI thioredoxin-like domains. The PDI variants have both cysteine-to-serine mutations in the N-terminal and/or C-terminal thioredoxin-like domains. Over-expression of wild-type PDI (PDI-wt), PDI-N, and PDI-C in HL1 stable individual clones were estimated by Western blot analysis. (C) Stable over-expression of PDI-wt but not of the N- and C-terminal mutants results in a significant protection from hypoxia-induced apoptosis. The HL1 cells were stable transfected with PDI-wt, PDI-N, PDI-C, or empty vector. Stable individual clones were incubated for the indicated periods in hypoxic conditions. The percentage of apoptotic cells was estimated by flow cytometric analysis. Values represent the mean ± SE of 5 independent experiments. *Significant differential apoptotic rate (Student t test, n = 5, p < 0.001) in normoxic and hypoxic cardiomyocytes at 24 h and 48 h. **Significant differential apoptotic rate (Student t test, n = 5, p < 0.001) in cells transfected with enzymatically inactive point mutants compared with the control in normoxic and hypoxic conditions at 24 h and 48 h.
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Figure 3 Hypoxia Induces the Endoplasmic Reticulum Stress Response Genes in HL1 Cardiomyocytes, and Zinc-Superoxide Dismutase-1 Is a Target of the Anti-Apoptotic Action of PDI
(A) Total RNA of hypoxia-treated murine HL1 cells was analyzed by quantitative reverse transcriptase polymerase chain reaction for the expression levels of PDI, Ero1, Grp94, binding protein (BIP), and the transcription factors PERK, IRE1-alpha, and ATF6-alpha. *Significant differential expression (Student t test, n = 4, p < 0.05) after exposure to hypoxia. (B) Hydrogen peroxide (H2O2) release was measured in HL1 cells by horseradish peroxidase-enhanced chemiluminescence. Given are the mean ± SD from 3 independent measurements. *Significant differential expression (Student t test, n = 5, p < 0.05). Abbreviations as in Figure 1.
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Figure 4 PDI Over-Expression Has a Protective Effect in the In Vivo Acute MI Mouse Model
Adenoviral vectors expressing both PDI and green fluorescent protein (GFP) (AdPDI/GFP) or GFP only (Adnull/GFP) were administered into the ventricular wall into C57/BL6 mice. A set of animals were killed 48 h after adenoviral administration to determine the amount of transgene expression. The remaining animals were subjected to left coronary artery ligation 48 h after viral administration. One week after the surgical procedure, mice were killed for analysis. (A) Adenovirus mediated over-expression of PDI-wt in C57/BL6 mice. Mice were killed 48 h after adenoviral injections. Western-blot analyses of GFP and PDI expression were performed with total lysates of homogenized myocardial tissue. (B) Immunohistochemical analysis of GFP and PDI expression in the injected hearts (original magnification x20). (C) Analysis of infarct extension, both in terms of extension and thickness, in AdPDI/GFP-injected hearts with respect to the controls. (D) Analysis of the apoptotic rate in injected hearts with respect to the controls. (E) Echocardiographic analysis of the left ventricular end-diastolic diameter (LVEDD) parameter in AdPDI/GFP-injected hearts with respect to the controls. (F) Echocardiographic analysis of the left ventricular end-systolic diameter (LVESD) parameter in AdPDI/GFP-injected hearts with respect to the controls. Abbreviations as in Figures 1 and 2.
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Figure 5 Histopathological Analysis of the Infarcted Mice Hearts
(A) Hematoxylin/eosin picture of the infarcted heart injected with AdPDI/GFP: the area of the wall of the left ventricle affected by the infarct is indicated by a box (original magnification x10). (B) Hematoxylin/eosin picture of the infarcted heart injected with AdPDI/GFP: the involvement of the wall in terms of thickness is indicated by a heavy black line (original magnification x20). (C) Hematoxylin/eosin picture of the infarcted heart injected with Adnull/GFP: the area of the wall of the left ventricle interested by the infarct is indicated by a box (original magnification x10). (D) Hematoxylin/eosin picture of the infarcted heart injected with the vector alone: the involvement of the wall in terms of thickness is indicated by a bold line (original magnification x20). (E) Examples of TUNEL staining in the infarcted hearts injected with AdPDI/GFP and with Adnull/GFP (original magnification x40). (F) Examples of the double staining TUNEL-muscle actin to detect only the apoptotic cardiomyocytes (original magnification x40). Abbreviations as in Figure 1.
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