Minocycline inhibits caspase activation and reactivation, increases the ratio of XIAP to smac/DIABLO, and reduces the mitochondrial leakage of cytochrome C and smac/DIABLO
Tiziano M. Scarabelli, MD, PhD*,*,
Anastasis Stephanou, PhD ,
Evasio Pasini, MD ,
Gianluca Gitti, BSc,
Paul Townsend, PhD,
Kevin Lawrence, PhD,
Carol Chen-Scarabelli, MSc||,
Louis Saravolatz, MD ,
David Latchman, PhD, DSc,
Richard Knight, MD, PhD¶ and
Julius Gardin, MD*
* Division of Cardiology, Detroit, Michigan, USA
Division of Internal Medicine, St. John Hospital and Medical Center, Detroit, Michigan, USA
Institute of Child Health, University College London, London, England, UK
Cardiovascular Pathophysiology Research Centre, S. Maugeri Foundation, IRCCS Gussago, Italy
|| Division of Cardiovascular Surgery, Jackson Memorial Hospital, University of Miami, Miami, Florida, USA
¶ Department of Cystic Fibrosis, National Heart and Lung Institute, Imperial College London, London, England, , UK
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Figure 1 Quantification of necrosis and apoptosis by flow cytometry in primary cultures of neonatal (A) and adult (B) myocytes. Data are expressed as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 vs. ischemic/reperfused (I/R) control (Ctrl). Open bars = necrosis; solid bars = apoptosis. MNC = minocycline.
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Figure 2 Hemodynamic measurements in the non-ischemic isolated rat heart perfused with minocycline (MNC) (10–5 to 10–7 M) for 1 h. Changes in left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) are shown in A and B, respectively. Results are expressed as mean ± SD. Mechanical function of the isolated rat heart during ischemia/reperfusion (I/R), with and without pretreatment with minocycline (C to E). CTRL = control; Solid triangles = LVSP; open squares = LVEDP.
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Figure 3 Infarct size, expressed as a percentage of myocardial risk zone (A), and post-ischemic release of creatine phosphokinase (CPK) (B) in ischemic/reperfused (I/R) control (Ctrl) hearts and hearts exposed to I/R, following ex vivo and in vivo treatment with either minocycline (Mnc) or tetracycline (Tcn). Data are expressed as mean ± SD. ***p < 0.001 versus non-ischemic control hearts. Infarcted areas, assessed by triphenyl-tetrazolium chloride exclusion, in I/R control (C) and in vivo minocycline-treated (D) hearts. The corresponding color-enhanced images (white = infarcted area; green = area at risk; red = non-ischemic zone) are depicted in E (Ctrl hearts) and F (Mnc-treated hearts), respectively. Percentages of TUNEL (G) and cleaved active caspase-3 (C3) (H) positive endothelial cells (EC) and cardiac myocytes (CM) in control hearts exposed or unexposed to I/R, and in I/R hearts pretreated ex vivo and in vivo with either Mnc or Tcn. Values are averages of three independent experiments ±SD. ***p < 0.001 vs. ischemic Ctrl hearts.
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Figure 4 Myocardial sections from control and treated hearts stained by TUNEL and propidium iodide. Myocytes and endothelial cells were selectively identified by specific anti-desmin (C and F) and von Willebrand (D and G) labeling, respectively. Non-ischemic control hearts exhibit no yellow TUNEL-positive cells (A and B). In ischemic/reperfused (I/R) control hearts, TUNEL-positivity was consistently observed both in cardiomyocytes (C) and endothelial cells (E). In vivo treatment with minocycline (Mnc) significantly reduced the magnitude of apoptosis in both cell types (F and H). Original magnification: x400.
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Figure 5 Reverse transcriptase-polymerase chain reaction (A) and Western blots (B) of procaspase-1, -3, -7, -8, -9, -12, XIAP, and Smac/DIABLO RNA and protein levels in cardiac tissues from hearts, exposed and unexposed, to ischemic/reperfusion (I/R), with and without in vivo treatment with minocycline. Corresponding densitometric analyses are shown at the bottom of each panel. Values are represented as relative fold change of the control value set to 1. Statistical analysis (*p < 0.05; **p < 0.01; ***p < 0.001) was performed versus control hearts (C) either unexposed (non-ischemic hearts treated in vivo with minocycline [Mnc]) or exposed to I/R (ischemic hearts pretreated in vivo with minocycline). (C) Caspase-3, -7, -8, and -9 enzymatic activity in tissue extracts from non-ischemic and ischemic control hearts and hearts pretreated in vivo with minocycline and exposed to I/R. Data are expressed as mean ± SD. ***p < 0.001 vs. control hearts. (D) Expression levels of cytochrome c and Smac/DIABLO proteins in mitochondrial and cytosolic fractions from control hearts exposed and unexposed to I/R, with and without in vivo treatment with minocycline. HSP60 and actin were used as internal controls for the mitochondrial and cytosolic fractions, respectively. The correspondent densitometric evaluation is shown at the bottom of the panel. Values are represented as relative fold change of the control value set to 1. Statistical analysis was performed as stated above. The reverse transcription-polymerase chain reaction and Western data are representative of five experiments performed in individual animals, and the in vivo treatment was performed as described in the Methods section and the legend to Figure 3.
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