Tiziano Scarabelli, MD, PhD, FAHA
Associate Professor FTA of Internal Medicine and Pharmacology; Wayne State University School of Medicine.
Director, Center for Heart & Vessel Preclinical Studies; Director, Basic Cardiovascular Research; St John Hospital & Medical Center.

421 E. Canfield
Detroit, MI 48201
Phone: 313-343-4559
Fax: 313-343-3912
email: tiziano.scarabelli@wayne.edu

RESEARCH INTERESTS

Apoptosis is a form of cell suicide, which contributes, together with necrosis, to the cardiac cell loss following ischemia/reperfusion injury (heart attack). Our work focuses on the occurrence of apoptotic cell death in different cell types during ischemia/reperfusion injury, the initiating pathways leading to activation of the apoptotic process, as well as the clinical potential of new cardioprotective agents.

Endothelial cells undergo apoptosis prior to cardiac cells
We first described the occurrence of apoptosis in different cell types in the isolated Langendorff-perfused rat heart (see Figure 1) exposed to ischemia/reperfusion injury. Apoptosis was seen mainly in endothelial cells, only after the onset of reperfusion. In addition, using a three-step immunocytochemical technique, we showed that the first cells to appear as apoptotic in the very early stage of reperfusion were those in the coronary vasculature, followed, in a later phase of reperfusion, by cardiac myocytes. Furthermore, the number of positive myocytes decreased with increasing distance from the positive vessel, suggesting that cells of the coronary vasculature may actively contribute to the death of myocytes by releasing pro-apoptotic mediators. 

Different apoptotic pathways lead to cell death endothelial and cardiac cells
The apoptotic cascade can be initiated either by mitochondrial damage and activation of caspase-9, or by death receptor ligation, which leads to activation of caspase-8. Specific inhibitors of caspase-8 and caspase-9 were used in order to estimate the relative contribution of the two main apoptotic initiating pathways in inducing apoptosis both in endothelial cells and cardiac myocytes in the two phases of ischemia reperfusion. Cleavage of caspase-9 was observed primarily in endothelial cells. Conversely, caspase-8 cleavage was only found in cardiomyocytes, where it progressively rose throughout reperfusion. Consistent with this finding, addition of a specific caspase-9 inhibitor to the perfusate before ischemia prevented endothelial apoptosis, whilst pre-ischemic infusion of a specific Caspase-8 inhibitor affected only myocyte apoptosis.

Urocortin: the endogenous peptide of cardioprotection
Other experiments were carried out de vivo in order to investigate the hemodynamic, bioenergetic and cytoprotective effects of Urocortin, a 40 amino acid member of the Corticotropin Releasing Hormone family, highly expressed in the cardiovascular system. This endogenous cardiac peptide was administered pre-ischemia, pre-ischemia and during reperfusion, and during reperfusion only, to isolated perfused rat hearts exposed to I/R. Significant, and in some treatment groups, complete recovery of end diastolic pressure and developed pressure was observed, together with reduction in endothelial and myocyte cell death. In the groups receiving urocortin, significant recovery of high-energy phosphate reservoirs was also seen. Since the cytoprotective and functional benefits are still produced when urocortin is given only at reperfusion, our data suggest that urocortin may be useful clinically in the management of myocardial infarction, especially when associated with a reduction of the ejection fraction.

Iatrogenic ischemia/reperfusion injury in the human heart
During cardiopulmonary bypass, the cardioplegic arrest and subsequent reperfusion inevitably expose the heart to an iatrogenic ischemia/reperfusion injury. This background encouraged us to evaluate the occurrence of apoptosis and the relative contribution of its signalling pathways in human myocytes from patients exposed to cardiopulmonary bypass, warm blood cardioplegia, and subsequent reperfusion. Furthermore, we investigated whether the above surgical ischemia/reperfusion injury modifies the cardiac expression of urocortin, as well as its potential involvement as a salvage mechanism. 
Our study showed for the first time that warm blood cardioplegia induces apoptotic cell death in cardiac myocytes. This myocyte apoptosis, which was shown to involve co-localisation between TUNEL and caspase-3 positive staining, appears to be mainly sustained by the mitochondrial caspase-9-mediated pathway. We demonstrated moreover that urocortin expression is increased only in those myocytes, which are not apoptotic, suggesting that endogenous urocortin can also protect the human myocardium from IRI.

Minocycline as a new cardioprotective agent
We also investigated the cardioprotective effects of minocycline in primary cultures of both neonatal and adult cardiac myocytes as well as in the intact heart. Minocycline is a second-generation tetracycline with proven safety that is used in humans for the treatment of acne and urethritis as well as of severe chronic inflammatory diseases. Our report showed that minocycline significantly reduced the post-ischemic occurrence of necrotic and apoptotic cell death, with normalization of developed and diastolic pressure. In regard to its antiapoptotic mechanism of action, we observed that minocycline reduced the expression level of initiator caspases, increased the ratio of XIAP to Smac/DIABLO at both the mRNA and protein level, and prevented the mitochondria-mediated release of cytochrome c and Smac/DIABLO. These synergistic actions dramatically reduced the post-ischemic induction of caspase activity associated with cardiac ischemia/reperfusion injury. Owing to its safety record and multiple novel mechanisms of action, minocycline may be a valuable cardioprotective agent to ameliorate the cardiac dysfunction and cell loss associated with acute and chronic ischemia reperfusion injury.

Oral supplementation with mixed essential amino acids
In patients undergoing coronary operations, one mechanism of cardiac adaptation to the iatrogenic ischemia/reperfusion injury associated with cardioplegic arrest, is the uptake of amino acids (AA), which was shown to correlate with oxygen consumption. Based on these premises, we investigated whether oral supplementation with mixed AA may protect the rat heart exposed to ischemia/reperfusion injury. Our study documented that long-term oral supplementation with mixed L-amino acids (AA) attenuated the extent of both necrotic and apoptotic cell death following ischemia/reperfusion injury, and induced a significant post-ischemic recovery of cardiac function. This cardioprotective action was achieved, at least in part, through preservation of the energy-producing properties of mitochondria. More specifically, we showed that ATP content and rate of ATP production in isolated mitochondria were reduced by over 75% in ischemic/reperfused control hearts after 2 hours of reperfusion, and returned toward values of non-ischemic control group in hearts supplemented with AA before undergoing ischemia/reperfusion. Furthermore, the oxygen consumption rate in myocardial skinned bundles, was markedly reduced in ischemia/reperfused control hearts and almost normalized in AA-treated hearts (approximately 20% and 93% of the value for normoxic hearts). As expected, this extraordinary recovery in high-energy stores paralled a reduction in both necrotic and apopotic cell death, finally resulting in enhanced hemodynamic recovery of cardiac function following ischemia/reperfusion injury.

Ephedra intoxication in the human heart
Ephedra, also known as “ma huang, ephedra sinica, ephedrine, sida cordifolia, and epitonin”, has been used in China for thousands of years to treat asthma and other respiratory disorders. This herb has been reported to stimulate the sympathetic nervous system and the heart, and suppress appetite. The reported effects have resulted in wide marketing of this substance in dietary supplements for weight loss, as well as for enhanced athletic performance. However, supplements containing ephedra or its alkaloid derivative, ephedrine, have been linked to serious adverse events, including seizure, stroke, and cardiovascular effects, ranging from hypertension and myocardial infarction to sudden death. We recently described the case of a 45-year-old woman who died of cardiovascular collapse while taking Ephedra. Tissue analysis revealed non-specific degenerative alterations in the myocardium (lipofuscin accumulation, basophilic degeneration and vacuolation of myocytes, as well as myofibrillary loss), associated with myocyte apoptosis, caspase activation, and extensive cleavage of miofibrillary proteins a-actin, a-actinin, and cardiac troponin T. Healthcare professionals are therefore urged to warn their patients about the risk of serious adverse effects, which may follow ephedra intake.

Figure 1. Primary cultures of neonatal cardiac cells stained with propidium iodide and TUNEL. Two of the six cells, showing yellow nuclear staining, are undergoing apoptotic cell death.

Figure 2. TUNEL positivity detected in endothelial cells specifically labeled by an anti-von Willebrand factor antibody.

Figure 3. Co-localization of TUNEL (yellow nuclear staining) and cleaved active form of caspase-3 (cytosolic positive labeling).
 

Figure 4. Endothelial cell showing chromatin margination and condensation, which are typical morphological hallmarks of apoptotic cell death.

Figure 5. The isolated Langendorff-perfused rat heart
 
 

Selected publications
 

• T.M. Scarabelli, N.B. Rayment, T.J. Cooper, R.A. Knight, B. Brar, D.S. Latchman, G.F. Baxter, D.M. Yellon. Quantitative assessment of cardiac myocyte apoptosis in tissue sections using the fluorescence-based tecnique enhanced with counterstains. Journal of Immunological Methods 228:23-28, 1999.
• T.M. Scarabelli, A. Stephanou, N.B. Rayment, E. Pasini, L. Comini, S. Curello, R. Ferrari, R.A Knight, D.S. Latchman. Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischaemia/reperfusion injury. Circulation 104(3):253-6, 2001
• T.M. Scarabelli, A. Stephanou, E. Pasini, L. Comini, R. Raddino, R. Ferrari, R.A. Knight, D.S. Latchman. Different signaling pathways induce apoptosis in endothelial cells and cardiac myocytes during ischaemia/reperfusion. Circ Res 90(6):745-8, 2002
• T.M. Scarabelli, E. Pasini, A. Stephanou, L. Comini, S. Curello, R. Raddino, R. Ferrari, R.A. Knight, D.S. Latchman. Urocortin promotes hemodynamic and bioenergetic recovery and improves cell survival in the isolated rat heart exposed to ischemia/reperfusion. J Am Coll Cardiol 40(1):155-61, 2002.
• TM Scarabelli, A Stephanou, E. Pasini, G. Gitti, P Townsend, K Lawrence, C Chen-Scarabelli, L Saravolatz, D Latchman, R Knight, J Gardin. 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. J Am Coll Cardiol. 43(5):865-74, 2004. 
• TM Scarabelli, E. Pasini, G. Gitti, A Stephanou, C Chen-Scarabelli, L Saravolatz, R Knight, J Gardin. Nutritional supplementation with mixed essential amino acids enhances myocyte survival preserving mitochondrial functional capacity during ischemia/reperfusion injury. Am J Cardiol. 93(8A):35A-40A, 2004. 
• TM Scarabelli, E. Pasini, G. Ferrari, M. Ferrari, A. Stephanou, K. Lawrence, P. Townsend, C. Chen-Scarabelli, G. Gitti, L Saravolatz, D Latchman, R. Knight, J. Gardin. Warm blood cardioplegic arrest induces mitochondrial-mediated cardiomyocyte apoptosis associated with increased urocortin expression in viable cells. J Thorac Cardiov Surg. 2004 Sep;128(3):364-71, 2004. 
• Chen-Scarabelli C, Scarabelli TM. Turning necrosis into apoptosis: the exacting task that can enhance survival. Am Heart J. 2004 Aug;148(2):196-9.
• C. Chen-Scarabelli, T.M. Scarabelli. Fungal endocarditis due to Scopulariopsis. Ann Intern Med. 139(9):W77, 2003. 
• T.M. Scarabelli, R. Knight. Urocortins: Take them to heart. Current Curr Med Chem Cardiovasc Hematol Agents. 2004 Oct;2(4):335-342.
• Chen-Scarabelli C, Scarabelli TM. Neurocardiogenic syncope. BMJ. 2004 Aug 7;329(7461):336-41.
• Scarabelli TM, R Gottlieb. Functional and clinical repercussions of myocyte apoptosis in the multi-faceted damage by ischemia/reperfusion injury: old and new concepts after ten years of contributions. Cell Death Differ. 2004 Dec;11 Suppl 2:S144-52.
• C Chen-Scarabelli, S Hughes, G Landon, P Rowley, N Lawson, L Saravolatz, J Gardin, TM Scarabelli. A case of fatal ephedra intake associated with lipofuscin accumulation, caspase activation and cleavage of myofibrillary proteins. Eur J Heart Fail. 2005 Aug;7(5):927-30.