Protection of myocardium by cyclosporin a and insulin: in vitro simulated ischemia study in human myocardium

doi:10.1016/S0003-4975(03)00830-0

The Annals of Thoracic Surgery

Volume 76, Issue 4, October 2003, Pages 1240-1245

https://doi.org/10.1016/S0003-4975(03)00830-0 Get rights and content

Abstract

Background

The efficacy of myocardial protection by cyclosporin A (CSA) and insulin was tested in human right atrial myocardial slices subjected to simulated ischemia and reoxygenation.

Methods

Slices of right atrial trabeculae were obtained from patients undergoing elective cardiac surgery. Trabeculae were incubated with oxygenated glucose containing phosphate buffered saline (O₂, G-PBS). After 30 minutes of stabilization the sections were exposed to 90 minutes of simulated ischemia (N₂, PBS without glucose) followed by 90 minutes reoxygenation (O₂, G-PBS). Cyclosporin A (0.2 μmol/L) or insulin (5 mU/mL) was added during the stabilization period prior the ischemia. Cell viability was measured by using 3-[4.5 dimethylthiazol 2-yl]-2,5-diphenyltetrazolium bromide (MTT), which is cleaved by active mitochondrial dehydrogenases of living cells.

Results

The viability of untreated slices (control) was 30.45% ± 2.5% versus 52.65% ± 4.4% in the CSA treated slices, p less than 0.001. The extent of protection by CSA was affected by oral antiglycemic drugs (glibenclamide). The effect obtained by CSA was inhibited by 5-hydroxydecanoate (5HD), a specific blocker of mitochondrial K_ATP channels. Protection of the myocardial slices with insulin appears to be superior and not affected by the medication before surgery. This protection was maximal when insulin was present during both preischemic equilibration and reoxygenation periods (68.9% ± 9.3% viability with insulin versus 33.2% ± 6.9% in the control, p < 0.001).

Conclusions

Protection of right atrial trabeculae slices with insulin is superior to that obtained with CSA and is independent of preoperative medication.

Section snippets

Material and methods

Cyclosporin A was purchased from Calbiochem (La Jolla, CA). The 5-hydroxydecanoic acid sodium salt (5HD) was purchased from Research Biologicals (Natick, MA). The 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and diazoxide were obtained from Sigma (Sigma-Aldrich Co, St. Louis, MO). Regular human insulin (100 U/mL) was purchased from Lilly France S.A. (Fegersheim, France).

Effect of cyclosporin A on viability of human right atrial slices

Human right atrial slices exposed to simulated ischemia and reoxygenation benefit from cyclosporin A treatment. The viability of the slices from untreated controls was 30.45% ± 2.5% versus 52.65% ± 4.4% in the CSA treated slices (p < 0.001; Fig 1a). Subgroup analysis revealed attenuated effect of CSA on atrial slices collected from patients that were treated with oral glibenclamide (K_ATP blocker) preoperatively (36% ± 5% for untreated controls versus 42.1% ± 5.4% for CSA, p > 0.05; Fig 1, b).

Comment

The present study has demonstrated the possibility of protecting human myocardium from simulated ischemic injury by CSA or insulin. Cyclosporin A was efficient when added during the preischemic period in part of the cohort; right atrial specimens obtained from noninsulin-dependent diabetes patients treated with glibenclamide did not respond well to CSA in vitro treatment. Insulin protected effectively slices exposed to simulated ischemia and reoxygenation from all the patients. Our study

Acknowledgements

This study was partially supported by the Aaron Beare Foundation.

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This study does not only define the mechanism of CsA but also rule out CsA sensitivity as a characteristic of the MPTP. CsA could have evolved as the first MPTP inhibitor for the treatment of diseases but the result of clinical trials was not encouraging (Cung et al., 2015), albeit other factors (such as but not limited to high molecular weight) waylaying it from being a drug (Schneider et al., 2003; Shanmuganathan et al., 2005). Genetic ablation of the Ppif gene revealed the importance of CyPD; mice lacking this gene prevented IR-induced cell death, more specifically, isolated mitochondria (from liver heart and brain) obtained was resistant to permeability transition.
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Antiinflammatory therapy is the principal therapy for most rheumatic diseases with the primary therapeutic goal being to reduce the destructive effects of the inflammatory process without causing systemic side effects due to overimmunosuppression. The heart is one of the primary organs affected by the side effects of these agents through both direct (myocardial injury) and indirect (hypertension, hyperlipidemia, etc.) actions that can result in myocardial injury in a number of different ways. Cyclophosphamide has direct cardiotoxic effects and can result in heart failure. Prednisone has secondary side effects including hypertension and hyperlipidemia, which can cause and/or accelerate coronary artery disease, and if a myocardial infarction happens can result in heart failure due to ischemic injury. Conversely, prednisone and other agents are often used for the therapy of inflammatory processes of the heart and in that setting can actually preserve left ventricular function and result in an improvement in ejection fraction. Finally, cyclosporine, which was the principal immunosuppressive agent responsible for the vast increase in organ transplantation, has resulted in many patients undergoing heart transplantation safely that otherwise would never have received a heart. This chapter will review the positive and negative cardiac properties of the traditional antiinflammatory agents prednisone, azathioprine, methotrexate, cyclosporine, and cyclophosphamide.
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The inhibition of MPTP opening as a therapeutic approach has been reported to be effective in the human heart tissue, an important step in the process for translating cardioprotection into the clinical setting (Table 1). Schneider et al. [153] found that pre-treatment with CsA protected slices of human right atrial appendage tissue (harvested from CABG patients) against simulated acute IRI [153]. Using human atrial trabeculae and atrial cardiomyocytes (isolated from patient right atrial appendage tissue) subjected to simulated acute IRI, we demonstrated that the administration of CsA at re-oxygenation improved the recovery of baseline contractile function and reduced cell death, respectively [154].
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Nevertheless, as mentioned previously, acute inhibition of CypD in myocardial IR injury remains a practical target for reducing infarct size and for improving cardiac functions. Although the cardioprotective effects of CypD inhibition in humans were already demonstrated [143,144], our group was the first to report a ground-breaking proof-of-concept clinical study in a small group of ST segment elevation myocardial infarction (STEMI) patients. Intravenous injection of a single dose of CsA just prior to reperfusion considerably reduced the blood levels of creatine kinase and troponin I as compared to placebo group.
Reperfusion is characterized by a deregulation of ion homeostasis and generation of reactive oxygen species that enhance the ischemia-related tissue damage culminating in cell death. The mitochondrial permeability transition pore (mPTP) has been established as an important mediator of ischemia–reperfusion (IR)-induced necrotic cell death. Although a handful of proteins have been proposed to contribute in mPTP induction, cyclophilin D (CypD) remains its only bona fide regulatory component. In this review we summarize existing knowledge on the involvement of CypD in mPTP formation in general and its relevance to cardiac IR injury in specific. Moreover, we provide insights of recent advancements on additional functions of CypD depending on its interaction partners and post-translational modifications. Finally we emphasize the therapeutic strategies targeting CypD in myocardial IR injury. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".
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The influence of mPTP inhibition on cell survival was also preserved in aged hearts. A number of studies confirm protection via mPTP inhibition in young tissue (Halestrap et al., 1997; Liu et al., 2011), and cyclosporin A protects human myocardium in vitro (Schneider et al., 2003) and in vivo (Piot et al., 2008). However, Liu et al. recently reported cyclosporin A is ineffective in aged rat hearts (Liu et al., 2011), and its effects may also be reduced by obesity or diabetes (Huhn et al., 2010).
Changes in cytoprotective signaling may influence cardiac aging, and underpin sensitization to ischemic insult and desensitization to ‘anti-ischemic’ therapies. We tested whether age-dependent shifts in ischemia–reperfusion (I–R) tolerance in murine and human myocardium are associated with reduced efficacies and coupling of membrane, cytoplasmic and mitochondrial survival-signaling. Hormesis (exemplified in ischemic preconditioning; IPC) and expression of proteins influencing signaling/stress-resistance were also assessed in mice. Mouse hearts (18 vs. 2–4 mo) and human atrial tissue (75 ± 2 vs. 55 ± 2 yrs) exhibited profound age-dependent reductions in I–R tolerance. In mice aging negated cardioprotection via IPC, G-protein coupled receptor (GPCR) agonism (opioid, A₁ and A₃ adenosine receptors) and distal protein kinase c (PKC) activation (4 nM phorbol 12-myristate 13-acetate; PMA). In contrast, p38-mitogen activated protein kinase (p38-MAPK) activation (1 μM anisomycin), mitochondrial ATP-sensitive K⁺ channel (mK_ATP) opening (50 μM diazoxide) and permeability transition pore (mPTP) inhibition (0.2 μM cyclosporin A) retained protective efficacies in older hearts (though failed to eliminate I–R tolerance differences). A similar pattern of change in protective efficacies was observed in human tissue. Murine hearts exhibited molecular changes consistent with altered membrane control (reduced caveolin-3, cholesterol and caveolae), kinase signaling (reduced p70 ribosomal s6 kinase; p70s6K) and stress-resistance (increased G-protein receptor kinase 2, GRK2; glycogen synthase kinase 3β, GSK3β; and cytosolic cytochrome c). In summary, myocardial I–R tolerance declines with age in association with dysfunctional hormesis and transduction of survival signals from GPCRs/PKC to mitochondrial effectors. Differential changes in proteins governing caveolar and mitochondrial function may contribute to signal dysfunction and stress-intolerance.
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Cyclosporin A is an immunosuppressor that prolongs graft survival but its use is limited by cardiotoxicity. The effects of cyclosporin A on several functional and biological characteristics were thus evaluated in rat cardiomyocytes in normal conditions and in a substrate-free, hypoxia–reoxygenation model of ischemia–reperfusion. Cyclosporin A (100 and 1000 ng/ml) did not induce cardiocytotoxicity in basal conditions. Simulated ischemia gradually decreased and then blocked the spontaneous electromechanical activity. Cyclosporin A at 100 and 1000 ng/ml permitted the maintenance of electromechanical functions that were abolished in control cells. Cyclosporin A also improved the post-“ischemic” functional recovery. Cyclosporin A reduced the “ischemia”-induced lactate dehydrogenase and troponine I releases and the successive rises in heat shock protein mRNA observed after “ischemia” and reoxygenation. Moreover, cyclosporin A improved the resumption of the mitochondrial function. To conclude, cyclosporin A displayed a direct, pleiotropic protection of isolated cardiomyocytes against physiological, metabolic, structural and stress signaling changes induced by ischemia–reperfusion mimicked in vitro.

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Original article: cardiovascularProtection of myocardium by cyclosporin a and insulin: in vitro simulated ischemia study in human myocardium

Abstract

Background

Methods

Results

Conclusions

Section snippets

Material and methods

Effect of cyclosporin A on viability of human right atrial slices

Comment

Acknowledgements

J Mol Cell Cardiol

Lancet

Cell

J Mol Cell Cardiol

J Mol Cell Cardiol

J Mol Cell Cardiol

J Mol Cell Cardiol

J Am Coll Cardiol

Ann Thorac Surg

Preconditioning with ischemiaa delay of lethal cell injury in ischemic myocardium

Circulation

Ischemic preconditioning as an adjunct to crystalloid or blood cardioplegia for myocardial protection in routine coronary surgery

Thorac Cardiovasc Surg

Protection by cyclosporin A from cardiac ischemia-reperfusion damage

Exp Clin Cardiol

Dinitrophenol, cyclosporin A, and trimetazidine modulate. Preconditioning in the isolated rat heartsupport for a mitochondrial role in cardioprotection

Cardiovasc Res

Glucose-insulin-potassium therapy for treatment of acute myocardial infarctionan overview of randomized placebo-controlled trials

Circulation

Original article: cardiovascular
Protection of myocardium by cyclosporin a and insulin: in vitro simulated ischemia study in human myocardium