Effects of hyperlipidemia and statins on cardioprotective signaling

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Caitlin Gilmore
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Péter Ferdinandy Cardiovascular Research Group,

Budapest; Pharmahungary Group, Szeged, Budapest,

1 Effects of hyperlipidemia and statins on cardioprotective signaling HFA-ISHR 212 Belgrade Péter Ferdinandy Cardiovascular Research Group, University of Szeged and Semmelweis University, Budapest; Pharmahungary Group, Szeged, Budapest, Hungary Szeged Budapest

2 Why do we still not have cardioprotective drugs? - Ischemic heart disease in humans is a complex disorder caused by or associated with cardiovascular risk factors - Preclinical development of cardioprotective drugs as well as preand postconditioning techniques were mainly done in healthy young adult animal models of ischemic heart disease

Cardioprotection by pre- and postconditioning: effect of co-morbidities and risk factors Ischemia/reperfusion Preconditioning Pharmacol Rev, 59:418-458, 27 infarction dysfunction arrhythmias

3 Cardioprotection by pre- and postconditioning: effect of co-morbidities and risk factors Ischemia/reperfusion Preconditioning Pharmacol Rev, 59: , 27 infarction dysfunction arrhythmias Postconditioning < 12 min: classic hours: delayed Cardioprotection Cardioprotection is diminished in the presence of risk factors: hyperlipidemia, diabetes, heart failure, nitrate tolerance, aging, etc Infarct size Cardiac function Arrhythmias Szilvassy et al, JMCC 1995 Ferdinandy et al, JMCC, 1997 reviews: Ovize et al, CVR, 21 Ferdinandy et al, Pharmacol Rev, 27 Ferdinandy et al, TiPS, 1998

Risk factors and co-morbidities that may influence cardioprotective mechanisms Hyperlipidemia & atherosclerosis - antihyperlipidemic drugs

4 Risk factors and co-morbidities that may influence cardioprotective mechanisms Hyperlipidemia & atherosclerosis - antihyperlipidemic drugs (e.g. statins) Diabetes - sensory neuropathy - antidiabetic drugs (KATP modulators) Obesity Aging Drugs used in ischemic heart disease - nitrates and nitrate tolerance - beta blockers - ACE inhibitors Uremia (renal failure) Postinfarction remodeling and heart failure Ferdinandy et al, Pharmacol Rev, 27

5 The first observations: hyperlipidemia but not atheroscerosis interferes with preconditioning Pacing-induced preconditioning, rabbits hyperlipidemia 3 control precon atherosclerosis (weeks) Szilvassy et al, JMCC 1995 Ferdinandy et al, JMCC, % cholesterol diet Normal diet

6 The infarct size limiting effect of pre- and postconditioning is lost in hearts of hyperlipidemic rats Infarct size (%) Ischemia/Reperfusion Preconditioning Postconditioning 6 normal cholesterol-diet (12 wk) 3 I/R PRE POST I/R PRE POST Kupai et al, Am J Physiol, 29 Giricz et al, J Pharmacol Exp Ther, 26

7 ST-segment elevation (mv) Preconditioning is attenuated in hypercholesterolemic patients during coronary angioplasty Normocholesterolemic patients Hypercholesterolemic patients time (s) time (s) Intracoronary ST-segment elevation during 3 subsequent coronary occlusions induced by balloon inflations 12 s 12 s 12 s Ungi et al, Chest 25

8 Hyperlipidemia interferes with cardioprotective mechanisms: pro & contra Major publications Pre / postcon Szilvassy et al, JMCC, 1995 Ferdinandy et al, JMCC, 1997 Ferdinandy et al, Mol Cell Biochem, 1998 Szilvassy et al, J Cardiovasc Pharm 1998 Ueda et al, JACC, 1999 Tang et al, Basic Res Cardiol, 24 Tang et al, Circulation 25 Precon Rabbit Rat Rat Rabbit Rabbit Rabbit Rabbit Species Primary end-point Inhibited by hyperchol? ST segment Infarct marker Infarct marker ST segment Infarct size Infarct size Infarct size Clinical trials of cardioprotective drugs have failed. Giricz et al, JPET, 26 Rat Infarct size Kyrikiades Clinical et al, 22 trials with postconditioning PCI patients are ST-segment not convincing. Ungi et al, Chest, 25 PCI patients ST-segment Due to the presence of multipe risk factors? Iliodromitis et al, Atherosclerosis, 26 Kupai et al, Am J Physiol, 29 Lauzier et al, J Heart Lung Transplant, 29 Iliodromitis et al, Basic Res Cardiol, 21 Kremastinos et al, Atherosclerosis, 2 Iliodromitis et al, Atherosclerosis, 26 Jung et al, Clin Exp Hypertens, 2 Li et al, Ann Thorac Surg, 21 Tokuno et al. ATVB, 22 Postcon Precon Rabbit Rat LDLr mice Rabbit Rabbit Rabbit Rabbit ApoE/LDLr mice ApoE/LDLr mice Infarct size Infarct size Infarct size Infarct size Infarct size Infarct size Infarct size Infarct size Infarct size Ferdinandy et al, Pharmacol Rev, 27; Ferdinandy, Br J Pharmacol, 23 Yes Yes No

9 Pre- and postconditioning is preserved in renal failure Cardiovasc Res (21) 87 (suppl 1): S135 Sarkozy et al, submitted, 212

10 How does hyperlipidemia interact with cardioprotective mechanisms?

11 Cholesterol-diet decreases NO level by increasing oxidative/nitrosative stress in rat & ApoB TG mice hearts Superoxide fomation Dityrosine formation (pmol/min/mg) NO + O 2 - ONOO -.3 Cardiac NO 1 Cardiac O 2-2 Cardiac ONOO Cont Cholest Cont Cholest Cont Cholest Csont et al, Cardiovasc Res, 27 Onody et al, Cardiovasc Res, 23

12 Protein S-nitrosylation in hyperlipidemic vs normal hearts hyperlipidemia-induced nitrosylation Long-chain specific acyl-coa dehydrogenase, mitochondrial hyperlipidemia-induced denitrosylation ATP synthase subunit epsilon, mitochondrial Pyruvate kinase isozymes M1/M2 Hyaluronan and proteoglycan link protein 2 Electron transfer flavoprotein subunit alpha, mitochondrial Acetyl-CoA acetyltransferase, mitochondrial Long-chain-fatty-acid-CoA ligase 1 Sarcoplasmic reticulum calcium ATPase 3 nitrosylation denitrosylation metabolic enzymes 9 2 structural proteins 9 2 Actin, gamma-enteric smooth muscle Fatty acid-binding protein, adipocyte Csonka et al, in preparation 212

13 Preconditioning decreases peroxynitrite - matrix metalloproteinase signaling: inhibition by hyperlipidemia MMP-2 relase & activation (zymography, density) NO + O 2 - ONOO - MMP-2 activation before ischemia early reperfusion kda Active pro-mmp kda Active MMP2 5 # Control Control Pre Chol Chol Pre Control Control Pre Chol Chol Pre Giricz et al, JPET, 26

14 Pharmacological inhibition of MMP-2 is cardioprotective in both normal and hyperlipidemic heart Infarct size (%) Control Cholesterol-diet µm.25 µm ILOMASTAT Pre.1 µm.25 µm ILOMASTAT Pre Giricz et al, JPET, 26

15 Nitrotyrosine (ng/mg prot) 72 kda MMP-2 activity Early activation of peroxynitrite MMP is a trigger signal for postconditiong: inhibition by cholesterol-diet NO + O 2 - ONOO - MMP-2 activation Ischemia/Reperfusion Postconditioning 5 4 Peroxynitrite 8 6 MMP Cont Cont Post Chol Chol Post Cont Cont Post Chol Chol Post Kupai et al, Am J Physiol, 29

intercaleted disks PRE normal-diet normal-diet cholesterol-diet 2.5 2. 1.5 1.

16 Mitochondrial Cx43 (band desity, arbitrary units) Hyperlipidemia is associated with redistribution of both sarcolemmal and mitochondrial Cx43 I/R Cx43 in intercaleted disks PRE normal-diet normal-diet cholesterol-diet cholesterol-diet.5 C I/R PRE C I/R PRE Görbe et al, Am J Physiol, 211

17 Infarct size (%) Hyperlipidemia interferes with NO-cGMP-PKG signaling in rat hearts Band density Control Normal diet Cholesterol diet 8-Br cgmp SNAP BNP NO BNP 15 kda 75 kda cytosolic GC particulate GC normal hyperlipidemic cgmp PKG cardioprotection PKG dimer (inactive) PKG monomer Giricz et al, Br J Pharmacol, 28 Görbe et al, Basic Res Cardiol, 21

18 Autophagy is decreased in the heart of hypercholesterolemic rats LC3 control hypercholesterolemic control hypercholesterolemic Beclin LC3-II/actin ratio,6,4,2, CON HC Beclin/actin ratio,8,6,4,2, CON HC Giricz et al, in preparation, 212

19 The mtor pathway is activated in the heart of hypercholesterolemic rats PI3K11α control hypercholesterolemic p-s6 S6 p-s6/s6 ratio 1,2 1,,8,6,4,2, control hypercholesterolemic CON HC CIII PI3K PI3K/actin ratio CIII PI3K/actin ratio,5,4,3,2,1,,8,6,4,2, CON CON HC HC Giricz et al, in preparation, 212

20 The mevalonate pathway in cardioprotection: statins may interfere with cardioprotective mechanisms? HMG-CoA HMG-CoA reductase Mevalonte Isopentenyl-PP Statins Cholesterol Geranyl-PP Dolichol Farnesyl-PP Cholesterol biosynthesis Ubiquinone (Coenzyme Q) Protein prenylation Modified from Goldstein and Brown, Nature, 343:425-43, 199

21 Interaction of lovastatin with pre- and postconditioning Rat I/R Pre Post I/R Pre Post Vehicle I/R Pre Post I/R Pre Post Chronic Lovastatin (15 mg/kg for 12 days) Acute Lovastatin (5 um/l) Kocsis et al, Am J Physiol, 28

22 P p42 density (arbitrary unit) Q9 mg/1mg wet tissue P- Akt density (arbitrary unit) Interaction of lovastatin with cardioprotective signaling phospho total Akt p44/p42 HMG-CoA reductase HMG-CoA Statin Vehicle Chronic Acute Veh Chron Acute lovastatin lovastatin Mevalonate 12 Farnesol-PP Coenzyme Q 8 4 Cholesterol Vehicle Chronic lovastatin Acute lovastatin Vehicle Chronic lovastatin Acute lovastatin Kocsis et al, Am J Physiol, 28

23 Protein prenylation (ng/mg protein) Interaction of atorvastatin with cardiac cellular signalling HMG-CoA HMG-CoA reductase Statin Protein farnesylation Protein geranylgeranylation Mevalonate Farnesol-PP Protein prenylation 1 8 Cholesterol Vehicle 1 mg atorvastatin Vehicle 1 mg atorvastatin Szücs et al, in preparation 212

24 Pre- and postconditioning is lost in nitrate-tolerance Csont et al, 212 in preparation

postconditioning involves dramatic changes in cardiac gene expression

25 One dominant pathway or multiple ones in cardioprotection? Importance of omics approach cardioprotection by preconditioning and postconditioning involves dramatic changes in cardiac gene expression profile (Onody et al, FEBS Lett, 23; Csonka et al, in preparation 212)

26 Effects of hyperlipidemia on cardiac gene expression profile (DNA, mirna, and protein microarray studies in rodent hearts) Microarray and RT-PCR studies Overexp. repressed 32 genes, rat - hyperchol vs. normal Reference Puskas et al, FEBS Lett, antibodies, rat - hyperchol vs. normal 1 3 unpublished 16 oxidative stress genes - hyperchol vs. normal enos 36 mirna, rat - hyperchol vs. normal mirna, rat - Statin vs normal genes, ZDF rat - Metabolic dis vs. normal Phox4, MMP9 Kocsis et al, Med Sci Monit, 21 Varga et al, in preparation, 212 Szücs et al, in preparation 212 Sárközy et al, in preparation

27 mirna expression change in hearts from cholesterol-fed vs. control rats Cardiac microrna expression pattern is altered due to hyperlipidemia: microrna-25 QRT-PCR fold change 2 Poster session 3 P852 microrna-25 1,5-1 1,5-2 rno-mir Varga et al, in preparation 212

28 Take home messages: Hyperlipidemia interact with endogenous cardioprotection by pre- and postconditioning Hyperlipidemia interferes with cardioprotective signalling pathways: NO-cGMP-PKG, peroxynitrite-mmp, mevalonate pathway, Cx43 translocation, survival kinases, gene expression, etc Cardioprotective statins and nitrates may interact with cardioprotective mechanisms (thereby limiting their efficacy and safety?) For successful development of cardioprotective therapies, there is a critical need for preclinical studies that examine their cardioprotective efficacy in the presence of risk factors Occult cardiotoxicity of medications: inhibition of cardioprotective mechanisms and ischemic tolerance? See for reviews: Ferdinandy et al, Pharmacol Rev, 27 Ovize et al, Cardiovasc Res, 21

Sárközy János Pálóczy Veronika Fekete Judit Pipis Szilvia Török JL University, Giessen, Germany: Rainer Shculz Kerstin

29 Cardiovascular Research Group, University of Szeged, and Pharmahungary Group, Szeged & Budapest, Hungary: Tamás Csont Csaba Csonka Anikó Görbe Péter Bencsik Zoltán Giricz Krisztina Kupai Gabriella Fodor Gergő Szücs Zoltán Varga Márta Sárközy János Pálóczy Veronika Fekete Judit Pipis Szilvia Török JL University, Giessen, Germany: Rainer Shculz Kerstin Boengler Cardiff University, Cardiff, UK: Gary F. Baxter Dwain S. Burley University of Alberta, Edmonton, Canada: Richard Schulz Manoj Lalu

FUNCTIONAL AND MOLECULAR EFFECTS OF HYPERCHOLESTEROLEMIA ON THE MYOCARDIUM: THE ROLE OF CONNEXIN-43 AND MICRORNA-25. Summary of PhD.

FUNCTIONAL AND MOLECULAR EFFECTS OF HYPERCHOLESTEROLEMIA ON THE MYOCARDIUM: THE ROLE OF CONNEXIN-43 AND MICRORNA-25 Summary of PhD. Thesis Zoltán Varga Supervisor: Tamás Csont Cardiovascular Research Group