Highlights of the Congress - Basic Science -

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1 Highlights of the Congress - Basic Science - Myocyte function and ion homeostasis Frank R. Heinzel, Graz

2 Predisposition Trigger Cardiomyocyte Excitability/ Arrhythmias Na + ECC channels pumps Ca 2+ receptors ETC Structural Remodeling Contraction Metabolism Gene regulation

3 P975: Robin,, Raddatz. University of Lausanne, Switzerland. Autocrine/paracrine signaling via A1 receptor is arrhythmogenic in the developing heart. Cardiomyocyte channels receptors pumps Excitability/ Arrhythmias Na + ECC Contraction Ca 2+ ETC Structural Remodeling Gene regulation Metabolism

4 Adenosine-mediated arrhythmias and ERK2 phosphorylation MODEL: spont. beating hearts from 4 days old chick embryos H o Extracellular adenosine (ADO) and inosine (INO) are generated from ATP and can simultaneously act in an autocrine/paracrine manner and induce arrhythmias. Intracellular ATP AMP ADO myocyte 1 INO myocyte 2 ATP Carrier ATP AMP ADO AOPCP Extracellular ent Equilibrative Nucleoside Transporter Dipyridamole 1µM INO Autocrine Adenosine Receptor A1 Adenosine Receptor A1 Paracrine ENT blockade (dipy) induced ERK2 phosphorylation which was prevented by A 1 AR inhibition (DPCPX). Robin,..,Raddatz. University of Lausanne, Switzerland

5 155: Pavlovic,, Shattock. King s College London, Rayne Institute, London, UK. Phospholemman-dependent regulation of the cardiac Na/K -ATPase activity is modulated by inhibitor-1 sensitive type-1 phosphatase in both normal physiology and heart failure Cardiomyocyte channels receptors pumps Excitability/ Arrhythmias Na + ECC Ca 2+ ETC Structural Remodeling Contraction Metabolism Gene regulation

6 Inhibitor-1 amplifies b-adrenergic NKA activity via phosphatase-1 deactivation PLB PLM Protein/actin level Ip (pa/pf) b agonist b-1 NKA camp PKA stimulation S 68 inhibition I-1c Actin PLM Ser-68 Adenoviral expression of activated I-1 increases NKA activity Ad Western blotting (PLM Ser-68) Ad-I-1c 2.0 * Ad-GFP 0 Ad-I-1c/GFP PLM Ser Patch clamp (NKA current) * stimulation SR T 35 SER CA Inhibitor-1 S 16 PP-1 inhibition I-1 co-regulates NKA and SERCA during adrenoceptor stimulation (via PLM and PLB) Actin PLM Ser-68 PLB Ser-16 I-1 Thr-35 Iso (10nm) Ad Ad-I-1 Pavlovic,, Shattock. King s College London, UK.

7 PLB Inhibitor-1 is deactivated in failing hearts PLM b agonist b-1 NKA Human Heart homogenates: ICM (n=8) NF (n=8) DCM (n=8) camp PKA stimulation SR T 35 SER CA stimulation Inhibitor-1 S 16 S 68 inhibition PP-1 inhibition CSQ SERCA NKA a-1 PP-1 I-1 I-1 Thr-35 PLB PLB Ser-16 PLM PLM Ser-68 NF F F NKA and SERCA activity reduced Na elevated and ejection fraction reduced - SERCA expression unchanged - NKA expression unchanged - PP1 expression unchanged - I-1 expression reduced - I-1 activation reduced - PLB expression unchanged - PLB Ser16 reduced - PLM expression unchanged - PLM Ser68 reduced Pavlovic,, Shattock. King s College London, UK.

8 Szuts,.., Varró. Department of Pharmacology, University of Szeged, Hungary Altered Expression of Kir2.x and two-pore ion channels are responsible for Changed I K1 Current in Dilated Cardiomyopathy Cardiomyocyte channels receptors pumps Excitability/ Arrhythmias Na + ECC Contraction Ca 2+ ETC Structural Remodeling Gene regulation Metabolism

9 Regulators of cardiac excitability: I K1 (Kir2.x) Two-pore K-channels(K 2P ) TWIK, TASK-1 Szüts,.., Varró. Department of Pharmacology, University of Szeged, Hungary

10 I K1 tended to be increased in human DCM Szüts,.., Varró. Department of Pharmacology, University of Szeged, Hungary

11 SAP97, an anchoring protein of Kir2.x, was also downregulated Szüts,.., Varró. Department of Pharmacology, University of Szeged, Hungary

12 Szüts,.., Varró. Department of Pharmacology, University of Szeged, Hungary

13 P898: Kohlhaas,, Maack. Saarland University Hospital, Germany. Mitofusin 2 tethers mitochondria to the sarcoplasmic reticulum and controls calcium transmission and mitochondrial redox state Cardiomyocyte channels receptors Excitability/ Arrhythmias Na + ECC Ca 2+ ETC Structural Remodeling Contraction Metabolism Gene regulation

14 Mitofusin 2 Linker protein between SR and mitochondria in cardiac myocytes MCU MCU Ca 2+ Ca 2+ Mfn2 Mfn1 Ca 2+ Ca 2+ RyR RyR RyR Mfn2 Mfn2 Mfn2 Mfn2 Mfn1-KO Mfn2-KO Kohlhaas,, Maack. Saarland University Hospital, Germany

15 Mfn2 deficiency impairs the spatial association of mitochondria to the SR WT Mfn2-KO Kohlhaas,, Maack. Saarland University Hospital, Germany In cooperation with Gyorgy Csordas, Thomas Jefferson University, Philadelphia, USA

16 Decreased mitochondrial Ca 2+ accumulation in Mfn2-KO myocytes MCU 5 Hz 0.5 Hz 0.5 Hz Isoproterenol 30 nm Mfn2-KO Diastolic [Ca 2+ ] m (F/F 0 ) inset WT Mfn2-KO mitoch. matrix Ca Time (s) IMM Min 0-3 F/F Hz Iso * sec WT Mfn2-KO *p<0.05 Kohlhaas,, Maack. Saarland University Hospital, Germany cytosol Ca 2+

17 Impaired bioenergetic feedback response in Mfn2-KO myocytes Isoproterenol NAD(P)H (%) FAD + (%) Hz 0.5 Hz 0.5 Hz ** ** WT Mfn2-/- [Ca 2+ ] m (F/F 0 ) e - NADH Mfn2-KO I 0.5 Hz Iso Q II # NAD + III WT sec Mfn2-/- NAD(P)H / FAD ** Time (s) Kohlhaas,, Maack. Saarland University Hospital, Germany Krebs Cycle + Ca 2+ FADH 2 FAD +

18 P1181: Benoist,, White. Institute of Membrane and Systems Biology, University of Leeds (UK) Electrophysiological remodelling and defective calcium handling are associated with alternans in rats with right ventricular failure. Cardiomyocyte channels receptors pumps Excitability/ Arrhythmias Na + ECC Ca 2+ ETC Structural Remodeling Contraction Metabolism Gene regulation

19 T-wave amplitude (mv) 0.5 mv Results In monocrotaline-treated rats with RV failure, alternans were traceable from the in vivo ECG (1), to the RV APD of isolated hearts (2) and to the Ca 2+ transient of isolated RV myocytes (3) T-wave alternans L S L S L S L S 500 ms In Vivo ECG Beat number 2. Discordant APD alternans a b APD80 Beat 2 - Beat 1 (ms) Region a Region b Ex Vivo Isolated heart Beat 1 Beat 2 Beat 1 Beat 2 20 ms ΔF/F 0 3. Ca 2+ transient alternans Cell length (µm) Fluorescence ratio (a.u.) Time (ms) Isolated RV myocytes Benoist et al. (2012) Am J Physiol Heart Circ Physiol In Press doi: / ajpheart

20 Spark-mediated leak (ΔF/F 0.µm 3.s -1 ) Results Mechanisms involved in alternans and arrhythmia generation in the failing RV of monocrotalinetreated rats include: Steep RV APD and conduction Dysfunction of RV sarcoplasmic Increased RV APD dispersion velocity restitutions reticulum Ca 2+ uptake and release A FAIL A CON FAIL CON B FAIL CON B CON *** FAIL Benoist et al. (2012) Am J Physiol Heart Circ Physiol In Press doi: / ajpheart

21 P456: Steinwascher,, Jaquet. Research Laboratory Molecular Cardiology, University Clinics St.-Josef Hospital & BG Bergmannsheil, Bochum Germany Effects of β-adrenoceptor subtypes in adult rat cardiomyocytes altered by expression of ctni mutant R145G. Cardiomyocyte channels receptors Excitability/ Arrhythmias Na + ECC Ca 2+ ETC Structural Remodeling Contraction Metabolism Gene regulation

22 ardiac Troponin I-R145G leads to HCM in patients and high susceptibility to sudden cardiac death in young athletes PKA-dependent phosphorylation of ctni due to β-ar stimulation enhances relaxation of myofibrils Does the mutation affect the β-adrenergic response? ardiac Troponin I-R145G overexpression Adult rat Shuttle vector Heart Recombinant adenovirus Isolated cardiomyocytes Overexpression of ctni mutant Steinwascher,, Jaquet. Research Laboratory Molecular Cardiology, University Clinics St.-Josef Hospital & BG Bergmannsheil, Bochum Germany Methods: 1. Contractions (Edge detection) 2. Calcium transients (Indo- 1 fluorescence) 3. Phosphorylation status (Western blots, ProQ/SYPRO)

23 rel. TnI phosph. (change vs. WT) Sarcomere length Sarcomere length ctni-r145g expression effects Contractile parameters suppressed Rescued by β 1 - but not β- or β 2 -AR stimulation Ca 2+ transient prolonged Rescued by β 1 - but not β- or β 2 -AR stimulation Basal phosphorylation of ctni, ctnt, cmybp-c and MLC-2 seems elevated Phosphorylation of Ser22/23 (PKA) is similarly lower in TG vs. WR after either β-ar stimulation Mismatch between phospho-state and acute rescuing β 1 -response Differential (phopho-) regulation of [Ca] and myofilaments basal adren. stim. Schematic overlay of single contractions basal b b 1 b 2 Steinwascher,, Jaquet. Research Laboratory Molecular Cardiology, University Clinics St.-Josef Hospital & BG Bergmannsheil, Bochum Germany

24 Cardiomyocyte Excitability/ Arrhythmias Na + ECC channels pumps Ca 2+ receptors ETC Structural Remodeling Contraction Metabolism Gene regulation

25 MAGNESIUM OROTATE INDUCES CARDIOPROTECTION AGAINST GLOBAL AND REGIONAL ISCHAEMIA IN RAT HEARTS REGARDLESS THE TIMING OF ADMINISTRATION S. Mirica 1, G. Gheorghiu 1, O. Duicu 1, A. Anechitei 1, O. Fira-Mladinescu 1, D. Angoulvant 2, D. Muntean 1 (1) Department of Pathophysiology, University of Medicine and Pharmacy, Timisoara, Romania (2) Hôpital Trousseau, EA 4245-UFR Médecine, Université François Rabelais, Tours, France AIM: To assess the importance of timing for magnesium orotate (Mg-Or) associated cardioprotection in 2 experimental settings: global ischaemia (GI, 1 mm) in isolated hearts and regional ischaemia (RI, 0.01 mg/kg) in either early (E, 2 min before the onset of reperfusion) or delayed (D, 3 min after the onset of reperfusion) administration. MATERIAL AND METHODS: Experimental protocols for: A. In vitro experiments B. In situ experiments Infarct size was assessed by the triphenyltetrazolium chloride staining and expressed as percentage of area at risk.

26 RESULTS Fig. 1. Representative samples of TTC staining from isolated hearts experiments. Fig. 2. Representative samples of TTC staining from in situ hearts experiments. CONCLUSION: The presence of Mg-Or throughout the postischaemic reperfusion elicited a significant anti-infarct protection in both experimental settings regardless the timing of administration.