磺酰脲类受体2型依赖的KATP通道在缺血预适应中的调控作用ppt课件

1、Regulation and Roles of the Sulfonylurea Receptor 2-Based KATP Channels in Ischemic Preconditioning Bin Ye1, Jie-lin Pu1, Tomoyki Wada1, Jason Sims2, Stacie Kroboth1, Douglas Stoller1, Elizabeth McNally3, Jonathan Makielski1, Nian-Qing Shi1* 1Department of Medicine, University o

2、f Wisconsin, Madison, WI USA 2Department of Pharmacology, University of Wisconsin, Madison, WI USA 3Department of Medicine, The University of Chicago, Chicago, IL USA The Cellular and Molecular Arrhythmia Research ProgramIon Channels Play Pivotal Roles in Cardiac Functions and Protection Situate on

3、plasma membrane Contain multiple transmembrane helixes Opening of ion channels is within milliseconds Impact the electrophysiological state of the cell Mutations in ion channels are linked to many cardiovascular diseases (i.e. Long QT syndrome) Noma (1983): Sarcolemmal KATP Inoue et al. (1991): Mito

4、chondrial KATPK+MitochondrionK+K+Plasma membraneKATP channels open in ischemia like metabolic sensors of the cellCardiac ATP-sensitive Potassium Channels (KATP)Reviewed by Shi, Ye, Makielski (2005) JMCC 39:51N1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17CWalker AWalker B Sulfonylurea Receptor (ATP-bindi

5、ng cassette protein)SUR2A (Major cardiac splice variant) SUR2B (Major smooth muscle splice variant)175 kDaRKR M1 M2P loopNCRKR Pore(K+ inward rectifier)Kir6.248 kDaStructure of Cardiac KATP ChannelsTMD0 TMD1 TMD2 Ischemia is a disease condition where the heart has insufficient levels of oxygen and b

6、lood supply Acute myocardial infarction is a life-threatening event that can cause sudden death and heart failure Blocking KATP channels during ischemia abolishes cardioprotection (Gross & Auchampach, 1992) The opening of KATP can re-polarize the cell membrane, reduce calcium entry and decrease

7、ATP consumption MitoKATP is more likely to confer cardioprotection (Garlid et al., 1996 & 1997) but its molecular nature is not completely understoodIschemia and KATP ChannelsmitoKATPReviewed by Gross et al., 2003 IPC is a natural phenomenon where brief periods of ischemia provide in vivo protec

8、tion from subsequent lethal ischemia that can cause infarction Acute IPC: where the protective period lasts for few hours after the preconditioning stimulus Delayed IPC (SWOP): where the protective period can last for 24-72 h after the preconditioning stimulus Ischemic Preconditioning (IPC)Reviewed

9、by Yellon & Downey, 2003Stimulus-Memory-ProtectionTo evaluate the responses of SUR2 mutant mice in ischemia and IPC To understand the regulation and roles of SUR2 variants in IPCPurpose of This StudyHypothesis: Losing the regulatory subunit of cardiac KATP leads to reduced cardiac protectionChut

10、kow, Pu, Wada, Makielski, Burant, McNally. PNAS (2001) 98:11760Kakkar, Ye, Shi, Makielski, McNally et al., Cir Res (2006) 98:675SUR2 Knockout Mutant MiceExon12-16Glibenclamide (a KATP blocker) action sitesThe Glibenclamide-Sensitive KATP Current is Absent in the Mutant Myocytes and VSM Cells-10-8-6-

11、4-20-10-8-6-4-20Current amplitude (pA) Current amplitude (pA)1000 msWTMutantChutkow et al (2001) PNAS 98:11760010203040506070809010033%74% of Patches Containing IKATPA Glibenclamide-Insensitive KATP Current (IKATPn) is recorded in KO mice KO (n=15)01020304050607080 Amplitude (pA)5410.012.35.4 WT (n=

12、19)0 pA0 pAPu*, Ye*, McNally, Makielski, Shi (2021) JMCC 44:188 1 s2 pA SUR2 mutantIKATPn is More Sensitive to ATPKinetics of IKATPn Differ from the Conventional IKATPMean burst duration (ms) WT SUR2 Mutant 44.29.520.61.8 WT SUR2 Mutant pS WT SUR2 Mutant PO1.20.540.130.490.12Duration Time

13、 (ms)Total number of burstsWTSUR2 MutantIKATPn is Glibenclamide-InsensitiveSummary of Part I The conventional glibenclamide-sensitive KATP activity is absent in SUR2 mutant mice A novel ATP-sensitive, glibenclamide-insensitive KATP activity is recorded in SUR2 mutant mice This IKATPn has distinct ch

14、annel kinetics, ATP sensitivity, and pharmacological properties from the conventional IKATPNC1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17*BNJ-1T1BNJ-UBNJ-2BNJ-39 (2A-specific)BNJ-40 (2B-specific)New SUR2 AntibodiesTMD0 TMD1 TMD2SUR1NBD1NBD2NBD1Walker AWalker BBNJ-17BNJ-14 155 kDa220110Na/K VDAC1 COXIVA

15、TPase Nav1.5 HCN4150 T1 BNJ-2 BNJ-39 BNJ-40Cut off at 100-kDa68 28 kDaBNJ-39BNJ-40 T1 BNJ-2 BNJ-39 BNJ-406828Cut off at 100-kDakDa150WTSUR2 MutantNovel SUR2 Short Variants are Detected in Cardiac Sarcolemmal Fractions1406828Na/K VDAC1 COXIVATPase1730kDa110Na/K VDAC1 COXIVATPase1730kDa1102855kDa68Hea

16、rt Brain 120BNJ-2 BNJ-39 BNJ-4055 BNJ-2 BNJ-39 BNJ-40kDaNovel SUR2 Short Variants are Detected in Mitochondrial FractionsYe, Kroboth, Wada, McNally, Makielski, Shi. 2021Summary of Part II A panel of new SUR2 antibodies are generated with tested specificity against SUR2 splice variants and isoforms I

17、n addition to a 150-kDa SUR2 long variant, novel SUR2 short variants are detected in WT plasma membrane In SUR2 mutant mice, the 150-kDa SUR2 long variant is absent suggesting it confers the glibenclamide-sensitive KATP activity The SUR2 short variants are intact in the mutant suggesting that they m

18、ay confer the glibenclamide-insensitive KATP activityMutant WTIIIIIIWTWTMutMutNormalized cell death rate (%) II III II III (n=6) (n=5)*nsP0.0546%22% 27%24%Ye*, Wada*, Sims, Kroboth, Pu, Stoller, McNally, Makielski, Shi. 2021 Ringer NaCN Ringer Ringer Ringer Ringer NaCN Ringer 2-DG+Ringer RingerRecov

19、ery Preconditioning Resting Ischemia Reperfusion 30 min10 min 60 min 30 min 30 min Ringer Ringer Ringer Ringer RingerIIIIIISimulated Ischemia and Metabolic Preconditioning in Isolated Myocytes of Male MiceStoller, Kakkar, Shi, Makielski, McNally. (2007) JMCC 43:445p0.001 I II I IIInfarct size (%I-AA

20、R)*ns(n=10) (n=5) (n=8) (n=5)WTWTMutMut54% 32% 30%27%WTSUR2 mutant3 min 5 minStabilization Reperfusion Ischemia Reperfusion 60 min 40 min 24 min 20 min 60 min 40 min 20 minStabilization Preconditioning Ischemia ReperfusionIIILangendorff Perfused Heart Model in Male Mice01020304050Infarct Size I-AAR%

21、 WT WT Mut Mut I II I II (n=10, P0.01)38%25%23%19%Whole Mouse Ischemia/IPC Model in Male Mice 4 minII Ischemia Reperfusion I Preconditioning Resting 30 min 90 min10 min 30 min 90 min10 min 30 min 90 min 3 minRegulation of SUR2 Variants During Acute IPC in Male MiceBCL2T1BNJ-39BNJ-40GAPDHHSP70WTUntre

22、ated IPC Cut off at100-kDa26kDa681502828 4070SUR2 mutant Untreated IPC Cut off at100-kDaSummary of Part III All models agree that SUR2 male mutant mice are protected from ischemia without the need of preconditioning The degree of protection in the mutant is similar to that recorded in the WT precond

23、itioned mice IPC only induced marginal improvements but not significant The 150-kDa SUR2 long variant and the 28-kDa SUR2B short variant are significantly down-regulated in WT preconditioned hearts, these forms are absent in the mutant The 68-kDa SUR2A short variant is significantly up-regulated in the mutantConclusions An unconventional glibenclamide-insensitive IKATPn is found in SUR2 mutant mice Novel SUR2 short variants are detected in SUR2 mutant mice The SUR2 short forms may serve as hemi-ABC transporters to regulate channel functions Levels of SUR2 are associated with c