磺酰脲類受體2型依賴的KATP通道在缺血預(yù)適應(yīng)中的調(diào)控作用英文PPT課件

1、 Noma (1983): Sarcolemmal KATP Inoue et al. (1991): Mitochondrial KATPK+MitochondrionK+K+Plasma membraneKATP channels open in ischemia like metabolic sensors of the cellCardiac ATP-sensitive Potassium Channels (KATP)第1頁/共28頁Reviewed by Shi, Ye, Makielski (2005) JMCC 39:51N1 2 3 4 5 6 7 8 9 10 11 12

2、13 14 15 16 17CWalker AWalker B Sulfonylurea Receptor (ATP-binding 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第2頁/共28頁 Ischemia is a d

3、isease condition where the heart has insufficient levels of oxygen and blood 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 KA

4、TP can re-polarize the cell membrane, reduce calcium entry and decrease 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第3頁/共28頁 IP

5、C is a natural phenomenon where brief periods of ischemia provide in vivo protection 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

6、-72 h after the preconditioning stimulus Ischemic Preconditioning (IPC)Reviewed by Yellon & Downey, 2003Stimulus-Memory-Protection第4頁/共28頁To evaluate the responses of SUR2 mutant mice in ischemia and IPC To understand the regulation and roles of SUR2 variants in IPCPurpose of This StudyHypothesi

7、s: Losing the regulatory subunit of cardiac KATP leads to reduced cardiac protection第5頁/共28頁Chutkow, 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 site

8、s第6頁/共28頁The Glibenclamide-Sensitive KATP Current is Absent in the Mutant Myocytes and VSM Cells-10-8-6-4-20-10-8-6-4-20Current amplitude (pA) Current amplitude (pA)1000 msWTMutantChutkow et al (2001) PNAS 98:11760第7頁/共28頁010203040506070809010033%74% of Patches Containing IKATPA Glibenclamide-Insens

9、itive KATP Current (IKATPn) is recorded in KO mice KO (n=15)01020304050607080 Amplitude (pA)5410.012.35.4 WT (n=19)0 pA0 pAPu*, Ye*, McNally, Makielski, Shi (2008) JMCC 44:188 第8頁/共28頁1 s2 pA SUR2 mutantIKATPn is More Sensitive to ATP第9頁/共28頁Kinetics of IKATPn Differ from the Conventional IKATPMean

10、burst duration (ms) WT SUR2 Mutant 44.29.520.61.8 WT SUR2 Mutant pS WT SUR2 Mutant PO23.71.123.11.20.540.130.490.12Duration Time (ms)Total number of bursts第10頁/共28頁WTSUR2 MutantIKATPn is Glibenclamide-Insensitive第11頁/共28頁Summary of Part I The conventional glibenclamide-sensitive KATP activity is abs

11、ent in SUR2 mutant mice A novel ATP-sensitive, glibenclamide-insensitive KATP activity is recorded in SUR2 mutant mice This IKATPn has distinct channel kinetics, ATP sensitivity, and pharmacological properties from the conventional IKATP第12頁/共28頁NC1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17*BNJ-1T1BNJ

12、-UBNJ-2BNJ-39 (2A-specific)BNJ-40 (2B-specific)New SUR2 AntibodiesTMD0 TMD1 TMD2SUR1NBD1NBD2NBD1Walker AWalker BBNJ-17BNJ-14第13頁/共28頁 155 kDa220110Na/K VDAC1 COXIVATPase 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-kDakDa150WTS

13、UR2 MutantNovel SUR2 Short Variants are Detected in Cardiac Sarcolemmal Fractions第14頁/共28頁1406828Na/K VDAC1 COXIVATPase1730kDa110Na/K VDAC1 COXIVATPase1730kDa1102855kDa68Heart Brain 120BNJ-2 BNJ-39 BNJ-4055 BNJ-2 BNJ-39 BNJ-40kDaNovel SUR2 Short Variants are Detected in Mitochondrial FractionsYe, Kr

14、oboth, Wada, McNally, Makielski, Shi. 2008第15頁/共28頁Summary of Part II A panel of new SUR2 antibodies are generated with tested specificity against SUR2 splice variants and isoforms In addition to a 150-kDa SUR2 long variant, novel SUR2 short variants are detected in WT plasma membrane In SUR2 mutant

15、 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 may confer the glibenclamide-insensitive KATP activity第16頁/共28頁Mutant WTIIIIIIWTWTMutMutNormalized cell death rate (%)

16、II III II III (n=6) (n=5)*nsP0.0546%22% 27%24%Ye*, Wada*, Sims, Kroboth, Pu, Stoller, McNally, Makielski, Shi. 2008 Ringer NaCN Ringer Ringer Ringer Ringer NaCN Ringer 2-DG+Ringer RingerRecovery Preconditioning Resting Ischemia Reperfusion 30 min10 min 60 min 30 min 30 min Ringer Ringer Ringer Ringe

17、r RingerIIIIIISimulated Ischemia and Metabolic Preconditioning in Isolated Myocytes of Male Mice第17頁/共28頁Stoller, Kakkar, Shi, Makielski, McNally. (2007) JMCC 43:445p0.001 I II I IIInfarct size (%I-AAR)*ns(n=10) (n=5) (n=8) (n=5)WTWTMutMut54% 32% 30%27%WTSUR2 mutant3 min 5 minStabilization Reperfusi

18、on Ischemia Reperfusion 60 min 40 min 24 min 20 min 60 min 40 min 20 minStabilization Preconditioning Ischemia ReperfusionIIILangendorff Perfused Heart Model in Male Mice第18頁/共28頁01020304050Infarct Size I-AAR% WT WT Mut Mut I II I II (n=10, P0.01)38%25%23%19%Whole Mouse Ischemia/IPC Model in Male Mi

19、ce 4 minII Ischemia Reperfusion I Preconditioning Resting 30 min 90 min10 min 30 min 90 min10 min 30 min 90 min 3 min第19頁/共28頁Regulation of SUR2 Variants During Acute IPC in Male MiceBCL2T1BNJ-39BNJ-40GAPDHHSP70WTUntreated IPC Cut off at100-kDa26kDa681502828 4070SUR2 mutant Untreated IPC Cut off at1

20、00-kDa第20頁/共28頁Summary 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 preconditioned mice IPC only induced marginal improvements but not significant T

21、he 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 mutant第21頁/共28頁Conclusions An unconventional glibenclamide-insensitiv

22、e 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 cardioprotection suggesting that they are in the mechanistic pathway of IPC第22頁/共28頁N 1 2 3 4 17CNBD2Hybrid TMDA Rare Intra-Exonic Splici