mri-and-pet-in-mouse-models-of-myocardial-infarction

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

University of Cambridge

&#x6E7E; 58-2667 (&#x6E7E;-58) &#x76F4;&#x63A5;&#x6FC0;&#x6D3B;&#x53EF;&#x6EB6;&#x6027;&#x9E1F;&#x82F7;&#x9170;&#x73AF;&#x5316;&#x9176;&#x65E0;&#x8010;&#x7684;&#x4E00;&#x6C27;&#x5316;&#x6C2E; (NO)-&#x72EC;&#x7ACB;&#x7684;&#x65B9;&#x5F0F;&#x53CA;&#x8840;&#x6D41;&#x52A8;&#x529B;&#x5B66;&#x7684;&#x5F71;&#x54CD;&#x662F;&#x76F8;&#x4F3C;&#x7684;&#x785D;&#x9178;&#x7518;&#x6CB9;&#x3002;&#x6211;&#x4EEC;&#x6D4B;&#x8BD5;&#x662F;&#x5426;&#x6E7E; 58 &#x80FD;&#x4F7F;&#x5154;&#x548C;&#x5927;&#x9F20;&#x5FC3;&#x8010;&#x6897;&#x6B7B;&#x65F6;&#x7F3A;&#x8840;&#x6027;&#x4FAE;&#x8FB1;&#x7684;&#x7ED3;&#x5C3E;&#x7ED9;&#x51FA;&#x3002;

&#x6E7E; 58-2667&#xFF0C;&#x72EC;&#x7ACB;&#x4E00;&#x6C27;&#x5316;&#x6C2E;&#x7684;&#x9E1F;&#x82F7;&#x9170;&#x73AF;&#x5316;&#x9176;&#x6FC0;&#x6D3B;&#x5242;&#xFF0C;&#x836F;&#x7406;&#x5B66; post-conditions &#x5154;&#x548C;&#x5927;&#x9F20;&#x7684;&#x5FC3;&#x3002;

BAY 58-2667 (BAY-58) directly activates soluble guanylyl cyclase without tolerance in a nitric oxide (NO)-independent manner, and its haemodynamic effect is similar to that of nitroglycerin. We tested whether BAY-58 could make both rabbit and rat hearts resistant to infarction when given at the end of an ischaemic insult.

BAY 58-2667, a nitric oxide-independent guanylyl cyclase activator, pharmacologically post-conditions rabbit and rat hearts.

58-2667 BAY (baie-58) directement active guanylyl cyclase soluble sans tol&eacute;rance dans un oxyde nitrique (non)-mani&egrave;re ind&eacute;pendante et son effet h&eacute;modynamique est similaire &agrave; celle de la nitroglyc&eacute;rine. Nous avons v&eacute;rifi&eacute; si BAY-58 pourrait faire tant de lapin et de rat coeur r&eacute;sistant aux infarctus du myocarde lorsqu&#39;il est administr&eacute; &agrave; la fin d&#39;une insulte isch&eacute;mique.

BAIE 58-2667, un activateur d&#39;ind&eacute;pendante de l&#39;oxyde nitrique guanylyl cyclase, post-conditions pharmacologiquement coeurs de lapin et le rat.

BAY 58-2667 (BAY-58) direkt aktiviert Guanylatzyklase l&ouml;sliche Guanylatcyclase ohne Toleranz in einer Stickstoffmonoxid (NO)-unabh&auml;ngiger Weise und deren h&auml;modynamischen Auswirkungen ist &auml;hnlich dem von Nitroglyzerin. Wir haben getestet, ob BAY-58 Kaninchen und Ratte Herz best&auml;ndig gegen Infarkt, wenn am Ende eine isch&auml;mische Beleidigung machen k&ouml;nnte.

BAY 58-2667, ein Stickoxid-unabh&auml;ngige Guanylatzyklase Guanylatcyclase Aktivator, post-conditions pharmakologisch Kaninchen und Ratte Herz.

58-2667 BAY (baia-58) si attiva direttamente solubile guanil ciclasi senza tolleranza in un ossido nitrico (NO)-modo indipendente e il suo effetto emodinamico &egrave; simile a quella della nitroglicerina. Abbiamo testato se BAY-58 potrebbe rendere sia coniglio e il topo cuori resistente all&#39;infarto quando alla fine di un insulto ischemico.

BAY 58-2667, un attivatore di ossido nitrico-indipendente guanil ciclasi, post-conditions farmacologicamente cuori di coniglio e il topo.

&#x30D9;&#x30A4; 58-2667 (&#x6E7E;-58) &#x76F4;&#x63A5;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x53EF;&#x6EB6;&#x6027;&#x30B0;&#x30A2;&#x30CB;&#x30EB;&#x9178;&#x30B7;&#x30AF;&#x30E9;&#x30FC;&#x30BC;&#x306F;&#x4E00;&#x9178;&#x5316;&#x7A92;&#x7D20; (NO) &#x306E;&#x8010;&#x6027;&#x306E;&#x306A;&#x3044;-&#x72EC;&#x7ACB;&#x3057;&#x305F;&#x65B9;&#x6CD5;&#x3068;&#x305D;&#x306E;&#x8840;&#x884C;&#x52D5;&#x614B;&#x52B9;&#x679C;&#x30CB;&#x30C8;&#x30ED;&#x30B0;&#x30EA;&#x30BB;&#x30EA;&#x30F3;&#x3068;&#x540C;&#x69D8;&#x3067;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x3001;&#x30A6;&#x30B5;&#x30AE;&#x3068;&#x30E9;&#x30C3;&#x30C8;&#x306E;&#x5FC3;&#x306B;&#x6897;&#x585E;&#x865A;&#x8840;&#x6027;&#x306E;&#x4FAE;&#x8FB1;&#x306E;&#x7D42;&#x308F;&#x308A;&#x306B;&#x4E0E;&#x3048;&#x3089;&#x308C;&#x305F;&#x3068;&#x304D;&#x8010;&#x6E7E; 58 &#x3092;&#x4F5C;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x304B;&#x3069;&#x3046;&#x304B;&#x30C6;&#x30B9;&#x30C8;&#x3002;

&#x6E7E;&#x3001;&#x7A92;&#x7D20;&#x9178;&#x5316;&#x7269;&#x72EC;&#x7ACB;&#x30B0;&#x30A2;&#x30CB;&#x30EB;&#x9178;&#x30B7;&#x30AF;&#x30E9;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x5316;&#x306F;&#x3001;58 2667&#x5E74;&#x85AC;&#x7406;&#x5B66;&#x7684;&#x30A6;&#x30B5;&#x30AE;&#x3068;&#x30E9;&#x30C3;&#x30C8;&#x306E;&#x5FC3;&#x3092;&#x4E8B;&#x5F8C;&#x6761;&#x4EF6;&#x3057;&#x307E;&#x3059;&#x3002;

&#xBCA0;&#xC774; 58-2667 (&#xBCA0;&#xC774;-58) &#xC9C1;&#xC811; &#xC0B0;&#xD654; &#xC9C8;&#xC18C; (NO)&#xC5D0; &#xD5C8;&#xC6A9; &#xC624;&#xCC28; &#xC5C6;&#xC774; &#xB179;&#xB294; guanylyl cyclase &#xD65C;&#xC131;&#xD654;-&#xB3C5;&#xB9BD; &#xBC29;&#xC2DD;&#xC73C;&#xB85C; &#xACE0;&#xC758; haemodynamic &#xD6A8;&#xACFC; &#xB2C8;&#xD2B8;&#xB85C; &#xAE00; &#xB9AC;&#xC138; &#xB9B0; &#xBE44;&#xC2B7;&#xD569;&#xB2C8;&#xB2E4;. &#xD1A0;&#xB07C;&#xC640; &#xC950; &#xB9C8;&#xC74C;&#xC5D0; &#xAC15;&#xD55C; &#xACBD;&#xC0C9; ischaemic &#xBAA8;&#xC695;&#xC758; &#xB05D;&#xC5D0; &#xC8FC;&#xC5B4;&#xC9C4; &#xBCA0;&#xC774; 58 &#xB9CC;&#xB4E4; &#xC218; &#xC788;&#xB294;&#xC9C0; &#xC5EC;&#xBD80;&#xB97C; &#xD14C;&#xC2A4;&#xD2B8; &#xD569;&#xB2C8;&#xB2E4;.

&#xBCA0;&#xC774; 58-2667, &#xC9C8;&#xC18C;&#xC758; &#xC0B0;&#xD654;&#xBB3C; &#xB3C5;&#xB9BD;&#xC801;&#xC778; guanylyl cyclase &#xD65C;&#xC131;&#xAE30; pharmacologically &#xD1A0;&#xB07C;&#xC640; &#xC950; &#xB9C8;&#xC74C; post-conditions.

58-2667 BAY (Bah&iacute;a-58) activa directamente ciclasa soluble guanylyl sin tolerancia en un &oacute;xido n&iacute;trico (NO)-manera independiente y su efecto hemodin&aacute;mico es similar a la de la nitroglicerina. Probamos si BAY-58 podr&iacute;a hacer tanto conejo y la rata corazones resistente al infarto cuando al final de un insulto isqu&eacute;mico.

Bah&iacute;a 58-2667, un activador de &oacute;xido n&iacute;trico-independiente guanylyl ciclasa, farmacol&oacute;gicamente post-conditions corazones de conejo y la rata.

Pre- and postconditioning depend on the activation of adenosine receptors (ARs) at the end of the index ischemia. The aim of this study was to determine which receptor subtypes must be activated. In situ mouse hearts underwent 30 min of regional ischemia, followed by 2 h of reperfusion. As expected, either ischemic postconditioning (6 cycles of 10 s of reperfusion and 10 s of coronary occlusion) or infusion of the selective A(2b) adenosine receptor (A(2b)AR) agonist BAY60-6583 (BAY60) for 60 min, starting 5 min before reperfusion reduced infarct size in wild-type C57Bl/6N mice. Protection from either was abolished by the selective A(2b)AR antagonist MRS-1754, confirming a role for A(2b)AR. Additionally, the coadministration of ischemic postconditioning and a selective A(2a)AR antagonist led to the loss of protection as well. 5'-Ectonucleotidase (CD73) is thought to be necessary for the production of adenosine during ischemia. As predicted, ischemic postconditioning did not protect CD73 knockout mice. Selective agonists of either A(2b)AR (BAY60) or A(2a)AR (CGS-21680), as well as the coadministration of ischemic postconditioning and BAY60, also failed to protect hearts of the CD73 knockout mice. But the nonselective A(1)/A(2)AR agonist 5'-(N-ethylcarboxamido)adenosine (NECA) was protective, suggesting that the activation of multiple AR subtypes might be required. The coadministration of CGS-21680 and BAY60 also elicited profound protection, indicating that two AR subtypes, A(2a) and A(2b), must be simultaneously activated for protection to occur.

Both A2a and A2b adenosine receptors at reperfusion are necessary to reduce infarct size in mouse hearts.

A2b adenosine receptors in cardioprotection: timing is everything.

&#x5DF2;&#x7ECF;&#x53D6;&#x5F97;&#x7684;&#x7F3A;&#x8840;&#x9884;&#x5904;&#x7406;&#x4FDD;&#x62A4;&#x662F;&#x4F9D;&#x8D56;&#x4E8E; A(2B) &#x817A;&#x82F7;&#x53D7;&#x4F53; (&#x5728;&#x5154;&#x548C;&#x9F20;&#x6807;&#x5FC3;&#x548C;&#xFF0C;&#x53EF;&#x4EE5;&#x9884;&#x89C1;&#x7684;&#x662F;&#xFF0C;A A(2B)AR) (2B) AR &#x6FC0;&#x52A8;&#x5242;&#x4FDD;&#x62A4;&#x4ED6;&#x4EEC;&#x3002;&#x4F46;&#x5B83;&#x662F;&#x6709;&#x4E89;&#x8BAE;&#x7684;&#x662F;&#x5426;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x672C;&#x8EAB;&#x5B9E;&#x9645;&#x4E0A;&#x8868;&#x8FBE; (2B) &#x4EBA;&#x624D;&#x57F9;&#x8BAD;&#x4E2D;&#x5FC3;&#x3002;&#x672C;&#x7814;&#x7A76;&#x62A5;&#x544A;&#x6D4B;&#x8BD5;&#x662F;&#x5426; (2B) AR &#x53EF;&#x4EE5;&#x8868;&#x660E;&#x5BF9;&#x9F20;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x3002;&#x5927;&#x9F20;&#x79BB;&#x7684;&#x4F53;&#x5FC3;&#x810F;&#x7ECF;&#x5386;&#x4E86; 30 &#x5206;&#x949F;&#x7684;&#x7F3A;&#x8840;&#x548C;&#x518D;&#x704C;&#x6CE8;&#x7684; 120 &#x5206;&#x949F;&#x3002;&#x9AD8;&#x9009;&#x62E9;&#x6027;&#x3001; &#x5355;&#x5143;&#x683C; permeant A (2B) AR &#x6FC0;&#x52A8;&#x5242; BAY60 6583 &#xFF08;500 nM&#xFF09; &#x5728;&#x63A7;&#x5236;&#x5FC3;&#x4E2D;&#x7684;&#x98CE;&#x9669;&#x533A; 40.4 &plusmn; 2.0%&#x81F3; 19.9 &plusmn; 2.8%&#xFF0C;&#x663E;&#x793A; (2B) AR &#x4FDD;&#x62A4;&#x4EE5;&#x53CA;&#x5927;&#x9F20;&#x5FC3;&#x810F;&#x518D;&#x704C;&#x6CE8;&#x51CF;&#x5C11;&#x6897;&#x585E;&#x6CE8;&#x5165;&#x4E86;&#x3002;&#x6B64;&#x5916;&#xFF0C;BAY60 6583 &#x51CF;&#x5C11;&#x79BB;&#x4F53;&#x7684;&#x5927;&#x9F20;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x9499;&#x8BF1;&#x5BFC;&#x7EBF;&#x7C92;&#x4F53;&#x901A;&#x900F;&#x3002;(2B) AR &#x86CB;&#x767D;&#x53EF;&#x4EE5;&#x8868;&#x660E;&#x5728;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x4E2D;&#xFF0C;&#x514D;&#x75AB;&#x5370;&#x8FF9;&#x6CD5;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x5728;&#x4F7F;&#x7528;&#x5B9A;&#x91CF;&#x9006;&#x8F6C;&#x5F55;-&#x805A;&#x5408;&#x9176;&#x94FE;&#x53CD;&#x5E94;&#x7684;&#x5355;&#x4E2A;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x4E2D;&#x627E; (2B) AR &#x7684;&#x6D88;&#x606F;&#x3002;&#x5947;&#x602A;&#x7684;&#x662F;&#xFF0C;&#x514D;&#x75AB;&#x8367;&#x5149;&#x663E;&#x5FAE;&#x955C;&#x6CA1;&#x6709;&#x663E;&#x793A; (2B) AR &#x5BF9;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x7684;&#x7EC6;&#x80DE;&#x8D28;&#x819C;&#xFF0C;&#x4F46;&#x800C;&#x662F;&#x5728;&#x7EC6;&#x80DE;&#x5185;&#x7684;&#x5730;&#x70B9;&#x3002;&#x5728;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x4E2D; co-staining &#x4E0E; MitoTracker &#x7EA2;&#x8272;&#x663E;&#x793A; (2B) AR &#x5DF2;&#x672C;&#x5730;&#x5316;&#x5230;&#x7EBF;&#x7C92;&#x4F53;&#x3002;&#x4ECE;&#x5927;&#x9F20;&#x5FC3;&#x810F;&#x6D3B;&#x68C0;&#x6216;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x7EBF;&#x7C92;&#x4F53;&#x5206;&#x6570;&#x7684;&#x5370;&#x8FF9;&#x5206;&#x6790;&#x663E;&#x793A; (2B) AR &#x5E26;&#x5F3A;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x672C;&#x7814;&#x7A76;&#x62A5;&#x544A;&#x8868;&#x660E;&#x6FC0;&#x6D3B; (2B) AR &#x5F3A;&#x70C8;&#x662F;&#x5927;&#x9F20;&#x5FC3;&#x810F;&#x5FC3;&#x808C;&#x4FDD;&#x62A4;&#x5E76;&#x6291;&#x5236;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#xFF0C;&#x5728;&#x8F6C;&#x578B;&#x6BDB;&#x5B54; (2B) AR &#x8868;&#x793A;&#x5355;&#x4E2A;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x5947;&#x602A;&#x7684;&#x662F;&#xFF0C;(2B) AR &#x90FD;&#x5B58;&#x5728;&#x6216;&#x9644;&#x8FD1;&#x7EBF;&#x7C92;&#x4F53;&#x800C;&#x4E0D;&#x662F;&#x4F5C;&#x4E3A;&#x808C;&#x819C;&#x4E0A;&#x5176;&#x4ED6;&#x817A;&#x82F7;&#x53D7;&#x4F53;&#x3002;(2B) AR &#x4FE1;&#x53F7;&#x4F20;&#x9012;&#x601D;&#x60F3;&#xFF0C;&#x5BFC;&#x81F4;&#x7EBF;&#x7C92;&#x4F53;&#x8F6C;&#x6BDB;&#x5B54;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#xFF0C;&#x56E0;&#x4E3A;&#x8FD9;&#x79CD;&#x65B9;&#x4FBF;&#x7684;&#x4F4D;&#x7F6E;&#x53EF;&#x80FD;&#x662F;&#x91CD;&#x8981;&#x7684;&#x3002;

&#x80DE;&#x5185;&#x7684;&#x672C;&#x5730;&#x5316;&#x817A;&#x82F7; A2B &#x53D7;&#x4F53;&#x5728;&#x5927;&#x9F20;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x7684;&#x8BC1;&#x636E;&#x3002;

Protection achieved by ischemic preconditioning is dependent on A(2B) adenosine receptors (A(2B)AR) in rabbit and mouse hearts and, predictably, an A(2B)AR agonist protects them. But it is controversial whether cardiomyocytes themselves actually express A(2B)AR. The present study tested whether A(2B)AR could be demonstrated on rat cardiomyocytes. Isolated rat hearts experienced 30 min of ischemia and 120 min of reperfusion. The highly selective, cell-permeant A(2B)AR agonist BAY60-6583 (500 nM) infused at reperfusion reduced infarct size from 40.4 Â± 2.0% of the risk zone in control hearts to 19.9 Â± 2.8% indicating that A(2B)AR are protective in rat heart as well. Furthermore, BAY60-6583 reduced calcium-induced mitochondrial permeability transition in isolated rat cardiomyocytes. A(2B)AR protein could be demonstrated in isolated cardiomyocytes by western blotting. In addition, message for A(2B)AR was found in individual cardiomyocytes using quantitative RT-PCR. Surprisingly, immunofluorescence microscopy did not show A(2B)AR on the cardiomyocyte's sarcolemma but rather at intracellular sites. Co-staining with MitoTracker Red in isolated cardiomyocytes revealed A(2B)AR are localized to mitochondria. Western blot analysis of a mitochondrial fraction from either rat heart biopsies or isolated cardiomyocytes revealed a strong A(2B)AR band. Thus, the present study demonstrates that activation of A(2B)AR is strongly cardioprotective in rat heart and suppresses transition pores in isolated cardiomyocytes, and A(2B)AR are expressed in individual cardiomyocytes. However, surprisingly, A(2B)AR are present in or near mitochondria rather than on the sarcolemma as are other adenosine receptors. Because A(2B)AR signaling is thought to result in inhibition of mitochondrial transition pores, this convenient location may be important.

Evidence for an intracellular localization of the adenosine A2B receptor in rat cardiomyocytes.

Protection atteint par un pr&eacute;conditionnement isch&eacute;mique d&eacute;pend de r&eacute;cepteurs de l&#39;ad&eacute;nosine A(2B) (A(2B)AR) en lapin et souris c&oelig;urs et, comme pr&eacute;vu, un A prot&egrave;ge agoniste AR (2 b). Mais il est controvers&eacute; que cardiomyocytes s&#39;exprimer r&eacute;ellement un AR. (2 b) La pr&eacute;sente &eacute;tude v&eacute;rifi&eacute; si un AR (2 b) a pu &ecirc;tre d&eacute;montr&eacute;e sur des cardiomyocytes de rat. C&oelig;urs de rats isol&eacute;s a connu 30 min d&#39;isch&eacute;mie et 120 min de reperfusion. La tr&egrave;s s&eacute;lective, cellule perm&eacute;able A agoniste d&#39;AR (2 b) BAY60-6583 (500 nM) infus&eacute; &agrave; la taille de l&#39;infarctus reperfusion r&eacute;duite de 40,4 &plusmn; 2,0 % de la zone &agrave; risque dans les coeurs t&eacute;moins &agrave; 19,9 &plusmn; 2,8 % indiquant qu&#39;un AR (2 b) sont protecteurs dans le coeur de rat aussi bien. En outre, BAY60-6583 r&eacute;duit de transition de perm&eacute;abilit&eacute; mitochondriale induits par le calcium dans les cardiomyocytes isol&eacute;s de rats. Une prot&eacute;ine de AR (2 b) peut &ecirc;tre d&eacute;montr&eacute;e dans les cardiomyocytes isol&eacute;s par western blot. En outre, message d&#39;un AR (2 b) a &eacute;t&eacute; trouv&eacute; dans les cardiomyocytes individuels &agrave; l&#39;aide de RT-PCR quantitative. &Eacute;tonnamment, la microscopie par immunofluorescence ne montre pas un AR (2 b) sur le sarcolemme de la cardiomyocyte, mais plut&ocirc;t aux sites intracellulaires. Co-Staining avec MitoTracker rouge dans les cardiomyocytes isol&eacute;s ont r&eacute;v&eacute;l&eacute; un AR (2 b) sont localis&eacute;es aux mitochondries. Analyse par Western blot de la fraction mitochondriale de biopsies de coeur de rat ou de cardiomyocytes isol&eacute;s ont r&eacute;v&eacute;l&eacute; une forte une bande d&#39;AR (2 b). Par cons&eacute;quent, la pr&eacute;sente &eacute;tude d&eacute;montre que l&#39;activation d&#39;un AR (2 b) est fortement cardioprotecteurs dans le coeur de rat et supprime les pores de la transition dans les cardiomyocytes isol&eacute;s et un AR (2 b) sont exprim&eacute;s dans les cardiomyocytes individuels. Cependant, &eacute;tonnamment, un AR (2 b) sont pr&eacute;sentes dans les mitochondries &agrave; proximit&eacute; plut&ocirc;t que sur le sarcolemme comme sont les autres r&eacute;cepteurs de l&#39;ad&eacute;nosine. Parce qu&#39;une signalisation d&#39;AR (2 b) est probablement le r&eacute;sultat dans l&#39;inhibition de mitochondrial transition pores, cet emplacement peut &ecirc;tre important.

T&eacute;moignage pour une localisation intracellulaire du r&eacute;cepteur de l&#39;ad&eacute;nosine A2B dans les cardiomyocytes de rat.

Schutz durch isch&auml;mische Vorkonditionierung erreicht ist abh&auml;ngig von A(2B) Adenosin-Rezeptoren (A(2B)AR) in Hase und Maus Herzen und, wie vorherzusehen war, ein A (2B)-AR-Agonist sch&uuml;tzt sie. Aber es ist umstritten, ob Cardiomyocytes sich schnelles tats&auml;chlich ein AR (2 b). Die vorliegende Studie gepr&uuml;ft, ob eine (2B)-AR Ratte Cardiomyocytes nachgewiesen werden konnte. Isolierter Rattenherzen erlebten 30 min von Ischemia und 120 min der Reperfusion. Die hochselektive, Zelle-permeant-A (2B)-AR-Agonisten BAY60-6583 (500 nM) bei Reperfusion reduziert Infarktgr&ouml;&szlig;e von 40,4 &plusmn; 2,0 % von der Zone in Steuerelement Herzen auf 19,9 &plusmn; 2,8 % anzeigt, dass eine AR (2B) sind zum Schutz in Ratte Herz als auch als Infusion verabreicht. Dar&uuml;ber hinaus reduziert BAY60-6583 Kalzium-induzierten mitochondrialen Permeabilit&auml;t &Uuml;bergang in isolierten Ratte Cardiomyocytes. Ein AR-Protein (2B) konnte in den isolierten Cardiomyocytes durch westliche Beflecken nachgewiesen werden. Dar&uuml;ber hinaus fand Nachricht f&uuml;r ein AR (2 b) in einzelne Cardiomyocytes mit quantitativen RT-PCR. &Uuml;berraschend, zeigen Immunfluoreszenz-Mikroskopie keine AR (2 b) auf die Cardiomyocyte Sarkolemm sondern an intrazellul&auml;re Websites. Co-Staining mit MitoTracker rot in den isolierten Cardiomyocytes ergab eine AR (2B) sind auf Mitochondrien lokalisiert. Western-Blot-Analyse der mitochondrialen Dezimalbruch von Ratte Herz Biopsien oder isolierte Cardiomyocytes ergab eine starke Band AR (2 b). So zeigt die vorliegende Studie, dass Aktivierung &uuml;ber eine (2B)-AR ist stark kardioprotektive in Ratte Herz &Uuml;bergang Poren in den isolierten Cardiomyocytes unterdr&uuml;ckt und eine AR (2 b) werden in den einzelnen Cardiomyocytes ausgedr&uuml;ckt. &Uuml;berraschenderweise eine AR (2 b) sind jedoch in oder in der N&auml;he von Mitochondrien, anstatt auf dem Sarkolemm als andere Adenosin-Rezeptoren sind vorhanden. Weil ein (2B)-AR-Signalisierung gedacht ist, zu einer Hemmung der mitochondrialen &Uuml;bergang Poren f&uuml;hren, sein Diese vorteilhafte Lage wichtig.

Beweise f&uuml;r eine intrazellul&auml;re Lokalisation des Adenosin A2B-Rezeptors in Ratte Cardiomyocytes.

Protezione ottenuta dal precondizionamento ischemico dipende dai recettori dell&#39;adenosina A(2B) (A(2B)AR) nel coniglio e cuori del mouse e, prevedibilmente, un agonista AR (2B) li protegge. Ma &egrave; controverso se cardiomyocytes si esprimono in realt&agrave; un AR (2B). Lo studio presente ha testato se una AR (2B) potrebbe essere dimostrata su ratto cardiomyocytes. Cuori del ratto isolato sperimentato 30 min di ischemia e 120 min di riperfusione. La A altamente selettiva, cella-permeant (2B) agonista AR BAY60-6583 (500 nM) infuso a riperfusione ridotta di infarct da 40,4 &plusmn; 2.0% della zona di rischio nei cuori di controllo 19,9 &plusmn; 2,8% che indica che una AR (2B) sono protettivi nel cuore del ratto. Inoltre, BAY60-6583 ridotto transizione di permeabilit&agrave; mitocondriale indotta da calcio in cardiomiociti isolati di ratto. Una proteina AR (2B) potrebbe essere dimostrata in cardiomiociti isolati da macchiarsi occidentale. Inoltre, il messaggio per una AR (2B) &egrave; stato trovato nei cardiomyocytes individuale mediante RT-PCR quantitativa. Sorprendentemente, microscopia di immunofluorescenza non ha mostrato una AR (2B), il sarcolemma di cardiomyocyte ma piuttosto presso siti intracellulari. Co-Staining con MitoTracker Red in cardiomiociti isolati ha rivelato una AR (2B) sono localizzate ai mitocondri. Analisi Western blot di una frazione mitocondriale da biopsie di cuore di ratto o cardiomiociti isolati hanno rivelato una forte un gruppo di AR (2B). Cos&igrave;, il presente studio dimostra che l&#39;attivazione di una AR (2B) &egrave; fortemente cardioprotettivi nel cuore del ratto e sopprime i pori di transizione in cardiomiociti isolati, e una AR (2B) sono espressi nei cardiomyocytes individuali. Tuttavia, sorprendentemente, una AR (2B) sono presenti in o vicino mitocondri, piuttosto che sul sarcolemma come sono altri recettori dell&#39;adenosina. Perch&eacute; una segnalazione di AR (2B) &egrave; pensata per causare inibizione dei pori di transizione mitocondriale, questa posizione pu&ograve; essere importante.

Prove per una localizzazione intracellulare del recettore dell&#39;adenosina A2B in cardiomiociti di topo.

&#x865A;&#x8840;&#x30D7;&#x30EC;&#x30B3;&#x30F3;&#x30C7;&#x30A3;&#x30B7;&#x30E7;&#x30CB;&#x30F3;&#x30B0;&#x306B;&#x3088;&#x3063;&#x3066;&#x9054;&#x6210;&#x306E;&#x4FDD;&#x8B77;&#x3067;&#x3059; A(2B) &#x30A2;&#x30C7;&#x30CE;&#x30B7;&#x30F3;&#x53D7;&#x5BB9;&#x4F53;&#x306B;&#x4F9D;&#x5B58; (&#x30A6;&#x30B5;&#x30AE;&#x304A;&#x3088;&#x3073;&#x30DE;&#x30A6;&#x30B9;&#x5FC3;&#x3068;&#x3001;&#x4E88;&#x60F3;&#x901A;&#x308A;&#x3001;A A(2B)AR) (2 b) AR &#x30A2;&#x30B4;&#x30CB;&#x30B9;&#x30C8;&#x306F;&#x305D;&#x308C;&#x3089;&#x3092;&#x4FDD;&#x8B77;&#x3057;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x81EA;&#x8EAB;&#x304C;&#x5B9F;&#x969B;&#x306B; &#xFF08;2 b&#xFF09; &#x7740;&#x8868;&#x73FE;&#x304B;&#x3069;&#x3046;&#x304B;&#x306F;&#x7269;&#x8B70;&#x3092;&#x91B8;&#x3059;&#x672C;&#x7814;&#x7A76;&#x3092;&#x30C6;&#x30B9;&#x30C8; (2 b) AR &#x306F;&#x30E9;&#x30C3;&#x30C8;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x306B;&#x793A;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x304B;&#x3069;&#x3046;&#x304B;&#x3002;&#x30E9;&#x30C3;&#x30C8;&#x5FC3;&#x81D3;&#x306B;&#x304A;&#x3051;&#x308B;&#x865A;&#x8840;&#x30FB;&#x518D;&#x704C;&#x6D41;&#x306E; 120 &#x5206;&#x306E; 30 &#x5206;&#x3092;&#x7D4C;&#x9A13;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x9AD8;&#x9078;&#x629E;&#x6027;&#x30FB; &#x30BB;&#x30EB;&#x4E0B;&#x6D41; A (2 b) AR &#x30A2;&#x30B4;&#x30CB;&#x30B9;&#x30C8; BAY60 6583 (500 nM) &#x304B;&#x3089; 40.4 &#x306E; &plusmn; 2.0% &#x30EA;&#x30B9;&#x30AF;&#x306E;&#x30BE;&#x30FC;&#x30F3;&#x5236;&#x5FA1;&#x306E;&#x5FC3;&#x306E;&#x524A;&#x6E1B;&#x518D;&#x704C;&#x6D41;&#x6897;&#x585E;&#x30B5;&#x30A4;&#x30BA; (2 b) AR &#x304C;&#x30E9;&#x30C3;&#x30C8;&#x306E;&#x5FC3;&#x306B;&#x3082;&#x4FDD;&#x8B77;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059; 19.9 &plusmn; 2.8 &#xFF05; &#x306B;&#x6CE8;&#x5165;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;BAY60 6583 &#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x8A98;&#x5C0E;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x900F;&#x904E;&#x6027;&#x9077;&#x79FB;&#x30E9;&#x30C3;&#x30C8;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x306E;&#x6E1B;&#x5C11;&#x3002;(2 B) AR &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x5206;&#x96E2;&#x306B;&#x304A;&#x3051;&#x308B;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x306F;&#x897F;&#x90E8;&#x306E;&#x3057;&#x307F;&#x304C;&#x4ED8;&#x304F;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x793A;&#x3055;&#x308C;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x30E1;&#x30C3;&#x30BB;&#x30FC;&#x30B8; (2 b) ar &#x5B9A;&#x91CF;&#x7684; RT-PCR &#x6CD5;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x500B;&#x3005; &#x306E;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x304C;&#x898B;&#x3064;&#x304B;&#x308A;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3002;&#x9A5A;&#x3044;&#x305F;&#x3053;&#x3068;&#x306B;&#x3001;&#x86CD;&#x5149;&#x9855;&#x5FAE;&#x93E1; (2 b) AR &#x5FC3;&#x7B4B;&#x306E;&#x7B4B;&#x9798;&#x304C;&#x7D30;&#x80DE;&#x5185;&#x306E;&#x30B5;&#x30A4;&#x30C8;&#x3067;&#x306F;&#x306A;&#x304F;&#x3092;&#x793A;&#x3055;&#x306A;&#x304B;&#x3063;&#x305F;&#x3002;MitoTracker &#x8D64;&#x3068;&#x5206;&#x96E2;&#x306B;&#x304A;&#x3051;&#x308B;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE; co-staining (2 b) AR &#x306F;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306B;&#x5C40;&#x5728;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30E9;&#x30C3;&#x30C8;&#x5FC3;&#x751F;&#x691C;&#x307E;&#x305F;&#x306F;&#x5B64;&#x7ACB;&#x3057;&#x305F;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x753B;&#x5206;&#x306E;&#x897F;&#x90E8;&#x306E;&#x3057;&#x307F;&#x306E;&#x5206;&#x6790;&#x306F;&#x5F37;&#x529B;&#x306A; (2 b) AR &#x30D0;&#x30F3;&#x30C9;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;(2 b) AR &#x306E;&#x6D3B;&#x6027;&#x5316;&#x304C;&#x5F37;&#x304F;&#x5FC3;&#x4FDD;&#x8B77;&#x306B;&#x304A;&#x3051;&#x308B;&#x30E9;&#x30C3;&#x30C8;&#x5FC3;&#x81D3;&#x306B;&#x3042;&#x308A;&#x5206;&#x96E2;&#x5FC3;&#x7B4B;&#x8EE2;&#x79FB;&#x6BDB;&#x7A74;&#x3092;&#x6291;&#x5236;&#x3057;&#x3066; (2 b) AR &#x306F;&#x500B;&#x3005; &#x306E;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x306E;&#x8868;&#x73FE;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x9A5A;&#x3044;&#x305F;&#x3053;&#x3068;&#x306B;&#x3001;(2 b) AR &#x304C;&#x4ED6;&#x30A2;&#x30C7;&#x30CE;&#x30B7;&#x30F3;&#x53D7;&#x5BB9;&#x4F53;&#x7B4B;&#x9798;&#x3068;&#x3057;&#x3066;&#x3067;&#x306F;&#x306A;&#x304F;&#x307E;&#x305F;&#x306F;&#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x306E;&#x8FD1;&#x304F;&#x3002;(2 B) AR &#x30B7;&#x30B0;&#x30CA;&#x30EA;&#x30F3;&#x30B0; &#x30DF;&#x30C8;&#x30B3;&#x30F3;&#x30C9;&#x30EA;&#x30A2;&#x9077;&#x79FB;&#x6BDB;&#x7A74;&#x306E;&#x6291;&#x5236;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x3068;&#x8003;&#x3048;&#x3089;&#x308C;&#x308B;&#x306E;&#x3067;&#x3001;&#x3053;&#x306E;&#x4FBF;&#x5229;&#x306A;&#x5834;&#x6240;&#x306F;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;

&#x30E9;&#x30C3;&#x30C8;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x30A2;&#x30C7;&#x30CE;&#x30B7;&#x30F3; A2B &#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x7D30;&#x80DE;&#x5185;&#x5C40;&#x5728;&#x5316;&#x306E;&#x8A3C;&#x62E0;&#x3002;

&#xD5C8; &#xD608; &#xC131; &#xB298;&#xC5B4;&#xC9C4; &#xC5EC; &#xBCF4;&#xD638; A(2B) &#xC544;&#xB370;&#xB178;&#xC2E0; &#xC218;&#xC6A9; &#xCCB4;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4; (&#xD1A0;&#xB07C;&#xC640; &#xB9C8;&#xC6B0;&#xC2A4; &#xB9C8;&#xC74C;&#xACFC;, &#xC608;&#xC0C1; &#xB300;&#xB85C;, A A(2B)AR) (2B) &#xC544;&#xCE78;&#xC18C; &#xAE38; &#xD56D; &#xC81C;&#xB294; &#xADF8;&#xB4E4;&#xC744; &#xBCF4;&#xD638; &#xD569;&#xB2C8;&#xB2E4;. &#xD558;&#xC9C0;&#xB9CC; &#xC2A4;&#xC2A4;&#xB85C; cardiomyocytes &#xC2E4;&#xC81C;&#xB85C; &#xC775;&#xC2A4;&#xD504;&#xB808;&#xC2A4; (2B) &#xC544;&#xCE78;&#xC18C; &#xC5EC;&#xBD80; &#xB17C;&#xB780; &#xD604;&#xC7AC;&#xC758; &#xC5F0;&#xAD6C; (2B) &#xC544;&#xCE78;&#xC18C; &#xC950; cardiomyocytes&#xC5D0; &#xC2DC;&#xC5F0; &#xB420; &#xC218; &#xC788;&#xB294;&#xC9C0; &#xC5EC;&#xBD80;&#xB97C; &#xD14C;&#xC2A4;&#xD2B8; &#xD569;&#xB2C8;&#xB2E4;. &#xACA9;&#xB9AC; &#xB41C; &#xC950; &#xB9C8;&#xC74C; &#xAD6D; &#xC18C; &#xBE48; &#xD608;&#xACFC; reperfusion&#xC758; 120 &#xBD84; 30 &#xBD84;&#xC744; &#xACBD;&#xD5D8; &#xD588;&#xB2E4;. &#xB9E4;&#xC6B0; &#xC120;&#xD0DD;&#xC801;, &#xC140; permeant A (2B) &#xC544;&#xCE78;&#xC18C; &#xAE38; &#xD56D; &#xC81C; BAY60-6583 (500 nM) 19.9 &plusmn; 2.8% (2B) &#xC544;&#xCE78;&#xC18C; &#xBCF4;&#xD638; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xC950; &#xB9C8;&#xC74C;&#xC5D0; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xC81C;&#xC5B4; &#xB9C8;&#xC74C;&#xC5D0; &#xC704;&#xD5D8; &#xC601;&#xC5ED;&#xC758; 40.4 &plusmn; 2.0%&#xC5D0;&#xC11C; &#xAC10;&#xC18C; reperfusion &#xACBD;&#xC0C9; &#xD06C;&#xAE30;&#xC5D0; &#xC8FC;&#xC785; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, BAY60-6583 &#xACA9;&#xB9AC; &#xB41C; &#xC950; cardiomyocytes&#xC5D0; &#xCE7C;&#xC298; &#xC720;&#xB3C4; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC; &#xB9AC;&#xC544; &#xCE68;&#xD22C;&#xC131; &#xC804;&#xD658; &#xAC10;&#xC18C;. (2B) &#xC544;&#xCE78;&#xC18C; &#xB2E8;&#xBC31;&#xC9C8; &#xC11C;&#xC591; blotting&#xC5D0; &#xC758;&#xD574; &#xACA9;&#xB9AC; &#xB41C; cardiomyocytes&#xC5D0; &#xC785;&#xC99D; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xBA54;&#xC2DC;&#xC9C0; (2B) &#xC544;&#xCE78;&#xC18C; &#xC591;&#xC801; RT-PCR&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xAC1C;&#xBCC4; cardiomyocytes&#xC5D0; &#xCC3E;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB180;&#xB78D;&#xAC8C;&#xB3C4;, cardiomyocyte&#xC758; sarcolemma &#xD558;&#xC9C0;&#xB9CC; &#xC138;&#xD3EC;&#xB0B4; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0; &#xC624;&#xD788;&#xB824; &#xBA74;&#xC5ED; &#xD615;&#xAD11; &#xAC80;&#xC0AC; &#xD604;&#xBBF8;&#xACBD; &#xAC80;&#xC0AC; &#xBC95; (2B) &#xC544;&#xCE78;&#xC18C; &#xD45C;&#xC2DC; &#xD558;&#xC9C0; &#xC54A;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. &#xACA9;&#xB9AC; &#xB41C; cardiomyocytes&#xC5D0; MitoTracker &#xB808;&#xB4DC; co-staining (2B) &#xC544;&#xCE78;&#xC18C; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544;&#xC5D0; &#xC9C0;&#xC5ED;&#xD654;&#xB294; &#xBC1D;&#xD614;&#xB2E4;. &#xC950; &#xC2EC;&#xC7A5; biopsies &#xB610;&#xB294; &#xACA9;&#xB9AC; cardiomyocytes&#xC5D0;&#xC11C; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xBD84;&#xC218;&#xC758; &#xC11C;&#xCABD; &#xC624; &#xC810; &#xBD84;&#xC11D; (2B) &#xC544;&#xCE78;&#xC18C; &#xBC34;&#xB4DC; &#xAC15;&#xB825;&#xD55C; &#xACF5;&#xAC1C;. &#xB530;&#xB77C;&#xC11C;, &#xD604;&#xC7AC;&#xC758; &#xC5F0;&#xAD6C; (2B) &#xC544;&#xCE78;&#xC18C; &#xAC1C;&#xBCC4; cardiomyocytes&#xC5D0; &#xD45C;&#xD604; &#xB429;&#xB2C8;&#xB2E4; (2B) AR&#xC758; &#xD65C;&#xC131;&#xD654; cardioprotective &#xC950; &#xB9C8;&#xC74C;&#xC5D0; &#xAC15;&#xB825; &#xD558; &#xAC8C;&#xB294;, &#xC808;&#xC5F0;&#xB41C; cardiomyocytes&#xC5D0; &#xC804;&#xD658; &#xC228; &#xAD6C;&#xBA4D;&#xC744; &#xD45C;&#xC2DC; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;&#xC744; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC758;&#xC678;&#xB85C;, (2B) &#xC544;&#xCE78;&#xC18C; &#xC788;&#xB294;&#xC9C0; &#xB610;&#xB294; &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC;&#xB9AC; &#xC544; &#xC778;&#xADFC;&#xC73C;&#xB85C; sarcolemma&#xC5D0;&#xB294; &#xB2E4;&#xB978; &#xC544;&#xB370;&#xB178;&#xC2E0; &#xC218;&#xC6A9; &#xCCB4; &#xBCF4;&#xB2E4;&#xB294;. &#xBBF8;&#xD1A0; &#xCF58; &#xB4DC; &#xB9AC;&#xC544; &#xC804;&#xD658; &#xC228; &#xAD6C;&#xBA4D;&#xC758; &#xC800;&#xD574; &#xB420; &#xAC83;&#xC73C;&#xB85C; &#xC0DD;&#xAC01; &#xB429;&#xB2C8;&#xB2E4; (2B) &#xC544;&#xCE78;&#xC18C; &#xC2E0;&#xD638;,&#xC774; &#xD3B8;&#xB9AC;&#xD55C; &#xC704;&#xCE58; &#xC911;&#xC694; &#xD55C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC950; cardiomyocytes&#xC5D0; A2B &#xC544;&#xB370;&#xB178;&#xC2E0; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC138;&#xD3EC;&#xB0B4; &#xC9C0;&#xC5ED;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xC99D;&#xAC70;.

Protecci&oacute;n mediante precondicionamiento isqu&eacute;mico es dependiente de los receptores de adenosina A(2B) (A(2B)AR) en conejo y rat&oacute;n corazones y, como era de esperar, un agonista de AR (2B) protege. Pero es pol&eacute;mico si cardiomiocitos expresan realmente un ar (2B). El presente estudio prob&oacute; si un AR (2B) se pudo demostrar en cardiomiocitos de rata. Coraz&oacute;n aislado de rata hab&iacute;a experimentado 30 minutos de isquemia y 120 min de la reperfusi&oacute;n. La c&eacute;lula-Florescente, altamente selectiva A (2B) agonista de AR BAY60-6583 (500 nM) infundido en la reperfusi&oacute;n reducida tama&ntilde;o del infarto de 40,4 &plusmn; 2.0% de la zona de riesgo en los corazones de control a 19,9 &plusmn; 2.8% lo que indica que un AR (2B) tienen un efecto protector en el coraz&oacute;n de rata as&iacute;. Adem&aacute;s, BAY60-6583 reduce la transici&oacute;n de la permeabilidad mitocondrial inducida por calcio en cardiomiocitos aislado de rata. Una prote&iacute;na de AR (2B) podr&iacute;a demostrarse en cardiomiocitos aislados por western blot. Adem&aacute;s, mensaje para un AR (2B) fue encontrado en los cardiomiocitos individuales usando RT-PCR cuantitativa. Sorprendentemente, microscopia de la inmunofluorescencia no mostraron un AR (2B) en el sarcolema de los cardiomiocitos sino sitios intracelulares. Co-staining con MitoTracker rojo en cardiomiocitos aislados revel&oacute; un AR (2B) se localizan en las mitocondrias. An&aacute;lisis de Western blot de una fracci&oacute;n mitocondrial de las biopsias de coraz&oacute;n de rata o cardiomiocitos aislados revel&oacute; una fuerte una banda de AR (2B). As&iacute;, el presente estudio demuestra que la activaci&oacute;n de un AR (2B) es fuertemente cardioprotector en coraz&oacute;n de rata y suprime los poros de transici&oacute;n en los cardiomiocitos aislados, y un AR (2B) se expresan en los cardiomiocitos individuales. Sin embargo, sorprendentemente, un AR (2B) est&aacute;n presentes en o cerca de las mitocondrias en lugar de en el sarcolema como son otros receptores de adenosina. Porque una se&ntilde;alizaci&oacute;n de AR (2B) se piensa para resultar en inhibici&oacute;n de los poros de transici&oacute;n mitocondrial, esta ubicaci&oacute;n puede ser importante.

Evidencia de una localizaci&oacute;n intracelular del receptor de la adenosina A2B en cardiomiocitos de rata.

To demonstrate and validate a late gadolinium enhancement (LGE) imaging protocol, optimized for tissue viability assessment in interventional mouse models of myocardial infarction.

A fast protocol for infarct quantification in mice.

Protein kinase G type I (PKGI) plays a critical role in survival signaling of pre- and postconditioning downstream of cardiac cGMP. However, it is unclear whether PKGI exerts its protective effects in the cardiomyocyte or if other cardiac cell types are involved, and whether nitric oxide (NO) metabolism can target cardiomyocyte mitochondria independently of cGMP/PKGI. We tested whether protection against reperfusion injury by ischemic postconditioning (IPost), soluble guanylyl cyclase (sGC) activation and inhibition, adenosine A(2B) receptor (A(2B)AR) agonist, phosphodiesterase type-5 (PDE-5) inhibitor, or mitochondria-targeted S-nitrosothiol (MitoSNO) was affected by a cardiomyocyte-specific ablation of the PKGI gene in the mouse (CMG-KO). In situ hearts underwent 30 min of regional ischemia followed by 2 h of reperfusion. As expected, in CMG-CTRs all interventions at early reperfusion lead to profound infarct size reduction: IPost (six cycles of 10-s reperfusion and 10-s coronary occlusion) with or without treatment with the sGC inhibitor ODQ, treatment with the specific sGC activator BAY58-2667 (BAY58), the selective A(2B)AR agonist BAY60-6583 (BAY60), PDE-5 inhibitor sildenafil, and MitoSNO. MitoSNO accumulates within mitochondria, driven by the membrane potential, where it generates NO· and S-nitrosates thiol proteins. In contrast, the hearts of CMG-KO animals were not protected by BAY58 and sildenafil, whereas the protective effects of IPost, IPost with ODQ, BAY60, and MitoSNO were unaffected by the lack of PKGI. Taken together, PKGI is important for the protection against ischemia reperfusion injury afforded by sGC activation or PDE-5 inhibition. However, the beneficial effects of IPost, activation of the A(2B)AR, as well as the direct effects via mitochondrial S-nitrosation do not depend on PKGI in cardiomyocytes.

Protection through postconditioning or a mitochondria-targeted S-nitrosothiol is unaffected by cardiomyocyte-selective ablation of protein kinase G.

Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.

Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I.

The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.

Using exomarkers to assess mitochondrial reactive species in vivo.

The glycation of protein and nucleic acids that occurs as a consequence of hyperglycaemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs following the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal and disruption to mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear due to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycaemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycaemia in both cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging.

Carmen Methner

Department of Medicine

University of Cambridge

MRI and PET in Mouse Models of Myocardial Infarction