Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Wyss Institute for Biologically Inspired Engineering at Harvard University

Embryonic stem (ES) cells offer unprecedented opportunities for in vitro drug discovery and safety assessment of compounds. Cardiomyocytes derived from ES cells enable development of predictive cardiotoxicity models to increase the safety of novel drugs. Heterogeneity of differentiated ES cells limits the development of reliable in vitro models for compound screening. We report an innovative and robust approach to isolate ES-derived cardiomyocytes using laser microdissection and pressure catapulting (LMPC). LMPC cells were readily applied onto 96-well format in vitro pharmacology assays. The expression of developmental and functional cardiac markers, Nkx 2.5, MLC2V, GATA-4, Connexin 43, Connexin 45, Serca-2a, cardiac alpha actin, and phospholamban, among others, was confirmed in LMPC ES-derived cardiomyocytes. Functional assays exhibited cardiac-like response to increased extracellular calcium (5.4 mM extracellular Ca2+) and L-type calcium channel antagonist (1 microM nifedipine). In conclusion, laser microdissection and pressure catapulting is a robust technology to isolate homogeneous ES-derived cell types from heterogeneous populations applicable to assay development.

Embryonic stem cells in predictive cardiotoxicity: laser capture microscopy enables assay development.

Inhibition of the delayed-rectifier potassium channel current, human ether-a-go-go (hERG), by pharmaceutical agents can lead to acquired long QT syndrome and the generation of potentially lethal arrhythmias and sudden death. There remains an unmet need for higher-throughput assays to screen compounds in preclinical development for the potential to block hERG and cause QT prolongation. We evaluated the rubidium efflux assay for its ability to determine block of the hERG potassium channel. hERG-transfected human embryonic kidney-293 cells were cultured on 96-well assay plates and loaded with rubidium ion by incubating in media in which potassium was replaced by 5.4 mM Rb+. Cells were exposed to test compounds and then depolarized with a K+ channel opening buffer containing 50 mM K+. The supernatant was removed, and cells were lysed using 0.1% Triton X-100. Concentration-response curves were generated for test agents by determining the Rb+ efflux using a flame atomic absorption spectrometer. Multiple trials with cisapride yielded 50% inhibitory concentration values between 308.1 +/- 11 nM to 456.3 +/- 24 nM for inhibition of Rb+ efflux and a Z factor of 0.80 +/- 0.07 (n = 5 plates, 12 wells per plate). The values for inhibition of the hERG channel exhibited a rightward shift in potency as compared to those measured using electrophysiological techniques. In addition, we evaluated 19 blinded compounds at 10 microM in the Rb+ efflux assay, and compared results to those using patch clamp electrophysiology and the dofetilide displacement binding assay. The dofetilide displacement binding assay yielded a good correlation with electrophysiological measurements of hERG block. The rubidium efflux assay lacked sensitivity to consistently identify significant channel blockade. In conclusion, the rubidium efflux assay provides a higher-throughput means to identify potent hERG channel blocking agents, but lacks the sensitivity required to accurately determine the potency of blockade.

Evaluation of the rubidium efflux assay for preclinical identification of HERG blockade.

&#x4EE5;&#x524D;&#x7684;&#x7814;&#x7A76;&#x8868;&#x660E;&#x589E;&#x52A0;&#x7684;&#x8102;&#x80AA;&#x9178;&#x6444;&#x53D6;&#x548C; MHC-FATP &#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20;&#xFF0C;&#x53BB;&#x8102;&#x80AA;&#x9178;&#x4EE3;&#x8C22;&#x7684;&#x8FD0;&#x8F93;&#x86CB;&#x767D; (FATP) &#x5728;&#x63A7;&#x5236;&#x4E0B;&#x5FC3; 1 &alpha;-&#x808C;&#x7403;&#x86CB;&#x767D;&#x91CD;&#x94FE; &#xFF08;&alpha;-MHC&#xFF09; &#x542F;&#x52A8;&#x5B50;&#x3002;&#x7EC4;&#x7EC7;&#x591A;&#x666E;&#x52D2;&#x6210;&#x50CF;&#x548C;&#x8840;&#x6D41;&#x6D4B;&#x91CF;&#x663E;&#x793A;&#x8212;&#x5F20;&#x529F;&#x80FD;&#x4E0D;&#x5168;&#xFF0C;&#x6CA1;&#x6709;&#x6536;&#x7F29;&#x529F;&#x80FD;&#x7684;&#x53D8;&#x5316;&#x3002;&#x8FD9;&#x91CC;&#x7684;&#x5B9E;&#x9A8C;&#x76F4;&#x63A5;&#x6D4B;&#x8BD5; MHC FATP &#x5C0F;&#x9F20;&#x7684;&#x8212;&#x5F20;&#x529F;&#x80FD;&#x969C;&#x788D;&#x53CD;&#x6620;&#x4E86;&#x53D7;&#x635F;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x6536;&#x7F29;&#x529F;&#x80FD;&#x7684;&#x5047;&#x8BF4;&#x3002;&#x4F53;&#x5916;&#x6210;&#x50CF;&#x7684;&#x5B64;&#x7ACB;&#x7684;&#x5FC3;&#x5BA4;&#x808C;&#x7EC6;&#x80DE;&#x663E;&#x793A;&#x5E73;&#x5747;&#x8212;&#x5F20;&#x808C;&#x8282;&#x957F;&#x5EA6;&#x662F;&#x5927;&#x5927;&#x7684;&#x6210;&#x4EBA; MHC-FATP (P &lt; 0.01&#xFF09; &#x6BD4;&#x91CE;&#x751F;&#x578B; (WT) &#x7EC6;&#x80DE; (1.79 + /-0.01 &#x4E0E; 1.84+/-0.01 microm&#xFF09; &#x8F83;&#x77ED;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x677E;&#x5F1B;&#x7387; &#xFF08;dL/dt) &#x662F;&#x663E;&#x8457; (P &lt; 0.05) MHC FATP &#x6BD4;&#x6162; WT &#x808C; (1.58 + /-0.09 &#x4E0E; 1.92+/-0.13 microm/s&#xFF09;&#xFF0C;&#x800C;&#x8FD9;&#x4E24;&#x4E2A;&#x5206;&#x6570;&#x7684;&#x7F29;&#x77ED;&#x548C;&#x6536;&#x7F29;&#x7387;&#x4E0D;&#x662F;&#x4E0D;&#x540C;&#x3002;40 &#x82F1;&#x56FD;/L 2&#xFF0C;3-butadionemonoxime &#xFF08;&#x975E;&#x7279;&#x5F02;&#x6027; ATPase &#x6291;&#x5236;&#xFF0C;&#x653E;&#x677E;&#x808C;&#x52A8;&#x86CB;&#x767D;-&#x808C;&#x7403;&#x86CB;&#x767D;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#xFF09; &#x7684;&#x5E94;&#x7528;&#x7A0B;&#x5E8F;&#x589E;&#x52A0;&#x8212;&#x5F20;&#x808C;&#x8282;&#x957F;&#x5EA6;&#x7684;&#x957F;&#x5EA6;&#x76F8;&#x540C;&#xFF0C;&#x5EFA;&#x8BAE;&#x5728;&#x5176;&#x4F59;&#x90E8;&#x5206;&#x6FC0;&#x6D3B;&#x5DF2;&#x5FC3;&#x808C;&#x808C;&#x7403;&#x86CB;&#x767D;&#x91CD;&#x94FE; FATP WT &#x548C; MHC-FATP &#x808C;&#x3002;&#x76F4;&#x63A5;&#x7684;&#x80DE;&#x5185;&#x7ECF;&#x6D4B;&#x91CF;&#x53D1;&#x73B0;&#xFF0C;&#x8212;&#x5F20; [Ca(2+)](i) &#x4E0D;&#x53D8;&#x7684; MHC FATP &#x808C;&#x7EC6;&#x80DE;&#x548C;&#x9499;&#x53BB;&#x9664;&#x7387;&#x662F;&#x610F;&#x5916;&#x7684; MHC-FATP WT &#x808C;&#x7EC6;&#x80DE;&#x6BD4;&#x66F4;&#x5FEB;&#x3002;&#x6B64;&#x5916;&#xFF0C;MHC-FATP &#x548C; WT &#x808C;&#x7EC6;&#x80DE;&#x8212;&#x5F20;&#x808C;&#x8282;&#x957F;&#x5EA6;&#x5E76;&#x6CA1;&#x6709;&#x53D7;&#x5230;&#x5F71;&#x54CD;&#xFF0C;&#x7531;&#x80DE;&#x5916;&#x7ECF;&#x53BB;&#x9664;&#x6216;&#x4E0E;&#x7ECF;&#x87AF;&#x5408;&#x5242; BAPTA &#x7684;&#x7EC6;&#x80DE;&#x7ECF;&#x7F13;&#x51B2; &#xFF08;100 &#x6469;&#x5C14;/&#x5347;)&#xFF0C;&#x6307;&#x793A;&#x9AD8;&#x67B6;&#x80DE;&#x7ECF;&#x4E0D;&#x662F;&#x57FA;&#x7840;&#x53D7;&#x635F; MHC FATP &#x5FC3;&#x5BA4;&#x8212;&#x5F20;&#x529F;&#x80FD;&#x3002;&#x529F;&#x80FD;&#x8BC4;&#x4F30;&#x76AE;&#x80A4;&#x808C;&#x7EC6;&#x80DE;&#xFF0C;&#x4F46;&#x662F;&#xFF0C;&#x8868;&#x660E;&#x8BE5;&#x808C;&#x4E1D;&#x7ED3;&#x6784;&#x7ECF;&#x654F;&#x611F;&#x6027;&#x663E;&#x8457;&#x4E0A;&#x5347;&#x76F8;&#x6BD4; WT&#xFF0C;&#x5FC3;&#x5BA4;&#x808C;&#x7EC6;&#x80DE;&#x7684; MHC-FATP&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x751F;&#x5316;&#x5B9E;&#x9A8C;&#x8868;&#x660E;&#x5728; MHC-FATP&#xFF0C;&#x76F8;&#x6BD4; WT &#x8111;&#x5BA4;&#xFF0C;&#x8FD9;&#x53EF;&#x80FD;&#x6709;&#x52A9;&#x4E8E; MHC FATP &#x808C;&#x7EC6;&#x80DE;&#x4E2D;&#x89C2;&#x5BDF;&#x5230;&#x7684;&#x6162;&#x677E;&#x5F1B;&#x7387; &beta;-MHC &#x5F02;&#x6784;&#x4F53;&#x8868;&#x8FBE;&#x589E;&#x52A0;&#x3002;&#x96C6;&#x4F53;&#xFF0C;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E; MHC FATP &#x8111;&#x5BA4;&#xFF0C;&#x662F;&#x7C7B;&#x4F3C;&#x4E8E;&#x7CD6;&#x5C3F;&#x75C5;&#x5FC3;&#x810F;&#x4E2D;&#x89C2;&#x5BDF;&#x5230;&#xFF0C;&#x8102;&#x8D28;&#x4EE3;&#x8C22;&#x7D0A;&#x4E71;&#x5BFC;&#x81F4;&#x8212;&#x5F20;&#x529F;&#x80FD;&#x53D7;&#x635F;&#xFF0C;&#x4E3B;&#x8981;&#x53CD;&#x6620;&#x4E86;&#x808C;&#x4E1D;&#x7ED3;&#x6784;&#x529F;&#x80FD;&#x7684;&#x53D8;&#x5316;&#xFF0C;&#x800C;&#x4E0D;&#x662F;&#x6539;&#x53D8;&#x7684;&#x7ECF;&#x5FAA;&#x73AF;&#x3002;

Ca2 +-&#x8212;&#x5F20;&#x808C;&#x8282;&#x957F;&#x5EA6;&#x548C;&#x5F1B;&#x8C6B;&#x52A8;&#x529B;&#x5B66;&#x4E2D;&#x7684;&#x8102;&#x6BD2;&#x6027;&#x7CD6;&#x5C3F;&#x75C5;&#x5FC3;&#x808C;&#x75C5;&#x7684;&#x5C0F;&#x9F20;&#x6A21;&#x578B;&#x7684;&#x72EC;&#x7ACB;&#x53D8;&#x5316;&#x3002;

Previous studies demonstrated increased fatty acid uptake and metabolism in MHC-FATP transgenic mice that overexpress fatty acid transport protein (FATP)1 in the heart under the control of the alpha-myosin heavy chain (alpha-MHC) promoter. Doppler tissue imaging and hemodynamic measurements revealed diastolic dysfunction, in the absence of changes in systolic function. The experiments here directly test the hypothesis that the diastolic dysfunction in MHC-FATP mice reflects impaired ventricular myocyte contractile function. In vitro imaging of isolated adult MHC-FATP ventricular myocytes revealed that mean diastolic sarcomere length is significantly (P<0.01) shorter than in wild-type (WT) cells (1.79+/-0.01 versus 1.84+/-0.01 microm). In addition, the relaxation rate (dL/dt) is significantly (P<0.05) slower in MHC-FATP than WT myocytes (1.58+/-0.09 versus 1.92+/-0.13 microm/s), whereas both fractional shortening and contraction rates are not different. Application of 40 mmol/L 2,3-butadionemonoxime (a nonspecific ATPase inhibitor that relaxes actin-myosin interactions) increased diastolic sarcomere length in both WT and MHC-FATP myocytes to the same length, suggesting that MHC-FATP myocytes are partially activated at rest. Direct measurements of intracellular Ca(2+) revealed that diastolic [Ca(2+)](i) is unchanged in MHC-FATP myocytes and the rate of calcium removal is unexpectedly faster in MHC-FATP than WT myocytes. Moreover, diastolic sarcomere length in MHC-FATP and WT myocytes was unaffected by removal of extracellular Ca(2+) or by buffering of intracellular Ca(2+) with the Ca(2+) chelator BAPTA (100 micromol/L), indicating that elevated intracellular Ca(2+) does not underlie impaired diastolic function in MHC-FATP ventricular myocytes. Functional assessment of skinned myocytes, however, revealed that myofilament Ca(2+) sensitivity is markedly increased in MHC-FATP, compared with WT, ventricular cells. In addition, biochemical experiments demonstrated increased expression of the beta-MHC isoform in MHC-FATP, compared with WT ventricles, which likely contributes to the slower relaxation rate observed in MHC-FATP myocytes. Collectively, these data demonstrate that derangements in lipid metabolism in MHC-FATP ventricles, which are similar to those observed in the diabetic heart, result in impaired diastolic function that primarily reflects changes in myofilament function, rather than altered Ca(2+) cycling.

Ca2+-independent alterations in diastolic sarcomere length and relaxation kinetics in a mouse model of lipotoxic diabetic cardiomyopathy.

Pr&eacute;c&eacute;dentes &eacute;tudes d&eacute;montr&eacute;es accrue d&#39;acide gras absorption et le m&eacute;tabolisme chez les souris transg&eacute;niques de MHC-FATP qui surexpriment les acides gras de transport prot&eacute;ique (FATP) 1 en plein sous le contr&ocirc;le du promoteur de la cha&icirc;ne lourde de myosine-alpha (alpha-MHC). Des mesures h&eacute;modynamiques et d&#39;imagerie Doppler tissulaire a r&eacute;v&eacute;l&eacute; la dysfonction diastolique, en l&#39;absence de changements dans la fonction systolique. Les exp&eacute;riences ici tester directement l&#39;hypoth&egrave;se que la dysfonction diastolique chez les souris de MHC-FATP refl&egrave;te la fonction contractile myocyte ventriculaire avec facult&eacute;s affaiblies. Imagerie in vitro d&#39;isol&eacute;es adulte MHC-FATP myocytes ventriculaires ont r&eacute;v&eacute;l&eacute; que sarcom&egrave;res diastolique moyenne est significativement (P &lt; 0,01) plus court que dans les cellules de type sauvage (WT) (1,79 +/-0.01 versus 1.84+/-0.01 microm). En outre, le taux de relaxation (dL/dt) est significativement (P &lt; 0.05) plus lent &agrave; MHC-FATP que WT myocytes (1,58 +/-0,09 versus 1.92+/-0.13 microm/s), alors que les deux taux fractionnaire de raccourcissement et contraction n&#39;est pas diff&eacute;rents. Application de 40 mmol/L 2, 3-butadionemonoxime (un inhibiteur ATPase non sp&eacute;cifique qui d&eacute;tend les interactions actine-myosine) a augment&eacute; de sarcom&egrave;res diastolique dans les myocytes fois WT et MHC-FATP &agrave; la m&ecirc;me longueur, ce qui sugg&egrave;re que les myocytes MHC-FATP sont partiellement activ&eacute;s au repos. Ordonner des mesures de Ca(2+) intracellulaires ont r&eacute;v&eacute;l&eacute; que diastolique [Ca(2+)](i) est inchang&eacute;e dans les myocytes de MHC-FATP et du taux d&#39;extraction de calcium est subitement plus rapide &agrave; MHC-FATP que WT myocytes. En outre, sarcom&egrave;res diastolique dans les myocytes MHC-FATP et WT n&#39;est pas affect&eacute;e par le retrait de Ca(2+) extracellulaire ou par mise en m&eacute;moire tampon de Ca(2+) intracellulaire avec le ch&eacute;lateur Ca(2+) BAPTA (100 micromol/L), ce qui indique que Ca(2+) intracellulaire &eacute;lev&eacute;e ne pas sous-tendent alt&eacute;ration de la fonction diastolique dans les myocytes ventriculaires MHC-FATP. L&#39;&eacute;valuation fonctionnelle des myocytes d&eacute;pouill&eacute;es, cependant, a r&eacute;v&eacute;l&eacute; que myofilaments Ca(2+) sensibilit&eacute; est augment&eacute;e &agrave; MHC-FATP, compar&eacute;e &agrave; WT, cellules ventriculaires. En outre, des exp&eacute;riences biochimiques ont d&eacute;montr&eacute; une expression accrue de l&#39;isoforme MHC-b&ecirc;ta &agrave; MHC-FATP, par rapport aux ventricules WT, qui contribue probablement &agrave; la vitesse de relaxation plus lente observ&eacute;e dans les myocytes de MHC-FATP. Collectivement, ces r&eacute;sultats d&eacute;montrent que les d&eacute;rangements dans le m&eacute;tabolisme des lipides dans les ventricules FATP-MHC, qui sont semblables &agrave; ceux observ&eacute;s dans le coeur diab&eacute;tique, provoquer dysfonctionnement diastolique qui refl&egrave;te principalement les changements dans la fonction de myofilaments, plut&ocirc;t que Ca(2+) alt&eacute;r&eacute; de cyclisme.

Ca2 +-ind&eacute;pendantes alt&eacute;ration cin&eacute;tique sarcom&egrave;re diastolique de longueur et de la relaxation dans un mod&egrave;le murin de la cardiomyopathie diab&eacute;tique lipotoxic.

Fr&uuml;here Studien nachgewiesenen erh&ouml;hten Fetts&auml;ure Aufnahme und Stoffwechsel im MHC-FATP transgenen M&auml;usen, die Fetts&auml;ure &uuml;berexprimiert transport Protein (FATP) 1 im Herzen unter der Kontrolle des Projekttr&auml;gers Alpha-Myosin heavy Chain (Alpha-MHC). Gewebe Doppler imaging und h&auml;modynamische Messungen ergeben diastolische Dysfunktion, keine &Auml;nderungen im systolischen Funktion. Die Experimente hier testen direkt die Hypothese, dass die diastolische Dysfunktion bei MHC-FATP M&auml;usen gest&ouml;rte ventrikul&auml;re Muskelfaser kontraktilen Funktion widerspiegelt. In-vitro-Bildgebung der isoliert Erwachsenen MHC-FATP ventrikul&auml;re Myozyten offenbart, dass mittlere diastolische Sarkomer L&auml;nge erheblich (P &lt; 0,01) k&uuml;rzer als in den Wildtyp (WT) Zellen (1.79 +/-0,01 gegen&uuml;ber 1.84+/-0.01 Microm). Dar&uuml;ber hinaus ist die Entspannung-Rate (dL/dt) erheblich (P &lt; 0,05) langsamer in MHC-FATP WT Myocytes (1,58 +/-0,09 gegen&uuml;ber 1.92+/-0.13 Microm/s), w&auml;hrend beide gebrochene Verk&uuml;rzung und Kontraktion Preise nicht unterschiedlich sind. Anwendung der 40 Mmol/L 2, 3-Butadionemonoxime (eine unspezifische ATPase-Inhibitor, der Aktin-Myosin-Interaktion entspannt) erh&ouml;hte diastolische Sarkomer L&auml;nge in WT und MHC-FATP Myocytes auf dieselbe L&auml;nge, was darauf hindeutet, dass MHC-FATP Myocytes teilweise im Ruhezustand aktiviert werden. Direkte Messungen des intrazellul&auml;ren Ca(2+) offenbart das diastolische [Ca(2+)](i) ist unver&auml;ndert in den MHC-FATP Myocytes und die Kalzium-Entfernung betr&auml;gt in MHC-FATP unerwartet schneller WT Myocytes. Dar&uuml;ber hinaus diastolische Sarkomer L&auml;nge in den MHC-FATP und WT Myocytes verschont durch Entfernung der extrazellul&auml;ren Ca(2+) oder Pufferung der intrazellul&auml;ren Ca(2+) mit der Ca(2+)-Chelatbildner BAPTA (100 Mikromol/L), darauf hinweist, dass erh&ouml;hte intrazellul&auml;re Ca(2+) nicht zugrunde liegen diastolische Funktion in MHC-FATP ventrikul&auml;ren Myozyten beeintr&auml;chtigt. Funktionelle Beurteilung der hellh&auml;utigen Myocytes, ergab jedoch, dass Myofilament Ca(2+) Empfindlichkeit in MHC-FATP, WT, ventrikul&auml;re Zellen gegen&uuml;ber deutlich gestiegen ist. Biochemische Experimente zeigten dar&uuml;ber hinaus erh&ouml;hte Expression von der Beta-MHC-Isoform in MHC-FATP, verglichen mit WT Ventrikel, die wahrscheinlich die langsamer Entspannung beobachtet in den MHC-FATP Myocytes beitr&auml;gt. Diese Daten gemeinsam zu demonstrieren, dass Derangements im Fettstoffwechsel in MHC-FATP Ventrikel, die &auml;hnlich wie bei zuckerkranken Herzen sind, beeintr&auml;chtigt diastolische Funktion f&uuml;hren, die vor allem Ver&auml;nderungen im Myofilament-Funktion, widerspiegelt, anstatt ver&auml;nderte Ca(2+) Radfahren.

Ca2 +-unabh&auml;ngige Ver&auml;nderungen in der diastolische Sarkomer L&auml;nge und Entspannung Kinetics in einem Mausmodell der Lipotoxic Diabetische Kardiomyopathie.

Precedenti studi ha dimostrato aumento degli acidi grassi assorbimento e metabolismo in topi transgenici MHC-FATP che iperesprimono acidi grassi trasporto della proteina (FATP) 1 nel cuore sotto il controllo del promotore della catena pesante della miosina alfa (alfa-MHC). Tissue Doppler imaging ed emodinamico misurazioni hanno rivelato la disfunzione diastolica, in assenza di cambiamenti nella funzione sistolica. Gli esperimenti qui direttamente testare l&#39;ipotesi che la disfunzione diastolica nei topi MHC-FATP riflette del miocita ventricolare alterata funzione contrattile. Imaging in vitro dell&#39;isolato adulto MHC-FATP miociti ventricolari ha rivelato che la lunghezza media diastolica sarcomero &egrave; significativamente (P &lt; 0,01) pi&ugrave; corta in cellule di wild-type (WT) (1,79 + /-0.01 versus 1.84+/-0.01 microm). Inoltre, il tasso di rilassamento (dL/dt) &egrave; significativamente (P &lt; 0.05) pi&ugrave; lento nel MHC-FATP di miociti WT (1,58 + /-0,09 versus 1.92+/-0.13 microm/s), considerando che entrambi tassi di contrazione e accorciamento frazionali non sono diversi. Applicazione di 40 mmol/L 2,3-butadionemonoxime (un aspecifico ATPasi inibitore che rilassa le interazioni actina-miosina) aumentata lunghezza del sarcomero diastolica in miociti WT sia MHC-FATP la stessa lunghezza, suggerendo che MHC-FATP miociti sono parzialmente attivati a riposo. Dirigere le misurazioni di Ca(2+) intracellulare ha rivelato che diastolica [Ca(2+)](i) &egrave; invariato in miociti MHC-FATP e il tasso di rimozione del calcio &egrave; inaspettatamente pi&ugrave; veloce nel MHC-FATP di miociti WT. Inoltre, fu influenzato dalla rimozione dei Ca(2+) extracellulare o di buffer di Ca(2+) intracellulare con il chelante Ca(2+) BAPTA lunghezza sarcomero diastolica in miociti MHC-FATP e WT (100 micromol/L), che indica che Ca(2+) intracellulari elevati non sottostanno alterata funzione diastolica nei miociti ventricolari MHC-FATP. Valutazione funzionale dei miociti scuoiati, tuttavia, hanno rivelato che miofilamenti Ca(2+) sensibilit&agrave; sono marcatamente aumentata di MHC-FATP, confrontato con WT, cellule ventricolari. Inoltre, esperimenti biochimici hanno dimostrato maggiore espressione dell&#39;isoforma MHC-beta nel MHC-FATP, rispetto ai ventricoli WT, che probabilmente contribuisce al pi&ugrave; lento tasso di relax osservato nei miociti MHC-FATP. Collettivamente, questi dati dimostrano che i disordini nel metabolismo dei lipidi nei ventricoli MHC-FATP, che sono simili a quelle osservate nel cuore diabetico, risultano in alterata funzione diastolica che riflette principalmente le modifiche in funzione dei miofilamenti, piuttosto che Ca(2+) alterato in bicicletta.

Ca2 +-indipendente alterazioni della cinetica di lunghezza e rilassamento diastolico sarcomero in un modello murino di cardiomiopatia diabetica lipotoxic.

&#x4EE5;&#x524D;&#x306E;&#x7814;&#x7A76;&#x5B9F;&#x8A3C;&#x5897;&#x52A0;&#x8102;&#x80AA;&#x9178;&#x53D6;&#x308A;&#x8FBC;&#x307F;&#x3068;&#x8102;&#x80AA;&#x9178;&#x30CE;&#x30B6;&#x30F3; MHC FATP &#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF; &#x30DE;&#x30A6;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B;&#x4EE3;&#x8B1D;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; (FATP) 1 &#x306E;&#x5236;&#x5FA1;&#x4E0B;&#x3067;&#x5FC3;&#x306B;&#x30A2;&#x30EB;&#x30D5;&#x30A1; &#x30DF;&#x30AA;&#x30B7;&#x30F3;&#x91CD;&#x9396; &#xFF08;&#x30A2;&#x30EB;&#x30D5;&#x30A1;-MHC&#xFF09; &#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x306E;&#x8F38;&#x9001;&#x3057;&#x307E;&#x3059;&#x3002;&#x30C9;&#x30C3;&#x30D7;&#x30E9;&#x30FC;&#x7D44;&#x7E54;&#x753B;&#x50CF;&#x53CA;&#x3073;&#x8840;&#x884C;&#x52D5;&#x614B;&#x8A08;&#x6E2C;&#x53CE;&#x7E2E;&#x6A5F;&#x80FD;&#x306E;&#x5909;&#x66F4;&#x304C;&#x306A;&#x3044;&#x5834;&#x5408;&#x3001;&#x62E1;&#x5F35;&#x6A5F;&#x80FD;&#x969C;&#x5BB3;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x3053;&#x3067;&#x306E;&#x5B9F;&#x9A13;&#x306F;&#x76F4;&#x63A5; MHC FATP &#x30DE;&#x30A6;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B;&#x62E1;&#x5F35;&#x6A5F;&#x80FD;&#x969C;&#x5BB3;&#x969C;&#x5BB3;&#x5FC3;&#x7B4B;&#x53CE;&#x7E2E;&#x6A5F;&#x80FD;&#x3092;&#x53CD;&#x6620;&#x3059;&#x308B;&#x3053;&#x3068;&#x4EEE;&#x8AAC;&#x3092;&#x30C6;&#x30B9;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;In vitro &#x3067;&#x306E;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x306E;&#x5B64;&#x7ACB;&#x5FC3;&#x5BA4;&#x7B4B;&#x304C;&#x660E;&#x3089;&#x304B;&#x306B;&#x610F;&#x5473;&#x62E1;&#x5F35;&#x671F;&#x7B4B;&#x7BC0;&#x9577;&#x3092;&#x5927;&#x5E45;&#x306B;&#x306F;&#x30A2;&#x30C0;&#x30EB;&#x30C8; MHC FATP (P &lt; 0.01) &#x91CE;&#x751F;&#x578B; &#xFF08;WT&#xFF09; &#x7D30;&#x80DE; (1.79 &plusmn; 0.01 &#x5BFE; 1.84+/-0.01 microm&#xFF09; &#x3067;&#x3088;&#x308A;&#x77ED;&#x3044;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x5927;&#x5E45;&#x306B;&#x7DE9;&#x548C;&#x7387; (dL/dt) &#x3067;&#x3042;&#x308B; (P &lt; 0.05) &#x4E21;&#x65B9;&#x5C0F;&#x6570;&#x306E;&#x77ED;&#x7E2E;&#x3068;&#x53CE;&#x7E2E;&#x7387;&#x304C;&#x7570;&#x306A;&#x308B;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x306B;&#x5BFE;&#x3057; WT myocytes &#xFF08;1.58&#xFF09; &plusmn; 0.09 1.92+/-0.13 microm/s &#x5BFE;&#x3088;&#x308A; MHC FATP &#x3067;&#x9045;&#x3044;&#x3002;40 &#x30E2;&#x30EB;/L &#x306E; 2, 3-butadionemonoxime &#xFF08;&#x30A2;&#x30AF;&#x30C1;&#x30F3;-&#x30DF;&#x30AA;&#x30B7;&#x30F3;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x7DE9;&#x548C;&#x3001;&#x975E;&#x7279;&#x7570;&#x7684; atp &#x30A2;&#x30FC;&#x30BC;&#x963B;&#x5BB3;) &#x306E;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x62E1;&#x5F35;&#x671F;&#x7B4B;&#x7BC0;&#x9577; WT &#x3068; MHC FATP &#x5FC3;&#x7B4B;&#x7D30;&#x80DE; MHC FATP &#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x304C;&#x5B89;&#x9759;&#x6642;&#x306B;&#x90E8;&#x5206;&#x7684;&#x306B;&#x6D3B;&#x6027;&#x5316;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x304C;&#x540C;&#x3058;&#x9577;&#x3055;&#x3067;&#x5897;&#x52A0;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x7D30;&#x80DE;&#x5185;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x306E;&#x6E2C;&#x5B9A;&#x304C;&#x660E;&#x3089;&#x304B;&#x306B;&#x305D;&#x306E;&#x62E1;&#x5F35;&#x671F;&#x8840;&#x5727;&#x306E;&#x76F4;&#x63A5; [&#x3055;&#x305B;&#x305F; MHC FATP myocytes &#x3067;&#x5909;&#x66F4;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3057;&#x3001;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x9664;&#x53BB;&#x7387;&#x304C;&#x3088;&#x308A; WT myocytes MHC-FATP &#x3067;&#x4E88;&#x671F;&#x305B;&#x305A;&#x306B;&#x9AD8;&#x901F;&#x3067;&#x3059;&#x3002;&#x307E;&#x305F;&#x3001;&#x62E1;&#x5F35;&#x671F;&#x8840;&#x5727;&#x306E;&#x7B4B;&#x7BC0;&#x9577; MHC FATP &#x3068; WT &#x306E;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x3067;&#x306F;&#x7D30;&#x80DE;&#x5916;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x9664;&#x53BB;&#x306B;&#x3088;&#x308B;&#x307E;&#x305F;&#x306F;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0; &#x30AD;&#x30EC;&#x30FC;&#x30C8;&#x5264;&#x3068;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D30;&#x80DE;&#x5185;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x306E;&#x30D0;&#x30C3;&#x30D5;&#x30A1;&#x30EA;&#x30F3;&#x30B0;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x308B;&#x3067;&#x3042;&#x3063;&#x305F; &#xFF08;100 micromol/L&#xFF09;&#x3001;MHC FATP &#x5FC3;&#x5BA4;&#x7B4B;&#x306E;&#x53CE;&#x7E2E;&#x80FD;&#x969C;&#x5BB3;&#x8005;&#x9AD8;&#x67B6;&#x306E;&#x7D30;&#x80DE;&#x5185;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x306A;&#x3044;&#x6839;&#x5E95;&#x306B;&#x3042;&#x308B;&#x3057;&#x306A;&#x3044;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x76AE;&#x3092;&#x5265;&#x304C;&#x308C;&#x305F; myocytes &#x306E;&#x6A5F;&#x80FD;&#x8A55;&#x4FA1;&#x306F;&#x305F;&#x3060;&#x3057;&#x3001;&#x305D;&#x306E;&#x30D5;&#x30A3;&#x30E9;&#x30E1;&#x30F3;&#x30C8; &#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x611F;&#x53D7;&#x6027; MHC-FATP WT &#x3067;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x8457;&#x3057;&#x304F;&#x5897;&#x52A0;&#x3067;&#x3059;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x307E;&#x305F;&#x3001;&#x751F;&#x5316;&#x5B66;&#x5B9F;&#x9A13;&#x3067; MHC - WT &#x5FC3;&#x5BA4;&#x3068;&#x6BD4;&#x8F03;&#x53EF;&#x80FD;&#x6027;&#x304C; MHC FATP myocytes &#x3067;&#x89B3;&#x6E2C;&#x3055;&#x308C;&#x305F;&#x9045;&#x3044;&#x7DE9;&#x548C;&#x7387;&#x306B;&#x8CA2;&#x732E;&#x3059;&#x308B; FATP&#x3001;&#x30D9;&#x30FC;&#x30BF; MHC &#x30A2;&#x30A4;&#x30BD; &#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x306E;&#x767A;&#x73FE;&#x306E;&#x5897;&#x52A0;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x7DCF;&#x79F0;&#x3057;&#x3066;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x7CD6;&#x5C3F;&#x75C5;&#x6027;&#x306E;&#x4E2D;&#x5FC3;&#x306B;&#x89B3;&#x5BDF;&#x3068;&#x4F3C;&#x3066;&#x3044;&#x307E;&#x3059;, MHC FATP &#x5FC3;&#x5BA4;&#x306B;&#x304A;&#x3051;&#x308B;&#x8102;&#x8CEA;&#x4EE3;&#x8B1D;&#x306E;&#x304B;&#x304F;&#x4E71;&#x4E3B;&#x306B;&#x7DAD;&#x6A5F;&#x80FD;&#x306E;&#x5909;&#x66F4;&#x3092;&#x53CD;&#x6620;&#x62E1;&#x5F35;&#x671F;&#x8840;&#x5727;&#x306E;&#x6A5F;&#x80FD;&#x969C;&#x5BB3;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3059;&#x5909;&#x66F4;&#x3055;&#x308C;&#x305F;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x3092;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x3067;&#x306F;&#x306A;&#x304F;&#x3002;

Ca 2 +-&#x72EC;&#x7ACB;&#x5909;&#x5316;&#x306B;&#x304A;&#x3051;&#x308B;&#x62E1;&#x5F35;&#x671F;&#x7B4B;&#x7BC0;&#x306E;&#x9577;&#x3055;&#x3068;&#x30EA;&#x30E9;&#x30AF;&#x30BC;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306E;&#x52D5;&#x614B; lipotoxic &#x7CD6;&#x5C3F;&#x75C5;&#x6027;&#x5FC3;&#x7B4B;&#x75C7;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x30E2;&#x30C7;&#x30EB;&#x3067;&#x3002;

&#xC774;&#xC804; &#xC5F0;&#xAD6C; &#xAC80;&#xC99D; &#xB41C; &#xD5A5;&#xC0C1; &#xB41C; &#xC9C0;&#xBC29; &#xC0B0; &#xC131; &#xD1B5;&#xD48D; &#xAD00; &#xBC0F; &#xC9C0;&#xBC29;&#xC0B0; overexpress FATP MHC &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC0DD;&#xC950;&#xC5D0;&#xC11C; &#xB300;&#xC0AC; &#xC54C;&#xD30C; myosin &#xBB34;&#xAC70;&#xC6B4; &#xCCB4;&#xC778; (&#xC54C;&#xD30C;-MHC) &#xBC1C;&#xAE30;&#xC778;&#xC758; &#xB2E8;&#xBC31;&#xC9C8; (FATP) 1 &#xD1B5;&#xC81C; &#xD558;&#xC5D0; &#xC804;&#xC1A1; &#xD569;&#xB2C8;&#xB2E4;. &#xB3C4;&#xD50C;&#xB7EC; &#xC870;&#xC9C1;&#xC758; &#xC774;&#xBBF8;&#xC9D5; &#xBC0F; hemodynamic &#xCE21;&#xC815; &#xD655;&#xC7A5;&#xAE30; &#xBD80;&#xC804;&#xC744;, &#xC2EC;&#xC7A5; &#xC218;&#xCD95; &#xAE30;&#xB2A5;&#xC5D0; &#xBCC0;&#xD654; &#xC5C6;&#xC774; &#xACF5;&#xAC1C; &#xD588;&#xB2E4;. &#xC5EC;&#xAE30; &#xC2E4;&#xD5D8; &#xC9C1;&#xC811; MHC FATP &#xC0DD;&#xC950;&#xC5D0;&#xC11C; &#xD655;&#xC7A5;&#xAE30; &#xBD80;&#xC804; &#xC7A5;&#xC560; &#xC2EC; &#xC2E4; myocyte &#xC218;&#xCD95; &#xAE30;&#xB2A5;&#xC744; &#xBC18;&#xC601; &#xD558;&#xB294; &#xAC00;&#xC124;&#xC744; &#xD14C;&#xC2A4;&#xD2B8; &#xD569;&#xB2C8;&#xB2E4;. &#xC0DD;&#xCCB4; &#xC678;&#xC5D0;&#xC11C; &#xC774;&#xBBF8;&#xC9D5; &#xC808;&#xC5F0; &#xC131;&#xC778; MHC FATP &#xC2EC; &#xC2E4; myocytes &#xD3C9;&#xADE0; &#xD655;&#xC7A5;&#xAE30; sarcomere &#xAE38;&#xC774; &#xD06C;&#xAC8C; &#xACF5;&#xAC1C; (P &lt; 0.01) &#xC57C;&#xC0DD;-&#xD0C0;&#xC785; (WT) &#xC140; (1.84+/-0.01 microm &#xB300; 0.01 &plusmn; 1.79) &#xBCF4;&#xB2E4; &#xC9E7;&#xC740;. &#xB610;&#xD55C;, &#xC774;&#xC644; &#xC18D;&#xB3C4; (dL/dt)&#xB294; &#xD06C;&#xAC8C; (P &lt; 0.05) MHC FATP&#xC5D0;&#xC11C; &#xBCF4;&#xB2E4; &#xB290;&#xB9B0; WT myocytes (1.92+/-0.13 microm/s &#xB300; 0.09 + 1.58) &#xBAA8;&#xB450; &#xC18C;&#xC218; &#xB2E8;&#xCD95; &#xBC0F; &#xC218;&#xCD95; &#xC18D;&#xB3C4; &#xB2E4;&#xB978;. 40 M m o l/L 2, 3 butadionemonoxime (&#xB294; &#xC77C;&#xBC18; ATPase &#xC5B5;&#xC81C;&#xC81C; &#xB9D0;&#xB77C; myosin &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC744; &#xC644;&#xD654; &#xD558;&#xB294;)&#xC758; &#xC751;&#xC6A9; MHC FATP myocytes &#xB098;&#xBA38;&#xC9C0;&#xC5D0;&#xC11C; &#xBD80;&#xBD84;&#xC801;&#xC73C;&#xB85C; &#xD65C;&#xC131;&#xD654;&#xB294; &#xC81C;&#xC548; &#xAC19;&#xC740; &#xAE38;&#xC774; WT&#xC640; MHC FATP myocytes&#xC5D0;&#xC11C; &#xD655;&#xC7A5;&#xAE30; sarcomere &#xAE38;&#xC774; &#xC99D;&#xAC00;. &#xCE21;&#xC815; &#xC138;&#xD3EC;&#xB0B4; ca(2+)&#xC758; &#xACF5;&#xAC1C;&#xB294; &#xD655;&#xC7A5;&#xAE30; &#xC9C1;&#xC811; [Ca(2+)](i) MHC FATP myocytes&#xC5D0;&#xC11C; &#xBCC0;&#xACBD; &#xBC0F; &#xCE7C;&#xC298; &#xC81C;&#xAC70; &#xC18D;&#xB3C4;&#xAC00; &#xC608;&#xAE30;&#xCE58; &#xC54A;&#xAC8C; MHC FATP&#xC758; WT myocytes &#xBCF4;&#xB2E4; &#xB354; &#xBE60;&#xB978;. &#xB610;&#xD55C;, MHC FATP&#xC640; WT myocytes&#xC5D0;&#xC11C; &#xD655;&#xC7A5;&#xAE30; sarcomere &#xAE38;&#xC774; &#xC138;&#xD3EC; &#xC678; ca(2+)&#xC758; &#xC81C;&#xAC70;&#xC5D0; &#xC758;&#xD574; &#xB610;&#xB294; Ca(2+) chelator BAPTA&#xC640; &#xC138;&#xD3EC;&#xB0B4; ca(2+)&#xC758; &#xBC84;&#xD37C;&#xB9C1;&#xC5D0; &#xC758;&#xD574; &#xC601;&#xD5A5;&#xC744; &#xBC1B;&#xB294; (100 micromol/L), MHC FATP &#xC2EC; &#xC2E4; myocytes &#xD655;&#xC7A5;&#xAE30; &#xAE30;&#xB2A5; &#xC7A5;&#xC560;&#xAC00; &#xB098;&#xD0C0;&#xB0B4;&#xB294; &#xB192;&#xC740; &#xC138;&#xD3EC;&#xB0B4; Ca(2+) &#xAE30;&#xCD08; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098; &#xD53C;&#xBD80; myocytes&#xC758; &#xAE30;&#xB2A5; &#xD3C9;&#xAC00; &#xADF8; myofilament Ca(2+) &#xAC10;&#xB3C4; MHC-FATP, WT, &#xC2EC; &#xC2E4; &#xC138;&#xD3EC;&#xC5D0; &#xBE44;&#xD574; &#xD604;&#xC800; &#xD558; &#xAC8C; &#xC99D;&#xAC00; &#xBC1D;&#xD614;&#xB2E4;. &#xB610;&#xD55C;, &#xC0DD;&#xD654;&#xD559; &#xC2E4;&#xD5D8; &#xBCA0;&#xD0C0; MHC isoform MHC-FATP, WT &#xC2EC; &#xC2E4;&#xC5D0; &#xBE44;&#xD574; MHC FATP myocytes&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB41C; &#xB290;&#xB9B0; &#xC774;&#xC644; &#xC18D;&#xB3C4;&#xC5D0; &#xAE30;&#xC5EC; &#xAC00;&#xB2A5;&#xC131;&#xC5D0; &#xC99D;&#xAC00; &#xC2DD; &#xC2DC;&#xC5F0;. &#xC0E8; &#xB2E4;, &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; &#xBCF4;&#xC5EC; &#xC8FC;&#xB85C; myofilament &#xAE30;&#xB2A5;&#xC5D0; &#xBCC0;&#xD654;&#xB97C; &#xBC18;&#xC601; &#xD558;&#xB294; &#xC7A5;&#xC560;&#xAC00; &#xC2EC;&#xC7A5; &#xD655;&#xC7A5; &#xAE30;&#xB2A5; &#xADC0;&#xCC29;&#xB418;&#xB294; &#xB2F9;&#xB1E8;&#xBCD1; &#xC2EC; &#xD63C;&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB41C; &#xAC83;&#xACFC; &#xC720;&#xC0AC;&#xD55C;, MHC FATP &#xC2EC;&#xC5D0;&#xC11C; &#xC9C0;&#xC9C8; &#xB300;&#xC0AC;&#xC5D0; &#xD63C;&#xB780; &#xBCF4;&#xB2E4;&#xB294; &#xBCC0;&#xACBD; &#xB41C; Ca(2+) &#xC0AC;&#xC774;&#xD074;&#xB9C1;.

Ca2 +-lipotoxic &#xB2F9;&#xB1E8;&#xBCD1; &#xC2EC;&#xC7A5; &#xADFC;&#xC721; &#xBCD1; &#xC99D;&#xC758; &#xB9C8;&#xC6B0;&#xC2A4; &#xBAA8;&#xB378;&#xC758; &#xD655;&#xC7A5;&#xAE30; sarcomere &#xAE38;&#xC774; &#xD734;&#xC2DD; &#xC18D;&#xB3C4; &#xB860;&#xC73C;&#xB85C; &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xBCC0;&#xACBD;.

Anteriores estudios demostrada del &aacute;cido graso mayor absorci&oacute;n y metabolismo en ratones transg&eacute;nicos MHC-FATP que sobreexpresan &aacute;cidos grasos transportan prote&iacute;na (FATP) 1, en el coraz&oacute;n bajo el control del promotor alfa-miosina de cadena pesada (alfa-MHC). Mediciones hemodin&aacute;micas e im&aacute;genes Doppler tejido revelaron disfunci&oacute;n diast&oacute;lica, en ausencia de cambios en la funci&oacute;n sist&oacute;lica. Los experimentos aqu&iacute; directamente probar la hip&oacute;tesis de que la disfunci&oacute;n diast&oacute;lica en ratones MHC-FATP refleja la funci&oacute;n contr&aacute;ctil del miocito ventricular deteriorada. Proyecci&oacute;n de imagen in vitro del aislado adultos MHC-FATP miocitos ventriculares revelaron que la longitud del sarc&oacute;mero diast&oacute;lica media es significativamente (P &lt; 0.01) m&aacute;s corto que en las c&eacute;lulas wild-type (WT) (1.79 +/-0.01 versus 1.84+/-0.01 microm). Adem&aacute;s, la tasa de relajaci&oacute;n (dL/dt) es significativamente (P &lt; 0.05) m&aacute;s lento en MHC-FATP que WT miocitos (1,58 + /-0.09 versus 1.92+/-0.13 microm/s), Considerando que ambas tasas fraccionales de acortamiento y contracci&oacute;n no son diferentes. Aplicaci&oacute;n de 40 mmol/L 2, 3-butadionemonoxime (un inespec&iacute;fico ATPasa inhibidor que relaja la interacci&oacute;n actina-miosina) aument&oacute; la longitud del sarc&oacute;mero diast&oacute;lica en miocitos WT tanto MHC-FATP a la misma longitud, lo que sugiere que los miocitos MHC-FATP parcialmente se activan al resto. Directa mediciones de CA intracelular revelaron que diast&oacute;lica [Ca(2+)](i) es sin cambios en miocitos MHC-FATP y la tasa de eliminaci&oacute;n de calcio es inesperadamente m&aacute;s en MHC-FATP WT miocitos. Por otra parte, era inafectada por el retiro de CA extracelular o de almacenamiento en b&uacute;fer de CA intracelular con el quelante de CA BAPTA longitud del sarc&oacute;mero diast&oacute;lica en miocitos MHC-FATP y WT (100 micromol/L), indicando que no subyacen a elevada CA intracelular deteriorada la funci&oacute;n diast&oacute;lica en miocitos ventriculares MHC-FATP. Evaluaci&oacute;n funcional de miocitos sin piel, sin embargo, revel&oacute; que Miofilamento CA sensibilidad se incrementa notablemente en MHC-FATP, en comparaci&oacute;n con el peso, las c&eacute;lulas ventriculares. Adem&aacute;s, experimentos bioqu&iacute;micos demostraron mayor expresi&oacute;n de la isoforma beta-MHC en MHC-FATP, de comparado con ventr&iacute;culos WT, que probablemente contribuye a la relajaci&oacute;n m&aacute;s lenta en miocitos MHC-FATP. Colectivamente, estos datos demuestran que atentamente en el metabolismo lip&iacute;dico en ventr&iacute;culos MHC-FATP, que son similares a los observados en el coraz&oacute;n diab&eacute;tico, causar disfunci&oacute;n diast&oacute;lica que refleja principalmente los cambios en funci&oacute;n de los miofilamentos, en lugar de ciclismo CA alterada.

Ca2 +-independientes alteraciones en la cin&eacute;tica de longitud y relajaci&oacute;n diast&oacute;lica sarc&oacute;mero en un modelo murino de miocardiopat&iacute;a diab&eacute;tica lipotoxic.

Inhibition of the human ether-a-go-go-related gene (hERG) potassium channel by pharmaceutical agents can lead to acquired long QT syndrome and the generation of potentially lethal arrhythmias. Higher throughput automated patch clamp systems, such as PatchXpress, can greatly increase the speed and capacity of evaluation of pharmaceutical compounds for hERG blocking activity. A factor that may affect the IC(50) value of a compound measured in this system is the composition of the multi-well compound plate. Hydrophobic compounds may adsorb to the surfaces of multi-well plates resulting in a reduction in the effective concentration of the compound delivered to the cell and altered IC(50) values. In the present study, we investigated the effects of four different compound plates--glass vials, non-binding polystyrene, hydrophilic polystyrene, and polystyrene--on determination of IC(50)s for four compounds--sotalol, dofetilide, cisapride, and bepridil--which ranged in hydrophobicity. In addition, we investigated the effects of incubation time in the compound plate on determination of IC(50)s. hERG currents were measured using the PatchXpress 7000A Automated Parallel Patch Clamp System (Molecular Devices Corporation; Sunnyvale, CA) and hERG channels stably expressed in HEK293 cells. The results suggest that more hydrophobic compounds may adsorb to non-binding polystyrene, hydrophilic, and polystyrene compound plates versus glass plates, especially with increasing time on the plates, resulting in altered IC(50) values.

Effect of compound plate composition on measurement of hERG current IC(50) using PatchXpress.

There is considerable evidence to support a role for lipotoxicity in the development of diabetic cardiomyopathy, although the molecular links between enhanced saturated fatty acid uptake/metabolism and impaired cardiac function are poorly understood. In the present study, the effects of acute exposure to the saturated fatty acid, palmitate, on myocardial contractility and excitability were examined directly. Exposure of isolated (adult mouse) ventricular myocytes to palmitate, complexed to bovine serum albumin (palmitate:BSA) as in blood, rapidly reduced (by 54+/-4%) mean (+/-SEM) unloaded fractional cell shortening. The amplitudes of intracellular Ca(2+) transients decreased in parallel. Current-clamp recordings revealed that exposure to palmitate:BSA markedly shortened action potential durations at 20%, 50%, and 90% repolarization. These effects were reversible and were occluded when the K(+) in the recording pipettes was replaced with Cs(+), suggesting a direct effect on repolarizing K(+) currents. Indeed, voltage-clamp recordings revealed that palmitate:BSA reversibly and selectively increased peak outward voltage-gated K(+) (Kv) current amplitudes by 20+/-2%, whereas inwardly rectifying K(+) (Kir) currents and voltage-gated Ca(2+) currents were unaffected. Further analyses revealed that the individual Kv current components I(to,f), I(K,slow) and I(ss), were all increased (by 12+/-2%, 37+/-4%, and 34+/-4%, respectively) in cells exposed to palmitate:BSA. Consistent with effects on both components of I(K,slow) (I(K,slow1) and I(K,slow)(2)) the magnitude of the palmitate-induced increase was attenuated in ventricular myocytes isolated from animals in which the Kv1.5 (I(K,slow)(1)) or the Kv2.1 (I(K,slow)(2)) locus was disrupted and I(K,slow)(1) or I(K,slow2) is eliminated. Both the enhancement of I(K,slow) and the negative inotropic effect of palmitate:BSA were reduced in the presence of the Kv1.5 selective channel blocker, diphenyl phosphine oxide-1 (DPO-1).Taken together, these results suggest that elevations in circulating saturated free fatty acids, as occurs in diabetes, can directly augment repolarizing myocardial Kv currents and impair excitation-contraction coupling.

Palmitate attenuates myocardial contractility through augmentation of repolarizing Kv currents.

We describe the systematic optimization, focused on the improvement of CV-TI, of a series of CCR2 antagonists. This work resulted in the identification of 10 (((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone) which possessed a low projected human dose 35-45mg BID and a CV-TI=3800-fold.

Discovery of ((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone, PF-4254196, a CCR2 antagonist with an improved cardiovascular profile.

A mini-microscope was developed for in situ monitoring of cells by modifying off-the-shelf components of a commercial webcam. The mini-microscope consists of a CMOS imaging module, a small plastic lens and a white LED illumination source. The CMOS imaging module was connected to a laptop computer through a USB port for image acquisition and analysis. Due to its compact size, 8 Ã— 10 Ã— 9 cm, the present microscope is portable and can easily fit inside a conventional incubator, and enables real-time monitoring of cellular behaviour. Moreover, the mini-microscope can be used for imaging cells in conventional cell culture flasks, such as Petri dishes and multi-well plates. To demonstrate the operation of the mini-microscope, we monitored the cellular migration of mouse 3T3 fibroblasts in a scratch assay in medium containing three different concentrations of fetal bovine serum (5, 10, and 20%) and demonstrated differential responses depending on serum levels. In addition, we seeded embryonic stem cells inside poly(ethylene glycol) microwells and monitored the formation of stem cell aggregates in real time using the mini-microscope. Furthermore, we also combined a lab-on-a-chip microfluidic device for microdroplet generation and analysis with the mini-microscope and observed the formation of droplets under different flow conditions. Given its cost effectiveness, robust imaging and portability, the presented platform may be useful for a range of applications for real-time cellular imaging using lab-on-a-chip devices at low cost.

A mini-microscope for in situ monitoring of cells.

Polydimethylsiloxane (PDMS) has numerous desirable properties for fabricating microfluidic devices, including optical transparency, flexibility, biocompatibility, and fabrication by casting; however, partitioning of small hydrophobic molecules into the bulk of PDMS hinders industrial acceptance of PDMS microfluidic devices for chemical processing and drug development applications. Here we describe an attractive alternative material that is similar to PDMS in terms of optical transparency, flexibility and castability, but that is also resistant to absorption of small hydrophobic molecules.

Clear castable polyurethane elastomer for fabrication of microfluidic devices.

'Organs-on-chips' are microengineered biomimetic systems containing microfluidic channels lined by living human cells, which replicate key functional units of living organs to reconstitute integrated human organ-level pathophysiology in vitro. These microdevices can be used to test efficacy and toxicity of drugs and chemicals, and to create in vitro models of human disease. Thus, they potentially represent low-cost alternatives to conventional animal models for pharmaceutical, chemical and environmental applications. Here we describe a protocol for the fabrication, microengineering and operation of these microfluidic organ-on-chip systems. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin porous flexible membrane, along with two full-height, hollow vacuum chambers on either side; this requires ∼3.5 d to complete. To create a 'breathing' lung-on-a-chip that mimics the mechanically active alveolar-capillary interface of the living human lung, human alveolar epithelial cells and microvascular endothelial cells are cultured in the microdevice with physiological flow and cyclic suction applied to the side chambers to reproduce rhythmic breathing movements. We describe how this protocol can be easily adapted to develop other human organ chips, such as a gut-on-a-chip lined by human intestinal epithelial cells that experiences peristalsis-like motions and trickling fluid flow. Also, we discuss experimental techniques that can be used to analyze the cells in these organ-on-chip devices.

Microfabrication of human organs-on-chips.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Improving the effectiveness of preclinical predictions of human drug responses is critical to reducing costly failures in clinical trials. Recent advances in cell biology, microfabrication and microfluidics have enabled the development of microengineered models of the functional units of human organs - known as organs-on-chips - that could provide the basis for preclinical assays with greater predictive power. Here, we examine the new opportunities for the application of organ-on-chip technologies in a range of areas in preclinical drug discovery, such as target identification and validation, target-based screening, and phenotypic screening. We also discuss emerging drug discovery opportunities enabled by organs-on-chips, as well as important challenges in realizing the full potential of this technology.

Organs-on-chips at the frontiers of drug discovery.

Here we describe the development of a human lung 'small airway-on-a-chip' containing a differentiated, mucociliary bronchiolar epithelium and an underlying microvascular endothelium that experiences fluid flow, which allows for analysis of organ-level lung pathophysiology in vitro. Exposure of the epithelium to interleukin-13 (IL-13) reconstituted the goblet cell hyperplasia, cytokine hypersecretion and decreased ciliary function of asthmatics. Small airway chips lined with epithelial cells from individuals with chronic obstructive pulmonary disease recapitulated features of the disease such as selective cytokine hypersecretion, increased neutrophil recruitment and clinical exacerbation by exposure to viral and bacterial infections. With this robust in vitro method for modeling human lung inflammatory disorders, it is possible to detect synergistic effects of lung endothelium and epithelium on cytokine secretion, identify new biomarkers of disease exacerbation and measure responses to anti-inflammatory compounds that inhibit cytokine-induced recruitment of circulating neutrophils under flow.

Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro.

Studies on hematopoiesis currently rely on animal models because in vitro culture methods do not accurately recapitulate complex bone marrow physiology. We recently described a bone marrow-on-a-chip microfluidic device that enables the culture of living hematopoietic bone marrow and mimics radiation toxicity in vitro. In the present study, we used this microdevice to demonstrate continuous blood cell production in vitro and model bone marrow responses to potential radiation countermeasure drugs. The device maintained mouse hematopoietic stem and progenitor cells in normal proportions for at least 2 weeks in culture. Increases in the number of leukocytes and red blood cells into the microfluidic circulation also could be detected over time, and addition of erythropoietin induced a significant increase in erythrocyte production. Exposure of the bone marrow chip to gamma radiation resulted in reduction of leukocyte production, and treatment of the chips with two potential therapeutics, granulocyte-colony stimulating factor or bactericidal/permeability-increasing protein (BPI), induced significant increases in the number of hematopoietic stem cells and myeloid cells in the fluidic outflow. In contrast, BPI was not found to have any effect when analyzed using static marrow cultures, even though it has been previously shown to accelerate recovery from radiation-induced toxicity in vivo. These findings demonstrate the potential value of the bone marrow-on-a-chip for modeling blood cell production, monitoring responses to hematopoiesis-modulating drugs, and testing radiation countermeasures in vitro.

Modeling Hematopoiesis and Responses to Radiation Countermeasures in a Bone Marrow-on-a-Chip.

The recent advent of microphysiological systems - microfluidic biomimetic devices that aspire to emulate the biology of human tissues, organs and circulation in vitro - is envisaged to enable a global paradigm shift in drug development. An extraordinary US governmental initiative and various dedicated research programs in Europe and Asia have led recently to the first cutting-edge achievements of human single-organ and multi-organ engineering based on microphysiological systems. The expectation is that test systems established on this basis would model various disease stages, and predict toxicity, immunogenicity, ADME profiles and treatment efficacy prior to clinical testing. Consequently, this technology could significantly affect the way drug substances are developed in the future. Furthermore, microphysiological system-based assays may revolutionize our current global programs of prioritization of hazard characterization for any new substances to be used, for example, in agriculture, food, ecosystems or cosmetics, thus, replacing laboratory animal models used currently. Thirty-six experts from academia, industry and regulatory bodies present here the results of an intensive workshop (held in June 2015, Berlin, Germany). They review the status quo of microphysiological systems available today against industry needs, and assess the broad variety of approaches with fit-for-purpose potential in the drug development cycle. Feasible technical solutions to reach the next levels of human biology in vitro are proposed. Furthermore, key organ-on-a-chip case studies, as well as various national and international programs are highlighted. Finally, a roadmap into the future is outlined, to allow for more predictive and regulatory-accepted substance testing on a global scale.

Anthony Bahinski

Wyss Institute for Biologically Inspired Engineering at Harvard University

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device