This work received support through Deutsche Forschungsgemeinschaft SFB 1002 (subprojects A05 and B05 to S.E.L.) and KFO 155 (subproject 4 to S.E.L.), a Halbach Foundation award to S.E.L. supporting E.W.; a grant from the German Cardiac Society to S.B.; and a DAAD exchange program supporting T.K. as visitor at the University of Maryland. The research leading to these results has received funding from the European Community&#8217;s Seventh Framework Program FP7/2007-2013 under grant agreement No. HEALTH-F2-2009-241526, EUTrigTreat (to S.E.L.). S.E.L. is a principal investigator of the German Center of Cardiovascular Research (DZHK).

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The authors declare that no conflict of interest exists.

בתאי שריר משורטטים בריאים, מבני קרום צינורי עם אוריינטציות &quot;רוחביים&quot; (T-tubules) בניצב לציר התא העיקרי הם בשפע. כתוצאה מכך, T-tubules מתאפיינים כהרחבות מתמשכת של תאי השריר &quot;לרוחב&quot; עיקרי קרום פני השטח (sarcolemma), אשר עמוק לחדור cytosol לכיוון מרכז התא. התפקיד הפיזיולוגי של T-tubules רציף עם קרום פני השטח החיצוני הוא הצימוד החשמלי המהיר של תאים תאיים מרוחקים שהוקמו על ידי תחומים מגע אברון SER לאורך גדול יחסית לב תא שריר על ידי צימוד קרבה ננומטריים של L-סוג מופעל מתח Ca נפח 2 + ערוצים (Cav1.2) פנימה הנוכחי (אני Ca) הפעלת קולטן ryanodine הסמוך משחרר (RyR2) SER Ca 2 +. בmyocytes חדרית (VM), אנשי הקשר לא רציף קרום (&quot;צמתים&quot;) בין תחומים SER junctional וT-tubules הם חשבו לשלוט אלפי nanodomains Ca 2 + שחרור תאיים הבודד בכל תא 1. לכל תחום קשר נתון, חלקי קרום זה לצד זה כל אחד מT-אבובית וSER ההיקפי (junctional) הם כ 15 ננומטר קרוב זה לזה, ומכאן שהוגדר כnanodomain. וכך, subspaces cytoplasmic פרט הקטן ביותר הפרדה המאפשרת התנהגויות תא תאים אוטונומיים למחצה. כאשר פוטנציאל פעולה נכנסת מפעיל ערוצי Cav1.2 בT-tubules של מכונות וירטואליות, Ca קטן יחסית 2 + פנימה נוכחי יהיה במהירות להגדיל את ריכוז subspace Ca 2 + S [Ca 2 +] בnanodomain בגודל attoliter 1. בשלב בא, העלייה [Ca 2 +] S מפעילה Ca 2 + קולטנים ryanodine -gated (RyR2) בתוך קרבת ננומטר בצומת קרום SER זה לצד זה, ותהליך צימוד זה מתרחש לאורך כל הזוג חשמליnanodomains myocyte ד. RyR2s להתרחש אשכולות מרובים ערוצים צפופים כמו עם stoichiometry משוער של ערוץ Cav1.2 1 ל5-10 ערוצי RyR2 2. מאז SER-to-cytosol [Ca 2 +] שיפוע תלול מאוד (יחס 10 4: 1) וRyR2s לתפקד כגבוהות מוליכות Ca 2 + ערוצי שחרור באשכולות בשילוב פונקציונלי, תוצאות הפעלת RyR2 בCa גדול כמותית 2 + לשחרר נוכחי מתחומים T-אבובית יחד junctional SER הגדלת subspace המקומי S [Ca 2 +] ל100 מיקרומטר או גבוהים יותר בטווח של 1-2 3,4 האלפיות השני. זה התנהגות הגברה אות לב גם נקראת Ca 2 + מושרה שחרור Ca 2 + (CICR). יחדיו, T-tubules הם מבני קרום חיוניים שבמהירות להפעיל אותות שחרור Ca 2 + באמצעות אנשי קשר SER nanodomain junctional וCICR תא רחב במהלך עירור והתכווצות צימוד (EC). בנוסף לT-tubules, אבובית הציריתשל (A-tubules) עם מקביל אורינטציה שונה באופן משמעותי לציר התא הראשי (אורך) תועד על ידי מיקרוסקופי אלקטרונים (EM), confocal ומחקרי מיקרוסקופיה 2 פוטונים. למשל, סריג רציף תא רחב של-tubules בין myofibrils מחובר עם T-tubules ליד Z-קווי sarcomere הוצג על ידי קליעים נותבים תאיים וההדמיה EM פן ניסיונות קבועים מכונות וירטואליות 5 של. באמצעות מכתים והעמסת צמוד dextran תאי ולחיות הדמיה 2 פוטונים של עכברוש מכונות וירטואליות, רשת אבובית 3D רשתי מורכבת הייתה דמיינה בהיקף של ~ 60% T-tubules ו~-tubules 40% 6. מחקר זה לא רק הוביל להדמיית 3D של-tubules בשפע, אלא גם להבנה שחתכה להדמיה EM מוגבל מטבעו לניתוח של רשתות קרום מורכבות ודינמיות כמו המערכת רוחבית צירית אבובית (מידות). כתוצאה מכך, הדמיה תא חי confocal של קרומי מידות מוכתמות ישירות עם di-8-ANNEPS פותח. אם תא חירשתות מידות מנותחות שינוי פורייה, את המראה הרגיל של רכיבי T-אבובית בחלל קרוב Z-קווי sarcomere בא לידי הביטוי על ידי כוח ספקטרום ההרכב מאזור של אותות משורטטים 7. אסטרטגית ניתוח עקיפה זה נעשתה שימוש כדי לזהות שינויים אזוריים תא רחב בסדירות מרכיב מידות במודלים של מחלות 7. לדוגמא, בתיווך shRNA junctophilin-2 עקומות למטה נגרמו אי ספיקת לב והמחסור בחלבון המסוים איזופורם הביאו ארגון מחדש T-אבובית עם nanodomain Ca תפקוד לקוי של שחרור 2 + 8. לאחרונה הרחיבו את הניתוח של רשתות קרום מידות באמצעות גישות כמותיות ישירות ועוד יותר על ידי מיקרוסקופ superresolution התא החי של רכיבי מידות בודדים בעכבר המכונות הווירטואליות באמצעות דלדול פליטה מאולץ (STED) nanoscopy 9. רזולוציית תמונה ננומטריים אפשרה לניתוח ישיר של רכיבי מידות פרט קטנים יותר, אשר משוער הפצת 50:50 של רוחבילעומת אוריינטציות אבובית ציריות, כמותית המאשר שני מרכיבים בשפע עדיין בכיוון באופן דיפרנציאלי בודדים מידות בלבבות עכבר בריאים 9. אסטרטגיות אלה יהיו מתוארים נוספים בסעיף הפרוטוקול להלן. בעוד התפקיד הפיזיולוגי של הרכיבים-אבובית השופעים בלב המבוגר נשאר מסתורי, מחקרי EM תעדו מבני קרום SER הקשורים-tubules מציע Ca אנדוגני 2 + nanodomains שחרור בפן ניסיונות ועכברוש מכונות וירטואליות 5,10. ניתוח Confocal של Cav1.2 וRyR2 מצא רמה גבוהה של colocalization בצמתים-אבובית 10. מאז ~ 20% מ2 + ניצוצות Ca ספונטניים בחולדת מכונות וירטואליות מקורו יחסית רחוקות מפסי Z-קו שבו T-tubules מתרחש בדרך כלל, טיעון אחד היה שnanodomains-אבובית קשורה אכן קיימת ומתפקד כ2 + אתרי שחרור Ca 11,12. מעניין, היווצרות T-אבובית וoc ההתבגרותבני כלאיים רק לאחר לידה, ומקביל לצמיחה של תאי לב, למשל, דרך נביטת invaginations sarcolemmal המבשר בP5 ולא בשלה מסועפים מכלולי רשת המידות בP10 בעכברים 13. נראה כי Junctophilin-2 הוא חשוב במיוחד עבור התבגרות רשת מידות לאחר לידה מאז shRNA לדפוק למטה מנע העיגון של קרומי T-אבובית לצומת SER מוביל לשחרור מושהה Ca 2 + וארגון מידות פתולוגיים בקנה אחד עם ארכיטקטורות-אבובית לא בשלה שלטו ב 13 מכונות וירטואליות. תצפיות אלה עשויות סופו של דבר להוביל להוכחה של הקונספט שT-tubules ליצור באמצעות תהליכי invagination הקרום ואילו-tubules עשוי מורף באמצעות מנגנונים תאיים נוספים או אפילו חלופיים 14. אפיון של מידות שינויי קרום במחלות לב הפך אזור מחקר חשוב לשאלות pathophysiological. דיווחים ראשוניים במודל כלב של failu לב הנגרם על ידי קצבמחדש הראה הפסד של T-tubules ונוכחי Cav1.2 (אני Ca) 15. מודל חזיר של קרדיומיופתיה איסכמית הראה מופחת צפיפות T-אבובית ותיאום מופחת של Ca 2 + תאיים לשחרר 16. תוך שימוש במודל החולדה באופן ספונטני עם יתר לחץ דם (SHR) של אי ספיקת לב, אובדן של T-tubules היה קשור בצימוד nanodomain מופחת של Cav1.2 וRyR2 על ידי המנגנון המוצע של &quot;RyR2 orphaning&quot; 7. הפסד של T-tubules גם הוכח במכונות וירטואליות אנושיות מדגימות קרדיומיופתיה איסכמית, מורחבות, וhypertrophic 17. יתר על כן, עלייה ב- tubules דווחה בסעיפי רקמות של קרדיומיופתיה מורחבת האנושית 18. בעקבות אוטם שריר לב, שהראינו מנגנון ההפרש של ארגון מחדש מידות בעכבר מכונות וירטואליות עם ירידה משמעותית של T-tubules בניגוד לעליות של רכיבים-אבובית 9. חשוב לציין, לעומת זאת קרום מקומי השתפרו מושגים באמצעות superre תא החימיקרוסקופיה STED פתרון אפשרה ניתוח מרכיבים כמותיים מפורט מדידות ישירות, אשר הראו ריבוי משמעותי של-tubules עם עליות הכוללות של אורך רשת מידות ומסתעפים מורכבות 9. יתר על כן, הם הראו שאימון גופני יכול להפוך שיפוץ T-אבובית בחולדות לאחר אוטם שריר לב 19 וכי טיפול resynchronization לב עלול להוביל להיפוך שיפוץ של T-tubules בכלבים עם tachypacing המושרה אי ספיקת לב פרוזדורים 20. יחדיו, מחקרים הן באדם חולה וחית מכונות וירטואליות כמו גם התערבויות טיפוליות פוטנציאליות לטעון נהנים מהנהלים בידוד תא באיכות גבוהה ובאסטרטגיות ניתוח כמותי מפורטות כפי שתוארו בפרוטוקול ותוצאות סעיפים שלהלן. יתר על כן, כפי שהודגם לאחרונה על ידי משטח לרוחב לעומת סחר בקרום מידות של ערוץ KATP isoforms 21, חשוב לשקול מ פרוזדוריםyocytes (AMS) כביולוגי מובחן, כמו גם מודל תא לב השוואתי לעומת מכונות וירטואליות. T-tubules תועדו לאחרונה בכבשים ואנושי 22 AMS. ראיות הנוכחיות מצביעות על כך שכמה T-tubules קיימים בתאים בבוקר ובדרך כלל ביונקים גדולים יותר כמו כבשים ובני אדם, אך לא במכרסמים קטנים 23. בניגוד למכונות וירטואליות, באדמיניסטרטיבים שחרור תאיים Ca 2 + מופיע להתרחש מpropagating פני תא על ידי דיפוזיה לכיוון מרכז התא וכתוצאה מכך Ca מרחב ובזמן מסומן 2 + הדרגתיים 23. במסגרת זו נראה חשוב להבהיר מנגנונים של Ca 2 + איתות תאית חוסר יציבות לצורות מחלה נפוצות כגון פרפור פרוזדורי 24. לסיכום, שני בידוד AM וVM התא וכל ללב בריא וחולה בדרך מועסק פרוטוקולים. רק אם בידוד תא נעשה כראוי כמו להישפט על ידי תיעוד מיקרוסקופי של איכות תא מספיק, דגימות בבוקר וVM צריכה להיות carried קדימה לניתוח מידות כמותי. בהתאם לכך, בסעיפי הפרוטוקול הבאים תלויים באופן קריטי בתא באיכות גבוהה מבודד מעכבר או מינים אחרים ואחריו מיקרוסקופיה תא החי לנתח קרומי מידות ללא פגע. כפי שציין בעבר, אפיון של ממברנות מידות הוא תחום מחקר מאתגר עם נטייה לחפצי קיבעון והכנת 6, שינויי קרום בשל שינויים האוסמוטי, ומגבלות רזולוציה של מיקרוסקופ אור הקונבנציונלית 9. נציין, כי פרוטוקולי המדינה של-the-art האחרונים לבידודה של אדמיניסטרטיבים אנושיים הדמיה Ca 2 + ותיקון מהדק ושל עכברוש מכונות וירטואליות לתרבית תאים כבר פורסמו בעבר בכתב עת זה 25,26.

<table border="0" cellpadding="0" cellspacing="0" width="1552">
	<tbody>
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			<td height="22" style="height:22px;width:304px;">Chemicals and enzymes</td>
			<td style="width:376px;"></td>
			<td style="width:113px;"></td>
			<td style="width:759px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">2,3-Butanedione monoxime</td>
			<td style="width:376px;">Sigma-Aldrich, Munich, Germany</td>
			<td style="width:113px;">B0753</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Bovine calf serum</td>
			<td style="width:376px;">Thermo Scientific, Schwerte, Germany</td>
			<td style="width:113px;">SH30073</td>
			<td>Triple 0.1 &#181;m sterile filtered.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">CaCl2</td>
			<td style="width:376px;">Sigma-Aldrich, Munich, Germany</td>
			<td style="width:113px;">21115</td>
			<td style="width:759px;">Diluted 1:10 in MQ water to obtain 100 mM CaCl2 stock concentration.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Collagenase type II</td>
			<td style="width:376px;">Worthington via Cell Systems, Troisdorf, Germany</td>
			<td style="width:113px;">on request</td>
			<td>Enzymatic activity depends on individual collagenase batches. Collagenase II&#160; and other enzyme activities (Caseinase, Clostripain, Tryptic) can be assessed in the &#34;collagenase lot selection tool&#34;. Determine cell yield and quality individually for each new lot of collagenase.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Glucose</td>
			<td style="width:376px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:113px;">HN06.1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Heparin</td>
			<td style="width:376px;">Rotexmedica, Trittau, Germany</td>
			<td style="width:113px;">PZN-03862340</td>
			<td>Diluted in 0,9 % NaCl and injected subcutaneuosly in abdominal skin.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">HEPES</td>
			<td style="width:376px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:113px;">9105.4</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Forene 100% (V/V)</td>
			<td style="width:376px;">Abbott, Libertyville, IL, USA</td>
			<td style="width:113px;">B506</td>
			<td>Active agent: isoflurane, 250 ml. Use approximately 2 Vol% in air/oxygen dispenser instrument.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">KCl</td>
			<td style="width:376px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:113px;">6781.3</td>
			<td style="width:759px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">KH2PO4</td>
			<td style="width:376px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:113px;">3904.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Laminin (2 mg/ml)</td>
			<td style="width:376px;">BD Biosciences, Heidelberg, Germany</td>
			<td style="width:113px;">354232</td>
			<td>Lamination is described under step 2.1.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">MgCl2 &#183; 6 H2O</td>
			<td style="width:376px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:113px;">2189.2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">MgSO4 &#183; 7 H2O</td>
			<td style="width:376px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:113px;">8283.2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Na2HPO4 &#183; 2 H2O</td>
			<td style="width:376px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:113px;">4984.2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">NaHCO3</td>
			<td style="width:376px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:113px;">HN01.1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Taurin</td>
			<td style="width:376px;">Carl Roth, Karlsruhe, Germany</td>
			<td style="width:113px;">4721.2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;"></td>
			<td style="width:376px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Dyes</td>
			<td style="width:376px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Di-8-ANEPPS</td>
			<td>Molecular Probes, Life Technologies, Darmstadt, Germany</td>
			<td style="width:113px;">D-3167</td>
			<td>Stock solution 2 mM in DMSO</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Trypan blue</td>
			<td style="width:376px;">Sigma-Aldrich, Munich, Germany</td>
			<td style="width:113px;">T8154</td>
			<td>Trypan blue is gently mixed 1:1 via tip-cut 1 ml plastic pipette with cell suspension prior to cell counting in Neubauer cytometer.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;"></td>
			<td style="width:376px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Langendorff perfusion setup</td>
			<td style="width:376px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Circulation thermostat</td>
			<td style="width:376px;">Lauda, Lauda-K&#246;nigshofen, Germany</td>
			<td style="width:113px;"></td>
			<td>Please refer to Louch et al. (JMCC 2011). Heat up thermostat und buffers in perfusion tubing to 37 &#176;C 15 min prior to use.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Flexible silicone tubing Tygon for peristaltic pump</td>
			<td style="width:376px;">VWR, Darmstadt, Germany</td>
			<td style="width:113px;">224-2252</td>
			<td rowspan="2">Tubing needs to be changed regularly.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Flexible silicone tubing Tygon for thermostat</td>
			<td style="width:376px;">VWR, Darmstadt, Germany</td>
			<td style="width:113px;">228-4340</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:304px;">Heating coil surroundung perfusion tubing</td>
			<td style="width:376px;">Rettberg, G&#246;ttingen, Germany</td>
			<td style="width:113px;">custom-made</td>
			<td style="width:759px;">Heating coil and tubing needs to be cleaned thoroughly via MQ water after using. Do not use detergents. Glass components should be bathed regularly&#160; in 10 mM NaOH overnight.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Peristaltic pump</td>
			<td style="width:376px;">Ismatec, Wertheim, Germany</td>
			<td style="width:113px;">ISM830</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Three way stop cock Discofix C Luer Lock 10 cm</td>
			<td style="width:376px;">Braun, Melsungen, Switzerland</td>
			<td style="width:113px;">16500C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Three way stop cock Discofix 3SC</td>
			<td style="width:376px;">Braun, Melsungen, Switzerland</td>
			<td style="width:113px;">4095146</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;"></td>
			<td style="width:376px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Instruments</td>
			<td style="width:376px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">42 mm glass coverslips</td>
			<td style="width:376px;">Menzel Gl&#228;ser via Thermo Scientific, Schwerte, Germany</td>
			<td style="width:113px;">on request</td>
			<td>0.13 - 0.16 mm thickness</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Cannula 21G</td>
			<td style="width:376px;">Becton, Dickinson and Company, Franklin Lake, NY, USA</td>
			<td style="width:113px;">304432</td>
			<td>Cut to a length of ~ 5 mm, roughened with sandpaper.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Coverslips for Neubauer cytometer 24 x 24 mm</td>
			<td style="width:376px;">Menzel Gl&#228;ser via Thermo Scientific, Schwerte, Germany</td>
			<td style="width:113px;">on request</td>
			<td>0.38 - 0.42 mm thickness</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Graefe forceps, 0.5 mm tips, slight curve</td>
			<td style="width:376px;">Fine Science Tools, Heidelberg, Germany</td>
			<td>11151-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">LSM 710 NLO</td>
			<td style="width:376px;">Carl Zeiss, Jena, Germany</td>
			<td style="width:113px;"></td>
			<td>63x 1.4 NA oil objective</td>
		</tr>
		<tr height="124">
			<td height="124" style="height:124px;width:304px;">Neubauer improved cytometer</td>
			<td style="width:376px;">Labor Optik, Friedrichsdorf, Germany</td>
			<td style="width:113px;">1100000</td>
			<td style="width:759px;">Counting procedure: Wipe cytometer and coverslip provided with the counting chamber with 70 % ethanol. Press coverslip gently on the counting chamber so that the two glass surfaces are in contact and Newton&#39;s rings can be observed. Subsequently, 10 - 20 &#181;l cell suspension can be applied to the edge of the coverslip to be sucked into the void by capillary action. Count the intact vs. defect myocytes using the squares of the cytometer grid which reflects 100 nl. Repeat counting procedure on the second grid provided on the cytometer. Calculate the density of cells in your original cell suspension by taking account of any dlutions and counting shortcuts.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">POC-R2 Imaging Chamber</td>
			<td style="width:376px;">Pecon, Erbach, Germany</td>
			<td style="width:113px;"></td>
			<td>Cell suspension volume: 800 &#181;l; desired plating density: ~ 1000 AM and ~ 10,000 VM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Spring scissors, 8 mm blades straight, blunt</td>
			<td style="width:376px;">Fine Science Tools, Heidelberg, Germany</td>
			<td style="width:113px;">15025-10</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Student dumont #7 forceps, inox</td>
			<td style="width:376px;">Fine Science Tools, Heidelberg, Germany</td>
			<td>91197-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Student iris scissors, curved, 11.5 cm</td>
			<td style="width:376px;">Fine Science Tools, Heidelberg, Germany</td>
			<td>91461-11</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Student iris scissors, straight, 11.5 cm</td>
			<td style="width:376px;">Fine Science Tools, Heidelberg, Germany</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Student surcigal scissors, straight, sharp, 12 cm</td>
			<td style="width:376px;">Fine Science Tools, Heidelberg, Germany</td>
			<td>91402-12</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:304px;">Tissue forceps, 1x2 teeth, slim, 10 cm</td>
			<td style="width:376px;">Fine Science Tools, Heidelberg, Germany</td>
			<td>11023-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of tubular membrane networks in cardiac myocytes from atria and ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

Watch this Scientific Journal Video about Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles at JoVE.com

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

בmyocytes לב, מבני קרום צינורי ליצור רשתות תאיות. אנו מתארים מותאמים פרוטוקולים לבידוד i) של myocytes מלב העכבר כוללים בקרת איכות, ii) מכתים תא חי למיקרוסקופ פלואורסצנטי המדינה של-the-art, וiii) ניתוח תמונה ישירה לכמת את מורכבות רכיב והפלסטיות של קרום תאיים רשתות.

ניתוח של Tubular ממברנה רשתות בלב myocytes מאטרייה וחדרי לב

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

analysis-tubular-membrane-networks-cardiac-myocytes-from-atria

Research

JoVE Journal

Bioengineering

20.4K Views.  Heart Research Center Goettingen.  בmyocytes לב, מבני קרום צינורי ליצור רשתות תאיות. אנו מתארים מותאמים פרוטוקולים לבידוד i) של myocytes מלב העכבר כוללים בקרת איכות, ii) מכתים תא חי למיקרוסקופ פלואורסצנטי המדינה של-the-art, וiii) ניתוח תמונה ישירה לכמת את מורכבות רכיב והפלסטיות של קרום תאיי...

Video: Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Surface פלזמה ליתוגרפיה דפוסים של יצירת רשתות Cell

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

כימות של פונקציה הגלובלית הדיאסטולי על ידי ניתוח המבוסס על דוגמנות Kinematic של Transmitral זרימה באמצעות הפורמליזם מילוי Parametrized הדיאסטולי

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

הארה תהודת אנרגיה העבר לחקר שינויי קונפורמציה בחלבונים בממברנה שבאו לידי ביטוי בתאי יונקים

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

ניתוח התכווצות האוטומטי של רקמת לב Engineered האנוש להקרנת בטיחות תרופת לב

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

ליבה/פגז הדפסה פיגומים קפלים להנדסת רקמות של מבנים צינורי

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

הדמיה אופטית של מודציטים מבודדים המוח החדרית

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

הכנת רקמות לב מהונדסות בצורת רשת נגזר תאי iPS אנושיים לתיקון שריר הלב Vivo

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

מדידה פוטנציאלית לפעולה אופטית חד-תאית בקרדיומיוציטים שמקורם בתאי גזע מושרים על ידי תאי גזע אנושיים

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

פיברובלסט נגזר רקמת חיבור מהונדסת אנושית עבור יישומי הקרנה

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

The present protocol describes the generation of Drosophila melanogaster expressing eNpHR2.0 or ReaChR opsins in the heart for OCT imaging and optogenetic heart pacing. Detailed instructions for Drosophila OCT imaging and heart beating modulation, including the simulation of restorable heart arrest, bradycardia, and tachycardia in live animals at different developmental stages, are reported.