מארק ווזניאק לשימוש במחלקה לסלולרית וציוד צילום וידאו בביולוגיה התפתחותית. תא ההדמיה האוניברסיטה ונדרבילט משותף למשאב. עבודה זו נתמכת על ידי מענק AHA 12PRE10950005 לERP, מענק AHA 11GRNT7690040 לבנק דיסקונט למשכנתות, NIH מענק R01 HL037675 לבנק דיסקונט למשכנתות. בנק דיסקונט למשכנתאות הוא יו&quot;ר גלדיס פ Stahlman במחקר לב וכלי דם.

להבטיח שכל ההליכים כרוכים בבעלי חיים מאושרים על ידי השימוש בבעלי החיים המתאימים וטיפול גוף. .1 הכינו חוצצי המלאי מראש <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן מניית 2 L Ca 2 + של Tyrode החופשי (טבלת 1). התאם את רמת החומציות ל7.4 עם NaOH. </li><li style=";text-align:right;direction:rtl"> הכן מניית 2 L 1.2 של מ&quot;מ Ca 2 + Tyrode (טבלת 2). התאם את רמת החומציות ל7.4 עם NaOH. </li></ol> .2 ביום של הניסוי, הכינו זלוף, העברה, ועיכול חוצצים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן חיץ זלוף 150 מ&quot;ל בCa 2 + של Tyrode החופשי (לוח 3) ולסנן דרך פילטר 0.2 מיקרומטר. </li><li style=";text-align:right;direction:rtl"> הכן 35 מ&quot;ל Ca 2 + העברה חופשית &quot;חוצץ&quot; (לוח 4) ו25 מ&quot;ל Ca 2 + המכיל &quot;הצפת B&quot; (לוח 5). לסנן כל דרך מסנן 0.2 מיקרומטר. </li><li style=";text-align:right;direction:rtl"> הכן את הפתרונות של 0.06, 0.24, 0.6, ו1.2 מ&quot;מ Ca 2 + על ידי הצפת ערבוב וB ללוח 6. </li><li style=";text-align:right;direction:rtl"> הכן חיץ עיכול 25 מ&quot;ל על ידי המסת אנזימי עיכול ב25 מ&quot;ל חיץ זלוף (לוח 7) המבוסס על משקל הגוף (BW) של העכבר. לסנן דרך פילטר 0.2 מיקרומטר לפני השימוש. </li></ol> .3 הגדרת ניסוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרכיב מנגנון זרימת Langendorff קבוע, זמין באופן מסחרי או באמצעות משאבה, צינורות, וסליל מחליף חום peristaltic כדי לאפשר קצב זרימה של 3ml / דקה של perfusate המחומם ל37 מעלות צלזיוס. סכמטי פשוט מסופק בסקירת 2011 על ידי בל, et al 18. </li><li style=";text-align:right;direction:rtl"> הגדר טמפרטורת אמבט מים במחזור (~ 47 ° C) של מנגנון Langendorff כך יצוא שמהצינורית הוא על 37 מעלות צלזיוס בקצב זרימה של 3 מ&quot;ל / דקה. </li><li style=";text-align:right;direction:rtl"> הפעל 70% אתנול באמצעות מערכת זלוף במשך 15 דקות, ואחריו מסוג ultrapure אני מים 100 מ&quot;ל. חיץ זלוף להפעיל באמצעות התוכנה למ &#39;במשך 5 דקות תוך ביטול בועות אוויר. </li><li style=";text-align:right;direction:rtl"> לעקר את כל הכלים כירורגיים עם שיטה רצויה. </li><li style=";text-align:right;direction:rtl"> צור צינורית על ידי הצמדת קטע קצר (מ&quot;מ על 3) PE-50 צינורות לקצה מתאם Luer-בדל 23 G. טיפ חום של צינורות כדי ליצור שפה אשר לאב העורקים יהיו ממוצבת. </li></ol> בידוד .4 Cardiomyocyte <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הזרק עכבר עם 0.2 מ&quot;ל פתרון הפרין (1,000 IU / ml), באמצעות הזרקת intraperitoneal. </li><li style=";text-align:right;direction:rtl"> אחרי 5 דקות, להרדים עכבר עם isofluorane. כשהרדים (אין תגובה לקמצוץ רגל חזקה) באופן מלא, לרסס חזה עם EtOH 70%. פתח את החזה, לכרות במהירות הלב, נזהר שלא לפגוע באב העורקים, ומקום ב4 ° C חיץ זלוף. השתמש במלקחיים מעוקלים לתפוס ולהרים תחת הלב. זה יוצר חלל לקצץ קבצים מצורפים מאחורי הלב עם מספריים עדינים. </li><li style=";text-align:right;direction:rtl"> Cannulate הלב בעדינות על ידי אחיזה בקצה של אב העורקים עם שני זוגות מלקחיים בסדר ומושך openi בזהירותng של אב העורקים מעבר לשפה של הצינורית. ודא את הקצה של הצינורית הוא לא עבר שסתום אב העורקים ולתוך החדר, כמו זה יהיה לעכב זלוף של הלב באמצעות העורקים הכליליים. </li><li style=";text-align:right;direction:rtl"> אבטח את אב העורקים לצינורית על ידי קשירת לולאה של 5-0 תפר משי סביב אב העורקים מייד מעל השפה של הצינורית. </li><li style=";text-align:right;direction:rtl"> הערה: שלבי 4.2 דרך 4.4 אמורים להסתיים במהירות האפשרית לעיכול באיכות הטובה ביותר. </li><li style=";text-align:right;direction:rtl"> הערה: הלב יכול להיות תלוי ישירות על גבי הצינורית כבר מחוברת למכשירי Langendorff עם זורם חיץ זלוף. עם זאת, תלוי הלב על צינורית שמחוברת למזרק קטן של חיץ זלוף תחת מיקרוסקופ לנתיחה כבר מצא להיות מוצלח ביותר. זה מאפשר כמות קטנה של חיץ זלוף כדי שתועבר, צופה לסליקת עורקים הכליליים והבטחת לב מחוברת היטב לצינורית. הצינורית לאחר מכן נתלתה על Langendorff עם הפעלת מאגר זלוף. </li><li style=";text-align:right;direction:rtl"> תנקבהלב עם חיץ זלוף החופשי Ca 2 + בזרימה של 3 מ&quot;ל / דקה במשך 2-3 דקות. </li><li style=";text-align:right;direction:rtl"> לעבור לאנזים חיץ עיכול ותנקב ל7-10 דקות, לב יהפוך רך יותר וקל יותר בצבע. </li><li style=";text-align:right;direction:rtl"> הערה: זמן עיכול יכול להיות מותאם המבוסס על פעילות אנזים וגודל לב. </li><li style=";text-align:right;direction:rtl"> ברגע שהלב הוא מוחשי רפוי, להוציא את הלב מהצינורית ומניחים בצלחת P60 סטרילי עם הצפת (Ca 2 + חיץ העברה חופשית). </li><li style=";text-align:right;direction:rtl"> הסר אטריה וכלי גדול. הסר חדר ממני אם תרצה בכך. עם מלקחיים, מפריד את החדר לחתיכות קטנות. כמה פעמים פיפטה עם טפטפת העברה 5 מ&quot;ל סטרילי כדי לפזר עוד תאים. </li><li style=";text-align:right;direction:rtl"> סנן את ההשעיה התא לתוך צינור חרוטי 50 מ&quot;ל דרך מסנן רשת ניילון 250 מיקרומטר. </li><li style=";text-align:right;direction:rtl"> לאפשר ההשעיה עד גלולה במשך כ 10 דקות. </li><li style=";text-align:right;direction:rtl"> העבר את הכדור לפתרון 0.06 מ&quot;מ Ca 2 +. לאפשר לתאי גלולה עבור 10 דקות. תאים בריאים גלולה, בעוד שרוב התאים מתים יצופו. </li&gt; <li style=";text-align:right;direction:rtl"> לשאוב supernatant, ולהעביר את הכדור לפתרון 2 + מ&quot;מ Ca 0.24. </li><li style=";text-align:right;direction:rtl"> חזור 10 דגירה דקות, שאיפה, ולהעביר באמצעות שני 2 + פתרונות Ca שנותרו עד תאים בפתרון 1.2 מ&quot;מ. בשלב זה, רוב התאים צריכים להיות myocytes מוט בצורה. </li></ol> .5 ארעיים התכווצות וסידן מדידה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הערה: שתי התכווצות וארעית סידן ניתן למדוד בו זמנית. </li><li style=";text-align:right;direction:rtl"> הכן פורע-2 פתרון PM על ידי resuspending בDMSO נטול מים לריכוז של 1 מיקרוגרם / μl. פתרון הפורע-02:00 יכול להיות מאוחסן בתא ייבוש ב-20 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> 1 טען מ&quot;ל של תאים תלויים עם μl 1 פורע-2 PM. לאפשר לתאים לשבת בחושך 10 דקות לאחר מכן supernatant לשאוב. שטוף פעמיים עם הפתרון של Ca 2 + Tyrode, מאפשר לתאי גלולה בחושך עבור 10 דקות בין שוטף. </li><li style=";text-align:right;direction:rtl"> מניחים coverslip זכוכית בהוספת שלב שמאפשרת PE המחומםrfusion / שאיפה ואלקטרודה צעדה עם ממריץ שדה myocyte. הוספת שלב מקום במיקרוסקופ ההפוכה להגדיר למדידת הקרינה, הוספת טיפה של שמן לעדשה אם באמצעות אובייקטיבי טבילת שמן. </li><li style=";text-align:right;direction:rtl"> הגדרת זלוף כובד המחומם עם פתרון 1.2 מ&quot;מ Ca 2 + של Tyrode כך פתרון על במה מגיע 37 מעלות צלזיוס. צרף שאיפה לואקום. חבר אלקטרודות. </li><li style=";text-align:right;direction:rtl"> הפעל את מערכת מיקרוסקופ, ולפתוח את תוכנת הקרינה ורכישת נתונים מידות תא ratiometric. </li><li style=";text-align:right;direction:rtl"> הוסף כמה טיפות של תאים עמוסים פורע-2 בבוקר ועד הוספת השלב, חסימת זלוף לרגע כדי לאפשר לתאים בריאים להתיישב. </li><li style=";text-align:right;direction:rtl"> השתמש אובייקטיבי 40X כדי לאתר תאים הרצויים. תא בריא צריך להיות מוט בצורה ולא באופן ספונטני להידבקות. צריך כאשר התא בקצב זה בעליל וחוזה אחיד ב5-20 V. </li><li style=";text-align:right;direction:rtl"> הערה: ניתוח פיזיולוגי דורש עיכול באיכות גבוהה. זה אפשרי שיהיה לי תאים שנראים בריא, אבל לא pa<html> ce גם לניתוח פיסיולוגי. פרוטוקול עיכול עשוי לדרוש עידון כדי לקבל איכות גבוהה, myocytes הבריא. בנוסף, לבבות מוטציה או חולים עשויים לדרוש שינויים בפרוטוקול. </li><li style=";text-align:right;direction:rtl"> התאימו סיבוב עדשה ושינוי צמצם כך התא רצוי הוא בשורה בצורה אופקית על מסך ורקע אינה גלויה. </li><li style=";text-align:right;direction:rtl"> יישר ברים גילוי קצה לכל קצה של myocyte ולהתאים סף. יישר זיהוי sarcomere על חלק מתא עם sarcomeres האחיד. ככל שאתה יכול לעשות זה בר, המודל המתמטי מדויק יותר של אורך sarcomere יהיה, כל עוד זה לא על שתי אוכלוסיות של sarcomeres. </li><li style=";text-align:right;direction:rtl"> תאי קצב ב2 הרץ ו5-20 V של 10-15 שניות לפני ההקלטה. </li><li style=";text-align:right;direction:rtl"> רשומה מעקב. אור הסביבה צריך להיות ממוזער להקלטה ארעית סידן הטוב ביותר. </li></ol> .6 ניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לנתח עקבות באמצעות הקרינה ratiometric ותוכנת רכישת נתונים מידות תא. </li></ol>כותרת &quot;&gt; 7. מחקרים נוספים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פרוטוקול זה מספק תאים עודפים שיכול לשמש למגוון רחב של ניסויים אחרים, כוללים: קיבעון וimmunostaining, הערכת השפעות ישירות סמים, תרבות טווח קצרה, ומיקרוסקופ כוח אטומי. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na+/K+-ATPase+a2-isoform+preferntially+modulates+Ca2++transients+and+sarcoplasmic+reticulum+Ca2++release+in+cardiac+myocytes.">Na+/K+-ATPase a2-isoform preferntially modulates Ca2+ transients and sarcoplasmic reticulum Ca2+ release in cardiac myocytes.</a> Cardiovascular Research. 95, 480-486 (2012).</li><li>Touchberry, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF23+is+a+novel+regulator+of+intracellular+calcium+and+cardiac+contractility+in+addition+to+cardiac+hypertophy.">FGF23 is a novel regulator of intracellular calcium and cardiac contractility in addition to cardiac hypertophy.</a> American Journal of Physiology - Endocrinology and Metabolism. , (2013).</li><li>Helmes, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titin+Determines+the+Frank-Starling+Relation+in+Early+Diastole.">Titin Determines the Frank-Starling Relation in Early Diastole.</a> The Journal of General Physiology. 121, 97-110 (2003).</li><li>Bell, R. M., Mocanu, M. M., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+heart+perfusion:+The+Langendorff+technique+of+isolated+heart+perfusion.">Retrograde heart perfusion: The Langendorff technique of isolated heart perfusion.</a> Journal of Molecular and Cellular Cardiology. 50, 940-950 (2011).</li><li>Flynn, J. M., Santana, L. F., Melov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Cell+Transcriptional+Profiling+of+Adult+Mouse+Cardiomyocytes.">Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes.</a> J Vis Exp. (58), e3302 (2011).</li></ol>

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בידוד של שריר לב באיכות דורש רמה מסוימת של תרגול ואופטימיזציה. פרוטוקול זה מכיל שלבים עיקריים שיכול להשפיע על התוצאה של העיכול באופן משמעותי. אלה יש לשקול בזהירות בעת ביצוע ופתרון הבעיות בפרוטוקול. הזמן מהסרת הלב לcannulation וזלוף צריך להיות ממוזער, באופן אידיאלי פחות מח...

Cardiomyocytes provide the contractile force for the heart. Each myocyte contains organized cytoskeletal and contractile elements essential to contraction. The heart not only contains cardiomyocytes but also fibroblasts, connective tissue, and modified myocytes such as Purkinje fibers. Changes in any or all of these cell types can be seen in heart failure, making determination of the ultimate cause of heart dysfunction difficult. Decreased ventricular contractility of a failing heart is preceded by varying degrees of fibrous tissue build-up and hypertrophy and dysfunction of the cardiomyocytes1. Arrhythmias can be caused by dysfunction in the conducting cells2, fibrosis interrupting cardiac conduction3, or changes in the expression of ion channels in cardiomyocytes1,4. Indeed, these problems often co-exist in a complicated fashion.
Isolating and studying cardiomyocytes allows one to examine contractile and electrical dysfunction of individual myocytes in the context of overall heart function. Techniques to isolate individual cardiac myocytes were developed over 40 years ago in an effort to better study physiology of this &#8216;workhorse&#8217; cell of the heart. Isolation techniques have been improved to increase yield and quality of cells, with the vital development of coronary perfusion via cannulation of the aorta, first performed in 19705. This article describes cardiomyocyte isolation followed by measurement of contractility and calcium transients using ratiometric fluorescence and cell dimensioning data acquisition software. In both genetic and environmental models of heart disease, utilizing this protocol provides key information about cell contractility, sarcomeric contractility and relaxation, calcium transients, and cytoskeletal disruption. This system is often used to test the effect of drugs on isolated cardiomyocytes6,7. We employ the system to compare contractility and calcium transients of cardiomyocytes between wild type mice and mice with a mutation leading to dilated cardiomyopathy.
Studying individual cardiac myocytes has numerous advantages over other commonly used methods to study cardiac anatomy and physiology. Echocardiography and electrocardiography provide information about the contractile function and electrical conduction of the heart overall. Neither of these methods explains the cause or nature of the dysfunction below the level of the whole organ. Measuring contractility of individual cardiomyocytes using edge-detection and sarcomere length algorithms can demonstrate that changes within the contractile cell of the heart are present within global heart dysfunction. Measuring calcium transients demonstrates the influence of changes in ion flux at a cellular level to contractile dysfunction or arrhythmias.
In addition to complementing whole organ studies, this protocol provides cells easily stained for sub-cellular components. It is possible to see cytoskeletal changes by staining tissue sections of the whole heart; however, one is only able to visualize a cross section of cells. A whole cardiomyocyte is thicker than the typical tissue section, and given the arrangement and length of the cardiomyocytes, it is difficult to see whole cells in a single section. Isolated cardiomyocytes can be fixed immediately and stained for a variety of cytoskeletal elements and ion channels. Confocal images can be compiled into full thickness z-stacks of the cell. Additionally, staining isolated cardiac myocytes allows for staining of components that are impossible to visualize in sections, such as the membrane invaginations known as t-tubules.
Cultured cell lines of cardiac myocytes do exist, some of which have similar transcriptional profiles and phenotypic features to cardiac myocytes. Some, such as the HL-1 cell line, even retain some sarcomeric organization and rudimentary contractile ability8. Despite these qualities, cultured cells lack the &#8220;box car&#8221; shape and t-tubule structure of cardiomyocytes and have less sarcomeric organization. These components are essential to the function of cardiomyocytes in vivo. In addition, using cultured cells allows genetic manipulation but not in vivo physiologic manipulation. Isolated cardiomyocytes retain their cellular organization long enough to study but are still easily imaged and even transduced9. 
Isolated cardiomyocytes are extremely versatile, providing a substrate to identically analyze cells from varied experimental conditions. Isolation and physiological analysis of myocytes have been used by countless labs for a variety of experiments including studying the effect of drugs6,7 or small molecules10, environmental stressors11, infection12, illness13, or genetic mutation14,15 on contractility and/or calcium transients and studying the regulation of contractility and calcium release within a cardiomyocyte15-17. With the application of any of these stressors, changes in heart function could be due to any combination of changes in the cardiomyocytes themselves and changes in the surrounding environment of the heart, such as scarring, changes in conductivity, or changes to the extracellular matrix. This is an ideal protocol for answering research questions regarding individual myocyte structure and function in the context of heart failure from any cause.
Although it has countless advantages over other techniques, physiological analysis of isolated cardiomyocytes is not ideal for every research question. Isolation is a terminal procedure, thus cardiomyocytes can only be assessed at one time in the mouse&#8217;s life. This can be partially overcome using cohorts and sacrificing individual mice along the course of illness development or by monitoring the mouse to determine whether it has clinically significant heart failure before isolation. Although this protocol can determine whether cardiomyocyte dysfunction has occurred, one cannot draw the conclusion that this dysfunction is the root cause of heart failure without additional experimental information. It is possible that cardiomyocyte dysfunction itself is secondary to other changes in the heart. Despite these challenges, studying cardiomyocytes offers valuable information in determining the nature of dysfunction in the heart, especially when combined with other experiments. 
The following protocol is adapted from one provided by Dr. Chee Lim, Vanderbilt University. Although contractility and calcium flux data are highly reproducible and useful in comparing hearts with genetic or environmental manipulation, isolation itself remains a technique dependent protocol, requiring optimization. Minimizing the time between removing the heart from the mouse and perfusing the heart via aortic cannulation is essential for a quality digestion. In addition, digestion time and enzyme concentration can be optimized for the highest quality myocytes. Steps likely requiring optimization, along with suggestions for optimization, are noted below.

<table><tbody><tr><td>NaCl</td><td>RPI</td><td>S23020</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P-3911</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M-8266</td><td></td></tr><tr><td>HEPES</td><td>EM Science</td><td>S320</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S5011</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>C79</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>2,3-Butanedione-monoxime (BDM)</td><td>Sigma</td><td>B-0753</td><td></td></tr><tr><td>Taurine</td><td>Sigma</td><td>T-0625</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A-7030</td><td></td></tr><tr><td>Collagenase B</td><td>Roche Diagnostics</td><td>1-088-823</td><td></td></tr><tr><td>Collagenase D</td><td>Roche Diagnostics</td><td>1-088-882</td><td></td></tr><tr><td>Protease XIV</td><td>Sigma</td><td>P-5147</td><td></td></tr><tr><td>Heparin</td><td>APP Pharmaceuticals, LLC</td><td>401586D</td><td></td></tr><tr><td>Isofluorane</td><td>Butler Schein</td><td>11695-6776-2</td><td></td></tr><tr><td>Fura-2 AM</td><td>Teflabs</td><td>103</td><td></td></tr><tr><td>DMSO &amp;ndash; anhydrous</td><td>Sigma</td><td>276855</td><td></td></tr><tr><td>IonOptix cell pacing and fluorescent analysis system</td><td>IonOptix</td><td></td><td></td></tr><tr><td>250 &amp;mu;m nylon mesh filter</td><td>Sefar America</td><td>Lab Pak 03-250/50</td><td></td></tr><tr><td>23 G Luer-stub adaptor</td><td>Becton Dickinson</td><td>1482619E</td><td></td></tr><tr><td>PE-50 tubing</td><td>Becton Dickinson</td><td>427410</td><td></td></tr></tbody></table>

isolation and physiological analysis of mouse cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

ברגע שההתכווצות והעתקים חולפים נאספות (איור 2), הנתונים מנותחים בקלות עם התוכנה המתאימה. המעקב כל, או החלקים מן יכול להיות בממוצע (איור 3). התכווצות יכולה להיות מנותחת במגוון של דרכים. לתפקוד הסיסטולי, אחד יכול להעריך את סדר הגודל של התכווצות עם קיצור ?...

Watch this Scientific Journal Video about Isolation and Physiological Analysis of Mouse Cardiomyocytes at JoVE.com

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

בידוד ופיזיולוגי ניתוח של שריר לב העכבר

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

isolation-and-physiological-analysis-of-mouse-cardiomyocytes

Research

JoVE Journal

Biology

22.9K Views.  Vanderbilt University.  Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause. 

Video: Isolation and Physiological Analysis of Mouse Cardiomyocytes

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

בידוד והתרבות של שריר לב עכבר בילוד

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

בידוד, תרבות ואפיון פונקציונלי של Cardiomyoctyes מאוס למבוגרים

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

בידוד של פרפור מיוציטים מעכברים למבוגרים

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Medicine

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

בידוד והתרבות של שריר לב מאוס למבוגרים לתא איתות ו<em&gt; במבחנה</em&gt; לב היפרטרופיה

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

בידוד, תרבות התמרה של cardiomyocytes עכבר למבוגרים

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

שיטות הבידוד, תרבות האפיון פונקציונלי של myocytes צומת Sinoatrial של עכברים בוגרים

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

שיטה פשוטה ויעילה לבודד באופן עקבי קרדיומיוציטים עכבר

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

בידוד של קרדיומיוציטים חדריים אנושיים מפרוסות שריר הלב ויסטום-לחתוך

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

בידוד ותרבות בו זמנית של מיוציטים פרך, מיוציטים חדריים, ולא מיוציטים מלב עכבר בוגר

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

בידוד ופיזיולוגי ניתוח של שריר לב העכבר

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