<p class="jove_content"&gt;이 작품은 NIH 부여 P20RR018766 및 알코올 연구 ABMRF / 재단의 교부금에 의해 지원되었다.</p

<ol>
	<li>Chakraborty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+system:+a+vital+link+between+metabolic+syndrome+and+inflammation.">Lymphatic system: a vital link between metabolic syndrome and inflammation.</a> Ann. N. Y. Acad. Sci. 1207, 94-94 (2010).</li><li>Zawieja, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+biology+and+the+microcirculation:+past,+present+and+future.">Lymphatic biology and the microcirculation: past, present and future.</a> Microcirculation. 12, 141-141 (2005).</li><li>Benoit, J. N., Zawieja, D. C., Goodman, A. H., Granger, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+intact+mesenteric+lymphatic+pump+and+its+responsiveness+to+acute+edemagenic+stress.">Characterization of intact mesenteric lymphatic pump and its responsiveness to acute edemagenic stress.</a> Am. J. Physiol. 257, H2059-H2059 (1989).</li><li>Davis, M. J., Davis, A. M., Ku, C. W., Gashev, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+constriction+and+dilation+of+isolated+lymphatic+vessels.">Myogenic constriction and dilation of isolated lymphatic vessels.</a> Am. J. Physiol. Heart. Circ. Physiol. 296, H293-H293 (2009).</li><li>Dougherty, P. J., Davis, M. J., Zawieja, D. C., Muthuchamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sensitivity+and+cooperativity+of+permeabilized+rat+mesenteric+lymphatics.">Calcium sensitivity and cooperativity of permeabilized rat mesenteric lymphatics.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R1524-R1524 (2008).</li><li>Fay, F. S., Shlevin, H. H., Granger, W. C., Taylor, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aequorin+luminescence+during+activation+of+single+isolated+smooth+muscle+cells.">Aequorin luminescence during activation of single isolated smooth muscle cells.</a> Nature. 280, 506-506 (1979).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+myosin+light+chain+phosphorylation+decreases+rat+mesenteric+lymphatic+contractile+activity.">Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, 726-726 (2009).</li><li>Karaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+movements,+distribution,+and+functions+in+smooth+muscle.">Calcium movements, distribution, and functions in smooth muscle.</a> Pharmacol. Rev. 49, 157-157 (1997).</li><li>Ratz, P. H., Berg, K. M., Urban, N. H., Miner, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+smooth+muscle+calcium+sensitivity:+KCl+as+a+calcium-sensitizing+stimulus.">Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus.</a> Am. J. Physiol. Cell. Physiol. 288, C769-C769 (2005).</li><li>Somlyo, A. P., Somlyo, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++sensitivity+of+smooth+muscle+and+nonmuscle+myosin+II:+modulated+by+G+proteins,+kinases,+and+myosin+phosphatase.">Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase.</a> Physiol. Rev. 83, 1325-1325 (2003).</li><li>Souza-Smith, F. M., Kurtz, K. M., Molina, P. E., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+mesenteric+collecting+lymphatic+pump+function+following+acute+alcohol+intoxication.">Adaptation of mesenteric collecting lymphatic pump function following acute alcohol intoxication.</a> Microcirculation. 17, 514-514 (2010).</li><li>Breslin, J. W., Yuan, S. Y., Wu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-C+alters+barrier+function+of+cultured+lymphatic+endothelial+cells+through+a+VEGFR-3-dependent+mechanism.">VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism.</a> Lymphat. Res. Biol. 5, 105-105 (2007).</li><li>Shirasawa, Y., Benoit, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretch-induced+calcium+sensitization+of+rat+lymphatic+smooth+muscle.">Stretch-induced calcium sensitization of rat lymphatic smooth muscle.</a> Am. J. Physiol. Heart. Circ. Physiol. 285, H2573-H2573 (2003).</li><li>Imtiaz, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacemaking+through+Ca2++stores+interacting+as+coupled+oscillators+via+membrane+depolarization.">Pacemaking through Ca2+ stores interacting as coupled oscillators via membrane depolarization.</a> Biophys. J. 92, 3843-3843 (2007).</li><li>Muller, J. M., Davis, M. J., Kuo, L., Chilian, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+coronary+endothelial+cell+Ca2++concentration+during+shear+stress-+and+agonist-induced+vasodilation.">Changes in coronary endothelial cell Ca2+ concentration during shear stress- and agonist-induced vasodilation.</a> Am. J. Physiol. 276, 1706-1706 (1999).</li><li>Ferrusi, I., Zhao, J., van Helden, D., von der Weid, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclopiazonic+acid+decreases+spontaneous+transient+depolarizations+in+guinea+pig+mesenteric+lymphatic+vessels+in+endothelium-dependent+and+-independent.">Cyclopiazonic acid decreases spontaneous transient depolarizations in guinea pig mesenteric lymphatic vessels in endothelium-dependent and -independent.</a> 286, H2287-H2287 (2004).</li></ol>

관심 없음 충돌 선언하지 않습니다.

방법의 소설 조합은 림프 혈관의 펌핑 본질을 연구하기 위해 고용되었다. 동시에 변화 측정 기능 [칼슘 2 +] i와 펌핑 lymphatics의 직경은 칼슘 2의 상대적인 기여도 + 종속 및 CA 2 림프 수축성주기를 지배하는 전체 메커니즘에 + - sensitizing 신호 경로. 연구를 허용합니다 림프 수축성주기 톤 이상 겹쳐 phasic 수축으로 구성되어 있습...

<table border="1"><tbody><tr><td> 1. 닮은 5 배 증권 </td></tr><tr><td 화학 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 양 </td></tr><tr><td> 나트륨 염화물 </td><td> EMD </td><td> SX0420 - 3 </td><td> 35g </td></tr><tr><td> 염화칼륨 </td><td> JT 베이커 </td><td> 3040 </td><td> 1.75 g </td></tr><tr><td> 염화칼슘 </td><td> 시그마 </td><td> C - 3881 </td><td> 1.47 g </td></tr><tr><td> 마그네슘 황산 </td><td> 시그마 </td><td> M - 9397 </td><td> 1.44 g </td></tr><tr><td> 무균 필터링 물 </td><td> N / A </td><td> N / A </td><td> 1 L에 지참 </td></tr><tr><td> 4 autoclaved 병 및 상점으로 살균 필터 ° C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 2. 맙스 버퍼 </td><td></td><td></td><td></td></tr><tr><td> 화학 </td><td> 회사 </TD&gt; <td> 카탈로그 번호 </td><td> 양 </td></tr><tr><td> 맙스 </td><td> 시그마 </td><td> M3183 </td><td> 125.6 g </td></tr><tr><td> 무균 필터링 물 </td><td> N / A </td><td> N / A </td><td> 1 L에 지참 </td></tr><tr><td> 4 autoclaved 병 및 상점으로 살균 필터 ° C </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 3. 알부민 생리 식염수 (APSS) </td></tr><tr><td> 화학 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 양 </td></tr><tr><td> 닮은 주식 (5 배) </td><td> N / A </td><td> N / A </td><td> 200 ML </td></tr><tr><td> 맙스 버퍼 </td><td> N / A </td><td> N / A </td><td> 5 ML </td></tr><tr><td> 인산 나트륨 </td><td> 시그마 </td><td> S - 9638 </td><td> 0.168 g </td></tr><tr><td> 나트륨 Pyruvate </td><td> 시그마 </td><td> P5280 </td><td> 0.22 g </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) 나트륨 샐티 </td><td> 시그마 </td><td> ED2SS </td><td> 0.0074 g </td></tr><tr><td> 포도당 </td><td> 시그마 </td><td> G7528 </td><td> 0.901 g </td></tr><tr><td> 알부민, 소의 </td><td> USB </td><td> 10,856 </td><td> 10g </td></tr><tr><td> 무균 필터링 물 </td><td> N / A </td><td> N / A </td><td> 1 L에 지참 </td></tr><tr><td> 37 7.4로 산도를 조절 ° C, 4 autoclaved 병 및 저장에 다음 살균 필터 ° C. </td></tr></tbody></table> 표 1. 특정 시약이 사용됩니다. 4 시에 보관 ° C.

measurement of cytosolic ca2+ in isolated contractile lymphatics

림프 혈관은 액체 항상성 유지 중앙 순환에 lipids를 제공하고, 잠재적으로 해로운 항원에 대한 감시 시스템 역할, 점막 면역과 적응 면역 반응 1 최적화 다기능 교통 시스템을 구성. 림프는 맹인 종단 초기 lymphatics를 입력하고, 다음 큰 수집 lymphatics에서 압력 구배에 대한 수송이다 중간 유체로부터 형성됩니다. 각 수집 림프는 역류를 방지 뾰족한 끝이 둘있는 밸브로 구분 lymphangions라는 세그먼트의 일련의 구성됩니다. 각 lymphangion는 중앙 혈액 순환이 방향 압력 구배에 대한 림프을 향한 추진력을 얻습니다 수축성 사이클을 가지고 있습니다. 이 phasic 수축성 패턴은 수축기 및 diastolic 단계와 심장주기에 유사하고, 낮은 수축 주파수 4. 또한, 림프 평활근은 음색을 생성하고 myogenic 수축과 팽창을 표시 I각각 luminal 압력 증가와 감소, 5 N 응답. lymphatics의 phasic와 토닉 수축성을 모두 지원하는 분자 메커니즘의 하이브리드 따라서 제안하고 있습니다. 평활근의 수축은 일반적으로 cytosolic 칼슘 2 + 농도 규제 ([칼슘 2 +] I) 칼슘 2 플러스 감성 +, 세포 6 주변 환경의 변화에 대한 응답으로 수축성 요소. [칼슘 2 +] 제가 + 플라즈마 막 리간드 또는 전압 문이 칼슘 2 + 채널과 + 내부 상점에서 칼슘 2의 릴리스와 이해를 통해 칼슘 2의 운동의 조합에 의해 결정됩니다. Cytosolic 칼슘 2 + calmodulin에 바인딩 및 마이 오신 조명 체인으로 효소를 활성화 (MLC) 차례로 MLC는 굴지 - 마이 오신 - 중재 수축 8 선도 phosphorylates 키나제 (MLCK). 그러나, 칼슘이 경로의 감도 <sup&gt; 2 +는 MLC 인산 가수 분해 효소 (MLCP) 9 조정할 수 있습니다. MLCP 활동 Rho 키나제 (바위)과 마이 오신의 인산 가수 분해 효소 억제제 단백질 CPI - 17에 의해 규제됩니다. 여기, 우리는 칼슘 2 + - 의존과 칼슘이 림프 평활근의 수축의 + - sensitizing 메커니즘을 연구하기 위해 절연, perfused lymphatics에서 시간이 지남에 따라 [+ 칼슘 2] 나는 변화를 평가하는 방법을 제시한다. 절연 쥐 mesenteric 수집 lymphatics를 사용하여 우리는 [+ 칼슘 2] I 및 수축성 활동에 스트레칭 유발 변화를 공부했습니다. 격리된 림프 모델은 압력, 유량 및 욕조 솔루션의 화학 성분이 밀접하게 제어할 수있는 장점을 제공합니다. [칼슘 2 +] 제가 ratiometric, CA 2 + 바인딩 염색 Fura - 2 lymphatics 로딩에 의해 결정되었다. 이러한 연구는 phasic 조절 다른 분자 메커니즘을 연구의 광범위한 문제에 대한 새로운 접근 방법을 제공합니다림프 평활근의 수축 토닉 대 수축.

Watch this Scientific Journal Video about Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics at JoVE.com

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

우리는 cytosolic CA를 평가하는 접근 방법을 소개합니다<sup&gt; 2 +</supCA를 공부 격리 lymphatics에&gt; 집중<sup&gt; 2 +</sup&gt; 종속 및 CA<sup&gt; 2 +</sup림프 평활근의 수축&gt; - sensitizing 메커니즘.

Cytosolic CA의 측정 2 + 절연 수축성 Lymphatics에

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

measurement-of-cytosolic-ca2-in-isolated-contractile-lymphatics

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

13.5K 조회수.  Louisiana State University Health Sciences Center. 우리는 cytosolic CA를 평가하는 접근 방법을 소개합니다 2 + 집중 2 + 종속 및 CA 2 + - sensitizing 메커니즘.

비디오: Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes의 빠른 칼슘 Fluxes 측정

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

연구

생물학

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.

절연, 문화, 그리고 성인 마우스 된 심근의 기능적 특성

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

고립된 림프관의 칼슘 및 수축률에 대한 실시간 분석

Real-time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i).
This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i&#160;and diameter measurements continuously in real time.
The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

저자 스포트라이트: 림프부종 및 고혈압 연구의 혁신적인 방법

This protocol describes a method to simultaneously measure cytosolic, free calcium [Ca2+]i and vessel diameter in contracting lymph vessels in real time and then calculate absolute Ca2+ concentrations as well as contractility/rhythmicity parameters. This protocol can be used to study Ca2+ and contractile dynamics across a variety of experimental conditions.

격리된 가압된 림프관에서 Absolute, Cytosolic, Free Ca2+ 및 해당 수축성의 실시간 평가

The lymphatic vasculature, now often referred to as &#34;the third circulation,&#34; is located in many vital organ systems. A principal mechanical ...

면역학 및 감염병학

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

유동 세포 계측법에 의한 신장 상피 세포에서 세포 내 칼슘 측정

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

마우스의 심장 수축력의 조사에 대 한 격리 유두 근육의 수축력 측정

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

배양 된 평활근 세포의 근질 그물 자리 내 칼슘 변동 FRET 측정을 기반으로 공 촛점 이미지

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial malfunction in the diseased heart remains to be fully clarified. Recent compelling evidence demonstrates that changes in the myofilament Ca2+ sensitivity affect intracellular Ca2+ homeostasis and ion channel activities in cardiac myocytes, the essential mechanisms responsible for the cardiac action potential and contraction in healthy and diseased hearts. Indeed, activities of ion channels and transporters underlying cardiac action potentials (e.g., Na+, Ca2+ and K+ channels and the Na+-Ca2+ exchanger) and intracellular Ca2+ handling proteins (e.g., ryanodine receptors and Ca2+-ATPase in sarcoplasmic reticulum (SERCA2a) or phospholamban and its phosphorylation) are conventionally measured to evaluate the fundamental mechanisms of cardiac excitation-contraction (E-C) coupling. Both electrical activities in the membrane and intracellular Ca2+ changes are the trigger signals of E-C coupling, whereas myofilament is the functional unit of contraction and relaxation, and myofilament Ca2+ sensitivity is imperative in the implementation of myofibril performance. Nevertheless, few studies incorporate myofilament Ca2+ sensitivity into the functional analysis of the myocardium unless it is the focus of the study. Here, we describe a protocol that measures sarcomere shortening/re-lengthening and the intracellular Ca2+ level using Fura-2 AM (ratiometric detection) and evaluate the changes of myofilament Ca2+ sensitivity in cardiac myocytes from rat hearts. The main aim is to emphasize that myofilament Ca2+ sensitivity should be taken into consideration in E-C coupling for mechanistic analysis. Comprehensive investigation of ion channels, ion transporters, intracellular Ca2+ handling, and myofilament Ca2+ sensitivity that underlie myocyte contractility in healthy and diseased hearts will provide valuable information for designing more effective strategies of translational and therapeutic value.

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

Myofilament 칼슘의 평가<sup&gt; 2 +</sup&gt; 감도 근본적인 심장 여진 수축 커플 링

Heart failure and cardiac arrhythmias are the leading causes of mortality and morbidity worldwide. However, the mechanism of pathogenesis and myocardial ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Cytosolic CA의 측정^{2 +} 절연 수축성 Lymphatics에

요약

더 많은 비디오 탐색

이 비디오의 챕터

Cardiomyocytes의 빠른 칼슘 Fluxes 측정

절연, 문화, 그리고 성인 마우스 된 심근의 기능적 특성

격리된 가압된 림프관에서 Absolute, Cytosolic, Free Ca²⁺ 및 해당 수축성의 실시간 평가

격리된 가압된 림프관에서 Absolute, Cytosolic, Free Ca²⁺ 및 해당 수축성의 실시간 평가

격리된 가압된 림프관에서 Absolute, Cytosolic, Free Ca²⁺ 및 해당 수축성의 실시간 평가

유동 세포 계측법에 의한 신장 상피 세포에서 세포 내 칼슘 측정

마우스의 심장 수축력의 조사에 대 한 격리 유두 근육의 수축력 측정

배양 된 평활근 세포의 근질 그물 자리 내 칼슘 변동 FRET 측정을 기반으로 공 촛점 이미지

Myofilament 칼슘의 평가

골격 근육 생검에서 Myofibrils를 분리하고 나노 뉴턴 해상도 포스 트랜스듀서로 수축 기능을 결정합니다.