54Detection, quantification and visualization of ryanodine receptor phosphorylation in human atrial myocytes using a novel ratiometric immunofluorescent analysis
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Purpose: Calcium release through the ryanodine receptor (RyR2) plays a central role in the regulation of cardiac contraction and rhythm, and is modulated by RyR2 phosphorylation. However, little is known about the distribution of phosphorylated RyR2s in isolated cardiac myocytes and the purpose of the present study was to develop an immunofluorescent ratio-metric approach to quantify and visualize the spatial distribution of phosphorylated RyR2s.
Methods: Forty-nine human atrial myocytes from nine patients were labeled with anti-phospho Ser-2808 (Red) and anti-RyR2 (Green) antibodies and detected automatically using a custom made algorithm based on: 1) Enhancing the contrast of both green and red-labeled images using a histogram stretching intensity transformation. 2) Removing background noise by using an adaptive median filter that estimates the noise level. 3) Enhancing the location of all green-labeled RyRs with a 2D Gaussian filter with a standard deviation of 0.5 microns followed by segmentation using a multilevel watershed algorithm. 4) Eliminating non-specific staining by setting the maximal RyR diameter to 1.2 microns. 5) Detection of red-labeled ser2808 phosphorylated RyR2s by checking if a cluster of red pixels was present at the location of each green labeled RyR2. Red clusters that overlapped at least 20% of the area of a green-labeled RyR2 were accepted if the normalized intensity of the overlapping area was above 15%.
Results: Superimposing detected RyR2s on the original confocal image revealed a detection efficiency near 95%, and visual inspection of superimposed original confocal images confirmed that all red ser2808 clusters coincided with a green RyR2. Moreover, stimulation of RyR2 phosphorylation with the beta-adrenergic agonist was used as a positive control, and it significantly increased the ser2808:total RyR2 ratio from 0.32±0.03 to 0.52±0.06 (p<0.01).
Conclusion: We have developed a novel immunofluorescent ratiometric approach that allows quantifying and visualizing RyR2 phosporylation in isolated human atrial myocytes. This approach may also apply to other proteins with a punctate distribution that are modulated by phosphorylation, and should be useful to study how disease affects the distribution of such proteins under basal or phosphorylating conditions.Keywords:
Atrial myocytes
Summary— Ro 22‐9194 reduced the Na + current in the atrial myocytes as well as ventricular myocytes in a tonic block fashion. Ro 22‐9194 had a higher affinity to the inactivated state Na + channels (Kd 1 = 3.3 μM in atrial myocytes, Kd 1 = 10.3 μM in ventricular myocytes) than to those in the rested state (Kd R = 91 μM in atrial myocytes, Kd R = 180 μM in ventricular myocytes), which indicated that Ro 22‐9194 had a higher affinity to the Na + channels in atrial myocytes than in ventricular myocytes. Ro 22‐9194 shifted the inactivation curve in the hyperpolarized direction in both atrial and ventricular myocytes. These findings suggest that Ro 22‐9194 more strongly inhibited the Na + channel of the atrial myocytes of the diseased hearts with the depolarized membranes potentials than the Na + channels in ventricular myocytes.
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Atrium (architecture)
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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.
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Cardiovascular physiology
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Objective To explore the optium method of isolating single human atrial myocytes and its effect factors.Methods Single human atrial myocytes from patients with various ages and heart diseases-pediatric congenital heart diseases(PCHD),coronary heart diseases(CHD),valvular heart diseases(VHD) were prepared with a two-step enzymatic method.These myocytes were observed by light microscope to show the shape,striation,refraction,quiescence and adherence and tested by the whole cell patch clamp technique to record muscarinic-gated atrial potassium currents(IK,Ach).Results The viability of the rod-shaped,striated,refractive,quiescent and adherent myocytes ranged from 30% to 50%.IK,Ach currents were recorded the highest in the myocytes from patients with PCHD(9/10 cells),second from patients with CHD(6/10 cells),third from patients with VHD(3/10 cells).Conclusion Two-step enzymatic method can be used to the isolation of single human atrial myocytes,the whole cell patch clamp technique is helpful to the identification of cell activity.The viability of single human atrial myocytes can be affected by enzymes disassemble,ages and kind of diseases.
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In canine right atrial hypertrophy, the cross-sectional area (Axs) of right atrial myocytes increases, whereas the Axs of the broader interatrial band myocytes does not. In the current study, myocyte reconstructions showed that right atrial myocyte length increased in proportion to Axs in right atrial hypertrophy. On the other hand, mean interatrial band myocyte length in both normal and right atrial hypertrophy dogs was roughly inversely proportional to mean Axs, as expected if interatrial band myocyte volume was constant. Plotting mean Axs vs. myocyte length for individual interatrial band myocytes revealed a distribution whose border defined a maximal volume curve; many myocytes were well beneath that curve. Mononuclear myocytes (generally diploid) were limited by a 65,000-micrometer 3 curve, which many binuclear myocytes (generally tetraploid) surpassed; myocyte ploidy thus constrained myocyte volume. However, because many mononuclear and binuclear myocytes had lower volumes, their failure to hypertrophy cannot be attributed to attainment of the maximal volume possible for their ploidy.
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目的将在心脏的 myocytes 在信号小径由浆液和在染色体十上删除的磷酸酶和 tensin 相当或相同的事物(PTEN ) 的角色导致了的有教养的老鼠的肥大上学习 simvastatin 的效果。方法有教养的新生的 Sprague-Dawley (SD ) 老鼠心脏的 myocytes 与 15% 胎儿的牛的浆液被对待，或没有 simvastatin 的浆液，或不同 consentrations。图象分析系统被用来测量心脏的 myocytes 表面区域。myocytes 的蛋白质合成被测量经由[3H ] 白氨酸加入方法。atrial natriuretic 的表示水平在 myocytes 的肽(ANP ) mRNA 与反向的抄写聚合酶链反应(RT-PCR ) 被决定。在心脏的 myocytes 的 PTEN 的 mRNA 和蛋白质表示层次分别地与 RT-PCR 和西方的污点被调查。在 24 个小时的结果，心脏的 myocytes 表面区域在 15% 浆液组是显著地更高的(1611.16
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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.
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Cardiovascular physiology
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