Optimization of Fluorescent Labeling for<i> In Vivo</i> Nanoimaging of Sarcomeres in the Mouse Heart

Kobirumaki-Shimozawa, Fuyu; Togo, Shinji; Oyama, Kotaro; Kushida, Yasuharu; Terui, Takako; Ishiwata, Shin’ichi; Fukuda, Norio

doi:10.1155/2018/4349170

Cited by 5 publications

(16 citation statements)

References 13 publications

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“…First, we confirmed that the myocardial membrane systems, including the intercellular junction, sarcolemma, and T-tubules, can be visualized by CellMask in the LV of the arrested mouse heart in vivo (Figure 2A, Movie S1) (as in References [24,25]). Given the nature of CellMask in that it enables fast and uniform labeling of the plasma membrane in virtually all living cells, it is reasonable to consider that the CellMask-stained ID in Figure 2A consists of the GJ, perinexii, adherens junctions, and desmosomes (see Figure 1A and Introduction).…”

Section: Resultssupporting

confidence: 68%

“…Myocardial membranes, including T-tubules, were stained by CellMaskTM Orange (CellMask, Life Technologies Inc., Tokyo, Japan) according to the method used in our previous study [24,25]. CellMask was dissolved in PBS (-) (termed "CellMask solution"), and administered to anesthetized open-chest, temperature-regulated mice (see below) via a small piece of gauze placed on the left ventricular (LV) surface.…”

Section: Cellmask Treatment In the Heart Of Living Mice In Vivomentioning

confidence: 99%

“…The adenovirus vector (ADV) was constructed based on our previous studies [25,26]. Recombinant adenoviruses encoding mouse α-actinin-3-AcGFP (Genbank/EMBL/DDBJ accession no.…”

Section: α-Actinin-acgfp Expression In the Heart Of Living Mice In Vivomentioning

confidence: 99%

“…The details of the microscopic system for in vivo nano-imaging have been described in our previous studies [25][26][27]. In brief, an upright microscope (BX-51WI, Olympus Co., Tokyo, Japan) combined with a Nipkow confocal scanner (CSU21, Yokogawa Electric Co., Tokyo, Japan) and an electron multiplying CCD camera (iXonEM+, Andor Technology Ltd., Belfast, Northern Ireland) were used at a 512 × 512 (or 512 × 170) pixel resolution at an exposure time of 28 (or 9.8) ms. A 60× water immersion lens (LUMPLFLN 60 × W, N/A 1.00, Olympus Co., Tokyo, Japan) was used to visualize the LV surface.…”

Section: Microscopic Systemmentioning

confidence: 99%

“…Nano-imaging was performed in accordance with our previous studies [25][26][27]. The anesthetized and ventilated open-chest mouse was placed on a custom-made microscope stage (250 × 350 mm), and the animal was warmed to 38 • C throughout imaging.…”

Section: In Vivo Nano-imagingmentioning

confidence: 99%

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Real-Time In Vivo Imaging of Mouse Left Ventricle Reveals Fluctuating Movements of the Intercalated Discs

et al. 2020

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Myocardial contraction is initiated by action potential propagation through the conduction system of the heart. It has been thought that connexin 43 in the gap junctions (GJ) within the intercalated disc (ID) provides direct electric connectivity between cardiomyocytes (electronic conduction). However, recent studies challenge this view by providing evidence that the mechanosensitive cardiac sodium channels Nav1.5 localized in perinexii at the GJ edge play an important role in spreading action potentials between neighboring cells (ephaptic conduction). In the present study, we performed real-time confocal imaging of the CellMask-stained ID in the living mouse heart in vivo. We found that the ID structure was not rigid. Instead, we observed marked flexing of the ID during propagation of contraction from cell to cell. The variation in ID length was between ~30 and ~42 μm (i.e., magnitude of change, ~30%). In contrast, tracking of α-actinin-AcGFP revealed a comparatively small change in the lateral dimension of the transitional junction near the ID (i.e., magnitude of change, ~20%). The present findings suggest that, when the heart is at work, mechanostress across the perinexii may activate Nav1.5 by promoting ephaptic conduction in coordination with electronic conduction, and, thereby, efficiently transmitting excitation-contraction coupling between cardiomyocytes.

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Section: Resultssupporting

confidence: 68%

Section: Cellmask Treatment In the Heart Of Living Mice In Vivomentioning

confidence: 99%

“…The adenovirus vector (ADV) was constructed based on our previous studies [25,26]. Recombinant adenoviruses encoding mouse α-actinin-3-AcGFP (Genbank/EMBL/DDBJ accession no.…”

Section: α-Actinin-acgfp Expression In the Heart Of Living Mice In Vivomentioning

confidence: 99%

Section: Microscopic Systemmentioning

confidence: 99%

Section: In Vivo Nano-imagingmentioning

confidence: 99%

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Real-Time In Vivo Imaging of Mouse Left Ventricle Reveals Fluctuating Movements of the Intercalated Discs

et al. 2020

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Synchrony of sarcomeric movement regulates left ventricular pump function in the in vivo beating mouse heart

Kobirumaki-Shimozawa

Togo

Oyama

et al. 2021

Journal of General Physiology

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Sarcomeric contraction in cardiomyocytes serves as the basis for the heart’s pump functions. It has generally been considered that in cardiac muscle as well as in skeletal muscle, sarcomeres equally contribute to myofibrillar dynamics in myocytes at varying loads by producing similar levels of active and passive force. In the present study, we expressed α-actinin–AcGFP in Z-disks to analyze dynamic behaviors of sequentially connected individual sarcomeres along a myofibril in a left ventricular (LV) myocyte of the in vivo beating mouse heart. To quantify the magnitude of the contribution of individual sarcomeres to myofibrillar dynamics, we introduced the novel parameter “contribution index” (CI) to measure the synchrony in movements between a sarcomere and a myofibril (from −1 [complete asynchrony] to 1 [complete synchrony]). First, CI varied markedly between sarcomeres, with an average value of ∼0.3 during normal systole. Second, when the movements between adjacent sarcomeres were asynchronous (CI < 0), a sarcomere and the ones next to the adjacent sarcomeres and farther away moved in synchrony (CI > 0) along a myofibril. Third, when difference in LV pressure in diastole and systole (ΔLVP) was lowered to <10 mm Hg, diastolic sarcomere length increased. Under depressed conditions, the movements between adjacent sarcomeres were in marked asynchrony (CI, −0.3 to −0.4), and, as a result, average CI was linearly decreased in association with a decrease in ΔLVP. These findings suggest that in the left ventricle of the in vivo beating mouse heart, (1) sarcomeres heterogeneously contribute to myofibrillar dynamics due to an imbalance of active and passive force between neighboring sarcomeres, (2) the force imbalance is pronounced under depressed conditions coupled with a marked increase in passive force and the ensuing tug-of-war between sarcomeres, and (3) sarcomere synchrony via the distal intersarcomere interaction regulates the heart's pump function in coordination with myofibrillar contractility.

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Cardiomyocyte sarcomere length variability: Membrane fluorescence versus second harmonic generation myosin imaging

Lookin

Tombe

Boulali

et al. 2023

Journal of General Physiology

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Sarcomere length (SL) and its variation along the myofibril strongly regulate integrated coordinated myocyte contraction. It is therefore important to obtain individual SL properties. Optical imaging by confocal fluorescence (for example, using ANEPPS) or transmitted light microscopy is often used for this purpose. However, this allows for the visualization of structures related to Z-disks only. In contrast, second-harmonic generation (SHG) microscopy visualizes A-band sarcomeric structures directly. Here, we compared averaged SL and its variability in isolated relaxed rat cardiomyocytes by imaging with ANEPPS and SHG. We found that SL variability, evaluated by several absolute and relative measures, is two times smaller using SHG vs. ANEPPS, while both optical methods give the same average (median) SL. We conclude that optical methods with similar optical spatial resolution provide valid estimations of average SL, but the use of SHG microscopy for visualization of sarcomeric A-bands may be the “gold standard” for evaluation of SL variability due to the absence of optical interference between the sarcomere center and non-sarcomeric structures. This contrasts with sarcomere edges where t-tubules may not consistently colocalize to Z-disks. The use of SHG microscopy instead of fluorescent imaging can be a prospective tool to map sarcomere variability both in vitro and in vivo conditions and to reveal its role in the functional behavior of living myocardium.

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Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart

Cited by 5 publications

References 13 publications

Real-Time In Vivo Imaging of Mouse Left Ventricle Reveals Fluctuating Movements of the Intercalated Discs

Real-Time In Vivo Imaging of Mouse Left Ventricle Reveals Fluctuating Movements of the Intercalated Discs

Synchrony of sarcomeric movement regulates left ventricular pump function in the in vivo beating mouse heart

Cardiomyocyte sarcomere length variability: Membrane fluorescence versus second harmonic generation myosin imaging

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