In vivo cardiac nano-imaging: A new technology for high-precision analyses of sarcomere dynamics in the heart

Togo, Shinji; Hirokawa, Erisa; Kobirumaki-Shimozawa, Fuyu; Oyama, Kotaro; Shintani, Seine A.; Terui, Takako; Kushida, Yasuharu; Tsukamoto, Seiichi; Fujii, Teruyuki; Ishiwata, Shin’ichi; Fukuda, Norio

doi:10.1016/j.pbiomolbio.2016.09.006

Cited by 12 publications

(23 citation statements)

References 63 publications

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“…It is, however, more likely that collagenase treatment increased the accessibility of ADV to myocytes, resulting in an increase in the influx of Ca 2+ into the cells, based on the mechanism described above. It is well established that, in the heart, intracellular Ca 2+ overload in myocytes results in the occurrence of arrhythmias due to depolarization of cellular membranes via influx of Na + by means of the Na + -Ca 2+ exchanger ([5] and references therein). Therefore, although the collagenase treatment ( Figure 2 ) (or a large volume / a high viral particles of ADV) increases the expression fraction of AcGFP-expressing cardiomyocytes (in other words, enhances the ease of finding of fluorescently labeled cells), Ca 2+ -overloaded myocytes are likely to affect the heart's conductance system, thereby affecting the precision of the SL analysis.…”

Section: Discussionmentioning

confidence: 99%

“…The details of the microscopic system for in vivo nanoimaging have been described in our previous studies [5, 6]. 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 (EMCCD) 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 water immersion lens, either 60× (LUMPLFLN 60XW, N/A 1.00, Olympus Co.), 40× (LUMPLFLN 40XW, N/A 0.80, Olympus Co.), or 20× (XLUMPLFLN 20XW, N/A 1.00, Olympus Co.), and also a 2× lens (XLFluor 2X/340, N/A 0.14, Olympus Co.) were used to visualize the LV surface.…”

Section: Methodsmentioning

confidence: 99%

“…Nanoimaging was performed as in our previous studies [5, 6]. Namely, the anesthetized open-chest mouse under ventilation was placed on a custom-made microscope stage (200 × 250 mm 2 ), and the animal was warmed at 38°C throughout imaging.…”

Section: Methodsmentioning

confidence: 99%

“…In 2014, Aguirre and colleagues captured images of the fluorescence-labeled T-tubules in the mouse heart, via two-photon microscopy following reconstruction of the original images [4]. However, the confocal system combined with a two-photon microscope has substantial limitations in the imaging of consistently working cardiac muscle in vivo , because the image acquisition time is extremely long compared with the physiologically relevant heartbeat frequencies of rodents (as discussed in [5]). In order to solve the shortcomings of these previous studies, we recently developed a new microscopic system for high-precision measurements of SL in mice via expression of α -actinin-AcGFP in the left ventricle (LV) [6].…”

Section: Introductionmentioning

confidence: 99%

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

Kobirumaki-Shimozawa

Togo

Oyama

et al. 2018

BioMed Research International

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The present study was conducted to systematically investigate the optimal viral titer as well as the volume of the adenovirus vector (ADV) that expresses α-actinin-AcGFP in the Z-disks of myocytes in the left ventricle (LV) of mice. An injection of 10 μL ADV at viral titers of 2 to 4 × 1011 viral particles per mL (VP/mL) into the LV epicardial surface consistently expressed α-actinin-AcGFP in myocytes in vivo, with the fraction of AcGFP-expressing myocytes at ~10%. Our analysis revealed that SL was ~1.90-2.15 μm upon heart arrest via deep anesthesia. Likewise, we developed a novel fluorescence labeling method of the T-tubular system by treating the LV surface with CellMask Orange (CellMask). We found that the T-tubular distance was ~2.10-2.25 μm, similar to SL, in the healthy heart in vivo. Therefore, the present high-precision visualization method for the Z-disks or the T-tubules is beneficial to unveiling the mechanisms of myocyte contraction in health and disease in vivo.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Kobirumaki-Shimozawa

Togo

Oyama

et al. 2018

BioMed Research International

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“…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%

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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Impact of stretch on sarcomere length variability in isolated fully relaxed rat cardiac myocytes

Lookin,

Boulali,

Cazorla

et al. 2023

Pflugers Arch - Eur J Physiol

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The contractility of cardiac muscle is greatly affected by preload via the Frank-Starling Mechanism (FSM). It is based on the preload-dependent activation of sarcomeres -the elementary contractile units in muscle cells. Recent ndings show a natural variability in sarcomere length (SL) in resting cardiomyocytes that, moreover, is altered in an actively contracting myocyte. SL variability may contribute to the FSM but it remains unresolved whether the change in the SL variability is regulated by activation process per se or simply by changes in cell stretch, i.e. average SL.To separate the roles of activation and SL, we characterized SL variability in isolated fully relaxed rat ventricular cardiomyocytes (n = 12) subjected to a longitudinal stretch with the carbon ber (CF) technique. Each cell was tested in three states: without CF attachment (control, no preload), with CF attachment without stretch, and with CF attachment and ~ 10% stretch of initial SL. The cells were imaged by transmitted light microscopy to retrieve and analyze individual SL and SL variability off-line by multiple quantitative measures like coe cient of variation or median absolute deviation.We found that CF attachment without stretch did not affect the extent of SL variability and averaged SL.In stretched myocytes, the averaged SL signi cantly increased while the SL variability remained unchanged. This result clearly indicates that the non-uniformity of individual SL is not sensitive to the average SL itself in fully relaxed myocytes. We conclude that SL variability per se does not contribute to the FSM in the heart.

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In vivo cardiac nano-imaging: A new technology for high-precision analyses of sarcomere dynamics in the heart

Cited by 12 publications

References 63 publications

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

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

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

Impact of stretch on sarcomere length variability in isolated fully relaxed rat cardiac myocytes

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