Abstract
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.
Highlights
Recent advances in microscopic optics have enabled measurements of sarcomere length (SL) at high spatial and temporal resolution in living cultured or isolated cardiac cells (e.g., [1, 2])
An increase in the viral titer to 2 × 1012 particles per mL increased the fraction of the AcGFP-expressing myocytes to ∼20-30%; this caused marked shortening of sarcomeres (Figure 1(c); Table 1)
We found in the present study that CellMask was effective for the in vivo measurement of the T-tubular distance, via treatment on the left ventricle (LV) epicardial surface
Summary
Recent advances in microscopic optics have enabled measurements of sarcomere length (SL) at high spatial and temporal resolution in living cultured or isolated cardiac cells (e.g., [1, 2]). Efforts have been made to visualize and analyze the dynamics of sarcomeres as well as cardiomyocytes in the heart in vivo. In 2014, Aguirre and colleagues captured images of the fluorescence-labeled Ttubules in the mouse heart, via two-photon microscopy following reconstruction of the original images [4]. In order to solve the shortcomings of these previous studies, we recently developed a new microscopic system for highprecision measurements of SL in mice via expression of 훼actinin-AcGFP in the left ventricle (LV) [6] 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]).
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