Real-time imaging of cardiac muscle in vivo

A number of studies have been conducted in tissues and cells to elucidate the molecular mechanisms of myocardial contraction. However, because of many differences between in vitro and in vivo conditions, the dynamics of myocardial sarcomere contractions in living animals is not yet understood. In the present study, we developed a novel system allowing us to conduct real-time imaging of single sarcomeres in the beating heart in vivo. First, anti-α-actinin antibody-quantum dots (QDs) were transfected from the surface of the beating heart of the rat in vivo. The striated patterns with ~2.00 μm intervals were observed after perfusion under fluorescence microscopy, and an electron microscopic observation confirmed the presence of QDs in and around the T-tubules and Z-disks, but primarily in the T-tubules, within the first layer of cardiomyocytes of the left ventricular wall. Then, we expressed GFP at sarcomeric Z-disks (α-actinin) by using the adenovirus vector system in living mice, and conducted real-time imaging of the movement of single sarcomeres under fluorescence microscopy. SL was found to be ~2.00 μm in the isolated heart during diastole. This value is close to what was obtained previously by others in rats under a similar experimental condition using various experimental techniques, i.e., in X-ray diffraction (Yagi et al., 2004) and two-photon imaging (Bub et al., 2010). Moreover, we successfully observed striations of cardiac muscle and measured the length of single sarcomeres in the open-chest mouse under anesthesia at 10 nm precision. It was found that SL was ~1.70 and ~2.00 μm during systole and diastole, respectively.

Likewise, we demonstrated that microscopic heat pulses induced contraction in intact cardiomyocytes of the rat. The temperature increase, ΔT, required for inducing contraction of cardiomyocytes was dependent upon the ambient temperature; that is, ΔT at physiological temperature was lower than that at room temperature. Ca2+ transients were not detected during the course of contraction. We confirmed that the contractions of skinned cardiomyocytes were induced by the heat pulses even in Ca2+-free solutions. We consider that this heat pulse-induced Ca2+-decoupled contraction technique has the potential to stimulate heart and skeletal muscles in a manner different from the conventional electrical stimulations.

Publications

Higuchi S, Tsukasaki Y, Fukuda N, Kurihara S, Fujita H., Thin filament-reconstituted skinned muscle fibers for the study of muscle physiology. J Biomed Biotechnol., 2011;2011:486021.
Oyama K, Mizuno A, Shintani SA, Itoh H, Serizawa T, Fukuda N, Suzuki M, Ishiwata S., Microscopic heat pulses induce contraction of cardiomyocytes without calcium transients. Biochem Biophys Res Commun., 2012;417:607.
Kobirumaki-Shimozawa F, Oyama K, Serizawa T, Mizuno A, Kagemoto T, Shimozawa T, Ishiwata S, Kurihara S, Fukuda N., Sarcomere imaging by quantum dots for the study of cardiac muscle physiology. J Biomed Biotechnol., 2012 in press.