No abstract

As the dynamic properties of cardiac sarcomeres are markedly changed in response to a length change of even ∼0.1 μm, it is imperative to quantitatively measure sarcomere length (SL). Here we show a novel system using quantum dots (QDs) that enables a real-time measurement of the length of a single sarcomere in cardiomyocytes. First, QDs were conjugated with anti-α-actinin antibody and applied to the sarcomeric Z disks in isolated skinned cardiomyocytes of the rat. At partial activation, spontaneous sarcomeric oscillations (SPOC) occurred, and QDs provided a quantitative measurement of the length of a single sarcomere over the broad range (i.e., from ∼1.7 to ∼2.3 μm). It was found that the SPOC amplitude was inversely related to SL, but the period showed no correlation with SL. We then treated intact cardiomyocytes with the mixture of the antibody-QDs and FuGENE HD, and visualized the movement of the Z lines/T tubules. At a low frequency of 1 Hz, the cycle of the motion of a single sarcomere consisted of fast shortening followed by slow relengthening. However, an increase in stimulation frequency to 3-5 Hz caused a phase shift of shortening and relengthening due to acceleration of relengthening, and the waveform became similar to that observed during SPOC. Finally, the anti-α-actinin antibody-QDs were transfected from the surface of the beating heart in vivo. The striated patterns with ∼1.96-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. Therefore, QDs are a useful tool to quantitatively analyze the movement of single sarcomeres in cardiomyocytes, under various experimental settings.

We here review the use of quantum dots (QDs) for the imaging of sarcomeric movements in cardiac muscle. QDs are fluorescence substances (CdSe) that absorb photons and reemit photons at a different wavelength (depending on the size of the particle); they are efficient in generating long-lasting, narrow symmetric emission profiles, and hence useful in various types of imaging studies. Recently, we developed a novel system in which the length of a particular, single sarcomere in cardiomyocytes can be measured at ~30 nm precision. Moreover, our system enables accurate measurement of sarcomere length in the isolated heart. We propose that QDs are the ideal tool for the study of sarcomere dynamics during excitation-contraction coupling in healthy and diseased cardiac muscle.

Laser irradiation has developed into a novel technique of non-invasive stimulation in cardiac and neural tissues. However, physical parameters for the laser irradiation-induced cardiac contractions have not been clarified, because various physicochemical reactions, such as photochemical and photothermal effects, are triggered in this process. Here we studied the effects of laserinduced local temperature changes on the functions of isolated cardiomyocytes. We demonstrated previously that a microscopic heat pulse (DT = 0.2 C for 2 sec) induces a Ca 2þ burst in cancer cells (HeLa cells) at a body temperature (Tseeb et al., HFSP J., 2009), with the mechanism similar to that of rapid cooling contracture in skeletal and cardiac muscles. In the present study, we generated microscopic heat pulses by focusing infrared laser light in extracellular solution near adult rat cardiomyocytes. We found that a microscopic heat pulse (DT = 5 C for 0.5 sec) induces contractions at basal temperature of 36 C. At 25 C, larger DT was required to induce contractions. When 2.5 Hz heat pulses were repeatedly applied, we observed oscillatory contractions of cardiomyocytes. Different from contractions induced by electric stimulation, Ca 2þ transients were not detected during the contraction. Likewise, heat pulses induced contractions of skinned cardiomyocytes in Ca 2þ-free solution in the presence of ATP. These results demonstrate that heat pulses can regulate cardiac contractions without any involvement of Ca 2þ dynamics, by directly activating the actomyosin interaction. Hence, our microheating technique may be useful for stimulating the beating of failing hearts without causing abnormal Ca 2þ dynamics.