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.

SPOC (spontaneous oscillatory contraction) is a characteristic state of the contractile system of striated (skeletal and cardiac) muscle that exists between the states of relaxation and contraction. For example, Ca-SPOCs occur at physiological Ca levels (pCa ∼6.0), whereas ADP-SPOC occurs in the virtual absence of Ca (pCa ≥ 8; relaxing conditions in the presence of MgATP), but in the presence of inorganic phosphate (Pi) and a high concentration of MgADP. The concentration of Mg-ADP necessary for SPOC is nearly equal to or greater than the MgATP concentration for cardiac muscle and is several times higher for skeletal muscle. Thus, the cellular conditions for SPOC are broader in cardiac muscle than in skeletal muscle. During these SPOCs, each sarcomere in a myofibril undergoes length oscillation that has a saw-tooth waveform consisting of a rapid lengthening and a slow shortening phase. The lengthening phase of one half of a sarcomere is transmitted to the adjacent half of the sarcomere successively, forming a propagating wave (termed a SPOC wave). The SPOC waves are synchronized across the cardiomyocytes resulting in a visible wave of successive contractions and relaxations termed the SPOC wave. Experimentally, the SPOC period (and therefore the velocity of SPOC wave) is observed in demembranated cardiomyocytes and can be prepared from a wide range of animal hearts. These periods correlate well with the resting heartbeats of a wide range of mammals (rat, rabbit, dog, pig and cow). Preliminary experiments showed that the SPOC properties of human cardiomyocytes are similar to the heartbeat of a large dog or a pig. This correlation suggests that SPOCs may play a fundamental role in the heart. Here, we briefly summarize a range of SPOC parameters obtained experimentally, and relate them to a theoretical model to explain those characteristics. Finally, we discuss the possible significance of these SPOC properties in each and every heartbeat.

Background: Left ventricular wall motion is depressed in patients with dilated cardiomyopathy (DCM). However, whether or not the depressed left ventricular wall motion is caused by impairment of sarcomere dynamics remains to be fully clarified. Methods and Results: We analyzed the mechanical properties of single sarcomere dynamics during sarcomeric auto-oscillations (calcium spontaneous oscillatory contractions [Ca-SPOC]) that occurred at partial activation under the isometric condition in myofibrils from donor hearts and from patients with severe DCM (New York Heart Association classification III-IV). Ca-SPOC reproducibly occurred in the presence of 1 μmol/L free Ca 2+ in both nonfailing and DCM myofibrils, and sarcomeres exhibited a saw-tooth waveform along single myofibrils composed of quick lengthening and slow shortening. The period of Ca-SPOC was longer in DCM myofibrils than in nonfailing myofibrils, in association with prolonged shortening time. Lengthening time was similar in both groups. Then, we performed Tn (troponin) exchange in myofibrils with a DCM-causing homozygous mutation (K36Q) in cTnI (cardiac TnI). On exchange with the Tn complex from healthy porcine ventricles, period, shortening time, and shortening velocity in cTnI-K36Q myofibrils became similar to those in Tn-reconstituted nonfailing myofibrils. Protein kinase A abbreviated period in both Tn-reconstituted nonfailing and cTnI-K36Q myofibrils, demonstrating acceleration of cross-bridge kinetics. Conclusions: Sarcomere dynamics was found to be depressed under loaded conditions in DCM myofibrils because of impairment of thick-thin filament sliding. Thus, microscopic analysis of Ca-SPOC in human cardiac myofibrils is beneficial to systematically unveil the kinetic properties of single sarcomeres in various types of heart disease.

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.

The asynchronous, indirect flight muscles (IFM) of Drosophila are characterized by a high passive stiffness and exceptionally fast myosin kinetics, two attributes that enhance power output to sustain flight. Flightin is an IFM-specific, 20kDa myosin rod-binding protein required for normal thick filament stiffness, sarcomere integrity, and flight. Previously, we showed that a COOH-terminal truncation of flightin (fln DeltaC44 ) decreased myofilament lattice order and myosin kinetics, resulting in lower oscillatory power output and flightlessness. Here, we investigate the function of the flightin N-terminal 62 amino acids by creating transgenic Drosophila (fln DeltaN62 ) expressing a truncated flightin. fln DeltaN62 flies were flight impaired (flight index: 2.850.1 vs. 4.250.4 for fln DeltaN62 vs. fln þ rescued null control) despite having a normal wing-beat frequency (19554 vs. 19852 Hz for fln þ ). Mechanical analysis of skinned IFM fibers showed that the flightin N-terminal truncation reduced passive, active, and rigor stiffness without affecting cross-bridge kinetics (frequency of maximum power: 20557 vs. 21757 Hz for fln þ ). fln DeltaN62 fibers produced approximately half the isometric tension (passive: 0.950.1 vs. 1.750.3 kN/m 2 , active: 0.850.1 vs. 1.550.2 kN/m 2 , rigor: 1.150.2 vs. 3.150.4 kN/m 2 ) and maximum oscillatory power output (38.054.6 vs. 89.559.6 W/m 3 ) as fln þ fibers. Moreover, about 60% of the fln DeltaN62 fibers tore in rigor, demonstrating mechanical failure near isometric tension values that were sustained by fln þ fibers. Fourier transform analysis of crosssectional electron micrographs revealed that the flightin N-terminal truncation compromised myofilament lattice crystallinity and reduced inter-thick filament spacing by 10% (44.151.3 vs. 49.750.4 nm). These results indicate that the flightin N-terminal region enhances myofilament lattice order and mechanical integrity, which in turn is required for effective force transmission, normal oscillatory power output, and flight.

mm, P < 0.001). MCT n = 25 cells; CON n = 18 cells, 2 way RM ANOVA. We conclude that the shorter resting SL in MCT myocytes is due to the formation of Ca 2þ -independent cross-bridges, we speculate that these are formed in response to disturbances in cellular metabolism, by mechanisms currently under investigation. Supported by the BHF and MRC

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