In Situ Measurements of Crossbridge Dynamics and Lattice Spacing in Rat Hearts by X-Ray Diffraction

Abstract

Background-Synchrotron radiation has been used to analyze crossbridge dynamics in isolated papillary muscle and excised perfused hearts with the use of x-ray diffraction techniques. We showed that these techniques can detect regional changes in rat left ventricle contractility and myosin lattice spacing in in situ ejecting hearts in real time. Furthermore, we examined the sensitivity of these indexes to regional ischemia. Methods and Results-The left ventricular free wall of spontaneously beating rat hearts (heart rate, 290 to 404 bpm) was directly exposed to brief high-flux, low-emittance x-ray beams provided at SPring-8. Myosin mass transfer to actin filaments was determined as the decrease in reflection intensity ratio (intensity of 1,0 plane over the 1,1 plane) between end-diastole and end-systole. The distance between 1,0 reflections was converted to a lattice spacing between myosin filaments. We found that mass transfer (mean, 1.71Ϯ0.09 SEM, nϭ13 hearts) preceded significant increases in lattice spacing (2 to 5 nm) during systole in nonischemic pericardium. Left coronary occlusion eliminated increases in lattice spacing and severely reduced mass transfer (PϽ0.01) in the ischemic region. Conclusions-Our results suggest that x-ray diffraction techniques permit real-time in situ analysis of regional crossbridge dynamics at molecular and fiber levels that might also facilitate investigations of ventricular output regulation by the Frank-Starling mechanism. Key Words: ischemia Ⅲ myocardial contraction Ⅲ myosin Ⅲ radiography D espite the history of studies on crossbridge dynamics, lower photon counts and poorer quality of diffraction patterns obtained from cardiac muscle than skeletal and insect flight muscles 1-3 have limited progress with cardiac muscle until recently. 4,5 Some of us used third-generation synchrotron radiation (SPring-8, Japan Synchrotron Radiation Research Institute) to determine x-ray diffraction patterns in excised, perfused rat hearts while moving systematically across the left ventricular (LV) equator from the epicardium through to the ventricular cavity. 6 X-ray diffraction patterns of cardiac muscle produce 2 equatorial-position reflections from the lattice-like arrangement of its protein elements. 2 Mass transfer of myosin heads to actin during contraction is inferred from a decrease in the integrated 1,0 reflection intensity (I 1,0 , lattice plane containing only thick myosin filaments) and an increase in 1,1 reflection intensity (I 1,1 , plane with thick myosin and thin actin filaments). 7 The myocardial intensity ratio (defined as I 1,0 /I 1,1 ) is minimal in the rigor state and maximal in a quiescent state. 1,2,6,8 Furthermore, the distance between 1,0 reflection peaks (d 1,0 spacing) represents the myosin lattice spacing, which is inversely related to sarcomere length in isolated fibers 5 as static myocytes maintain a constant cell volume. Whether decreases in myofilament spacing contribute to increasing Ca 2ϩ sensitivity and increased probability of crossbridge formation at longe...

Impact of osmotic compression on sarcomere structure and myofilament calcium sensitivity of isolated rat myocardium

“…Such a mechanism can explain the abrupt transitions that we observed in myofilament calcium sensitivity, I 1,1 /I 1,0 intensity ratio, and interfilament spacing at low levels of applied osmotic compression. A difficulty with this view, however, is that it conflicts with recent studies (30,39) that measured myofilament lattice spacing and I 1,1 /I 1,0 intensity ratio in whole hearts. Both studies reported a diastolic (relaxed) I 1,1 /I 1,0 intensity ratio of ϳ0.4, equivalent to the compressed patterns we observed in the skinned fibers, but the reported myofilament lattice spacing was very different between the two studies: ϳ42 nm (30) and ϳ37.5 nm (39).…”

Synchrotron Radiation Imaging for Advancing Our Understanding of Cardiovascular Function

“…By exposing a finely collimated SR X-ray beam (dimensions, 0.2×0.2 mm) to the whole heart, it is now possible to record the diffraction peaks in a myocardial region without averaging over multiple heart beats at a time resolution of 15 to 20 ms. [78][79][80] The time resolution is not limited by the available flux but mainly by radiation damage. Studies of a whole heart started with the isolated rat heart.…”

“…This initial study showed that the interfilament spacing does not differ significantly at different layers of the wall. 80 Studies of an isolated rat heart have expanded to more physiological studies of an open-chest in situ rat heart 78,79 and a closed-chest mouse heart. 81 The open-chest heart preparation allows the researcher to investigate regional differences in cross-bridge dynamics, because the myocardial regions exposed to the beam can be determined.…”

Clinical step onward with X-ray dark-field imaging and perspective view of medical applications of synchrotron radiation in Japan

“…Diffraction patterns were recorded for time resolved studies in isolated perfused hearts [24] and in situ [25]. Results suggest that it is possible to assign the origin of reflections in the diffraction diagram from the whole heart [24].…”

“…Results suggest that it is possible to assign the origin of reflections in the diffraction diagram from the whole heart [24]. Moreover, X-ray diffraction techniques permit real-time in situ analysis of regional crossbridge dynamics at the molecular and fiber levels for examination of regional ischemia [25].…”