拍動する心臓の位相ターゲティング迅速凍結固定と組織学的解析による収縮状態依存性サルコメアダイナミクスの解明

The heart is a functional syncytium consisting of numerous cardiomyocytes that repetitively exhibit coordinated contractions/relaxations. However, the extent to which myocyte sarcomere arrangements in the heart differ across beats is unknown. To examine this, we conducted cardiac phase-targeting rapid cryofixation of Langendorff-perfused rat hearts. We adjusted the timepoint of cryogen exposure to the electrically paced heart and observed phase-dependent differences in the sarcomere length (SL) of subepicardial myocytes by α-actinin immunohistochemistry, namely a significantly shorter SL during systole than during diastole. We detected spatially inhomogeneous SL distributions by generating a heatmap of the myocardium. For peak systole the SL heatmap exhibited nearly uniform SL shortening within and among the individual myocytes with some myocardia exhibiting nonuniform SLs. During diastole, the heart showed predominant SL elongation, which was also accompanied by patchy distributions of locally short-SL regions, reflecting inhomogeneous SLs. This SL inhomogeneity was attenuated by pharmacological relaxation by 2,3-butanedione monoxime. The heatmap of the rapidly-frozen heart during ventricular fibrillation also revealed inhomogeneous SLs within and among individual myocytes. Overall, cardiac phase-targeting cryofixation unveiled in-depth behaviors on SL in the heart. Our cryofixation strategy will open a new horizon to clarify precise spatiotemporal changes in sarcomere structures and understand cardiac functions.