Fast Measurement of Sarcomere Length and Cell Orientation in Langendorff-Perfused Hearts Using Remote Focusing Microscopy

Abstract

Objective: We developed a method for measuring SL and regional cell orientation using remote focusing microscopy, an emerging imaging modality that can capture light from arbitrary oblique planes within a sample. Methods and Results:We present a protocol that unambiguously and quickly determines cell orientation from user-selected areas in a field of view by imaging 2 oblique planes that share a common major axis with the cell. We demonstrate the effectiveness of the technique in establishing single-cell SL in Langendorff-perfused hearts loaded with the membrane dye di-4-ANEPPS. Conclusions:

Attenuated sarcomere lengthening of the aged murine left ventricle observed using two-photon fluorescence microscopy

“…2D) and endocardial to epicardial regional differences (11,28,30). The mean L s values determined for subepicardial cardiomyocytes in our preparation at 0 mmHg (young, 2.01 m; and aged, 2.02 m) fit well within the range (1.93 to 2.09 m) determined in unpressurized intact rat hearts (5,6) or fixed hearts maintained at 0 mmHg filling pressure (11,28,30). Upon left ventricular filling, the pressure versus L s relationship of hearts from young mice studied here (0 -40 mmHg L s range, 2.01 to 2.36 m) was also similar to that reported in classic measurements of dog hearts fixed ex vivo (0 -30 mmHg L s range, 1.93 to 2.33 m) (30) and from reconstructions of L s dynamics in vivo at physiological filling pressures (28).…”

“…Angle of the longitudinal cell plane relative to the imaging plane, particularly with respect to rotation around the middle (width) axis of the cell, affects apparent L s as described in detail by Bub et al (6) and Botcherby et al (5). To determine this angular component, we performed optical sectioning (z sectioning) of subepicardial cardiomyocytes over a distance of 20 -25 m (section thickness of 1 or 2 m) to determine the number of optical sections separating longitudinal edges of cardiomyocytes.…”

“…For example, during submaximal Ca 2ϩ activation of isolated cardiomyocytes (7,9,13) or trabecular/papillary muscle preparations (17,19,32), contractile force can increase severalfold through the range in L s from ϳ1.70 to ϳ2.20 m. While force production as a function of L s has been well defined in isolated cardiac muscle preparations, there is a paucity of information concerned with how left ventricular filling pressure alters L s of cardiomyocytes in the intact living heart. Fluorescent labeling of the t-tubule network gives accurate indication of z-line spacing and therefore L s in cardiomyocytes of the rat heart (5,6). However, these latter studies examined L s in the absence of ventricular filling pressure.…”

Optimization of Fluorescent Labeling for In Vivo Nanoimaging of Sarcomeres in the Mouse Heart

“…However, taking into consideration the careful analysis by the group of Bub [10, 11] combined with our SL analysis based on the Gaussian fitting, the overestimation is ~2% in the present study, because the length of in-focus images in the image plane (i.e., L min in [10, 11]) was longer than ~70  μ m (e.g., Figures 1(a) and 1(b) ). It can therefore be considered that the influence of the myocyte angle on SL is practically negligible in the present experimental setting.…”

Intravital imaging of cardiac function at the single-cell level

“…Intravital confocal and two-photon microscopy have been used in combination with fluorescence molecular imaging probes in cancer research, immunology, and the neurosciences to reveal biological processes at the cellular level in living organisms (2). Application of intravital techniques for imaging the beating heart in rodent models has been significantly limited by motion from cardiac contraction and respiration, and most studies as a result have used noncontracting Langendorf heart preparations (3)(4)(5)(6)(7)(8)(9)(10) or transplanted heart models (11). These model systems do not allow investigation of cardiomyocyte biology in the native heart under physiologic conditions.…”