Myocardial Contraction

After 20–30 minutes of washout, gradual warming after cold cardioplegia to 37°C, tissue recovery, and stabilization, the wedges were stained with 4 μM di-4-ANEPPS (Molecular Probes, Eugene, OR), a membrane potential-sensitive fluorescent dye having no known electrophysiological effects and used widely in optical mapping studies in hearts of many species.
We immobilized the wedges with 10 μM Blebbistatin (Tocris Bioscience, Ellisville, MO) which inhibits the adenosine triphosphatases (ATPases) associated with class II myosin isoforms in an actin-detached state, and thus successfully blocks cardiac contraction without any effect on electrical activity, including ECG parameters, atrial and ventricular effective refractory periods, and atrial and ventricular activation patterns in many mammalian species.21 (link), 22 (link) We used microelectrode recordings to validate the effect of Blebbistatin in the human ventricle (see Online Supplement).
The wedges were paced at the endocardium by 5–10 ms pulses at 2 × diastolic current thresholds at a pacing cycle length (CL) ranging from 4,000ms to the ventricular functional refractory period. Two Ag/AgCl electrodes were immersed into the superfusion solution, one at the epicardial and the other at the endocardial side, to document the transmural pseudo-ECG (Fig. 1C).
An optical mapping system23 with a 100×100 pixels resolution MiCAM Ultima-L CMOS camera (SciMedia, USA Ltd., CA) collected the fluorescent light from an area of 2–3 cm by 2–3 cm (Fig. 1B) on the cut-exposed transmural surface of the wedge. Optical action potentials (APs) were recorded from the transmural optical field of view (20×20 to 30×30 mm²) with a spatial resolution of 200–300 μm/pixel at a rate of 1,000 frames/s (Fig. 1C). The fluorescent signals were amplified, digitized, and visualized during experiment using specialized software (SciMedia, USA Ltd., CA)

To facilitate volumetric coverage of the entire LV as well as relatively high spatial and temporal resolution compared to previous 3D myocardial tagging studies (10 (link)–13 (link)), a free-breathing navigator-gated method was employed for 3D cine DENSE data acquisition. Also, to minimize echo time (TE) and increase signal-to-noise ratio (SNR) compared to an echo-planar approach, data were sampled using a spiral k-space trajectory (16 ). The specific design details of the sequence are shown in Fig. 1. The navigator echo, formed using two orthogonal slice selective 90o and 180o RF pulses (17 (link)), was acquired at the end of the cardiac cycle, so as not to interfere with displacement encoding or imaging during the onset of myocardial contraction. The navigator data were used to accept or reject the DENSE data acquired in the subsequent heart beat. Immediately following R-wave detection and just before application of the initial displacement-encoding pulses, a fat suppression RF pulse was applied. The purpose of this pulse was to suppress the contribution of fat to the displacement-encoded magnetization to be stored along the longitudinal axis, resulting in fat suppression for stimulated-echo images throughout the cardiac cycle (18 (link)). The displacement-encoding module was applied next, and supported either simple or balanced multi-point displacement encoding (for optimal phase SNR (19 (link))), and two- or three-point phase cycling for suppression of additional artifact-generating echoes (20 ). The displacement-encoding module was followed by successive (multiphase) application of a readout module, which employed an interleaved stack-of-spirals trajectory to sample the 3D k-space after application of the DENSE unencoding gradients. DENSE displacement-encoding gradients were designed using the shortest possible time, and unencoding gradients were combined with phase-encoding gradients in the slice-select direction to minimize TE. Ramped flip angle was implemented to approximately equalize the SNR at all cardiac phases (21 ). Two 3D acquisitions with different TEs, where each used a single spiral interleave per 3D partition, were also acquired for field mapping and spiral off-resonance correction (deblurring).
Displacement-encoded phase images of the stimulated echo were reconstructed online. First, deblurring of the 3D stack-of-spirals data set was performed (22 ). Specifically, a Fourier transform (FT) was performed in the z direction, and then 2D off-resonance correction was performed partition-by-partition using linear fit coefficients calculated using the field maps (22 ). Two-dimensional in-plane gridding and FT were applied to the deblurred k-space data to obtain complex 3D image volumes. Cancellation of interference from artifact-generating echoes and isolation of the stimulated echo were achieved by combination of the phase cycling data sets (20 ). Decoding of the multi-point displacement-encoded data (19 (link)), integrated with phase-difference multi-coil reconstruction (23 ), was performed to obtain the displacement-encoded phase images in the x, y and z directions at each cardiac phase. The corresponding overall magnitude image at each cardiac phase was also calculated using the square root of the sum of the squares of stimulated echo data for all encoding directions and all coils.