Clin Res Cardiol (2023).

Role of microtubules in preserving the dynamic nanostructure of cardiomyocytes
J. Greiner1, D. Kaltenbacher1, T. Kok1, P. Kohl1, E. Rog-Zielinska1
1Institut für Experimentelle Kardiovaskuläre Medizin, Universitäts-Herzzentrum Freiburg - Bad Krozingen, Freiburg im Breisgau;
Structure and function of cardiomycytes (CM) are tightly interlinked. However, ultrastructural dynamics of CM during the cardiac action potential, including mechanical organelle deformation, are poorly understood. Dynamics of contracting CM are conventionally resolved using light microscopy, a modality with orders of magnitudes lower spatial resolution than electron-based imaging.

Here, we use action potential-synchronised high-pressure freezing to assess the ultrastructural dynamics during CM contraction with dual-axis electron tomography, resulting in a spatial resolution of (1.2 nm)3 and millisecond temporal resolution. CM were isolated from precision-cut left-ventricular rabbit tissue slices. We used pharmacological interventions (paclitaxel and colchicine) to stabilise and destabilise microtubules. CM were high-pressure-frozen at time intervals corresponding to rest and peak contraction, freeze-substituted, heavy metal-stained, resin-embedded, and cut into 200–300 nm sections. Then, CM fragments were imaged using electron tomography on a 300 kV transmission electron microscope. The resulting images were reconstructed and segmented utilising fully convolutional neural networks into 3D organelle models.

We developed custom software ('SegmentPuzzler') to proofread and correct automatic segmentations. Using this workflow, we generated 353 3D reconstructions of CM organelles, including the sarcoplasmic reticulum and the transverse-axial tubular system (Figure 1). We developed a portable, interactive browser-based visualization tool to foster a deeper comprehension of the otherwise unwieldy (TB-sized) image data and reconstructions. We analysed dyad dimensions, coupling distances, and deformation of the transverse tubular geometry in our reconstructions.

Our proof-of-principle study resolves the structural dynamics of CM in a nanoscopic, 3D, and millisecond-accurate manner. Precisely understanding the ultrastructure and its modulation thereof, ultimately in human CM under both physiological and pathophysiological conditions, is expected to advance our current understanding of the ultrastructural foundations of cardiac diseases, and their diagnosis and treatment.

Figure 1: Time-resolved 3D organelle model of the sarcoplasmic reticulum (yellow) and transverse-axial tubular system (green). Cardiomyocytes in this reconstruction were high-pressure-frozen in a relaxed state (0 ms offset to the action potential initiation). The reconstruction has a dimension of 3.14 µm x 3.14 µm x 180 nm and a voxel size of (1.2 nm)3.