Analyzing T-tubule Structure in Cardiomyocytes

The hearts from AAV-infected mice were isolated and perfused with FM4-64FX and subjected to in situ confocal microscopy. The t-tubule content, integrity and regularity were next analyzed in ImageJ following previously described STAR Methods.^{79 (link)} Briefly, intact mouse hearts were Langendorff-perfused at room temperature with Tyrode’s solution (NaCl 137, KCl 5.4, HEPES 10, Glucose 10, MgCl₂ 1, NaH₂PO₄ 0.33, pH adjusted to 7.4 with NaOH, oxygenated with 95% O₂ and 5% CO₂ during experiments), containing 10 μM FM4-64FX, a lipophilic fluorescence indicator of membrane structure (Thermo Fisher), for 20 min. The membrane structure of epicardial myocytes was analyzed in situ with confocal microscope (STELLARIS 8, Leica Microsystems). T-tubule images were next analyzed with IDL image analysis program (ITT VIS Inc., Colorado). Background noise in confocal images was filtered with a threshold value retrieved from image intensity histogram. T-tubule two-dimensional images were converted to frequency domain using the Fast Fourier Transformation function in IDL, so that it could be determined whether repeating patterns occur (T-tubule regularity) and how strong the repeating patterns are (T-tubule power).