Electron microscopy

Pregnant dams were sacrificed at E9.5, 11.5, 13.5 and E16.5 by cervical dislocation and uterine segments were rapidly removed and dissected in EM fixative (4% paraformaldehyde, 2% glutaraldehyde, 0.1 M Na cacodylate) such that the embryo was exposed to fixative within 40 seconds of maternal cervical dislocation.⁴ Postnatal animals were sacrificed by the same method and E16.5 to adult hearts were rapidly perfused with EM fixative via 26-gauge syringe inserted into the left ventricle while clamping the ascending aorta with forceps. Specimens were stored in fixative at 4°C, rinsed in 0.1 M Na cacodylate, post-fixed in 1 % osmium tetroxide, dehydrated in a graded series of ethanol, embedded into EPON/Araldite resin and polymerized at 70°C for two days. Thin sections were cut with a diamond knife at 70nm, placed onto nickel slot grids and stained with aqueous uranyl acetate and lead citrate. The grids were examined using a Hitachi 7650 TEM with an attached Gatan 11 megapixel Erlangshen digital camera. Low power images were obtained to identify random left ventricular cardiac myocytes with the cell and contractile apparatus orientation a longitudinal orientation in more mature cells. All cells had at least one nucleus to ensure that a more central plane of the cell was examined. Higher power images were then taken to make a virtual montage of the entire cell for morphometry. Nine left ventricular myocytes were analyzed for each age and genotype (3 hearts x 3 myocytes per heart). However, for E11.5 and E13.5 hearts, analysis was performed on 9 cells from the myocardial wall and 9 from the trabeculae; a subgroup analysis found little difference between the two regions, so these data were combined for final analyses.

Both low- and high-power images were analyzed in Fiji (ImageJ 1.53f51) using a Cintiq tablet (Wacom, Saitama, Japan) to outline perimeters of the cell, nucleus, myofibrils, and individual mitochondria to measure shape descriptors, and these data were transferred into Excel spreadsheets for calculations and combining data from all cells at each age and genotype, as described.¹³ These measurements include mitochondrial area (μm²), perimeter (μm), aspect ratio (major axis/minor axis), form factor (perimeter²/4π x surface area), and Feret diameter (longest distance (μm) between any two points within a mitochondrion).

Mitochondria also underwent subgroup analysis to determine differences in cytoplasmic, intermyofibrillar, perinuclear, and subsarcolemmal mitochondria within and between ages/genotypes. Cytoplasmic mitochondria were defined as mitochondria not associated with nuclei or the sarcolemma and not between myofibrils and were only observed at earlier stages of development. Intermyofibrillar mitochondria were those that were surrounded by myofibrils or closely associated with myofibrils. Perinuclear mitochondria were defined as those touching nuclei or within the defined perinuclear region; if myofibrils enclosed this region, then mitochondria were considered intermyofibrillar when the “perinuclear” extension narrowed to <2 mitochondria in width. Similarly, subsarcolemmal mitochondria were defined as lying close to the sarcolemma, bounded intracellularly by myofibrils and/or nuclei, but mitochondria near the sarcolemma were considered intermyofibrillar using the same definition as above for the boundary of perinuclear and intermyofibrillar mitochondria.