Cardiomyocyte contractility measurements

Left ventricular cardiomyocytes were isolated from cMyBP-C AAA, cMyBP-C DDD, and the control cMyBP-C transgenic tWt mice. Following heart excision, the aorta was cannulated on a Langendorff apparatus (Harvard Apparatus). The heart was perfused retrograde with Tyrode buffer (in mmol/L: 140 NaCl, 5.4 KCl, 0.33 NaH2PO4, 0.5 MgCl₂, 5 HEPES, and 11 D-glucose, pH 7.4) at 2.5 ml/min, 37°C for 4 minutes. The heart was then perfused with enzyme solution using the adult mouse cardiomyocyte isolation kit following the manufacturer’s instructions (Cellutron Life Technologies, ac-7034). After digestion, the heart was removed from the cannula and the left ventricle was gently teased apart. Cell suspensions were obtained by gentle pipetting. After spinning, cardiomyocytes were transferred to Tyrode buffer with Ca²⁺.

Cardiomyocyte contractility was measured using an IonOptix contractility system. Myocytes were loaded in the perfusion chamber of the microscope stage filled with perfusion buffer (Tyrode buffer with 1.8 mmol/L CaCl₂, 0.01% DMSO). After stabilization, the myocytes were perfused with perfusion buffer at room temperature. They were field stimulated with 12 V at 1.0 Hz with a 1.0 milliseconds pulse width using a pair of platinum wires placed on opposite sides of the chamber connected to a MyoPacer stimulator. Myocytes perfused with perfusion buffer were recorded at baseline, then perfused with compound for 3-4 minutes. Analysis was performed using the IonWizard software (IonOptix). For each test, an average of 10-15 sarcomere shortening traces was analyzed. Cells were selected that were beating and appeared grossly normal. Cells were not considered for recording if they were not contracting or, in very rare instances, appeared to be exhibiting an abnormally large reduction in sarcomere length (an abnormally large contraction). All recorded cells were analyzed. Cells with poor data quality were excluded. The remaining cells were recorded and exhibited a 7%-15% reduction in sarcomere length relative to its resting sarcomere length at baseline before treatments were given. Cells were isolated from 2 to 4 animals for each group. Neither the genotype nor the treatment disproportionately influenced cell exclusion.