Abstract 11948: A Standardized Method to Isolate and Purify Cardiomyocytes from Individual Mouse Hearts Irrespective of Postnatal Age

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Rational: Primary cardiomyocytes (CMs) are invaluable for understanding postnatal heart development and elucidating disease mechanisms in genetic and pharmacological models, however, a method to obtain freshly purified CMs at any postnatal age, without the need for different age-dependent isolation procedures and cell culture, is lacking. Objective: To develop a standardized method that allows rapid isolation and purification of CMs in high yield and viability from individual neonatal, infant, and adult mice. Methods and Results: Using a novel in situ aortic cannulation procedure optimized to allow cannulation of even the very small vessel of neonates [postnatal day 0-2 (P0-2)], hearts of C57BL/6J mice were subjected to Langendorff retrograde perfusion and enzymatic digestion. After subsequent tissue disaggregation and filtration, CMs were isolated in high yield (1.55-2.2 x 10 6 CMs/heart) and viability (~78%). The larger size of infant (P10 and P13) and adult (P70), but not neonatal CMs, compared to non-myocytes, allowed their further enrichment by differential centrifugation, before purification at the bench by bead-based immunomagnetic cell separation. This involved depletion of endothelial cells (for infant and adult preparations) or non-myocytes (neonatal preparations). Together, these procedures resulted in highly purified CMs (~95%) from hearts of all ages within 1 hour. Moreover, in situ fixation immediately after tissue digestion via coronary perfusion, preserved the cytoarchitecture of isolated CMs (~94% rod-shaped CMs at all postnatal ages), allowing capture of spindle-shaped neonatal cells undergoing mitosis, as well as enabling accurate quantitation of CM area and nucleation state. RNA-sequencing of CMs purified from one P2 male and female heart per litter (n=4 litters) showed distinct clustering by litters rather than by sexes; a finding consistent with cardiac size and shape being indistinguishable between P2 male (n=6) and female (n=6) hearts as determined by micro-computed tomography (X-ray 3D imaging). Conclusion: The procedures developed here provide a universal protocol for the rapid purification of high-quality CMs from individual hearts at any postnatal age, even those of neonates.

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