A modification of retrograde Langendorff-perfusion to increase the yield of Ca2+-tolerant murine atrial cardiomyocytes isolated from remodelled, fibrotic atria.

OBJECTIVE

Langendorff-perfusion is an established method to isolate cardiomyocytes from cardiac tissue for the investigation of cellular remodelling in cardiovascular diseases. Despite the existence of several protocols for isolation of murine atrial cardiomyocytes (aCM), extraction of a sufficient yield of Ca2+-tolerant aCM from remodelled, fibrotic murine atria with established protocols remains challenging. Here, we evaluate if a novel, simple modification of an established Langendorff-based protocol improves the isolation of aCM.

METHODS

The effect of an additional ventricular ligature during Langendorff-perfusion on the yield of isolated aCM was evaluated in mice with cardiomyocyte-specific overexpression of CREM-IbΔC-X, a mouse model of pronounced atrial dilatation and spontaneous onset atrial fibrillation compared to littermate controls.

RESULTS

Atria with overexpression of CREM-IbΔC-X exhibited dilatation, increased mass and enhanced fibrosis. From such remodelled atria, we observed significantly lower extractions both of total and morphologically intact aCM compared to wildtype. The additional ventricular ligature did not affect total aCM yield in wildtype, but in fibrotic atria; the total aCM count was significantly increased (26 ± 4 vs. 15 ± 3 aCM/10 µL). Importantly, the number of morphologically intact, Ca2+-tolerant aCM available for functional assessment from fibrotic atria was doubled with the modified protocol.

CONCLUSION

The modification of an established Langendorff-perfusion protocol for aCM isolation by an additional ventricular ligature is associated with a numerically higher yield of morphologically intact, Ca2+-tolerant aCM from remodelled atria. This may facilitate research on cellular electrophysiology in cardiomyocytes of murine models with atrial remodelling.