Research Project - Details

Analyzing signalling pathways and contractile functions using xCelligence

Although cardiac function of gene targeted animals can be assessed in vivo through echocardiography analysis, or on the organ level by Langendorff systems, investigating contractile parameters on the cellular level is usually limited by small sample size and high variability: e.g. electrophysiological studies of single cardiomyocytes using patch clamp techniques, measurement of calcium transients in single cells, or force measurements on isolated, skinned cardiac myocytes. In collaboration with Acea we are using the xCELLigence cardio-system to characterize contractile parameters of neonatal mouse cardiomyocyte cultures derived from gene knockout models in a high throughput, high replicate fashion.

Using the system

Cardiomyocytes of newborn mice were isolated and plated into cardio E-plates. Pilot experiments established optimal culture conditions. You can download a quick protocol here and view the isolation procedure on JOVE.

Variables for the optimization process:
  1. preplating for removal of fibroblasts
  2. coating conditions
  3. cell-density
  4. addition of proliferation inhibitors
  5. addition of chronotropic agents
Immunofluorescence analysis of neonatal cardiomyocytes cultured on xCELLigence cardio system plates confirms the growth of the cells and the successful re-establishment of the contractile apparatus.

IF image of cardiomyocytes on electrodes Immunofluorescence image of cardiomyocytes cultured on electrodes to record impedance changes. Fixed cells were stained with antibodies against alpha-actinin (red in overlay, cardiomyocyte marker), filamentous actin (green) and DAPI (blue, nuclei). Electrodes are outlined in white.
Brightfield microscopy video of cells cultured on xcell-eplates.

Impedance measurements

The beating behaviour of live cardiomyocytes is recorded label-free in the xCelligence cardio system . Impedance traces are later analysed for several parameters, like beating frequency or amplitude. Because cells are plated in 96-well plates, multiple conditions (cell-density, control vs. knockout cells, addition of small molecule activators/inhibitors) can be tested simultaneously.
For more information, go to Acea.

impedance trace (cell index)
Impedance traces of beating cardiomyocytes.

Project outlook

With our partners at Acea, we used the xCelligence system to achieve the following objectives:

  1. Analysis of wildtype and gene targeted neonatal knockout cardiomyocytes using the xCELLigence Cardio System to investigate early changes to contractile functions
  2. Characterization of small molecule inhibitors that are able to prevent or reverse pathological changes to cardiac functions
Results from this collaboration were presented at the Biophysics Conference in San Francisco (Feb 15-19, 2014).

Related manuscripts

  • MLP and CARP are linked to chronic PKCα signalling in dilated cardiomyopathy. Lange S, Gehmlich K, Lun AS, Blondelle J, Hooper C, Dalton ND, Alvarez EA, Zhang X, Bang ML, Abassi YA, Dos Remedios CG, Peterson KL, Chen J, Ehler E. Nat Commun. 2016 Jun 29;7:12120. doi: 10.1038/ncomms12120. PMID: 27353086