Third party funded individual grant
Start date : 01.01.2016
End date : 31.12.2017
The heart provides constant pumping activity and its cells are exposed to large tensile strain challenges during systolic pumping but moreover, during diastolic filling. In order to balance mechanical wall tension, pathological conditions of hypertension or increased afterload are answered by chronic organ and cellular remodeling, i.e. hypertrophy. Hypertrophic signaling in cardiomyocytes (CM) is complex and involves upregulation of mechanosensitive channels MSC, of which canonical transient receptor potential channels (TRPC) are crucial in mammals. It is assumed that aberrant activation of MSCs in general, and of TRPCs in particular, trigger Ca2+ influx and Ca2+-mediated hypertrophic signaling that leads to hypertrophy, but also to life-threatening arrhythmias due to Ca2+ overload. The link between TRPC channels, their definite mechanosensitivity, and Ca2+ entry during systole and diastole in heart failure has not been experimentally established, mainly because adequate mechatronics systems were missing. We have recently built a novel, isotropic cell stretch device to be used in conjunction with automated Ca2+ fluorescence microscopy during defined applied cellular stretch to single CMs. This novel device will be implemented in this collaborative project to:
1) perform surface tracking and fluo-4 Ca2+ fluorescence recordings of putative Ca2+ entry in single CMs coated on PDMS membranes subjected to isotropic radial stretch between 0% and 20%
2) perform experiments as in (1) in normal mouse CMs and CMs from a relevant cardiac hypertrophy model (transaortic constriction, TAC) as well as in an immortalised murine cell line of ventricular cardiomyocytes (HL-1)
3) study hypertrophic signaling in single CMs from control and TAC mice establishing TRPC isoforms (Western blots, Immuno-fluorescence), resting Ca2+ levels and Ca2+ transients during field-stimulation
4) evaluate isotropic stretch-induced Ca2+ entry in TAC-CMs towards mechanosensitivity using specific MSC blockers (GsMTx-4) and TRPC blockers (e.g. OAG)
5) assess a potential crosstalk between endothelium and ventricular CMs by pre-conditioning endothelial cells with various stretch regimes and assessing the mechano-response of Ca2+ entry in CMs acutely challenged with supernatant from such endothelial cells
Through the application of both active (field-stimulation) and passive (isotropic stretch) regimes in mechanically challenged CMs, the contribution of MSC towards early Ca2+-driven hypertrophic signaling will be clarified for the first time.