Third party funded individual grant
Start date : 01.01.2012
End date : 31.12.2013
The major aim of this project is to clarify to which extent MS channels in mammalian cardiomyocytes modulate both global and microdomain Ca2+ signalling properties during mechanical performance in health and disease states. Towards this aim we will: (i) record Ca2+ transients in intact mammalian cardiomyocytes during external field stimulation while contracting using Ca2+ fluorescence microscopy. Rat cardiomyocytes will be paced with external platinum wires at frequencies between 1 Hz and 3 Hz after loading cells with Ca2+ dyes (Fluo-4). Ca2+ transients will be recorded using confocal or multiphoton microscopes available; (ii) record confined Ca2+ spark activity in resting cardiomyocytes that are being stretched to up to 130% slack length using a novel single cardiomyocyte cell stretcher and force transducer. Passive elasticity modulus will be simultaneously obtained from these recordings. Ca2+ spark frequency and morphology under stretch conditions will be analyzed using automated image processing algorithms; (iii) determine the expression levels of transient receptor potential channels (TRPC1, TRPC3, TRPC6), all very likely to function as mechanosensitive channels (MSC) in the heart; (iv) record Ca2+ transients/sparks in the presence and absence of the specific blocker of MSC, GsMTx-4 (5 µM of the blocker) or commercially available pore-blocking anti-TRPC1, anti-TRPC3, anti-TRPC6 antibodies; (v) separate the impact of GsMTx-4 on external Ca2+-influx and intracellular Ca2+ release. This will reveal for the first time the relative contribution of MSC to physiological Ca2+ homeostasis. The specific anti-TRPC blocking antibodies will identify the channel in question; (vi) apply the recordings in (i)-(v) also to cardiomyocytes from septic rats that have received a non-lethal LPS challenge to clarify whether MSC, i.e. TRPC, are involved in septic cardiomyopathy.