| Popis: |
Optogenetics is a fast-developing technology, first applied to neuroscience research. A number of studies have begun to transfer the optogenetic approach to cardiovascular research, with a focus on electrical regulation of specific cell types in the heart. There is demand to develop tools to selectively modulate electrical activity in cardiomyocytes (CM) and to interfere with membrane currents in non-myocytes (NM) to advance our understanding of heterocellular-electrotonic coupling in vitro and in vivo. We here report on cell-type specific activation of co-cultured CM and NM using the light-gated cation channel ChR2. Furthermore, we tested three anion-selective channelrhodopsins (ACR) for their potential to inhibit action potential initiation and propagation.Neonatal hearts of the lines WT1-VSFP+;+, αMHC-VSFP+;+ and WT1-ChR2-H134R+;+ were isolated and digested. Cultured cells were transfected with cDNA of ACRs coupled to fluorescent marker proteins.In order to analyse heterocellular coupling we performed whole-cell patch-clamp recordings on CM cocultured with NM from the WT1-ChR2-H134R+;+ line. In a first set of experiments, pulsed ChR2 activation in NM evoked action potentials in patched CM, indicating direct electrical coupling.Next, we tested three different ACR for their potential to hyperpolarise cardiac cells (GtACR1-eGFP, iC++ mCherry, Phobos-mCherry). Transfected cells were patch-clamped in the current-clamp mode to follow light-induced changes in membrane voltage. Notably, ACR activation lead to hyperpolarisation in isolated cardiac NM and to depolarisation in CM.We would like to inhibit cardiac electrical activity by optogenetic hyperpolarisation of NM. As we have shown ACR photocurrents depolarise CM and evoke action potentials, different to the inhibitory effect reported in neurons. We expect that NM, which are connected to CM, have more negative potentials, resulting in depolarising ACR currents also in NM. Therefore, we are looking for a stronger tool to inhibit cardiac activity and plan to generate a light-activated K+-channel. |
