Method, system and composition for optically inducing cardiomyocyte contraction
The present invention provides a method, system and composition for screening drug candidates for cardiotoxicity and for novel drugs that effect cardiomyocyte contractility and function. The invention provides an efficient and reliable screening assay to detect the effect of new and potential drug candidates on cardiomyocyte calcium flux, membrane depolarization, and/or the propagation of action potentials.
1. A method of screening a drug candidate for cardiotoxicity comprising:
(a) providing a cocultured sample, comprising:
(i) at least one cardiomyocyte cell, and
(ii) at least one non-cardiomyocyte cell selected from the group consisting of a HeLa cell, a CHO cell and NIH/3T3 cell, wherein the at least one non-cardiomyocyte cell contains a heterologous nucleic acid encoding Channelrhodopsin1 (ChR1) or Channelrhodopsin2 (ChR2) light sensitive protein in addition to a heterologous nucleic acid encoding a connexin protein selected from the group consisting of Cx23, Cx25, Cx26, Cx29, Cx30, Cx30.2, Cx30.3, Cx31, Cx31.1, Cx31.9, Cx32, Cx33, Cx36, Cx37, Cx39, Cx40, Cx40.1, Cx42, Cx45, Cx46, Cx47, Cx50, Cx59, Cx62, or a combination thereof;
(b) contacting the sample of (a) with a drug candidate;
(c) optically activating the light sensitive protein in the at least one non-cardiomyocyte cell to induce an action potential in the at least one cardiomyocyte cell and produce an efflux of calcium cations from the non-cardiomyocyte cell thereby optically inducing cardiomyocyte contraction with the at least one non-cardiomyocyte cell; and
(d) measuring an effect of the efflux of cations on the cardiomyocyte, thereby identifying the drug candidate as cardiotoxic, wherein the method does not use an electric current to stimulate contraction of the at least one cardiomyocyte cell.
2. The method of claim 1 , wherein the non-cardiomyocyte cell is a HeLa cell.
3. The method of claim 1 , wherein the cardiomyocyte and non-cardiomyocyte cell are connected via a plurality of gap junctions, and arranged as a monolayer.
4. The method of claim 1 , wherein the sample is cocultured for at least 1, 2, 3, 4, or 5 days before contacting the sample with the drug candidate.
5. The method of claim 1 , wherein the heterologous nucleic acid further comprises an inducible promoter.
6. The method of claim 1 , wherein the non-cardiomyocyte cell is stably transformed with the heterologous nucleotide.
7. The method of claim 1 , wherein, the ratio of cardiomyocyte to non-cardiomyocyte cells is greater than about 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 50:1, 100:1, 500:1 or 1000:1.
8. The method of claim 1 , wherein the effect is selected from the group consisting of cardiomyocyte action potential, cardiomyocyte intracellular calcium level, velocity of conduction, or a combination thereof.
9. The method of claim 8 , wherein the effect is cardiomyocyte action potential or velocity of conduction.