METHOD, DEVICE AND KIT FOR DETERMINING CARDIAC CONDUCTION

Abstract

The invention relates to a method for determining cardiac conduction, especially the cardiac conduction of a test subject, comprising: (i) providing induced sinoatrial bodies (iSABs) comprising cardiac pacemaker cells; (ii) providing working-myocardium cardiomyocytes, especially working-myocardium cardiomyocytes of the test subject; (iii) measuring the cardiac conduction;
the induced sinoatrial bodies according to (i) and the working-myocardium cardiomyocytes according to (ii) being arranged in a spatially separated manner, but in conductive communication with one another.

Claims

1. A method for determining cardiac conduction, comprising: (i) providing induced sinoatrial bodies (iSABs) comprising cardiac pacemaker cells; (ii) providing working-myocardium cardiomyocytes; (iii) measuring the cardiac conduction; the induced sinoatrial bodies according to (i) and the working-myocardium cardiomyocytes according to (ii) being arranged in a spatially separated manner, but in conductive communication with one another.

2. The method as claimed in claim 1, comprising comparison of the cardiac conduction measured in (iii) with a reference value, the comparison preferably comprising (iv) administering a reference substance; (v) measuring the cardiac conduction in the presence of the reference substance according to (iv); (vi) comparing the values measured according to (iii) and the values measured according to (v).

3. The method as claimed in claim 2, wherein the reference substance is selected from the group of heart rate-lowering substances or from the group of heart rate-raising substances, preferably from the group consisting of isoprenaline, ZD-7288, zatebradine and ivabradine.

4. The method as claimed in claim 2, comprising (vii) washing out the reference substance according to (iv), (v); (viii) measuring the cardiac conduction; (ix) optionally comparing the values measured according to (viii) and the values measured according to (iii) or according to (v); or comparing the values measured according to (viii) and the values measured according to (iii) and the values measured according to (v).

5. A method for determining the effect of substances on cardiac conduction, comprising: (i) providing induced sinoatrial bodies (iSABs) comprising cardiac pacemaker cells; (ii) providing working-myocardium cardiomyocytes; (iii) optionally measuring the cardiac conduction; (iv) administering a substance to be investigated; (v) measuring the cardiac conduction in the presence of the substance to be investigated according to (iv); (vi) optionally comparing the cardiac conduction measured according to (iii) and the cardiac conduction measured according to (v); the induced sinoatrial bodies according to (i) and the working-myocardium cardiomyocytes according to (ii) being arranged in a spatially separated manner, but in conductive communication with one another.

6. A method for identifying substances which have an effect on cardiac conduction, comprising: (i) providing induced sinoatrial bodies (iSABs) comprising pacemaker cells; (ii) providing working-myocardium cardiomyocytes; (iii) optionally measuring the cardiac conduction; (iv) administering a substance to be investigated; (v) measuring the cardiac conduction; (vi) optionally comparing the cardiac conduction measured according to (iii) and the cardiac conduction measured according to (v); and optionally determining whether the substance to be investigated has an effect on cardiac conduction on the basis of the comparison according to (vi); the induced sinoatrial bodies according to (i) and the working-myocardium cardiomyocytes according to (ii) being arranged in a spatially separated manner, but in conductive communication with one another.

7. The method as claimed in claim 5, comprising (vii) washing out the substance to be investigated according to (iv) (vii) measuring the cardiac conduction; (viii) optionally comparing the values measured according to (vii) and the values measured according to (iii) or according to (v); or comparing the values measured according to (vii) and the values measured according to (iii) and the values measured according to (v); (ix) optionally determining whether the substance to be investigated has an effect on cardiac conduction on the basis of the comparison according to (viii).

8. The method as claimed in claim 1, wherein the working-myocardium cardiomyocytes according to (ii) comprise atrial cells or ventricular cells or atrial and ventricular cells, preferably (ii.1) atrial and (ii.2) ventricular cells, preference being given to the induced sinoatrial bodies according to (i), the atrial cells according to (ii.1) and the ventricular cells according to (ii.2) being arranged in a spatially separated manner, but in conductive communication with one another.

9. The method as claimed in claim 1, which is carried out in vitro.

10. The method as claimed in claim 1, wherein the induced sinoatrial bodies (iSABs) comprising pacemaker cells are generated from multipotent or pluripotent stem cells, preferably from pluripotent stem cells.

11. The method as claimed in claim 1, wherein the induced sinoatrial bodies (iSABs) comprising pacemaker cells are generated from nonhuman embryonic stem cells or nonhuman induced pluripotent stem cells or human induced pluripotent stem cells or parthenogenetic stem cells or spermatogonial stem cells, preferably from nonhuman embryonic stem cells or nonhuman induced pluripotent stem cells or human induced pluripotent stem cells.

12. A device for determining a cardiac conduction, comprising: (a) a first region 1 suitable for accommodating induced sinoatrial bodies (iSABs) comprising pacemaker cells; (b) a second region 2 suitable for accommodating working-myocardium cardiomyocytes; (c) a third region 3 which is suitable for allowing a conduction from the first region 1 into the second region 2.

13. The device for determining a cardiac conduction as claimed in claim 12, comprising: (a) a first region 1 containing induced sinoatrial bodies (iSABs) comprising pacemaker cells; (b) a second region 2 suitable for accommodating working-myocardium cardiomyocytes; (c) a third region 3 which is suitable for allowing a conduction from the first region 1 into the second region 2.

14. The device as claimed in claim 12, wherein the second region 2 according to (b) comprises: (b.1) a region 2.1 which is suitable for accommodating atrial cells; (b.2) a region 2.2 which is suitable for accommodating ventricular cells; the third region 3 according to (c) comprising: (c.1) a region 3.1 which is suitable for allowing a conduction from the first region 1 according to (a) into the region 2.1 according to (b.1); (c.2) a region 3.2 which is suitable for allowing a conduction from the region 2.1 according to (b.1) into the region 2.2 according to (b.2).

15. The device as claimed in claim 12, wherein the device of a material selected from the group consisting of glass, ceramic, plastic and a mixture of two or more of these materials, the plastic preferably being selected from the group consisting of polystyrene, polyethylene, polypropylene, polyethylene terephthalate and mixtures of two or more of these substances, preferably polystyrene.

16. A kit for determining a cardiac conduction, comprising (A) a device comprising: (a) a first region 1 suitable for accommodating induced sinoatrial bodies (iSABs); (b) a second region 2 suitable for accommodating working-myocardium cardiomyocytes; (c) a third region 3 which is in conductive communication with the first and the second region and is suitable for allowing a conduction from the first region 1 into the second region 2; (B) induced sinoatrial bodies (iSABs) comprising pacemaker cells.

17. The kit as claimed in claim 16, wherein the second region 2 according to (b) comprises: (b.1) a region 2.1 which is suitable for accommodating atrial cells; (b.2) a region 2.2 which is suitable for accommodating ventricular cells; the third region 3 according to (c) comprising: (c.1) a region 3.1 which is suitable for allowing a conduction from the first region 1 according to (a) into the region 2.1 according to (b.1); (c.2) a region 3.2 which is suitable for allowing a conduction from the region 2.1 according to (b.1) into the region 2.2 according to (b.2).

18. The kit as claimed in claim 16, wherein the device consists of a material selected from the group consisting of glass, ceramic, plastic and a mixture of two or more of these materials, the plastic preferably being selected from the group consisting of polystyrene, polyethylene, polypropylene, polyethylene terephthalate and mixtures of two or more of these substances, preferably polystyrene.

19. The use of a device as claimed in claim 12 for the in vitro evaluation of drugs or for the identification of substances which have an effect on cardiac conduction.

20. The use of a device as claimed in claim 12 for the diagnosis or prediagnosis, especially for the in vitro diagnosis or in vitro prediagnosis, of a disturbed cardiac conduction in a test subject or for the determination of the risk of a test subject developing a disturbed cardiac conduction.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0196] FIG. 1 shows schematically the structure of a device having a first region (1) suitable for accommodating induced sinoatrial bodies (iSABs); a second region (2) suitable for accommodating working-myocardium cardiomyocytes; and a third region (3) which is in conductive communication with the first and the second region and is suitable for allowing a conduction from the first region into the second region; in FIG. 1A, the third region (3) is a direct contact region between first region (1) and second region (2); in FIG. 1B, the third region (3) comprises a spatial extent along the axis of first and second region.

[0197] FIG. 2 shows schematically the structure of a device having a first region (1) suitable for accommodating induced sinoatrial bodies (iSABs); a second region (2) suitable for accommodating working-myocardium cardiomyocytes; and a third region (3), the region (2) comprising two regions 2.1 and 2.2, in which 2.1 is suitable for accommodating atrial cells and 2.2 is suitable for accommodating ventricular cells; the third region (3) comprises a region 3.1 and a region 3.2; in FIG. 2A, the third region (3.1) is a direct contact region between 1.1 and 2.1, and additionally 3.2 is a direct contact region between 2.1 and 2.2; in FIG. 2B, 3.1 and 3.2 comprise in both cases a spatial extent along the axis of regions (1), (2.1), (2.2).

[0198] FIG. 3 shows a prototype of a synchronized conductoid composed of murine iSABs coupled to murine ventricular cardiomyocytes; FIG. 3B shows the iSABs and ventricular cardiomyocytes, the iSABs (dotted border) being capable of coupling to ventricular cardiomyocytes (hatched arrow) and of inducing rhythmically synchronized contractions thereof with 300 bpm; FIG. 3A shows the corresponding deflections.

CITED LITERATURE

[0199] Jung, J. J., et al., Programming and isolation of highly pure physiologically and pharmacologically functional sinus-nodal bodies from pluripotent stem cells. Stem Cell Reports, 2014. 2(5): p. 592-605 [0200] Rimmbach, C., J. J. Jung, and R. David, Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection. J Vis Exp, 2015(96) [0201] DE 10 2013 114 671 A1 [0202] WO 2015/091157 A1

[0203] The present invention is illustrated in more detail by the following example.

Example

[0204] Coupling of iSABs and Ventricular Cardiomyocytes

[0205] Induced sinoatrial bodies (iSABs) were prepared as described in Jung et al., (2014) and Rimmbach et al., (2015). On day 28 of differentiation, rapidly (>300 bpm) and regularly beating iSABs were picked under a light microscope. The medium of the neonatal murine cardiomyocytes seeded on the day before by means of the Primary Cardiomyocyte Isolation Kit (Pierce, USA) and grown overnight was aspirated and iSAB medium was added. 10 iSABs were seeded per well of a 24-well plate. The plates were incubated at 37 C. and 5% CO2 for 24 h in order to give the iSABs sufficient time for growth. After a medium change, the plate was searched under a light microscope for grown iSABs. With the aid of Zeiss ZENblack software, both the frequency of the cardiomyocytes and that of the iSABs was determined and checked for synchronization.

[0206] On the basis of these results, it is evident that, by maintaining iSABs in coculture with ventricular cardiomyocytes, it was possible to generate therefrom first simple conductoid constructs, in this case still without spatial separation, for reconstructing cardiac conduction. This means that it was in principle also possible to provide evidence for the feasibility of the approach for medicament testing.