Active contribution of transplanted induced pluripotent stem cell-derived cardiomyocytes to left ventricular function in a guinea pig model

Abstract

Induced pluripotent stem cell (iPSC)-derived cardiomyocyte transplantation has evolved into a potential novel therapeutic option for heart failure patients in the past twenty years. It has shown efficacy in both small and large animal models. Because of these promising results, the first clinical studies have started in 2021. However, the mechanism of action is not understood. It is still unknown if the transplanted cardiomyocytes contribute to force generation of the left ventricle. In this work, I aimed to investigate if transplanted cardiomyocytes contribute to left ventricular function after transplantation. I hypothesized that selectively and reversibly switching off cardiomyocyte contractility only in engrafted cardiomyocytes will result in a drop in left ventricular function. Switching cardiomyocytes back on should result in recovery of cardiac function. This could indicate that the engrafted cells contribute to left ventricular force.

For this, I created four novel iPSC lines using CRISPR/Cas9 expressing artificial ion channels, hypothesizing that their activation will lead to electrical silencing and thereby switching off contractility. The four approaches were divided into two groups by their mode of activation. Two cell lines expressed a chemogenetic construct, meaning that the artificial cation (PSAM 5HT3) or anion (PSAM GlyR) channels can be activated by the small molecular substrate PSEM89S. The other two cell lines were chemo- and optogenetic cation (LMO4) and anion (iLMO4) channels, consisting of a luciferase coupled to an optogenetic channel. Coelenterazine, the luciferase substrate, is converted, producing light that activates the fused optogenetic channel. Alternatively, direct activation by light can be used. In iPSCs and differentiated cardiomyocytes, the four transgenes were expressed and functional. PSAM GlyR cardiomyocytes, cast into EHTs, could be stopped with PSEM89S, while PSAM 5HT3 EHTs were not affected. In the optogenetic lines, iLMO4 EHTs could be stopped using light pulses while LMO4 EHTs continued to beat and followed the light pulse frequency. Coelenterazine had unexpected detrimental effects on EHTs and was therefore not further used.

Hence, PSAM GlyR and iLMO4 were chosen for further thorough analysis. PSAM GlyR EHTs developed similarly to control EHTs. PSAM GlyR EHTs stopped to contract immediately upon PSEM89S application. iLMO4 EHTs showed lower force generation and were overall more heterogenous. iLMO4 EHTs could not be electrically paced reliably above 2.75 Hz compared to control EHTs which could be paced up to 4 Hz.

With two cell lines that could reversibly be switched off, we aimed to test our hypothesis in a guinea pig model. Beforehand, we conducted a pharmacokinetic study, which revealed that sufficient PSEM89S concentrations were difficult to reach in guinea pigs. Therefore, I set out to assemble a Langendorff system to assess cardiac function ex vivo. This allowed us to use iLMO4 cardiomyocytes because in vivo light application would have been impossible.

We then transplanted PSAM GlyR, iLMO4, or control cardiomyocytes (20x106) in a cryoinjury guinea pig model. Cardiomyocytes from all cell lines engrafted and showed similar graft sizes. Cardiomyocyte transplantation stabilized left ventricular function. Unexpectedly, PSEM89S had a negative inotropic and chronotropic effect on guinea pig hearts in the Langendorff system, regardless of the transplanted cell line. Therefore, we could not assess the degree of contribution of the engrafted cells to left ventricular function with this approach. However, after transplantation of iLMO4 cardiomyocytes, we observed an immediate decline in left ventricular function upon light application in the Langendorff system. Upon the termination of light application, the left ventricular function recovered. Interestingly, this effect was frequency-dependent, i.e., the contribution was smaller under electrical pacing at a higher frequency. This finding fits the in vitro results, where iLMO4 EHTs could not be paced reliably above 2.75 Hz.

In conclusion, I was able to show for the first time that transplanted cardiomyocytes can actively contribute to left ventricular function, which is the first step into understanding how cardiomyocyte transplantation can potentially benefit patients in the future.