In a recent study published in the journal Cell Stem Cell, researchers hypothesized that pacemaker-like activity of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) resulted in engraftment arrhythmias (EAs), which hampers the clinical use of cell-based therapy using hPSC-CMs for treatment of myocardial infarction (MI).

Study: Gene editing to prevent ventricular arrhythmias associated with cardiomyocyte cell therapy. Image Credit: FrentaN / Shutterstock

Background

The human heart does not have regeneration potential. So when non-contractile scar tissue takes the place of one billion adult CMs after one MI episode, this impairs heart function, which, in turn, could even lead to a heart attack or heart failure.

The discovery of hPSCs has opened new avenues for MI treatment. Note highly pure hPSC-CMs whose intra-myocardial transplantation forms new myocardium grafts in infarcted hearts follow pacing from the host's sinoatrial (SA) node. These are under intensive investigation as candidates for the regeneration of the human heart post-MI. However, EAs, a transitory but sustained ventricular tachycardia (VT), hinder the clinical use of hPSC-CMs-based cell therapy. Moreover, unlike adult ventricular CMs (vCMs), hPSC-CMs exhibit automaticity, the ability to spontaneously and rhythmically depolarize and trigger action potentials (APs).

So far, researchers have used mouse, rat, guinea pig, and non-human primate (NHP) models of subacute MI to examine the effects of transplantation of hPSC-CMs. During normal maturation of these cells, automaticity remains limited to specialized cells in the pacemaking system, and in small animal models, high heart rates often mask EAs. However, in large animal models with heart rates comparable to humans, i.e., 70 beats per minute [bpm], the transplantation of hPSC-CMs triggers EA within a week, likely arising from the graft-host electrical coupling.

EA-triggered VT typically lasts one month (on average), taking the heart rates of pigs and NHPs to up to 300 revolutions per minute (rpm), which, in some severe cases, could be lethal for pigs. However,  in most cases, EA wanes gradually as the graft matures. Since humans might not be able to tolerate rapid EA, it is crucial to identify strategies that control EA till the time their graft attains maturity.

Invasive electrophysiology studies in NHPs and pigs have also shown that a spontaneous aberrant impulse generated from the graft (because hPSC-CMs exhibit shorter AP duration due to more depolarized membrane potential) leads to EA rather than conduction defects (i.e., re-entry pathways). It appears to be related to a fetus-like gene expression profile for involved ion channels. Thus, overdrive pacing and direct current (DC) cardioversion cannot terminate EA.

About the study

The authors had previously shown that hPSC CMs transplanted into the infarcted heart of a rat matured to exhibit adult-like myofibril isoform organization. This model closely mimicked the maturation milieu that hPSC-CMs would experience inside humans, thus, had more clinical and physiological relevance. In this model, they performed laser-capture microdissection (LCM) followed by bulk ribonucleic acid-sequencing (RNA-seq) to characterize the genome-wide expression dynamics of hPSC-CMs during in vivo maturation, which took around 12 weeks.

To extract in vivo transplanted hiPSC-CMs from rat hearts, they transduced them with green fluorescent calmodulin (GCaMP3) before grafting. For comparisons, they analyzed hiPSC-CMs cultured long-term in vitro for up to one year. They also analyzed human-specific and rat-specific RNA-seq reads (or graft signals) separately.

Then, the researchers set out to generate hPSC-CMs with reduced automaticity like adult vCMs but with appropriate electrophysiological behavior, i.e., beating in response to electrical stimulation. To this end, they used CRISPR/Cas9 technology and systematically knocked out over expressive ion channel genes—singly and in combination.

The researchers characterized these hPSC-CMs in vitro and tested the impact of gene edits on EA in an in vivo model, the uninjured hearts of immunosuppressed Yucata' n minipigs, transplanted with 150 million hESC-CMs. They monitored their heart rate and rhythm with a  continuous electrocardiogram (ECG) system, which indicated their EA burden. Further, they determined the expression dynamics of all ion channel genes post-hPSC-CM transplantation to curate a list of EA mediators and to engineer their electrophysiology toward an adult-like phenotype.

Further, the researchers set out to understand the drivers of automaticity in human embryonic stem cell-derived cardiomyocytes (hESC-CMs) differentiated by modulating the WNT signaling pathway with small molecules. To assess the role of specific ion currents, the researchers genetically manipulated candidate genes encoding for ion channels likely playing an important role in hESC-CM automaticity based on available transcriptomic and pharmacological data, which helped them identify the minimum number of gene edits needed to nullify automaticity (or EA).

Results

Single, double, and triple gene edits reduced the heartbeat rate and destabilized the SA node rhythm, but they could not eliminate automaticity or EA in vitro and in vivo, respectively. A quadruple edit or simultaneous knockout (KO) of four genes, HCN4, CACNA1H, and SLC8A1, coupled with overexpression of KCNJ2 termed MEDUSA eliminated the automaticity of hESC-CMs in vitro (in >10 cell lines) without affecting the ability of these cells to fire APs when stimulated, i.e., the cells were quiescent yet excitable.

Relative to wild-type (WT) controls and the other cell lines, transplantation of post-quadruple-gene edited CMs did not result in sustained EA in vivo. Also, MEDUSA hESC-CMs engrafts were stable for three months, beat synchronously with the host myocardium, and exhibited markedly attenuated VTs. These modifications also prevented pigs' morbidity, mortality, and heart failure post-hPSC-CM transplantation.

Gene expression profiles showed stronger and faster maturation in vivo compared to in vitro culture, which lagged after one year in culture. Inducing a more adult-like ion-channel gene expression profile in hPSC-CMs reduced automaticity and, potentially, the burden of EA after transplantation. Interestingly, the study results highlighted the importance of Ca²⁺ trafficking in hESC-CM automaticity.

Conclusions

Further studies are needed to test whether MEDUSA hESC-CMs could work effectively post-transplantation into infarcted rat hearts. It is necessary because the researchers observed that MEDUSA cells were not completely quiescent. During heat shock or other stress conditions, they occasionally beat spontaneously.

Similarly, transplantation-induced stresses, such as ischemia or inflammation, could induce similar beating activity in vivo, which might govern the self-restricted EA episodes observed during the transplantation of high doses of MEDUSA cells. Nevertheless, this study showed a favorable profile of MEDUSA hESC-CMs compared to their WT counterparts. Moreover, combining less arrhythmogenic hESC-CMs with ivabradine might confer additional safety benefits during clinical trials.

To conclude, these results provided new insights into the mechanisms behind the automaticity of hESC-CMs. However, more work is warranted to determine whether MEDUSA hESC-CM could potentially restore systolic function, an advancement needed to safely revascularize the injured heart.

Journal reference:

• Gene editing to prevent ventricular arrhythmias associated with cardiomyocyte cell therapy, Silvia Marchiano, Kenta Nakamura, Hans Reinecke, Lauren Neidig, Michael Lai, Shin Kadota, Filippo Perbellini, Xiulan Yang, Jordan M. Klaiman, Leslie P. Blakely, Elaheh Karbassi, Paul A. Fields, Aidan M. Fenix, Kevin M. Beussman, Anu Jayabalu, Faith A. Kalucki, Jennifer C. Potter, Akiko Futakuchi-Tsuchida, Gerhard J. Weber, Sarah Dupras, Hiroshi Tsuchida, Lil Pabon, Lili Wang, Björn C. Knollmann, Steven Kattman, R. Scott Thies, Nathan Sniadecki, W. Robb MacLellan, Alessandro Bertero, Charles E. Murry, Cell Stem Cell 2023, DOI: https://doi.org/10.1016/j.stem.2023.03.010, https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(23)00081-4