Aims: Telomeres are protective caps found at the ends of linear chromosomes, which are synthesized by the enzyme telomerase. However, in adult mice and humans, telomerase is downregulated after birth thus leading to loss of telomeric repeats with each round of cell cycle. Critically short telomeres are known to contribute to aging-related diseases and short telomeres in cardiomyocytes (CMs) are strongly associated with several cardiomyopathies. However, it remains ambiguous whether short telomeres are the cause or the result of the disease in terminally differentiated, non-dividing CMs. Therefore, we aimed to delineate the correlation between short telomeres and CM dysfunction.

Methods & results: We utilized human induced pluripotent stem cells (hiPSCs) in a dish and further combined it with differentiation techniques to derive human CMs in vitro. We have already established that differentiating hiPSCs into CMs leads to shut down of telomerase activity. Hence, after hiPSCs are differentiated into CMs they become incapable of replenishing telomeres. We first utilized patient derived hiPSCs with heterozygous mutation in the telomerase reverse transcriptase (TERT) gene to study the ramifications of telomeropathies in the context of CMs. Our observations indicate that TERT haploinsufficiency is unable to recapitulate telomere shortening at the hiPSC stage. Therefore, we employed an inducible CRISPRi hiPSC line to modulate the TERT gene at the hiPSC stage, which allowed us to alter the telomere lengths as well which was validated by TRAP assay and telomere qFISH respectively. This enabled us to further generate hiPSC-derived CMs with long and short telomere lengths as confirmed by telomere qFISH analysis. We confirmed that shorter telomeres predispose CMs to stress-induced cell death via caspase assay. We also identified that reduced telomerase activity and shorter telomere lengths of hiPSCs negatively impact their differentiation towards CMs using qualitative and flow cytometric analysis. Single cell sequencing analyses further validated the observations that amongst the mesodermal lineage, it is specifically the CM lineage that is most affected due to loss of telomerase activity and telomeres at the hiPSC stage.

Conclusion: Collectively, our data suggests, that telomerase activity and intact telomeres are essential for CM differentiation and maintenance. The CRISPRi TERT hiPSCs system is an excellent platform to investigate both telomere biology in CMs and the mechanisms of telomere driven cardiovascular disorders. CRISPRi TERT hiPSCs are an ideal tool for drug screening purposes against telomeropathies in any hiPSC-derived cell type of choice.