AUTHORS: Suchi Raghunathan1, Jose Francisco Islas2, Brandon Mistretta3, Dinakar Iyer3, Liheng Shi4, Preethi H. Gunaratne3, Gladys Ko4, Robert J. Schwartz3*, and Bradley K. McConnell (co-senior author)1*
AFFILIATIONS: 1 Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204-5037, USA. 2 Department of Biochemistry and Molecular Medicine, Autonomous University of Nuevo León, Monterrey, Mexico. 3 Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001, USA. 4 Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843-4458, USA.
RUNNING TITLE: CPCs Reprogrammed into Cardiac Pacemaker-like Cells
ABSTRACT: We used a screening strategy to test for reprogramming factors for the conversion of human cardiac progenitor cells (CPCs) into pacemaker cells. Human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first transduced with the connexin 30.2 (CX30.2) mCherry reporter. Following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b. These findings suggest that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations and single transcription factors. Additionally, single-cell RNA sequencing of SHT5 mCherry+ cells revealed cellular enrichment of pacemaker specific genes including TBX3, KCNN4, CX30.2, and BMP2, as well as pacemaker specific potassium and calcium channels (KCND2, KCNK2, and CACNB1). In addition, similar to human and mouse sinoatrial node (SAN) studies, we also observed the down-regulation of NKX2.5. Patch-clamp recordings of the converted Pacemaker-like cells exhibited HCN currents demonstrated the functional characteristic of pacemaker cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of SAN dysfunction.
HIGHLIGHTS: SHOX2, HCN2, and TBX5 (SHT5) cocktail of transcription factors and channel protein reprogrammed CPCs into Pacemaker-like cells. The SHT5 factors resulted in upregulation of pacemaker specific gene expression and transcriptome expression, attributing the pacemaker phenotype to the cells. The SHT5 mCherry+ cells also exhibited the funny current via HCN4 channels, attributing the functional characteristic of pacemaker cells. Thus, the findings of this study show that the SHT5 combination of transcription factors can be used to reprogram CPCs into Pacemaker-like cells as a potential stem cell therapy for sick sinus syndrome (SSS) as well as for other cardiac conduction diseases.
ONE SENTENCE SUMMARY: The SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors and channel proteins can be used to reprogram CPCs into Pacemaker-like cells as a potential stem cell therapy for sick sinus syndrome (SSS).
ACKNOWLEDGEMENT: The research reported in this paper was supported in part by a grant from Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (to B.K.M.).
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*ADDRESS FOR CORRESPONDANCE: Bradley K. McConnell, PhD, FAHA, FCVS, Associate Professor of Pharmacology, Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, 4849 Calhoun Road, Health-2 (H2) Building, Room 5024, Houston, TX 77204-5037; Phone: 713-743-1218; Fax: 713-743-1232, Email: email@example.com