Abstract 17326: Blockade of Thrombospondin1 Attenuates Tgfβ Signaling and Enhances the Automaticity of Tbx18-Induced Pacemaker Cells

Abstract

Introduction: Transcription factor Tbx18 can reprogram chamber cardiomyocytes to induced pacemaker cells (iPMs). Transcriptomic analysis of Tbx18-iPMs identified Tgfβ pathway as a prominent signaling cascade in this transition. The activity of Tgfβ1 is regulated primarily in the extracellular domain where the secreted latent form must be modified to expose the active ligand. We hypothesize that thrombospondin1 (Tsp1) mediates the activation of the Tgfβ signaling by Tbx18. Methods: Neonatal rat ventricular myocytes (NRVMs) were transduced with adenoviral constructs expressing either human TBX18 or GFP. Results: Tsp1 transcript (Thbs1) level was higher in TBX18 iPMs compared to GFP-NRVMs (1.5±1.3 vs 0.6±0.6, p<0.05, n=4). Tsp1 concentration was higher in the conditioned media of TBX18 iPMs than in GFP-NRVMs (1220±794 vs 138±178 pg/mL, p<0.05, n=20). The population of Tsp1+ fibroblasts were significantly higher within TBX18 iPMs compared to GFP-NRVMs (45±7% vs 9±10%, p<0.05, n=3). We tested for cause-and-effect relationship between Tsp1 and Tgfβ signaling. Treatment of TBX18 iPMs with 20μM LSKL, a peptide inhibitor of Tsp1, attenuated secreted Tgfβ1 ligand concentration compared to DMSO-treated TBX18 iPMs (127±70 vs 214±44 pg/mL, p<0.05, n=20). Similarly, LSKL treatment reduced αSMA+ myofibroblast population in TBX18 iPMs at day 4 compared to untreated iPMs (10±6 vs 21±5%, p<0.05, n=3). Transcript levels of key Tgfβ signaling genes such as of Cola1, Tgfβ1, and Thbs1 were downregulated in LSKL-treated TBX18 iPMs compared to DMSO-treated iPMs. Functionally, LSKL treatment increased rates of single cell automaticity (125±13 vs 61±6 bpm) and the percentage of spontaneously beating cells (95±7 vs 62±17%, n=6, p<0.05) in TBX18-iPMs compared to DMSO-treated TBX18 iPMs at day 16 after gene transfer. The enhanced automaticity was evident by day 7 and remained stable for two weeks. Conclusions: Our data identify Tsp1 as the key mediator through which Tbx18 activates the Tgfβ pathway during iPM reprogramming. Inhibition of Tgfβ pathway may serve as a solution to attain durable pacing from TBX18-iPMs.