Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtypespecific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5 + progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols.

Background The clinical efficacy of migraine therapeutic agents directed towards the calcitonin-gene related peptide (CGRP) pathway has confirmed the key role of this axis in migraine pathogenesis. Three antibodies against CGRP – fremanezumab, galcanezumab and eptinezumab – and one antibody against the CGRP receptor, erenumab, are clinically approved therapeutics for the prevention of migraine. In addition, two small molecule CGRP receptor antagonists, ubrogepant and rimegepant, are approved for acute migraine treatment. Targeting either the CGRP ligand or receptor is efficacious for migraine treatment; however, a comparison of the mechanism of action of these therapeutic agents is lacking in the literature. Methods To gain insights into the potential differences between these CGRP pathway therapeutics, we compared the effect of a CGRP ligand antibody (fremanezumab), a CGRP receptor antibody (erenumab) and a CGRP receptor small molecule antagonist (telcagepant) using a combination of binding, functional and imaging assays. Results Erenumab and telcagepant antagonized CGRP, adrenomedullin and intermedin cAMP signaling at the canonical human CGRP receptor. In contrast, fremanezumab only antagonized CGRP-induced cAMP signaling at the human CGRP receptor. In addition, erenumab, but not fremanezumab, bound and internalized at the canonical human CGRP receptor. Interestingly, erenumab also bound and internalized at the human AMY1 receptor, a CGRP receptor family member. Both erenumab and telcagepant antagonized amylin-induced cAMP signaling at the AMY1 receptor while fremanezumab did not affect amylin responses. Conclusion The therapeutic effect of agents targeting the CGRP ligand versus receptor for migraine prevention (antibodies) or acute treatment (gepants) may involve distinct mechanisms of action. These findings suggest that differing mechanisms could affect efficacy, safety, and/or tolerability in migraine patients.

Background: ␣6␤3*-Nicotinic receptors (nAChRs) are physiologically important but difficult to express heterologously. Results: Influences of ␤3 subunits on ␣6␤3*-nAChR function are impacted by ␣6 subunit N-terminal domain loop E residues. Conclusion: There are unexpected roles for the complementary face of the nAChR ␣6 subunit in receptor function. Significance: Novel medicinals acting at new sites on ␣6␤3*-nAChRs could be useful antidepressants and/or smoking cessation aids.

Background/Aims: The effect of daily injections with genistein (naturally occurring phytoestrogen) on intestinal chloride (Cl^-) secretion was measured with Ussing chamber short circuit current (I_sc, µA/cm²), in C57BL/6J male and female mice, using 600 mg/kg genistein/day (600G), 300 mg/kg genistein/day (300G), 150 mg/kg genistein/day (150G) or genistein-free vehicle control (0G) for 1- or 2-weeks. Methods and Results: Injecting with 600G elicited significant increases in basal I_sc in females after 1-week (ñ70 µA/cm², n=15, p < 0.05) and in males after 2-weeks (ñ80 µA/cm², n=5, p < 0.05) compared to their 0G counterparts. Chloride-free ringer significantly reduced basal I_sc by 65% in 600G males and 72% in 600G females, suggesting that Cl^- was the major anion comprising the genistein-stimulated secretion. The forskolin-stimulated (10 µM) I_sc was significantly inhibited by the CFTR chloride channel inhibitors, glibenclamide (500 µM) and CFTR_inh-172 (100 µM) in 600G males and females, suggesting some contribution by genistein-dependent CFTR-mediated Cl^- secretion. We found no associated changes in intestinal morphology, nor change in total CFTR protein with 600G. There was a 5% increase in apical/subapical ratio in 600G males compared to controls (no change in females). Conclusion: These data suggest that male and female mice both exhibit increased Cl- secretion with 600G, however, the mechanisms mediating this are gender-dependent.

SUMMARY Gata4, Hand2, Mef2c, and Tbx5 (GHMT) can reprogram transduced fibroblasts into induced pacemaker-like myocytes (iPMs), but the underlying mechanisms remain obscure. Here, we explore the role of Hand2 in iPM formation by using a combination of transcriptome, genome, and biochemical as-says. We found many shared transcriptional signatures between iPMs and the endogenous sinoatrial node (SAN), yet key regulatory networks remain missing. We demonstrate that Hand2 augments chromatin accessibility at loci involved in sarcomere organization, electrical coupling, and membrane depolarization. Focusing on an established cardiac Hand2 cistrome, we observe selective reorganization of chromatin accessibility to promote pacemaker-specific gene expression. Moreover, we identify a Hand2 cardiac subtype diversity (CSD) domain through biochemical analysis of the N terminus. By integrating our RNA-seq and ATAC-seq datasets, we highlight desmosome organization as a hallmark feature of iPM formation. Collectively, our results illuminate Hand2-dependent mechanisms that may guide future efforts to rationally improve iPM formation.