Direct reprogramming of cardiomyocytes into cardiac Purkinje-like cells.

Currently, there are no treatments that ameliorate cardiac cell death, the underlying basis of cardiovascular disease. An unexplored cell type in cardiac regeneration is cardiac Purkinje cells; specialized cells from the cardiac conduction system (CCS) responsible for propagating electrical signals. Purkinje cells have tremendous potential as a regenerative treatment because they may intrinsically integrate with the CCS of a recipient myocardium, resulting in more efficient electrical conduction in diseased hearts. This study is the first to demonstrate an effective protocol for the direct reprogramming of human cardiomyocytes into cardiac Purkinje-like cells using small molecules. The cells generated were genetically and functionally similar to native cardiac Purkinje cells, where expression of key cardiac Purkinje genes such as CNTN2, ETV1, PCP4, IRX3, SCN5a, HCN2 and the conduction of electrical signals with increased velocity was observed. This study may help to advance the quest to finding an optimized cell therapy for heart regeneration.

Molecular Mechanisms of Class B GPCR Activation: Insights from Adrenomedullin Receptors.

Adrenomedullin (AM) is a 52 amino acid peptide that plays a regulatory role in the vasculature. Receptors for AM comprise the class B G protein-coupled receptor, the calcitonin-like receptor (CLR), in complex with one of three receptor activity-modifying proteins (RAMPs). The C-terminus of AM is involved in binding to the extracellular domain of the receptor, while the N-terminus is proposed to interact with the juxtamembranous portion of the receptor to activate signaling. There is currently limited information on the molecular determinants involved in AM signaling, thus we set out to define the importance of the AM N-terminus through five signaling pathways (cAMP production, ERK phosphorylation, CREB phosphorylation, Akt phosphorylation, and IP1 production). We characterized the three CLR:RAMP complexes through the five pathways, finding that each had a distinct repertoire of intracellular signaling pathways that it is able to regulate. We then performed an alanine scan of AM from residues 15-31 and found that most residues could be substituted with only small effects on signaling, and that most substitutions affected signaling through all receptors and pathways in a similar manner. We identify F18, T20, L26, and I30 as being critical for AM function, while also identifying an analogue (AM15-52 G19A) which has unique signaling properties relative to the unmodified AM. We interpret our findings in the context of new structural information, highlighting the complementary nature of structural biology and functional assays.

Identification of Small-Molecule Positive Modulators of Calcitonin-like Receptor-Based Receptors.

Class B G protein-coupled receptors are highly therapeutically relevant but challenges remain in identifying suitable small-molecule drugs. The calcitonin-like receptor (CLR) in particular is linked to conditions such as migraine, cardiovascular disease, and inflammatory bowel disease. The CLR cannot act as a cell-surface receptor alone but rather must couple to one of three receptor activity-modifying proteins (RAMPs), forming heterodimeric receptors for the peptides adrenomedullin and calcitonin gene-related peptide. These peptides have extended binding sites across their receptors. This is one reason why there are few small-molecule ligands that can modulate these receptors. Here we describe small molecules that are able to positively modulate the signaling of the CLR with all three RAMPs but are not active at the related calcitonin receptor. These compounds were selected from a ß-arrestin recruitment screen, coupled with rounds of medicinal chemistry to improve their activity. Translational potential is shown as the compounds can positively modulate cAMP signaling in a vascular cell line model. Binding experiments do not support an extracellular domain binding site; however, molecular modeling reveals potential allosteric binding sites in multiple receptor regions. These are the first small-molecule positive modulators described for the CLR:RAMP complexes.