Abstract Body: Connexin-43 (Cx43) is the main connexin isoform found in the heart. Cx43 monomers oligomerize as hexamers to form hemichannels at the plasma membrane of cardiomyocytes. Hemichannels from adjacent cells dock to form a gap junction channel (GJC), which are concentrated at the intercalated discs and are essential for proper heart functioning via electric and metabolic coupling. In many cardiac-associated pathologies such as Duchenne Muscular Dystrophy, Ischemia/Reperfusion Injury and hypertension, undocked Cx43 hemichannels are remodeled to the lateral side of cardiomyocytes, where they open upon cardiac stress. This aberrant hemichannel activity disrupts cardiac excitability, leading to membrane depolarization, prolonged action potentials, arrhythmogenesis and sudden death. Despite the clinical relevance of this process, there are currently no pharmacological tools that selectively modulate Cx43 hemichannels without affecting GJCs. To address this gap, we aimed to develop anti-Cx43 hemichannel nanobodies (nAbs), targeting the extracellular loops of Cx43 (EC1 and EC2). Peptides corresponding to both domains were used for llama immunization, resulting in a serum with high reactivity against the epitopes. The B-cells of the immunized llama were used as source of nAb-coding RNA to produce a nanobody-Phage display library. After three selection cycles, bacteria infected by EC2-binding nAb-Phages were individually cultivated, and recombinant EC2-nAb production was induced with IPTG. More than 80% of the tested colonies produced EC2-nAbs with at least a 3-fold higher reactivity towards EC2, compared to the control. To date, this strategy has yield seven distinct nAbs with the potential to recognize and prevent the opening of Cx43 undocked hemichannels.