Cardiovascular disease (CVD) is recognized as a major cause of death worldwide. Restriction of the blood supply leads to detrimental effects within the myocardium, which are paradoxically exacerbated upon reperfusion. Few therapeutic strategies are available up to date, yet none of the treatment is efficient enough to abolish ischemia/reperfusion (I/R) injury and provide efficient cardioprotection. An exaggerated inflammatory response is one of the key contributors to I/R injury. Activation of endothelial cells (ECs) enhances inflammation by releasing signaling molecules (e.g., ATP) through Pannexin1 (Panx1) channels. Panx1 is a ubiquitously- expressed protein that mediates paracrine and autocrine signaling and regulates a plethora of physiological and pathological processes. Panx1 has been shown to mediate inflammatory responses e.g., by contributing to leukocyte-endothelial adhesion or chemoattraction of leukocytes to the site of injury. Its role in the ischemic injury of several organs has been reported, yet its contribution to the cardiac I/R remains to be explored. Both genetic ablation and pharmacological inhibition of the Panx1 channel were proven beneficial as they decreased inflammatory response and mitigated ischemic injury in different organs. However, currently available blockers lack specificity and/or in vivo stability. Hence, a high-quality tool that specifically targets and blocks Panx1 channels is needed. As recently shown, the variable domain of the heavy chain camelids antibodies (nanobodies; Nbs) display unique features which promote their therapeutic application. Thanks to their small size and absence of the light chain in their structure, Nbs target cryptic epitopes with a high affinity without inducing the immunogenic reaction upon in vivo administration.

With the help of the Geneva Antibody Facility, we produced recombinant anti-Panx1 scFv’s (mini-antibodies) HRB459 and HRB462 that specifically recognized Panx1. Using mouse in vivo and ex vivo models, we studied the role of Panx1 in myocardial I/R. Finally, in collaboration with the Vrije Universiteit Brussels, a panel of anti-Panx1 nanobodies were generated that were further characterized in in vitro experiments. The two best candidates were applied in our models of I/R injury to study their cardioprotective potential.

With immunofluorescence staining, we confirmed that HRB462 and HRB459 specifically target Panx1 in murine cell lines and tissues specimens, respectively. We further used these tools to explore the role of Panx1 in cardiac I/R injury. We showed that Panx1 is expressed in cardiac ECs, neutrophils and cardiomyocytes. The ubiquitous knock-out of Panx1 gene in mice (Panx1-/-) decreased cardiac cell death following I/R without affecting neutrophil recruitment to the site of injury. A neutrophil-specific deletion of Panx1 did not evoke cardioprotection, suggesting that protective effects in Panx1-/- mice may involve cardiomyocytes rather than

inflammatory cells. Panx1-deficient mice had similar physiological cardiac function as wild-type (WT) mice but displayed lower sensitivity to ischemia and improved recovery of myocardial function after ex vivo I/R. Ischemic preconditioning abolished those effects, suggesting the involvement of mitochondria in th