Perrier, R. | bioRxiv Science

bioRxiv2025

Animal-free recombinant nanobody rescues HCN4 channel deficit in sinus node dysfunction

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) control cardiac and neuronal firing and their dysfunction leads to cardiac arrythmias (HCN4), epilepsy (HCN1) and chronic pain (HCN2). Prompted by the urgent need for HCN subtype-specific treatments, we screened a recombinant nanobody library in search of HCN4-specific binders. Here we show that nanobody 5 (NB5) binds to the extracellular side of HCN4 with high specificity and nanomolar affinity and activates the channel by a non-canonical electromechanical coupling path. In ex vivo and in vitro experiments, NB5 acts as an agonist of the pacemaker current If, increasing the firing rate of rabbit cardiac pacemaker myocytes and of human derived cardiomyocytes. Notably, NB5 rescued the effects of a LOF HCN4 mutation causing sinus node dysfunction in a patient. Our work illustrates that animal-free recombinant nanobodies have strong potential as next generation modulators for clinical application in symptomatic bradycardia.

biophysics↗

bioRxiv2024

Circadian regulation of CaV1.2 expression by RORalpha in the mouse heart

BackgroundIn addition to show autonomous beating rhythmicity, the physiological functions of the heart present daily periodic oscillations. Notably the ventricular repolarization itself varies throughout the circadian cycle which was mainly related to the periodic expression of K+ channels. However, the involvement of the L-type Ca2+ channel (CaV1.2 encoded by Cacna1c gene) in these circadian variations remains elusive. MethodsWe used a transgenic mouse model (PCa-luc) that expresses the luciferase reporter under the control of the cardiac Cacna1c promoter and analyzed promoter activity by bioluminescent imaging, qPCR, immunoblot, Chromatin immunoprecipitation assay (ChIP) and CaV1.2 activity. ResultsUnder normal 12:12h light-dark cycle, we observed in vivo a biphasic diurnal variation of promoter activities peaking at 9 and 19.5 Zeitgeber time (ZT). This was associated with a periodicity of Cacna1c mRNA levels preceding 24-h oscillations of CaV1.2 protein levels in ventricle (with a 1.5 h phase shift) but not in atrial heart tissues. The periodicity of promoter activities and CaV1.2 proteins, which correlated with biphasic oscillations of L-type Ca2+ current conductance, persisted in isolated ventricular cardiomyocytes from PCa-Luc mice over the course of the 24-h cycle, suggesting an endogenous cardiac circadian regulation. Comparison of 24-h temporal patterns of clock gene expressions in ventricles and atrial tissues of the same mice revealed conserved circadian oscillations of the core clock genes except for the retinoid-related orphan receptor gene (ROR), which remained constant throughout the course of a day in atrial tissues. In vitro we found that ROR is recruited to two specific regions on the Cacna1c promoter and that incubation with specific ROR inhibitor disrupted 24-h oscillations of ventricular promoter activities and CaV1.2 protein levels. Similar results were observed for pore forming subunits of the K+ transient outward currents, KV4.2 and KV4.3. ConclusionsThese findings raise the possibility that the ROR-dependent rhythmic regulation of cardiac CaV1.2 and KV4.2/4.3 throughout the daily cycle may play an important role in physiopathology of heart function.

physiology↗