Biology subjects

Mesirca, P.

Publications and source records attributed to Mesirca, P..

8 recordsLinked to original sources

bioRxiv2026

Validation of differentiated sinoatrial-like hiPSCs as a model of native sinus node myocytes

BackgroundHuman induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) constitute an attractive system for basic research and pharmacologic screening of new molecules of clinical interest. Numerous protocols aiming at differentiating atrial- or ventricular-like cardiomyocytes (hiPSC-CMs) are available. Conversely, only a few are available for obtaining patient-derived sinoatrial node-like pacemaker myocytes (PM-hiPSC-CMs). Here we validate a new protocol to differentiate mature PM-hiPSC-CMs as a model of native sinoatrial node (SAN) myocytes. MethodsWe generated PM-hiPSC-CMs through a 2D matrix-sandwich method promoting epithelial-to-mesenchymal transition and small molecule-based temporal modulation of Wnt signaling pathway. In addition, we treated our cells with triiodothyronine, dexamethasone and intracellular cyclic AMP (DTA) to enhance expression of proteins involved in intracellular Ca2+ handling. ResultsProteomic analyses showed expression of key SAN proteins in DTA-treated PM-hiPSC-CMs. Importantly, expression of proteins related to Ca2+ handling was increased in DTA-treated PM-hiPSC-CMs compared to untreated ones. DTA-treated PM-hiPSC-CMs displayed action potentials, ionic currents and intracellular Ca2+ dynamics typical of native SAN. In addition, pacemaker activity responded to both {beta}-adrenergic and muscarinic stimulation. ConclusionsOur data indicate that the differentiation protocol effectively generates PM-hiPSC-CMs with typical native human SAN features. This protocol may serve as a potential approach to generate PM-hiPSC-CMs from patients with history of sinoatrial node disfunction (SND) carrying different mutations in ion channels underlying pacemaking. In addition, these in vitro models of SND could be used for testing long-term vector-based gene therapeutic strategies to handle bradycardia.

cell biology↗

bioRxiv2025

The expression level of CACNA1C-encoded CaV1.2 is a tipping point between promotion and inhibition of dendritic growth in neurons

The CACNA1C gene encodes the CaV1.2 L-type voltage-gated calcium channel, which plays a crucial role in neuronal signaling. CACNA1C is a risk gene for psychiatric conditions involving disruption of neuronal connectivity such as schizophrenia, autism, and bipolar disorders. While genomic studies are consistently reinforcing the notion of CACNA1C as an important locus related to these diseases, the role of CaV1.2 channels in determining neuronal architecture is incompletely understood. Several studies pinpoint the L-type current (ICaL) as regulator of dendritic arborization development. ICaL lays upstream of competing cellular mechanisms leading to both inhibition and promotion of dendritic growth. How signal selectivity is achieved remains an open question. Here, we report that ICaL-dependent dendritic development of murine cultured hippocampal neurons relies on CaV1.2 and is determined by an equilibrium between the level of CaV1.2 protein expression and ICaL activity. Indeed, increasing ICaL enhances dendritic complexity only when CaV1.2 expression level is reduced. In contrast, when channel levels are at baseline, the CaV1.2-dependent growing signal is overcome by the elevation of the dendritic growth inhibiting CaMKII signaling beyond basal conditions. These findings suggest that CaV1.2 expression level acts as a molecular switch between dendritic growing and inhibiting signals. Consequently, altered CaV1.2 expression during early development may alter neuronal structure, potentially impairing neural network formation and increasing susceptibility to psychiatric disorders. SIGNIFICANCE STATEMENTL-type voltage-gated calcium channels (L-VGCCs) allow calcium influx upon membrane depolarization. In neurons, L-VGCCs play a crucial role in regulating gene transcription, synaptic plasticity, and membrane excitability. Our data demonstrate that the L-VGCCs CaV1.2 isoform is a key regulator of early dendritic development. Calcium influx via CaV1.2 is necessary for proper dendritic growth under basal conditions. However, enhanced calcium currents boost dendritic growth only when channel expression levels are reduced. When the amount of CaV1.2 is at baseline, current stimulation antagonizes the growing process by upregulating the CaMKII signaling. Our findings suggest that the number of CaV1.2 available determines whether channel activity leads to aberrant dendritic arborization by sizing the recruitment of growth inhibiting CaMKII cascade.

neuroscience↗

bioRxiv2025

A fast and tiny current as common generator of slow regular pacemaking in brain and heart

Slow pacemaking is a key physiological process in specific excitable cells, yet its underlying mechanism remains debated. Here we identify a conserved, voltage-dependent pacemaker current that is essential for slow, regular firing in both midbrain dopaminergic neurons and sinoatrial node myocytes. Conductance-based models incorporating this current reproduce stable pacemaking, requiring a fast-activating, small-amplitude current. This is further confirmed by dynamic-clamp experiments in dopaminergic neurons. Replacing the pacemaker current in a model with a voltage-independent conductance such as the non-selective sodium leak channels fails to sustain slow rhythmicity, highlighting the necessity for an adequate voltage dependence. Our results suggest a novel and shared biophysical mechanism for slow pacemaking in neuronal and cardiac systems.

physiology↗

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↗

bioRxiv2023

Optical control of cardiac rhythm by in vivo photoactivation of an ERG channel peptide inhibitor

RATIONALECardiac rhythm, conduction and synchronization of electrical activity require the coordinated action of different types of ion channels that differ according to transmural and regional specificities. Classical pharmacology affects these ion channels in a non-regionalized way which explains why treating arrhythmias, that often occur in specific foci, has often limited efficacy in addition to negative side-effects on non-targeted organs. Photopharmacology is an emergent technology that has the potential to counteract all the negative aspects of classical pharmacology by restricting drug activity in a spatio-temporal manner. OBJECTIVEWe tested the potential of photopharmacology in specifically regulating heart activity by using a caged derivative of a natural peptide inhibitor of the ERG channel, BeKm1. The peptide was uncaged and activity monitored in vitro on a cell line expressing the hERG channel, on human cardiomyocytes derived from iPS cells, and ex vivo and in vivo on zebrafish larvae and rat hearts. METHODS AND RESULTSCaged BeKm-1 is inactive and fully active upon uncaging. Uncaging of the peptide on human iPS-derived cardiomyocytes enlarges the action potential duration and triggers arrhythmias. Uncaging also triggers bradycardia and disturbs cardiac conduction within the atria in perfused rat hearts upon illumination. The potency of photopharmacology for cardiac electrical modulation was further validated in zebrafish larvae where illumination of the caged compound induces bradycardia and atrio-ventricular desynchrony. Finally, in anesthetized rats, illumination of the caged peptide in the right atria, containing the sino-atrial node, leads to bradycardia without arrhythmia. CONCLUSIONSThis report demonstrates that photopharmacology, using the caged peptide strategy, can be used for dynamically regulating cardiac electrical activity in vivo and that spatial illumination restriction can dissociate the bradycardic effect from the arrhythmic one. The technology is applicable to all kinds of cardiac ion channels and regions of interest to create arrhythmogenic models or investigate new clinical applications.

pharmacology and toxicology↗

bioRxiv2021

Characterization of cardiac pacemaker activity in the primate lemur Microcebus murinus.

BackgroundGray mouse lemurs (Microcebus murinus) are the smallest and most ancestral primates known. Their size falls in between that of mice and rats, and their genetic proximity to humans makes them an emerging model to study age-related neurodegeneration. Since mouse lemurs replicate similar senescence processes of humans, they constitute a useful model for studying cardiovascular dysfunctions. However, their cardiac physiology is unknown. Thus, we investigated the cardiac pacemaker activity generated by the sinoatrial node (SAN) of mouse lemurs, presenting the first characterization of heart automaticity in nonhuman primates. Methods and ResultsWe recorded cardiac automaticity in mouse lemurs and in their SAN tissues and pacemaker myocytes. Mouse lemurs have a heart rate (HR) in between those of mice and rats and a similar generation of the SAN electrical impulse. Their SAN myocytes express the main pacemaker currents at densities similar to mice: the hyperpolarization-activated current (If) and the L-type (Ica,L) and T-type (Ica,T) calcium currents. Conversely, their ventricular depolarization resembles that of large mammals and despite the small size of mouse lemurs, the total number of heartbeats in their life corresponds to what can be attained by humans. Using muscle-derived stem cells (MDSCs) from mouse lemurs, we also differentiated pacemaker-like (PML) cells showing spontaneous automaticity and expressing markers of native SAN myocytes (HCN4 and connexin-45). ConclusionsOur characterization of heart automaticity in Microcebus murinus provides new opportunities for comparative cardio-physiology studies in primates and with humans and for testing molecules that could modulate age-related dysfunctions of heart rate.

physiology↗

bioRxiv2018

Bradycardic mice undergo effective heart rate improvement after specific homing to the sino-atrial node and differentiation of adult muscle derived stem cells.

Current treatments for heart automaticity disorders still lack a safe and efficient source of stem cells to restore normal biological pacemaking. Since adult Muscle-Derived Stem Cells (MDSC) show multi-lineage differentiation in vitro including into spontaneously beating cardiomyocytes, we questioned whether they could effectively differentiate into cardiac pacemakers, a specific population of cardiomyocytes producing electrical impulses in the sino-atrial node (SAN) of adult heart. We show here that beating cardiomyocytes, differentiated from MDSC in vitro, exhibit typical characteristics of cardiac pacemakers: expression of markers of the SAN lineage Hcn4, Tbx3 and Islet1, as well as spontaneous calcium transients and hyperpolarization-activated "funny" current and L-type Cav1.3 channels. Pacemaker-like myocytes differentiated in vitro from Cav1.3-deficient mouse stem cells produced slower rate of spontaneous Ca2+ transients, consistent with the reduced activity of native pacemakers in mutant mice. In vivo, undifferentiated wild type MDSC migrated and homed with increased engraftment to the SAN of bradycardic mutant Cav1.3-/- within 2-3 days after systemic I.P. injection. The increased homing of MDSCs corresponded to increased levels of the chemokine SDF1 and its receptor CXCR4 in mutant SAN tissue and was ensued by differentiation of MDSCs into Cav1.3-expressing pacemaker-like myocytes within 10 days and a significant improvement of the heart rate maintained for up to 40 days. Optical mapping and immunofluorescence analyses performed after 40 days on SAN tissue from transplanted wild type and mutant mice showed MDSCs integrated as pacemaking cells both electrically and functionally within recipient mouse SAN. These findings identify MDSCs as directly transplantable stem cells that efficiently home, differentiate and improve heart rhythm in mouse models of congenital bradycardia.

cell biology↗

bioRxiv2018

Structure-guided design of a cell penetrating peptide preventing cAMP modulation of HCN channels

The auxiliary subunit TRIP8b prevents cAMP activation of HCN channels by antagonizing its binding to their cyclic-nucleotide binding domain (CNBD). By determining an NMR-derived structure of the complex formed by the HCN2 channel CNBD and a minimal TRIP8b fragment, TRIPnano, we show here a bipartite interaction between the peptide and CNBD which prevents cAMP binding in two ways: through direct competition for binding at the distal C-helix of the CNBD; and through an allosteric reduction in cAMP affinity induced by TRIP8b binding to the CNBD N-bundle loop. TRIPnano abolishes cAMP binding in all three isoforms, HCN1, HCN2 and HCN4 and can be used to prevent cAMP stimulation in native f-channels. Application of TRIP8bnano, or its delivery via a cell-penetrating sequence, in sinoatrial node myocytes, selectively inhibits beta-adrenergic stimulation of the native If current and mimics the physiological concentrations of acetylcholine leading to a 30% reduction in the spontaneus rate of action potential firing.

physiology↗