Biology subjects

Christoffels, V. M.

Publications and source records attributed to Christoffels, V. M..

6 recordsLinked to original sources

bioRxiv2026

TBX5 dosage governs ventricular cardiomyocyte maturation, specialization and dedifferentiation in vivo

Variation in transcription factor (TF) activity modulates traits and disease susceptibility, yet how such variation translates into cellular phenotype and organ function in vivo is not well established. We utilized AAV-mediated gene delivery to express the dosage-sensitive TF TBX5 across a physiologically plausible range in postnatal ventricular cardiomyocytes. Transcriptomic profiling revealed that TBX5 dosage-dependent cardiomyocyte states changed gradually and often non-monotonically across the TBX5 dosage spectrum. The lowest dosages induced cardiomyocyte hypertrophy and upregulated gene programs governing oxidative metabolism, calcium handling, and contractility. In contrast, mid-to-high dosages induced a ventricular conduction system-like transcriptional profile. Supraphysiological dosages triggered cardiomyocyte dedifferentiation, characterized by cardiomyocyte size reduction, cell cycle re-entry, metabolic reprogramming, and impaired ventricular ejection fraction. Furthermore, the ventricular state of an Nppa-Nppb deficiency model characterized by cardiac hypertrophy and reduced Tbx5 expression was partially normalized by TBX5 delivery. By defining the non-linear relationship of TBX5 activity level, cardiomyocyte state and cardiac function, our study reveals how TF dosage regulates the transitions from physiological maturation to specialized lineage acquisition and dedifferentiation at the cell and organ level in vivo.

developmental biology↗

bioRxiv2025

Adeno-associated virus (AAV)-TBX18 does not generate biological pacemaker activity, unlike AAV-Hcn2

Gene therapy-based biological pacemakers have been proposed as an alternative to their hardware-based counterparts. In this context, short-term ectopic expression of the T-box transcription factor 18 (TBX18) in the ventricle reportedly generated potent short-term pacemaker function in various animal models. Here, we investigated the effect of adeno-associated virus (AAV)-mediated long-term expression of TBX18, and compared the outcome to that of the pacemaker ion channel Hcn2. Our findings revealed that CMV-driven ectopic TBX18 expression in mouse hearts led to severe cardiac fibrosis. At lower, non-fibrogenic levels, TBX18 maintained its transcriptional function but failed to induce pacemaker phenotypes. TBX18-expressing cells showed suppressed expression of key working myocardial genes, but the pacemaker gene program was not induced. Electrophysiological studies showed abnormal automaticity in TBX18-expressing cells, combined with prolonged repolarization and various current changes. However, no hyperpolarization-activated funny current was detected. In a complete AV-block rat model, AAV-mediated Hcn2 expression induced robust ectopic pacemaker activity in the presence of isoproterenol, whereas TBX18 expression neither generated such activities, nor augmented Hcn2-mediated pacing. In conclusion, at functional non-fibrogenic levels, TBX18 is neither sufficient nor necessary to induce pacemaker activity. In contrast, Hcn2 generates reliable pacing, making it a more viable candidate for biological pacemaker development.

physiology↗

bioRxiv2025

AAV6-HCN4t-mediated biological pacing as a potential life-saving therapy for congenital complete heart block

Congenital complete heart block (CCHB) is a life-threatening condition in fetuses due to severe bradycardia. Maternal administration of {beta}-adrenergic agonists is used to increase fetal heart rates, but its effectiveness is limited and lost over time most likely due to insufficient expression of HCN channels in some individuals. We report the development of an injectable gene therapy that produces reliable cardiac pacemaker function in the presence of {beta}-adrenergic stimulation. Intramyocardial injection of adeno-associated viral serotype 6 vectors expressing HCN4t (AAV6-HCN4t) into the left ventricular apex significantly increased ectopic pacing frequency and heart rate in response to isoproterenol in rats with complete heart block, and this effect remained stable throughout the 4 weeks of follow-up. Injection of AAV6-HCN4t showed similar reliable biological pacing in complete heart block pigs. These results suggest that AAV6-HCN4t generates robust biological pacing in the presence of isoproterenol, providing the foundation for a potentially life-saving therapy for in utero CCHB.

physiology↗

bioRxiv2022

An atrial fibrillation-associated regulatory region modulates cardiac Tbx5 levels and arrhythmia susceptibility

Heart development and rhythm control are highly Tbx5 dosage-sensitive. TBX5 haploinsufficiency causes congenital conduction disorders, whereas increased expression levels of TBX5 in human heart samples has been associated with atrial fibrillation. We deleted the conserved mouse orthologues of two independent AF-associated genomic regions in the Tbx5 locus, one intronic (RE(int)-/-) and one downstream of Tbx5 (RE(down)-/-). In both lines we observed a modest (30%) increase of Tbx5 in the postnatal atria. To gain insight into the effects of slight dosage increase in vivo, we investigated the atrial transcriptional, epigenetic and electrophysiological properties of both lines. We observed induction of genes involved in development, ion transport and conduction, increased action potential duration and increased susceptibility to atrial arrhythmias. We identified an AF-associated variant in the human intronic regulatory region that increases transcriptional activity. Expression of the AF-associated transcription factor Prrx1 was induced in RE(int)-/- cardiomyocytes. We found that some of the transcriptional and functional changes in the atria caused by increased Tbx5 expression were normalized when reducing cardiac Prrx1 expression in RE(int)-/- mice, indicating an interaction between these two AF genes. We conclude that modest increases in expression of dose-dependent transcription factors, caused by common regulatory variants, significantly impact on the cardiac gene regulatory network and disease susceptibility.

molecular biology↗

bioRxiv2021

A single cell transcriptional roadmap for human pacemaker cell differentiation

Each heartbeat is triggered by the sinoatrial node, the natural pacemaker of the heart. Animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into "transitional", "tail" and "head" subtypes. However, the underlying molecular mechanisms are poorly understood. Here, we studied the differentiation of human induced pluripotent stem cells into pacemaker cardiomyocytes. Single cell RNA sequencing identified the presence of myocardial populations resembling subtypes present in the formed sinoatrial node, and in addition revealed a side population of (pro)epicardial cells. Time-course trajectory analysis uncovered a role for WNT signaling in determining myocardial versus proepicardial cell fate. We experimentally demonstrate that presence of WNT signaling prior to the branching point of a common progenitor enhances proepicardial cell differentiation at the expense of myocardial pacemaker cells. Furthermore, we uncover a role for TGF{beta} and WNT signaling in differentiation towards transitional and head pacemaker subtypes, respectively. Our findings provide new biological insights into human pacemaker differentiation, open avenues for complex disease modeling and inform regenerative approaches.

developmental biology↗

bioRxiv2020

Twisting of the heart tube during cardiac looping is a tbx5-dependent and tissue-intrinsic process

Organ laterality refers to the Left-Right (LR) asymmetry in disposition and conformation of internal organs, established in the developing embryo. The heart is the first organ to display visible LR asymmetries as it is positioned to the left side of the midline and undergoes rightward looping morphogenesis. Cardiac looping morphogenesis is tightly controlled by a combination of heart-intrinsic and -extrinsic mechanisms. As the mechanisms that drive cardiac looping are not well understood, we performed a forward genetic screen for zebrafish mutants with defective heart looping. We describe a new loss-of-function allele for tbx5a, which displays normal leftward positioning but defective rightward looping morphogenesis. By using live two-photon confocal imaging to map cardiomyocyte behavior during cardiac looping at a single-cell level we establish that during looping, ventricular and atrial cardiomyocytes rearrange in opposite directions towards the outer curvatures of the chambers. As a consequence, the cardiac chambers twist around the atrioventricular canal resulting in torsion of the heart tube, which is compromised in tbx5a mutants. Manipulations of cardiac looping by chemical treatment and ex vivo culture establishes that the twisting of the heart tube depends on intrinsic mechanisms and is independent from tissue growth by cell addition. Furthermore, the cardiac looping defect in tbx5a mutants is rescued in tbx5a/tbx2b double mutants, indicating that it requires proper tissue patterning. Together, our results establish that cardiac looping in zebrafish involves twisting of the chambers around the AV canal, which requires correct tissue patterning by Tbx5a.

developmental biology↗