han, m. | Search bioRxiv

bioRxiv2024

Interleaved intersectional strategy enables genetic lineage tracing with enhanced specificity

BACKGROUNDDual recombinases have been increasingly employed for enhanced precision in genetic targeting. A recent study utilizing an intersectional genetic approach through dual recombinases (Dre + CreER) has revealed that endocardium-derived fibroblasts (EndoFbs) play a pivotal role in cardiac fibrosis after pressure overload. However, this intersectional strategy has limitations, primarily due to ectopic genetic labeling of non-target cells within the adult heart by the constitutively active Dre recombinase. METHODSTo address this issue, we have developed an advanced, interleaved and intersectional reporter (IIR) strategy in this study. This IIR strategy leverages an inducible CreER to prevent inadvertent Dre-rox recombination during development or disease progression by designing an interleaved reporter to allow for more specific tracing of EndoFbs. Moreover, our IIR system also incorporates Diphtheria Toxin Receptor (DTR) in targeted cells, enabling functional characterization of these cells after genetic ablation. RESULTSEndoFbs were regionally distributed in the heart during homeostasis and proliferated preferentially in response to pressure overload, leading to cardiac fibrosis in defined regions. The IIR strategy enables the tracing of EndoFbs with a more prominent regional pattern and facilitates genetic ablation of EndoFbs through DT injection. In addition, we have applied this IIR strategy to specifically target fibroblasts derived from the epicardium (EpiFbs). Genetic lineage tracing of EpiFb reveals that their distribution pattern is complementary to that of EndoFbs in the adult heart. When a substantial number of EpiFbs were genetically ablated, EndoFbs could replace the loss of EpiFbs in some specific regions of hearts. CONCLUSIONSThe IIR strategy refines the precision of genetic lineage tracing while still employing the constitutively active Dre recombinase in tandem with inducible Cre. EndoFbs and EpiFbs are complementary in their distribution pattern in the heart, where EndoFbs have the potential to replace the loss of EpiFbs in some regions.

genetics↗

bioRxiv2020

Repression of Pdzrn3 is required for heart maturation and protects against heart failure

Heart failure is the final common stage of most cardiopathies. Cardiomyocytes connect with others via their extremities by intercalated disk protein complexes. This planar and directional organization of myocytes is crucial for mechanical coupling and anisotropic conduction of the electric signal in the heart. One of the hallmarks of heart failure is alterations in the contact sites between cardiomyocytes. Yet no factor on its own is known to coordinate cardiomyocyte polarized organization. We report enhanced levels of an ubiquitine ligase Pdzrn3 in diseased hypertrophic human and mouse myocardium, which correlates with a loss of cardiomyocyte polarized elongation. We provide evidence that Pdzrn3 has a causative role in heart failure. We found that cardiac Pdzrn3 deficiency protected against heart failure while over expression of Pdzrn3 in mouse cardiomyocytes during the first weeks of life, impaired postnatal cardiomyocyte maturation leading to premature death. Our results reveal a novel signaling pathway that controls a genetic program essential for heart maturation and maintenance of overall geometry, as well as the contractile function of cardiomyocytes, and implicates PDZRN3 as a potential therapeutic target for the prevention of human heart failure.

physiology↗