Tcf21-iCre does not sufficiently target adult cardiac fibroblasts after intraperitoneal delivery of tamoxifen

Cardiac fibroblasts (cFb) are critical mediators of fibrosis, a pathological process that leads to myocardial stiffening and eventually heart failure. Despite their importance, targeting fibroblast remains a challenge due to their heterogeneity and diverse embryonic origins. The transcription factor Tcf21 has been described as a marker for resident cardiac fibroblasts in the adult heart, offering the potential to specifically target those cells genetically using the Cre/loxP system.

 
We investigated the efficiency of Tcf21-driven iCre-mediated recombination of a floxed reporter gene in cardiac fibroblasts with and without transverse aortic constriction (TAC) surgery, a well-established model of pressure overload-induced cardiac fibrosis. A dual-fluorescent Cre-reporter mouse model (mTmG) was crossed with a mouse line expressing inducible Cre-recombinase (iCre) under the control of the Tcf21 promoter. The recombination was induced by tamoxifen administration (5 d i.p.) at 6 weeks of age and 2 weeks later cardiac fibroblasts from ventricular tissue were isolated. Alternatively, TAC surgery was performed 2 weeks after tamoxifen injections and cardiac fibroblasts were isolated after 2 weeks of TAC. The recombination efficiency was assessed at both mRNA and protein levels. Moreover, heart failure with preserved ejection fraction (HFpEF) was induced by administering a high fat diet and L-Name for 10 weeks.

Our data revealed that Tcf21 promoter activity is restricted to a small subset of cardiac fibroblasts in uninjured adult hearts. Consequently, tamoxifen-induced Tcf21-Cre-mediated recombination was observed only in a small fraction of fibroblasts, and some cells showed signs of incomplete recombination pointing towards low activity of the Tcf21 promoter. Following TAC surgery, we observed increased myocardial fibrosis; however, most fibrotic cells did not originate from previously labelled Tcf21+ fibroblasts. Furthermore, after the induction of HFpEF, TCF21-Cre mediated recombination was virtually absent in the myocardium.

This suggests that Tcf21-Cre-mediated recombination fails to target the fibroblast population that drives the fibrotic response after the induction of pressure overload. Consequently, this Cre-driver is not suitable to study the effects of loxP-dependent knockdown or overexpression of genes in cardiac fibroblasts in experimental models where the genetic model is induced prior to the intervention.

Future studies should explore alternative promoters or combinatorial approaches to more effectively target fibrogenic fibroblast populations involved in pathological remodelling. Enhancing our ability to manipulate these cells could pave the way to study potential targets for novel therapeutic interventions to mitigate fibrosis and preserve cardiac function.