Fibroblast-cardiomyocyte crosstalk as a regulator of the adaptive response of the neonatal mouse heart to pressure overload

Introduction: Heart disease is the most common cause of death worldwide. One potential reason is the insufficient regenerative capacity of the adult myocardium. In contrast, neonatal mice at postnatal day 0 (P0) display cardiac regenerative ability, which is lost shortly after birth. Earlier work of our group has shown that neonatal hearts at P0 can also adapt to pressure overload (POL), which is one of the known detrimental stimuli triggering heart failure in adults. This adaptive response at P0 is characterized by enhanced cardiomyocyte (CM) proliferation and angiogenesis with preserved cardiac function in the absence of fibrosis or hypertrophy. POL at P7, however, induces a maladaptive response characterized by CM hypertrophy and fibrosis with reduced cardiac function. Given the differences in interstitial fibrosis, which was only observed upon POL in P7 mice, we hypothesized that P0 and P7 cardiac fibroblasts (cFB) have different features and respond differently to POL, potentially underlying the adaptive and maladaptive responses of P0 vs P7 hearts. We have therefore characterized herein the cellular and molecular properties of cFB at P0 and P7 and their response to POL e.g. cFB-CM crosstalk.

Methods: POL was induced in wild type (CD1) mice by performing neonatal transverse aortic constriction (nTAC) at P0 and P7. cFB and CM were isolated 3 days after nTAC using magnetic activated cell sorting. Bulk RNAseq was performed to study their transcriptome and identify potential ligand-receptor interactions. Additionally, cFB were cultured, and their supernatant was collected to study cFB-CM crosstalk in vitro. For this purpose, primary neonatal mouse CM were treated with the supernatant of cFB from nTAC P0/P7 and control groups. Afterwards, immunohistological analyses were performed to assess the effect of cFB secretomes on CM hypertrophy, binucleation, proliferation as well as apoptosis. Next, we identified a factor that could be responsible for the observed effect and therefore tested its interaction and effect on CM to validate the identified cFB-CM crosstalk in vitro. Additionally, western blot and immunohistological analyses were performed to assess the corresponding receptor's expression in CM.

Results: Immunohistological analyses of CM treated with the cFB supernatant isolated from nTACP0 revealed enhanced cell cycle activity, pro-survival and anti-hypertrophic effects on CM. In contrast, cFB supernatant from nTACP7 had pro-hypertrophic, pro-apoptotic and pro-binucleation effects on CM. Transcriptomics identified a collagen signalling underlying cFB-CM crosstalk, which could be responsible for the observed effect and thus, responsible for the adaptive response of P0 hearts to POL. Testing of this crosstalk in vitro by culturing primary CM on collagen-coated plates confirmed its anti-apoptotic, anti-hypertrophic, and anti-binucleation and pro-proliferative effects on CM. We also discovered downregulation of the corresponding receptor with age and after nTACP7.

Conclusion: Our study revealed that cFB-CM crosstalk through collagen signalling plays a crucial role in the adaptive response of P0 and the maladaptive response of P7 hearts. Taking advantage of the use of recombinant proteins and blocking antibodies in vitro and in vivo, we are currently investigating the underlying mechanism.