Methods and Results. RNA expression of GADLORs was elevated in the myocardium and serum of heart failure patients. We investigated GADLOR expression in different organs in wild-type Bl/6J mice, and found higher expression levels in the brain, liver, heart, and kidney compared to other organs. Postnatal analysis revealed stable expression of GADLORs in brain, while their expression increased in heart and liver towards adulthood. Cellular expression analysis in the heart showed the highest GADLOR expression in cardiac macrophages, followed by FBs and ECs, whereas CMs showed the lowest expression level. Interestingly, GADLOR expression was strongly enriched in EVs derived from ECs and FBs, respectively, while macrophages did not release GADLORs in vesicles.

To determine the impact of endogenous GADLORs, mice with a systemic knock-out of both GADLORs (KO) were generated. Unchallenged KO mice were indistinguishable from their wild-type littermates (WT) based on appearance, body weight, heart weight, and baseline echocardiographic analysis, although liver weight was mildly increased in KO mice.

We next investigated whether GADLOR knock-out affects the response to cardiac pressure overload. As a consequence of transverse aortic constriction (TAC), deletion of GADLORs markedly ameliorated cardiac hypertrophy as shown by a reduced increase in cardiomyocyte area and hypertrophy-associated genes (Nppa, Nppb, Myh7/6 ratio). Additionally, GADLOR KO mice exerted less TAC-induced cardiac dysfunction and loss of compliance (Ejection fraction: 41.73 vs. 30.66%, Global longitudinal strain: -12.09% vs. -6.96%, KO vs. WT, p<0.0001, n=10) and were protected from heart failure-induced lung congestion. Additionally, myocardial capillarization during pressure overload was substantially increased in KO mice 2 weeks after TAC compared to WT mice. Interestingly, KO mice were strongly protected from extracellular matrix deposition (shown by Sirius red staining), and exerted significantly less induction of both collagen I and III at the mRNA level (approx. 2-fold vs. 6-fold increase KO vs. WT, p<0.05, n=5), indicating a massively improved fibrotic response and a preserved endothelial function. Despite the high abundance of GADLORs in cardiac macrophages observed under basal conditions, no difference in recruitment of CD45+ cells was observed between WT and KO mice after TAC.

RNA sequencing from cardiac tissue after TAC confirmed the downregulation of matrix gene expression (e.g. Col1a1, Comp, Fmod) in KO mice and additionally revealed a significant upregulation of genes related to fatty acid metabolism (e.g. Acat1, Echs1, Acadm), and downregulation of cardiac muscle contraction genes (e.g. Myh7, Cacna1c, Slc8a1) in KO mice compared to WT mice. Investigations are ongoing to decipher the molecular mechanism of the GADLOR effects.  

Conclusion. GADLORs are mainly expressed in non-cardiomyocytes of the heart and upregulated in human heart failure. In a pre-clinical model of pressure overload, deletion of GADLORs protects from cardiac fibrosis and preserves cardiac function.