(1) Background: Diabetic cardiomyopathy is a major health problem worldwide. CTRP9, a secreted glycoprotein, is mainly expressed in cardiac endothelial cells and becomes downregulated in mouse models of diabetes mellitus; (2) Methods: In this study, we investigated the impact of CTRP9 on early stages of diabetic cardiomyopathy induced by 12 weeks of high-fat diet; (3) Results: While the lack of CTRP9 in knock-out mice aggravated insulin resistance and triggered diastolic left ventricular dysfunction, AAV9-mediated cardiac CTRP9 overexpression ameliorated cardiomyopathy under these conditions. At this early disease state upon high-fat diet, no fibrosis, no oxidative damage and no lipid deposition were identified in the myocardium of any of the experimental groups. Mechanistically, we found that CTRP9 is required for insulin-dependent signaling, cardiac glucose uptake in vivo and oxidative energy production in cardiomyocytes. Extensive RNA sequencing from myocardial tissue of CTRP9-overexpressing and knock-out as well as respective control mice revealed that CTRP9 acts as an anti-inflammatory mediator in the myocardium. Hence, CTRP9 knock-out exerted more, while CTRP9-overexpressing mice showed less leukocytes accumulation in the heart during high-fat diet; (4) Conclusions: In summary, endothelial-derived CTRP9 plays a prominent paracrine role to protect against diabetic cardiomyopathy and might constitute a therapeutic target.

Cardiac fibrosis plays a crucial role in heart failure, but research in this field remains challenging due to heterogeneity of cardiac fibroblast populations. In the past years, several Cre-drivers have been proposed for cardiac fibroblast-specific deletion of floxed genes in adult mouse research models. Here, we evaluated whether Tcf21-iCre, a tamoxifen-inducible Cre recombinase (iCre) under the endogenous Tcf21 promoter, was able to successfully target two floxed loci (Hypoxia inducible factor 1a (Hif1a) and the fluorescent reporter locus mTmG) after intraperitoneal tamoxifen injections. Six-week-old mice received intraperitoneal tamoxifen injections on five consecutive days to induce Cre activity, and the recombination efficiency was measured in isolated fibroblasts via qPCR, Western Blot and fluorescence microscopy. Recombination efficiency was very low, with minimal reduction in expression of Hif1a. For the mTmG locus, both bi-allelic and mono-allelic recombination was detected, with mono-allelic events suggesting low Cre activity even within Tcf21-expressing fibroblasts. The addition of experimental models of heart failure with reduced or preserved ejection fraction as pro-fibrotic stimuli or an inclusion of an additional round or higher doses of tamoxifen injections did not improve recombination efficiency of the targeted alleles. Thus, Tcf21-iCre targets only a small proportion of fibrogenic fibroblasts following i.p. tamoxifen injection, limiting its usability in loss-of-function studies that require broad fibroblast coverage and highlighting the need for alternative Cre-drivers or delivery strategies to facilitate antifibrotic therapy development.