T cell-instructed extracellular matrix remodeling underlies diastolic dysfunction in cardiometabolic HFpEF (DGK-Forschungsstipendium)

Heart failure with preserved ejection fraction (HFpEF) presents a major health burden affecting >5% of people in the Western world. Rising prevalence and the lack of effective treatment make HFpEF one of the greatest unmet medical needs in modern medicine. The recent development of preclinical models of HFpEF led to the discovery that combined co-morbidities like obesity and hypertension cause systemic inflammation and persistent cardiotropic CD4⁺ T cell response. This occurs simultaneously with stiffening of the left ventricle (LV), which consequently impairs diastolic function. However, the mechanisms by which cardiac T cell infiltration causes diastolic dysfunction in this context remain incompletely understood. As the extracellular matrix (ECM) is a major contributor to myocardial stiffness, we hypothesized that T cell infiltration causes ECM remodeling in cardiometabolic HFpEF.

C57BL/6J mice were kept on a high fat diet (HFD, 60% of caloric intake) and received the hypertensive agent L-NAME (0.5 g/L in drinking water) for 5 weeks, while control mice were kept on a standard diet. After 5 weeks, we assessed systolic and diastolic cardiac function by Doppler echocardiography. The numbers of non-myocyte cell types in LV digests were quantified by flow cytometry and we evaluated the abundance and mechanical properties of the ECM using histological, biochemical and mechanical analyses. Finally, we used isolated primary splenic CD4⁺ T cells and cardiac fibroblasts from C57BL/6J mice to assess heterocellular crosstalk in vitro.

Mice receiving HFD+L-NAME showed a higher ratio of early:late mitral valve inflow velocity (E/A) and no significant differences in ejection fraction compared to standard-fed mice, indicative of diastolic dysfunction and preserved systolic function. Flow cytometry revealed an increased number of cardiac CD45⁺ leukocytes and CD4⁺ T cells in response to HFD and L-NAME compared to control mice. Despite no significant differences in deposited collagen probed by Picrosirius Red in LV sections, we found more ECM-derived peptides and higher abundance of the lysyl oxidase LOXL3 in the hearts of HFD and L-NAME treated mice compared to controls. Tensile testing of decellularized LV revealed higher stiffness of the ECM in the hearts of HFD and L-NAME treated mice. Strikingly, we found that ECM stiffness strongly correlated with E/A, positioning the ECM as an important determinant of diastolic function in this model. T cell-deficient mice (Tcra^-/-) hearts did not develop increased ECM stiffness and consequently preserved diastolic function in response to HFD and L-NAME. In vitro treatment of primary cardiac fibroblasts with conditioned media from splenic CD4+ T cell blasts resulted in increased expression of Loxl3. This was prevented by the addition of an interferon-γ neutralizing antibody. Moreover, Loxl3 enriched cardiac fibroblast culture media induced stiffening of decellularized ECM preparations ex vivo. This suggests that T cell-derived interferon-γ initiates ECM stiffening via the expression of LOXL3 in cardiac fibroblasts.

Taken together, our data demonstrates that diastolic dysfunction in cardiometabolic HFpEF is caused by T cell dependent sub-histological ECM remodeling through induction of LOXL3 secretion in cardiac fibroblasts that contributes to elevated ECM crosslinking. Ongoing studies are investigating if inhibition of lysyl oxidase activity protects mice from diastolic dysfunction in cardiometabolic HFpEF.