Depressed endothelial inflammatory signaling drives early diastolic dysfunction in klotho heterozygote swine

Background The anti-aging protein Klotho is decreased in chronic kidney disease, and this decrease is associated with coronary microvascular dysfunction (CMD) and heart failure (HF). However, the precise molecular events that occur within the coronary endothelium and the myocardium at with systemic Klotho deficiency, as well as their contribution to CMD and HF remain uncharacterized (1). Purpose To investigate the early molecular and functional changes occurring in the myocardium, with a particular focus on the coronary endothelium, during the initial phase of systemic Klotho deficiency. Methods A novel transgenic pig model with heterozygous deletion of the Klotho gene was generated. Wild-type littermates served as controls. At 10 months of age, cardiac function was assessed by cardiac ultrasound and vasodilator reserve was assessed through intracoronary injection of adenosine. Finally, single-nucleus RNA sequencing (snRNA-seq) was performed on left ventricular tissue (3 in each group) to characterize cell-type-specific transcriptional changes. The transcriptomic data were analyzed using the R package Seurat, enabling in-depth profiling of endothelial and cardiomyocyte populations. Results Echocardiographic assessment revealed a depressed E/A ratio in Klotho heterozygous (Klotho+/-) pigs compared to wild-type (WT) controls, indicative of diastolic dysfunction along with increased left ventricular end diastolic (LVDd) and systolic diameter (LVDs)(Fig1 a-c). Notably, coronary flow reserve was significantly depressed in the Klotho+/- pigs (Fig 1d). Single-nucleus RNA sequencing of left ventricular tissue identified substantial downregulation of endothelial adhesion molecules VCAM1 and ICAM1, along with the chemokine CXCL10, suggesting suppressed inflammatory signaling in coronary endothelium. Further analysis showed decreased expression of JunB, an AP-1 family transcription factor, in these cells. In cardiomyocytes, snRNA-seq revealed significant downregulation of STAT3, coupled with upregulation of extracellular matrix components including COL1A1, ASPN, and ECM2, alongside downregulation of the matrix metalloproteinase inhibitor TIMP2. These molecular changes indicate dysregulated matrix remodeling consistent with enhanced fibrosis, which was confirmed by histological staining for fibrotic tissue. Conclusions This study demonstrates that partial Klotho deficiency in a novel transgenic pig model leads to early diastolic dysfunction accompanied by suppressed inflammatory signaling in coronary endothelial cells as well as abnormal myocardial extracellular matrix remodeling. The downregulation of key endothelial adhesion molecules and chemokines alongside reduced STAT3 in cardiomyocytes suggests that Klotho plays a critical role in maintaining coronary endothelial immunomodulatory functions and cardiomyocyte signaling integrity. These results identify a novel, non-inflammatory endothelial pathology as an initiating event in heart disease.