Molecular alterations of human diabetic heart disease revealed using single nucleus RNA sequencing

Abstract Type 2 diabetes mellitus (T2D) increases the risk for heart failure. Whether mechanisms proposed to underlie cardiac structural and functional alterations in the rodent diabetic heart are recapitulated in humans remains poorly investigated. Here, we studied left ventricular samples of 8 subjects with T2D, preserved ejection fraction (63±5%) and no history of ischemic heart disease (Db-pEF), 7 subjects with T2D, reduced ejection fraction (26±9%) and ischemic heart disease (Db-ICM), and 15 non-diabetic individuals with normal ejection fraction (Non-Db-pEF) serving as controls. 1168 proteins were identified using label-free proteomics by LC-MS/MS. 146 were differentially expressed in Db-ICM, but only 66 in Db-pEF. Bulk RNA sequencing revealed differential expression of 1795 and 527 genes in Db-ICM or Db-pEF, respectively, with only 128 genes being commonly regulated. Pathway analysis revealed a significant regulation of pathways related to inflammation or extracellular matrix remodelling in Db-ICM and Db-pEF, however literally no enrichment in pathways previously related to diabetic cardiomyopathy. To characterize the cardiac cellular heterogeneity, we performed single nucleus RNA sequencing (snRNAseq) that showed no differences in cell frequencies between the groups. However, analysis of genes differentially expressed in cardiomyocytes of diabetic hearts unmasked enrichments in many pathways related to rodent diabetic cardiomyopathy not observed by proteomics or RNA sequencing, including insulin resistance, fatty acid oxidation, oxidative phosphorylation, oxidative stress, and various signaling pathways. Furthermore, interactome analysis revealed fewer cell-cell interactions in Db-pEF and Db-ICM compared to controls and implied differential activation of several signaling pathways to contribute to diabetic heart disease, including adiponectin, hepatocyte growth factor, and natural cell adhesion molecule signaling. Thus, we present the first comprehensive atlas of molecular and cellular alterations in the human diabetic heart, revealing recapitulation of many pathomechanisms previously proposed in animal studies. This analysis represents a decisive step towards understanding the pathology underlying human diabetic heart disease and may help develop future diagnostic and therapeutic strategies.