Chronic kidney disease induces left ventricular overexpression of the pro-hypertrophic microRNA-212

Abstract

Chronic kidney disease (CKD) is a public health problem that increases the risk of cardiovascular morbidity and mortality. Heart failure with preserved ejection fraction (HFpEF) characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction is a common cardiovascular complication of CKD. MicroRNA-212 (miR-212) has been demonstrated previously to be a crucial regulator of pathologic LVH in pressure-overload-induced heart failure via regulating the forkhead box O3 (FOXO3)/calcineurin/nuclear factor of activated T-cells (NFAT) pathway. Here we aimed to investigate whether miR-212 and its hypertrophy-associated targets including FOXO3, extracellular signal-regulated kinase 2 (ERK2), and AMP-activated protein kinase (AMPK) play a role in the development of HFpEF in CKD. CKD was induced by 5/6 nephrectomy in male Wistar rats. Echocardiography and histology revealed LVH, fibrosis, preserved systolic function, and diastolic dysfunction in the CKD group as compared to sham-operated animals eight and/or nine weeks later. Left ventricular miR-212 was significantly overexpressed in CKD. However, expressions of FOXO3, AMPK, and ERK2 failed to change significantly at the mRNA or protein level. The protein kinase B (AKT)/FOXO3 and AKT/mammalian target of rapamycin (mTOR) pathways are also proposed regulators of LVH induced by pressure-overload. Interestingly, phospho-AKT/total-AKT ratio was increased in CKD without significantly affecting phosphorylation of FOXO3 or mTOR. In summary, cardiac overexpression of miR-212 in CKD failed to affect its previously implicated hypertrophy-associated downstream targets. Thus, the molecular mechanism of the development of LVH in CKD seems to be independent of the FOXO3, ERK1/2, AMPK, and AKT/mTOR-mediated pathways indicating unique features in this form of LVH.

Selective Heart Irradiation Induces Cardiac Overexpression of the Pro-hypertrophic miR-212

“…Five-micrometer paraffin-embedded transverse cut sections of the formalin-fixed subvalvular area of the ventricles were stained with hematoxylin-eosin, in the samples collected at week 19. On these slides, myocardial fiber diameters were measured to verify the development of LVH as described previously (37, 38). Transverse transnuclear widths (cardiomyocyte diameter) were measured of 100 longitudinally oriented, mono-nucleated cardiomyocytes on left ventricle sections cut on the same plane (37, 38).…”

“…Five-micrometer paraffin-embedded transverse cut sections of the formalin-fixed subvalvular area of the ventricles were stained with picrosirius red staining in the samples collected at week 19, to assess cardiac fibrosis as described previously [Figure 1D; (16, 38)]. Histological slides were scanned with a Pannoramic P250 scanner (3D-Histech, Budapest, Hungary) and digital images at the magnification of ×40 and ×100 were captured.…”

“…Medium-size vessels and their perivascular connective tissue sheet, the subepicardial and subendocardial areas were avoided as best as possible. The picrosirius red dyed images were analyzed with an in-house developed program as described previously (16, 38). This program determines the proportion of red pixels of heart sections using two simple color filters.…”

“…Quantitative RT-PCR was performed with miR-specific primers to monitor miR expression as described earlier [Figure 1D; (22, 38)]. In the case of miR-212, RNA was isolated using Trizol reagent (Invitrogen, #15596-018) from heart tissue.…”

“…Target mRNAs of miR-212 associated with cardiac hypertrophy and heart failure were selected in the TargetScan database. Quantitative RT-PCR was performed with gene-specific primers to monitor mRNA expression as described previously [Figure 1D; Table 1; (38, 39)]. RNA was isolated using Qiagen RNeasy Fibrous Tissue Mini Kit (Qiagen, #74704) from heart tissue.…”