Heart failure induces significant changes in nuclear pore complex of human cardiomyocytes

Abstract

Aims: The objectives of this study were to analyse the effect of heart failure (HF) on several proteins of nuclear pore complex (NPC) and their relationship with the human ventricular function.

Methods and results: A total of 88 human heart samples from ischemic (ICM, n = 52) and dilated (DCM, n = 36) patients undergoing heart transplant and control donors (CNT, n = 9) were analyzed by Western blot. Subcellular distribution of nucleoporins was analysed by fluorescence and immunocytochemistry. When we compared protein levels according to etiology, ICM showed significant higher levels of NDC1 (65%, p<0.0001), Nup160 (88%, p<0.0001) and Nup153 (137%, p = 0.004) than those of the CNT levels. Furthermore, DCM group showed significant differences for NDC1 (41%, p<0.0001), Nup160 (65%, p<0.0001), Nup153 (155%, p = 0.006) and Nup93 (88%, p<0.0001) compared with CNT. However, Nup155 and translocated promoter region (TPR) did not show significant differences in their levels in any etiology. Regarding the distribution of these proteins in cell nucleus, only NDC1 showed differences in HF. In addition, in the pathological group we obtained good relationship between the ventricular function parameters (LVEDD and LVESD) and Nup160 (r = -0382, p = 0.004; r = -0.290, p = 0.033; respectively).

Conclusions: This study shows alterations in specific proteins (NDC1, Nup160, Nup153 and Nup93) that compose NPC in ischaemic and dilated human heart. These changes, related to ventricular function, could be accompanied by alterations in the nucleocytoplasmic transport. Therefore, our findings may be the basis for a new approach to HF management.

Figure 1. Influence of heart failure aetiology on the levels of nucleoporins.

NDC1, Nup155, Nup160, Nup153, Nup93 and TPR were analysed by Western blot techniques. The values from the CNT group were set to 100. The data are expressed as mean ± SEM in arbitrary units (optical density) of six independent experiments. ICM, ischaemic cardiomyopathy; DCM, dilated cardiomyopathy; CNT, control. **p<0.01, ***p<0.0001 vs. CNT group. ^##p<0.01, ICM vs. DCM group.

Figure 2. Relationships between Nup160 and NDC1 levels.

Subjects with ICM (A), DCM (B) and all patients (C). Values are normalized to β-actin and finally to CNT group.

Figure 3. Relationships between Nup160 levels and ventricular function parameters.

A) LVEDD (left ventricular end-diastolic diameter), B) LVESD (left ventricular end-systolic diameter) in HF patients group (ICM and DCM). Values are normalized to β-actin and finally to CNT group.

Figure 4. Effect of heart failure on cell distribution of some nucleoporins in left ventricular human cardiomyocytes.

Inmunofluorescence staining with and without DAPI of NDC1, Nup160 and Nup93 according to heart failure aetiology, control (CNT) (Figure A,C and E, fluorescence; Figure B, D and F, DAPI A–F), ischaemic (ICM) (Figure G, I and K, fluorescence; Figure H, J and L, DAPI G–L) and dilated (DCM) (Figure M, O and Q, fluorescence; Figure N, P and R, DAPI M–R) groups. Arrows shows the immunofluorescence due to lipofuscin particles. Scale bar = 10 µm.

Figure 5. Immunolocalization of NDC1 in human cardiomyocytes and western blot of nucleoporins in nuclear and cytosolic fraction.

(A) Electron micrograph, in all case, gold particles (10 nm) are over nuclear pore complex; in control (a), dilated (b) and ischaemic tissue (c). The labeling is increased in ischaemics. No labely of NDC1 was observed in other nuclear structure. Scale bar = 50 nm. (B) Western blot analysis of NDC1, Nup160 and Nup93 in nuclear (Nu) and cytosolic (Cy) fraction in controls and HF patients (ICM and DCM).