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Unfolded Protein Response and Mitochondria Crosstalk Drives PINK1 Downregulation and Mitochondrial Dysfunction in Pulmonary Endothelial Cells
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A2877 - Unfolded Protein Response and Mitochondria Crosstalk Drives PINK1 Downregulation and Mitochondrial Dysfunction in Pulmonary Endothelial Cells
Author Block: L. Tu, M. Bueno, L. Voltz, A. L. Mora; Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, United States.
Rationale: Mitochondrial dysfunction has been demonstrated as a key contributor to the development of pulmonary arterial hypertension (PAH). The crosstalk between mitochondria and endoplasmic reticulum (ER) is recognized, including the unfolded protein response (UPR) triggering changes in mitochondrial function. PINK1, one of the key regulators of mitochondrial homeostasis is transcriptionally repressed by activation of the UPR. Therefore, we examined how PINK1 deficiency and ER stress affects mitochondrial function in human pulmonary artery endothelial cells (PAECs) and smooth muscle cells (PASMCs).
Methods: PINK1 expression level was analyzed in isolated PAECs and PASMCs from PAH patients and healthy donors. PINK1 was then knocked down by siRNA in human primary PAECs and PASMCs cell lines. Mitochondrial respiration was compared between PINK1 deficient cells and control cells using the Seahorse extracellular flux analyzer. Cells were also treated with Tunicamycin to induce ER stress. Mitochondrial mass, membrane potential (MMP) and superoxide were measured by Mitotracker Deep red, Tetramethylrhodamine methyl ester (TMRM) and Mitosox respectively to compare the changes among different groups.
Results: ATF3, an ER stress-induced transcriptional repressor of PINK1, was highly upregulated and PINK1 expression was significantly reduced in PAECs but not in PASMCs of PAH patients compared to the control donors. In primary PAECs cell line, loss of PINK1 caused a significant decrease in mitochondrial maximal respiration and spare respiratory capacity, an increase in mitochondrial mass, and a decrease in MMP by 50%. In PASMCs, PINK1 deficiency did not affect mitochondrial respiration, but also caused an increase in mitochondrial mass and a decrease in MMP albeit to a smaller extent compared to the changes in PAECs. Gene expression analysis showed that similar to PAECs from PAH patients, Tunicamycin-treated PAECs upregulated ATF3 and severely downregulated PINK1 while no changes were observed in PASMCs. Tunicamycin-treated PAECs also showed a significant increase in mitochondrial mass and a reduction in MMP, similar to PINK1-deficient PAECs.
Conclusion: Unlike PASMCs, PAECs downregulated PINK1 in response to ER stress. Loss of PINK1 caused more severe mitochondrial dysfunctions in PAECs than in PASMCs, making PAECs more fragile and susceptible to further injuries. Hence, PINK1 deficiency in PAECs might increase the susceptibility to PAH.
Author Block: L. Tu, M. Bueno, L. Voltz, A. L. Mora; Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, United States.
Rationale: Mitochondrial dysfunction has been demonstrated as a key contributor to the development of pulmonary arterial hypertension (PAH). The crosstalk between mitochondria and endoplasmic reticulum (ER) is recognized, including the unfolded protein response (UPR) triggering changes in mitochondrial function. PINK1, one of the key regulators of mitochondrial homeostasis is transcriptionally repressed by activation of the UPR. Therefore, we examined how PINK1 deficiency and ER stress affects mitochondrial function in human pulmonary artery endothelial cells (PAECs) and smooth muscle cells (PASMCs).
Methods: PINK1 expression level was analyzed in isolated PAECs and PASMCs from PAH patients and healthy donors. PINK1 was then knocked down by siRNA in human primary PAECs and PASMCs cell lines. Mitochondrial respiration was compared between PINK1 deficient cells and control cells using the Seahorse extracellular flux analyzer. Cells were also treated with Tunicamycin to induce ER stress. Mitochondrial mass, membrane potential (MMP) and superoxide were measured by Mitotracker Deep red, Tetramethylrhodamine methyl ester (TMRM) and Mitosox respectively to compare the changes among different groups.
Results: ATF3, an ER stress-induced transcriptional repressor of PINK1, was highly upregulated and PINK1 expression was significantly reduced in PAECs but not in PASMCs of PAH patients compared to the control donors. In primary PAECs cell line, loss of PINK1 caused a significant decrease in mitochondrial maximal respiration and spare respiratory capacity, an increase in mitochondrial mass, and a decrease in MMP by 50%. In PASMCs, PINK1 deficiency did not affect mitochondrial respiration, but also caused an increase in mitochondrial mass and a decrease in MMP albeit to a smaller extent compared to the changes in PAECs. Gene expression analysis showed that similar to PAECs from PAH patients, Tunicamycin-treated PAECs upregulated ATF3 and severely downregulated PINK1 while no changes were observed in PASMCs. Tunicamycin-treated PAECs also showed a significant increase in mitochondrial mass and a reduction in MMP, similar to PINK1-deficient PAECs.
Conclusion: Unlike PASMCs, PAECs downregulated PINK1 in response to ER stress. Loss of PINK1 caused more severe mitochondrial dysfunctions in PAECs than in PASMCs, making PAECs more fragile and susceptible to further injuries. Hence, PINK1 deficiency in PAECs might increase the susceptibility to PAH.
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