Dysfunction of mitochondria Ca²⁺ uptake in cystic fibrosis airway epithelial cells

Abstract

In the genetic disease cystic fibrosis (CF), the most common mutation F508del promotes the endoplasmic reticulum (ER) retention of misfolded CF proteins. Furthermore, in homozygous F508del-CFTR airway epithelial cells, the histamine Ca²⁺ mobilization is abnormally increased. Because the uptake of Ca²⁺ by mitochondria during Ca²⁺ influx or Ca²⁺ release from ER stores may be crucial for maintaining a normal Ca²⁺ homeostasis, we compared the mitochondria morphology and distribution by transmission electron microscopy technique and the mitochondria membrane potential variation (ΔΨ_mit) using a fluorescent probe (TMRE) on human CF (CF-KM4) and non-CF (MM39) tracheal serous gland cell lines. Confocal imaging of Rhod-2–AM-loaded or of the mitochondrial targeted cameleon 4mtD3cpv-transfected human CF and non-CF cells, were used to examine the ability of mitochondria to sequester intracellular Ca²⁺. The present study reveals that (i) the mitochondria network is fragmented in F508del-CFTR cells, (ii) the ΔΨ_mit of CF mitochondria is depolarized compared non-CF mitochondria, and (iii) the CF mitochondria Ca²⁺ uptake is reduced compared non-CF cells. We propose that these defects in airway epithelial F508del-CFTR cells are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca²⁺ uptake.

Introduction

In the genetic disease cystic fibrosis (CF), mutations of cystic fibrosis transmembrane conductance regulator gene (CFTR), among them the deletion of phenylalanine at position 508 (F508del-CFTR), lead to chloride impermeability in many exocrine glands (salivary, airways, pancreatic) associated with dehydration and reduced volume of the final secretory fluid (Kerem et al., 1989, Quinton, 1990, Riordan et al., 1989, Rommens et al., 1989). CFTR is a cAMP-activated Cl⁻ channel and a regulator of the activity of other channels in the apical membrane of epithelial cells (Chabot et al., 1999, Stutts et al., 1995, Wei et al., 1999). The misfolded F508del-CFTR protein is trapped in the endoplasmic reticulum (ER) (Cheng et al., 1990) by multiple interactions with ER chaperone proteins (Helenius and Aebi, 2004, Norez et al., 2006b, Pind et al., 1994, Ware et al., 1995). In addition to this molecular defect, abnormal cytosolic Ca²⁺ signalling has been reported, whereas no apparent defect in cAMP signalling occurs in CF (Antigny et al., 2008, Ribeiro et al., 2005). In particular, cytosolic Ca²⁺ signalling appears to be exacerbated in homozygous F508del-CFTR epithelial cells (Antigny et al., 2008, Ribeiro et al., 2005, Tabary et al., 2006).

An important aspect of cytosolic Ca²⁺ signalling, infrequently studied in the CF field, is the role that mitochondria are playing as a Ca²⁺ sink to modulate cytosolic Ca²⁺ signalling as shown in excitable and non-excitable cells (Carafoli, 1987, Gunter and Pfeiffer, 1990, Babcock et al., 1997, Budd and Nicholls, 1996, Hoth et al., 1997, Ichas et al., 1997, Jouaville et al., 1995). Calcium as an activator of mitochondrial dehydrogenases (McCormack and Denton, 1990) could be an ideal signal to synchronize cell function and mitochondrial metabolism during stimulation by Ca²⁺-mobilizing stimuli. To support this hypothesis, previous and pioneer works revealed that mitochondria are dysfunctional in CF fibroblast cells and that the major site of increased Ca²⁺ signal in CF is mitochondria (Shapiro and Lam, 1987, Shapiro, 1989). The mitochondria from CF patients consume more oxygen than normal (Shapiro, 1988, Shapiro, 1989), respond differentially to inhibitors of mitochondrial function, express increased electron transport activity and altered kinetics of complex I (of the mitochondrial electron transport system) (Shapiro et al., 1982, Shapiro, 1989). In airway epithelia, mitochondria function as an intracellular Ca²⁺ buffering system and provide structural and compartmentalizing barriers that protect the airway epithelia against non-specific activation of Ca²⁺ modulated functions (Ribeiro et al., 2003). More recently, Il-1β induced mitochondria Ca²⁺ uptake was absent in CFTR-deficient IB3-1 cells compared to CFTR-corrected S9 cells (Tabary et al., 2006). In addition, the uptake of Ca²⁺ by mitochondria during calcium influx or calcium release from ER stores may be crucial for maintaining calcium homeostasis. In mitochondria, the uptake of Ca²⁺ is driven by the membrane potential and mediated by an electrogenic uniporter, the activity of which is triggered by a rise in [Ca²⁺]i and depends on the high inner mitochondrial membrane potential (ΔΨ_m). A recent study showed an exaggerated apoptosis in response to actinomycin D (Act D) and staurosporine in pancreatic duct and tracheal cystic fibrosis epithelial cells compared to non-CF cells (Rottner et al., 2007). Interestingly, the inhibition of CFTR by CFTR_inh-172, a selective CFTR inhibitor (Ma et al., 2002) increased the percentage of apoptotic cells in non-CF pancreatic duct PANC-1 cells and in CF pancreatic duct CFPAC-1 cells (Rottner et al., 2007). Moreover, in airway polymorphonuclear neutrophil (PMN) of CF patients the activity of caspase 3 is significantly greater than in PMN of non-CF patients (Tabary et al., 2006). Mitochondria also play key roles in apoptosis (Mayer and Oberbauer, 2003). Recently, two studies showed that in CF cells, two mitochondrial genes implicated in complex I subunits are in various ways regulated and are dependent on the chloride channel activity of CFTR (Taminelli et al., 2007, Valdivieso et al., 2007).

In this report we examined the Ca²⁺ homeostasis and the ability of mitochondria to sequester Ca²⁺ in human F508del-CFTR homozygous tracheal gland CF-KM4 cells (Kammouni et al., 1999) compared to non-CF tracheal serous gland epithelial MM39 cells (Merten et al., 1996). We studied the mitochondria morphology, the mitochondria membrane potential and measured the activity of caspase 3. Finally, we examined the mitochondrial Ca²⁺ buffering power in F508del-CFTR and non-CF cells.