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. 2015;11(2):385-402.
doi: 10.1080/15548627.2015.1009779.

Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease

Jeong-A Lim  1 , Lishu Li, Or Kakhlon, Rachel Myerowitz, Nina Raben
Affiliations

Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease

Jeong-A Lim et al. Autophagy. 2015.

Abstract

Mitochondria-induced oxidative stress and flawed autophagy are common features of neurodegenerative and lysosomal storage diseases (LSDs). Although defective autophagy is particularly prominent in Pompe disease, mitochondrial function has escaped examination in this typical LSD. We have found multiple mitochondrial defects in mouse and human models of Pompe disease, a life-threatening cardiac and skeletal muscle myopathy: a profound dysregulation of Ca(2+) homeostasis, mitochondrial Ca(2+) overload, an increase in reactive oxygen species, a decrease in mitochondrial membrane potential, an increase in caspase-independent apoptosis, as well as a decreased oxygen consumption and ATP production of mitochondria. In addition, gene expression studies revealed a striking upregulation of the β 1 subunit of L-type Ca(2+) channel in Pompe muscle cells. This study provides strong evidence that disturbance of Ca(2+) homeostasis and mitochondrial abnormalities in Pompe disease represent early changes in a complex pathogenetic cascade leading from a deficiency of a single lysosomal enzyme to severe and hard-to-treat autophagic myopathy. Remarkably, L-type Ca(2+)channel blockers, commonly used to treat other maladies, reversed these defects, indicating that a similar approach can be beneficial to the plethora of lysosomal and neurodegenerative disorders.

Keywords: AIFM1, apoptosis-inducing factor, mitochondrion-associated, 1; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DMEM, Dulbecco's modified Eagle's medium; EGTA, ethylene glycol-bis(2-aminoethylether)-N, N, N′, N′-tetraacetic acid; ERT, enzyme replacement therapy; GAA, glucosidase; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; LSD, lysosomal storage disease; MAP1LC3A/B (LC3), microtubule-associated protein 1 light chain 3 α/β; MOPS, 3-morpholinopropane-1-sulfonic acid; MitoG, MitoTracker Green; OMM, outer mitochondrial membrane; Pompe disease; RFP, red fluorescent protein; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; Ub, ubiquitinated; VDCC, voltage-dependent Ca2+ channel; autophagy; calcium; lysosome; mitochondria; mitophagy; α, acid.

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Figures

Figure 1.
Figure 1.
Morphological alterations in KO muscle cells. (A) Electron-microscopy of muscle biopsies (white part of gastrocnemius) from 5-mo-old WT and KO mice. Black arrows point to enlarged mitochondria with distorted cristae (middle and right panels and inset). Abnormal mitochondria are often found within the area of autophagic accumulation (white arrow; right panel). Bar = 1 μm. MitoTracker Red staining of myoblasts (B) reveals abnormal pattern in KO cells. CCCP, carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial-uncoupling reagent, was used at a concentration of 10 μM for 6 h. (C) Graphical representation of the images in (B); mitochondrial chain length in WT and KO myoblasts (n = 6). (D) Western blot analysis of whole muscle from 4- to 5-mo-old WT and KO mice with the indicated antibodies. (E) Graphical presentation of data in (D). Asterisks indicate P < 0.05.
Figure 2.
Figure 2.
Assessment of Ca2+ levels and flux in WT and KO muscle cells. (A) WT and KO myotubes (7 d in differentiation medium) were loaded with Fluo-4 dye and analyzed by confocal microscopy. The images show a significant increase in the steady-state level of cellular Ca2+ in the KO myotubes. Bar = 10 μm. (B) KO myotubes were treated with rhGAA at 5 μM for 4 d; the treatment resulted in efficient glycogen clearance (top; arrows point to glycogen deposition in untreated KO myotube) and a modest reduction of Ca2+ levels (bottom and (C) graphical representation of the images). Lysosomal glycogen in live cells was detected by the incorporation of fluorescent glucose derivative 2-NBDG [2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose] into glycogen. Bars = 10 μm. (D and E) Time-lapse experiment showing progressive influx of Ca2+ after its addition to the medium; the images, taken every 10 sec by confocal microscopy, illustrate a faster uptake of Ca2+ in the KO myotubes. Note the higher level of Ca2+ in the KO myotubes compared to the WT at the onset of the experiment. Ca2+ flux in WT and KO myotubes was calculated by conversion of the intensity of staining to molarity (nMol), which was then plotted versus time; the graph in (D) shows a much higher and sustained rise of Ca2+ in the KO. Asterisks indicate P < 0.05.
Figure 3.
Figure 3.
Assessment of Ca2+ levels and distribution in KO fibers and in a new cellular model of Pompe disease. (A) Confocal microscopy image of a live fiber derived from a 4-mo-old KO mouse that was loaded with green Fluo-4 dye. The image shows a bright “spotty” pattern of Ca2+ distribution similar to that typically seen in the KO fibers stained for lysosomal marker LAMP1 (left panel). To exclude the intralysosomal accumulation of Ca2+, the fibers were transfected in vivo with mCherry-LAMP1 to visualize lysosomes (red) prior to in vitro staining with the dye. The image (a single frame from the Z series presented in Video S3) shows only occasional overlap (yellow) between the 2 colors indicating that Ca2+ clusters are located primarily outside the lysosomes (right panel). The images are taken with the same laser intensity (n = 4; FDB muscles of each of the 2 hind limbs were electroporated). Bar = 20 μm. Images of additional patterns of Lamp1 expression in KO can be found in Fig. S2. (B) A new immortalized KO muscle cell line (JL12KO), which stably expresses mCherry-LAMP1 (left panel). Right panel shows JL12KO myotubes loaded with green Fluo-4 dye; again there is little overlap between the 2 colors. The image is a single frame from the Z series presented in Video S4. Bar = 10 μm.
Figure 4.
Figure 4.
Distribution of the subcellular Ca2+ in the WT and KO myotubes. (A) The cells were incubated with red mitochondrial Ca2+ indicator Rhod-2/AM and green cytosolic Ca2+ indicator Fluo-4/AM. The images show mostly different localization of the 2 dyes (insets) and a much stronger fluorescence in the KO cells. The images are taken with the same laser intensity. Bar = 10 μm. (B) The graph reflects an increase in mitochondrial Ca2+. Asterisk indicates P < 0.05.
Figure 5.
Figure 5.
Changes in mitochondrial physiological parameters in KO myotubes. WT and KO myotubes were analyzed on d 7 in differentiation medium. (A) Green JC-1 dye aggregates and becomes red when ΔΨm is preserved (top). There is a significant degree of mitochondrial depolarization in the KO myotubes (bottom). Bar = 10 μm. (B) Colabeling with TMRM, a potential sensitive fluorophore, and MitoTracker Green (MitoG; a potential-independent mitochondrial dye) confirms mitochondrial depolarization in the KO myotubes (arrowheads; TMRM-negative and MitoG-positive structures). Note that KO cells contain a subset of polarized mitochondria (TMRM and MitoG-double positive; arrow) with profoundly abnormal shape. (C). The graph reflects the extent of mitochondria depolarization in KO myotubes. (D) Incubation with CellROX® Green dye reveals an increase in the amount of ROS (quantified in E) in KO myotubes. DIC (differential interference contrast) images show the presence of myotubes (right panels). Bar = 20 μm. (F) The cell were incubated in the presence of H2O2 in the medium. KO myotubes are more vulnerable to oxidative stress compared to WT as shown by a significant loss of myotubes (lower right panel). (G) Graphical presentation of the data in F. Asterisks indicate P < 0.05.
Figure 6.
Figure 6.
Increased apoptosis in KO myotubes. (A) Western blot of total lysates (top) and nuclear and mitochondrial fractions of WT and KO myotubes grown in differentiation medium for 6 to 7 d. No changes in the levels of activated (cleaved) CASP3 products are detected in the KO cells (top). A slight increase in the amount of AIFM1 in the total lysates of the KO (top) is consistent with its decrease in the mitochondrial and increase in the nuclear fraction (bottom). The purity of the mitochondrial and nuclear fractions was shown by the presence of COX4I1 and HIST1H3A, respectively. (B) TUNEL assay shows absence of apoptotic nuclei (green) in WT myotubes and their presence in the diseased cells (arrows; lower panels). Bar = 20 μm.
Figure 7.
Figure 7.
Evaluation of ΔΨm, using JC-1 dye, in myotubes from Pompe patients. Human primary myoblasts were derived from a healthy individual (control; N) and 2 Pompe patients with the adult form of the disease (P#484 & P#542). Cultured myotubes (7 d in differentiation media) were loaded with the dye and analyzed by confocal microscopy. The dye became red upon entry into healthy mitochondria (control). Abnormal ΔΨm in the diseased cells manifests as a marked decrease in red fluorescence (middle and right panels) compared to control. Bar = 20 μm.
Figure 8.
Figure 8.
Impairment of bioenergetics in KO muscle. (A) Measurements of oxygen consumption rate of mitochondria from the skeletal muscle of 6- to 7-mo-old KO and WT mice. Measurements were made in the absence (state IV) and presence (state III) of ADP. Arrows indicate additions of mitochondria (mito) and 500 μM ADP. A significant reduction in respiration in the presence of ADP is observed in the KO mice. (B) Graph represents the mean oxygen consumption rate +/− SD from KO (n = 3) and WT (n = 3) mice. P < 0.05 (C) Measurements of ATP levels of mitochondria isolated from 7- to 10-mo-old KO (n = 3) and WT (n = 2) mice. Asterisk indicates P < 0.05.
Figure 9.
Figure 9.
Evaluation of autophagy in WT and KO myotubes. (A) Confocal images of WT and KO cells infected with virus containing LC3B (autophagosomal marker) linked to acid-sensitive GFP and acid-insensitive RFP tags. Both WT and KO cells show predominantly red fluorescence indicating that most autophagosomes reached lysosomes. Addition of chloroquine, a reagent that blocks autophagosomal-lysosomal fusion, leads to the extralysosomal location of autophagosomes (yellow) in both WT and KO cells. (B) Western blot of total lysates from WT and KO myotubes shows an increase in LC3-II levels in the KO (untreated [Non]; graph in C) and a diminished response to chloroquine (CQ) and bafilomycin A1 (BF) treatment in the KO cells (C; shown for chloroquine). The levels of SQSTM1 and Ub-proteins are increased in KO myotubes (B and D). Asterisks indicate P < 0.05. Note, that an autophagic defect was not detected in early passages of the KO cells. This discrepancy is not surprising; the variations in cell morphology and functions in older cultures of muscle cells have been observed by others.
Figure 10.
Figure 10.
Evaluation of mitophagy in WT and KO myotubes. Confocal images (A) and the Pearson correlation values (B) of WT and KO cells infected with virus containing OMM protein FIS1 linked to acid-sensitive GFP and acid-insensitive mCherry tags. CCCP treatment of the WT cells results in increased mitophagy as shown by the predominance of red only fluorescence (WT+CCCP) along with a significantly decreased red-green colocalization (compare WT to WT+CCCP in B). The KO cells exhibit a subset of mitochondria localized in the lysosomes (red fluorescence; increased mitophagy) and a subset of damaged mitochondria (as evidenced in this paper) localized extralysosomally (yellow). Note, that the degree of red-green colocalization is significantly decreased in the KO compared to WT (B; asterisks indicate P < 0.05). CCCP treatment of the KO cells (KO+CCCP) leads to profound mitochondrial damage (more than in the WT; red fluorescence) and a greater increase in mitophagy compared to WT cells (compare KO+CCCP to WT+CCCP). Bar = 10 μm. (C) An increase in the level of PARK2 is much more pronounced in KO muscle (gastrocnemius; white part) compared to that in KO myotubes. An increase in the amount of ubiquitinated (Ub) proteins is seen in the KO muscle. (D) Western blot of the mitochondrial fraction isolated from WT and KO muscle with the indicated antibodies; the levels of PINK1, PARK2, and Ub-proteins are increased in KO. COX4I1 was used as a loading control.
Figure 11.
Figure 11.
Reversibility of the phenotype by a Ca2+ channel blocker. (A and B) Western blot of total lysates from WT and KO myotubes shows a dramatic increase in the levels of the CACNB1. (C, D, and E) KO myotubes (d 4 in differentiation medium) were incubated with verapamil (Ver; 10 μM) for 24 h. The abnormal phenotype (left panels) in KO myotubes—increased intracellular Ca2+ and ROS production, and a loss of ΔΨm—was reversed to the normal phenotype (right panels) following treatment with the drug (middle panels). Bar = 10 μm. Asterisks indicate P < 0.05.
Figure 12.
Figure 12.
Reversibility of Ca2+ levels by verapamil in vivo. (A) Western blot of total lysates from WT and KO muscle shows a dramatic increase in the levels of the CACNB1. Top panel, short exposure (exp); middle panel, long exposure (exp). (B and C) Muscle fibers derived from verapamil-treated KO mice (KO + Ver; 25 mg/kg for 3 wk) show a significant decrease in Ca2+ levels in the areas free from autophagic buildup (arrows). Bar = 20 μm. Asterisk indicates P < 0.05.
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References

    1. Nunnari J, Suomalainen A. Mitochondria: in sickness and in health. Cell 2012; 148:1145-59; PMID:22424226; http://dx.doi.org/10.1016/j.cell.2012.02.035 - DOI - PMC - PubMed
    1. Hoppins S, Nunnari J. Cell biology. mitochondrial dynamics and apoptosis–the ER connection. Science 2012; 337:1052-4; PMID:22936767; http://dx.doi.org/10.1126/science.1224709 - DOI - PubMed
    1. Youle RJ, van der Bliek AM. Mitochondrial fission, fusion, and stress. Science 2012; 337:1062-5; PMID:22936770; http://dx.doi.org/10.1126/science.1219855 - DOI - PMC - PubMed
    1. Klionsky DJ. The molecular machinery of autophagy: unanswered questions. J Cell Sci 2005; 118:7-18; PMID:1561577919029340 - PMC - PubMed
    1. Klionsky DJ. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 2007; 8:931-7; PMID:1771235819029340 - PubMed

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