In Vitro Skeletal Muscle Model of PGM1 Deficiency Reveals Altered Energy Homeostasis.

Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1, according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.

Mitochondrial function and morphology are impaired in parkin-mutant fibroblasts.

OBJECTIVE: There are marked mitochondrial abnormalities in parkin-knock-out Drosophila and other model systems. The aim of our study was to determine mitochondrial function and morphology in parkin-mutant patients. We also investigated whether pharmacological rescue of impaired mitochondrial function may be possible in parkin-mutant human tissue. METHODS: We used three sets of techniques, namely, biochemical measurements of mitochondrial function, quantitative morphology, and live cell imaging of functional connectivity to assess the mitochondrial respiratory chain, the outer shape and connectivity of the mitochondria, and their functional inner connectivity in fibroblasts from patients with homozygous or compound heterozygous parkin mutations. RESULTS: Parkin-mutant cells had lower mitochondrial complex I activity and complex I-linked adenosine triphosphate production, which correlated with a greater degree of mitochondrial branching, suggesting that the functional and morphological effects of parkin are related. Knockdown of parkin in control fibroblasts confirmed that parkin deficiency is sufficient to explain these mitochondrial effects. In contrast, 50% knockdown of parkin, mimicking haploinsufficiency in human patient tissue, did not result in impaired mitochondrial function or morphology. Fluorescence recovery after photobleaching assays demonstrated a lower level of functional connectivity of the mitochondrial matrix, which further worsened after rotenone exposure. Treatment with experimental neuroprotective compounds resulted in a rescue of the mitochondrial membrane potential. INTERPRETATION: Our study demonstrates marked abnormalities of mitochondrial function and morphology in parkin-mutant patients and provides proof-of-principle data for the potential usefulness of this new model system as a tool to screen for disease-modifying compounds in genetically homogenous parkinsonian disorders.

Subunits of mitochondrial complex I exist as part of matrix- and membrane-associated subcomplexes in living cells.

Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved. To answer this question, we here applied submitochondrial fluorescence recovery after photobleaching to HEK293 cells expressing 6 GFP-tagged subunits selected on the basis of current CI assembly models. We established that each subunit was partially present in a virtually immobile fraction, possibly representing the holo-enzyme. Four subunits (NDUFV1, NDUFV2, NDUFA2, and NDUFA12) were also present as highly mobile matrix-soluble monomers, whereas, in sharp contrast, the other two subunits (NDUFB6 and NDUFS3) were additionally present in a slowly mobile fraction. In the case of the integral membrane protein NDUFB6, this fraction most likely represented one or more membrane-bound subassemblies, whereas biochemical evidence suggested that for the NDUFS3 protein this fraction most probably corresponded to a matrix-soluble subassembly. Our results provide first time evidence for the existence of CI subassemblies in mitochondria of living cells.

Amplitude modulation of nuclear Ca2+ signals in human skeletal myotubes: a possible role for nuclear Ca2+ buffering.

Video-rate confocal microscopy of Indo-1-loaded human skeletal myotubes was used to assess the relationship between the changes in sarcoplasmic ([Ca(2+)](S)) and nuclear ([Ca(2+)](N)) Ca(2+) concentration during low- and high-frequency electrostimulation. A single stimulus of 10 ms duration transiently increased [Ca(2+)] in both compartments with the same time of onset. Rate and amplitude of the [Ca(2+)] rise were significantly lower in the nucleus (4.0- and 2.5-fold, respectively). Similarly, [Ca(2+)](N) decayed more slowly than [Ca(2+)](S) (mono-exponential time constants of 6.1 and 2.5 s, respectively). After return of [Ca(2+)] to the prestimulatory level, a train of 10 stimuli was applied at a frequency of 1 Hz. The amplitude of the first [Ca(2+)](S) transient was 25% lower than that of the preceding single transient. Thereafter, [Ca(2+)](S) increased stepwise to a maximum that equalled that of the single transient. Similarly, the amplitude of the first [Ca(2+)](N) transient was 20% lower than that of the preceding single transient. In contrast to [Ca(2+)](S), [Ca(2+)](N) then increased to a maximum that was 2.3-fold higher than that of the single transient and equalled that of [Ca(2+)](S). In the nucleus, and to a lesser extent in the sarcoplasm, [Ca(2+)] decreased faster at the end of the stimulus train than after the preceding single stimulus (time constants of 3.3 and 2.1 s, respectively). To gain insight into the molecular principles underlying the shaping of the nuclear Ca(2+) signal, a 3-D mathematical model was constructed. Intriguingly, quantitative modelling required the inclusion of a satiable nuclear Ca(2+) buffer. Alterations in the concentration of this putative buffer had dramatic effects on the kinetics of the nuclear Ca(2+) signal. This finding unveils a possible mechanism by which the skeletal muscle can adapt to changes in physiological demand.