Muscle fibre mitochondrial [Ca2+ ] dynamics during Ca2+ waves in RYR1 gain-of-function mouse.

AIM: A fraction of the Ca2+ released from the sarcoplasmic reticulum (SR) enters mitochondria to transiently increase its [Ca2+ ] ([Ca2+ ]mito ). This transient [Ca2+ ]mito increase may be important in the resynthesis of ATP and other processes. The resynthesis of ATP in the mitochondria generates heat that can lead to hypermetabolic reactions in muscle with ryanodine receptor 1 (RyR1) variants during the cyclic releasing of SR Ca2+ in the presence of a RyR1 agonist. We aimed to analyse whether the mitochondria of RYR1 variant muscle handles Ca2+ differently from healthy muscle. METHODS: We used confocal microscopy to track mitochondrial and cytoplasmic Ca2+ with fluorescent dyes simultaneously during caffeine-induced Ca2+ waves in extensor digitorum longus muscle fibres from healthy mice and mice heterozygous (HET) for a malignant hyperthermia-causative RYR1 variant. RESULTS: Mitochondrial Ca2+ -transient peaks trailed the peak of cytoplasmic Ca2+ transients by many seconds with [Ca2+ ]mito not increasing by more than 250 nM. A strong linear relationship between cytoplasmic Ca2+ and [Ca2+ ]mito amplitudes was observed in HET RYR1 KI fibres but not wild type (WT). CONCLUSION: Our results indicate that [Ca2+ ]mito change within the nM range during SR Ca2+ release. HET fibre mitochondria are more sensitive to SR Ca2+ release flux than WT. This may indicate post-translation modification differences of the mitochondrial Ca2+ uniporter between the genotypes.

Persistent mdx diaphragm alterations are accompanied by increased expression and activity of calcium and muscle-specific proteins.

The mdx mouse model of Duchenne Muscular Dystrophy (DMD) presents sarcolemma instability and develops a mild multi-stage dystrophinopathy characterized by intense myonecrosis with inflammatory infiltrate at 4-weeks; muscular regeneration at 12-weeks and persistent fibrosis onwards. Mdx diaphragm muscle has a more severe phenotype with structural and functional deterioration that closely resembles the diaphragm impairment responsible for DMD human patients' morbidity. Herein, we compared calcium deposits, activity of calcium-related proteases, and expression of muscle-specific proteins in mdx diaphragm at 4-weeks and 12-weeks. We found increased calcium deposits mainly at 12-weeks, concomitant with high activity of calpains and matrix metalloprotease-9, but decreased expression of Myh4 (Myhc IIb) and Atp2a1 (SERCA1), and high expression of the myogenic regulatory factors Myod1 and Myog. Our results suggest that increased calcium deposits and persistent activity of calcium dependent proteases throughout the disease are involved in the degeneration and regeneration processes in the mdx diaphragm.

Comparative study of calcium and calcium-related enzymes with differentiation markers in different ages and muscle types in mdx mice.

Sarcolemma instability and increased calcium influx in muscle fibers are characteristics of the Duchenne muscular dystrophy. Excessive calcium activates calcium-dependent enzymes, such as calpains (CAPN) and matrix metalloproteases (MMP). Here, we analyzed calcium deposits, the activity of CAPN and MMP and the expression of Myh, SERCA and myogenic regulatory factors in different skeletal muscles during myonecrosis (4-weeks) and regeneration (12-weeks) phases of the mdx muscular pathology. Alizarin red staining was used to assess calcium deposits, casein and gelatin zymography were performed to evaluate CAPN and MMP activity, and qPCR was used to evaluate the expression of Myh, Capn, Atp2a1 and Atp2a2, Myod1 and Myog. We observed the following characteristics in mdx muscles: (i) calcium deposits almost exclusively in mdx muscles, (ii) lower CAPN1 activity in mdx muscles, (iii) higher CAPN2 activity in mdx muscles (only at 12 wks), (iv) autolyzed CAPN activity exclusively in mdx muscles, (v) lower expression of Capn1 and higher expression of Capn2 in mdx muscles; (vi) lower expression of Atp2a1 and Atp2a2 in mdx muscles, (vii) higher MMP (pre pro MMP2, pro MMP2, MMP2 and MMP9) activity in mdx muscles, (viii) MMP2 activity exclusively in mdx muscles at 12 wks, (ix) MMP9 activity exclusively in mdx muscles, (x) higher expression of Myog in mdx muscles at 12 wks, and (xi) lower expression of Myh (Myh7, Myh2, Myh1, Myh4) in mdx muscles, particularly Myh7 and Myh2. The collection of our results provides valuable information for a better characterization of mdx pathology phenotype.

Reduced mitochondrial respiration and increased calcium deposits in the EDL muscle, but not in soleus, from 12-week-old dystrophic mdx mice.

Mitochondria play an important role in providing ATP for muscle contraction. Muscle physiology is compromised in Duchenne muscular dystrophy (DMD) and several studies have shown the involvement of bioenergetics. In this work we investigated the mitochondrial physiology in fibers from fast-twitch muscle (EDL) and slow-twitch muscle (soleus) in the mdx mouse model for DMD and in control C57BL/10J mice. In our study, multiple mitochondrial respiratory parameters were investigated in permeabilized muscle fibers from 12-week-old animals, a critical age where muscle regeneration is observed in the mdx mouse. Using substrates of complex I and complex II from the electron transport chain, ADP and mitochondrial inhibitors, we found in the mdx EDL, but not in the mdx soleus, a reduction in coupled respiration suggesting that ATP synthesis is affected. In addition, the oxygen consumption after addition of complex II substrate is reduced in mdx EDL; the maximal consumption rate (measured in the presence of uncoupler) also seems to be reduced. Mitochondria are involved in calcium regulation and we observed, using alizarin stain, calcium deposits in mdx muscles but not in control muscles. Interestingly, more calcium deposits were found in mdx EDL than in mdx soleus. These data provide evidence that in 12-week-old mdx mice, calcium is accumulated and mitochondrial function is disturbed in the fast-twitch muscle EDL, but not in the slow-twitch muscle soleus.