Cecilia Paolini

Reorganized stores and impaired calcium handling in skeletal muscle of mice lacking calsequestrin-1

Calsequestrin (CS), the major Ca2+‐binding protein in the sarcoplasmic reticulum (SR), is thought to play a dual role in excitation–contraction coupling: buffering free Ca2+ increasing SR capacity, and modulating the activity of the Ca2+ release channels (RyRs). In this study, we generated and characterized the first murine model lacking the skeletal CS isoform (CS1). CS1‐null mice are viable and fertile, even though skeletal muscles appear slightly atrophic compared to the control mice. No compensatory increase of the cardiac isoform CS2 is detectable in any type of skeletal muscle. CS1‐null muscle fibres are characterized by structural and functional changes, which are much more evident in fast‐twitch muscles (EDL) in which most fibres express only CS1, than in slow‐twitch muscles (soleus), where CS2 is expressed in about 50% of the fibres. In isolated EDL muscle, force development is preserved, but characterized by prolonged time‐to‐peak and half‐relaxation time, probably related to impaired calcium release from and re‐uptake by the SR. Ca2+‐imaging studies show that the amount of Ca2+ released from the SR and the amplitude of the Ca2+ transient are significantly reduced. The lack of CS1 also causes significant ultrastructural changes, which include: (i) striking proliferation of SR junctional domains; (ii) increased density of Ca2+‐release channels (confirmed also by 3H‐ryanodine binding); (iii) decreased SR terminal cisternae volume; (iv) higher density of mitochondria. Taken together these results demonstrate that CS1 is essential for the normal development of the SR and its calcium release units and for the storage and release of appropriate amounts of SR Ca2+.

Multiple Regions of RyR1 Mediate Functional and Structural Interactions with α1S-Dihydropyridine Receptors in Skeletal Muscle

Excitation-contraction (e-c) coupling in muscle relies on the interaction between dihydropyridine receptors (DHPRs) and RyRs within Ca(2+) release units (CRUs). In skeletal muscle this interaction is bidirectional: alpha(1S)DHPRs trigger RyR1 (the skeletal form of the ryanodine receptor) to release Ca(2+) in the absence of Ca(2+) permeation through the DHPR, and RyR1s, in turn, affect the open probability of alpha(1S)DHPRs. alpha(1S)DHPR and RyR1 are linked to each other, organizing alpha(1S)-DHPRs into groups of four, or tetrads. In cardiac muscle, however, alpha(1C)DHPR Ca(2+) current is important for activation of RyR2 (the cardiac isoform of the ryanodine receptor) and alpha(1C)-DHPRs are not organized into tetrads. We expressed RyR1, RyR2, and four different RyR1/RyR2 chimeras (R4: Sk1635-3720, R9: Sk2659-3720, R10: Sk1635-2559, R16: Sk1837-2154) in 1B5 dyspedic myotubes to test their ability to restore skeletal-type e-c coupling and DHPR tetrads. The rank-order for restoring skeletal e-c coupling, indicated by Ca(2+) transients in the absence of extracellular Ca(2+), is RyR1 > R4 > R10 >> R16 > R9 >> RyR2. The rank-order for restoration of DHPR tetrads is RyR1 > R4 = R9 > R10 = R16 >> RyR2. Because the skeletal segment in R9 does not overlap with that in either R10 or R16, our results indicate that multiple regions of RyR1 may interact with alpha(1S)DHPRs and that the regions responsible for tetrad formation do not correspond exactly to the ones required for functional coupling.

Anesthetic- and heat-induced sudden death in calsequestrin-1-knockout mice

CaIsequestrin‐1 (CASQ1) is a moderate‐affinity, high‐capacity Ca2+‐binding protein in the sarcoplasmic reticulum (SR) terminal cisternae of skeletal muscle. CASQ1 functions as both a Ca2+‐binding protein and a luminal regulator of ryanodine receptor (RYR1)‐mediated Ca2+ release. Mice lacking skeletal CASQ1 are viable but exhibit reduced levels of releasable Ca2+ and altered contractile properties. Here we report that CASQ1‐null mice exhibit increased spontaneous mortality and susceptibility to heat‐and anesthetic‐induced sudden death. Exposure of CASQ1‐null mice to either 2% halo‐thane or heat stress triggers lethal episodes characterized by whole‐body contractures, elevated core temperature, and severe rhabdomyolysis, which are prevented by prior dantrolene administration. The characteristics of these events are remarkably similar to analogous episodes observed in humans with malignant hyperthermia (MH) and animal models of MH and environmental heat stroke (EHS). In vitro studies indicate that CASQ1‐null muscle exhibits increased contractile sensitivity to temperature and caffeine, temperature‐dependent increases in resting Ca2+, and an increase in the magnitude of depolarization‐induced Ca2+ release. These results demonstrate that CASQ1 deficiency alters proper control of RYR1 function and suggest CASQ1 as a potential candidate gene for linkage analysis in families with MH/EHS where mutations in the RYR1 gene are excluded.—Dainese, M.,Quarta, M., Lyfenko, A.D., Paolini, C., Canato, M., Reggiani, C., Dirksen, R.T., Protasi, F. Anesthetic‐and heat‐induced sudden death incalsequestrin‐1‐knockout mice. FASEB J. 23, 1710–1720 (2009)

Home-based functional electrical stimulation rescues permanently denervated muscles in paraplegic patients with complete lower motor neuron lesion

Background. Spinal cord injury causes muscle wasting and loss of function, which are especially severe after complete and permanent damage to lower motor neurons. In a previous cross-sectional study, long-standing denervated muscles were rescued by home-based functional electrical stimulation (h-bFES) training. Objective. To confirm results by a 2-year longitudinal prospective study of 25 patients with complete conus/cauda equina lesions. Methods. Denervated leg muscles were stimulated by h-bFES using a custom-designed stimulator and large surface electrodes. Muscle mass, force, and structure were determined before and after 2 years of h-bFES using computed tomography, measurements of knee torque during stimulation, and muscle biopsies analyzed by histology and electron microscopy. Results. Twenty of 25 patients completed the 2-year h-bFES program, which resulted in (a) a 35% cross-sectional increase in area of the quadriceps muscle from 28.2 ± 8.1 to 38.1 ± 12.7 cm 2 (P < .001), a 75% increase in mean diameter of muscle fibers from 16.6 ± 14.3 to 29.1 ± 23.3 μm (P < .001), and improvements of the ultrastructural organization of contractile material; and (b) a 1187% increase in force output during electrical stimulation from 0.8 ± 1.3 to 10.3 ± 8.1 N m (P < .001). The recovery of quadriceps force was sufficient to allow 25% of the subjects to perform FES-assisted stand-up exercises. Conclusions. Home-based FES of denervated muscle is an effective home therapy that results in rescue of muscle mass and tetanic contractility. Important immediate benefits for the patients are the improved cosmetic appearance of lower extremities and the enhanced cushioning effect for seating.

Calsequestrin-1: a new candidate gene for malignant hyperthermia and exertional/environmental heat stroke

Malignant hyperthermia (MH) and exertional/environmental heat stroke (EHS) in humans present as similar life threatening crises triggered by volatile anaesthetics and strenuous exercise and/or high temperature, respectively. Many families (70–80%) diagnosed with MH susceptibility (MHS), and a few with EHS, are linked to mutations in the gene for the ryanodine receptor type‐1 (RyR1), Ca2+ release channel of the sarcoplasmic reticulum (SR) of skeletal muscle and a key protein in excitation–contraction (EC) coupling. However, mutations in the RyR1 gene are not found in all MH families, suggesting that alternative genes remain to be identified. In our laboratory we have recently characterized a novel knockout model lacking skeletal muscle calsequestrin (CASQ1), a SR Ca2+‐binding protein that modulates RyR1 function, and investigated whether these mice present a MH/EHS‐like phenotype. Ablation of CASQ1 results in remodelling of the EC coupling apparatus and functional changes, which in male mice causes a striking increase in the rate of spontaneous mortality and susceptibility to trigger MH‐like lethal episodes in response to halothane and heat stress. The demonstration that ablation of CASQ1 results in MH‐ and EHS‐like lethal episodes validates CASQ1 as a viable candidate gene for linkage analysis in MH and EHS families where mutations in RyR1 are excluded.

Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease

The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3‐kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15‐deficient mouse tissues are competent for LC3‐positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7−/− mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15−/− mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases.

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200–500 μM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 μs) gating events and insensitivity to Ca2+. Addition of ryanodine restores Ca2+-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

A mutation in the CASQ1 gene causes a vacuolar myopathy with accumulation of sarcoplasmic reticulum protein aggregates

A missense mutation in the calsequestrin‐1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high‐affinity Ca2+‐binding sites of CASQ1 and alters the kinetics of Ca2+ release in muscle fibers. Expression of the mutated CASQ1 protein in COS‐7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron‐dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca2+ release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.

Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice

Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content.

Lessons from calsequestrin-1 ablation in vivo: much more than a Ca2+ buffer after all

Calsequestrin type-1 (CASQ1), the main sarcoplasmic reticulum (SR) Ca2+ binding protein, plays a dual role in skeletal fibers: a) it provides a large pool of rapidly-releasable Ca2+ during excitation–contraction (EC) coupling; and b) it modulates the activity of ryanodine receptors (RYRs), the SR Ca2+ release channels. We have generated a mouse lacking CASQ1 in order to further characterize the role of CASQ1 in skeletal muscle. Contrary to initial expectations, CASQ1 ablation is compatible with normal motor activity, in spite of moderate muscle atrophy. However, CASQ1 deficiency results in profound remodeling of the EC coupling apparatus: shrinkage of junctional SR lumen; proliferation of SR/transverse-tubule contacts; and increased density of RYRs. While force development during a twitch is preserved, it is nevertheless characterized by a prolonged time course, likely reflecting impaired Ca2+ re-uptake by the SR. Finally, lack of CASQ1 also results in increased rate of SR Ca2+ depletion and inability of muscle to sustain tension during a prolonged tetani. All modifications are more pronounced (or only found) in fast-twitch extensor digitorum longus muscle compared to slow-twitch soleus muscle, likely because the latter expresses higher amounts of calsequestrin type-2 (CASQ2). Surprisingly, male CASQ1-null mice also exhibit a marked increased rate of spontaneous mortality suggestive of a stress-induced phenotype. Consistent with this idea, CASQ1-null mice exhibit an increased susceptibility to undergo a hypermetabolic syndrome characterized by whole body contractures, rhabdomyolysis, hyperthermia and sudden death in response to halothane- and heat-exposure, a phenotype remarkably similar to human malignant hyperthermia and environmental heat-stroke. The latter findings validate the CASQ1 gene as a candidate for linkage analysis in human muscle disorders.