Constitutive assembly of Ca2+ entry units in soleus muscle from calsequestrin knockout mice

Abstract

Calcium (Ca2+) entry units (CEUs) are junctions within the I band of the sarcomere between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of the transverse (T)-tubule. CEUs contain STIM1 and Orai1 proteins, the molecular machinery of store-operated Ca2+ entry (SOCE). In extensor digitorum longus (EDL) fibers of wild-type (WT) mice, CEUs transiently assemble during acute exercise and disassemble several hours thereafter. By contrast, calsequestrin-1 (CASQ1) ablation induces a compensatory constitutive assembly of CEUs in EDL fibers, resulting in enhanced constitutive and maximum SOCE that counteracts SR Ca2+ depletion during repetitive activity. However, whether CEUs form in slow-twitch fibers, which express both the skeletal CASQ1 and the cardiac CASQ2 isoforms, is unknown. Herein, we compared the structure and function of soleus muscles from WT and knockout mice that lack either CASQ1 (CASQ1-null) or both CASQs (dCASQ-null). Ultrastructural analyses showed that SR/T-tubule junctions at the I band, virtually identical to CEUs in EDL muscle, were present and more frequent in CASQ1-null than WT mice, with dCASQ-null exhibiting the highest incidence. The greater incidence of CEUs in soleus from dCASQ-null mice correlated with increased specific force production during repetitive, high-frequency stimulation, which depended on Ca2+ entry. Consistent with this, Orai1 expression was significantly increased in soleus of CASQ1-null mice, but even more in dCASQ-null mice, compared with WT. Together, these results strengthen the concept that CEU assembly strongly depends on CASQ expression and provides an alternative source of Ca2+ needed to refill SR Ca2+ stores to maintain specific force production during sustained muscle activity.

Intracellular Ca2+ signalling: unexpected new roles for the usual suspect

“…But other candidates presenting similar sensitivity to heat and ROS, such as TRP Ankyrin 1, are likely to be rapidly integrated in the arsenal of Ca 2+ -permeable channels that could be probed for their therapeutic potential. In conclusion, this Symposium, which also engendered may fruitful discussions and opened the way to new collaborations among the participants (including many foreigner guests), confirmed that Italian Physiology is at the forefront of research in Ca 2+ signalling, as also proven by many other oral and poster presentations of the meeting ( Martinotti et al, 2019 ; Nesher et al, 2019 ; Badone et al, 2021 ; Lazzarini et al, 2022 ; Michelucci et al, 2022 ; Sforna et al, 2022 ; Arici et al, 2023 ; Lia et al, 2023 ).…”

Loss of calpain 3 dysregulates store-operated calcium entry and its exercise response in mice

“…total T-tubule) length and T-tubule/SR stack contact length (i.e., length of the association between T-tubule and SR stack membranes) were measured in electron micrographs of nonoverlapping regions randomly collected from muscle fibers after staining of T-tubules with potassium ferrocyanide. For each specimen, 10–15 representative fibers were analyzed, and 5 micrographs at 10,000-12,000× magnification were taken for each fiber. The incidence of T-tubule/SR stack junctions (i.e., CEUs; 100 μm2) were determined from electron micrographs of nonoverlapping regions randomly collected by counting the number of CEUs per area of section (100 μm 2 ) as described previously (42). In WT muscles, 4-5 representative fibers were analyzed, while for each of the other samples, 15-20 representative fibers were analyzed, and 5 micrographs at 10,000-12,000× magnification were taken for each fiber.…”

“…iii) The incidence of T-tubule/SR stack junctions (i.e., CEUs; 100 μm2) were determined from electron micrographs of nonoverlapping regions randomly collected by counting the number of CEUs per area of section (100 μm 2 ) as described previously (42). In WT muscles, 4-5 representative fibers were analyzed, while for each of the other samples, 15-20 representative fibers were analyzed, and 5 micrographs at 10,000-12,000× magnification were taken for each fiber.…”

Loss of Calpain 3 dysregulates store‐operated calcium entry and its exercise response in mice

“…total T‐tubule) length and T‐tubule/SR stack contact length (i.e., length of the association between T‐tubule and SR stack membranes) were measured in electron micrographs of nonoverlapping regions randomly collected from muscle fibers after staining of T‐tubules with potassium ferrocyanide. For each specimen, 10–15 representative fibers were analyzed, and 5 micrographs at 10 000–12 000× magnification were taken for each fiber. The incidence of T‐tubule/SR stack junctions (i.e., CEUs; 100 μm 2 ) were determined from electron micrographs of nonoverlapping regions randomly collected by counting the number of CEUs per area of section (100 μm 2 ) as described previously 33 . In WT muscles, 4–5 representative fibers were analyzed, whereas for each of the other samples, 15–20 representative fibers were analyzed, and 5 micrographs at 10 000–12 000× magnification were taken for each fiber.…”