Tubular aggregates (TA) are skeletal muscle structures that arise from the progressive accumulation of sarcoplasmic reticulum (SR) proteins (1–5). Cytoplasmic aggregates of muscle fibers have been observed in both mice (6) and humans (3). These factors are relevant to aging and muscle disease processes. The pathophysiological mechanisms of many human neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are intrinsically related to protein aggregation and inclusion body formation (7). Thus, there is a strong link between condensate-forming proteins and age-related diseases, such as neurodegeneration and cancer (8).

In humans, TA are found in diseased skeletal muscles such as tubular aggregate myopathy (TAM) (9), Andersen–Tawil syndrome (10), and limb-girdle myasthenia (11). Major differences between TAM and TA are associated with other myopathies. TA are prevalent in both slow-twitch (type I) and fast-twitch (type II) muscle fibers in TAM, whereas they are only present in type II fibers as a secondary effect in other myopathies. TAM affects mostly men, and in isolated TA, the proportion of women presenting with them is greater, although men are generally more affected (12).

TA have also been found in wild-type aged mice of different strains within muscle fibers, especially type II, but only in male specimens (5), which makes these structures age-, fiber type- and sex-specific (1).

The effects of muscle regeneration on TA have not yet been reported. This study aimed to compare the presence of TA in normal and dystrophic aged muscles and assess whether TA negatively influences muscle function after induced regeneration.

In this study, we aimed to compare the capacity of the old muscle to regenerate, both in chronic model of degeneration, as observed in muscular dystrophies, as compared to acute degeneration, induced by muscle demage by electroporation. The number of positive dMyHCs was quantified to verify the efficiency of regeneration and formation of new muscle fibers in genetically degenerated dystrophin-deficient mice (Dmd^mdx) and in normal muscles under induced degeneration. As observed in young normal mice subjected to electrical muscle injury (17, 18), new fibers positive for dMyHC clearly increased at 5 dpe. dMyHC-positive fibers were reduced in number at 15 dpe and reached normal values after 30 dpe. The findings indicate that aged normal muscle preserves its regenerative capacity and formation of new muscle fibers in a pattern similar to that observed in young normal animals. In contrast, old Dmd^mdx mice showed a small increase in the number of positive new fibers. This result is different from the significantly greater prevalence of regenerated fibers observed in young Dmd^mdx mice (17%) (17).

TA inside aged muscle fibers are more likely to form during muscular physiological senescence. TA are repositories of proteins that have accumulated for a long time (several months in mice or years in humans). Acute injury, such as electroporation, or chronic injury, such as dystrophic state, leads to a degeneration-regeneration process in which severely damaged fibers are removed from muscle and satellite cells are recruited. The cells differentiate into myoblasts and fuse with each other to form new fibers or with a lesioned fiber to repair the fiber (19). In the present study, new fibers lacked TA. In the first round of experiments, both degeneration-regeneration groups of mice (Dmd^mdx and electroporated C57BL/6J) presented fewer fibers containing TA than the control group (NE C57BL/6J mice). These findings indicate that recently formed muscle fibers do not contain TA, possibly due to the short post-regeneration time.

The identification of TA is technically challenging. Previous studies using different staining protocols to visualize TA have generated inconsistent results (20). For example, TA stained positively with nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase (NADH-TR) (21) but negatively in another study (6). Details are lacking concerning the mechanism of TA genesis, chemical content, and functional consequences. However, some relevant knowledge is known. For example, SR proteins such as calsequestrin (1, 2, 5), Serca1 (1, 2, 6) and Stim1 (22, 23) are found in TA. All these SR proteins are involved in calcium homeostasis (2, 23). Schiaffino et al. (5) proposed the origin of TA in the SR, and compared protein aggregation in muscle with that in neurodegenerative diseases, and discussed the rearrangement of endoplasmic reticulum membranes in non-muscle cells in a similar form to that observed in muscle. The authors suggested that hypoxia led to the formation of TA. Orai1 plasma membrane calcium channel protein (23) was also identified in muscle TA (2, 6, 22). Due to the protein composition of TA, it is possible that correct calcium homeostasis influences TA formation. It would be interesting to verify whether the abnormal elevation of intracellular calcium concentration in dystrophin-deficient muscle, which contributes to disease progression in Duchenne muscular dystrophy (24), could be related to a lower amount of TA in Dmd^mdx dystrophic muscle.

TA have been demonstrated in different mouse strains (C57BL/6J, BALB/c, DBA/2, 129Sv, and 129Ola) since 5 months of age, with a subsequent increased prevalence within fibers to almost 80% in old gastrocnemius mice (18 months) (6). In addition, male mice fed a resveratrol-enriched diet from 12 to 18 months of age showed a reduction in TA and an improvement in capillarization per fiber (25). Despite the broad observation of TA in wild-type inbred mouse strains (6), TA has also been previously found in dystrophic mice (Lama2^dy model for congenital merosin-deficient muscular dystrophy 1A) (26). TA were observed in heterozygous, but not homozygous, mice; in the latter, degeneration-regeneration is more intense. In the present study, the presence of TA was observed in another dystrophic model (Dmd^mdx), specifically in old animals, but in a lesser quantity that observed in old NE C57BL/6J mice. These findings reinforce the hypothesis that TA requires time to accumulate in the sarcoplasm because the intense degeneration-regeneration process in dystrophic animals results in the formation of new fibers that do not have aggregate.

TA have also been observed in human muscle diseases. Funk et al. (12) reported TA in 103 myopathic patients with variable clinical findings and age of onset ranging from childhood to old age. TA presented in subsarcolemmal regions and stained positively for SERCA1 and SERCA2 in both fiber types. Interestingly, the TA stained positive for tau, a protein associated with neurodegenerative diseases. Jain et al. (9) reported four adult patients with TAM; TA appeared in type 1 fibers in two patients and in type 2 fibers in the other two. TA were positively stained by HE, GT, and NADH-TR but were not stained by SDH and Cox stains. All patients presented with muscle weakness. Vivekanandam et al. (10) reported TA (as subsarcolemmal structures) in two of five biopsies of Andersen–Tawil syndrome, which affects most patients with episodic muscle weakness. Limb-girdle myasthenia is another example of a disease in which TA could be present, with a reported patient with clinical signs at 5 years of age. TA in his muscle was revealed at 23 years of age by biopsy (11). In all these reported cases (9–12), TA did not seem to be the cause of the disease. The eventual contribution of TA to pathophysiology or even to the worsening of clinical signs has not yet been investigated.

The functional assessments in the present study measured the physical aptitude of aged mice with more or less TA within muscle fibers. The aim was to see if the absence of TA in new fibers could affect muscle function. However, we did not consider that this in vivo experimental design could be influenced by other changes in the tissue caused by the injury, such as loss of muscle mass and consequent loss of strength, fiber type change, and diminished motor units (motor neuron and the myofibers activated by it) (27, 28). The experimental design could not assess the presence of TA as the only variable among the analyzed groups. Moreover, the absence of differences in these tests may have been due to the limited number of animals used.

A possible strategy for TA reduction is exercise (22). Exercise improves health, especially for the elderly population. This has global importance, given that it is expected to be 1 billion people ≥60 years of age worldwide by 2030 (29). During aging, progressively increasing cellular death and malfunction result in sarcopenia and osteoporosis, featuring loss of muscle and bone mass, respectively (30). Locomotor skills can be impaired, reducing the autonomy of the older population in their daily activities. A better understanding of age-related dysfunctions, such as the aggregation of intracellular structures, would help in tracing targets for therapies and other strategies to improve healthier aging.

TA form over time, appears in aging normal murine muscles. TA reduction in injured conditions may be due to the degeneration-regeneration process in muscles, with loss of damaged muscle fibers and formation of new fibers that do not present protein aggregation. These new regenerated fibers do not improve the functiona capacity of the aged muscle.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was approved by Comissão de Ética no Uso de Animais em Pesquisa, Instituto de Biociencias, Universidade de São Paulo. The study was conducted in accordance with the local legislation and institutional requirements.

FV: Conceptualization, Data curation, Formal analysis, Validation, Writing – original draft, Writing – review & editing, Investigation, Methodology. AJ: Data curation, Investigation, Writing – review & editing. BS: Writing – review & editing, Methodology. IA: Methodology, Writing – review & editing. LC: Writing – review & editing. LF: Writing – review & editing, Methodology. LS: Methodology, Writing – review & editing. NS: Methodology, Writing – review & editing. MF: Writing – review & editing, Formal analysis. MV: Formal analysis, Writing – review & editing, Conceptualization, Data curation, Funding acquisition, Project administration, Supervision, Validation, Writing – original draft.