MiR-199-3p Enhances Muscle Regeneration And Ameliorates Aged Muscle And Muscular Dystrophy

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Parabiosis experiments suggest that molecular factors related to rejuvenation and aging circulate in the blood. Here, we show that miR-199-3p, which circulates in the blood as a cell-free miRNA, is significantly decreased in the blood of aged mice compared to young mice;and miR-199-3p has the ability to enhance myogenic differentiation and muscle regeneration. Administration of miR-199 mimics, which supply miR-199-3p, to aged mice resulted in muscle fiber hypertrophy and delayed loss of muscle strength. Systemic administration of miR-199 mimics to mdx mice, a well-known animal model of Duchenne muscular dystrophy (DMD), markedly improved the muscle strength of mice. Taken together, cell-free miR-199-3p in the blood may have an anti-aging effect such as ahypertrophic effect in aged muscle fibers and could have potential as a novel RNA therapeutic for DMD as well as age-related diseases. The findings provide us with new insights into blood-circulating miRNAs as age-related molecules.

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Vital functions, such as muscle force, operation of the nervous system, respiratory function, defense against infection, and resistance against diseases, decline with aging. Diseases and dysfunctions associated with such functional decline are increasing and becoming major problems in aging societies. Various changes, such as metabolic and gene regulation changes, occur in the body with agingl-3; and studying such changes may help to understand aging and provide strategies to deal with the problems facing aging societies. Biomolecules (including genes) related to aging have been identified and may be useful in aging research; e.g,β-galactosidase and plolnk4a expression are associated with cellular senescence4-6, and a mouse model lacking the klotho gene is known as an aging model with a number of phenotypes, resembling human premature aging.

Parabiosis is the union between two individuals that share a common vascular system, and can be generated experimentally by surgery8. Experiments with parabiosis, in which young and aged rodents were conjoined to share a common vascular system, showed that the aging process in young animals was accelerated, while aged animals were rejuvenated9-12. The findings strongly suggest that certain factors related to rejuvenation and aging are present, and circulating with blood in the vascular system;however, such circulating factors are not yet fully understoodl3.

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MicroRNAs (miRNAs) are 21-23-nucleotide-long small non-coding RNAs, which are processed from longer transcripts (pri-mary miRNAs) by digestion, with a microprocessor complex in the nucleus and Dicer in the cytoplasm, and incorporated into the RNA-induced silencing complex(RISC)14,15.MiRNA in RISC functions as a mediator in gene silencing, where translation of messenger RNAs (mRNAs) having partial complementarity to the miRNA is inhibited or mRNAs carrying nearly complementary sequences to the miRNA are digestedl4,15. Cistanche Extract Anti Radiation Thus, miRNAs play important roles in the gene regulation via sup-pressing their target gene expression. Thousands of miRNA genes have been found in animals and plants [see the microRNA database (miRBase): http://www.mirbase.org/index.shtml]. The expression profile of miRNAs has been examined, and tissue- and developmental stage-specific expression and disease-associated expression of miRNAs have been detectedl6-21. Gene regulation involving miRNAs is closely related to various vital functions and life phenomena, including diseases14,16,21-24

MiRNAs are present outside, as well as inside the cells25. For example, miRNAs are present in the blood as cell-free nucleotides and circulate in the vascular system2627. Such cell-free miRNAs (cf-miRNAs) are incorporated into small vesicles called extra-cellular vesicles or associated with proteins, thereby gaining protection from extracellular nucleases25,28. The presence of some cf-miRNAs may be significant; for example, significant associa-tions between cf-miRNAs and certain diseases have been detected26,27,29-31. Cf-miRNAs may reflect changes in the phy-sical condition; thus, such miRNAs may be potential biomarkers for monitoring life maintenance systems, as well as diseases.

The present study was conducted to examine associations between cf-miRNAs and aging, and cf-miRNAs in the blood of young and aged mice were investigated. The results revealed changes in cf-miRNAs between young and aged mouse blood. Among such miRNAs, miR-199-3p was markedly decreased in aged blood compared to young blood. Interestingly, miR-19-3p has the ability to enhance muscle differentiation and regeneration, and its administration to aged mice resulted in the muscle fiber expansion. When miR-199-3p was introduced into mdx mice, an animal model of Duchenne muscular dystrophy (DMD) the muscle strength of mice was markedly improved. The present study provides new insights into blood-circulating cf-miRNAs in terms of aging, latent ability, and medical applications.

Results

Cell-free miRNAs in the blood of young and aged mice. We investigated cf-miRNAs in the blood of young (6-week-old) and aged (>23-month-old) C57BL/6J mice, using DNA microarray to identify age-related differences. The profile of cf-miRNAs exhibited marked differences between young and aged mice, i.e, cf-miRNAs that are biased toward young and aged mouse blood were detected (Table 1). Of such miRNAs, myogenic miRNAs (e.g, miR-1 and miR-133)1833 were present in the miRNA group that is abundant in young blood; in other words, myogenic miRNAs were markedly less in aged blood (Table 1). cistanche herba The difference in the miRNAs between young and aged blood was confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR; Fig. la), and further reproduced using RNA sources extracted from small vesicles, so called exosomes, isolated from the plasma (Supplementary Fig.1).

Cell culture experiments, on the other hand, revealed that young mouse serum had the ability to induce myogenic differentiation, and the ability was stronger than that of aged mice (Fig. 1b). Insulin-like growth factor I (IGF-I), which is a major factor capable of inducing myogenic differentiation in the blood34,35, was examined; however, no significant difference was observed between young and aged blood (Fig. lc). Thus, factors other than IGF-I in young blood may be implicated in myogenic differentiation.

miR-199-3p is capable of strongly inducing myogenic differ-entiation. We investigated the effects of miRNAs abundant in young blood on myogenic differentiation. Synthetic miRNA mimics were introduced into C2C12 cells, a mouse myoblast cell line, and the expression of myogenic differentiation markers, the myogenin (Myog) and myosin heavy chain 1 (Myh1) genes, was examined. The results were unexpected and attracted our interest:miR-199-3p, which was predicted as a negative miRNA due to the little information on myogenic differentiation, markedly induced the expression of Myog and Myh1 (Fig. 2a, b). In addition, the induction of miR-199-3p was stronger than that of conventional myogenic miRNAs: the expression of myosin heavy chain in the presence of miR-199-3p was particularly remarkable (Fig. 2b, c).

To clarify the relationship between miR-199-3p and myogenic differentiation, we investigated cellular and extracellular (exoso-mal) miR-199-3p during myogenic differentiation, and also the effects of miR-199-3p inhibitors on the differentiation. The expression of endogenous (cellular)miR-199-3p was upregulated after the initiation of differentiation. Exosomal miR-19-3p was increased once and decreased thereafter (Supplementary Fig. 2a). Inhibitor treatment suppressed the expression of myogenic differentiation markers, Myog, Myh1, and creatine kinase (Ckm)(Supplementary Fig. 2b). cistanche penis growth Thus, the findings suggest that miR-199-3p participates in myogenic differentiation.

Designing miR-199 mimics. We designed miR-199 mimics such that the mimics can efficiently produce miR-199-3p as a func-tional mediator (guide strand) in gene silencing. The designed miRNA mimics were screened and miR199#4 was selected as a competent miR-199 mimic (Supplementary Fig. 3). Subsequently, miR199#4 was used in both in vivo and in vitro experiments.

Target genes of miR-199-3p in myogenic differentiation. To identify target genes for miR-199-3p under myogenic differ-entiation, we searched for candidate genes from the TargetScan database and focused on the Lin28b and Suz12 genes, both of which are expressed in C2C12 myoblasts. Lin28b and Suz12 possess putative miR-199-3p binding sequences in their 3'untranslated region (UTR; Fig. 3a). We demonstrated that the putative sequences are authentic binding sites for miR-19-3p, using the Luciferase reporter genes carrying the binding sequences and corresponding mismatched sequences (Fig. 3a, b). The Luciferase genes carrying the putative binding sequences were significantly suppressed by miR-199-3p [pLin28b-(81), pLin28-(983), and pSuz12-(973)], whereas the mismatched sequences, which possess base substitution mutations in the seed [pLin28b-(81)mut, region, abrogated its suppression effect [pSuz12-(973)mut]. pLin28-(983)mut, and When miR-199 mimics were introduced to C2Cl2 cells, the expression of Lin28b and Suz12 was consistently suppressed (Fig. 3c).To assess whether the suppression of Lin28b and/or Suz12 directly con-tributes to myogenic differentiation, specific inhibition of Lin28b and Suz12 was carried out by gene silencing, using RNA inter-ference (RNAi; Fig. 3d, e). As expected, the expression of myo-genic differentiation markers, such as Ckm,Myh1, and Myog was upregulated under Lin28b and Suz12 gene silencing (Fig. 3f, g).

Since Lin28b is an RNA-binding protein involved in processing of miRNAs3637, we investigated the effects of miR-19 mimics via Lin28b on myogenic miRNA processing. The level of miR-1, a major myogenic miRNA, was examined because processing of its precursor is regulated by Lin28b37. Matured miR-1 was significantly increased by miR-199 mimic treatment (Fig. 3h), suggesting that miR-199-3p can regulate the functional expres-sion of miR-1 through suppression of its target Lin28b. Taken together, the findings suggest that miR-199-3p is involved in myogenic differentiation through suppression of its target genes, Lin28b and Suz12.

Enhancement of muscle regeneration by miR199#4 in muscle injury mice. cistanche salsa benefits We examined the effects of an miR-199 mimic (miR199#4) in vivo using muscle injury models (Fig. 4a, b). Eight-week-old C57BL/6J mice were injected with BaClz to the tibialis anterior(TA) to induce muscle injury, and miR19#4 was administered to the damaged sites 24h later. Two days after miR199#4 administration, the expression of myogenic marker genes was examined. Myog expression was significantly increased and myogenic differentiation 1(Myod1) and myogenic factor 5 (Myf5) were also upregulated by miR199#4 administration (Fig. 4c). The data are consistent with the in vitro results described above (Fig. 2).

Immunohistochemical analysis of regenerating muscles was performed 9 days after miR199#4 administration. The cross-sectional areas of regenerating muscle fibers (myofibers), characterized by central nuclei, were examined (Fig. 4b). The mean cross-sectional area of regenerating myofibers treated with miR199#4 was larger than that of control myofibers treated with a non-silencing control RNA duplex (nsCont; Fig. 4d), with the difference approaching statistical significance. In addition to the mean cross-sectional area, the size-divided histogram of the cross-sectional area showed an increase in enlarged myofibers in the presence of miR199#4(Fig.4e).

Hypertrophic effect of miR199#4 on aged muscle fibers. We examined the effects of miR199#4 on aged mice(~24month-old). Aged mice exhibited a significant decrease in muscle miR-199-3p (Fig. 5a), as in blood-circulating cf-miR-19-3p, com-pared to young mice. We administered miR199#4 to the TA muscles of aged mice, and 2 days later the TA muscles were examined by immunohistochemical analysis (Fig. 5b). The mean cross-sectional area of myofibers treated with miR199#4 was significantly larger than that of control myofibers treated with nsCont (Fig. 5c). Consistent with this, the size-divided histogram analysis revealed a marked increase in enlarged myofibers in the presence of miR199#4(Fig.5d).

Since blood-circulating cf-miR-199-3p is reduced in aged mice, we introduced miR199#4 into the circulating blood of aged mice via the tail vain. After intravenous administration of miR19#4, the cross-sectional area of myofibers was examined and analyzed, as described above. The results, as shown in Fig. 5e, are consistent with the results of the local administration of miR199#4 to aged muscles (Fig. 5c, d).

It should be noted that there is a difference in enlarged muscle fibers between the muscle injury model and aged mice. Central nuclei are seen in regenerating muscle fibers (Fig. 4b), but such myofibers were hardly detected in aged mice treated with miR199#4 (Fig. 5b). This suggests that preexisting muscle fibers may become enlarged in aged mice following miR199#4 treatment. We hypothesized that another mechanism different from muscle regeneration might be involved in myofiber enlargement in aged mice, and proposed the involvement of the muscle atrophy pathway. cistanche tubulosa dosage reddit The Atroginl and MuRFI genes are closely related to muscle atrophy38, and their expression levels are significantly elevated in aged muscles compared to young muscles (Fig. 6a). We examined the level of Atroginl and MuRFI in muscles of aged mice after systemic administration of miR199#4. Atroginl and MuRFI were markedly reduced in various muscles by miR19#4 administration (Fig. 6b), suggesting that suppression of the muscle atrophy pathway by miR199#4 may cause myofiber expansion in aged mice.

MiR199#4 delays loss of muscle strength in aged mice. The effect of miR199#4 on muscle function in aged mice is of interest. The grip strength test was performed on aged mice before and after systemic administration of miR199#4. The experiment was performed in a double-blind manner;RNA injection and the 100% grip test were carried out by different experimenters without sharing i information. C57BL/6 mice (80-week-old)male were grip-tested, then 1 week later, they were injected intrave-nously with miR199#4 and nsCont, and grip-tested again. The change in muscle strength after miR199#4 injection was MiR199#4 improves the muscle strength of mdx mice. Since miR199#4 has a positive impact on muscles, we conducted an experiment to assess the effect of miR199#4 administration in mdx mice, a well-known DMD animal model32. Eight-week-old mdx mice were injected intravenously with miR199#4(1.6 mg/kg)twice (1 week interval), and the grip test was carried out 1 week after the last administration. The experiment was performed in a double-blind manner as shown in Fig.7. The results (Fig. 8a)show a significant improvement in muscle strength in mdx mice treated with miR199#4 compared to mdx mice treated with nsCont. In addition, significant decreases in creatine kinase (CK)activity and cell-free miR-1 were observed in the blood of mdx mice treated with miR199#4(Fig. 8b, c), which are possible signs of the disease improvement.

As a drug delivery reagent, atelocollagen is viscous and difficult to handle, and the mice treated with atelocollagen appeared to suffer negative effects due to the vehicle. Thus, we looked for alternatives to atelocollagen to improve our drug delivery system (DDS) and found DC-CHOL/DOPE cationic liposomes as a new DDS reagent that is equal or superior to atelocollagen (Supplementary Fig. 4). The drug distribution of miR199#4 with the cationic liposomes showed that the administered miR19#4 was delivered to the muscle and consistently taken up in large amounts in the liver (Supplementary Fig. 5). Extracellular miR199#4 delivered to the muscle may function as a mediator in gene silencing and contribute to the gene regulation involved in the improvement of muscle strength.

We attempted to identify biomolecules related to the improvement following miR199#4 administration. Various poly-peptides related to DMD were examined by western blotting;however, distinct signals related to to the improvement were not detected in this study (Supplementary Fig. 6). We will discuss this point further in the following section.

Discussion

Biomolecules change qualitatively and quantitatively with aging--3. Capturing such changes and studying them are vital for understanding aging, and may reveal strategies for dealing with problems in aging societies. Parabiosis studies in which young and aged animals are conjoined to share a common vascular system have provided important insights into aging and rejuvenation9-l2. The findings strongly suggest that there are rejuvenating and aging factors in young and aged circulating blood, respectively. Previous studies reported various molecules as candidates related to rejuvenation and aging, but the results are controversiall3.

Our in vitro cell culture experiments using young and aged sera are similar to parabiosis experiments (Fig. lb), and we found miR-199-3p, which changes with age, in circulating blood; the miRNA was identified to significantly decrease in aged blood (Table 1 and Fig. la).In addition, the expression of muscle (cellular)miR-199-3p markedly decreases in aged muscles com-pared to young muscles (Fig. 5a); thus, cf-miR-199-3p in the blood may correlate with muscle miR-199-3p. The decrease in expression of miR-199-3p with aging is also observed in mesenchymal stem cells derived from rhesus macaque bone marrow39. Therefore, the expression of cellular miR-19-3p may be under age-related control. In contrast to miR-19-3p, the expression of cellular myogenic miRNAs, such as miR-1 and miR-133, remain unchanged in young and aged muscles (Fig. 5a), but the level of their extracellular miRNAs in the blood is markedly reduced in aged mice (Fig. la). The difference between miR-199-3p and myogenic miRNAs(miR-1 and-133)may reflect the difference in cf-miRNA processing and/or in age-related control of cf-miRNAs between them. To elucidate this difference, further studies need to be conducted in the future.

Previous studies suggested that miR-199-3p participates in various cellular functions and vital phenomena via suppression of its target genes; e.g, the miRNA may promote the proliferation and migration of endothelial tip cells and modulate the onset of puberty, respectively, through downregulation of the targeted semaphorin-3A gene40 and through suppression of target genes (Cdc42,Map3k2,Map3k4,and Taok1)involved in the p38MAPK pathway41. The present study has found that miR-19-3p is involved in myogenic differentiation through suppression of its target genes, Lin28b and Suz12 (Fig. 3). Lin28b is an RNA-binding protein and inhibits the maturation of miRNAs, including miR-1, which is a muscle-specific miRNA and pro-motes myogenesis37. Suppression of Lin28b by miR-19-3p causes an increase in matured miR-1 (Fig. 3), which may allow myogenic differentiation to proceed.

Suzl2 is a core subunit of polycomb repressive complex 2 (PRC2), which participates in repressing chromatin42. Gene regulation of PRC2 is involved in stem cell maintenance, embryonic development, cell proliferation and differentiation, including myogenic differentiation42.PRC2 is present at silent muscle-specific loci, such as Myog and muscle creatine kinase (MCK) in undifferentiated myoblasts, but is dissociated from such loci in differentiated myotubes43. Gene silencing against Suz12 by RNAi causes the enhancement of myogenic gene expression(Fig. 3). Suppression of the enhancer of the zeste homolog 2 (Ezh2) protein, another core subunit of PRC2, also causes myogenic differentiation44. These findings suggest that inhibition of PRC2 formation due to suppression of its core subunits may trigger the creation of a genomic state for muscle differentiation. Thus, suppression of Suz12 by miR-199-3p may induce myogenic differentiation. Taken together, miR-19-3p may play an important role in myogenic differentiation through inhibition of two independent pathways involving Lin28b and Suzl2, which are each involved in the maintenance of the undifferentiated state of myoblasts.

The effect of miR-199-3p on myogenic differentiation has also been shown in vivo (Fig. 4). Muscle damage triggers cell pro-liferation and differentiation due to muscle regeneration. Intro-duction of miR199#4, which supplies miR-199-3p, to damaged sites enhanced muscle regeneration and expanded regenerating myofibers (Fig. 4). Myogenic gene expression was consistently upregulated under miR199#4 treatment (Fig. 4). From a practical viewpoint, miR199#4 might be effective and useful for surgical treatments.

When miR199#4 was administered to aged mice lacking blood-circulating miR-199-3p, the mice exhibited markedly expanded myofibers (Fig. 5) and delayed loss of muscle strength with aging (Fig. 7). The expanded myofibers in aged mice differ from regenerating myofibers, which are enlarged in the presence of miR199#4; the former is derived from preexisting myofibers, and the latter is from neo-myofibers (newborn myofibers). Thus, there are different, independent mechanisms in the muscle fiber enlargements. Our study suggests that (i) in the former case, inhibition of muscle atrophy due to suppression of Atroginl and MuRFI by miR199#4 treatment brings about enlargement of aged myofibers, and (ii) in the latter case, suppression of target genes, Lin28b and Suz12, causes enlargement of regenerating myofibers. Altogether, different (multiple) genes regulated by miR-19-3p may be involved in muscle hypertrophy in different situations. The findings also suggest that miR-199-3p may have at least an antiaging effect on muscle, and that miR199#4 supplying miR-199-3p may have potential as a novel RNA medicine for age-related muscle diseases, such as sarcopenia.

Based on the high efficacy of miR199#4 on muscles, we administered miR199#4 to mdx mice,a well-known DMD model, and obtained remarkable results: systemic administration of miR199#4 improved muscle strength in the mice by ~20%(Fig.8a). To understand the mechanism of the muscle strength recovery, we attempted to identify biomolecules that were improved (or influenced) by miR-199-3p. However, with the exception of improvements of blood CK and cf-miR-1 (Fig. 8b, c), we were unable to detect such improvements at the molecular level. This difficulty in detection may be due to the complex cell population, which consists of a mix of regenerating and dis-rupting muscle cells in mdx mice. Even if miR199#4 results in the improvement, the high background noise caused by the complex cell population would mask evidence of the improvement. To solve this problem, more extensive studies using a homogenous muscle cell population derived from mdx mice or focusing on various tissues, including muscle need to be carried out.

Although the molecular mechanism of the muscle strength recovery by miR199#4 remains unknown, the dose of miR19#4 for improved muscle strength is noteworthy. Two times admin-istration of ~1.6 mg/kg miR199#4 improved muscle strength in mdx mice. The effective dose appears to be lower than that of antisense oligonucleotides for exon-skipping against mutant dystrophin genes in vivo45-47. The reason why miR199#4 is capable of improving muscle strength at such lower doses may be due to its different mechanism of action compared with antisense oligonucleotides. The mechanism of action of miR19#4 is cata-lytic gene silencing. In contrast, the mechanism of antisense oli-gonucleotides is based on physical hybridization. Therefore, differences in the appropriate doses to show drug efficacy may be attributable to the different mechanisms of action.

The mechanism by which organs, tissues, or cells release miRNAs into the circulating blood is not yet fully understood, and the age-related regulation on the release of such miRNAs is also unclear. The present study showed that myogenic cf-miR-NAs, including cf-miR-199-3p circulate with the blood in the vascular system and decrease with aging. Such cf-miRNAs may be present in exosomes,ie, they are exosomal miRNAs.

Myogenic miRNAs participate in the myogenic differentiation, muscle regeneration, and maintenance of muscular homeostasis33. Previous studies reported that myogenic miRNAs, such as miR-1 and miR-133, were observed to increase in the blood in response to physical exercise4849. Based on these reports, active young mice may release more myogenic miRNAs into the blood than aged slow-moving mice, and such extracellular miRNAs, including cf-miR-199-3p may contribute to maintaining muscle homeostasis in young mice through autocrine action. However, the level of extracellular (exosomal) miRNA does not always correlate with the cellular miRNA level, and differences have been observed in myogenic miRNAs and miR-199-3p between young and aged mice (Figs. la and 5a) and during myogenic differentiation (Supplementary Fig. 2a). Thus, as another possibility, it is con-ceivable that age-related and differentiation-related regulation of exosome formation may affect cf-miRNA production; specifically, there may be differences in exosome formation between young and aged mice. In the context of these hypotheses, further study is needed to elucidate the age-related release of myogenic miRNAs into the blood.

Aside from the release mechanism, when young myogenic cf-miRNAs containing cf-miR-199-3p flow into the aged blood-circulating system from a young vascular system by parabiosis, the cf-miRNAs could trigger muscle activation and inhibition of muscle atrophy on the aged side; this concept is supported by evidence from aged mice intravenously injected with miR19#4 in this study (Figs. 5-7). Cf-miRNAs in the blood may be related to age-related homeostatic function and could represent altered homeostasis. When such cf-miRNAs are ectopically present, for example, when cf-miRNAs in young blood are transferred to the aged blood-circulating system, some cf-miRNAs may show rejuvenating effects on the aged side, i.e, they may produce antiaging effects. In contrast, cf-miRNAs in aged blood may represent altered homeostasis with aging. When such cf-miRNAs are transferred to the young blood-circulating system, some cf-miRNAs might trigger aging-related deterioration and might cause premature aging on the young side. Cf-miRNAs that are significantly increased in aged blood represent candidates that will require detailed mechanistic analysis to understand and prevent aging.

In conclusion, our current study has provided new insights into cf-miRNAs as age-related molecules, and revealed a new research direction to clarify the relationship between aging and cell-free functional RNAs,including cf-miRNAs.

This article is extracted from COMMUNICATIONS BIOLOGY | (2021) 4:427 | https://doi.org/10.1038/s42003-021-01952-2 | www.nature.com/commsbio