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. 2020 Dec 18;3(6):562–572. doi: 10.1089/crispr.2020.0077

Regenerating Urethral Striated Muscle by CRISPRi/dCas9-KRAB-Mediated Myostatin Silencing for Obesity-Associated Stress Urinary Incontinence

Huixing Yuan 1,2, Yajun Ruan 1,2, Yan Tan 1, Amanda B Reed-Maldonado 1,3, Yinwei Chen 1, Dehua Zhao 4, Zhao Wang 1, Feng Zhou 1, Dongyi Peng 1, Lia Banie 1, Guifang Wang 1, Jihong Liu 2, Guiting Lin 1, Lei S Qi 4, Tom F Lue 1,*
PMCID: PMC7757699  PMID: 33346712

Abstract

Overweight females are prone to obesity-associated stress urinary incontinence (OA-SUI), and there are no definitive medical therapies for this common urologic condition. This study was designed to test the hypothesis that regenerative therapy to restore urethral striated muscle (stM) and pelvic floor muscles might represent a valuable therapeutic approach. For the in vitro experiment, single-guide RNAs targeting myostatin (MSTN) were used for CRISPRi/dCas9-Kruppel associated box (KRAB)-mediated gene silencing. For the in vivo experiment, a total of 14 female lean ZUC-Leprfa 186 and 14 fatty ZUC-Leprfa 185 rats were used as control and CRISPRi-MSTN treated groups, respectively. The results indicated that lentivirus-mediated expression of MSTN CRISPRi/dCas9-KRAB caused sustained downregulation of MSTN in rat L6 myoblast cells and significantly enhanced myogenesis in vitro. In vivo, the urethral sphincter injection of lentiviral-MSTN sgRNA and lentiviral-dCas9-KRAB significantly increased the leak point pressure, the thickness of the stM layer, the ratio of stM to smooth muscle, and the number of neuromuscular junctions. Downregulation of MSTN with CRISPRi/dCas9-KRAB-mediated gene silencing significantly enhanced myogenesis in vitro and in vivo. It also improved urethral continence in the OA-SUI rat model.

Introduction

Obesity is one of the most challenging public health issues globally.1 It causes not only decreased quality of life but also a substantial economic burden for patients. Obese women are especially prone to stress urinary incontinence (SUI).2 Studies report that for every increase of five units of body mass index, the risk of incontinence increases by about 20–70%.3 Current treatments for obesity-associated SUI (OA-SUI) include weight loss, pelvic floor muscle training, injection of bulking agents, and urethral sling surgery.4 These interventions, however, do not treat the underlying pathophysiological causes of OA-SUI. Moreover, these treatments are often ineffective or difficult to comply with and in some cases may be associated with serious postoperative complications. A novel minimally invasive approach to the treatment of OA-SUI is needed. Although the underlying mechanisms of OA-SUI are not completely understood, we recently demonstrated that dysfunction of urethral striated muscle (stM) and pelvic floor muscle contributes to OA-SUI. In this study, we investigate the potential to regenerate these muscles to treat OA-SUI.

Working in concert, the urethral stMs, the urethral smooth muscles, and the vascular elements within the submucosa of the urethra ensure urethral sphincter closure and maintain continence. StM often undergoes atrophy in association with obesity. Obesity from chronic high-fat feeding in middle-aged mice was reported to decrease the myofiber area, satellite cells, and myonuclei of the gastrocnemius muscle.5 StM regeneration occurs in two main steps: activation of muscle stem cells and satellite cells and differentiation of these cells.6,7 Obesity contributes to muscle dysfunction by negatively impacting both of these critical steps in addition to increasing the expression of the myogenesis-inhibiting factor myostatin (MSTN). These effects contribute to the development of OA-SUI.

MSTN, also known as growth differentiation factor 8 (GDF-8), is a myokine. Myokines are proteins produced and released by myocytes that act on muscle cells' autocrine function. MSTN inhibits not only myogenesis but also muscle cell growth and differentiation. MSTN is a negative regulator of satellite cell proliferation and differentiation. Mutation of MSTN can cause muscle overgrowth and increase the diameter and number of muscle fibers.8,9 Several cellular signaling pathways are involved in myogenesis, including MSTN, TNF-like weak inducer of apoptosis (TWEAK), and Wnt/Frizzled pathways.10 In particular, MSTN and its receptor ActRIIB have been studied extensively as negative regulators of myoblast differentiation. Through the activation of its downstream targets, such as Smad2/3, MSTN inhibits myogenesis and promotes fibrosis. This may also occur through upstream activation of MSTN expression by the ERK1/2/p38/MAPK5 pathway. In both cases, dyslipidemia and/or hyperglycemia may play a major role, not just in impairing muscle-derived stem cells (MDSC) but also in triggering the overexpression of MSTN. The therapeutic potential of inhibitors, antibodies, and RNAi-mediated MSTN inhibition is being investigated.11,12 Currently, however, these approaches are limited to organisms that have the proper host machinery. These therapeutics can also produce significant off-target effects and toxicity, which further limits their applicability.13

There are several ways to inhibit MSTN. CRISPR-mediated interference (CRISPRi) provides a new tool for targeted endogenous gene repression in the genome without altering the DNA sequence.14–18 CRISPRi uses a nuclease-deactivated dCas9 (D10A&H840A) fused with a Kruppel associated box (KRAB) domain, which has the advantages of high repression efficiency (>80% on many genes), low cost, and easy operation. Compared to alternative technologies, the CRISPRi/dCas9-KRAB method avoids DNA damage induced by gene editing, is reversible, and is multiplexable, which means it can be utilized for gene regulation purposes in different cell types.19–23 To achieve efficient gene repression in MDSCs, we constructed single-guide RNAs (sgRNAs) targeting the MSTN gene using the Streptococcus pyogenes–derived CRISPRi/dCas9-KRAB gene silencing system. In our previous work, we successfully established fatty ZUC-Leprfa 185 (ZF) rats as a consistent and reliable animal model to study OA-SUI.24 To test the hypothesis that MSTN silence in the urethral sphincter could improve urethral function, we used lentivirus (LV) to mediate the MSTN CRISPRi/dCas9-KRAB gene silencing system in vitro in L6 cells and in vivo in Zucker rats.

Methods

sgRNA design, construct generation, and LV production

Four sgRNAs targeting the first exonic regions of the murine MSTN gene were designed. The CRISPR-ERA online algorithm was used to design and estimate on-target and off-target gene repression efficiency.25 The four MSTN targeting sgRNAs (1–4) were sgRNA-1: 5′-TGGCCCAGTGGATCTAAATG-3′, sgRNA-2: 5′- GTGCGTGGAGACAAAACACA-3′, sgRNA-3: 5′-TGTCCTCATTTAGATCCACT-3′, and sgRNA-4: 5′-GGCTGTGTAATGCGTGTGCG-3′.

MSTN-sgRNA expression plasmids were developed by inserting annealed MSTN sgRNA oligonucleotides into the lentiviral U6-based expression vector via digestion with BstXI and XhoI. Packaging of dCas9-KRAB (fused with mCherry via a cleavable p2A linker driven by a doxycycline-inducible TRE3G promoter; Clontech, Mountain View, CA) and MSTN-sgRNA (which also encodes BFP) expression constructs into lentiviral particles (pCMV-dR8.91 lentiviral packaging plasmid, pMD2.G lentiviral packaging plasmid) was conducted, as previously reported.14 For in vitro experiments, we added 1 μg/mL doxycycline to the medium to induce expression of dCas9-KRAB. For in vivo experiments, we administered 1 μg/kg doxycycline to the animals to induce dCas9-KRAB expression. LV-expressing dCas9-KRAB and the MSTN-sgRNA were produced by transfecting HEK293T packaging cell lines according to the method of Qi et al.26 After 72 h, the supernatant was collected and enriched for the recombinant viruses by incubating with a virus precipitation solution PEG-it™ (System Biosciences, Palo Alto, CA) for 16 h at 4°C.27 The recombinant LV was recollected by centrifuging the samples at 1500 g for 45 min at 4°C. The resultant LVs were named LV-dCas9-KRAB, LV-MSTN-sgRNA1, LV-MSTN-sgRNA2, LV-MSTN-sgRNA3, and LV-MSTN-sgRNA4. A scrambled sgRNA LV was also developed. The LVs were used for both in vitro and in vivo delivery.

Cell culture and transductions

Rat myoblast L6 cells were used in this experiment. The cells were divided into three groups: (1) control, (2) induction, and (3) induction + CRISPRi/MSTN. In the control and induction groups, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 2% horse serum (HS), respectively. In the induction + CRISPRi/MSTN group, 2 × 105 L6 cells were seeded on each six-well plate and cultured in DMEM containing 10% FBS. The following day, the medium was replaced by fresh differentiation medium containing 8 mg/mL polybrene (Sigma–Aldrich, St. Louis, MO) before transducing the cells with 500 μL LV-dCas9-KRAB and LV-MSTN-sgRNA1-4 (4 × 106 transduction units/mL). One day after transduction, the medium was replaced by fresh DMEM with differentiation medium, and 5 days after transduction, cells were harvested for myotube formation assay and gene expression assay. The mRNA and protein expression of MSTN were assayed with reverse transcription polymerase chain reaction and Western blot, as previously reported.28–30 The expression of muscle-specific transcription factors myogenin D (MyoD), myogenin G (MyoG), and myosin heavy chain (MHC) was also assayed with Western blot. Myotube formation assay was conducted by fixing cells with ice-cold methanol and staining for striated myosin heavy chain (MHC;1:500; Abcam, Inc., Cambridge, MA) and MyoG (1:500; Abcam, Inc.), followed by microscopy and photography analysis, as previously reported.31

Animal experiments

All rats were obtained from Charles River Laboratories (Wilmington, MA). The animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee at our University. Fourteen female ZF) and 14 female Zucker lean (ZUC-Leprfa 186; ZL) rats at 12 weeks old were used. ZF and ZL rats were randomly grouped into control or CRISPRi/MSTN cohorts with an allocation ratio of 1:1. The groups were named ZL control, ZL CRISPRi/MSTN, ZF control, and ZF CRISPRi/MSTN (n = 7). The ZL control group and ZF control group received one urethral injection of 600 μl saline. The ZL CRISPRi/MSTN and ZF CRISPRi/MSTN groups received one urethral injection of LV-MSTN-sgRNA2 (300 μL) and LV-dCas9-KRAB (300 μL) in a total volume of 600 μL. Four weeks after the injection, all rats underwent conscious cystometry and leak point pressure (LPP) measurement. The rats were subsequently sacrificed, and tissues were harvested.

Technique of gene injection into the urethral sphincter

After the rat was anesthetized with isoflurane, a lower abdominal incision was made, and the periurethral tissue was exposed. A dose of 300 μL LV-MSTN-sgRNA2 (4 × 106 TU/mL) and 300 μL LV-dCas9-KRAB (4 × 106 TU/mL) or saline was injected into the external urethral sphincter and periurethral tissue with a 21G needle along the urethral wall at 3 and 9 o'clock toward 6 o'clock (Fig. 1A).

FIG. 1.

FIG. 1.

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Localization of dCas9-mCherry and sgRNA-BFP in the urethral wall after local injection. (A) Injection (inj) of LVs. The 21G needle was injected along the urethral wall at 3 and 9 o'clock site toward the 6 o'clock direction, and LVs were delivered. (B) and (C) dCas9-mCherry (red) and sgRNA-BFP were mainly expressed within the urethral stM and less within the urethral SM. sgRNA, single-guide RNA; LV, lentivirus; stM, striated muscle; SM, smooth muscle.

Conscious cystometry and LPP measurement

Conscious cystometry was performed, as previously described.24,32 For LPP measurement, the anesthetized rats were placed in a supine position, and a lower abdominal incision was made to expose the bladder. At the level of zero pressure, the bladder was filled with saline through a bladder catheter connected to a pressure transducer, and bladder volume was measured. The bladder was filled to half capacity. The bladder was tested by manually increasing bladder pressure until a leak occurred, at which time the external vesicle pressure was rapidly removed. Peak bladder pressure was recorded as the LPP. This procedure was repeated three to four times in each rat, and the average pressure was calculated.

Tissue immunofluorescence staining

After the experiment was performed, immunofluorescence staining of the urethra was performed, as previously described.33 In brief, the tissue sections were incubated overnight with anti-myosin skeletal heavy chain (MHC; 1:500, Mouse [MY-32] [ab51263]; Abcam, Inc.) and anti-α-smooth muscle actin (α-SMA; 1:1,000, Mouse; Abcam, Inc.). Secondary antibodies used included Alexa-488- and Alexa-594-conjugated antibodies (1:500; Invitrogen, Carlsbad, CA). Nuclei were stained with 4′,6-diamidino-2-phenylindole. Neuromuscular junctions (NMJs) were stained with α-bungarotoxin conjugated with Alexa-488 (1:500; Invitrogen) followed by phalloidin conjugated with Alexa-594 (1:500; Invitrogen). For image analysis, five randomly selected fields per slide were photographed and recorded using a Retiga imaging camera and ACT-1 software (Nikon Instruments, Melville, NY). Image-Pro Plus (Media Cybernetics, Rockville, MD) was used to quantify differential staining.

Statistical analysis

Data were analyzed with GraphPad Prism v5 (GraphPad Software, San Diego, CA). All data are shown as means ± standard deviation (SD). One-way analysis of variance followed by Tukey's post hoc test was used for multiple comparisons.

Results

Optimized sgRNA for CRISPRi/MSTN

To induce efficient repression of the murine MSTN gene, different MSTN-targeting sgRNAs were designed. The four sgRNAs mainly targeted the first exonic regions of the murine MSTN gene (Fig. 2A). LVs LV-dCas9-KRAB, LV-MSTN-sgRNA1, LV-MSTN-sgRNA2, LV-MSTN-sgRNA3, and LV-MSTN-sgRNA4 were successfully developed. We infected L6 myoblast cells with LVs expressing four MSTNS-sgRNAs and the dCas9-KRAB protein to screen for the most effective sgRNA. After treatment with the LV-mediated MSTN CRISPR/dCas9-KRAB system, all MSTN-sgRNA were shown to silence MSTN gene expression (p < 0.05), and maximal silence (80.1%) of MSTN protein expression was achieved by MSTN-sgRNA2 (Fig. 2). Compared to the induction group, the myotubes in the CRISPRi/MSTN group were significantly longer and wider (p < 0.05). Of the four MSTN-sgRNAs, MSTN-sgRNA2 was the most effective, while scrambled sgRNA LV did not enhance the myogenesis. Since MSTN-sgRNA2 resulted in the strongest MSTN interference in L6 cells, subsequent experiments were performed utilizing MSTN-sgRNA2.

FIG. 2.

FIG. 2.

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CRISPRi/MSTN enhanced myogenesis in vitro. sgRNAs targeting the first exonic regions of the murine MSTN gene were designed (A). L6 cells were untreated (ctrl), treated with 2% HS (Ind), or treated with 2% HS + LV-dCas9 and LV-MSTN-sgRNA (Ind+sgRNA). Five days later, L6 cells were stained for MHC (green), and the length and width of the myotubes was determined. *p < 0.05 vs. Ind (B). The cells were harvested for RNA and protein isolation. MSTN mRNA was detected by reverse transcription polymerase chain reaction. Results are shown as the mean ± SD of three independent experiments (C). Proteins were separated by SDS-PAGE. MSTN protein was detected by Western blot using relative antibody. Beta-actin protein was also detected as protein loading control using β-actin antibody. Relative MSTN protein was determined by quantifying Western blot membrane band intensity. Results are shown as the mean ± SD of three independent experiments (D). *p < 0.05 vs. ctrl; **p < 0.05 vs. Ind. CRISPRi, CRISPR-mediated interference; MSTN, myostatin; HS, horse serum; MHC, myosin heavy chain; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Silencing MSTN with CRISPRi/MSTN enhanced myogenesis in vitro

L6 myoblast cells were transfected with LV-dCas9-KRAB and LV-MSTN-sgRNA2. Mean transduction efficiencies were 75.6 ± 3.4% and 77.4 ± 3.1%, respectively. No viral toxicity in cells was observed during transduction. The MSTN gene was not expressed in L6 cells without induction, while medium containing 2% HS (induction medium) induced MSTN expression and myotubes formation. Five days after treatment with the CRISPRi/MSTN system, larger and more robust myotubes were formed compared to the induction control (Fig. 3A–D; p < 0.05). Interestingly, compared to the induction control group, the CRISPRi/MSTN group showed significantly lower MSTN protein levels (p < 0.05), while MHC, MyoD, and MyoG were significantly upregulated (p < 0.05; Fig. 3E and F).

FIG. 3.

FIG. 3.

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Silencing MSTN with CRISPRi/MSTN enhanced myogenesis in vitro. L6 cells were not treated (ctrl), treated with 2% HS (Ind), or treated with 2% HS, LV-dCas9, and LV-MSTN-sgRNA2 (Ind + CRISPRi). Five days later, the cells were stained for MHC (green) and MyoG (red), and the length and width of the myotubes was determined. *p < 0.05 vs. ctrl and Ind (A)–(D). The cells were harvested for protein. Proteins were separated by SDS-PAGE. MSTN, MHC, MyoG, and MyoD proteins were detected by Western blot using relative antibody. Beta-actin protein was also detected as protein loading control using β-actin antibody. Relative protein was determined by quantifying Western blot membrane band intensity. Results are shown as the mean ± SD of three independent experiments. *p < 0.05 vs. ctrl; **p < 0.05 vs. Ind. (E) and (F). MyoG, myogenin G.

Primary outcomes of CRISPRi/MSTN on voiding function in ZF rats in vivo

After the injection of LV-MSTN-sgRNA2 and LV-dCas9-KRAB, the mCherry signal of dCas9-KRAB and the BFP signal of sgRNA were localized in the urethral wall, with most contained within the urethral stM (Fig. 1B and C). No abnormal behavior was observed in any of the rats at any time during the study. No hematuria or bloody stool was seen. No clinical signs of pain, irritation, or injury were observed from general expression, activities, or attention to the treated area. Surveillance revealed normal activity with proper response to the stimulation of CRISPRi/MSTN treatment, adequate vitality of movement, satisfactory food and water consumption, normal social interaction, and normal usage of the accessories in the cage. For the baseline data, there was no effect on the body weight of either the ZL or the ZF group after the treatment.

During each urination, the bladder pressure in the ZL group increased from the baseline level to about 60 cm H2O. In contrast, the bladder pressure in the ZF group increased from the baseline level to 25 cm H2O and with continuous leakage and no obvious capacity. For the primary outcome, cystometry revealed that LPP decreased to 33.07 ± 3.13 cm H2O in the ZF rats compared to 66.77 ± 3.26 cm H2O (p = 0.001) in the ZL rats. After treatment, ZF CRISPRi/MSTN groups had significantly improved voiding parameters with increased LPP (47.23 ± 3.50 cm H2O; p = 0.010). No significant changes were noted in the treated ZL rats (Fig. 4).

FIG. 4.

FIG. 4.

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Voiding function affected by CRISPRi/MSTN in vivo. The body weight of ZF (n = 7) and ZL rats (n = 7). *p < 0.05 ZF vs. ZL group (A). Compared to ZL rats, bladder pressure in the ZF rats increased less after saline was injected into the bladder, and in many ZF rats, there was no discrete void but rather continuous leakage (B). LPPs in ZF rats were much lower than in ZL rats (C). After treatment, LPPs in ZF CRISPRi rats (n = 7) were much higher than that in ZF rats (N = 7). *p < 0.05 ZF vs. ZL group; **p < 0.05 ZF vs. ZF CRISPRi group (D). ZF, fatty ZUC-Leprfa 185 rats; ZL, lean ZUC-Leprfa 186 rats; LPP, leak point pressure.

CRISPRi/MSTN regenerated impaired urethral stM in female ZF rats

To study secondary outcomes of the LV-mediated expression of CRISPRi/MSTN system on the urethral muscular structure, the urethral tissues were stained with MHC (green) and a-SMA (red) to differentiate stM from smooth muscle. The results revealed a much higher ratio of stM to smooth muscle in ZL, ZL CRISPRi/MSTN, and ZF CRISPRi/MSTN rats than that in ZF rats. In addition, the individual muscular fibers were longer and the stM layers were thicker in the CRISPRi/MSTN rats than in the control rats (p < 0.05; Fig. 5). Meanwhile, the expression of MSTN within the urethral wall was checked, and silencing by CRISPRi/dCas9-KRAB was confirmed (Fig. 6).

FIG. 5.

FIG. 5.

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Cross-section of mid-urethral segments for urethral stM and SM. (A) Urethral stM and Sm in ZL, ZL CRISPRi/MSTN, ZF, and ZF CRISPRi/MSTN groups. (B) Compared to ZF rats, the ratio of stM to SM was higher in ZL, ZL CRISPRi/MSTN, and ZF CRISPRi/MSTN groups (p < 0.05). (C) Compared to ZF rats, ZL, ZL CRISPRi/MSTN, and ZF CRISPRi/MSTN rats have longer stM fiber length (p < 0.05). (D) Compared to ZF rats, ZL, ZL CRISPRi/MSTN, and ZF CRISPRi/MSTN rats had more srM layers (p < 0.05).

FIG. 6.

FIG. 6.

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Decreased urethral MSTN expression with the CRISPRi/MSTN system. The urethral sections were stained for MSTN (red), and muscles with Pha (green). (A) Mean fluorescence intensity AU of MSTN expression in ZF rats was higher than in ZL rats. After treatment, mean fluorescence intensity AU of MSTN expression in ZF CRISPRi/MSTN rats was lower than in ZF rats. *p < 0.05 ZF vs. ZL group; **p < 0.05 ZF vs. ZF CRISPRi/MSTN group (B). Pha, phalloidin; AU, arbitrary unit.

CRISPRi/MSTN increased NMJs in urethral stM

A NMJ is a chemical synapse formed by contact between a motor neuron and a muscle fiber. The NMJ allows for the neuron to send signals to the muscle fiber to trigger muscular contraction. In this study, we calculated the number of NMJs in the stM of the rat urethra. Under high power ( × 400), the NMJs are shown with a white arrow. The number of NMJs was much reduced in ZF rats compared to the ZL rats (6.7 ± 1.2 vs. 12.7 ± 1.5, p = 0.03). After treatment, the number of urethral NMJs increased significantly in the ZF CRISPRi/MSTN rats to 13.5 ± 1.6 (p < 0.05; Fig. 7).

FIG. 7.

FIG. 7.

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Increased NMJs in the urethra of ZF rats with the LV-mediated CRISPRi/MSTN system. Female fat urethral NMJs stained with a-bungarotoxin (a-BTX; green) and muscles stained with Pha (red) (A). There were fewer NMJs in ZF rats than in ZL rats. After treatment, NMJs in ZF CRISPRi/MSTN rats increased significantly in number compared to ZF rats. *p < 0.05 ZF vs. ZL group; **p < 0.05 ZF vs. ZF CRISPRi/MSTN group (B). NMJ, neuromuscular junctions.

Discussion

Urinary incontinence is a serious quality of life health problem that affects more than 13 million women in the United States.34 Epidemiological studies show that obesity is an important risk factor for urinary incontinence. Longitudinal cohort studies show specifically that overweight women have a higher risk of urinary incontinence, which is referred to clinically as OA-SUI. Our previous studies show that obesity leads urethral muscular atrophy, muscular deformation, and lipid deposition within the muscle cells of the stM of the urethra sphincter.24,32 In addition, our previous work shows that muscle stem/progenitor cells can be activated (using microenergy acoustic pulses [MAP]) for urethral stM regeneration, thus improving urinary function.35,36 StMs were found throughout the main portion of the female rat urethra and were most prominent in the mid-urethra.37 Our research demonstrated that MAP treatment activates tissue-resident stem cells within the pelvic floor and urethral muscle in situ via the FAK-AMPK and Wnt/β-catenin signaling pathway.38

Satellite cells are adult myogenic stem cells that can repair damaged muscle. Extensive research demonstrates that the enduring capacity for muscle regeneration requires efficient satellite cell expansion, their differentiation to produce myoblasts that can reconstitute damaged fibers, and their self-renewal to replenish the muscle stem cell pool for subsequent cycles of injury and repair. During muscle regeneration, the activation of satellite cells is the initial step of myotube formation.39 If we were able harness the ability to activate endogenous stem cells and to influence stem cell differentiation, this would provide powerful, potentially curative treatments for many human diseases. This ability is the basis of the technological innovations we are developing for focally enhancing muscle regeneration to treat OA-SUI. In 2017, we successfully isolated urethral striated MDSCs (uMDSCs), which differentiate into myotubes in vitro.40 In recent research, we established that MAP-treated uMDSCs have increased expression of MHC and MyoG, a transcriptional activator that plays a role in muscle differentiation.41 Myogenesis is negatively regulated by MSTN and reduced expression of MSTN is required in the early phase of stM regeneration.42,43 Therefore, inhibition of the negative regulator MSTN is a promising approach to enhance muscle regeneration.

Our in vitro experiments also found that after downregulating MSTN gene expression in L6 cells, the expression of MyoD and MyoG genes was upregulated, which could promote the differentiation of myoblast cells into myotubes. The MSTN knockout mice showed a biomyogentic effect of muscle fiber proliferation and hypertrophy, leading to a significant increase in muscle weight.44 On the contrary, the MSTN overexpressed mice can become extremely emaciated.45 The MSTN knockout mice can improve muscle volume via enhanced muscle regeneration and reduced fibrosis.46 This indicates that MSTN can inhibit the proliferation and differentiation of muscle cells in vivo. In addition, there is some evidence that muscular atrophy can upregulate MSTN expression in skeletal muscle,47 while long-term endurance training can downregulate MSTN expression in skeletal muscle.48

This study utilized a recent technology developed by Dr. Qi on CRISPRi.14,15 The CRISPRi system requires a minimal set of only two molecules: a dCas9 deactivated nuclease protein fused with a transcriptional repressor (KRAB) and custom-designed sgRNAs. Targeted gene repression via dCas9-KRAB/sgRNA can achieved up to 80–90% with high specificity.14,49,50 In this study, we constructed a LV-mediated expression of MSTN CRISPRi/dCas9-KRAB system and used it to transduce rat L6 cells and the urethral sphincter of female ZL and ZF rats. All four MSTN sgRNAs mainly target the first exonic regions of the MSTN gene and enhanced myogenesis, with sgRNA#2 producing the best effects. To check the effect of this system in vivo, the LVs of dCas9-KRAB and sgRNA were co-injected into the urethral wall, as described in Figure 1A. The MSTN CRISPRi/dCas9-KRAB system successfully downregulated the expression of MSTN and resulted in significant increases in MyoD, MyoG, and MHC.

Very recently, we reported that MAP treatment restored micturition function in ZF rats via activation of muscle stem/progenitor cells to regenerate the urethral sphincter muscle.35,36 With the goal of finding alternative, potentially more effective, efficient ways to accomplish this task, we applied CRISPRi/MSTN system in the OA-SUI animal model in vivo. Strikingly, 4 weeks after the delivery of CRISPRi/MSTN system in the urethral tissue, the LPP significantly improved by twofold in the ZF rats. Voiding function also significantly improved. Histologic studies revealed that the urethral stM regenerated in the CRISPRi/MSTN-treated animals. The ratio of stM to smooth muscle was much higher in the ZL, ZL CRISPRi/MSTN, and ZF CRISPRi/MSTN rats than in the ZF rats. In addition, muscular fibers were longer and stM layers were thicker in CRISPRi/MSTN rats than in control rats (p < 0.05). Also, nerve innervation improved, as there are more NMJs in the ZF CRISPRi/MSTN rats (p < 0.05).

This study has several limitations. First, we tested only four MSTN sgRNAs. Second, there was a small animal sample size in each of our groups. Also, the combination of MAP and CRISPRi/MSTN was not tested in the current study. We plan to perform this study in the future to investigate the potential for both therapies further.

Conclusion

Downregulation of MSTN with CRISPRi/dCas9-KRAB-mediated gene repression significantly enhanced myogenesis both in vitro and in vivo and improved urethral continence in the ZF rat OA-SUI animal model. This may be a potential therapeutic approach for OA-SUI.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

The research reported in this publication was supported by NIDDK of the National Institutes of Health under award number R56DK105097 and 1R01DK105097-01A1.

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