Inhibition of prolyl 4-hydroxylase decreases muscle fibrosis following chronic rotator cuff tear

Article focus

• We used a translationally relevant rat model to assess the ability of a pharmacological inhibitor of Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases, GSK1120360A, to reduce fibrosis and improve healing following a rotator cuff tear.

Key messages

• Inhibition of HIF prolyl 4-hydroxylases improved mechanical properties of the enthesis.

• Inhibition of HIF prolyl 4-hydroxylases reduced fibrosis without improving contractile function of muscles.

Strengths and limitations

• Strength: Detailed analysis of changes in muscle fibre contractile function and size, muscle extracellular matrix accumulation and gene expression, and enthesis mechanics.

• Limitation: We only measured properties at a single time point and did not evaluate changes over time.

Introduction

Tears of the rotator cuff are one of the most frequent and debilitating joint injuries, and are often associated with fibrosis, muscle atrophy, and fat infiltration within and around muscle fibres, collectively referred to as ‘fatty degeneration’.¹ Surgical repair of torn cuff muscles does not decrease fatty degeneration, and in some cases lipid infiltration and fibrosis continue to persist despite successful repair of the torn tendon.¹ Fibrosis can increase the passive stiffness of the muscle extracellular matrix (ECM),² and lead to increased shearing and damage of the muscle fibre sarcolemma.³ The combination of increased tissue stiffness and membrane damage can render the muscle more prone to further injury and re-tear, which complicates rehabilitation and prevents the full restoration of function.⁴ Identifying approaches that reduce fibrosis and promote muscle regeneration are therefore likely to speed up the recovery and improve the quality of life in patients who undergo surgical repair of chronically torn rotator cuff muscles.

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that regulates the cellular and molecular responses to hypoxia, or low oxygen conditions. Under normoxic conditions, HIF-1α is hydroxylated on two proline residues by the HIF-prolyl 4-hydroxylases (PHDs), which then lead to the subsequent ubiquitination and rapid degradation of HIF-1α.⁵ In conditions of hypoxia, there is a reduction in the enzymatic activity of PHDs, which then allows HIF-1α to accumulate, translocate to the nucleus and direct the expression of target genes.⁵ HIF-1α induces the expression of several genes which have beneficial effects on the regeneration and protection of many organ systems including neural tissue, heart, lung, and skeletal muscle.⁶ In a skeletal muscle ischaemic injury model, compared with normoxic conditioned muscles, preconditioning of muscles in a hypoxic environment improved contractile force production, calcium influx, and recovery after an ischaemic insult.⁷ While hypoxia has been shown to positively affect the regeneration of several different types of tissues, inducing functional hypoxia in vivo can be difficult to achieve, which has led to the development of pharmacological inhibitors of PHD.⁶ In renal and cardiac injury models, treatment with PHD inhibitors reduced inflammation and fibrosis, and improved tissue regeneration.^8,9 However, the effects of PHD inhibition on the treatment of skeletal muscle injuries are not known.

As chronically torn rotator cuff muscles often experience substantial inflammation and fibrosis, and the use of PHD inhibitors has shown promise in the amelioration of these conditions in other tissue injury models, our objective was to test the ability of a PHD inhibitor, GSK1120360A,⁹ to enhance rotator cuff regeneration following a chronic tear. We hypothesised that the targeted inhibition of PHD using GSK1120360A would decrease fibrosis, increase muscle fibre and specific force production, and increase tendon to bone strength in chronically torn rotator cuff muscles that underwent surgical repair. To test this hypothesis, we treated rats with GSK1120360A following the repair of chronically torn rotator cuff muscles, and measured changes in supraspinatus muscle collagen content, contractility, fibre type distribution and size, the expression of genes involved in fibrosis, lipid accumulation, atrophy and inflammation, and the mechanical properties of the enthesis two weeks following surgical repair.

Materials and Methods

Results

At two weeks following repair, muscle mass in the 3 mg/kg group was 18% greater than in controls, and 12% greater in the 10 mg/kg group compared with controls (Fig. 1a) but there was no dose-dependent increase in muscle mass. There were no changes between groups for relative protein content (Fig. 1b). There were few changes in muscle fibre type distribution between groups, with the exception of the percentage of hybrid type fast IIB/IIX fibres, which exhibited a greater than 50% increase in frequency in the 10 mg/kg group compared with the 0 mg/kg and 3 mg/kg groups (Fig. 1c). There were also very few differences in muscle fibre CSA between groups (Fig. 1d), with a 53% increase in type IIX fast fibre CSA in the 10 mg/kg group compared with the 0 mg/kg group, and no differences were observed between the two treatment groups.

Fig.

Muscle mass, protein content and fibre type distribution and size: (a) muscle mass of supraspinatus muscles, (b) relative protein content of supraspinatus muscles, (c) muscle fibre type distribution and (d) muscle fibre type cross-sectional area (CSA) from 0 mg/kg, 3 mg/kg, and 10 mg/kg groups. Values are mean and standard error. Differences between groups were tested using a one-way analysis of variance (α = 0.05) followed by Fisher’s Least Significant Difference post hoc sorting. *, significantly different from 0 mg/kg (p < 0.05).

For fibre contractility, there were no significant differences between muscle fibre CSA (Fig. 2a). However, there was a 25% decrease in F_o for the 10 mg/kg group compared with controls (Fig. 2b), but no differences between the 3 mg/kg group and controls (Fig. 2b). Additionally, there was no differences in sF_o between the 0 mg/kg and 3 mg/kg groups (Fig. 2c), but the 10 mg/kg group exhibited a 15% and 10% decrease in sF_o compared with the 0 mg/kg and 3 mg/kg groups, respectively (Fig. 2c). With regards to muscle fibrosis, there was roughly a 50% reduction in hydroxyproline content in the treated groups compared with the 0 mg/kg group, but no dose-dependent decrease in fibrosis between treatment groups (Fig. 3a). Representative wheat germ agglutinin stained images, that identify the ECM, from each group are presented in Figure 3b.

Fig.

Permeabilised muscle fibre size and contractility: (a) permeabilised fibre cross-sectional area (CSA), (b) maximum isometric force (F_o) and (c) specific force (sF_o) of 0 mg/kg, 3 mg/kg, and 10 mg/kg groups. Values are mean and standard error. Differences between groups were tested using a one-way analysis of variance (α = 0.05) followed by Fisher’s Least Significant Difference post hoc sorting. *, significantly different from 0 mg/kg; #, significantly different from 3 mg/kg (p < 0.05).

Fig.

Hydroxyproline (Hyp) content and wheat germ agglutinin (WGA) stained muscle sections: (a) Hyp content from 0 mg/kg, 3 mg/kg, and 10 mg/kg groups. Values are mean and standard error. Differences between groups were tested using a one-way analysis of variance (α = 0.05) followed by Fisher’s Least Significant Difference post hoc sorting. *, significantly different from 0 mg/kg (p < 0.05). (b) Representative WGA stained images to identify extracellular matrix left to right from 0 mg/kg, 3 mg/kg, and 10 mg/kg groups (50μm).

There was a reduction in the expression of several genes involved in muscle fibrosis in the treated groups compared with controls (Fig. 4a), including collagen 1α2 (COL1α2), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-14 (MMP14), TIMP1, TIMP2, Tenomodulin, and fibroblast-specific protein-1 (FSP-1). For genes related to inflammation (Fig. 4b), there was a significant decrease in the pro-inflammatory interleukin IL-6 in the treated groups compared with controls. There was also an increase in the expression of the anti-inflammatory interleukin, IL-10, in the 10 mg/kg group compared with 0 mg/kg, but no differences from 3 mg/kg. There was a reduction in the pan-macrophage marker, CD68, in the 3 mg/kg group compared with controls, but no change in the 10 mg/kg group from other groups. Lastly, the anti-inflammatory macrophage marker, CD168, was elevated in the treated groups compared with controls.

Fig.

Gene expression in muscles from 0 mg/kg, 3 mg/kg, and 10 mg/kg groups. Transcripts related to (a) extracellular matrix accumulation and maintenance, (b) inflammation and macrophage accumulation, (c) lipid synthesis and storage, and (d) muscle atrophy and autophagy. Values are mean and standard error. Differences between groups were tested using a one-way analysis of variance (α = 0.05) followed by Fisher’s Least Significant Difference post hoc sorting. *, significantly different from 0 mg/kg; #, significantly different from 3 mg/kg (p < 0.05).

For genes related to lipid accumulation and storage (Fig. 4c), there was a reduction in the expression of several genes in the treated groups compared with controls, including peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, perilipin 1, and fat-specific protein 27 (FSP27). Additionally, there was an induction in the expression of several genes in the treated groups compared with controls, including glycerol-3-phosphate acyltransferase 4 (GPAT4), diacylglycerol O-acyltransferase 1 (DGAT1), adipose triglyceride lipase, and perilipin 5. PHD inhibitor treatment also had an effect on the expression of genes related to muscle atrophy and remodelling (Fig. 4d). There was an increase in the expression of several genes in the treated groups compared with controls, including the E3 ubiquitin ligases, atrogin-1 and MuRF-1, vascular endothelial growth factor, and the autophagy-related transcripts, beclin-1, Vps34, and Atg16L1. The autophagy-related transcript Atg5 was also significantly reduced in the 10 mg/kg compared with the 3 mg/kg group, although both treated groups were upregulated compared with the 0 mg/kg group.

Finally, we measured the biomechanical properties of repaired shoulders. There was a reduction in tendon CSA in the 3 mg/kg group compared with the 0 mg/kg group, but no other significant changes in CSA (Fig. 5a). There were no changes in absolute load to failure (Fig. 5b), but displacement to failure was elevated in the treated groups compared with controls (Fig. 5c). There was a significant increase in peak stiffness for the 3 mg/kg group compared with the 0 mg/kg group but no differences between the 10 mg/kg group and 0 mg/kg group, nor were there any dose-dependent changes in stiffness between treatment groups (Fig. 5d). This was also the case for maximum stress to failure, where the 3 mg/kg group was increased from 0 mg/kg, and no other differences were measured (Fig. 5e).

Fig.

Enthesis biomechanical failure testing values in 0 mg/kg, 3 mg/kg, and 10 mg/kg groups: (a) tendon cross-sectional area (CSA), (b) maximum load to failure, (c) maximum displacement to failure, (d) peak stiffness, and (e) maximum stress. Values are mean and standard error. Differences between groups were tested using a one-way analysis of variance (α = 0.05) followed by Fisher’s Least Significant Difference post hoc sorting. *, significantly different from 0 mg/kg (p < 0.05).

Discussion

Currently our ability to successfully treat chronic rotator cuff tears is limited by the poor quality of the muscle following repair. In particular, chronic inflammation and fibrosis play a large role in increasing the probability of failure of structurally successful repairs.^1,2 Due to the lack of viable treatment options to treat muscle and tendon quality following chronic rotator cuff tears,¹ and the potential for the activation of HIF-1α in augmenting muscle recovery,⁶ the use of PHD inhibitors holds attractive therapeutic potential to treat these injuries. In this study, the use of the PHD inhibitor, GSK1120360A, dramatically reduced fibrosis and inflammation within torn rotator cuff muscles, and increased the stiffness and stress to failure of the enthesis, with the 3 mg/kg dose overall demonstrating the most favourable changes.

Decreasing fibrosis in chronic tears of the rotator cuff has the potential to improve both functional recovery and patient quality of life, as fibrosis leads to muscle stiffness and a greater susceptibility to re-tear.^2,4 In this study, following systemic administration of GSK1120360A, there was a 50% decrease in muscle hydroxyproline content after rotator cuff repair. This reduction in hydroxyproline content corresponded with lower levels of expression of type I collagen, MMP2, MMP14, and the fibroblast proliferation markers, tenomodulin and FSP-1. PHD inhibition was also associated with an improvement in mechanical properties of the enthesis. For both doses of GSK1120360A, there was an improvement in displacement to yield, a clinically important parameter for predicting long-term successful repair. The 3 mg/kg group also had an increase in peak stiffness and maximum stress to yield, suggesting an improvement in the organisation and quality of the tendon ECM. The inhibition of PHD may therefore improve the treatment of chronic rotator cuff tears not only by improving muscle recovery by reducing fibrosis, but also by promoting the development of a strong, mature enthesis.

Inflammation and lipid accumulation are associated with rotator cuff tears and are thought to contribute to impaired regeneration and recovery.¹ Although the use of non-steroidal anti-inflammatory drugs or cox-2 inhibitor anti-inflammatory medications inhibits rotator cuff regeneration,¹⁶ we previously reported that the combined inhibition of the cyclooxygenase (COX)-1, COX-2 and 5-lipoxygenase pathways with licofelone decreased fibrosis, and increased load to failure of the enthesis.¹⁰ In the current study, inhibition of PHD reduced the expression of the pro-inflammatory cytokine IL-6 and increased the expression of the anti-inflammatory cytokine IL-10. Macrophages, which are important regulators of inflammation, also accumulate after rotator cuff tear,^12,13 and PHD inhibition increased the expression of the anti-inflammatory M2 macrophage marker CD168. Increased inflammation in many models of skeletal muscle disease can also be caused by excess availability of lipid. In the case of type 2 diabetes mellitus, the accumulation of fatty acids and lipid intermediates within muscle fibres leads to an increase in the expression of several pro-inflammatory genes that have both local and systemic effects,¹⁷ and increasing the expression of triglyceride synthesis and storage enzymes reduces this inflammation.¹⁸ In the current study, the triglyceride storage enzymes, GPAT4 and DGAT1, were elevated in the PHD inhibitor-treated group. Additionally, perilipin 1 and FSP27, which are markers of pro-inflammatory lipid droplets and are highly upregulated following rotator cuff tear,^12,13 were downregulated following PHD inhibition. PHD inhibition also resulted in an increase in perilipin 5 expression, which indicates lipid within the cell is likely being used for metabolic purposes rather than the synthesis of pro-inflammatory signalling molecules.¹⁹ Overall, these results indicate that PHD inhibition may be beneficial to long-term muscle recovery by reducing prolonged inflammation following chronic rotator cuff tears.

In addition to severe inflammation and fibrosis, chronic atrophy is a hallmark of rotator cuff tears. The two most common biochemical pathways that regulate muscle fibre atrophy are the ubiquitin-proteasome pathway and the autophagy pathway.²⁰ For the ubiquitin-proteasome pathway, the E3 ubiquitin ligases atrogin-1 and MuRF-1 direct the polyubiquitination and subsequent degradation of damaged proteins through the 26S proteasome.²⁰ Autophagy is a biochemical pathway in which organelles and proteins are targeted to the autolysosome for breakdown.²⁰ There are several important components of the autophagy pathway, but Atg16L1, Atg5, beclin-1, and Vps34 are some of the key components of this process, and are commonly used as markers of autophagy.^13,20 For both pathways, proteins are broken down into amino acids which are eventually returned to the cytosol to be used to synthesise new proteins.²⁰ Although these processes are linked to atrophy and are thought to be detrimental to muscle function, they serve as important contributors to muscle amino acid availability and protein turnover. We and others have previously demonstrated that chronic rotator cuff tears are associated with increased expression of transcripts related to autophagy, but not E3 ubiquitin ligases atrogin-1 and MuRF-1, which are key rate-limiting enzymes in skeletal muscle protein degradation.^13,21,22 In the current study, the E3 ubiquitin ligases atrogin-1 and MuRF-1, and the autophagy transcripts, Atg16L1, Atg5, beclin-1, and Vps34, were elevated in both groups that received the PHD inhibitor compared with control muscles. Despite the increase in expression of these transcripts, with the exception of a modest increase in type IIX muscle fibre CSA in the 10 mg/kg group, we did not observe changes in muscle fibre CSA or relative protein content with GSK1120360A treatment. HIF-1α can directly stimulate autophagy, which is thought to contribute to cell survival in hypoxia by reducing energy demands of the tissue.²³ The relationship between HIF-1α accumulation and autophagy also likely influenced muscle fibre force production, with the 3 mg/kg dose of GSK1120360A demonstrating the ability to increase autophagy without decreasing force production, while the 10 mg/kg dose led to excessive autophagy that decreased force production. Combined, these results suggest that inhibition of PHD activity at the 3 mg/kg dose was able to reduce fibrosis and inflammation without negatively affecting muscle function or quality.

There are several limitations in this study. We use a rat model of chronic rotator cuff tear and repair due to similar anatomical features to human shoulders, but the rat model does not develop fatty degeneration to the extent observed in humans.^13,24,25 A single time point of two weeks following repair was chosen to evaluate intermediate changes in regeneration, and did not assess the effect of PHD inhibition on muscle function and biochemical measures at different times following repair. We also did not evaluate an acute tear and repair model, which might be informative. Given that we did not use a genetic model of HIF-1α manipulation, we were unable to determine the effects of the inhibitor on tendon to bone healing versus direct muscle regeneration. For our single-fibre contractility measurements, we only measured the contractility of type II fibres. However, we demonstrate in this study and elsewhere¹³ that rat rotator cuff muscles contain primarily type II fibres, and there are no appreciable differences in sF_o between type II isoforms.²⁶ Despite these limitations, we believe that this study provided important insight into the role of PHD in the regeneration of chronically torn rotator cuff muscles following repair.

Fibrosis, lipid accumulation, and inflammation are debilitating to long-term functional recovery following rotator cuff tear. Current treatment strategies for patients with massive chronic tears do not reverse fatty degeneration, and persistent atrophy and fibrosis can increase the susceptibility of the repaired muscle to re-tear. Although PHD inhibition reduced fibrosis and inflammation, and improved mechanical properties of the bone-tendon interface, the treatment did not result in improvements in all of our functional measures and biomarkers. This study served as an important step in our understanding of the mechanisms behind muscle atrophy, inflammation, and fibrosis following rotator cuff tears. Although further studies are necessary, we identified HIF-1α as a potentially important pharmacological target for the treatment of chronic fibrotic and inflammatory muscle diseases, and it may be beneficial for acute connective tissue injuries in which muscle atrophy has not yet occurred.