New insights about myofibrillar myopathies: the role of metalloproteinases 2 and 9 in the pathogenesis

Background There are few reports suggesting that gene expression and activation of various matrix metalloproteinases (MMPs) are deregulated. MMP-2 and MMP-9 represent the two MMPs, which degrade type IV collagen, the component of basement membrane. Methods We analyzed the involvement of gelatinases, MMP-2 and MMP-9, in the pathogenesis of myofibrillar myopathy (MFM). Muscle specimens from 23 patients well diagnosed with MFM, were immunostained by MMP-2 and MMP-9. We analyzed qualitatively the immunoexpression in three compartments: subsarcolemmal (SSC), intracytoplasmic (ICC) and perinuclear (PNC). Results 95,7% and 100% samples showed MMP-2 and MMP-9 upregulation ICC, respectively. PNC showed MMP-2 (82,6%) and MMP-9 (8,7%) regulation (p<0.001). SSC and ICC did not present statistical significance. There was no correlation between mutated gene and immunohistochemical pattern distribution. Our results suggest that MMP-2 and/or MMP-9 could participate in the pathomechanism of MFM, causing damage of sarcomere and deposition of protein aggregates.


Conclusion
Our results suggest that MMP-2 and/or MMP-9 could participate in the pathomechanism of MFM, causing damage of sarcomere and deposition of protein aggregates.

Background
Since the discovery of metalloproteinases and its functions in 1962, they have been identified as extracellular matrix (ECM) proteinases responsible for transforming the matrix and degrading its components [1]. However, the discovery of an intracellular function of MMP-2 challenged this idea corroborating that MMP-2 not only repairs the ECM but also acts intracellularly in ischemia-reperfusion in myocardium by degrading troponin I, contributing therefore, to cardiac dysfunction [1,2,3,4].
Regarding the role of MMPs in some myopathies, there are few reports suggesting that stimulation of various MMPs and also gene expression is deregulated. Whilst MMP-2 and MMP-9 are likely involved in the pathology of dystrophin-deficient skeletal muscle [5,6], MMP-9 is implicated in the inflammatory 3 myopathies during the process of elimination of necrotic fibers and muscle degeneration and MMP-2 may be associated with the process of reconstruction of ECM during muscle regeneration and fiber growth [7,8].
Myofibrillar myopathies (MFM), a heterogeneous group of hereditary or sporadic skeletal and cardiac muscle disorders of sarcomere, are still a challenge in terms of our understanding about the pathogenesis [9]. Many different components are involved in the cascade of events occurring in muscle fibers in these disorders, as mutant proteins, a defective ubiquitin -proteasome system and oxidative stress [10]. Nevertheless, there are no previous studies correlating the activation of MMPs and MFM.
In order to determine other components engaged in this pathogenic process we decided to examine the involvement of the gelatinases MMP-2 and MMP-9 in in muscle fibers in MFM.

Methods
Muscle biopsies from 23 well-characterized patients with a previous diagnosis of MFM were studied.
The diagnosis was obtained by clinical findings, morphological and ultrastructural aspects of muscle biopsies, and genetic studies. Control muscles specimens from 4 patients showing minimal and no specific findings in muscle biopsy were concurrently examined. We collected demographic data (gender, age of onset and age of diagnosis), gene mutated and specific mutations in order to correlate them with immunocytochemistry results.
Single immunocytochemistry: Five-micrometer serial sections from muscle specimens, stored in a freezer at -80°, were incubated in acetone at 4°C for 10 minutes, washed in 0.05 mol/L Tris-buffered saline (pH 7.5) for 15 minutes, and fixed for 30 minutes with a blocking reagent containing 2% bovine serum albumin and 5% normal goat serum or 5% horse serum. Sections were then incubated overnight at 4°C with one of the following antibodies: goat polyclonal IgG against human MMP-9 (dilution 1:50; Santa Cruz, Santa Cruz, CA, USA) or rabbit polyclonal IgG against human MMP-2 (dilution 1:400; Chemicon, Temecula, CA, USA). These affinity-purified antibodies are well characterized and do not cross-react with each other. After being washed for 30 minutes in Trisbuffered saline, the sections were incubated with biotinylated goat anti-rabbit IgG for MMP-2 or biotinylated horse anti-goat IgG for MMP-9, followed by fluorescent isothiocyanate avidin D.
We analyzed qualitatively the MMP-2 and MMP-9 muscle immunoexpression from all patients, including controls by optic microscopy fluorescence in three different compartmentssubsarcolemmal (SS), intracytoplasmic (IC), and perinuclear (PN) -classifying them as expressed (+) or no expressed (-). In order to correlate the histopathological and immunocytochemical findings, fivemicrometer serial sections were also stained with hematoxilyn and eosin simultaneously.

Statistical analysis
To compare the immunostaining pattern and its correlation with demographic and genetic data, we performed the McNemar non-parametric test. For the tests, we used the SPSS 17.0 softwares for Windows. The significance level used for the tests was 5% [11]. Qualitative variables were presented using absolute frequency and relative frequency. In order to compare MMP-2 and MPP-9 regulation among the genetic groups, the Chi-square test was used. The level of significance adopted in this analysis was p <0.05. The statistical program used was Stata version 11.0.

Results
Demographic features and molecular diagnosis from these patients are shown in Table 1 The MMP-2 and MMP-9 muscle immunoexpression pattern distributions are presented in Table 1 and In terms of MMP-2 and MMP-9 expression, PN compartment showed a significant difference between MMP-2 expressed (82.6%) and MMP-9 expressed (8.7%) (p<0.001). In contrast, SS and IC compartments did not present statistical significance between MMP-2 and 9 expression (Graphic 1, Table 1) however, the protein aggregates showing expression (+) varied from one patient to another.
In addition, the same protein aggregate was expressed by MMP-9 but not by MMP-2, and vice-versa in the same section (Figure 2b). Also, the immunodistribution of MMPs were similar in most patients in relation to different genetic groups (FLNC, DES, MYOT, ZASP, CRYAB and NMF) (p>0.05).

Discussion
The pathogenesis and etiology of myofibrillar myopathy is not yet completed elucidated. Destruction of the myofibrillar network starting at the z-disk is supposed to be the first stage in the possible mechanism participating in the sarcomere's damage process.
Our study was a descriptive analysis about the behavior of MMP-2 and 9 in different compartments of the skeletal muscle fiber (Figure 1). Particularly, MMP-2 presented significant expression (+) in the PN compartment (p <0.001) in relation to MMP-9. By contrast, MMP-9 expression (+) was predominantly in the SS compartment demonstrating a possible influence of this gelatinase in ECM. In relation to IC compartment, our results were intriguing: both MMPs were expressed (+) in different regions of sarcoplasm. The same protein aggregate was expressed (+) by MMP-9 but not by MMP-2, and viceversa in the same section (Figure 2b). MMP-9 is normally expressed during early embryonic developmental while in normal adult tissues, it is not present suggesting that MMP-9 are increased upon tissue injury in order to repair damage, release growth factors and modulation of ECM. By contrast, the continuous expression of MMP-9 will contribute to inflammation and tissue destruction in different diseases [5]. Choi and Dalakas demonstrated that upregulation of MMP-2 and 9 in the muscle membrane of patients with s-IBM and PM may cause an active role in disarrangement of the integrity of the muscle fibers by the autoinvasive cytotoxic T cells [7]. In addition, increased proinflammatory cytokines in mdx mice, 6 appear to be some of the potential stimuli for up-regulation in MMP-9 levels in skeletal and cardiac muscle in muscular dystrophy [12].
In relation to MMP-2, it is constitutively expressed by many cell types including skeletal muscle and has a minimal regulation at the transcriptional level therefore, the regulation process takes place at the post-transcriptional level. Ogura According to previous reports, MMP-2 is demonstrated in several subcellular compartments, including the thin and thick myofilaments, close to the Z disks of the cardiac sarcomere, the cytoskeleton, nuclei, mitochondria, and caveolae observed in cardiac sarcomere after injury induced by ischemia [4,13].
In view of the aforementioned aspects of MMPs there would be another component of cell signaling that could be part of the protein aggregate deposit and MMPs expressed would be just an epiphenomenon? Or why not, those MMPS expressed (PN, IC and SS) could not be protection factor to muscle fiber?
We believe that both MMPs may participate actively in the pathological cascade leading to muscle membrane injury. MMP-9 comes from extracellular space into intracellular space and MMP-2 comes from intracellular space into extracellular space (Figures 1, 2a). Several intracellular processes are related to MMP-2 as cleaving specific proteins inside of myocytes thereafter reducing contractile function [4].
The mutations of known genes responsible for MFM will lead to an absence or dysfunction of coded proteins, causing mechanical instability of the sarcomere and, consequently, injury to the subcellular fiber compartments (ECM, sarcolemma, sarcoplasm, nucleus, and endoplasmic reticulum). The mechanical stress due to this instability would lead to MMP-2 upregulation with consequent destruction of the sarcomere, causing accumulation of proteins in the sarcoplasm (aggregates).

Conclusion
This study was the first one to analyze the role of MMPs in the pathogenesis of MFM. Our results showed that MMP-2 and 9 are expressed in different compartments of skeletal muscle while in control muscles they were not demonstrated. Therefore, they could participate in the pathomechanism of 7 MFM, causing damage of sarcomere and deposition of protein aggregates.
More studies are necessary to clarify this new concept. Our study provides only preliminary qualitatively data about the role of MMPs in order to better understand the pathogenesis of this set of

Availability of data and material
The datasets used and / or analyzed during the current study will be available from Professor Alves de Siqueira Carvalho on a reasonable request

Competing interests
None of the authors has any conflict of interest to disclose

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