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Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy.

Mehuron T, Kumar A, Duarte L, Yamauchi J, Accorsi A, Girgenrath M - Skelet Muscle (2014)

Bottom Line: We found that DyW mice show significantly impaired myogenesis and high levels of apoptosis as early as postnatal week 1.Interestingly, fibronectin was found to be the predominant fibrous protein of the extracellular matrix in early postnatal development.On the basis of our results, we believe that therapies that may reduce apoptosis and stabilize the homeostasis of extracellular matrix proteins may have increased efficacy if started at a very early age.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Health Sciences, Boston University, 635 Commonwealth Avenue, Boston, MA 02215, USA.

ABSTRACT

Background: MDC1A is a congenital neuromuscular disorder with developmentally complex and progressive pathologies that results from a deficiency in the protein laminin α2. MDC1A is associated with a multitude of pathologies, including increased apoptosis, inflammation and fibrosis. In order to assess and treat a complicated disease such as MDC1A, we must understand the natural history of the disease so that we can identify early disease drivers and pinpoint critical time periods for implementing potential therapies.

Results: We found that DyW mice show significantly impaired myogenesis and high levels of apoptosis as early as postnatal week 1. We also saw a surge of inflammatory response at the first week, marked by high levels of infiltrating macrophages, nuclear factor κB activation, osteopontin expression and overexpression of inflammatory cytokines. Fibrosis markers and related pathways were also observed to be elevated throughout early postnatal development in these mice, including periostin, collagen and fibronectin gene expression, as well as transforming growth factor β signaling. Interestingly, fibronectin was found to be the predominant fibrous protein of the extracellular matrix in early postnatal development. Lastly, we observed upregulation in various genes related to angiotensin signaling.

Methods: We sought out to examine the dysregulation of various pathways throughout early development (postnatal weeks 1-4) in the DyW mouse, the most commonly used mouse model of laminin-deficient muscular dystrophy. Muscle function tests (stand-ups and retractions) as well as gene (qRT-PCR) and protein levels (western blot, ELISA), histology (H&E, picrosirius red staining) and immunohistochemistry (fibronectin, TUNEL assay) were used to assess dysregulation of matricelluar protieins.

Conclusions: Our results implicate the involvement of multiple signaling pathways in driving the earliest stages of pathology in DyW mice. As opposed to classical dystrophies, such as Duchenne muscular dystrophy, the dysregulation of various matricellular proteins appears to be a distinct feature of the early progression of DyW pathology. On the basis of our results, we believe that therapies that may reduce apoptosis and stabilize the homeostasis of extracellular matrix proteins may have increased efficacy if started at a very early age.

No MeSH data available.


Related in: MedlinePlus

Gene expression analysis using quantitative RT-PCR, along with TUNEL immunostaining, show an increase in the myogenic program and cell death. (A) Terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick-end labeling (TUNEL) staining shows an increased number of apoptotic nuclei in Lama2DyW (DyW) muscles at each of the four time points. Desmin costaining was used to delineate differentiated myogenic cells. Arrows point to those cells that are both desmin-positive (red) and TUNEL-positive (green). (DyW is also referred to as KO, Knockout.) (B) Quantitative analysis of the TUNEL staining shows the highest incidence of apoptosis at postnatal week 1, which decreased by 50% at week 3. (C) Gene analysis of MyoD shows the highest expression for both groups early in life, followed by decreases over time. DyW mice had significantly increased expression compared to age-matched wild-type (WT) mice at all time points (1 week: P < 0.05, 2 weeks: P < 0.001, 3 weeks: P < 0.0001 and 4 weeks: P < 0.001, all by two-way analysis of variance (ANOVA) (n = 5 for all groups). (D) Myogenin expression shows a similar trend of having the highest expression early on, and DyW levels were also increased compared to all age-matched WT groups (1 week: P < 0.01, 2 weeks: P < 0.0001, 3 weeks: P < 0.005 and 4 weeks: P < 0.0001, all by two-way ANOVA (n = 5 for all groups). (E) Embryonic myosin heavy chain (eMYHC) expression was significantly elevated in the DyW mice at each of the four time points (1 week: P < 0.0005, 2 weeks: P < 0.0001, 3 weeks: P < 0.005 and 4 weeks: P < 0.0001, all by two-way ANOVA (n = 5). Asterisks indicate statistical significance between age-matched DyW and WT animals, hash tags indicate significant changes within the DyW group and Deltas indicate significant changes within the WT group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. These statistical representations also apply other symbols (Δ, #). Bar = 100 μm.
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Figure 3: Gene expression analysis using quantitative RT-PCR, along with TUNEL immunostaining, show an increase in the myogenic program and cell death. (A) Terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick-end labeling (TUNEL) staining shows an increased number of apoptotic nuclei in Lama2DyW (DyW) muscles at each of the four time points. Desmin costaining was used to delineate differentiated myogenic cells. Arrows point to those cells that are both desmin-positive (red) and TUNEL-positive (green). (DyW is also referred to as KO, Knockout.) (B) Quantitative analysis of the TUNEL staining shows the highest incidence of apoptosis at postnatal week 1, which decreased by 50% at week 3. (C) Gene analysis of MyoD shows the highest expression for both groups early in life, followed by decreases over time. DyW mice had significantly increased expression compared to age-matched wild-type (WT) mice at all time points (1 week: P < 0.05, 2 weeks: P < 0.001, 3 weeks: P < 0.0001 and 4 weeks: P < 0.001, all by two-way analysis of variance (ANOVA) (n = 5 for all groups). (D) Myogenin expression shows a similar trend of having the highest expression early on, and DyW levels were also increased compared to all age-matched WT groups (1 week: P < 0.01, 2 weeks: P < 0.0001, 3 weeks: P < 0.005 and 4 weeks: P < 0.0001, all by two-way ANOVA (n = 5 for all groups). (E) Embryonic myosin heavy chain (eMYHC) expression was significantly elevated in the DyW mice at each of the four time points (1 week: P < 0.0005, 2 weeks: P < 0.0001, 3 weeks: P < 0.005 and 4 weeks: P < 0.0001, all by two-way ANOVA (n = 5). Asterisks indicate statistical significance between age-matched DyW and WT animals, hash tags indicate significant changes within the DyW group and Deltas indicate significant changes within the WT group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. These statistical representations also apply other symbols (Δ, #). Bar = 100 μm.

Mentions: TUNEL staining of TA muscle sections showed that there was an elevated number of apoptotic nuclei in DyW animals throughout the early development time points. Representative images of 2- and 4-week-old animals are shown in Figure 3A. We demonstrate that both mature muscle fibers and cells within the interstitium undergo apoptosis in these tissues. Importantly, we observed that at earlier time points, a subset of those TUNEL-positive nuclei in the interstitial space are also desmin-positive (one of the earliest markers for differentiated myogenic cells). This is in contrast to older tissues, where TUNEL-positive nuclei were mostly limited to the mature myofibers and desmin-negative interstitial cells. Quantitation of TUNEL-positive nuclei shows that apoptosis was significantly increased in the DyW mice compared to the WT mice at all time points (P < 0.001 by two-way ANOVA (n = 3)) and also that it was most abundant in DyW mice at weeks 1 and 2 and became less prevalent by weeks 3 and 4 (P < 0.0001 by two-way ANOVA (n = 3)) (Figure 3B). We observed few TUNEL-positive nuclei in the WT mice in the first 2 weeks and virtually none at subsequent time points.


Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy.

Mehuron T, Kumar A, Duarte L, Yamauchi J, Accorsi A, Girgenrath M - Skelet Muscle (2014)

Gene expression analysis using quantitative RT-PCR, along with TUNEL immunostaining, show an increase in the myogenic program and cell death. (A) Terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick-end labeling (TUNEL) staining shows an increased number of apoptotic nuclei in Lama2DyW (DyW) muscles at each of the four time points. Desmin costaining was used to delineate differentiated myogenic cells. Arrows point to those cells that are both desmin-positive (red) and TUNEL-positive (green). (DyW is also referred to as KO, Knockout.) (B) Quantitative analysis of the TUNEL staining shows the highest incidence of apoptosis at postnatal week 1, which decreased by 50% at week 3. (C) Gene analysis of MyoD shows the highest expression for both groups early in life, followed by decreases over time. DyW mice had significantly increased expression compared to age-matched wild-type (WT) mice at all time points (1 week: P < 0.05, 2 weeks: P < 0.001, 3 weeks: P < 0.0001 and 4 weeks: P < 0.001, all by two-way analysis of variance (ANOVA) (n = 5 for all groups). (D) Myogenin expression shows a similar trend of having the highest expression early on, and DyW levels were also increased compared to all age-matched WT groups (1 week: P < 0.01, 2 weeks: P < 0.0001, 3 weeks: P < 0.005 and 4 weeks: P < 0.0001, all by two-way ANOVA (n = 5 for all groups). (E) Embryonic myosin heavy chain (eMYHC) expression was significantly elevated in the DyW mice at each of the four time points (1 week: P < 0.0005, 2 weeks: P < 0.0001, 3 weeks: P < 0.005 and 4 weeks: P < 0.0001, all by two-way ANOVA (n = 5). Asterisks indicate statistical significance between age-matched DyW and WT animals, hash tags indicate significant changes within the DyW group and Deltas indicate significant changes within the WT group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. These statistical representations also apply other symbols (Δ, #). Bar = 100 μm.
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Figure 3: Gene expression analysis using quantitative RT-PCR, along with TUNEL immunostaining, show an increase in the myogenic program and cell death. (A) Terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick-end labeling (TUNEL) staining shows an increased number of apoptotic nuclei in Lama2DyW (DyW) muscles at each of the four time points. Desmin costaining was used to delineate differentiated myogenic cells. Arrows point to those cells that are both desmin-positive (red) and TUNEL-positive (green). (DyW is also referred to as KO, Knockout.) (B) Quantitative analysis of the TUNEL staining shows the highest incidence of apoptosis at postnatal week 1, which decreased by 50% at week 3. (C) Gene analysis of MyoD shows the highest expression for both groups early in life, followed by decreases over time. DyW mice had significantly increased expression compared to age-matched wild-type (WT) mice at all time points (1 week: P < 0.05, 2 weeks: P < 0.001, 3 weeks: P < 0.0001 and 4 weeks: P < 0.001, all by two-way analysis of variance (ANOVA) (n = 5 for all groups). (D) Myogenin expression shows a similar trend of having the highest expression early on, and DyW levels were also increased compared to all age-matched WT groups (1 week: P < 0.01, 2 weeks: P < 0.0001, 3 weeks: P < 0.005 and 4 weeks: P < 0.0001, all by two-way ANOVA (n = 5 for all groups). (E) Embryonic myosin heavy chain (eMYHC) expression was significantly elevated in the DyW mice at each of the four time points (1 week: P < 0.0005, 2 weeks: P < 0.0001, 3 weeks: P < 0.005 and 4 weeks: P < 0.0001, all by two-way ANOVA (n = 5). Asterisks indicate statistical significance between age-matched DyW and WT animals, hash tags indicate significant changes within the DyW group and Deltas indicate significant changes within the WT group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. These statistical representations also apply other symbols (Δ, #). Bar = 100 μm.
Mentions: TUNEL staining of TA muscle sections showed that there was an elevated number of apoptotic nuclei in DyW animals throughout the early development time points. Representative images of 2- and 4-week-old animals are shown in Figure 3A. We demonstrate that both mature muscle fibers and cells within the interstitium undergo apoptosis in these tissues. Importantly, we observed that at earlier time points, a subset of those TUNEL-positive nuclei in the interstitial space are also desmin-positive (one of the earliest markers for differentiated myogenic cells). This is in contrast to older tissues, where TUNEL-positive nuclei were mostly limited to the mature myofibers and desmin-negative interstitial cells. Quantitation of TUNEL-positive nuclei shows that apoptosis was significantly increased in the DyW mice compared to the WT mice at all time points (P < 0.001 by two-way ANOVA (n = 3)) and also that it was most abundant in DyW mice at weeks 1 and 2 and became less prevalent by weeks 3 and 4 (P < 0.0001 by two-way ANOVA (n = 3)) (Figure 3B). We observed few TUNEL-positive nuclei in the WT mice in the first 2 weeks and virtually none at subsequent time points.

Bottom Line: We found that DyW mice show significantly impaired myogenesis and high levels of apoptosis as early as postnatal week 1.Interestingly, fibronectin was found to be the predominant fibrous protein of the extracellular matrix in early postnatal development.On the basis of our results, we believe that therapies that may reduce apoptosis and stabilize the homeostasis of extracellular matrix proteins may have increased efficacy if started at a very early age.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Health Sciences, Boston University, 635 Commonwealth Avenue, Boston, MA 02215, USA.

ABSTRACT

Background: MDC1A is a congenital neuromuscular disorder with developmentally complex and progressive pathologies that results from a deficiency in the protein laminin α2. MDC1A is associated with a multitude of pathologies, including increased apoptosis, inflammation and fibrosis. In order to assess and treat a complicated disease such as MDC1A, we must understand the natural history of the disease so that we can identify early disease drivers and pinpoint critical time periods for implementing potential therapies.

Results: We found that DyW mice show significantly impaired myogenesis and high levels of apoptosis as early as postnatal week 1. We also saw a surge of inflammatory response at the first week, marked by high levels of infiltrating macrophages, nuclear factor κB activation, osteopontin expression and overexpression of inflammatory cytokines. Fibrosis markers and related pathways were also observed to be elevated throughout early postnatal development in these mice, including periostin, collagen and fibronectin gene expression, as well as transforming growth factor β signaling. Interestingly, fibronectin was found to be the predominant fibrous protein of the extracellular matrix in early postnatal development. Lastly, we observed upregulation in various genes related to angiotensin signaling.

Methods: We sought out to examine the dysregulation of various pathways throughout early development (postnatal weeks 1-4) in the DyW mouse, the most commonly used mouse model of laminin-deficient muscular dystrophy. Muscle function tests (stand-ups and retractions) as well as gene (qRT-PCR) and protein levels (western blot, ELISA), histology (H&E, picrosirius red staining) and immunohistochemistry (fibronectin, TUNEL assay) were used to assess dysregulation of matricelluar protieins.

Conclusions: Our results implicate the involvement of multiple signaling pathways in driving the earliest stages of pathology in DyW mice. As opposed to classical dystrophies, such as Duchenne muscular dystrophy, the dysregulation of various matricellular proteins appears to be a distinct feature of the early progression of DyW pathology. On the basis of our results, we believe that therapies that may reduce apoptosis and stabilize the homeostasis of extracellular matrix proteins may have increased efficacy if started at a very early age.

No MeSH data available.


Related in: MedlinePlus