Microbiopsy Sampling for Examining Age-Related Differences in Skeletal Muscle Fiber Morphology and Composition.

Introduction: The increasingly popular microbiopsy is an appealing alternative to the more invasive Bergström biopsy given the challenges associated with harvesting skeletal muscle in older populations. Parameters of muscle fiber morphology and composition derived from the microbiopsy have not been compared between young and older adults. Purpose: The purpose of this study was to examine muscle fiber morphology and composition in young (YM) and older (OM) males using the microbiopsy sampling technique. A secondary aim was to determine if specific strength is associated with serum levels of C-terminal agrin fragment [CAF; an indicator of neuromuscular junction (NMJ) degradation]. Methods: Thirty healthy, YM (n = 15, age = 20.7 ± 2.2 years) and OM (n = 15, age = 71.6 ± 3.9 years) underwent ultrasound imaging to determine whole-muscle cross-sectional area (CSA) of the vastus lateralis and rectus femoris as well as isometric and isokinetic (60°â‹…s-1 and 180°â‹…s-1) peak torque testing of the knee extensors. Microbiopsy samples of the vastus lateralis were collected from 13 YM and 11 OM, and immunofluorescence was used to calculate CSA and proportion of type I and type II fibers. Results: Peak torque was lower in OM at all velocities (p ≤ 0.001; d = 1.39-1.86) but only lower at 180°â‹…s-1 (p = 0.003; d = 1.23) when normalized to whole-muscle CSA. Whole-muscle CSA was smaller in OM (p = 0.001; d = 1.34), but atrophy was not present at the single fiber level (p > 0.05). Per individual, ∼900 fibers were analyzed, and type I fiber CSA was larger (p = 0.05; d = 0.94) in OM which resulted in a smaller type II/I fiber CSA ratio (p = 0.015; d = 0.95). CAF levels were not sensitive to age (p = 0.159; d = 0.53) nor associated with specific strength or whole-muscle CSA in OM. Conclusion: The microbiopsy appears to be a viable alternative to the Bergström biopsy for histological analyses of skeletal muscle in older adults. NMJ integrity was not influential for age-related differences in specific strength in our healthy, non-sarcopenic older sample.

Regulation of fiber-specific actin expression by the Drosophila SRF ortholog Blistered.

Serum response factor (SRF) has an established role in controlling actin homeostasis in mammalian cells, yet its role in non-vertebrate muscle development has remained enigmatic. Here, we demonstrate that the single Drosophila SRF ortholog, termed Blistered (Bs), is expressed in all adult muscles, but Bs is required for muscle organization only in the adult indirect flight muscles. Bs is a direct activator of the flight muscle actin gene Act88F, via a conserved promoter-proximal binding site. However, Bs only activates Act88F expression in the context of the flight muscle regulatory program provided by the Pbx and Meis orthologs Extradenticle and Homothorax, and appears to function in a similar manner to mammalian SRF in muscle maturation. These studies place Bs in a regulatory framework where it functions to sustain the flight muscle phenotype in Drosophila Our studies uncover an evolutionarily ancient role for SRF in regulating muscle actin expression, and provide a model for how SRF might function to sustain muscle fate downstream of pioneer factors.

Myogenesis in Drosophila melanogaster: Dissection of Distinct Muscle Types for Molecular Analysis.

Drosophila is a useful model organism for studying the molecular signatures that define specific muscle types during myogenesis. It possesses significant genetic conservation with humans for muscle disease causing genes and a lack of redundancy that simplifies functional analysis. Traditional molecular methods can be utilized to understand muscle developmental processes such as Western blots, in situ hybridizations, RT-PCR and RNAseq, to name a few. However, one challenge for these molecular methods is the ability to dissect different muscle types. In this protocol we describe some useful techniques for extracting muscles from the pupal and adult stages of development using flight and jump muscles as an example.

Functional redundancy and nonredundancy between two Troponin C isoforms in Drosophila adult muscles.

We investigated the functional overlap of two muscle Troponin C (TpnC) genes that are expressed in the adult fruit fly, Drosophila melanogaster: TpnC4 is predominantly expressed in the indirect flight muscles (IFMs), whereas TpnC41C is the main isoform in the tergal depressor of the trochanter muscle (TDT; jump muscle). Using CRISPR/Cas9, we created a transgenic line with a homozygous deletion of TpnC41C and compared its phenotype to a line lacking functional TpnC4 We found that the removal of either of these genes leads to expression of the other isoform in both muscle types. The switching between isoforms occurs at the transcriptional level and involves minimal enhancers located upstream of the transcription start points of each gene. Functionally, the two TpnC isoforms were not equal. Although ectopic TpnC4 in TDT muscles was able to maintain jumping ability, TpnC41C in IFMs could not effectively support flying. Simultaneous functional disruption of both TpnC genes resulted in jump-defective and flightless phenotypes of the survivors, as well as abnormal sarcomere organization. These results indicated that TpnC is required for myofibril assembly, and that there is functional specialization among TpnC isoforms in Drosophila.

Arrest is a regulator of fiber-specific alternative splicing in the indirect flight muscles of Drosophila.

Drosophila melanogaster flight muscles are distinct from other skeletal muscles, such as jump muscles, and express several uniquely spliced muscle-associated transcripts. We sought to identify factors mediating splicing differences between the flight and jump muscle fiber types. We found that the ribonucleic acid-binding protein Arrest (Aret) is expressed in flight muscles: in founder cells, Aret accumulates in a novel intranuclear compartment that we termed the Bruno body, and after the onset of muscle differentiation, Aret disperses in the nucleus. Down-regulation of the aret gene led to ultrastructural changes and functional impairment of flight muscles, and transcripts of structural genes expressed in the flight muscles became spliced in a manner characteristic of jump muscles. Aret also potently promoted flight muscle splicing patterns when ectopically expressed in jump muscles or tissue culture cells. Genetically, aret is located downstream of exd (extradenticle), hth (homothorax), and salm (spalt major), transcription factors that control fiber identity. Our observations provide insight into a transcriptional and splicing regulatory network for muscle fiber specification.

The Drosophila Z-disc protein Z(210) is an adult muscle isoform of Zasp52, which is required for normal myofibril organization in indirect flight muscles.

The Z-disc is a critical anchoring point for thin filaments as they slide during muscle contraction. Therefore, identifying components of the Z-disc is critical for fully comprehending how myofibrils assemble and function. In the adult Drosophila musculature, the fibrillar indirect flight muscles accumulate a >200 kDa Z-disc protein termed Z(210), the identity of which has to date been unknown. Here, we use mass spectrometry and gene specific knockdown studies, to identify Z(210) as an adult isoform of the Z-disc protein Zasp52. The Zasp52 primary transcript is extensively alternatively spliced, and we describe its splicing pattern in the flight muscles, identifying a new Zasp52 isoform, which is the one recognized by the Z(210) antibody. We also demonstrate that Zasp52 is required for the association of α-actinin with the flight muscle Z-disc, and for normal sarcomere structure. These studies expand our knowledge of Zasp isoforms and their functions in muscle. Given the role of Zasp proteins in mammalian muscle development and disease, our results have relevance to mammalian muscle biology.

Extradenticle and homothorax control adult muscle fiber identity in Drosophila.

Here we identify a key role for the homeodomain proteins Extradenticle (Exd) and Homothorax (Hth) in the specification of muscle fiber fate in Drosophila. exd and hth are expressed in the fibrillar indirect flight muscles but not in tubular jump muscles, and manipulating exd or hth expression converts one muscle type into the other. In the flight muscles, exd and hth are genetically upstream of another muscle identity gene, salm, and are direct transcriptional regulators of the signature flight muscle structural gene, Actin88F. Exd and Hth also impact muscle identity in other somatic muscles of the body by cooperating with Hox factors. Because mammalian orthologs of exd and hth also contribute to muscle gene regulation, our studies suggest that an evolutionarily conserved genetic pathway determines muscle fiber differentiation.

Differential requirements for Myocyte Enhancer Factor-2 during adult myogenesis in Drosophila.

Identifying the genetic program that leads to formation of functionally and morphologically distinct muscle fibers is one of the major challenges in developmental biology. In Drosophila, the Myocyte Enhancer Factor-2 (MEF2) transcription factor is important for all types of embryonic muscle differentiation. In this study we investigated the role of MEF2 at different stages of adult skeletal muscle formation, where a diverse group of specialized muscles arises. Through stage- and tissue-specific expression of Mef2 RNAi constructs, we demonstrate that MEF2 is critical at the early stages of adult myoblast fusion: mutant myoblasts are attracted normally to their founder cell targets, but are unable to fuse to form myotubes. Interestingly, ablation of Mef2 expression at later stages of development showed MEF2 to be more dispensable for structural gene expression: after myoblast fusion, Mef2 knockdown did not interrupt expression of major structural gene transcripts, and myofibrils were formed. However, the MEF2-depleted fibers showed impaired integrity and a lack of fibrillar organization. When Mef2 RNAi was induced in muscles following eclosion, we found no adverse effects of attenuating Mef2 function. We conclude that in the context of adult myogenesis, MEF2 remains an essential factor, participating in control of myoblast fusion, and myofibrillogenesis in developing myotubes. However, MEF2 does not show a major requirement in the maintenance of muscle structural gene expression. Our findings point to the importance of a diversity of regulatory factors that are required for the formation and function of the distinct muscle fibers found in animals.

Analysis of skeletal muscle development in Drosophila.

The Drosophila system has been invaluable in providing important insights into mesoderm specification, muscle specification, myoblast fusion, muscle differentiation, and myofibril assembly. Here, we present a series of Drosophila protocols that enable the researcher to visualize muscle precursors and differentiated muscles, at all stages of development. In doing so, we also highlight the variety of techniques that are used to create these findings. These protocols are directly used for the Drosophila system, and are provided with explanatory detail to enable the researcher to apply them to other systems.

Purification of cardiac cells from Drosophila embryos.

Recent genome-level innovations have enabled the identification of the entire cadre of genes that are expressed in specific tissues at particular developmental times. However, to be informative as to how individual cell types develop, this process relies upon the successful and efficient purification of cells for a particular tissue. Here, we describe a method to isolate cardiac cells from Drosophila embryos. We generated transgenic embryos in which a cardiac-specific enhancer of the Sulphonylurea receptor (Sur) gene drove expression of the green fluorescent protein (GFP) gene. Homogenized embryos were subjected to fluorescence activated cell sorting (FACS), resulting in approximately 50,000 cardiac cells purified. The prevalence of cardiac cells in the purified population was high, based upon a significant enrichment for cardiac-specific marker genes, including Sur and Toll. This enrichment also enabled the identification of cardiac-specific alternatively spliced isoforms of the Zasp66 gene. In the future, this approach can be used to describe the cardiac transcriptome of Drosophila at distinct stages of embryonic development.

Hsp27 is persistently expressed in zebrafish skeletal and cardiac muscle tissues but dispensable for their morphogenesis.

Constitutive expression of Hsp27 has been demonstrated in vertebrate embryos, especially in developing skeletal and cardiac muscle. Results of several previous studies have indicated that Hsp27 could play a role in the development of these tissues. For example, inhibition of Hsp27 expression has been reported to cause defective development of mammalian myoblasts in vitro and frog embryos in vivo. In contrast, transgenic mice lacking Hsp27 develop normally. Here, we examined the distribution of Hsp27 protein in developing and adult zebrafish and effects of suppressing Hsp27 expression using phosphorodiamidate morpholino oligonucleotides (PMO) on zebrafish development. Consistent with our previous analysis of hsp27 messenger RNA expression, we detected the protein Hsp27 in cardiac, smooth, and skeletal muscle of both embryonic and adult zebrafish. However, embryos lacking detectable Hsp27 after injection of antisense hsp27 PMO exhibited comparable heart beat rates to that of control embryos and cardiac morphology was indistinguishable in the presence or absence of Hsp27. Loss of Hsp27 also had no effect on the structure of the skeletal muscle myotomes in the developing embryo. Finally, embryos injected with antisense hsp27 and scrambled control PMO displayed equal motility. We conclude that Hsp27 is dispensable for zebrafish morphogenesis but could play a role in long-term maintenance of heart and muscle tissues.

Cardiac gene regulatory networks in Drosophila.

The Drosophila system has proven a powerful tool to help unlock the regulatory processes that occur during specification and differentiation of the embryonic heart. In this review, we focus upon a temporal analysis of the molecular events that result in heart formation in Drosophila, with a particular emphasis upon how genomic and other cutting-edge approaches are being brought to bear upon the subject. We anticipate that systems-level approaches will contribute greatly to our comprehension of heart development and disease in the animal kingdom.

Cardiac expression of the Drosophila Transglutaminase (CG7356) gene is directly controlled by myocyte enhancer factor-2.

The myocyte enhancer factor-2 (MEF2) family of transcription factors plays key roles in the activation of muscle structural genes. In Drosophila, MEF2 accumulates at high levels in the embryonic muscles, where it activates target genes throughout the mesoderm. Here, we identify the Transglutaminase gene (Tg; CG7356) as a direct transcriptional target of MEF2 in the cardiac musculature. Tg is expressed in cells forming the inflow tracts of the dorsal vessel, and we identify the enhancer responsible for this expression. The enhancer contains three binding sites for MEF2, and can be activated by MEF2 in tissue culture and in vivo. Moreover, loss of MEF2 function, or removal of the MEF2 binding sites from the enhancer, results in loss of Tg expression. These studies identify a new MEF2 target in the cardiac musculature. These studies provide a possible mechanism for the activation of transglutaminase genes.

Myocyte enhancer factor 2 and chorion factor 2 collaborate in activation of the myogenic program in Drosophila.

The process of myogenesis requires the coordinated activation of many structural genes whose products are required for myofibril assembly, function, and regulation. Although numerous reports have documented the importance of the myogenic regulator myocyte enhancer factor 2 (MEF2) in muscle differentiation, the interaction of MEF2 with cofactors is critical to the realization of muscle fate. We identify here a genomic region required for full MEF2-mediated activation of actin gene expression in Drosophila, and we identify the zinc finger transcriptional regulator chorion factor 2 (CF2) as a factor functioning alongside MEF2 via this region. Furthermore, although both MEF2 and CF2 can individually activate actin gene expression, we demonstrate that these two factors collaborate in regulating the Actin57B target gene in vitro and in vivo. More globally, MEF2 and CF2 synergistically activate the enhancers of a number of muscle-specific genes, and loss of CF2 function in vivo results in reductions in the levels of several muscle structural gene transcripts. These findings validate a general importance of CF2 alongside MEF2 as a critical regulator of the myogenic program, identify a new regulator functioning with MEF2 to control cell fate, and provide insight into the network of regulatory events that shape the developing musculature.

Regulation of stress-induced intracellular sorting and chaperone function of Hsp27 (HspB1) in mammalian cells.

In vitro, small Hsps (heat-shock proteins) have been shown to have chaperone function capable of keeping unfolded proteins in a form competent for Hsp70-dependent refolding. However, this has never been confirmed in living mammalian cells. In the present study, we show that Hsp27 (HspB1) translocates into the nucleus upon heat shock, where it forms granules that co-localize with IGCs (interchromatin granule clusters). Although heat-induced changes in the oligomerization status of Hsp27 correlate with its phosphorylation and nuclear translocation, Hsp27 phosphorylation alone is not sufficient for effective nuclear translocation of HspB1. Using firefly luciferase as a heat-sensitive reporter protein, we demonstrate that HspB1 expression in HspB1-deficient fibroblasts enhances protein refolding after heat shock. The positive effect of HspB1 on refolding is completely diminished by overexpression of Bag-1 (Bcl-2-associated athanogene), the negative regulator of Hsp70, consistent with the idea of HspB1 being the substrate holder for Hsp70. Although HspB1 and luciferase both accumulate in nuclear granules after heat shock, our results suggest that this is not related to the refolding activity of HspB1. Rather, granular accumulation may reflect a situation of failed refolding where the substrate is stored for subsequent degradation. Consistently, we found 20S proteasomes concentrated in nuclear granules of HspB1 after heat shock. We conclude that HspB1 contributes to an increased chaperone capacity of cells by binding unfolded proteins that are hereby kept competent for refolding by Hsp70 or that are sorted to nuclear granules if such refolding fails.

Cloning, characterization, and heat stress-induced redistribution of a protein homologous to human hsp27 in the zebrafish Danio rerio.

Hsp27 is a small heat shock protein (shsp) regulating stress tolerance and increasingly thought to play roles in tissue homeostasis and differentiation. The zebrafish Danio rerio is an important model for the study of developmental processes, but little is known regarding shsps in this animal. Here, we report the sequence, expression, regulation, and function of a zebrafish protein (zfHsp27) homologous to human Hsp27. zfHsp27 contains three conserved phosphorylatable serines and a cysteine important for regulation of apoptosis, but it lacks much of a C-terminal tail domain and shows low homology in two putative actin interacting domains that are features of mammalian Hsp27. zfHsp27 mRNA is most abundant in adult skeletal muscle and heart and is upregulated during early embryogenesis. zfHsp27 expressed in mammalian fibroblasts was phosphorylated in response to heat stress and anisomycin, and this phosphorylation was prevented by treatment with SB202190, an inhibitor of p38 MAPK. Expression of zfHsp27 and human Hsp27 in mammalian fibroblasts promoted a similar degree of tolerance to heat stress. zfHsp27 fusion proteins entered the nucleus and associated with the cytoskeleton of heat stressed cells in vitro and in zebrafish embryos. These results reveal conservation in regulation and function of mammalian and teleost Hsp27 proteins and define zebrafish as a new model for the study of Hsp27 function.

HSP27 protects AML cells against VP-16-induced apoptosis through modulation of p38 and c-Jun.

OBJECTIVE: To investigate 1) the signal transduction pathways affected by heat shock protein 27 (HSP27) expression; and 2) the expression and regulation of HSP27 in acute myeloid leukemia (AML). MATERIALS AND METHODS: RNA interference studies for HSP27 in leukemic TF-1 cells were used to investigate the effects on downstream signal transduction and apoptosis after VP-16 and CD95/Fas treatment. HSP27 expression and activation was investigated in AML blasts through Western blot analysis. RESULTS: RNA interference for HSP27 resulted in a twofold increase in VP-16-induced apoptosis, which was preceded by enhanced p38 and c-Jun phosphorylation and a twofold increased cytochrome c release into the cytoplasm. DAXX co-immunoprecipitated with HSP27, suggesting an inhibitory role of HSP27 in VP-16-mediated activation of the ASK1/p38/JNK pathway. CD95/Fas-induced apoptosis, however, was unaffected by HSP27 siRNA, due to upregulation of HSP27. Although HSP27 was highly expressed and phosphorylated in primitive monocytic AML blasts (M4-M5, 91%, n=11) and undetectable in myeloid blasts (M1-M2, n=5), VP-16-mediated apoptosis correlated moderately with HSP27 expression. This is likely due to the co-expression of p21Waf1/Cip1, which is in the majority of the monocytic AML M4-M5 blasts constitutively localized in the cytoplasm. Overexpression of cytoplasmic p21 inhibited the enhanced p38 phosphorylation after HSP27 RNAi, suggesting a predominant anti-apoptotic role of p21 over HSP27. CONCLUSION: 1) HSP27 inhibits VP-16-mediated phosphorylation of p38 and c-Jun, cytochrome c release, and subsequent apoptosis; 2) HSP27 is expressed and activated in monocytic AML blasts; 3) cytoplasmic expression of p21 compensates for the lack of HSP27.

Heat shock protein 70 or heat shock protein 27 overexpressed in human endothelial cells during posthypoxic reoxygenation can protect from delayed apoptosis.

Overexpression of heat shock protein (Hsp) 70 and Hsp27 in vivo was proclaimed as a potential tool in therapy of ischemia-reperfusion injury. However, it was so far not known whether these Hsps can beneficially act when increased in cells just at the stage of postischemic reperfusion. This issue was examined in a model of ischemia-reperfusion stress when cultures of endothelial cells (EC) from human umbilical vein were infected with virus-based vectors expressing Hsp70 or Hsp27, or Hsp56, or green fluorescent protein (GFP) and exposed to 20 hours of hypoxia followed by reoxygenation. The infection was performed either 10 hours before hypoxia or immediately after hypoxia, or at different time points of reoxygenation. Only low cell death was detected during hypoxia, but later, up to 40% of the treated cells died via caspase-dependent apoptosis between 6 and 12 hours of reoxygenation. The percentage of apoptotic cells was 1.6- to 3-fold greater in Hsp56- and GFP-infected EC than in Hsp70- or Hsp27-infected EC. The last 2 groups exhibited a lesser extent of procaspase-9 and procaspase-3 activation within 6-9 hours of reoxygenation. The cytoprotective effects of overexpressed Hsp70 and Hsp27 were observed not only in the case of infection before hypoxia but also when EC were infected at the start of reoxygenation or 1-2 hours later. An increase in the Hsp70 and Hsp27 levels in infected EC correlated well with their resistance to apoptosis under reoxygenation. These findings suggest that overexpression of Hsp70 or Hsp27, if it occurs in the involved cells at the early stage of postischemic reperfusion, can still be cytoprotective.

Distribution, phosphorylation, and activities of Hsp25 in heat-stressed H9c2 myoblasts: a functional link to cytoprotection.

The behavior of the endogenous heat shock protein 25 (Hsp25) in heat-stressed rat H9c2 myoblasts was studied. After mild or severe heating, this protein became less extractable with Triton X-100 and displayed characteristic immunofluorescence patterns, namely (1) granules in the nucleus, and (2) association with F-actin bundles in the cytoplasm. The intranuclear granulation of Hsp25 and its association with F-actin were sensitive to drugs affecting Hsp25 phosphorylation (cantharidin, sodium orthovanadate, SB203580, SB202190). Isoform analysis of Hsp25 translocated to the nucleus-free cytoskeletal fraction revealed only mono- and biphosphorylated Hsp25 and no unphosphorylated Hsp25. Transfected luciferase with initial localization in the nucleosol became colocalized with the Hsp25-containing granules after a heat shock treatment that denatured the enzyme in the cells. The association of Hsp25 with actin filaments after a mild heat stress conferred protection from subsequent F-actin-damaging treatments with cytochalasins (D and B) or severe heat stress. We hypothesize that (1) the binding of heat-denatured nucleosolic proteins to the Hsp25 contained in specific granular structures may serve for the subsequent chaperoning or degradation of the bound proteins, and (2) the actin cytoskeleton is stabilized by the direct targeting of phosphorylated Hsp25 to microfilament bundles.