Histone H1 is released from myonuclei and present in rimmed vacuoles with DNA in inclusion body myositis

1. Introduction 

Sporadic inclusion body myositis (s-IBM) is a main cause of muscular disability in people over 50 years old and is refractory to therapies. The diagnostic criteria of s-IBM on muscle biopsy are (1) inflammatory exudates surrounding non-necrotic fibers, (2) rimmed vacuoles, which are rounded or irregularly polygonal spaces in fibers with basophilic granules, and (3) congophilic inclusions in light microscopy or filaments that are 15–20nm in diameter in electron microscopy [1], [2]. In addition to these pathological findings, several studies showed distinct myonuclear alterations in s-IBM. Firstly, under electron microscopy, filamentous inclusions were sometimes detected in myonuclei as well as in the cytoplasm. Rarely, these inclusions appeared to be released from nuclei into the cytoplasm with breaks in the nuclear membrane [1]. Secondly, a study showed abnormal expression of single-stranded DNA binding protein of nuclear origin in muscle cytoplasm [3]. Thirdly, perinuclear localizations of nuclear transcription factor and nucleus-oriented protein kinases suggested inhibition of nucleocytoplasmic transport [4], [5]. Two groups showed that rimmed vacuoles might originate from the breakdown of the nucleus [3], [6], although most of the studies hypothesized that rimmed vacuoles are autophagic and composed of lysosomes [7], [8]. If rimmed vacuoles are of nuclear origin, the basophilic materials in rimmed vacuoles should be components of the nucleus.

Histones represent basophilic nuclear proteins. In inactive chromatin, the DNA is complexed to histones and forms nucleosomes. A nucleosome is an octamer of four pairs of the core histones H2A, H2B, H3, and H4, around which double-stranded DNA is wound. Histone H1 binds to the linker DNA that connects the individual nucleosomes. It is essential for the generation of the highly condensed chromatin structure and plays a pivotal role in gene regulation [9]. H1 is rich in arginine and lysine residues, which makes it highly basic [10].

In the current study, we examined histones in s-IBM by immunohistochemistry. We show that H1-positive products often delineated vacuolar boundaries in s-IBM. In addition, we detected H1 beyond intact nuclear membranes, which suggests release of nuclear components. The localization of H1 was usually different from that of phosphorylated Elk-1 (pElk-1), which is a nuclear protein, but shows extranuclear localization in abnormal fibers in IBM [5].

2. Materials and methods 

3. Results 

3.1. Expression of histone H1 in s-IBM 

A proportion of fibers harbored one or more immunopositive rings of H1 in s-IBM (Fig. 1). With eosin counter-staining, the inside spaces were lucent. Therefore, the positive rings corresponded to the vacuolar boundaries. Some vacuoles contained H1-positive closed rings, while others harbored broken circle or several dots of H1-positive immunoreaction. The table shows the relative percentage of fibers with H1-positive vacuoles vs. total vacuolated fibers in each patient with s-IBM. Approximately 60% of vacuolated fibers contained H1-positive rings or other H1-positive remnants. In immunohistochemistry of histones H2A, H2B, H3, and H4, rare vacuoles harbored distinctly positive deposits.

View Large Image Download to PowerPoint

Fig. 1. Localization of histone H1 in vacuolated fibers in sporadic inclusion body myositis (s-IBM). Immunoperoxidase method. Counter-stained with eosin. Distinct H1-positive vacuolar rims are seen (arrows). Bar=20μm.

3.2. Triple-fluorescence study of H1, emerin, and DNA in s-IBM 

In triple-fluorescence study of H1, emerin, and DAPI, H1-positive rings or deposits usually associated with the nuclear membrane protein and DNA (Fig. 2A and B). Strong vacuolar H1-positive reaction was detected inside or on emerin-positive reaction in a proportion of vacuoles. Attenuated DNA reaction was accompanied or partially co-localized with H1-positive materials (Fig. 2A and B). Moderate or strong H1-positive reaction that appeared to be leaking beyond emerin-positive lines was found in some other vacuoles (Fig. 2C–E). This cytoplasmic release of H1 was also observed in some morphologically preserved nuclei (Fig. 2F and G).

View Large Image Download to PowerPoint

Fig. 2. Triple-color fluorescence study in s-IBM. Row A, B: Localizations of DNA (blue), emerin (green), and histone H1 (red). In the left column, their merged pictures are shown. In the second column, the blue signals are enhanced to visualize weak circular DNA signals at the periphery of vacuoles (x). Histone H1-positive circles are found on the inner surface of emerin-boundaries and accompanied by DNA. The presence of the three nuclear components indicates that these vacuoles originate from the nucleus. C: A merging picture and H&E on the same section. Some cytoplasmic H1 materials appear to correspond to the basophilic radiations from the rimmed vacuole. D, E: A merging picture (top left), localizations of DNA (blue), emerin (green), and histone H1 (red) in vacuoles. The figures suggest that these vacuoles are altered nuclei with histone H1 released into the cytoplasm. Unlike intact nuclei, a DNA signal in each vacuole is ring-shaped. Fig. 2D is the central part of Fig. 2C. F, G: Cytoplasmic H1-release from nuclei in s-IBM. The cytoplasmic H1-localizations are similar to the faint basophilic leaks from the nuclei in H&E. The figures suggest that cytoplasmic H1-release occurs in an early phase of the nuclear disintegration. H: Localizations of emerin (red), pElk-1 (green), and DNA (blue). Deposits of pElk-1 are found outside of emerin–DNA complexes. Some products of pElk-1 are present at vacuolar peripheries. The differential localization of pElk-1 from the emerin–H1–DNA complex may correspond to the hypothesis that the nuclear import of pElk-1 may be prevented [5] while H1 is released from the nucleus. Bar=20μm.

The subsequent H&E after the fluorescence study showed that emerin-positive products in vacuoles often appeared to correspond to the basophilic lines in H&E (Fig. 2C). A region of cytoplasmic H1-positive reaction often corresponded to basophilic lakes around vacuoles or nuclei in H&E (Fig. 2C–G). H1-positive products within vacuoles sometimes correlated to inclusions within vacuoles. Control sections stained with H&E alone without immunostainings contained intra- and extra-vacuolar basophilic materials as well as basophilic lines.

3.3. Localization of pElk-1 vs. histone H1/emerin 

Deposits of pElk-1 were often localized in vacuoles without H1 or emerin (Fig. 2H). This means that vacuoles are positive either for emerin–histone H1 complex or pElk-1. To calculate vacuolated fibers that were positive for histone H1 or pElk-1, we immunostained muscle sections with anti-histone H1 and pElk-1 antibodies simultaneously. The results indicated that approximately 74% of vacuolated fibers harbored vacuoles positive for either H1 or pElk-1 (see Table 1).

Table 1. Percentages of rimmed vacuoles, muscle fibers with H1-positive vacuoles, those with pElk-1-positive vacuoles, and those with either of the two in 11 patients with sporadic inclusion body myositis

		Mean±SD (%)	Range (%)
	Vacuolated fibers/total fibers	6.75±2.15	4.23–11.28
	Histone H1 in vacuolated fibers∗	59.13±11.76	35.70–72.73
	pElk-1 in vacuolated fibers∗	60.24±6.33	50.00–69.81
	H1 or pElk-1 in vacuolated fibers∗	73.88±7.47	54.55–79.25

∗ Number of fibers harboring immunopositive vacuoles vs. the number of total vacuolated fibers.

3.4. Findings in controls 

DMRV showed the same results as s-IBM (Fig. 3). In DMRV, we detected H1-positive vacuoles in 61.6–70.0% of vacuolated fibers and pElk-1-positive vacuoles in 56.1–63.8% of vacuolated fibers. H1 were accompanied with emerin and DNA. Cytoplasmic H1 release was observed. Vacuoles negative for H1 often contained deposits of pElk-1 in DMRV. As for other controls, up to five fibers in a whole section contained vacuoles rimmed by H1-positive products in three of three patients with OPMD, nine of 18 patients with myositis controls. When compared with H&E, those H1-positive vacuoles seemed to correspond to rimmed vacuoles. Vacuoles in acid maltase deficiency, myopathy with autophagic vacuoles, and hypokalemic myopathy were negative for histone H1. For reviewing H1 release from nuclei in controls, we performed a triple-fluorescence study of H1, emerin, and DNA in every patient. Distinct cytoplasmic H1 release, as in s-IBM, was detected in a few myonuclei in a whole section in several patients with polymyositis or dermatomyositis, but not in the others.

View Large Image Download to PowerPoint

Fig. 3. Triple-color fluorescence study in distal myopathy with rimmed vacuoles (left) and H&E of the same sections (right). A: H1 (red), emerin (green), and DNA (blue). B: H1 (red), pElk-1 (green), and DNA (blue). The results are compatible with those in s-IBM. Note that basophilic rims and inclusions in rimmed vacuoles correspond to some H1-positive products, emerin or pElk-1. These figures suggest that rimmed vacuoles are nuclei undergoing degeneration. Bar=20μm.

4. Discussion 

We have shown that a significant proportion (approximately 60%) of vacuoles in s-IBM were rimmed by histone H1-positive products, and like nuclei, the H1-positive products were accompanied by emerin and DNA. In addition, in triple-color fluorescence study, vacuoles negative for H1 frequently contained immunopositive deposits of nuclear transcription factor pElk-1 and an immunoperoxidase study of H1 plus pElk-1 indicated that about three-fourths of vacuolated fibers were positive either for H1 or pElk-1. Therefore, our study strongly supports the hypothesis that vacuoles in s-IBM originate from the nucleus [3]. Vacuoles negative for H1 or pElk-1 might contain other nuclear remnants. It is, however, still possible that these vacuoles are derived from non-nuclear structures and that vacuoles in IBM have multiple origins. We sometimes encountered vacuoles rimmed by broken emerin-lines, which suggests nuclear membrane breakdown, as in a previous study [6]. However, cytoplasmic release of H1 usually appeared to occur beyond intact emerin-boundaries, but not through broken spaces. Moreover, we sometimes found cytoplasmic release of H1 from morphologically preserved nuclei. The results indicate that release of nuclear components occurs in an early phase of nuclear disintegration. In s-IBM muscle fibers, we found the most marked abnormal distribution of H1 among histones, probably because H1 shows dynamic behavior to regulate chromatin folding and gene expression, while core histones are integral components of chromatin fibers [9]. As histone H1 plays a dominant role in transcriptional regulation by chromatin remodeling [12], abnormal H1-expression may alter transcription and thereby might cause expression of various ectopic proteins detected in s-IBM [13]. Cytoplasmic release of H1, but not other histones, has been observed in a type of apoptosis in an experimental study of cultured human cells: apoptosis induced by stimuli causing DNA double-stranded breaks such as X-ray irradiation, but not other apoptotic stimuli, releases histone H1 into the cytoplasm [14]. Therefore, the cytoplasmic H1 release in s-IBM might indicate that some apoptotic stimuli causing DNA double-stranded breaks induce the s-IBM pathology.

We found deposits of nuclear transcription factor pElk-1 in vacuoles, which is additional evidence of nuclear disintegration in s-IBM. Unlike H1, we rarely encountered figures that suggest release of pElk-1 beyond the emerin-border. Instead, the pElk-1 positive inclusions were usually found isolated from nuclei. The results might indicate prompt gathering in clusters of pElk-1 after release of the nuclei and delivery to the processing locations, such as the proteasome [15] or lysosome [7], [8], which is abnormally upregulated in s-IBM muscle fibers. However, we think it more probable that formation of pElk-1 positive inclusions is due to inhibition of protein transport from the cytoplasm to the nucleus after translation and posttranslational phosphorylation of the protein. Elk-1 belongs to the E 26-specific (Ets) family of proteins. A study showed that a member of the Ets protein family, Elf-1, is transferred from the cytoplasm to the nucleus upon phosphorylation and other posttranslational modifications in hematopoietic cells [16]. The inhibition of nucleocytoplasmic protein transport in s-IBM has been suggested in the deposition of cyclin-dependent kinase-5 (CDK5) and extracellular signal-regulated kinase (ERK) in s-IBM muscle fibers [4], [5]. We hypothesize that release of nuclear components such as histone H1 and inhibition of the nucleocytoplasmic protein transport occur concurrently in s-IBM and that these abnormalities may be due to dysfunction of the nuclear envelope.

The nuclear envelope consists of inner and outer membranes. Exchange of the contents of the cytoplasm and the nucleus takes place only through the nuclear pore complex [17]. Several studies indicated that dysfunction of nuclear pores results in release of nuclear components into the cytoplasm and disables nucleocytoplasmic transport. For example, poliovirus increases bidirectional permeability for nuclear and cytoplasmic proteins and inhibits active nucleocytoplasmic transport [18]. This abnormality is caused by the proteolysis of some components on the nuclear pore complex, performed by the viral 2Apro protease activity [19]. Interestingly, muscles with postpoliomyelitis muscular atrophy sometimes show similar pathological alterations, including rimmed vacuoles, congo-red positivity, and tubulofilaments [20]. In addition to the virus, injection of an antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of proteins and RNA in experiments with Xenopus oocytes [21]. We therefore speculate that myonuclear abnormality of s-IBM resides in the nuclear pore complex or its associated nuclear envelope components. As ample evidence suggests that autoimmune mechanisms play a pivotal role in the pathogenesis of s-IBM [1], [2], it is necessary to investigate the relationship between the autoimmunity and myonuclear degeneration.

Distal myopathy with rimmed vacuoles (DMRV) shows rimmed vacuoles and tubulofilaments in muscles as s-IBM, but it lacks inflammation [22]. In DMRV, we observed H1-positive rims, cytoplasmic H1 release, and pElk-1 positive deposits in a similar magnitude as s-IBM. The result suggests that a close pathogenic mechanism is exerted both upon s-IBM and DMRV. DMRV and allelic disorder hereditary inclusion body myopathy (HIBM) have a mutation in GNE, which encodes an enzyme involved in the sialic acid biosynthetic pathway [23], [24], [25]. The GNE enzyme is present in the Golgi apparatus and the nucleus and it may be a nucleocytoplasmic shuttling protein [26]. Therefore, one hypothesis is that the mutated protein might be harmful to the nuclear membranes when shuttling through them. Alternatively, the enzyme dysfunction might affect glycosylation of nuclear pore protein, the function of which is regulated by a specific glycosylation cascade [27]. Although a much less amount than s-IBM or DMRV, a few rimmed vacuoles in OPMD and some other controls were positive for histone H1 and they may also be nuclear in origin. In OPMD, intranuclear aggregates consisting of mutated poly(A) binding protein 2 and poly(A) RNA [28] may cause nuclear injuries. Conversely, non-rimmed vacuoles such as those in acid maltase deficiency were negative for H1.

We commenced this study with the hypothesis that histones, which are basophilic proteins rich in the nucleus, might comprise vacuolar rims. The comparative study with immunofluorescence and subsequent H&E staining suggest that histone H1 corresponds to some, if not all, basophilic granules and streams found in or around vacuoles. We suspect that histone H1 and other nuclear proteins comprise basophilia in rimmed vacuoles.