Novel vectors expressing anti-apoptotic protein Bcl-2 to study cell death in Semliki Forest virus-infected cells

Semliki Forest virus (SFV, Alphavirus) induce rapid shut down of host cell protein synthesis and apoptotic death of infected vertebrate cells. Data on alphavirus-induced apoptosis are controversial. In this study, the anti-apoptotic bcl-2 gene was placed under the control of duplicated subgenomic promoter or different internal ribosome entry sites (IRES) and expressed using a novel bicistronic SFV vector. The use of IRES containing vectors resulted in high-level Bcl-2 synthesis during the early stages of infection. Nevertheless, in infected BHK-21 cells translational shutdown was almost complete by 6 h post-infection, which was similar to infection with appropriate control vectors. These results indicate that very early and high-level bcl-2 expression did not have a protective effect against SFV induced shutdown of host cell translation. No apoptotic cells were detected at those time points for any SFV vectors. Furthermore, Bcl-2 expression did not protect BHK-21 or AT3-neo cells at later time points, and infection of BHK-21 or AT3-neo cells with SFV replicon vectors or with wild-type SFV4 did not lead to release of cytochrome c from mitochondria. Taken together, our data suggest that SFV induced death in BHK-21 or AT3-neo cells is not triggered by the intrinsic pathway of apoptosis.
1
Introduction
Semliki Forest virus (SFV) is a positive-stranded RNA virus in Alphavirus genus (family Togaviridae), a widely distributed group of human and animal pathogens (Strauss and Strauss, 1994). SFV genomic RNA (so-called 42S RNA) is approximately 11.5 kb long and encodes four non-structural proteins designated nsP1–4; these are involved in viral RNA synthesis. The remaining proteins form the virus capsid and envelope and are not essential for virus replication.
After virus entry, the 42S RNA is translated into a large non-structural polyprotein, which is processed to form an early and subsequently a late replicase complex (Strauss and Strauss, 1994). The early replicase mediates synthesis of the negative-stranded RNA complementary to the genomic 42S RNA. Minus strands are used by the late replicase as templates for the synthesis of new positive strand 42S RNA, and for transcription of subgenomic mRNAs encoding the structural proteins.
The structural genes of SFV are not required for replication and can be removed or replaced with a polylinker and/or with foreign gene sequences. This property forms the basis of the SFV-based replicon vector systems (Liljestrom and Garoff, 1991; Smerdou and Liljestrom, 1999). SFV-based replicon vectors mediate high-level expression of heterologous proteins. However, as with virus, vectors cause shutdown of cellular biosynthesis and induce apoptotic death (Glasgow et al., 1997, 1998; Scallan et al., 1997). This precludes long-term foreign gene expression, and several attempts to reduce the cytotoxicity of alphavirus vectors have been made (Fazakerley et al., 2002; Lundstrom et al., 2003, 2001; Perri et al., 2000).
The anti-apoptotic gene bcl-2 is an antagonist of the intrinsic mitochondrial pathway of apoptosis (for reviews see Ashe and Berry, 2003; Cory and Adams, 2002; Tsujimoto and Shimizu, 2000). Bcl-2 can prevent release of cytochrome c from mitochondria, thus, precluding the apoptotic cascade (Kluck et al., 1997; Yang et al., 1997). Bcl-2 can block apoptosis induced by several viruses, including influenza virus and reovirus (Nencioni et al., 2003; Rodgers et al., 1997). Existing data on Bcl-2 in SFV- or Sindbis virus-induced apoptosis are contradictory. On one hand it has been shown that alphavirus-induced apoptosis of baby hamster kidney (BHK) cells, Chinese hamster ovary cells, rat insulinoma cells and rat prostatic adenocarcinoma (AT3) cells can be prevented by over-expression of Bcl-2 (Levine et al., 1993; Lundstrom et al., 1997; Mastrangelo et al., 2000; Scallan et al., 1997). Similarly, a Sindbis virus expressing Bcl-2 produces reduced encephalitis in infected mice (Levine et al., 1996). That Bcl-2 expression can block apoptosis, suggests involvement of intrinsic pathway of apoptosis. In contrast, other studies using rat embryo fibroblasts and monocyte cell lines overexpressing Bcl-2 failed to detect a protective effect against alphavirus-induced apoptosis (Grandgirard et al., 1998; Murphy et al., 2001).
The aim of this study was to determine whether expression of anti-apoptotic Bcl-2 directly from SFV-based replicon vectors in BHK-21 cells could be used to prolong co-expression of marker proteins from a bicistronic SFV replicon. Using the SFV1 vector system (Liljestrom and Garoff, 1991), the bcl-2 gene was placed either under the control of a duplicated SFV subgenomic promoter or an internal ribosome entry site (IRES). It is possible that expression of Bcl-2 from the subgenomic promoter occurs too late to prevent cell death. Expression from an IRES element within the genomic RNA should be more rapid. We tested two different IRES elements, the Encephalomyocarditis virus IRES (EMCV-IRES) and the crucifer-infecting tobamovirus IRES (CR-IRES). The latter is a 148-nt element, which precedes the CR coat protein gene and displays IRES activity across all kingdoms (Dorokhov et al., 2002). Using this novel approach we demonstrate that early Bcl-2 expression does not protect SFV-infected BHK-21 cells from alphavirus-induced translational shutdown or cell death. Moreover, our results indicate that SFV-induced cell death in BHK-21 cells does not involve the release of cytochrome c from mitochondria, and most likely does not occur by the apoptotic intrinsic pathway.
2
Materials and methods
2.1
Plasmid construction
The BamHI-XmaI multicloning site of the pSFV1 replicon (Liljestrom and Garoff, 1991) was replaced with a BamHI, ApaI, ClaI, AvrII, NruI, NsiI and XmaI multicloning site; the resulting construct was designated as pSFV-PL. The spliced sequences encoding the mouse Bcl-2 alpha protein (locus AAA37282), the EMCV-IRES (pIRES2-EGFP; BD Clontech) and the 148 bp CR-IRES (Ivanov et al., 1997) were amplified by PCR, cloned and verified by sequence analysis. Each IRES was fused to the Bcl-2 coding sequence and cloned into NsiI-XmaI digested pSFV-PL vector; obtained constructs were designated as pSFV-EMCV-bcl2 and pSFV-CR-bcl2. To create constructs expressing Bcl-2 protein from the duplicated subgenomic promoter, the IRES from pSFV-EMCV-bcl2 was replaced by an oligonucleotide duplex representing the minimal SFV subgenomic promoter (Hertz and Huang, 1992); the resulting construct was designated pSFV-PR-bcl2. The d1EGFP reporter gene (BD Clontech) was amplified by PCR, sequenced and cloned into pSFV-PL, pSFV-EMCV-bcl2, pSFV-CR-bcl2 and pSFV-PR-bcl2 vectors treated with ClaI-NsiI. Resulting constructs were designated as pSFV-PL-d1EGFP, pSFV-d1EGFP-EMCV-bcl2, pSFV-d1EGFP-CR-bcl2 and pSFV-d1EGFP-PR-bcl2, respectively (Fig. 1). Sequences and primers are available upon request.
To construct SFV replicons expressing mutated chromoprotein HcRed (from the reef coral Heteractis crispa), HcRed was PCR amplified (from pHcRed1-N1; BD Clontech), cloned and sequenced. The sequence encoding Bcl-2 from pSFV-d1EGFP-EMCV-bcl2, pSFV-d1EGFP-CR-bcl2 and pSFV-d1EGFP-PR-bcl2 was replaced with HcRed to give constructs pSFV-d1EGFP-EMCV-HcRed, pSFV-d1EGFP-CR-HcRed and pSFV-d1EGFP-PR-HcRed (Fig. 1).
To obtain constructs used for viability analysis under puromycin selection, the sequence encoding d1EGFP from pSFV-PL-d1EGFP, pSFV-d1EGFP-EMCV-bcl2, pSFV-d1EGFP-CR-bcl2 and pSFV-d1EGFP-PR-bcl2 was replaced by that of puromycin acetyltransferase (Pac), and constructs were designated pSFV-PL-Pac, pSFV-Pac-EMCV-bcl2, pSFV-Pac-CR-bcl2 and pSFV-Pac-PR-bcl2.
To generate infectious RNA, constructs were linearised by SpeI digestion and in vitro transcription was carried out as previously described (Karlsson and Liljestrom, 2003).
2.2
Cells and viruses
BHK-21 cells were grown in Glasgow's Minimal Essential Medium containing 5% foetal calf serum, 0.3% tryptose phosphate broth, 0.1 U/ml penicillin and 0.1 μg/ml streptomycin. AT3-neo and AT3-bcl2 cells were grown in Roswell Park Memorial Institute-1640 medium containing 10% foetal calf serum, 0.1 U/ml penicillin and 0.1 μg/ml streptomycin. All cells were grown at 37 °C in a 5% CO2 atmosphere. SFV4 was derived from the infectious cDNA clone pSP6-SFV4 (Liljestrom et al., 1991).
2.3
Transfection and collection of virus-like particles (VLPs)
BHK-21 cells were co-transfected with equal amounts of vector and helper RNA (Liljestrom and Garoff, 1991). Helper RNA encodes the structural proteins under subgenomic promoter. Transfected cells were grown at 28 °C for 72 h and the VLPs collected, concentrated, purified and titrated as described by Karlsson and Liljestrom (2003). Although the replicase encoded by the replicon vector will amplify both RNAs, helper RNA is not packed into VLPs due to a missing packaging signal. To determine if any replication-proficient viruses were formed due to the recombination between replicon vector RNA and helper RNA, batches of VLPs were tested as described by Smerdou and Liljestrom (1999). All infections with VLPs were carried out in BHK-21 cells at a multiplicity of infections 10 (moi = 10) for 1 h at 37 °C.
2.4
Metabolic labelling
BHK-21 cells in 35 mm diameter plates were infected with SFV VLPs as described above. Infected cells were washed twice with PBS, once with methionine and cysteine free Dulbecco's Modified Eagle Medium followed by 30 min labelling with 50 μCi/ml of [35S]methionine and [35S]cysteine (RedivuePRO-MIX, Amersham Biosciences). After labelling, cells were washed with PBS, lysed in Laemmli buffer and analyzed by SDS-PAGE. Gels were dried under vacuum and exposed to film.
2.5
Immunoblot analysis
BHK-21 cells were infected and samples collected as described above. After SDS-PAGE, proteins were transferred to a nitrocellulose membrane, probed with rabbit polyclonal antisera against SFV nsP1, EGFP (in-house), HcRed (Clontech), with a mouse monoclonal antibody against Bcl-2 (Santa Cruz Biotechnology, Inc.) or with a mouse monoclonal antibody against beta-actin (C4) (Cruz Biotechnology, Inc.) and visualized by ECL immunoblot detection kit (Amersham Life Science).
2.6
Analysis of bicistronic SFV vector cytotoxicity in BHK-21 cells
Cytotoxicity of SFV vectors was analyzed as described by Garmashova et al., 2006. Replicon RNA was obtained from pSFV-PL-Pac, pSFV-Pac-EMCV-bcl2, pSFV-Pac-CR-bcl2 and pSFV-Pac-PR-bcl2. 106 BHK-21 cells were electroporated with 5 μg of RNA. Cells were seeded into wells (growth area 2.0 cm2 per well; Cellstar, Greiner bio-one plates) and selected with puromycin (10 μg/ml) from 6 h post-transfection. Viable adherent cells were determined at 6, 24, 48 and 72 h post-transfection using Trypan blue (Flow Laboratories).
The viability of infected cells was also analyzed by WST-1 assay (Roche). The assay is based on the reduction of WST-1 to a water-soluble formazan dye by viable cells. BHK-21 cells were seeded in 96-well plates (7 × 103 cells/well), grown for 18 h and infected with VLPs containing recombinant replicons at moi = 10. Control cells were mock-infected. Infected cells were analyzed 6, 24 or 48 h post-infection (p.i.) by adding 10 μl of WST-1 to each well, followed by incubating the plate for 1 h and measuring the change in color intensity at 450 nm in a microplate reader.
2.7
Immunofluorescence microscopy
Cells were grown on cover slips and infected for selected times, mock-infected cells were used as controls. Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with cold methanol for 7 min at −20 °C. Cells were then washed with PBS, blocked in the 3% BSA-PBS and incubated for 1 h with primary antibody (mouse anti-cytochrome c monoclonal antibody (BD Pharmingen) or rabbit polyclonal antibody against SFV nsP1). Then the cells were washed again with PBS and incubated with a AlexaFluor 568 (Invitrogen) or Cy3 conjugated secondary antibody for 1 h, washed three times with PBS and air-dried. Staurosporine (final concentration 0.5 μM) was added to mock-infected cells 1–2 h before fixing to induce release of cytochrome c from mitochondria. Samples were analyzed on an Olympus U-RFL-TX microscope or a Bio-Rad MRC-1024 confocal microscope.
2.8
Analysis of the viability of AT3 cells infected with SFV VLPs
3 × 106 AT3-neo or AT3-bcl2 cells were infected with VLPs in serum-free RPMI1640 media supplemented with 0.2% BSA. The amount of VLPs used for infections corresponded to a moi = 20 for BHK-21 cells. At 12 h p.i., EGFP positive cells were separated using a BD FACSAria cell sorter; EGFP-positive cells were seeded in 24-well plates. Viable cells were determined at 24 and 48 h p.i. using Trypan blue (Flow Laboratories).
3
Results
3.1
Expression of d1EGFP and Bcl-2 using bicistronic SFV vectors
To study the expression of foreign proteins using the bicistronic replicons, BHK-21 cells were infected with SFV-d1EGFP-CR-bcl2, SFV-d1EGFP-EMCV-bcl2 and SFV-d1EGFP-PR-bcl2 VLPs and in addition, with monocistronic SFV-PL-d1EGFP VLPs. Samples were collected at 2, 4, 6, 8, 12 and 24 h p.i. and analyzed by immunobloting. Expression of SFV nsP1 was generally detectable by 2 h p.i. and increased up to 8–12 h p.i. (Fig. 2a–e). The amount of nsP1 was approximately equal for all bicistronic vectors (Fig. 2b–d). Expression of d1EGFP was also detectable by 2-4 h post-infection (Fig. 2a–d) and was highest in SFV-PL-d1EGFP infected cells (Fig. 2a). Major differences were observed with Bcl-2 expression, which was detected on immunoblots as two bands (Fig. 2b–d). Bcl-2 expression was strongest and earliest (2 h p.i.) in SFV-d1EGFP-EMCV-bcl2 infected cells (Fig. 2c). In cells infected with SFV-d1EGFP-CR-bcl2 (Fig. 2b), expression of Bcl-2 was detected 4 h p.i. Expression of Bcl-2 was also found in cells infected with SFV-d1EGFP-PR-bcl2 (Fig. 2d) although levels were lower compared to IRES containing constructs.
To assess the ability of the bicistronic vectors to express proteins in general, Bcl-2 sequences were replaced with non-cytotoxic HcRed. The resulting replicons SFV-d1EGFP-EMCV-HcRed, SFV-d1EGFP-CR-HcRed and pSFV-d1EGFP-PR-HcRed were used to infect BHK-21 cells. There was no significant difference in the expression of nsP1 and d1EGFP proteins between bicistronic vectors also expressing HcRed or Bcl-2 (Fig. 2e for SFV-d1EGFP-EMCV-HcRed, data not shown for SFV-d1EGFP-CR-HcRed and pSFV-d1EGFP-PR-HcRed). Expression of HcRed was strongest for SFV-d1EGFP-EMCV-HcRed (detectable by Western blotting by 4 h p.i.; Fig. 2e). This is in agreement with previous observations suggesting that the highest expression levels of the second target proteins are achieved with SFV-d1EGFP-EMCV vectors. The apparent delay in detection of protein expression (2 h p.i. for Bcl-2 versus 4 h p.i. for HcRed) is most likely due to the quality of the HcRed antibody (Clontech).
Thus, vectors containing IRES elements expressed Bcl-2 earlier and to higher levels. Presence of a duplicated subgenomic promoter or IRES element following the Bcl-2 encoding sequence did not have any major effect on the time-course of infection, however, differences in expression levels of the first marker protein, d1EGFP, were observed.
3.2
Effects of infections by recombinant SFV VLPs on host cell protein synthesis
Metabolic labelling was used to study the effects of infection by SFV VLPs on host cell protein synthesis. Infected BHK-21 cells were pulse labelled at 2, 4, 6, 8, 12 and 24 h p.i. In contrast to mock-infected cells, where protein synthesis is ongoing (Fig. 3A), translation in infected cells was rapidly inhibited (Fig. 3B–E). Shutdown of host protein synthesis was detected as early as 4 h p.i. (Fig. 3B–E). High-level and continuous expression of Bcl-2 was visible in cells infected with SFV-d1EGFP-EMCV-bcl2 (Fig. 3D). In cells infected with other bicistronic vectors, expression of Bcl-2 could only be detected by Western blot (Fig. 2b–d). These results indicate that high-level, continuous expression of Bcl-2 from a bicistronic replicon vector does not protect against shut-off of host cell protein synthesis.
3.3
Survival of cells infected with Bcl-2 expressing bicistronic replicons
It has been shown that similar to wild-type virus, alphavirus based replicon vectors induce apoptotic death of infected vertebrate cells (Glasgow et al., 1997, 1998; Scallan et al., 1997) and that over-expression of Bcl-2 can protect cells against alphavirus-induced apoptosis (Lundstrom et al., 1997; Scallan et al., 1997).
To study the effect of Bcl-2 expression from bicistronic vectors on cell death, BHK-21 cells were transfected with SFV-PL-Pac, SFV-Pac-EMCV-bcl2, SFV-Pac-CR-bcl2 and SFV-Pac-PR-bcl2 replicons or mock-transfected, and puromycin selection was applied 6 h post-transfection. Almost all mock-transfected cells died within 24 h of adding puromycin (Fig. 4a). The transfected cells survived longer and the percentage of viable adherent cells was found to be similar at the selected time points (Fig. 4a). Most of the transfected cells were dead 3 days post-transfection regardless of the replicon. Thus, our results demonstrate that high-level expression of Bcl-2 does not protect BHK-21 cells against SFV induced death. Similar results were also obtained when Annexin-V PE conjugate or propidium iodide were used to label apoptotic and necrotic cells (data not shown).
The WST-1 assay, used to analyze cell viability, measures mitochondrial activity in cells. Results obtained with this assay were similar to cell survival experiments: expression of Bcl-2 from bicistronic vectors (SFV-d1EGFP-CR-bcl2, SFV-d1EGFP-EMCV-bcl2 or SFV-d1EGFP-PR-bcl2) did not provide any protective effect (Fig. 4b). In fact, viability of cells infected with bicistronic replicons at 48 h p.i. was reduced, compared to cells infected with SFV-PL-d1EGFP (Fig. 4b). Infection with SFV-d1EGFP-EMCV-bcl2 caused the most important reduction in cell viability, which is in accordance with our previous data. (Fig. 4a). This tendency was also seen when cells infected with SFV-d1EGFP-EMCV-HcRed, SFV-d1EGFP-CR-HcRed and SFV-d1EGFP-PR-HcRed VLPs were analyzed by WST-1 assay (data not shown), indicating that not Bcl-2 expression, but rather the second gene expression unit (especially EMCV-IRES) is responsible for this effect. Taken together, our results suggest that Bcl-2 expression does not have a detectable effect on the viability of SFV-infected BHK-21 cells.
3.4
SFV infection does not induce release of cytochrome c from mitochondria
The anti-apoptotic effect of Bcl-2 is connected to its ability to prevent the release of cytochrome c from mitochondria (Kluck et al., 1997; Yang et al., 1997). Apoptosis induced by death receptors or by ER stress bypasses this mitochondrial pathway and is, therefore, relatively insensitive to protection by Bcl-2 (Scaffidi et al., 1998). The finding that high-levels of Bcl-2 expression did not have a protective effect against SFV induced death suggests that apoptosis in SFV-infected BHK-21 cells may not involve the mitochondrial pathway. To determine whether the mitochondrial pathway is activated in SFV-infected BHK cells, localization of cytochrome c was visualized by immunofluorescence in BHK-21 cells infected with the SFV-PL-d1EGFP 4 and 24 h p.i. Localization of cytochrome c in mock-infected BHK-21 cells was mitochondrial as evidenced by dotty localization (Fig. 5A). In cells, treated with the non-selective protein kinase inhibitor staurosporine, cytochrome c staining was diffuse, demonstrating its release from mitochondria (Fig. 5B). In cells infected with SFV-PL-d1EGFP, the cytochrome c staining maintained a granular pattern 4 h p.i. (Fig. 5C and D), indicating that when shut-off of cellular gene expression starts cytochrome c was not released from mitochondria. Furthermore, the cytochrome c localization pattern was unchanged even at 24 h p.i. (Fig. 5E and F). We conclude that induction of cell death in SFV replicon-infected BHK-21 cells does not involve the release of mitochondrial cytochrome c. This result is consistent with the absence of a protective effect of Bcl-2.
SFV replicons lack the coding sequences for structural proteins. To analyze the role of the structural proteins in the release of cytochrome c from mitochondria, BHK-21 cells were infected with SFV4 (moi = 1) and localization of cytochrome c was determined at 4, 12 and 24 h p.i. By 4 h p.i. infected cells did not release cytochrome c (Fig. 6A and B). At 12 and 24 h p.i. detection of cytochrome c became more difficult due to extensive cytopathic effects. This includes cell rounding and re-localization of mitochondria into the perinuclear region (Fig. 6C–F); all these effects were more extensive than in SFV-PL-d1EGFP infected cells (Fig. 5). Nevertheless, the localization pattern of cytochrome c remained dotty indicating that its mitochondrial localization was preserved (Fig. 6C–F). Taken together, these results indicate that for both replicon vector and SFV4 infection, BHK-21 cells do not release cytochrome c from the mitochondria.
3.5
Bcl-2 expression does not protect SFV-infected AT3-neo cells against cell death
To analyze whether the absence of cytochrome c release and lack of the protection against SFV induced cell death are specific to BHK-21 cells, two lines of AT3 cells, AT3-neo (Cepko et al., 1984) and AT3-Bcl2 (Levine et al., 1993) cells were also characterized.
Firstly, AT3-neo cells were infected with SFV4 and cytochrome c distribution was determined. Infected AT3-neo cells showed extensive virus-induced cytopathic effects but as with BHK-21 cells no detectable release of cytochrome c (Fig. 7). Similarly, no detectable release of cytochrome c was observed in AT3-neo or AT3-bcl2 cells infected with SFV-d1EGFP-EMCV-bcl2 (Fig. 8A–D and I–L) or SFV-PL-d1EGFP VLPs (Fig. 8E–H and M–P). Therefore, we conclude that the inability of SFV to induce cytochrome c release from mitochondria was not a BHK-21 cell line-restricted phenomenon.
Secondly, the viability of the AT3-neo and AT3-bcl2 cells, infected with SFV VLPs was analyzed. It should be noted that we were unable to obtain highly efficient infection of the AT3-neo and especially the AT3-bcl2 cells by use of the SFV VLPs. Typically, up to 20% of AT3-neo and 10% of AT3-bcl2 cells were infected when the amount of VLPs, corresponding to moi = 20 in BHK-21 cells, was used for infection; using larger amounts of VLPs did not result in higher percentages of infected cells. Thus, we conclude that AT3 cells are not as susceptible for SFV infection as BHK-21 cells and sorting of EGFP positive (i.e. infected) cells before the viability analysis was required. This revealed that, in the case of AT3-neo cells, expression of Bcl-2 by any SFV replicon vector did not provide any protective effect and almost all infected cells were dead by 48 h p.i. (Fig. 9a). Thus, the results were highly similar to those obtained in BHK-21 cells. In contrast, we found that almost all AT3-bcl2 cells, infected with SFV VLPs, were viable at 24 h p.i. and over the next 24 h numbers of viable cells rapidly increased in all samples. This indicates efficient cell division (Fig. 9b). Since cell sorting ruled out the possibility of non-infected cells being present in analyzed samples, we concluded that AT3-bcl2 cells, infected with SFV replicons were either able to recover from infection or, alternatively, established a persistent infection. Our observation that surviving AT3-bcl2 cells rapidly lost EGFP fluorescence favours the first option. Expression of Bcl-2 by SFV replicons had little or no effect on survival and subsequent division of infected AT3-bcl2 cells. All results taken together we conclude that not bcl-2 expression as such but some other property of AT3-bcl2 cells is likely responsible for this phenomenon.
4
Discussion
Previously published data on mechanisms of alphavirus-induced cell death are controversial (Glasgow et al., 1997; Griffin, 2005; Li and Stollar, 2004). On the one hand, induction of apoptotic cell death via the death receptor pathway has been suggested (Li and Stollar, 2004; Nava et al., 1998); on the other hand, it has been shown that over-expression of the anti-apoptotic Bcl-2 can protect against alphavirus-induced apoptosis and pathogenesis (Levine et al., 1993; Lundstrom et al., 1997; Mastrangelo et al., 2000; Scallan et al., 1997). However, in these studies different alphaviruses, strains, cell lines and experimental systems were used. It is possible that mechanisms, by which cell death is induced may differ between viruses, cells etc.
In this study, shut-off of cellular translation and cell death in SFV-infected BHK-21 cells were studied using a novel bicistronic replicon vector system. This avoids transient or stable expression of Bcl-2 in host cells. In transiently transfected cells, cell metabolism can be seriously altered by the transfection procedure (Lepik et al., 2003). Stable expression of Bcl-2, known to have oncogenic properties (Reed, 1994; Reed et al., 1991), may lead to adaptation of the cell, which in turn might affect virus replication and the outcome of infection. It is also important to mention that both transient and stable cellular expression are mediated by cellular RNA polymerase II and are, therefore, subjected to the alphavirus-induced transcriptional shutdown, which starts early in infection.
In this study, Bcl-2 was expressed using novel vectors containing IRES elements. The use of IRESs has significant advantages over other systems. The first important advantage, as shown by our results (Fig. 2), is that IRES-mediated expression of Bcl-2 can take place directly from incoming genomic RNA. In contrast, expression from the subgenomic promoter requires replication and is activated later on in infection. Early expression of Bcl-2 may be crucial since it is not known when and how cell death is induced. It is possible that expression of Bcl-2 from the subgenomic promoter is too late to protect from cell death. Another important advantage of IRES-mediated expression is the level of expression, which is significantly higher than from the minimal subgenomic promoter (Fig. 2). It is also important to mention that at least for the EMCV-IRES, synthesis of Bcl-2 was resistant to inhibition of cellular translation (Fig. 3D).
Detection of Bcl-2 protein by immunoblotting in BHK-21 cells infected with VLPs of SFV1 vectors harboring bcl-2 revealed a lower molecular band, which increased in intensity proportional to Bcl-2 expression. This is presumably protein expressed from an internal initiation site (Tsujimoto and Croce, 1986). The truncated protein could also be the product of Bcl-2 cleavage by endogenous caspase-3, resulting in curtailed Bcl-2 with proapoptotic activity (Cheng et al., 1997; Kirsch et al., 1999). The latter localizes to mitochondria and causes the release of cytochrome c, thus, promoting further caspase activation as a part of a positive feedback loop (Kirsch et al., 1999). It has been shown that alphaviruses induce apoptosis in Bcl-2-overexpressing cells by caspase-mediated proteolytic inactivation of Bcl-2 (Grandgirard et al., 1998). Viral capsid protein was suggested to trigger activation of the cell death machinery. However, in this study neither release of cytochrome c nor increase in the proportion of truncated product due to feedback loop action was observed. Infection of cells with VLPs eliminated the possibility that capsid protein alone triggers apoptosis.
Simultaneous expression of two target proteins was achieved with all the bicistronic SFV expression vectors constructed in this study. The expression level of d1EGFP was slightly reduced for bicistronic vectors, compared to the monocistronic SFV-PL-d1EGFP, independent of the nature of the second gene (bcl-2 or HcRed) (Fig. 2). Interference between the native subgenomic promoter and the duplicated promoter or IRES element may have influenced expression from the first promoter. It is also possible that presence of the IRES elements had some effect on replication. However, infectivity of bicistronic vectors was not altered. Taken together, these results indicate that different IRES elements can be used to construct novel and efficient bicistronic SFV vectors.
In this study, we show that early and high-level of Bcl-2 expression, achieved with bicistronic SFV replicon vectors, did not have a detectable effect on host protein synthesis shut-off (Fig. 3) or cell death (Fig. 4). This cannot be attributed to the delayed expression of bcl-2 since its high expression levels were observed at early time points (Fig. 2). These findings are coherent with our data suggesting that infection of BHK-21 cells with SFV replicons or with SFV4 does not cause the release of cytochrome c from mitochondria (Figs. 5 and 6). This effect was not restricted to BHK-21 cells since similar results were also obtained for AT3-neo (Figs. 7 and 8) and AT3-bcl2 (Fig. 8) cells. The release of cytochrome c in SFV-infected BHK-21 or AT3-neo cells did not even occur at late time points. This explains the lack of protective effect by Bcl-2 expression against cell death in infected cells, as observed by us.
At the same time, the mechanism of cell death in infected cells remains unknown. We can rule out that binding of virions to the cellular receptor and/or virus internalisation have crucial roles, since induction of cell death does not depend on whether the cells are infected with virus or transfected by infectious transcripts. It seems most likely that cell death is induced by some non-mitochondrial pathway. This is consistent with previous data, which suggested that apoptosis in alphavirus infected cells uses the death receptor pathway (Nava et al., 1998; Li and Stollar, 2004). However, our findings do not exclude the possibility that cell death in SFV-infected BHK-21 cells may be triggered by the ER pathway.
A delay of cell death in SFV-infected AT3 cell line expressing bcl-2 has been reported (Scallan et al., 1997). Our results confirm that the AT3-bcl2 cell line is remarkably resistant to infection by SFV VLPs. However, our data is more coherent with the hypothesis that these cells are not just delaying cell death but are also capable to recover from SFV infection. The finding that Bcl-2 expression by SFV replicons did not have any effect on the survival of AT3-neo cells suggests that resistance of the AT3-bcl2 cells to the SFV infection most likely represents an indirect effect of Bcl-2. The Bcl-2 gene is potent oncogene and its constitutive expression significantly changes cell cycle and gene expression. It is indeed evident from data presented in Fig. 9 that AT3-bcl2 cells grow much more rapidly than AT3-neo cells. It could be hypothesized that constitutive expression of Bcl-2 in AT3-bcl2 cells may have resulted in changes protecting cells from virus infection. The most likely candidates for roles of these factors may be the components of the innate immune system and/or host factor(s) like rat zinc-finger antiviral protein, which provides resistance against alphavirus infection (Bick et al., 2003).


Biomechanical Analysis of Reducing Sacroiliac Joint Shear Load by Optimization of Pelvic Muscle and Ligament Forces

Effective stabilization of the sacroiliac joints (SIJ) is essential, since spinal loading is transferred via the SIJ to the coxal bones, and further to the legs. We performed a biomechanical analysis of SIJ stability in terms of reduced SIJ shear force in standing posture using a validated static 3-D simulation model. This model contained 100 muscle elements, 8 ligaments, and 8 joints in trunk, pelvis, and upper legs. Initially, the model was set up to minimize the maximum muscle stress. In this situation, the trunk load was mainly balanced between the coxal bones by vertical SIJ shear force. An imposed reduction of the vertical SIJ shear by 20% resulted in 70% increase of SIJ compression force due to activation of hip flexors and counteracting hip extensors. Another 20% reduction of the vertical SIJ shear force resulted in further increase of SIJ compression force by 400%, due to activation of the transversely oriented M. transversus abdominis and pelvic floor muscles. The M. transversus abdominis crosses the SIJ and clamps the sacrum between the coxal bones. Moreover, the pelvic floor muscles oppose lateral movement of the coxal bones, which stabilizes the position of the sacrum between the coxal bones (the pelvic arc). Our results suggest that training of the M. transversus abdominis and the pelvic floor muscles could help to relieve SI-joint related pelvic pain.
Introduction
The human body uses an ingenious 3-D framework of bones, joints, muscles, and ligaments for posture and movement. In upright posture, the trunk load passes the sacroiliac joints (SIJ). The orientation of the SIJ surfaces, however, is more or less in line with the direction of loading, which induces high shear forces between sacrum and coxal bones.34 The SIJ have a strong passive, viscoelastic ligamentous system for providing stability. These ligaments are vulnerable for creep during constant trunk load and need to be protected against high SIJ shear forces.19 From a biomechanical point of view, an active muscle corset that increases the compression force between the coxal bones and the sacrum could protect the ligamentous system and support the transfer of trunk load to the legs and vice versa. Interlocking of the SIJ may be promoted by transversely oriented muscles, e.g., M. transversus abdominis, M. piriformis, M. gluteus maximus, M. obliquus externus abdominis, and M. obliquus internus abdominis, which has been described as self-bracing.33–35 However, due to the complex lines of action of (counteracting) muscles and ligaments in the pelvic region, it is difficult to demonstrate the contribution of transversely oriented muscles to SIJ stability, in vitro as well as in vivo.
In the past, a number of biomechanical models of the lumbosacral region (spine and pelvis) have been developed to study the aetiology of low back pain (LBP) in relation to (over)loading of the lumbar spine1, 9, 11, 36 and the pelvis.10,27 Most of these models dealt with mechanical stability in terms of muscle3,4,18 and compression forces between (lumbar) vertebrae.2,8,12,20,22, 28 A different approach is to relate LBP to overloading of SIJ and nearby ligaments, for example the iliolumbar ligaments.24,30 The load transfer through the SIJ was studied using a static, 3-D biomechanical simulation model based on the musculoskeletal anatomy of the trunk, pelvis, and upper legs.15 This simulation model calculates forces in muscles, ligaments, and joints that are needed to counterbalance trunk weight and other external forces. It was shown that this simulation model underestimated antagonistic muscle activity, but a good agreement was found for agonist muscle activity. The number of passive structures in the model was small, for example no joint capsules were incorporated. Therefore, the model was only valid for postures in which none of the joints were near an end position. The aim of the present study was to determine which muscles have to become active in the 3-D pelvic simulation model when there is an imposed reduction of the vertical SIJ shear force.
Materials and Methods
The 3-D Simulation Model
The present study was performed using the validated, 3-D simulation model as described by Hoek van Dijke et al.15 The model is based on the musculoskeletal anatomy of the trunk, pelvis, and upper legs, including muscle and ligament attachment sites, cross-sectional areas of muscles and the direction of muscle, ligament, and joint reaction forces. The geometry of this model was based on structures extracted from MRI slices and from previously published data on lumbar spine8 and upper leg4,17 geometry. Figure 1a illustrates, in frontal and median view, the bones on which muscle and ligament forces act in the simulation model. These are the lowest thoracic vertebra, five lumbar vertebrae, the sacrum, the left and right coxal bones and the left and right femurs. The vertebrae are treated as a single structure. The arrangement of the bones depends on the static posture for which muscle forces are calculated, for example standing with or without trunk flexion. A description of the model equilibrium, its optimization scheme and validation of some of the parameters is presented in the Appendix. In the present study, we focus on the compression and shear forces in the SIJ. These forces are represented as perpendicular vectors. The normal vector of the SIJ surface has an oblique direction (xyz = 0.365, ±0.924, 0.114). Compression force is defined along this normal vector and can only vary in magnitude. One of the two components of the SIJ shear force was defined in the YZ-plane (xyz = 0, ±0.123, 0.992). This force is denoted as the vertical SIJ shear force. Directions of the SIJ compression force and the SIJ vertical shear force in the YZ-plane are shown in Fig. 1a, left panel. Figure 1b shows the vectors representing the most important muscle and ligament forces in the pelvic region superimposed on the bones. Bone shapes are for illustration purpose only; they are not part of the simulation model. In total, the model contains 100 vectors for muscle forces, 8 vectors for ligament forces, and 22 vectors for joint forces; see Table 1 for a list of all the structures.
Figure 1Panel (a) shows the bones on which the muscles, ligaments and joint reaction forces act in the frontal plane (left) and the median plane (right): the lowest thoracic vertebra, five lumbar vertebrae, the sacrum, the left and right coxal bones, and the left and right femurs. The coordinate system is defined with the origin halfway between the rotation centers of the hip joints. Axes: x posterior, y left, z vertical. Panel (b) shows, superimposed on the bones, the vectors representing the most important force components in the frontal plane (left) and the median plane (right), see also Table 1. The labels in this panel refer to a selection of the muscle structures listed in Table 1Table 1List of muscles, ligaments, and joints and the number of vectors describing the forces used in the simulation model on transferring trunk load from lumbar spine via the pelvis to the upper legs (unilateral), see also Fig. 1Name of structureNumber of elementsRemark1M. adductor brevis2Upper and lower muscle2M. adductor longus13M. adductor magnus3Upper, middle and lower muscle4M. biceps femoris15M. coccygeus1Pelvic floor muscle6M. iliococcygeus1Pelvic floor muscle7M. pubococcygeus1Pelvic floor muscle8M. gemellus inferior19M. gemellus superior110M. gluteus maximus2Femur—sacrum and ilium muscle11M. gluteus maximus facia2Ilium—femur and trunk muscle12M. gluteus medius3Upper, middle, and lower muscle13M. gluteus minimus3Upper, middle, and lower muscle14M. gracilis115M. iliacus116M. longissimus117M. iliocostalis118M. multifidus119M. obliquus externus abdominis2Ventral and dorsal muscle20M. obliquus internus abdominis2Ventral and dorsal muscle21M. obturatorius externus122M. obturatorius internus123M. pectineus124M. piriformis125M. psoas2Upper and lower muscle26M. quadratus femoris127M. quadratus lumborum5Sacrum—rib12, L1, L2, L3 and L4 muscle28M. rectus abdominis129M. rectus femoris130M. sartorius131M. semimembranosus132M. semitendinosus133M. tensor fasciae latae134M. transversus abdominis1AIliolumbar ligament1Transversal planeBPosterior sacroiliac ligament1Transversal planeCSacrospinal ligament1DSacrotuberous ligament1IL5-S1 joint3Shear (two directions) and compressionIISI joint3Shear (two directions) and compressionIIIHip joint3Shear (two directions) and compressionIVKnee joint3Shear (two directions) and compressionVPubic symphysis1Compression
Simulations and Data Analyses
A first simulation, with the model in standing posture and a trunk weight of 500 N, showed that the vertical shear SIJ force was 563 N on each side of the sacrum. To find the muscles that promote sacroiliac joint stability, the maximum value for the vertical SIJ shear force was decreased in steps of 30 N (∼5% of the initial vertical SIJ shear force). Theoretically, lowering of the imposed vertical SIJ shear force to 0 N could induce a non-physiological equilibrium between muscle, ligament, and joint forces. In addition, when the model was set in 30° flexion, the force in the iliolumbar, the sacrotuberal, and the posterior sacroiliac ligaments was 250 N. This value was set as a maximum physiological ligament force in the simulation model in the upright position to prevent overloading of the pelvic ligaments that were implemented in the model. The following criteria were defined to warrant a physiological solution for muscle and ligament forces.Muscle tension must not exceed 240 kPa16;Lowering of the maximum vertical SIJ shear force must result in reduction of the total SIJ shear force (combination of vertical and horizontal shear);Ligament force must not exceed 250 N.
A muscle was included for further analysis when it produced at least 15% of the maximum muscle stress during the simulation. For all muscles, the maximum muscle stress depended on the calculated minimum muscle stress (see optimization criterion 1 in the Appendix). Two muscle groups were analyzed separately: (1) the muscles that increased at least 80% in force after the first simulation step and (2) the muscles that increased at least 10 times in force after completion of the simulation series.
Results
Table 2 summarizes the muscle (de)activation pattern when the maximum vertical SIJ shear force was stepwise decreased. Initially, vertical SIJ shear force was 563 N (on each side of the sacrum) at a trunk load of 500 N. The angle between the normal direction of the SIJ surface and the direction of the total SIJ reaction force was 81°, indicating that mainly vertical shear force acted through the SIJ, see Fig. 2a. Force equilibrium was mainly achieved by activation of M. abdominal oblique (internus and externus), M. iliacus, M. psoas, M. rectus abdominis, M. rectus femoris, M. tensor fasciae latae, and loading of the sacrotuberous ligament.
Table 2Summary of the structures that stabilize the sacroiliac joints in terms of lowered shearReduction of sacroiliac shearInitialPreset value (N)SIJ (vertical shear)563533503473443413383353323StructuresForce (N)M. adductor longus9181818189M. coccygeus1111241020M. iliococcygeus1111241020M. pubococcygeus126M. gluteus medius (lower)711131415274430M. gluteus medius (middle)58111010102941M. gluteus medius (upper)4290M. gluteus minimus (lower)333347M. gluteus minimus (middle)51011111151012M. gluteus minimus (upper)8171818175111625M. iliacus4747505458688885102M. obliquus externus abdominis2118171412M. obliquus internus abdominis15202023273429M. obturatorius externus1544458M. pectineus8181818186M. piriformis1826282522M. psoas (lower)64504627M. rectus abdominis272729313437517683M. rectus femoris343436394249646250M. sartorius16171819162214M. tensor fasciae latae313133353844588579M. transversus abdominis3556721325382Iliolumbar ligament53250250Posterior sacroiliac ligament26496273147250250250Sacrospinal ligament3815015914714513210674Sacrotuberous ligament20615121SIJ (compression)92121130142154229473607633SIJ (horizontal shear)−132−141−160−154−142−154−208−226−233Total SIJ shear579551528497465441436419398Angle of SIJ reaction force (°)817876747263433532Maximum muscle tension (kPa)37373942455369125247Included are those muscles that produced at least 15% of the maximum muscle stress after each simulationThe muscles printed italic increased at least 80% in force after the first simulation step. The muscles printed bold increased at least 10 times in force after completion of the simulation seriesFigure 2Directions of the force in the frontal plane exerted by the right ilium through the SIJ on the sacrum as a reaction to trunk load, Ftrunk. Panel (a): initial loading condition without limitation of the vertical shear component (563 N, see under “initial” in Table 2). This condition led to loading of the sacrotuberal ligaments, Fsacrotuberal lig. (solid thick line). Panel (b): loading condition with the vertical shear component preset at a 120 N lower level than the initial value (see under 443 N in Table 2). This condition led to loading of the sacrospinal ligaments, Fsacrospinal lig. (solid thick line). Panel (c): loading condition with the vertical shear component preset at a 240 N lower level than the initial value (see under 323 N in Table 2). In this situation, SIJ compression force increased by ∼400%, mainly by M. transversus abdominis, Ftransversus abdominis, and the pelvic floor, Fpelvic floor, muscle forces. The location of these muscles is schematically drawn by the thick solid lines, including the M. pubococcygeus, the M. iliococcygeus and the M. coccygeus (as drawn from the mid to the lateral position). It also led to loading of the iliolumbar ligaments to the maximum allowed force Filiolumbar lig. of 250 N, (solid thick line). 3-D images copyright of Primal Pictures Ltd. http://www.primalpictures.com
When the maximum vertical SIJ shear force was decreased from 563 to 443 N in steps of 30 N, the SIJ compression force increased by about 70%. Force equilibrium was obtained, amongst others, by activation of some of the muscles with a hip flexion component (M. adductor longus, M. iliacus, M. pectineus, and M. sartorius, M. rectus femoris) and some of the counteracting hip extensors (MM. gluteus medius and minimus and M. piriformis). Most of these muscles became (more) active after we lowered the maximum vertical SIJ shear force by 30 N. This led to unloading of the sacrotuberous ligaments and loading of the sacrospinal ligaments. The angle between the normal direction of the SIJ surface and the direction of the total SIJ reaction force was reduced to 72°, indicating that a combination of reduced vertical SIJ shear and increased SIJ compression could balance the trunk load on the sacrum, see Fig. 2b.
Further stepwise reduction of the vertical SIJ shear force resulted in a sharp rise of the maximum muscle stress. The simulation series ended with exceeding the maximum physiological muscle stress when the vertical SIJ shear force was decreased to about 60% of its initial value. Surprisingly, activation of some of the hip flexors and extensors had decreased or even disappeared. This was not the case for the MM. gluteus medius and minimus. In this simulation, force equilibrium was obtained by activation of the transversely oriented M. transversus abdominis (ventral to the SIJ) and the pelvic floor muscles, i.e., the M. coccygeus, the M. iliococcygeus, and the M. pubococcygeus (caudal to the SIJ). This resulted in further reduction of the angle between the normal direction of the SIJ surface and the direction of the total SIJ reaction force to 35°, see Fig. 2c. This indicates that the SIJ compression force, which increased by about 400% and the reduced vertical SIJ shear force, now clamped the sacrum between the coxal bones, see Fig. 2c. The MM. gluteus medius and minimus contributed to some extent to this increased compression due to a distinct force component in the transverse direction. To maintain force equilibrium, increased SIJ compression led to loading of the iliolumbar and posterior sacroiliac ligaments to the preset maximum value of 250 N.
Discussion
In the present study, the simulation model predicted muscle and ligament forces in the pelvic region when there was an imposed reduction of the vertical SIJ shear force. Initially, the forces acting through the SIJ were mainly vertical shear forces, see Fig. 2a. These forces were not only caused by trunk load, but also by muscles that acted in the longitudinal direction of the spine, for example the M. psoas and M. rectus abdominis. As a result of the forward bending moment, the sacrotuberous ligament was loaded. This large ligament protects the SIJ against excessive flexion of the sacrum relative to the coxal bones. The controlled reduction of the vertical SIJ shear force with 30 N forced some muscles that act as hip flexors and hip extensors to become active. Due to their transverse orientation, especially the MM. gluteus medius and minimus and M. piriformis contributed to the increased compression force between the coxal bones and the sacrum. However, these muscles did not contribute enough to self-bracing of the SIJ, because the total force through the SIJ still mainly acted in vertical direction. When the vertical SIJ shear was further reduced to about 60% of its initial value, the simulation model predicted that self-bracing mainly resulted from the transverse muscles ventrally (M. transversus abdominis) and caudally (pelvic floor) to the SIJ. In this situation, some of the hip flexors and extensors reduced in activity, for example the M. piriformis. Although the M. piriformis has a transverse orientation and crosses the SIJ, its contribution was minimized by the simulation program because this muscle also induces vertical SIJ shear force. The pelvic floor muscles, the M. coccygeus and M. pubo-, and iliococcygeus, contribute to the stabilization with respect to the sacrum. It has been suggested that this stabilization by force closure has an analogy with a classical stone arc.33 When sideways displacement of both ends of the arc is opposed, mechanical equilibrium of the stones is achieved by compression forces and not by shear forces. In the pelvis, the pelvic floor muscles may help the coxal bones to support the sacrum by compression forces, while shear forces between sacrum and coxal bones are minimized, see Fig. 3. Note that the SI compression force is defined as the force acting perpendicular to the SIJ surface. Therefore, decreasing or increasing this force will not alter the shear forces. The articular surfaces of the SIJ are irregular which results in bony interdigitation in the SI joint space. SIJ shear force calculated in the simulation model thus reflects the combination of real joint friction and friction due to this intermingling of bones. The real joint friction forces may be extremely small considering the extremely low coefficients of friction between the articular surfaces. The majority of the shear force is effectuated as normal contact pressures due to the bony interdigitation in the SI joint space. It was not possible to calculate the percentage of shear in terms of joint friction force. This requires a more detailed description of the SIJ surfaces.
Figure 3Analogy of pelvic bones supporting the trunk with a classical stone arc. The M. transversus abdominis and the pelvic floor muscles caudal to the SIJ mainly oppose lateral movement of the coxal bones. Spinal loading is transferred mainly by compression forces through the SIJ to the coxal bones and further down to the legs. 3-D images copyright of Primal Pictures Ltd. http://www.primalpictures.com
The simulation model predicts that simultaneous contraction of the M. transversus abdominis and pelvic floor muscles, i.e., the M. coccygeus, the M. iliococcygeus, and the M. pubococcygeus, contribute to lowering of the vertical SIJ shear forces, increasing of the SIJ compression and hence increasing of the SIJ stability. We emphasize that this simulation model was set up to estimate the forces acting in the pelvic region under static conditions and that the outcome of the simulations must be interpreted with caution.5 Nevertheless, in a previous study co-contraction was shown of pelvic muscles and M. transversus abdominis.26 This result and the prediction of our simulation model suggest that a protective mechanism against high SIJ shear forces may exist in humans. This mechanism has been investigated in vivo and in vitro. The contribution of the M. transversus abdominis to SIJ stability was shown in an in vivo study in patients with LBP.25 An in vitro study in embalmed human pelvises showed that simulated pelvic floor tension increased the stiffness of the pelvic ring in female pelvises.23 It is worthwhile to further investigate the contribution of both muscle groups simultaneously, not only during stiffness measurements of the SIJ but also during lumbo-pelvic stability tests based on increased intra-abdominal pressure (IAP). It was shown that the pelvic floor muscles, in combination with abdominal muscles and the diaphragm, may control and/or sustain IAP to increase lumbar spine stability as well.7,14
In the present study, the ligament forces were not allowed to exceed 250 N. The distribution between muscle and ligament forces depended on the maximum muscle stress as formulated in the first optimization scheme as presented in the Appendix. Increasing the maximum ligament forces might result in a lower maximum muscle stress, which could lead to a different muscle activation pattern to stabilize the SIJ. A small sensitivity test, however, showed that when the ligament forces were allowed to exceed the 250 N up to 500 N and in a next step up to 750 N, the model calculated a similar muscle activation pattern. The outcome of the present study also depended on the choice of optimization criteria and the magnitude of the cross-sectional areas of the muscles. The influence of different criteria was previously investigated for muscle forces in the leg.21 Indeed, various choices led to different calculated forces, but the obtained solutions were qualitatively similar, as was the case in our model. When we developed the model, other optimization criteria were also tested, for example minimization of the sum of muscle forces. However, minimization of the sum of squared muscle stresses yielded the most plausible solutions. The model cannot account for anatomical variations or detailed variation in muscle attachment sites. Obviously, direct comparison between the model predictions and the outcome of in vivo force measurements in the SIJ are not available, so there is no data to confirm the outcome of the present study. Nevertheless, EMG recordings of (superficial) abdominal and back muscles in various postures showed higher M. abdominal oblique internus activity when standing upright than resting on one leg and tilting the pelvic backwards.33 This muscle is considered as one of the self-bracing muscles of the SIJ. It was hypothesized that when standing on one leg, the shear load on the contralateral SIJ is diminished. Posterior tilt of the pelvis with less lumbar lordosis may than lead to less M. psoas major muscle load on the spine meaning less shear load on the SIJ. These findings indirectly support our findings that transversely oriented muscles reduce SIJ shear forces. We emphasize that the present model served as a tool to investigate the general relations between muscle and ligament forces in the pelvic region. The present simulations results may lead to the development of a new SIJ stabilizing training-program to reduce pain induced by high SIJ shear forces. The effectiveness of such a program, however, can only be tested with an intervention study.
The simulation model predicted unloading of the sacrotuberous and loading of the iliolumbar and posterior sacroiliac ligaments when the vertical SIJ shear was forced to reduce. This loading of the dorsal ligaments resulted from the absence of transversely oriented muscles at the dorsal side of the SIJ to counterbalance activation of the M. transverse abdominis at the ventral side of the SIJ. Loading of the iliolumbar ligament has been related to LBP.24 It was shown that in sitting position, the stepwise backward movement of an erect trunk (from upright position into a slouch) resulted in forward flexion of the spine combined with backward tilt of the sacrum relative to the pelvis.32 It was shown, that this movement into a sudden or sustained slouch might cause loading of the well-innervated iliolumbar ligaments near failure load.31 The co-contraction that exists between the deep abdominal M. transversus abdominis and the deep back extensor M. multifidus presumably retains lumbo-pelvic stability.13 In the future, we intend to extend the model with co-contraction between the M. transversus abdominis, the M. multifidus, and the pelvic floor muscles to study prevention of (over)loading of pelvic ligaments at different static postures.
Conclusions
Effective stabilization of the SIJ is essential in transferring spinal load via the SIJ to the coxal bones and the legs. A biomechanical analysis of the upright standing posture showed that activation of transversely oriented abdominal M. transversus abdominis and pelvic floor, i.e., M. coccygeus and M. pubo- and iliococcygeus muscles would be an effective strategy to reduce vertical SIJ shear force and thus to increase SIJ stability. The force equilibrium in this situation induced loading of the iliolumbar and posterior sacroiliac ligaments. The M. transversus abdominis crosses the SIJ and clamps the sacrum between the coxal bones. Moreover, the pelvic floor muscles oppose lateral movement of the coxal bones, which stabilizes the position of the sacrum (the pelvic arc).

sacroiliac joints
pelvis
pelvic floor muscles
static forces
human posture