Chloroquine improves the response to ischemic muscle injury and increases HMGB1 after arterial ligation

Abstract

Objective: We have previously shown that exogenous administration of the nuclear protein high mobility group box 1 (HMGB1) improves angiogenesis after tissue ischemia. Antagonizing HMGB1 prolongs muscle necrosis and deters regeneration. In this study, we evaluated HMGB1 expression in peripheral arterial disease (PAD) and the mechanisms that promote its release in a murine model of hindlimb ischemia. Specifically, we investigated how chloroquine (CQ), a commonly employed disease-modifying antirheumatic drug, promotes HMGB1 release from muscle. We hypothesized that CQ could increase HMGB1 locally and systemically, allowing it to mediate recovery from ischemic injury.

Methods: Muscle biopsies were performed on patients undergoing lower extremity surgery for non-PAD-related disease as well as for claudication and critical limb ischemia. Clinical symptoms and ankle-brachial indices were recorded for each patient. HMGB1 was detected in muscle sections using immunohistochemical staining. Unilateral femoral artery ligation was performed on both wild-type and inducible HMGB1 knockout mice. Wild-type mice were administered intraperitoneal CQ 2 weeks before and after femoral artery ligation. Laser Doppler perfusion imaging was used to determine perfusion recovery. Serum and tissue levels of HMGB1 were measured at designated time points. In vitro, cultured C2C12 myoblasts were treated with increasing doses of CQ. HMGB1, autophagosome formation, p62/SQSTM1 accumulation, caspase-1 expression and activity, and lactate dehydrogenase levels were measured in supernatants and cell lysates.

Results: Nuclear expression of HMGB1 was prominent in patients with claudication and critical limb ischemia (P < .05) compared with controls. CQ-treated mice had elevated serum HMGB1 and diffuse HMGB1 staining in muscle (P < .01). In wild-type mice, CQ treatment resulted in higher laser Doppler perfusion imaging ratios in the ischemic limb at 7 days (P < .03) and less fat replacement after 2 weeks (P < .03). In cultured myoblasts, CQ induced autophagosome accumulation, inhibited p62/SQSTM-1 degradation, and activated caspase-1.

Conclusions: HMGB1 is prominently expressed in PAD muscle but mostly confined to the nucleus. Our in vivo data suggest that HMGB1 mobilization into the sarcoplasm and serum can be increased with CQ, possibly through caspase-1-mediated pathways. Whereas HMGB1 can be released by many cell types, these studies suggest that the muscle may be an important additional source that is relevant in PAD.

Figure 1. HMGB1 expression in peripheral arterial disease (PAD)

HMGB1 staining in human skeletal muscle was performed on serial muscle sections obtained from patients A) without PAD or with chronic limb ischemia in the form of B) claudication or C) critical limb ischemia (CLI). Images were obtained using an Olympus FV1000 scanning confocal microscope with a 40X objective. D) Quantification of HMGB1 integrated density was performed using Image J analysis software on color separated images obtained from human samples *P<.03, N=5–7 patients/group, ANOVA. Standard hematoxylin and eosin staining was performed to assess the general architecture of muscles from E) control, F) claudication and G) CLI patients. Scale bar = 50μm

Figure 2. CQ effects on muscle and serum expression of HMGB1

Anterior tibialis muscle from mice pretreated for two weeks with intraperitoneal phosphate buffered saline (PBS) or chloroquine (CQ) before and 7 days after femoral artery ligation (FAL) is shown. The HMGB1 staining and quantification were performed on muscle sections from mice sacrificed 7 days after arterial ligation. Sections were stained for HMGB1 using primary and Cy-3 conjugated secondary antibody (red). Muscle actin (green) and DAPI (blue) are also shown to highlight the fibers and nuclei, respectively. Images were obtained using an Olympus FV1000 scanning confocal microscope with a 40X objective. Merged images (A and C) as well as color-separated images that represent HMGB1 staining (B and D) are shown. Arrows represent areas of HMGB1 staining in both nuclear and sarcoplasmic areas. E) Quantification of HMGB1 integrated density was calculated using Image J analysis using color-separated images. *P< .01, N=4–6 mice/group, t-test. F) Serum was obtained from cardiac puncture at the time of sacrifice before or 1 and 7 days following femoral artery ligation. ELISA was performed on serum samples to assess HMGB1 concentration. *P<.01 CQ to PBS, N= 5–7mice/group, t-test at each time point. G) LDPI was performed to assess perfusion ratios in ischemic/nonischemic limbs following FAL in mice treated with PBS or CQ before and after FAL. *P<0.01 day 1 to baseline and day 7 to day 1,** p<0.03 day 7 CQ to day 7 PBS; N=4–6 mice/group, t-test comparing PBS and CQ. Scale bar = 50μm

Figure 3. Perfusion recovery in HMGB1KO mice

A) Laser Doppler perfusion imaging (LDPI) is shown from wild type C57Bl/6J and inducible HMGB1 knockout (iHMGB1KO) mice before, and 1, 7 and 14 days following femoral artery ligation (FAL). (+) indicates the ligated limb. (-) indicates the nonligated limb with uninterrupted perfusion. B) The mean ischemic/nonischemic perfusion ratio was compared between groups at each time point using t-test. *P<.01, N=3–4 mice/group, t-test iHMGBKO to C57Bl/6J

Figure 4. Histologic findings following arterial ligation with and without chloroquine (CQ)

Hematoxylin and eosin (H&E) staining demonstrates fat replacement (arrow) regenerating muscle (asterisk) and mature muscle (arrowhead) in PBS (A) and CQ (B) treated mice after 14 days. Regenerating myocytes are identified by their circular shape and centrally located nuclei. Mature myocytes are identified by peripheral nuclei. Fat is identified by rounded, septate and clear spaces. CD31 (green), and CD45 (red) antigen detection was determined using immunohistochemistry, and imaged on an Olympus FV1000 confocal microscope. Mean Fluorescence Intensity (MFI) was determined for each section for the right and left hindlimbs. The right, ischemic hindlimbs are shown for PBS (C) and CQ (D) treated mice. E) Quantification of fat replacement between PBS and CQ treated groups was calculated by measuring the area of fat and expressing it as a percentage of the total muscle area. Four-six images per animal were used to determine percent fat replacement. **P<.03; N=5–6 mice/group, t-test. Mature myocytes were not quantified but predominated in CQ treated mice. F) Quantification of CD31 Mean Fluorescent Intensity (MFI) *P<0.03, PBS vs. CQ, N=4–6 amice/group, t-test. G) Quantification of CD45 MFI *P<0.03, ischemic vs. nonischemic, N=4–6 mice/group, t-test. Scale bar = 25μm

Figure 5. Effects of chloroquine (CQ) on cultured C2C12 myoblasts

A) ELISA was used to determine HMGB1 concentration in the supernatant of CQ treated C2C12 myoblasts. *P<.05, **p<.005 compared to no CQ; N=3 experiments performed in triplicate, t-test. B) Western blot of C2C12 myoblast lysates (B) and supernatants (C) after exposure to CQ at designated concentration for 18 hours. Lysates were assessed for P62/SQSTM1, LC3I-II conversion, HMGB1 and actin expression. Supernatants were concentrated and evaluated for HMGB1 expression. D) WB showing procaspase-1 and activated caspase-1 (20Kd subunit) from C2C12 myoblasts treated with CQ at designated concentration. E) LC3II and F) activated caspase-1 expression normalized to β-actin in response to CQ is shown relative to untreated cells. *P<.05; N=3 experiments performed in triplicate, t-test.

Figure 6. Caspase-1 and LDH activity in cultured C2C12 myoblasts exposed to chloroquine (CQ)

A) Images depicting C2C12 myoblast morphology in culture after exposure to CQ for 18 hours showing maintenance of cell numbers up to 25 μM. B) Caspase-1 activity assays were performed on CQ treated cell lysates and expressed as a ratio to untreated (0) samples. *P<.03, **P<.005; N=4 experiments, t-test to no treatment C) Lactate dehydrogenase (LDH) assays to assess cell damage was performed on supernatants and expressed as a function of maximum lysis and cell leakage which is a positive control. Numbers indicate CQ concentration. *P<.05; **P<.01, N=3 experiments performed in triplicate, t-test to maximally lysed samples.

Figure 7. Mechanisms linking autophagy, HMGB1 and inflammasome signaling

A proposed mechanism linking autophagy, inflammasome activation and HMGB1 release from myoblasts is shown. It is known that HMGB1 requires acetylation to leave the nucleus, which can be facilitated by histone deacetylases and JAK-STAT signaling. It is also known that in other cell types, inflammasomes activate caspase-1 and initiates release of HMGB1 as well as IL1-β and IL-18. Lysosomal disruption with agents like CQ may initiate inflammasome activation, and promote the release of HMGB1 from myoblasts. This is a novel area of investigation in PAD.