Inflammatory Caspase Activity Mediates HMGB1 Release and Differentiation in Myoblasts Affected by Peripheral Arterial Disease

Abstract

Introduction: We previously showed that caspase-1 and -11, which are activated by inflammasomes, mediate recovery from muscle ischemia in mice. We hypothesized that similar to murine models, inflammatory caspases modulate myogenicity and inflammation in ischemic muscle disease. Methods: Caspase activity was measured in ischemic and perfused human myoblasts in response to the NLRP3 and AIM2 inflammasome agonists (nigericin and poly(dA:dT), respectively) with and without specific caspase-1 or pan-caspase inhibition. mRNA levels of myogenic markers and caspase-1 were assessed, and protein levels of caspases-1, -4, -5, and -3 were measured by Western blot. Results: When compared to perfused cells, ischemic myoblasts demonstrated attenuated MyoD and myogenin and elevated caspase-1 mRNA. Ischemic myoblasts also had significantly higher enzymatic caspase activity with poly(dA:dT) (p < 0.001), but not nigericin stimulation. Inhibition of caspase activity including caspase-4/-5, but not caspase-1, blocked activation effects of poly(dA:dT). Ischemic myoblasts had elevated cleaved caspase-5. Inhibition of caspase activity deterred differentiation in ischemic but not perfused myoblasts and reduced the release of HMGB1 from both groups. Conclusion: Inflammatory caspases can be activated in ischemic myoblasts by AIM2 and influence ischemic myoblast differentiation and release of pro-angiogenic HMGB1. AIM2 inflammasome involvement suggests a role as a DNA damage sensor, and our data suggest that caspase-5 rather than caspase-1 may mediate the downstream mediator of this pathway.

Keywords: caspase-5; inflammasomes; myoblasts; peripheral arterial disease.

Figure 1

Myoblasts harvested from ischemic muscle had attenuated differentiation compared to those from perfused segments. Myoblasts harvested from proximal (P) and distal (D) segments of muscle affected by PAD (N = 4) were compared to those from perfused controls (C) (N = 2) for markers of muscle or fibroblast origin, and differentiated over five days. (A,D) MyoD (* p < 0.03, ** p < 0.002, distal vs. proximal and control, respectively); (B,E) TE-7 (* p < 0.03, distal vs. control). Myocyte fusion index (MFI); (C,F) calculated by dividing the number of nuclei within multinucleated MF20-positive cells (>2) by the total number of nuclei (* p = 0.03, ischemic vs. control). Images are representative. Scale bar = 10 μM.

Figure 2

Differential mRNA expression suggested decreased myogenic differentiation and increased inflammatory caspase in ischemic myoblasts. Perfused (N = 2) and ischemic myoblasts (N = 3) were allowed to proliferate for 24 h and to differentiate for 5 or 10 days. RNA was extracted and probed for markers associated with muscle differentiation as well as inflammatory caspase-1. (A) MyoD; (B) myogenin; (C) Myh2; (D) IGF1; (E) caspase-1; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Cleaved caspase-5 and gasdermin D were elevated in ischemic myoblasts. Western blot was performed in perfused (N = 2) and ischemic myoblasts (N = 6) to determine the expression of caspase-1, caspase-3, gasdermin D, and cleaved caspase-5. Actin was used as a loading control. Normalization. kDa are shown. Relative density of each band compared to that of actin and calculated using ImageJ. (A) Western blot image of caspase-1, caspase-3, and gasdermin D. (B) Quantification of caspase-1 (NS). (C) Quantification of caspase-3 (NS). (D) Quantification of gasdermin D (* p < 0.03). (E) Cleaved caspase-5 Western blot with arrows denoting cleaved products. (F) Quantification of cleaved caspase-5 (p < 0.03, N = 3 perfused, 6 ischemic).

Figure 4

AIM2 expression was elevated and promoted inflammatory caspase activity in ischemic myoblasts. Expression of AIM2 and caspase-5 (total) were calculated in perfused and ischemic myoblasts using immunofluorescence. DAPI was used to detect nuclei, and mean intensity per cell was calculated using ImageJ for a total of 5 images per cell group (N = 2 control, N = 3–4 ischemic). (A,C) AIM2 immunofluorescence and quantification (**** p < 0.0001); (B,D) caspase 5 (*** p < 0.001). Luminescence assays were used to detect inflammatory caspase activity induced by caspase-1, -4, and -5 in perfused and ischemic cells with and without either caspase-1-specific or additional pan-caspase inhibition. Cells were additionally exposed to (E) poly(dA:dT), an AIM2 agonist (* p < 0.05, *** p < 0.001) and (F) nigericin, an NLRP3 agonist. Scale bar = 10 μM. (G) Detection of pro-and cleaved caspase 1 and caspase 5 by Western blots performed with perfused and ischemic myoblast with and without poly(dA:dT). ** p < 0.002 (N = 2 perfused, 4 ischemic).

Figure 5

Pan-caspase inhibition attenuated myocyte differentiation in ischemic myoblasts and reduced HMGB1 release during myocyte fusion. Perfused and ischemic (proximal and distal) myoblasts were induced to differentiate in the presence or absence of Z-VAD-FMK. (A) Cells were immunostained with antibodies against MF20, which detects myosin heavy chain 2, a marker of differentiation. Nuclei were detected using DAPI, and the myocyte fusion index (MFI) was calculated. (B) MFI in perfused and ischemic cells with and without Z-VAD-FMK (* p < 0.05, *** p < 0.001, **** p < 0.001). (C) Supernatants were collected and subjected to ELISA to determine HMGB1 concentration (* p < 0.03, ** p < 0.001). (D) Incucyte live cell imaging was performed to assess expression of apoptotic markers over time, with and without Z-VAD-FMK, and final count was assessed after five days (* p < 0.03, ** p < 0.01). Scale bar = 10 μM.