The inflammasome promotes adverse cardiac remodeling following acute myocardial infarction in the mouse

Abstract

Acute myocardial infarction (AMI) initiates an intense inflammatory response that promotes cardiac dysfunction, cell death, and ventricular remodeling. The molecular events underlying this inflammatory response, however, are incompletely understood. In experimental models of sterile inflammation, ATP released from dying cells triggers, through activation of the purinergic P2X7 receptor, the formation of the inflammasome, a multiprotein complex necessary for caspase-1 activation and amplification of the inflammatory response. Here we describe the presence of the inflammasome in the heart in an experimental mouse model of AMI as evidenced by increased caspase-1 activity and cytoplasmic aggregates of the three components of the inflammasome--apoptosis speck-like protein containing a caspase-recruitment domain (ASC), cryopyrin, and caspase-1, localized to the granulation tissue and cardiomyocytes bordering the infarct. Cultured adult murine cardiomyocytes also showed the inducible formation of the inflammasome associated with increased cell death. P2X7 and cryopyrin inhibition (using silencing RNA or a pharmacologic inhibitor) prevented the formation of the inflammasome and limited infarct size and cardiac enlargement after AMI. The formation of the inflammasome in the mouse heart during AMI causes additional loss of functional myocardium, leading to heart failure. Modulation of the inflammasome may therefore represent a unique therapeutic strategy to limit cell death and prevent heart failure after AMI.

Fig. 1.

Formation of the inflammasome in the granulation tissue and cardiomyocytes. Immunofluorescence shows overlap of cardiac actin (green) and the different components of the inflammasome [ASC (red, A–D), cryopyrin (red, E–H), and caspase-1 (red, I–L)] in the granulation tissue and border zone, 3 and 7 d after AMI (C, G, and K and D, H, and L, respectively), with minimal staining and overlap in the sham-operated mouse or the remote zone (A, E, and I and B, F, and J, respectively). Counterstaining with DAPI (blue). Original magnification 40×. (Scale bar, 20 μm.)

Fig. 2.

Cell type distribution of the inflammasome in acute myocardial infarction. The graph shows results of a semiquantitative assessment of ASC expression by immunofluorescence (examples in Fig. 1). Expression was quantified as intense (3+), moderate (2+), weak (1+), or no expression (0) (Materials and Methods) by three investigators separately and then averaged. Mean ± SEM values are reported (n = 3–4 per group).

Fig. 3.

Formation of the ASC inflammasome in isolated cardiomyocyte in culture. (A–C) Effects of LPS pretreatment (priming) and nigericin or ATP triggering on adult cardiomyocytes (HL-1) in culture. (A) Untreated cell culture. (B) Culture treated with LPS and nigericin. (C) Culture treated with LPS and ATP. ASC aggregates in the cytoplasm are evident in B and C (arrows). (D–F) Mean ± SEM of ASC staining expressed in a semiquantitative scale in which 1+ is mild expression and 3+ is intense expression (D), caspase-1 activity assessed using an enzymatic assay (N-Ac-Tyr-Val-Ala-Asp-CHO was used as caspase-1 inhibitor) (E), cell death assessed by either Trypan blue or TUNEL (F–H) in cultured adult HL-1 cardiomyocytes treated with LPS and triggered with nigericin, or ATP, or simulated ischemia. P2X7 inh refers to the use of a pharmacologic P2X7 inhibitor, PPADS (5 mM, equimolar to ATP), used as an ATP antagonist. Simulated ischemia refers to simulated ischemia in vitro obtained by exposing cells to hypoxia and an “ischemic” buffer. A caspase-1 inhibitor (casp-1 inh, benzyl-oxycarbonyl-Trp-Glu(OMe)-His-Asp(OMe)-fluoromethylketone) was added to determine whether cell death was caspase-1 dependent or not. *P < 0.05 vs. control conditions, ^†P < 0.05 for P2X7 inh vs. no treatment. Details of D–H are found in Materials and Methods.

Fig. 4.

Cryopyrin- and P2X7 targeted interventions in acute myocardial infarction. (A) Cryopyrin- and P2X7 targeted small interfering (si)RNAs administered intraperitoneally 24 h before surgery leading to a >80% reduction in target protein expression (Western blot) and a significant reduction in caspase-1 activity. Treatment with a pharmacological P2X7 inhibitor (PPADS 25 mg/kg intraperitoneally daily) provided a similar effect on caspase-1 activity. Values are expressed as mean ± SEM % of control values; n = 4–6 per group, *P < 0.05 vs. sham-operated mice. (B and C) Histology (Masson's trichrome) of cardiac left ventricle midsections of mice 7 d after AMI and treatment with vehicle (NaCl 0.9%) or a P2X7 inhibitor (PPADS), respectively. (D–F) Reductions in ASC expression (Western blot, day 3) and in infarct size (Masson's trichrome, day 7) and apoptosis (in situ end labeling of DNA fragmentation, day 7), and in mice treated with a P2X7 inhibitor (PPADS) vs. vehicle (NaCl 0.9%) after AMI. Values are expressed as mean ± SEM; n = 4–6 per group, *P < 0.05 vs. sham-operated mice, ^†P < 0.05 vs. control AMI. (G and H) Monodimensional echocardiography recordings obtained 7 d after AMI in mice treated with NaCl 0.9% (control) or a P2X7 inhibitor (PPADS), respectively. (I–N) Reductions in left ventricular enlargement (end-diastolic and end-systolic diameters) in mice 7 d after AMI treated with a P2X7 inhibitor (PPADS) vs. vehicle, without any difference in left ventricular hypertrophy. (O–R) Similar reductions in left ventricular enlargement (end-diastolic and end-systolic diameters) in mice 7 d after AMI treated with silencing RNA targeted to cryopyrin and P2X7 but not with scrambled siRNA sequences. Values are expressed as mean ± SEM; n = 6–8 per group, *P < 0.05 vs. sham-operated mice, ^†P < 0.05 vs. control AMI.

Fig. 5.

Central role of the inflammasome in acute myocardial infarction. Simplified scheme proposing the central roles of the P2X7 receptor and the ASC/cryopyrin inflammasome in the promotion of heart failure after AMI. ATP and other cellular debris released during ischemic myocardial necrosis activates the membrane P2X7 receptor channel and leads to cryopyrin activation by facilitating K⁺ efflux from the cell. Activated cryopyrin recruits the ASC scaffolding protein and procaspase-1 allowing for homodimerization and autocatalytic activation of caspase-1. Active caspase-1 cleaves pro–IL-1β and other proinflammatory cytokines into the active isoforms thus amplifying the inflammatory response (sterile inflammation). Active caspase-1 also triggers cell death, further contributing to the additional loss of functional myocardium and subsequent heart failure following AMI.