Table 4.

Evidence of pyroptosis in MI: reports from in vivo studies.

Disease Condition	Model	Pyroptotic marker				IS%	LV function	Cardiac injury	Inflammatory marker/ROS	Other cell death marker/Relevant findings	Interpretation	Ref.
Ischemia		GSDMD		Caspase-1	Others
		Active form	Full form
10 min	C57BL/6 mice	–	↔	↔	↔ NLRP3 ↔ Caspase-4	–	–	–	↔ IL-1β ↔ IL-18	↔ CF genes ↔ DEGs, DEPs of immune and apoptosis ↔ Caspase-3	GSDMD was activated in both canonical and non-canonical pathways, other molecular changes were adapted after 6 h of MI, with inflammatory cells infiltration. They continued to increase in a time dependent manner, leading to cardiac fibroblast activation at 72 h.	42, 43
1 h		–	↔	↑	↔ NLRP3 ↔ Caspase-4	–	–	–	↔ IL-1β ↔ IL-18	↔ CF genes ↔ DEGs, DEPs of immune and apoptosis ↔ Caspase-3
6 h		–	↑	↑	↑ NLRP3 ↑↑ Caspase-4	–	–	–	↑↑ IL-1β ↔ IL-18 ↑ Inflammatory cells ↑ %CD8	↔ CF genes ↑ DEGs, DEPs of immune and apoptosis ↔ Caspase-3
24 h		–	↑	↑	↑ NLRP3 ↑ Caspase-4	–	↓ %EF	–	↑↑ IL-1β ↑ IL-18 ↑ Inflammatory cells ↑ %CD8 ↑ Monocyte ↑ M0- macrophage	↔ CF genes ↑ DEGs, DEPs of immune and apoptosis ↑ Caspase-3
72 h		–	↑↑	↑↑	↑ NLRP3 ↑↑ Caspase-4	–	↓ %EF	–	↑ IL-1β ↑↑ IL-18 ↑↑Inflammatory cells ↑ %CD8 ↑ Monocyte ↑ M0- Macrophage	↑ CF genes ↑ DEGs, DEPs of immune and apoptosis ↑↑ Caspase-3
12 h	C57BL/6 mice	↑	–	↑	↑ ASC ↑ NLRP3	–	↓ %EF ↓ %FS ↑ LVIDd ↑ LVIDs	–	↑ IL-1β ↑ IL-18	↓ GDF11 ↓ HOXA3 ↑ Mitochondrial damage	MI activated GSDMD through GDF11/HOXA3 in canonical pathway, leading to pyroptosis, mitochondrial damage, and LV dysfunction.	42, 43
	C57BL/6 mice + GDF11 overexpression vs. WT-MI	↓	–	↓	↓ ASC ↓ NLRP3	–	↑ %EF ↑ %FS ↓ LVIDd ↓ LVIDs	–	↓ IL-1β ↓ IL-18	↑ Myofibrils arrangement ↓ Mitochondrial damage
24 h	C57BL/6 mice	↑	↑	↑	↑ NLRP3	↑	↓ %EF ↓ %FS	↑ LDH	↑ IL-1β ↑ IL-18	↑ DNA fragmentation ↑ Mitochondrial swelling ↑ Cytoplasmic membrane pore formation	MI activated GSDMD in canonical pathway, leading to pyroptosis, mitochondrial damage, and LV dysfunction.	42, 43
24 h	C57BL/6 mice	↑	–	↑	↑ NLRP3	↑	↓ %EF ↓ %FS	↑ LDH	↑ TLR4 ↑ NF-κB ↑ IL-1β ↑ IL-18	–	MI activated GSDMD in canonical pathway, leading to pyroptosis, and LV dysfunction.	42, 43
1 day	SD rats	↑	↑	↑	↑ NLRP3	↑	↓ %EF ↓ %FS ↑ LVIDd ↑ LVIDs		↑ ROS ↑ TAK1 ↑ p-JNK ↑ IL-1β ↑ IL-18	↔ CVF	MI increased oxidative stress, activated TAK1/JNK pathway, and activated GSDMD in canonical pathway, resulting in pyroptosis and LV dysfunction. The deterioration of LV function was in a time dependent manner, and increase of collagen volume was observed only at 7 days post–MI.	42, 43
3 days		↑	↑	↑	↑ NLRP3	↑↑	↓↓ %EF ↓↓ %FS ↑↑ LVIDd ↑↑ LVIDs		↑↑ ROS ↑ TAK1 ↑ p-JNK ↑ IL-1β ↑ IL-18	↔ CVF
7 days		↑	↑	↑	↑ NLRP3	↑↑↑	↓↓↓ %EF ↓↓↓ %FS ↑↑↑ LVIDd ↑↑↑ LVIDs		↑↑↑ ROS ↑ TAK1 ↑ p-JNK ↑ IL-1β ↑ IL-18	↑ CVF
72 h	SD rats	↑	↔	↑	↑ ASC ↑ NLRP3	↑	↓ %EF ↓ %FS ↑ LVIDd ↑ LVIDs	↑ cTnI ↑ LDH	↑ TLR4 ↑ MYD88 ↑ NF-κB ↑ IL-1β ↑ IL-18	↓ ATP ↓ ADP ↓ AMP ↓ ATP/ADP ratio ↓ Total adenine nucleotide	MI activated GSDMD in canonical pathway, leading to pyroptosis, ATP depletion, and LV dysfunction.	42, 43
3 days	C57BL/6 mice	↑	↑	↑	↑ ASC ↑ NLRP3	–	↓ %EF ↓ %FS ↑ LVIDd ↑ LVIDs ↑ LVvold ↑ LVvols	–	↑ MDA ↑ ROS ↓ GSH-PX ↓ SOD ↑ IL-1β ↑ IL-18	↑ Heart RBP4 ↔ Serum RBP4 ↔ Retinol ↔ Retinyl ester	MI increased oxidative stress to activate GSDMD through RBP in canonical pathway, leading to pyroptosis and LV dysfunction. However, serum RBP4, retinol or retinyl ester did not change following MI.	42, 43
	C57BL/6 mice + sh-RBP4 vs. WT + MI	↓	↓	↓	↓ ASC ↓ NLRP3	↓	↑ %EF ↑ %FS ↓ LVIDd ↓ LVIDs ↓ LVvold ↓ LVvols	↓ ANP ↓ BNP ↓ MHC-7 ↓ LDH	↓ IL-1β ↓ IL-18	↓ Heart RBP4
3 days	WT-FVB/NJ mice	↑	–	↑	–	↑	↓ %EF ↓ %FS ↑ LVIDs ↑ LVvols (3 days) ↑ LVIDd ↑ LVvold (7 days)	–	–	↑ CLMP	MI increased CLMP expression to prevent pyroptosis and LV dysfunction following MI. Knockdown CLMP led to more serious myocardial injury through promoting non-NLRP3-dependent pyroptosis but not necroptosis or parthanatos.	42, 43
	CLMP+/− FVB/NJ mice vs. WT + MI	↑↑	–	↑↑	↔ NLRP3	↑↑	↓↓ %EF ↓↓ %FS (14 days) ↑↑ LVIDd ↑↑ LVIDs ↑↑ LVvols ↑↑ LVvold (7 days)	↑ LDH	↑ IL-1β ↑ MPO ↑ Ly6g	↓ CLMP ↔ RIPK3 ↔ CaMKII ↔ PARP
1 week	C57BL/6 mice	↑	–	↑	↑ ASC ↑ NLRP3	↑	–	–	↑ IL-1β ↑ IL-18	↑ PCSK9	Upregulation of PCSK9 involved in the activation of GSDMD in canonical pathway to induce cardiac pyroptosis, which was confirmed by genetic inhibition.	42, 43
	PCSK9−/− C57BL/6 mice vs. WT-MI	↓	↔	↓	↓ ASC ↓ NLRP3	–	–	↓ LDH	↓ IL-1β ↓ IL-18	–
1 week	C57BL/6 mice	↑ GSDMD-N per GSDMD-FL ratio		↑	↑ ASC ↑ NLRP3	↑	↓ %EF ↓ %FS ↑ LVIDd ↑ LVIDs ↑ LVvols ↑ LVvold		↑ p-NF-κB/NF-κB ↑ nuNF-κB ↓ cytoNF-κB ↑ IL-1β ↑ IL-18 ↑ IL-6 ↑ TNF-α ↑ iNOS ↑ MAC-3+ CLEC5A+ ↑ MAC-3+ iNOS+	↑ CLEC5A	Upregulation of CLEC5A involved in the activation of GSDMD in canonical pathway to induce cardiac pyroptosis, cytokine release, and LV dysfunction, which was confirmed by genetic inhibition.	54
	C57BL/6 mice + sh-CLEC5A vs. WT-MI	↓ GSDMD-N per GSDMD-FL ratio	↓	↓ ASC ↓ NLRP3	↓	↑ %EF ↑ %FS ↓ LVIDd ↓ LVIDs ↓ LVvols ↓ LVvold		↓ p-NF-κB/NF-κB ↓ nuNF-κB ↑ cytoNF-κB ↓ IL-1β ↓ IL-18 ↓ IL-6 ↓ TNF-α ↓ iNOS ↓ MAC-3+ CLEC5A ↓ MAC-3+ iNOS+	↓ CLEC5A
1 week	F344 rats	↑	–	↑	↑ ASC ↑ NLRP3	↑	↓ %EF ↑ LVIDd ↑ Heart weight/tibial length	–	↑ IL-1β ↑ IL-18	↑ TUNEL ↑ Cell swelling↑ Irregular nuclear arrangement ↑ Neutrophil infiltration	MI activated GSDMD in canonical pathway, leading to pyroptosis, LV dysfunction, and LV remodeling.	42, 43
4 weeks	SD rats	↑ GSDMD-N per GSDMD-FL ratio		↑	↑ ASC ↑ NLRP3	–	↓ %EF ↓ %FS ↑ LVIDd ↑ LVIDs	↑ BNP	↑ MDA ↑ •OH ↑ IL-1β	↑ Mitochondrial damage ↑ Fibrosis	MI increased oxidative stress to activate GSDMD in canonical pathway, leading to pyroptosis, mitochondrial damage, LV remodeling, and LV dysfunction.	18
CME induction (Post CME 3 days)	C57BL/6 mice		↑ GSDMD-N per GSDMD-FL ratio	↑	↑ NLRP3	↑	↓ %EF ↓ %FS	↑ LDH	↑ SDHA ↑ SDHB ↑ ROS ↑ IL-1β	↑ Collagen deposit ↑ Vacuolated and malformed mitochondria	CME increased oxidative stress and GSDMD in canonical pathway, leading to pyroptosis, mitochondrial dysfunction, LV remodeling, and LV dysfunction.	43

·OH: hydroxyl radical; ADP: adenosine di-phosphate; AMP: adenosine monophosphate; ASC: apoptosis-associated speck-like protein containing a caspase recruitment domain; ATP: adenosine triphosphate; BNP: B-type natriuretic peptide; CaMKII: calcium/calmodulin-dependent protein kinase II; CD8: T cell CD8; CF: cardiac fibroblasts; CLEC5A: C-type lectin membrane 5 A; CLMP: CXADR-like membrane protein; CME: coronary microembolization; CVF: collagen volume fraction; cyto NF-κB: the expression of NF-κB in cytosol; DEGs: differentially expressed genes; DEPs: differentially expressed proteins; DNA: deoxyribonucleic acid; %EF: left ventricular ejection fraction; %FS: left ventricular fractional shortening; GDF11: growth differentiation factor 11; GSDMD: Gasdermin D; GSDMD-FL: full-length Gasdermin D; GSDMD-N: N-terminal Gasdermin D fragment; HOXA3: homeobox A3; I: ischemia by coronary artery ligation; IL: interleukin; iNOS: inducible Nitric oxide synthase; IS%: infarct size/area at risk; LDH: lactate dehydrogenase; LV: left ventricle; LVIDd: LV internal dimension at end-diastole; LVIDs: LV internal dimension at end-systole; LVvold: LV volume at end-diastole; LVvols: LV volume at end-systole; Ly6g: lymphocyte antigen six complex locus G6D; MAC-3: macrophage marker MAC-3; MDA: malondialdehyde; MI: myocardial ischemia; MPO: myeloperoxidase; MYD88: myeloid differentiation factor 88; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; nu NF-κB: the expression of NF-κB in nuclues; NLRP3: NACHT, LRR and PYD domains-containing protein 3; nu p65: nuclear NF-κB-p65; p-JNK: phosphorylated Jun N-terminal kinase; PARP: Poly (ADP-ribose) polymerase; PCSK9: proprotein convertase subtilisin/Kexin type 9; PCSK9^−/−: PCSK9 knockdown; R: reperfusion; RBP4: retinol-binding protein 4; RIPK3: receptor-interacting protein kinase 3; ROS: reactive oxygen species; SD rats: Sprague–Dawley rats; SDHA: succinate dehydrogenase complex flavoprotein subunit A; SDHB: succinate dehydrogenase complex flavoprotein subunit B; SOD: superoxide dismutases; TAK1: transforming growth factor-β-activated kinase 1; TLR4: toll-like receptor 4; TNF-α: tumor necrosis factor alpha; TXNIP: thioredoxin-interacting protein; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; WT: wild type.