Table 2.

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

Condition/Hypoxia (h)	Model	Pyroptotic marker				Inflammatory marker /ROS	Cell viability /Toxicity	Other cell death marker/Relevant finding	Interpretation	Ref.
		GSDMD		Caspase-1	Others
		Active form	Full form
2	H9C2 cells	↑	–	↑	↑ NLRP3	↑ TLR4 ↑ NF-κB ↑ IL-1β ↑ IL-18	↑ LDH	–	Hypoxia activated GSDMD in canonical pathway, leading to cardiomyoblast pyroptosis.	38
4	NRCMs	↑ GSDMD-N per GSDMD-FL ratio		↑	↑ ASC ↑ NLRP3	↑ IL-1β	↓ Cell viability ↑ LDH	↑ PI	Hypoxia activated GSDMD in canonical pathway, leading to cardiomyocyte pyroptosis.	18
6	H9C2 cells	↑	–	↑	↑ ASC ↑ NLRP3	↑ IL-1β ↑ IL-18	↓ Cell viability	↑ TUNEL	Hypoxia activated GSDMD in canonical pathway, leading to cardiomyoblast pyroptosis.	39
6	NMVCMs	↑	↑	↑	↑ ASC ↑ NLRP3	↑ IL-18 ↑ MDA ↑ ROS ↓ GSH-PX ↓ SOD	↓ Cell viability ↑ LDH	↑ RBP4 ↑ PI	Upregulation of RBP4 involved in GSDMD activation in canonical pathway inducing cardiomyocyte injury and pyroptosis during hypoxia, which was confirmed by both genetic and pharmacological inhibition.	40
	NMVCMs + RBP4 overexpression	↑↑	↑↑	↑↑	↑↑ ASC ↑↑ NLRP3	↑↑ IL-18 ↑↑ MDA ↑↑ ROS ↓↓ GSH-PX ↓↓ SOD	↓↓ Cell viability ↑↑ LDH	↑↑ RBP4 ↑↑ PI
	NMVCMs + sh-RBP4 vs. hypoxia	↓	↓	↓	↓ ASC ↓ NLRP3	↓ IL-18 ↓ MDA ↓ ROS ↑ GSH-PX ↑ SOD	↑ Cell viability ↓ LDH	↓ RBP4 ↓ PI
	NMVCMs + RBP4 overexpression + si-NLRP3 vs. RBP4 overexpression + hypoxia	↓	↓	↓	↓ ASC ↓ NLRP3	↓ IL-18	↓ LDH	↔ RBP4 ↓ PI
	NMVCMs + RBP4 overexpression + MCC950 (NLRP3 inhibitor) vs. RBP4 overexpression + hypoxia	↓	↓	↓	↓ ASC ↓ NLRP3	↓ IL-18	↓ LDH	↔ RBP4 ↓ PI
12	NMCMs	↑	–	↑	↑ ASC ↑ NLRP3	–	↓ Cell viability	↓ GDF11 ↓ HOXA3	Downregulation of GDF11/HOXA3 involved in GSDMD activation in canonical pathway to induce cardiomyocyte injury and pyroptosis during hypoxia, which was confirmed by genetic overexpression.	41
	NMCMs + GDF11 overexpression vs. hypoxia	↓	–	↓	↓ ASC ↓ NLRP3	–	↑ Cell viability	–
	NMCMs + HOXA3 overexpression vs. hypoxia	↓	–	↓	↓ ASC ↓ NLRP3	–	–	–
24	NMCMs	↑	↑	↑	↑ NLRP3	↑ IL-1β ↑ IL-18	–	↑ TUNEL ↑ PI	Hypoxia activated GSDMD in canonical pathway, leading to cardiomycyte pyroptosis.	27
48 h	Primary cardiomyocyte	–	–	–	–	–	↓ Cell viability	↑ PCSK9	Upregulation of PCSK9 induced oxidative stress which led to GSDMD activation in canonical pathway to induce cardiomyocyte injury and pyroptosis during hypoxia, which was confirmed by genetic inhibition.	30
	PCSK9−/− HL-1 cells	↓	–	↓	↓ ASC ↓ NLRP3	↓ IL-1β ↓ ROS	↓ LDH	–
	HL-1 cells + hrPCSK9 vs. hypoxia	↑	–	↑	↑ ASC ↑ NLRP3	↑ IL-1β ↑ ROS	↑ LDH	–
	HL-1 cells + PCSK9CRISPRavs. hypoxia	↑	–	↑	↑ ASC ↑ NLRP3	↑ IL-1β ↑ ROS	↑ LDH	–
12 (+TNF-α)	H9C2 cells	↑	–	↑	↑ NLRP3	↑ NOX4 ↑ ROS	↓ Cell viability ↑ LDH	↓ SIRT1 ↑ PI	Hypoxia with TNF-α increased oxidative stress, and reduced SIRT1 to activate GSDMD in canonical pathway resulting in cardiomyoblast injury and pyroptosis.	42,43
		↑ GSDMD-N per GSDMD-FL ratio
OGD 36 h	H9C2 cells	↑	↑	↑	↑ ASC ↑ NLRP3	↑ p-NF-κB ↑ ROS ↓ SOD	↑ LDH	–	OGD increased oxidative stress to activate GSDMD in canonical pathway, leading to cardiomyoblast pyroptosis.	42, 43
H2O2 1, 2, 3 h	AMVCMs	↑	↑	–	–	–	–	–	Oxidative stress induced the activation of GSDMD, which is an executioner of cardiomyocyte pyroptosis.	42, 43

AMVCMs: adult mouse ventricular cardiomyocytes; ASC: apoptosis-associated speck-like protein containing a caspase recruitment domain; GDF11: growth differentiation factor 11; GSDMD: Gasdermin D; GSDMD-FL: full-length Gasdermin D; GSDMD-N: N-terminal Gasdermin D fragment; GSH-PX: glutathione peroxidase; H: hypoxia; H/R: hypoxia/reoxygenation; H₂O₂: hydrogen peroxide; HCEMCs: human cardiac microvascular endothelial cells line; HOXA3: homeobox A3; IL: interleukin; LDH: lactate dehydrogenase; MDA: malondyaldehyde; NF-κB: nuclear factor κ-light-chain-enhancer of activated B cells; NLRP3: NACHT, LRR and PYD domains-containing protein 3; NMCMs: neonatal mouse cardiomyocytes; NMVCMs: neonatal mouse ventricular cardiomyocytes; NOX4: nicotinamide adenine dinucleotide phosphate oxidase 4; NRCMs: neonatal rat cardiomyocytes; OGD: oxygen–glucose deprivation; PCSK9: proprotein convertase subtilisin kexin type 9; PI: propidium iodide; R: reoxygenation; RBP4: retinol-binding protein 4; ROS: reactive oxygen species; sh: short hairpin; si-: short interfering; SIRT: sirtuin; SOD: superoxide dismutases; TLR4: toll-like receptor 4; TNF-α: tumor necrosis factor alpha; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.