Table 4 |.

Experiments to address criteria for establishing MMP causality

Criterion addressed	Experiment	Establishes
1	qRT-PCR, immunoblotting, in situ hybridization, and immunohistochemistry of plasma, infarct tissue, or isolated cells	Changes in MMP levels after MI and which cells express the MMP
2	Gene deletion or overexpression, pharmacological agents (MMP inhibitors), and other blocking strategies (antisense oligonucleotides and small interfering RNA) Outputs: left ventricular physiology and remodelling, cell physiology, and gene or protein expression	MMP is in the signalling pathway
3	Stimulate naive cardiac cells with secretome of post-MI, MMP9-modified cells, use proteomics to identify secretome constituents, and use blocking antibodies to assign cause and effect relationships Outputs: cell physiology and gene or protein expression	Intercellular communication is paracrine
	Cell co-cultures	Intercellular communication requires direct contact
4	Proteomics (matridomics and degradomics) to identify MMP substrates, surface plasmon resonance binding assays for kinetics assessment; might need to consider complexity of effects when two or more MMPs and two or more substrates are involved, which mimics in vivo setting	MMP substrate profile and hierarchy of substrate preferences and MMP preferences
	Infuse substrate fragments to mimic MMP effects Outputs: in vitro and in vivo signalling and cell physiology, left ventricular physiology and remodelling	Substrate is downstream of MMP, and proteolysis is necessary and sufficient to recapitulate MMP phenotype

MI, myocardial infarction; MMP, matrix metalloproteinase; qRT-PCR, quantitative real-time polymerase chain reaction.