NLRP3 inflammasome activation during myocardial ischemia reperfusion is cardioprotective.

BACKGROUND: The innate immune receptor NLRP3 recognizes tissue damage and initiates inflammatory processes through formation multiprotein complexes with the adaptor protein ASC and caspase-1, i.e. NLRP3 inflammasomes, which through cleavage of pro-IL-1ß mediates release of bioactive IL-1ß. We hypothesized that NLRP3 mediates tissue damage during acute myocardial infarction (MI) and sought to investigate the mechanisms herein in an experimental MI model in mice. METHODS AND RESULTS: The left coronary artery (LCA) of WT, NLRP3(-/-) and ASC(-/-) mice of both genders was ligated for 30 min followed by 3 or 24 h reperfusion. For pre-conditioning studies, the TLR2 agonist Pam3CSK4 or PBS was injected intraperitoneally 60 min prior to LCA ligation. For mechanistic investigations, blood plasmas and left ventricle tissues were collected, and a hypothesis-driven selection of protein or mRNA targets was investigated. Surprisingly, hearts from NLRP3-deficient mice featured larger infarct size than WT mice (p = 0.0048). In general, there were only modest changes with no significant pattern in myocardial infiltration of neutrophils and macrophages and systemic and myocardial cytokine expression between the three genotypes. Preconditioning with the TLR2 agonist Pam3CSK4 induced Akt phosphorylation and reduced infarct size in WT but not NLRP3 -or ASC -deficient hearts. CONCLUSION: Absence of NLRP3 results in increased myocardial infarct size after in vivo ischemia reperfusion, seemingly due to dysfunction of the cardioprotective RISK pathway. Our data imply that NLRP3 contributes to cardio-protection during I/R and do not support a role for NLRP3 or ASC inhibition in the management of acute MI including revascularization therapy.

Mitochondrial DNA damage and repair during ischemia-reperfusion injury of the heart.

Ischemia-reperfusion (IR) injury of the heart generates reactive oxygen species that oxidize macromolecules including mitochondrial DNA (mtDNA). The 8-oxoguanine DNA glycosylase (OGG1) works synergistically with MutY DNA glycosylase (MYH) to maintain mtDNA integrity. Our objective was to study the functional outcome of lacking the repair enzymes OGG1 and MYH after myocardial IR and we hypothesized that OGG1 and MYH are important enzymes to preserve mtDNA and heart function after IR. Ex vivo global ischemia for 30min followed by 10min of reperfusion induced mtDNA damage that was removed within 60min of reperfusion in wild-type mice. After 60min of reperfusion the ogg1(-/-) mice demonstrated increased mtDNA copy number and decreased mtDNA damage removal suggesting that OGG1 is responsible for removal of IR-induced mtDNA damage and copy number regulation. mtDNA damage was not detected in the ogg1(-/-)/myh(-/-), inferring that adenine opposite 8-oxoguanine is an abundant mtDNA lesion upon IR. The level and integrity of mtDNA were restored in all genotypes after 35min of regional ischemia and six week reperfusion with no change in cardiac function. No consistent upregulation of other mitochondrial base excision repair enzymes in any of our knockout models was found. Thus repair of mtDNA oxidative base lesions may not be important for maintenance of cardiac function during IR injury in vivo. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease."

Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts.

In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability (R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, ß-actin and 18S rRNA showed stable expression per-unit-weight tissue and a positive correlation with infarct size (R = 0.61 and R = 0.77, P < 0.05 for both analyses). The amount of total RNA extracted per-unit-weight tissue did not differ between groups despite wide variation in infarct size (7.1-50.1%). When ß-actin expression was assessed using four different normalization strategies, GAPDH and geNorm provided appropriate per-unit-living expression, while 18S and total RNA resulted in marked underestimations. In studies of ischemic tissues, we recommend using geometric averaging of carefully selected reference genes for normalization of real-time RT-PCR data. A marked shift in the mRNA/rRNA ratio renders rRNA as useless for normalization purposes.

Mode of perfusion influences infarct size, coronary flow and stress kinases in the isolated mouse heart.

AIM: The isolated, retrogradely perfused heart (modified Langendorff model) is a widely used method in experimental heart research. The presence of an intraventricular balloon is necessary to get functional measurements. We have previously shown that the balloon induces phosphorylation of some suggested cardioprotective mitogen-activated protein kinases (MAPK): P38-MAPK, ERK 1/2 and JNK. We hypothesized that the balloon could influence cardioprotection, protect against ischaemia reperfusion injury and interfere with coronary flow. METHODS AND RESULTS: Isolated mouse hearts were perfused for 5, 10, 20, 40 and 60 min with a balloon in the left ventricle. We found a wavelike phosphorylation of all MAPK while AKT displayed a gradual dephosphorylation when compared to non-perfused hearts. Hearts were subjected to 20 min of stabilization with or without the balloon, followed by 35 min of ischaemia and 120 min of reperfusion. Although the MAPK were phosphorylated, the infarcts were larger in the balloon group. When the balloon was present during the entire protocol, compared to removal at the end of ischaemia, the infarct size was also larger, especially in the endocardial layer. The balloon reduced post-ischaemic endocardial coronary flow, despite a higher average flow, indicating a hyperperfused epicard. Blocking the balloon-induced ERK 1/2 phosphorylation during stabilization did not affect infarct size. The effect of post-conditioning was influenced by the balloon, showing reduced infarct size when the balloon was present. CONCLUSION: The balloon used for pressure measurements may contributes to cell death possibly by reducing endocardial coronary flow.