Assembly and activation of NLRP3 inflammasome complex in mitochondria of failing canine hearts
H N Sabbah, R C Gupta, D E LanfearAbstract
Background
Increased levels of pro-inflammatory cytokines is a key feature of chronic heart failure (HF) and an important driver of interstitial fibrosis and disease progression. We previously showed that mitochondrial damage-associated molecular patterns (mDAMPs) are increased in patients and dogs with chronic HF. mDAMPs can activate NLRP3 (NLR family pyrin domain containing 3), a cytosolic pattern recognition receptor. Once activated, NLRP3 translocates to mitochondria and recruits ASC (apoptosis-associated protein containing a caspase recruitment domain) and caspase-1 to form the inflammasome complex that triggers the release of inflammatory cytokines.
Purpose
This study tested the hypothesis that the inflammasome complex constituent proteins levels of NLRP3, ASC and caspase-1 are increased in mitochondria of LV myocardium of chronic HF dogs compared to normal (NL) dogs.
Methods
Mitochondrial membrane fractions were isolated from LV tissue of 7 normal (NL) dogs and 7 dogs with chronic HF produced by multiple intracoronary microembolizations (LV ejection fraction ~35%). Western blot analysis was performed to quantify the levels of NLRP3, ASC, and caspase-1 in mitochondrial fractions. Results were expressed in densitometric units (du) and normalized to porin, a mitochondrial membrane protein that is unchanged in HF.
Results
Protein levels of NLRP3, ASC, and caspase-1, normalized to porin, were significantly elevated in HF compared to NL dogs. NLRP3 levels increased from 0.07 ± 0.01 in NL to 0.17 ± 0.01 in HF (p<0.01), representing a 2.4-fold increase. ASC levels showed a more pronounced change, rising from 0.03 ± 0.01 in NL to 0.16 ± 0.01 in HF, a 5.3-fold increase (p<0.001). Caspase-1 levels increased from 0.07 ± 0.01 in NL to 0.17 ± 0.02 in HF (p = 0.001), a 2.4-fold increase.
Conclusion
Increased mitochondrial levels of NLRP3, ASC, and Caspase-1 indicate the assembly and activation of the NLRP3 inflammasome. This represents a critical convergence point between cellular danger sensing and mitochondrial stress with profound implications for a sustained pro-inflammatory state, increased reactive fibrosis and cell death that culminates in progressive worsening of the HF state.