Muscle Ring Finger‐1 Knockout (MuRF1−/−) Mice are Resistant to LPS‐induced Cardiac Dysfunction Due to Decreased NF‐κB Activity

Abstract

Sepsis is associated with a high mortality (up to 40%) in hospitalized patients. Cardiac dysfunction plays a critical role in the outcome of severe sepsis, and sepsis‐targeted cardiac therapies are lacking. Fortunately, recent studies have identified the role of Toll‐like receptors, cytokines, and NF‐kappaB signaling in sepsis‐associated heart failure. We recently identified that the muscle‐specific ubiquitin ligase MuRF1 (Muscle Ring Finger1) inhibits peroxisome proliferator‐activated receptor alpha (PPARalpha) in cardiomyocytes by mediating its nuclear export in a ubiquitin‐dependent manner. We found that MuRF1 interacts with PPARalpha to mediate a multi‐monoubiquitination using three lysines surrounding a newly identified nuclear export sequence. Interestingly, MuRF1−/− mice have a five‐fold increase in cardiac PPARalpha activity. Since studies have shown that PPARalpha competitively inhibits NF‐kappaB and downstream inflammation, we hypothesized that MuRF1−/− mice would exhibit cardiomyocyte anti‐inflammatory responses through its competitive increase in PPARalpha activity. To test this, we performed echocardiography on MuRF1−/− and wildtype mice at baseline and 4, 24, 48, and 72 hours post‐lipopolysaccharide injection (2 mg/kg of LPS). LPS interacts with Toll‐like receptor 4 on cardiomyocytes and inflammatory cells, releasing cardiodepressant cytokines (e.g. TNFa, IL‐1, IL‐6) that activate NF‐kappaB. No mice died during the course of the 72 hour study and no cardiac differences were observed between groups at baseline. Four hours after LPS, wildtype mice exhibited significantly worse systolic function compared to MuRF1−/− mice (Fractional Shortening (FS)%=34.5+1.5 vs. 42.0+4.5). Wildtype LPS treated hearts exhibited significant wall thinning, while MuRF1−/− wall thickness did not change from baseline. MuRF1−/− LPS‐induced cardiac dysfunction was completely resolved by 72 hours (FS%=57.5+1.4), whereas wildtype hearts still exhibited residual dysfunction (FS%=48.4+1.9). Analysis by immunoblot (from hearts harvested 72 hours post LPS treatment) showed (active) P‐Ikappa Balpha was decreased in MuRF1−/− mice, indicating NF‐kB signaling was decreased in these mice. Similarly, inflammatory cytokine markers IL‐1alpha and IL‐1beta were decreased in MuRF1−/− mice, indicating MuRF1−/− mice had less inflammation. These data are in step with our hypothesis, demonstrating that MuRF1's regulation of PPARalpha may affect the heart's ability to respond to NF‐kappaB (due to enhanced nuclear PPARalpha activity inhibiting NF‐kappaB signaling). Therapeutic inhibition of the MuRF1‐PPARalpha interaction provides a muscle‐specific therapy that may prove beneficial in NF‐kappaB‐mediated cardiac dysfunction seen in sepsis, heart failure, and myocardial infarction.