1. Nuclear receptor mediated cardioprotective pathways in hibernating mammals.
Mammalian hibernation is an example of natural molecular and cellular adaptations to extreme environmental conditions. Hibernators such as arctic ground squirrels (AGS) depress their metabolic, heart, and respiratory rates as well as their core body temperature to enter a state called torpor during which they exhibit remarkable resistance to myocardial I/R injury and ventricular dysrhythmias normally associated with hypothermia. Interestingly, this winter cardioprotective phenotype is associated not only with suppression of metabolic demand, but also with a fuel shift from myocardial carbohydrate to fatty acid (FA) metabolism. The molecular events by which this precisely controlled increased reliance on myocardial FA utilization is cardioprotective in hibernators while it adversely impacts post-I/R cardiac efficiency and function in non-hibernators represent a significant knowledge gap. Using a clinically relevant model of surgical deep hypothermic circulatory arrest (DHCA) combined with targeted metabolomic and proteomic profiling, we are investigating how changes in hibernation-specific myocardial expression/activity of PPARα nuclear receptor and downstream target genes (FGF21) following cardiac surgery can provide cardioprotection by increasing myocardial fatty acid oxidation and inhibiting NF-κB regulated pro-inflammatory responses. Compared to rats, AGS display robust myocardial ischemic tolerance following DHCA (both reduced myocardial necrosis and apoptosis). Mechanistically this is accompanied by preservation of myocardial PPARα activity in AGS, which is significantly downregulated in the rat, and a metabolic phenotype consistent with the development of mitochondrial substrate flux “bottlenecks” in rats (myocardial accumulation of acylcarnitines and ceramides, organic acid profiles consistent with compromised citric acid cycle flux) compared to AGS. Furthermore, the hibernator cardioprotective phenotype is also associated with reduced myocardial NF-κB activity, reduced expression of downstream cytokines and neutrophil extravasation following cardiac surgery. We are characterizing changes in the abundance of the full spectrum of proteins expressed in the heart using proteomic analyses, as well as in the repertoire of inflammatory cells responsible for heart damage following cardiac surgery using flow cytometry analyses.
We feel that developing strategies to help ‘switch’ myocardial metabolism to resemble that naturally occurring in mammalian hibernators represents a transformative approach that could ultimately have an important positive impact in patients undergoing cardiac surgery and transplantation, as well as in victims of cardiac arrest, stroke, trauma and hypothermia, in addition to fundamentally advancing the field of nuclear receptor biology and myocardial substrate metabolism under extremes of physiology.
2. MicroRNAs as targets for perioperative cardioprotection.
MicroRNAs, an abundant class of posttranscriptional regulators of more than 30% of the human genome, are druggable targets for cardiovascular pathology. However, chemical and viral- mediated transfection of commercially-available miRNA modulators has been limited by toxicity and off- target effects. Furthermore, important gaps in knowledge remain in two areas that limit the translational applications of miRNA modulators in cardioprotection research. First, our understanding of the biological roles played by miRNAs in regulating myocardial responses to I/R, notably in the setting of planned global ischemia such as during cardiac surgery, remains rudimentary. Second, efficient functional in vivo delivery of miRNA modulators to the heart, specifically overexpressing therapeutic pre-miRNAs using non-viral based technologies, is still a major obstacle. We have identified and prioritized differentially expressed miRNAs in response to acute surgical I/R across multiple species and strains. Overexpression of one of those (miRNA-146b) in vitro and in vivo is cardioprotective by enhancing the endogenous inhibitory feedback on NF-κB activation through effects on the key regulatory elements IRAK1 and TRAF6, and showed effective nanoparticle-mediated delivery in a rat cardioplegic arrest model. We are now conducting functional genomic analyses of other top miRNAs and optimizing nanoparticle-mediated in vivo delivery protocols.
3. The role of Annexin A1 and its bioactive peptides in perioperative cardioprotection.
Annexin-A1 (ANXA1) is an endogenous glucocorticoid-regulated protein with potent inflammation- resolving properties, previously implicated in attenuating myocardial ischemia-reperfusion (I/R) injury via neutrophil-mediated mechanisms. We have developed a novel bioactive tripeptide (ANXA1sp) derived from the N-terminal domain of ANXA1 and shown it produces cardioprotection following surgical myocardial I/R in multiple species (rats, pigs) through direct inhibition of reperfusion-induced NF-kB activation in cardiomyocyes. We are studying the mechanisms by which ANXA1 attenuates I/R induced nuclear translocation of NF-kB p65, and optimizing the dosing and timing of ANXA1sp administration in preparation for first-in-man studies.