Mechanisms of mitochondrial dysfunction in sepsis-induced cardiomyopathy

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection (Singer 2016). It is a major global health problem, estimated to affect over 30 million people every year worldwide. It carries a high mortality (25-50%), significant long-term disability, and staggering economic costs. Multiple large-scale clinical trials have failed to yield any novel, effective therapeutic intervention, so management remains largely supportive.
Up to 70% of septic patients develop myocardial dysfunction (sepsis-induced cardiomyopathy, SIC), and this likely plays a pivotal role in disease progression and outcome (Zaky 2014). The pathophysiology of SIC is complex and multifactorial, but mitochondrial dysfunction appears to play a crucial role (Rudiger 2007). Cardiac mitochondria develop multiple structural and functional abnormalities during sepsis (Cimolai 2015). These changes are thought to result in a decrease in ATP production, increased production of reactive oxygen species, and activation of apoptotic pathways. Cardiac mitochondria can therefore be viewed both as a targets of sepsis and as potential amplifiers of sepsis-induced organ dysfunction. Of note, some authors (including my host lab) have suggested that these changes may potentially represent an adaptive response to prolonged stress aimed at protecting cell viability by slowing down cell metabolism ('hibernation') (Singer 2004). These opposing views underline the lack of a full understanding of the cellular pathways of SIC and their role in the clinical course of the disease.

My host lab has recently exposed naïve kidney slices to septic serum and shown a decrease in mitochondrial membrane potential and reduced NADH, and a rise in the production of mitochondrial reactive oxygen species. Interestingly, these changes occur within minutes and can be inhibited or reversed by mitochondrially-targeted antioxidants, suggesting a role of oxidative stress (Pollen 2016). My host lab has also observed similar changes in cardiomyocytes isolated from septic rats (Pinto 2017). In pilot studies I have replicated these findings in cardiomyoblasts exposed to septic serum.

My initial objective is to elucidate in vitro specific mechanisms that underlie these mitochondrial effects, assess their impact on cell viability and protection, and to identify potential biological targets that could be pharmacologically modulated in vivo. I propose to investigate these mechanisms by exposing primary murine adult cardiomyocytes to septic serum. The use of such in vitro models offers the advantage of teasing out the effect of circulating factors independent of alterations in blood flow. The serum will be collected and pooled from a long-term animal model (rat) of sepsis that is well established in the lab. Serum will be collected at acute (6h) and established (24h) disease timepoints. Cardiomyocytes will be cultured under different conditions, including the use of pacing and agonists to induce calcium cycling, and to simulate the increase in metabolic demand seen in early critical illness. Incubation with septic serum will be performed over short-term (1h) and long-term (12h) periods. Mitochondrial function and cell viability will be assessed primarily via confocal laser scanning microscopy, wide field high-content imaging, and high-resolution respirometry.

I then plan to translate significant findings back to the in vivo sepsis model, quantifying the biological targets identified, and assessing differences between survivor and non-survivor animals. Protein quantification in vitro and in vivo will be evaluated via Western Blot, with spectrophotometric assays used to measure activity.