Background: Paediatric heart failure is a severe disease with limited therapeutic options available. The predominant causes of paediatric heart failure are congenital structural malformations which cause progressive myocardial damage due to the abnormal physical forces associated with pumping blood. Some of the most severe congenital heart malformations are those where patients develop only a single ventricle and it is common for these patients to experience heart failure during childhood. A greater understanding of the causes and risk factors associated with paediatric heart failure is needed to develop novel precision therapies for improving myocardial function, which would greatly improve quality of life for these patients.
Methods: Blood samples were collected from patients with single ventricle congenital heart disease and clinical or subclinical heart failure to test for established and novel biomarkers of heart failure. Following identification of target pathways, further investigations into the implications of aberrant mechanical force and elevated oxidative stress were conducted in cardiomyocytes differentiated from induced-pluripotent stem cells (iPSC-CMs) and AC16 cardiomyocytes.
Results: Immunoassays of patient plasma revealed a significant correlation between elevation of plasma BNP levels and severity of heart failure, calculated by modified Ross score (R2=0.233, p > 0.0001) . BNP is released by the myocardium under abnormal cardiac load, suggesting that altered mechanical homeostasis of the heart contributes to heart failure. Interestingly, we did not find the same trend with other biomarkers for heart failure, suggesting heart failure is driven by mechanical loading. Next, we manipulated cellular micromechanics in iPSC-CMs and found that stiffness regulates cardiomyocyte protein expression, function and contractility. To search for novel biomarkers, we conducted proteomic analysis of plasma. Of 569 proteins identified, 97 were up-regulated and 6 down-regulated compared to patient controls. Pathway enrichment analysis highlighted oxidative stress pathways in the up-regulated protein population. Aberrant mechanical load can lead to oxidative stress by production of reactive oxygen species (ROS), leading to heart failure. Similarly, elevated oxidative stress can disrupt calcium handling and cause contractile dysfunction. To explore this connection further, we next generated a single ventricle cardiomyocyte cell model by pharmacologically manipulating the activity of gap junction protein, Connexin43; a mechanosensitive protein that regulates oxidative stress, and which genetic mutations are linked to single ventricle congenital heart disease. We found that pharmacological modulation of Connexin43 hemichannel function regulates oxidative stress in cardiomyocytes, both basally and in response to H2O2-induced oxidative stress (p > 0.05).
Conclusions: Paediatric heart failure shares some common hallmarks with adult heart failure, however, mechanical load may play a more prominent role. High levels of oxidative stress experienced by single ventricle patients are likely due to abnormal mechanical loading of the heart, particularly when linked to pathogenic mutations in complexes that regulate mechanical and oxidative homeostasis, such as Connexin43. Our results suggest that these interactions between genetics, mechanical strain and oxidative stress drive myocardial dysfunction and heart failure. Identification of key disease mechanisms will support the development of new treatments and inform better long-term care for patients and their families.