Contribution of Energy Metabolism to Cardiomyocyte Maturation
Creators: Persad, Kaya L
Publisher: name: University of Alberta Library
Year: 2023

<p>Proliferating cells, such as neonatal cardiomyocytes, have a high Warburg effect, which is a metabolic state in which there is high rates of glycolysis uncoupled from glucose oxidation under aerobic conditions. The Warburg effect is typically seen in cancerous cells and actively proliferating cells, such as fetal cardiomyocytes.  In the newborn period there is a decreased proliferation of cardiomyocytes accompanied by a decrease in glycolysis. This is followed by the maturation of cardiomyocytes. This change in energy metabolism in the newborn heart may be due to changes in energy substrate availability, such as an increase in circulating ketone levels. Ketones have the potential to modify the Warburg effect, either through altering glycolysis or glucose oxidation. Ketones, such as β-hydroxybutyrate (BOHB), are not only a fuel source for the heart, but also have cell signaling properties, including the endogenous inhibition of histone deacetylases (HDAC). HDAC2 knockout and knockdown studies in animals and cell cultures are known to increase differentiation and reduce proliferation of cancerous cells. The objective of this study is to determine if the Warburg effect influences maturation of cardiomyocytes, and whether ketones, affect this process. <br>
Objective 1: To determine if the Warburg effect influences maturation of cardiomyocytes, and whether ketones, which increase in the newborn period, affect this process. <br>
Objective 2: To understand how ketones influence maturation and the Warburg effect in proliferating cardiomyocytes.<br>
 Hypothesis: Ketones will promote cardiomyocyte maturation through decreases in glycolysis, and the subsequent decrease of the Warburg effect, and/or due to an inhibition of HDAC2 signalling.<br>
Methods: H9c2 cardiomyocytes were either proliferating or were differentiated (matured) for 7-days using 1uM trans-retinoic acid, in the presence or absence of 1 mM BOHB. The Warburg effect was then directly measured by assessing glycolysis and glucose oxidation. Ketone oxidation and fatty acid oxidation were also assessed. To assess if ketones were acting through metabolic pathways, siRNA was used to knockdown BOHB dehydrogenase 1 (BDH1) the first enzyme involved in BOHB oxidation, in proliferating H9c2 cardiomyocytes. Cells were then cultured in the presence or absence of 1 mM BOHB for 6 days, before assessing glycolysis, glucose oxidation, and ketone oxidation. To assess if ketones were acting through the endogenous inhibition of HDAC2, siRNA was used to knockdown HDAC2 in proliferating H9c2 cardiomyocytes. Cells were then cultured in the presence or absence of 1 mM BOHB for 6 days, before assessing glycolysis, glucose oxidation, and ketone oxidation.<br>
Results: The Warburg effect was high in proliferating cardiomyocytes and was decreased by 78% with maturation. This was not due to an increase in glucose oxidation, but rather due to a decrease in glycolysis in proliferating versus matured cells. Ketones decreased the Warburg effect by 28%. This was due solely to a decrease in glycolysis and not glucose oxidation. Adding ketones to proliferating cells also increased markers of cardiomyocyte maturation such as SERCA2 and PGC-1α.  Knocking down BDH1 did decrease ketone oxidation but was not accompanied by changes in glycolysis or glucose oxidative rates. Furthermore, ketones affected glycolysis similarly in BDH1 knockdown cells compared to controls in that there was a decrease in the Warburg effect. The knockdown of HDAC2 lead to a increase in glycolysis and glucose oxidation, with reductions in ketone oxidation. The addition of ketones led to a decrease in glycolysis comparable to untreated controls. <br>
Conclusion: Cardiomyocyte maturation is associated with a reduced Warburg effect, due exclusively to a decrease in glycolysis. Ketones contribute to the decrease in the Warburg effect, primarily by inhibiting glycolysis. The addition of BOHB increases maturation and decreases the Warburg effect in proliferating cardiomyocytes. BDH1 knockdown does not change the effects of ketones on glycolysis in proliferating cardiomyocytes. HDAC2 knockdown increases glycolysis in proliferating H9c2 cells, however this affect is abolished by the addition of ketones. This indicates that HDAC2 signaling may play a key role in ketones regulation of cardiomyocyte maturation. These results have potential implications in understanding the maturation of cardiomyocytes in the newborn period.</p>