The role of beta-hydroxybutyrate as inducer for adult cardiomyocyte proliferation during reprogramming in vivo
Authors: Cheng Y.Y , Prajnamitra R.P , Ma T.Y , Huang Y.H , Hsieh P.C.H 
Published: November 2020

Abstract

Background
Regional loss of myocardium is the dominant cause of heart failure due to coronary artery disease. In mammals, the response to injury shows some similarities with border zone myocytes remodeling, but little proliferation and repair occur in adult mammalian hearts. Several studies have identified some cell cycle-related genes essential for dramatically inducing neonatal CM proliferation, but the same effects cannot be replicated in adult CMs. This implies that there is an underlying mechanism (an inducer) that is yet to be discovered, that should first be activated in order to subsequently increase adult CM proliferation efficiently in vivo.

Purpose
To determine the inducer for the purpose of efficiently increasing adult CM proliferation, and to examine its therapeutic potential for heart regeneration after injury.

Methods
Transgenic mice (αMHC-rtTA-OSKM), whose adult CMs can be specifically induced by Yamanaka's OSKM factors, were generated to systemically examine the early stage of iPSC reprogramming after tetracycline treatment. These induced reprogramming CMs were further isolated for gene ontology analysis by microarray to determine the specific inducer for the purpose of efficiently increasing CM proliferation. Finally, the inducer was applied to a mouse model of myocardial infarction (MI) to confirm its therapeutic potential for heart regeneration after injury.

Results
OSKM expressions of αMHC-rtTA-OSKM were confirmed to only increase in CMs after tetracycline treatment in vivo, and BrdU+ CM% was more than 2-times higher in isolated adult CMs from αMHC-rtTA-OSKM mice treated with tetracycline for 2 days compared to PBS-treated mice. More H3P+/cTnT+ population% was also confirmed in this CM-disorganized reprogramming hearts compared to normal hearts. Microarray data revealed that the morphological changes of the reprogrammed CMs were due to a metabolic switch shown in gene ontology analysis. Thus, we profiled the metabolic switch in normal and CM reprogramming hearts by perfusing them with 13C-labeled metabolic substrates through the Langendorff ex-vivo working heart perfusion system. Through 13C NMR spectroscopy we determined that TCA cycle metabolites were 2-times lower while β-hydroxybutyrate were 2-times higher in the reprogrammed hearts compared to that in control hearts. Next, we surprisingly found that β-hydroxybutyrate supplement in MI mice at post-MI day 3 improved the heart function but showed the opposite effects when supplement was administered at post-MI day 7. Thus, the timing of β-hydroxybutyrate supplement is important for increasing adult CM proliferation for effectiveness of heart regeneration after MI.

Conclusion
This is the first study to identify the metabolism shift to increase β-hydroxybutyrate as the inducer for the purpose of efficiently increasing adult CM proliferation, which supports heart function improvement when treating at short period immediately after MI.

Funding Acknowledgement
Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): 2020 Academia Sinica Young Scholarship Travel Award