Abstract
Knockout of multifunction gene cysteine- and glycine-rich protein 3 (CSRP3) in cardiomyocytes (CMs) of mice leads to heart dilation, severely affecting its functions. In humans, CSRP3 mutations are associated with hypertrophic (HCM) and dilated cardiomyopathy (DCM). The absence of the CSRP3 expression produces unknown effects on in vitro neonatal CMs’ metabolism. The metabolome changes in culture media conditioned by CSRP3 knockout (KO-CSRP3), and wild type (WT) neonatal cardiomyocytes were investigated under untreated or after metabolic challenging conditions produced by isoproterenol (ISO) stimulation, by in vitro high-resolution proton magnetic resonance spectroscopy (1H-MRS)-based metabolomics. Metabolic differences between neonatal KO-CSRP3 and WT rats’ CMs were identified. After 72 h of culture, ISO administration was associated with increased CMs’ energy requirements and increased levels of threonine, alanine, and 3-hydroxybutyrate in both neonatal KO-CSRP3 and WT CMs conditioned media. When compared with KO-CSRP3, culture media derived from WT cells presented higher lactate concentrations either under basal or ISO-stimulated conditions. The higher activity of ketogenic biochemical pathways met the elevated energy requirements of the contractile cells. Both cells are considered phenotypically indistinguishable in the neonatal period of animal lives, but the observed metabolic stress responses of KO-CSRP3 and WT CMs to ISO were different. KO-CSRP3 CMs produced less lactate than WT CMs in both basal and stimulated conditions. Mainly, ISO-stimulated conditions produced evidence for lactate overload within KO-CSRP3 CMs, while WT CMs succeeded to manage the metabolic stress. Thus, 1H-MRS-based metabolomics was suitable to identify early inefficient energetic metabolism in neonatal KO-CSRP3 CMs. These results may reflect an apparent lower lactate transport and consumption, in association with protein catabolism.
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Acknowledgments
The authors would like to acknowledge the São Paulo Research Foundation (FAPESP) (grants 2011/19678-1; 2013/17368-0; 2014/22102-2; 2014/21646-9; 2018/20910-5) and Medical Sciences Graduate Program-CAPES/PROEX for financial support; LNBio-CNPEM (20992) for providing spectrometer and facilities; Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor) - University of Sao Paulo School of Medicine for providing cells and scientific assistance; and National Institute of Science and Technology Complex Fluids (INCT-FCX) for daily financial aid.
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ACB and VB equally contributed to cell experiments. ACB contributed with sample preparation, NMR experiments, data processing, statistical analysis, and biological interpretations and drafted the manuscript. LFG critically revised the manuscript. JAS, IVB, VB, and AMA contributed to the design of the research and cell experimental setup. All authors agree to be fully accountable for ensuring the integrity and accuracy of the work and read and approved the final manuscript.
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Bloise, A.C., dos Santos, J.A., de Brito, I.V. et al. Discriminating aspects of global metabolism of neonatal cardiomyocytes from wild type and KO-CSRP3 rats using proton magnetic resonance spectroscopy of culture media samples. In Vitro Cell.Dev.Biol.-Animal 56, 604–613 (2020). https://doi.org/10.1007/s11626-020-00497-8
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DOI: https://doi.org/10.1007/s11626-020-00497-8