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. 2021 Jan 21;11:1931. doi: 10.1038/s41598-021-81438-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Effects of 3 months CR, beta-blocker or diuretic on LV mitochondrial biogenesis, oxidative stress and apoptosis. (A) Representative Western blots and quantification of protein expression of Tfam, PGC-1alpha, a mitochondrial component of complex IV (Cox I), TOM20 and VDAC1 in LV tissue (n = 10 per group). GAPDH served as loading control. *p < 0.05; **p < 0.01. (B) Citrate synthase enzyme activity (units/mg protein) as a marker of mitochondrial content, cytochrome c oxidase (COX) activity and ATP content were analysed in LV tissue lysates (n = 8 per group). In addition, real-time PCR measurements of the relative copy number of mtDNA per diploid nuclear genome (n = 10 per group) were performed. *p < 0.05; **p < 0.01. (C) Thiobarbituric Acid Reactive Substances (TBARS) and protein carbonyl content were measured in LV tissue at the end of the study (n = 10 per group). *p < 0.05; **p < 0.01. (D) Quantification and representative Western blots of Bcl-2 and Bax protein in LV tissue. GAPDH served as loading control. LV Caspase 3 activity was measured in tissue lysates (n = 10 per group) by a colorimetric assay and is given as artificial units (A.U.). *p < 0.05; **p < 0.01. All data are mean ± SEM. Co = control, CR = caloric restriction, BB = beta-blocker, DF = diuretic. Full-size blots are shown in Suppl. Fig. 5.