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Label-free quantitative SWATH-MS proteomic analysis of adult myocardial slices in vitro after biomimetic electromechanical stimulation

ABSTRACT: Conventional in vitro cell cultures are relatively available and have been widely used in explaining many mechanisms, but their simple construction is not directly applied to the in vivo environment. A possible solution to overcome this problem is special in vitro model maintained with cardiac slices. Slices show a preserved architecture, multi-cellularity and physiology of the heart tissue. This approach creates a bridge between the gap in cellular and in vivo studies. This study demonstrated that it is possible to find protein biomarkers between rat myocardial slices which were stretched to sarcomere lengths (SL) within the physiological range of 1.8–2.4μm and slices cultured without electromechanical stimulation and with fresh myocardial slices.

INSTRUMENT(S): TripleTOF 5600+

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Heart

SUBMITTER: Aleksandra Bogucka

LAB HEAD: Paulina Czaplewska

PROVIDER: PXD034891 | Pride | 2022-08-25

REPOSITORIES: Pride

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Project description:Background: Studies in mice have shown that PPARÎ± is an important regulator of lipid metabolism in liver and a key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARÎ± in human liver. Here we set out to study the function of PPARÎ± in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARÎ± agonist Wy14643. Results: Quantitative PCR indicated that PPARÎ± is well expressed in human liver and human liver slices and that the classical PPARÎ± targets PLIN2, VLDLR, ANGPTL4, CPT1A and PDK4 are robustly induced by PPARÎ± activation. Transcriptomics analysis indicated that 617 genes were upregulated and 665 genes were downregulated by PPARÎ± activation (q value<0.05). Many genes induced by PPARÎ± activation were involved in lipid metabolism (ACSL5, AGPAT9, FADS1, SLC27A4), xenobiotic metabolism (POR, ABCC2, CYP3A5) or the unfolded protein response, whereas most of the downregulated genes were involved in immune-related pathways. Among the most highly repressed genes upon PPARÎ± activation were several chemokines (e.g. CXCL9-11, CCL8, CX3CL1, CXCL6), interferon Î³-induced genes (e.g. IFITM1, IFIT1, IFIT2, IFIT3) and numerous other immune-related genes (e.g. TLR3, NOS2, and LCN2). Comparative analysis of gene regulation by Wy14643 between human liver slices and primary human hepatocytes showed that down-regulation of gene expression by PPARÎ± is much better captured by liver slices as compared to primary hepatocytes. In particular, PPARÎ± activation markedly suppressed immunity/inflammation-related genes in human liver slices but not in primary hepatocytes. Finally, several putative new target genes of PPARÎ± were identified that were commonly induced by PPARÎ± activation in the two human liver model systems, including TSKU, RHOF, CA12 and VSIG10L. Conclusion: Our manuscript demonstrates the suitability and superiority of human liver slices over primary hepatocytes for studying the functional role of PPARÎ± in human liver. Our data underscore the major role of PPARÎ± in regulation of hepatic lipid and xenobiotic metabolism in human liver and reveal a marked immuno-suppressive/anti-inflammatory effect of PPARÎ± in human liver slices that may be therapeutically relevant for non-alcoholic fatty liver disease. Precision-cut liver slices, prepared from liver biopsies obtained from obese subjects undergoing bariatric surgery, were incubated with the peroxisome proliferator-activated receptor alpha (PPARÎ±) agonist Wy14643 or vehicle for 24hrs, after which gene expression was profiled by array.

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Label-free quantitative SWATH-MS proteomic analysis of adult myocardial slices in vitro after biomimetic electromechanical stimulation

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