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. 2018 Aug:121:163-172.
doi: 10.1016/j.yjmcc.2018.07.126. Epub 2018 Jul 19.

Quantitative temporal analysis of protein dynamics in cardiac remodeling

Affiliations

Quantitative temporal analysis of protein dynamics in cardiac remodeling

Daniel B McClatchy et al. J Mol Cell Cardiol. 2018 Aug.

Abstract

Cardiac remodeling (CR) is a complex dynamic process common to many heart diseases. CR is characterized as a temporal progression of global adaptive and maladaptive perturbations. The complex nature of this process clouds a comprehensive understanding of CR, but greater insight into the processes and mechanisms has potential to identify new therapeutic targets. To provide a deeper understanding of this important cardiac process, we applied a new proteomic technique, PALM (Pulse Azidohomoalanine in Mammals), to quantitate the newly-synthesized protein (NSP) changes during the progression of isoproterenol (ISO)-induced CR in the mouse left ventricle. This analysis revealed a complex combination of adaptive and maladaptive alterations at acute and prolonged time points including the identification of proteins not previously associated with CR. We also combined the PALM dataset with our published protein turnover rate dataset to identify putative biochemical mechanisms underlying CR. The novel integration of analyzing NSPs together with their protein turnover rates demonstrated that alterations in specific biological pathways (e.g., inflammation and oxidative stress) are produced by differential regulation of protein synthesis and degradation.

Keywords: Azidohomoalanine; Cardiac remodeling; Isoproterenol; Metabolic labeling; Proteomics.

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Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Figure 1:
Figure 1:
Confirmation of cardiac remodeling in ISO mouse model. A) Schematic workflow. Surgically implanted osmotic pumps delivered ISO (15 mg/kg/d) or saline (i.e., control) to mice for 14 days. The mice were fed a diet of either light AHA (L-AHA) or heavy AHA (H-AHA) for four days at different time points. Left ventricles (LVs) were dissected after labeling. B) HW/BW ratio and their 95% confidence intervals for each time point. The HW/BW ratio in the ISO treated mice increased continuously starting on Day 1, whereas the HW/BW ratios in the control mice did not change over 14 days remaining at 4.5. C) We monitored the cardiac function and displayed the ejection fraction (EF) for each day. Notably, the EF was elevated starting on Day 1 and remained elevated versus baseline on Day 0 due to the beta-adrenergic stimulation by ISO. As the EF was elevated during ISO treatment, the mice were not in heart failure but rather exhibited cardiac remodeling only. We observed a downward trend on Day 14 of ISO treatment indicating that prolonged beta-adrenergic stimulation (beyond 14 days) will eventually lead to cardiac heart failure. D) Mitochondrial complex I (ETC CI) activity was inhibited by ISO treatment over 14 days compared to baseline on day 0 demonstrating suppression of mitochondrial function at the level of ETC CI. The solid lines denote the regression. The dotted lines denote the 95% confidence interval. The dashed line denotes the trend. E) Mice model verification of cardiac remodeling. The levels of Galetin3 and GDF15 in total homogenates both control and ISO left ventricles were determined by western blotting.
Figure 2:
Figure 2:
Protein identification of Day 4 and Day 14 time points. A) The number of NSPs identified at Day 4 and Day 14 are similar. Average protein numbers of three replicates for each time point were plotted. Error bar represents standard deviation. The overlap of protein identifications of three biological replicates are from (B) Day-4 (replicate A, B, and C) analysis and (C) Day-14 (replicate E, F, and G) analysis. D) Significant NSPs (p< 0.05 using a one sample t- test) were plotted in a histogram with their fold change (ISO/control) on the x-axis. There were 93 and 75 proteins that were significantly different between the ISO and control hearts in Day 4 and Day 14, respectively.
Figure 3:
Figure 3:
NSP quantification A) Western blotting confirmation of MYBPC3, NDUFV1 and ApoA4 in NSP datasets. Taking GAPDH in input as internal standards, the three proteins showed consistent changes with the NSP datasets. N/A means the protein were not quantified at this time point. Bands were quantified using Image J software (National Institutes of Health, Bethesda, MD). B) Correlation of NSPs quantified at both acute and chronic analyses. The average of proteins ratios (ISO/control) calculated from 3 biological replicates of each time point were plotted to address correlation between the two datasets. C) NSPs quantified at both acute and chronic analyses. “*” represents an unquantifiable large change.
Figure 4:
Figure 4:
Functional CR network. The protein network and localization were analyzed by STRING and visualized by Cytoscape. The log ratio used in visualization was the average ratio of replicates. The white ellipse represents the protein Titin (TTN), which was found up-regulated at Day 4 of ISO but down-regulated at Day 14 of ISO.
Figure 5:
Figure 5:
Comparison of acute and chronic ISO treatment. A) Pathway analysis on NSPs with significant changes and unquantifiable large changes at acute and chronic time points. B) Significantly altered NSPs previously associated with human cardiac diseases or animal models. Manual literature curation associated quantified NSPs in this study with previous reports of human genetic mutations causing cardiac disease (red), biomarker of or association with human cardiac disease (black), or association with animal models of cardiac disease (pink). Arrows indicate an up- or down-regulation of the NSP with ISO in this study. C) Percentage of trends identified from the comparison of NSP ratios and protein turnover rates in response to ISO. Each trend is annotated with the top two significantly enriched pathways. The number in parentheses represents the p-value for pathway enrichment. D) Comparison of the absolute NSP changes associated with each of the four NSP/turnover trends. One-way ANOVA analysis was performed, and a significant (p < 0.0001) difference was observed between the four trends. A Bonferroni’s Multiple Comparison post-hoc test was then performed, and a significant (***p<0.001) difference was observed in the “down NSP/up turnover” trend and each of the other trends.

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