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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 9, 2004 as doi:10.1096/fj.04-2025fje. |
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Department of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA;
* Pfizer Global Research and Development, Ann Arbor, Michigan, USA; and
Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USA
2Correspondence: Department of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert B. Sabin Way, Cincinnati, OH 45267-0575, USA. E-mail: litsa.kranias{at}uc.edu
SPECIFIC AIMS
Since depressed sarcoplasmic reticulum (SR) Ca cycling is a hallmark of human and experimental heart failure, strategies to improve this impairment by decreasing phospholamban (PLN) activity have been implicated as promising therapeutic approaches. Indeed, ablation of the PLN gene in mice has been associated with greatly enhanced SR Ca cycling and cardiac contractility. To determine whether PLN ablation was associated with any modification of myocardial proteins, we performed a proteomics-based analysis of PLN knockout (PLN-KO) hearts using 2-D gel electrophoresis (2-DE) and mass spectrometry.
PRINCIPAL FINDINGS
Protein expression profiling
Ventricular proteins from PLN-KO and wild-type (WT) mice were processed in parallel, loaded onto pH 3–10 immobilized pH gradient (IPG) strips, and subjected to 2-DE. Image analysis software detected 
1100 spots on each SYPRO-stained gel and the images were analyzed for quantitative spot comparisons using Z3® 2-D software. Raw master gel (RMG) images were first generated for the WT and PLN-KO protein profiles by averaging three individual gel images that could display the differences in protein spot volume (Fig. 1
). Differential expression was calculated for each spot that could be matched between the two RMGs by Z3®. Comparison of the RMGs indicated that ablation of PLN was associated with a 2-fold increase in the expression of 34 protein spots and a 2-fold decrease in the expression of 26 proteins. Furthermore, 83 proteins were detected "exclusively" in WT and 51 proteins were exclusively detected in PLN-KO.
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Wild-type and PLN-KO protein samples were also run on narrow pH range, 18 cm IPG strips (pH ranges: 6–9, 4.0–5.0, 4.5–5.5, 5.0–6.0, and 5.5–6.7), which allowed for better resolution of protein spots than broad-range (pH 3–10) strips. Analysis of the 2-D images demonstrated that 1300 spots could be detected in each narrow-range gel; after overlaying the gel images from various pH ranges and subtracting common protein spots between overlapping pH ranges, 3300 distinct spots could be resolved between pH 4–7. The resulting composite overlay image of narrow-range gels between pH 4.0 to 6.7 allowed us to observe a 3-fold increase in total number of detectable protein spots vs. a single pH 4–7 or 3–10 IPG 18 cm strip. From pairwise analysis, 49 proteins were found to be significantly up-regulated and 26 proteins were found to be down-regulated upon PLN ablation. Among these changes, 18 proteins were detectable exclusively in the PLN-KO and 8 detectable exclusively in the WT hearts.
Protein spots with significant differences (>2-fold) in abundance between WT and PLN-KO were processed and subjected to MALDI-TOF or LC-MS/MS for identification. For a complete list of the successfully identified proteins, please refer to Table 1 of the full-text online version of the article at http://www.fasebj.org.
Post-translational modifications
Of particular importance in cardiac physiology is the phosphorylation state of myofilament and energy production proteins, since phosphorylation tightly regulates overall cardiac output and contractility. To determine whether increased or decreased phosphorylation of myocyte proteins was associated with ablation of PLN, isolated cardiomyocytes were labeled with 32Pi, processed for 2-DE, and subjected to autoradiography. Image analysis of the autoradiograms revealed a significant difference in phosphorylation of five protein spots. Phosphorylation of succinate CoA synthetase was decreased whereas that of MLC-2, pyruvate dehydrogenase, heat-shock protein-27, and 
B-crystallin was increased.
PLN ablation was also associated with post-translational modification and up-regulation of SCAD. We determined that the difference in protein patterns (see Fig. 2
) resulted from a change in the expression level and isoelectric point of the short chain acyl-CoA dehydrogenase (SCAD) upon PLN ablation. Mass spectrometry identified both spots in Fig. 2
as SCAD. Furthermore, 2-D Western blot analysis confirmed that portion of the SCAD migrated at a more acidic pI in the KO tissue while its molecular weight remained unchanged, consistent with findings from image analysis of stained 2-D gels. Finally, quantitative immunoblotting of 1-D gels with a polyclonal antibody to SCAD demonstrated it was up-regulated by 2-fold in PLN-KO ventricles. Since no isoform of SCAD has been identified so far, the pI shift suggested a post-translational modification of the enzyme.
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DISCUSSION AND SIGNIFICANCE
For the past decade, the PLN-KO mouse model has provided a great deal of insight into cardiac SR function and its role in regulating cardiac contractility. The wealth of information gained from studies in this model stands in contrast to our lack of understanding of the subcellular players maintaining the heightened basal cardiac performance over the long term without apparent negative pathophysiologic consequences. A combination of 2-DE, mass spectrometry, and autoradiography was used in this study in an attempt to map changes in ventricular protein expression of the hyperdynamic PLN-deficient hearts. These studies indicate that ablation of PLN is associated with a large number of previously unrecognized alterations in myocardial protein expression patterns that may contribute to or compensate for the cardiac phenotype upon PLN ablation.
The hyperdynamic state of the PLN-KO heart places great demand on the myocyte energy production system. Accordingly, our results suggest that the enzymes responsible for ß-oxidation of fatty acids, along with Krebs cycle enzymes, are increased in the KO ventricles. This suggests that cross-talk must exist between mitochondria and SR to maintain a balance between energy production and consumption. A schematic representation of the identified changes in energy production proteins is summarized in Fig. 3
. The increase in fatty acid ß-oxidation was evidenced by significant increases in the levels of both MCAD and SCAD enzymes. ETF and ETFß were up-regulated; these two proteins act as electron acceptors from dehydrogenases such as MCAD and SCAD. Up-regulation and post-translational modification of SCAD may represent a metabolic adaptation in response to heightened cellular energy utilization and contractility.
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To handle the increased load of acetyl-CoA that results from increased ß-oxidation, several enzymes of the Krebs cycle were up-regulated, including succinyl-CoA ligase, fumarase, and malate dehydrogenase (Fig. 3)
. Similarly, we observed a decrease in the phosphorylation of succinyl-CoA synthetase. The up-regulation of these enzymes would facilitate the transfer of high energy electrons to NAD and FADH in ATP synthesis. Thus, from the beginning of the pathway to the end, enzymes involved in ß-oxidation are up-regulated to more rapidly produce energy in the PLN-KO. In contrast to the increases in ß-oxidation, there was a decrease in several enzymes of the glycolysis pathway. Fructose-bisphosphate aldolase A, ß-enolase, and pyruvate dehydrogenase E1 were down-regulated, along with a 2-fold increase in the phosphorylation level of pyruvate dehydrogenase. Finally, such high levels of aerobic respiration would likely produce higher levels of oxygen free radical species. An increase in peroxiredoxin, antioxidant protein 2, and superoxide dismutase was observed in the PLN-KO, which may help maintain the reduction state of the cell, scavenge free radicals, and thus protect cellular components from oxidative damage by free radical species.
Ablation of PLN is associated with MLC-1 isoform switching and increased MLC-2v phosphorylation. The atrial isoform of MLC-1 (MLC-1a) was down-regulated whereas the ventricular isoform of MLC-1 (MLC-1v) was up-regulated in PLN-KO ventricles. Ventricular MLC-1a, which is normally suppressed after birth, was reported to accompany cardiac hypertrophy. MLC-2a was identified in the present study and its expression was increased. An increased phosphorylation level of MLC-2v may allow for higher organization of the sarcomeric architecture and increase Ca2+ sensitivity of the myofilaments, thereby increasing contractility.
Finally, we observed a 1.5- and 2.0-fold increase in the phosphorylation of HSP27 and B-crystallin, respectively. Earlier reports indicate that when these two homologous chaperone proteins are phosphorylated, oligomers of each protein dissociate and exert strong anti-apoptotic effects in the nucleus and mitochondria. Thus, the increased phosphorylation of the two chaperone proteins may ameliorate the chronic stress of maintaining enhanced basal contractile performance and prevent apoptosis in the PLN-KO hearts.
In conclusion, this study has uncovered two major differences in the PLN-KO cardiac proteome: 1) fatty acid ß-oxidation is up-regulated in an effort to meet the energetic demands of enhanced SR calcium cycling and hyperdynamic function; and 2) myofilament adaptations occur that may result in higher sarcomeric organization and efficient ATP utilization, which could potentially be anti-hypertrophic and anti-apoptotic in nature. These findings suggest that after PLN ablation, cross-talk or synergism occurs among SR Ca2+ cycling proteins, the contractile apparatus, and the mitochondrial ATP production machinery to maintain the physiological hyperdynamic steady state of contractility in PLN-KO hearts.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2025fje;
1 Both authors contributed equally to this study. 
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) whereas two terminal enzymes of glycolysis were down-regulated (
); this suggests a shift away from glycolysis as a major source of acetyl-CoA and toward fatty acid utilization. 2-PG: 2-phosphoglycerate; PEP: phosphoenolpyruvate; SCAD: short chain acyl-CoA dehydrogenase; MCAD: medium chain acyl-CoA dehydrogenase; ETFA and ETFB: electron transfer flavoprotein