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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 12, 2002 as doi:10.1096/fj.01-0574fje. |
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Departments of Physiology and
*
Anesthesiology, Heinrich-Heine-University Düsseldorf, Germany
2Correspondence: Department of Physiology Heinrich-Heine-University Düsseldorf P.O. Box 10 10 07, 40001 Düsseldorf, Germany. E-mail: decking{at}uni-duesseldorf.de
SPECIFIC AIMS
Despite apparent morphological uniformity within the left ventricular myocardium in terms of capillary, myofibril, and mitochondrial density, local energy turnover and myocardial blood flow (MBF) vary > 10-fold between individual areas and display a patchwork pattern that is stable at least for hours. To characterize the basis for this metabolic and functional heterogeneity, we compared myocardial protein expression of areas of low and high local blood flow by two-dimensional gel electrophoresis (2D-PAGE) and identified the proteins differentially expressed by mass spectrometry. We also tested whether local distribution of blood flow was stable over a period of 2 wk.
PRINCIPAL FINDINGS
1. Differential protein expression in areas of low and high myocardial blood flow
Local myocardial blood flow of awake beagle dogs was assessed by radioactive microspheres applied via a left atrial catheter. To obtain high spatial resolution (300 µl), the left ventricular free wall of the anesthetized animals was excised, frozen in liquid nitrogen, lyophilized, and cut into 198 ± 23 samples (n=7 hearts) of an average dry mass of 59.4 ± 19.4 mg. Approximately 6% of samples received a flow < 50% of the average and 
6% of all samples received > 150% of the mean flow, with 99% of all individual samples being in the range from 0.16 to 2.16 of the mean. There was thus up to a 14-fold difference in local flow between individual areas, demonstrating the substantial extent of spatial heterogeneity of myocardial blood flow.
In 30 low and 30 high blood flow samples (<50% or >150% of average flow, n=5 each from six hearts), local protein expression was analyzed by 2D-PAGE. Approximately 380 protein spots were suitable for quantitative analysis on each gel, with identical numbers of spots in both groups (low flow: 389±82, high flow: 370±75). Quantitative comparison of protein spot volumes revealed six proteins that showed an enhanced expression in regions of high local flow and another six that were enhanced in low-flow regions (Fig. 1
). Of the 12 differentially expressed protein species, 8 could be identified by nano-spray ESI-MS/MS. These proteins are predominantly related to 1) cardiac NO formation; 2) cardiac energy metabolism.
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2. Enhanced NO formation in areas of low energy turnover and flow
The protein displaying the greatest relative change in expression, spot 273, was identified to be dimethyl-L-arginine dimethylaminohydrolase 1 (DDAH1). DDAH1 catalyzes the hydrolytic cleavage of asymmetric dimethyl-L-arginine (ADMA), a potent endogenous inhibitor of NO synthase. Expression of DDAH1 was much more pronounced in low- vs. high-flow areas. This was true not only at the protein (+377%, P<0.001, Fig. 2
) but also at the mRNA level (+340%, P<0.01, Fig. 2
). To test whether the rise in DDAH1 results in increased ADMA degradation, tissue content of ADMA was analyzed by HPLC. This revealed that in low-flow samples ADMA was reduced to 5.5 µM, only 25% of the ADMA content of high-flow samples (P<0.01, Fig. 2
). In low-flow samples, the amount of L-arginine was reduced by 45% (59±17 vs. 107±45 µM, P<0.05). No differences in eNOS mRNA expression were observed between low- and high-flow samples as demonstrated by quantitative PCR (P<0.05, Fig. 2
). Based on the reduced concentration of ADMA, the potent inhibitor of NO synthase in the presence of identical levels of eNOS, the data strongly suggest enhanced NO formation in low-flow areas.
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3. Increased glycolysis and decreased fatty acid oxidation in areas of low turnover and flow
In low-flow areas, glycerinaldehyde-3-phosphate dehydrogenase (GAPDH) (spot 350) and phosphoglycerate kinase 2 (PGK2) (spot 252), both part of the glycolytic pathway, were increased by 88% and 100%, respectively (P<0.01); short chain 3-hydroxyacyl CoA dehydrogenase (HADH) (spot 302) and electron transfer flavoprotein beta subunit (ETF) (spot 371), both part of the fatty acid oxidation pathway, were reduced (-31% and -23%, resp, P<0.01) when compared to high-flow areas. The decreased capacity of the fatty oxidation pathway (HADH, ETF) in low-flow areas was associated with a significantly higher total lipid content (56±12 vs. 32±5 mg/g, P<0.001, each n=7 sample derived from two hearts). Low-flow areas were also characterized by a lower myoglobin content (spot 489, -23%, P<0.01).
To exclude that major differences in tissue composition may account for the differences observed in protein expression, mitochondrial and myofibrillar marker proteins were quantified, as well as marker proteins of plasma space and erythrocyte volume. No differences were observed in these structural markers. Only the intermediate filament desmin was differentially expressed (spot 134 and spot 135). Desmin, which is located at the Z-discs linking the myofibrils, was elevated in high-flow areas by 40% (P<0.05).
4. The spatial heterogeneity of myocardial blood flow is stable for weeks
To test whether the differences in protein expression are related to a long-term stability of local flow, different colored fluorescent microspheres were applied consecutively every 2nd day in the 2 wk preceding excision of the left ventricular wall. When relating local MBF on day 1 to that measured on day 13, a very close correlation was observed: r2 = 0.79–0.90 in the individual hearts. Also, when relating the local perfusion of each day of microsphere application to the flow measured on the final day, a close correlation was observed, r2 being 0.79 ± 0.07 on average. The spatial heterogeneity of MBF is thus stable at least for weeks.
CONCLUSIONS
Myocardial work, energy turnover, metabolism, and perfusion are known to be tightly coupled. Left ventricular areas differing by > threefold in local flow and energy turnover thus might be expected to display substantial differences in local gene and protein expression, finally translating into morphological changes. However, despite a temporally stable flow distribution, we detected no differences in vascular, mitochondrial, and myofibrillar protein markers, consistent with a homogeneous morphology of low- and high-flow regions reported in previous studies. This seems surprising considering recent reports by us and by others demonstrating substantial differences in local oxygen consumption between high- and low-flow areas. The present study identified 12 proteins (of 380) that displayed significant differences in their expression level. The proteins identified delineate a close link between local flow and energy turnover on the one, and NO and energy metabolism on the other hand. We suggest that the spatial heterogeneity of NO formation and substrate utilization may be key to a homeostatic mechanism whereby local O2 demand and O2 supply are balanced in areas of low local flow (Fig. 3
).
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NO formation
We found that DDAH1 (ADMA dimethylaminohydrolase) was increased by almost fourfold in low-flow regions on the protein and mRNA levels. This resulted in a reduction of its substrate ADMA by 75% in low-flow samples. ADMA is known as a potent endogenous inhibitor of all isoforms of NO synthase. Since the expression of eNOS is similar in high- and low-flow regions, the low tissue concentrations of ADMA in low-flow regions will result in less inhibition of eNOS and an increased NO formation, inducing a distinct vasodilatation in these regions. In line with this conclusion, blockade of NOS activity by L-NAME induced a greater decrease in flow in low-flow regions in an earlier study. Besides increased NO formation, decreased NO inactivation may further enhance local NO. We demonstrated recently that myoglobin, in addition to its function as oxygen carrier, serves as an effective scavenger of NO. In consequence, the reduced myoglobin content in low-flow regions observed in this study may diminish the rate of NO inactivation. The present data thus clearly indicate reduced inhibition of NOS, enhanced NO formation, and decreased NO inactivation in low-flow regions. It is well known that NO not only plays a decisive role in the control of coronary tone, but can also reduce myocardial oxygen consumption. Therefore, higher NO concentration may even contribute to the lower oxygen consumption and energy turnover of low-flow areas.
Energy metabolism
Low-flow areas displayed almost twofold higher levels of glycolytic enzymes (PGK2 and GAPDH), whereas enzymes involved in fatty acid catabolism (HADH and ETF) were down-regulated. The importance of glycolysis appears to be greater in low-flow areas whereas, conversely, in high-flow areas fatty acid oxidation may prevail. This apparent shift from ß-oxidation to glycolysis in low-flow regions would locally decrease the relative need for O2, since the oxidation of glucose requires less oxygen per mole of ATP produced. Thus, a higher capacity for glycolysis may facilitate the generation of sufficient energy in regions of low O2 supply. In high-flow regions, the enhanced capacity for ß-oxidation is consistent with a higher uptake of fatty acids previously observed and in line with the lower absolute lipid content measured. The greater capacity for fatty acid oxidation in high-flow areas will enhance energy supply in areas of elevated energy turnover. The increased expression of myoglobin might improve oxygen diffusion in regions of high blood flow and energy demand. Since local energy turnover in high-flow areas exceeds that of low-flow areas by at least threefold, not only fatty acid oxidation, but also the amount of glucose oxidized, may be higher than in low-flow areas. To elucidate the relative importance of glycolysis and fatty acid oxidation in low- and high-flow regions, tracer studies of substrate utilization are required.
This study is the first to characterize the molecular basis associated with the spatial heterogeneity of myocardial perfusion and metabolism. Our findings indicate major differences in NO formation and substrate utilization between areas of a persistent low/high local flow and energy turnover. In fact, they unravel a possible homeostatic mechanism by which areas of low local flow and thus limited O2 supply are protected (Fig. 3)
. In these areas, the local concentration of NO can be expected to be elevated due to impaired NO transport and metabolism (decreased myoglobin) and increased NO formation (increased ADMA dimethylaminohydrolase, less ADMA, and thus less NOS inhibition). The elevated NO will result in a greater contribution of NO to the maintenance of vascular tone and an increased O2 supply. The recent recognition that NO tonically reduces myocardial O2 consumption and enhances cardiac efficiency suggests that a higher NO concentration in low-flow areas may even contribute to their lower O2 consumption. The higher capacity for glycolysis in low-flow areas will reduce the O2 demand and consumption for a given ATP turnover. These factors together improve the balance between O2 supply and demand in areas receiving < 50% of the mean myocardial perfusion, thereby preventing local hypoxia or ischemia. The high temporal stability of local perfusion pattern suggests that external factors possibly related to myocardial fiber architecture and local strain predominantly govern the local energy demand. We would like to suggest that it is the local energy demand that is the primary force driving local energy turnover and myocardial blood flow, whereas gene and protein expression are modulated by local needs (Fig. 3)
.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0574fje; to cite this article, use FASEB J. (February 12, 2002) 10.1096/fj.01-0574fje 
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