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Cardiovascular Research 1998 38(3):727-735; doi:10.1016/S0008-6363(98)00056-X
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Super-normal Formula retention in hibernating myocardium: an ex-vivo study using the failing human heart

Oberdan Parodia,*, Renata De Mariaa, Roberto Testaa, Ettore Vitalib, Livia Ruffinic, Giovanna Palearib, Edoardo Grondab, Jonica Campoloa and Alessandro Pellegrinib

aC.N.R. Clinical Physiology Institute, Milan Section, Niguarda Hospital, Milan, Italy
bMedical and Surgical Cardio-Thoracic Department A. De Gasperis, Niguarda Hospital, Milan, Italy
cNuclear Medicine Service, Niguarda Hospital, Milan, Italy

* Corresponding author. Tel.: +39 (2) 6444 2605; Fax: +39 (2) 647 3407; E-mail: ifcnigmi@tin.it

Received 22 October 1997; accepted 27 January 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Objective: Although the relationship between delayed Formula distribution and blood flow in acutely ischemic and infarcted myocardium has been widely explored in the experimental setting, its behaviour in chronically hypoperfused dysfunctioning human myocardium has not yet been evaluated. Methods: In tissue samples of excised failing hearts taken from ischemic (IHD) patients and idiopathic dilated cardiomyopathy (IDC) controls, we evaluated the relationship between delayed Formula retention (4 h redistribution), blood flow (assessed by means of Formula -labelled human albumin microspheres injected during transplantation) and biochemically-assessed fibrosis. Formula activity was expressed as the percent of the activity in the region with highest flow and the least fibrosis. Results: Fibrosis and Formula activity were inversely related (r=–0.62, P=0.0001). In IDC controls, low flows corresponded to uniformly preserved Formula retention. In IHD, 46 segments with flows ≤0.60 ml·min–1·g–1 and 20 segments with flows >0.60 ml·min–1·g1 showed matching delayed Formula retention and flow values; in the remaining 27, there was a disproportionately high tracer accumulation in comparison with flow (flow/Formula mismatch). Despite significantly less fibrosis and lower flows, the mismatch segments showed significantly greater Formula activity than the segments with concordantly high tracer retention and flow values. Conversely, at equivalent flow rates, the mismatch regions had less fibrosis than the areas with concordantly depressed Formula activity and perfusion. Conclusions: This super-normal Formula retention in hibernating myocardium may indicate a mechanism of cell adaptation to chronic hypoperfusion.

KEYWORDS Heart failure; Transplantation; Radioisotopes; Perfusion; Coronary artery disease; Human


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
After intravenous Formula administration, initial tracer accumulation under conditions of normal or reduced coronary blood flow correlates with the extent of perfusion [1]in both normal and acutely ischemic or infarcted myocardium [2, 3]. Conversely, time-dependent Formula accumulation is thought to reflect the amount of viable myocardium capable of retaining the tracer [4]: hypoperfused areas with initially reduced Formula activity may normalize tracer content at a later time due to tracer redistribution, indicating preserved cell membrane integrity [5, 6]. This kinetic property of the isotope has been clinically applied to the identification of viable myocardium using post-stress/reinjection or rest/redistribution protocols [7–11]. In experimental studies, Formula redistribution has been shown to be primarily due to tracer clearance from the normal zone and delayed accumulation in the previously ischemic zone [12, 13]. This normalization of Formula activity in ischemic areas may occur despite the maintenance of a constantly reduced perfusion pressure, thus indicating that tracer redistribution does not require the restoration of normal blood flow in the territories that are underperfused during isotope injection [14–16]. Delayed Formula images of hypoperfused but still viable (hibernating) myocardium may therefore be a powerful signal of cell membrane integrity [17]. The mechanisms underlying the delayed accumulation and slow clearance rates of this tracer have been mainly evaluated in experimental models of acute ischemia and reperfusion [12–16], but neither Formula distribution in chronically hypoperfused dysfunctioning human myocardium (the main clinical application of Formula viability imaging), nor its relationship with blood flow, have yet been fully characterized. In this setting, delayed Formula accumulation may reflect alterations in myocardial metabolic processes that are still not fully understood in humans. An important factor for the myocardial handling of Formula is the activity of the Na, K-ATPase pump [18], whose action is related to transmembrane electrolyte gradients and sympathetic stimulation [19]. In patients with ischemic heart disease (IHD) and heart failure, cardiac adrenergic drive is increased, leading to the depletion of myocardial catecholamine stores [20, 21]; this chronic sympathetic hyperstimulation might enhance tissue Formula accumulation and lead to super-normal tracer uptake in the myocardial regions exposed to adrenergic hyperactivity.

The heart transplant procedure provides a unique experimental setting [22, 23]for the exploration of chronic hibernation in the failing human heart. In the present investigation, we used the ex-vivo analysis of failing human hearts excised at transplantation to verify the behaviour of delayed Formula distribution in chronically hibernating myocardium in comparison with necrotic and normally perfused territories, and assess its relationship with indirect estimates of increased cardiac adrenergic drive.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusions
 References
 
2.1 Selection and characterization of patients
The study involved eleven patients (ten men and one woman, with a mean age of 46±15 years; range: 19–61), who underwent orthotopic heart transplantation for refractory heart failure at the Cardiac Surgery Division of our Institution. Six of the patients had chronic IHD documented either by a history of previous myocardial infarction (five patients), or by evidence of diffuse critical coronary artery narrowing at coronary angiography; the myocardial infarctions were documented by symptoms, diagnostic ECG changes and increased serum enzyme levels, and confirmed by means of the histological analysis of the excised heart performed by an experienced cardiovascular pathologist who was unaware of the clinical findings. The remaining five patients had IDC diagnosed on the basis of the finding of a dilated, hypocontractile left and/or right ventricle in the absence of other forms of cardiac disease, and were used as control group.

IDC patients were younger than IHD ones (36±16 vs. 54±8 years, P=0.035).

All of the patients were being treated with furosemide and vasodilators (angiotensin-converting-enzyme inhibitors in ten, hydralazine in one). Preoperative treatment included digoxin in nine cases, amiodarone in four, and the phosphodiesterase inhibitor enoximone in two.

All of the patients showed severe ventricular function impairment: their mean cardiac index was 2.05±0.31·L min–1·m–2; mean left ventricular ejection fraction 0.26±0.08; and mean pulmonary wedge pressure 22±6 mmHg. The hemodynamic impairment was similar in the IDC and IHD cases.

The nature of the study and the reason for injecting radiolabeled human albumin microspheres were both explained to all of the patients, who subsequently gave their informed consent. The study protocol was approved by the local Ethics Committee for Human Research; the investigation was in accordance with the institutional guidelines and conforms with the principles outlined in the Declaration of Helsinki [24].

2.2 Radiotracer protocol
37 MBq (1 mCi) of Formula chloride (physical half-life: 73 h) were injected in a peripheral vein 4 h before surgery. In order to measure myocardial blood flow (MBF), 2.5–3 million human albumin microspheres labeled with 185 MBq (5 mCi) of Formula pertechnetate (physical half-life: 6 h) were used during the Shumway heart transplantation procedures [25]. Heart rate and blood pressure were measured at the time of both radionuclide injections. For the injection of the radiolabeled microspheres, a flared 3F polyethylene tube was placed in the left atrial appendage and subsequently flushed with heparinized saline. Before clamping, the ascending aorta was cannulated with a 3F polyethylene catheter connected to a 50 cm heparinized Vigon tube (1 ml) for the drawing of a microsphere reference sample by means of a peristaltic pump (P-1 Pharmacia, Uppsala, Sweden). Arterial blood sampling was started 10 s before tracer injection by activating the peristaltic pump to draw 6 ml/min for 3 min. All of the blood (18 ml) was collected into a 20 ml syringe connected to the peristaltic pump by a silicon tube. The entire procedure usually lasted no more than 10 min. Before harvesting, the heart was arrested using a standard cold (4°C) hyperkaliemic cristalloid solution infused through the ascending aorta.

2.3 Post-surgical tissue preparation
After the completion of the surgical protocol, the heart was excised and immediately transported to our nearby laboratory, where it was weighed and inspected for the presence of gross abnormalities. The main branches of the coronary arteries were sequentially cross-sectioned at 3 mm intervals in order to detect any critical stenoses; all of the segments with a reduced lumen and at least one normal segment were excised for histologic processing. The heart was subsequently divided from base to apex into four or five slices of equal thickness: a mid-ventricular slice was used for the determination of regional MBF and Formula uptake, which were both measured at the same time the biochemical assay was performed.

2.4 Regional MBF and Formula analysis
The slice was divided into nine transmural wedges, eight of which corresponded to the anterior, lateral, posterior and septal left ventricular walls, and one to the free wall of the right ventricle; the wedges were further subdivided into epicardial (outer half) and endocardial (inner half) specimens, leading to a total of 18 segments. These were then split into two parts: one for well counter determination and the other for biochemical analysis (see below).

Each specimen was placed into a vial to be weighed (average weight 1.3 g; range: 0.8–1.8 g) and counted. The blood and tissue Formula counts were obtained within 12 h of the tracer injection using a multichannel gamma counter with an energy window of 120–160 KeV; decay was corrected back to the time of injection. Regional MBF was determined using the method of Heymann and Rudolph [26]and expressed in ml·min–1·g–1. After allowing for Formula decay (at least ten half-lives), the samples were counted for Formula activity using energy windows of 65–90 and 120–160 KeV. The Formula contribution to the 120–160 KeV Formula photopeak was calculated after correction for decay, and then subtracted from the earlier Formula counts in order to obtain true Formula counts. Formula activity in each endocardial and epicardial wedge was expressed as a percentage of the activity in the ‘most normal’ region of each patient, which was defined as the segment with the highest MBF and the least fibrosis. Transmural activity was calculated by summing endocardial and epicardial counts (normalized per g of tissue), and expressed as a percentage of the one observed in the most normal region of each patient.

2.5 Quantitative determination of myocardial fibrosis and norepinephrine
Frozen tissue (300 mg) was homogenated and added to 3 ml 0.1 N perchloric acid containing 0.05% Na-metabisulfite with 0.1% EDTA as antioxidant and dihydroxybenzylamine as the internal standard.

The amount of collagen was determined by means of HPLC on the basis of the tissue concentration of 4-hydroxyproline, a collagen-specific iminoacid. The homogenate (2 ml) was added to 2 µL of HCl 12 N, containing norvaline and sarcosine as internal standards for primary and secondary aminoacids respectively; the mixture was hydrolysed for 70 h at 110°C in glass-stoppered tubes.

Five hundred ml of the hydrolysate were freeze-dried and dissolved in 500 µl of distilled water; after centrifugation at 18 000xg for five minutes in an ultracentrifuge (TL100, Beckman Instruments, Palo Alto, CA, USA), 5 µl of the supernatant were diluted with 1 ml of HCl 0.1 N. Ten-ml portions were derivatized in 55 µl of borate buffer 0.4 M, pH 10.2, with 2 µl of o-phthalaldehyde and 3-mercaptopropionic acid (OPA-3-MPA) (5061-3335 in borate buffer, Hewlett Packard GmbH, Waldbronn, Germany) in order to derivatize all of the primary aminoacids; after 3 min, 2 µL of 9-fluorenyl-methyl-chloroformate (FMOC) (5061-3337 in acetonitrile, Hewlett Packard GmbH, Waldbronn, Germany) were added to derivatize the secondary aminoacids and mixed for 2 min [27]; 10 µl of this mixture were injected into the HPLC system (Kontron Liquid Chromatograph, Kontron Instruments, Milan, Italy), which was equipped with a fluorescence detector (SFM 25 Programmable, Kontron Instruments). The excitation and emission wavelengths were respectively 340 and 450 nm for the primary, and 266 and 305 nm for the secondary aminoacids.

Binary solvent elution was carried out in a reversed phase C18 column (ProgelTM TSK, ODS-80 TS, 5 µm, 250 mm length, 4.6 mm internal diameter, Supelco, Bellafonte, PA, USA) at a flow rate of 0.75 ml/min at 40°C. The buffers were: A=0.02 M acetate buffer with 0.02% of triethylamine (pH 7.2); and B=0.1 M acetate buffer: acetonitrile: methanol (1:2:2). An acid hydrolysate of standard collagen (Sigma Chemical, St. Louis, MO, USA) was used for calibration purposes in order to derive the K constant as the ratio between the peak areas of hydroxyproline and the total aminoacids contained in the standard hydrolysate. The mean constant was 10.6±0.46. The reproducibility of the derivatization was calculated from the chromatographic peak areas of hydroxyproline (74.9±2, coefficient of variation 2.6%) and total aminoacids (736.2±24.7, coefficient of variation 3.3%).

For each sample, tissue fibrosis was calculated and expressed in percentage terms as the ratio between the collagen protein and total aminoacid peak areas (collagen protein plus non-collagen protein), multiplied by the K constant.

In order to determine the level of myocardial catecholamine stores, one ml of tissue homogenate was centrifuged at 100 000xg in a TL100 centrifuge for 10 min at 4°C, and the norepinephrine-containing supernatant was extracted at 4°C as follows: 10 mg of alumine (JWE000313, Millipore, Bedford, MA, USA) and 300 µl of 2 M TRIS/HCl buffer (pH 8.3) were added to 500 µl of the supernatant and stirred for 30 min. The slurry was centrifuged at 6000xg for 5 min, and the pellet (alumine) was washed thrice with 1 ml 5 mM TRIS/HCl buffer (pH 8.3). 100 µl 0.1 N perchloric acid was added to the alumine after 30 min of stirring and centrifugation at 6000xg. Five µl of the norepinephrine-containing supernatant were injected into the HPLC system (injector U6K, pump 510, Waters, Milford, MA, USA), which was equipped with an electrochemical detector (Coulochem Electrochemical Detector, model 5100A, ESA, Bedford, MA, USA) and a 5011 cell (ESA, Bedford, MA, USA) at –0.1 V and +0.35 V. Isocratic elution was carried out in a reversed phase C18 column (Supelcosil LC18, 5 µm, 250 mm length, 4.6 mm internal diameter, Supelco, Bellafonte, PA, USA) at 1.2 ml/min with a 5 mM citrate buffer (pH 4.5), using methanol (95:5) and 1.2 mM octansulphonic acid as an ion pair.

In order to avoid the confounding effect of the base-apex norepinephrine content gradient [20], care was taken to assess mid-ventricular slices obtained at the same level of the base-apex plane in all patients. The normal reference values for tissue norepinephrine concentrations in our laboratory are 1573 pg/mg for the left and 2312 pg/mg for the right ventricle.

2.6 Statistical analysis
Continuous data are expressed as mean values ±1 standard deviation. Student's t-test for unpaired data or one-way analysis of variance with post-hoc testing (Scheffe's test) and the chi-square test were used for normally distributed variables. For the variables showing a skewed distribution, the Wilcoxon, Mann–Whitney or Kruskall–Wallis non-parametric tests were used as appropriate. Formula uptake was correlated with the biochemical indices by means of linear regression analysis. A P value of <0.05 was considered significant. The StatviewTM SE statistical package (Abacus Concept, Berkeley, CA, USA) was used.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusions
 References
 
Mean diastolic arterial pressure and mean rate-pressure product at the time of Formula injection 4 h before the surgical procedure were about 20% higher than those measured at the time of microsphere injection performed during the surgical procedure (75±12 vs. 60±14 mmHg, P=0.009 and 10572±2444 vs. 8600±2300, P=0.04, respectively). Neither hemodynamic changes nor new electrocardiographic abnormalities were observed during and after microsphere injection.

3.1 Gross pathology
The five IDC control hearts showed moderate to severe left ventricular (3 cases) or biventricular (2 cases) dilatation, and normal coronary arteries.

Five of the six IHD hearts had myocardial scars involving the apex, anterolateral wall and anterior septum in four cases, and the apex and posterior wall in one. All of the IHD patients had lumen restriction of the left anterior descending coronary artery which was ≥70% in five; the right coronary artery was occluded in two patients, and showed a critical stenosis in one; the left circumflex was critically stenosed in five patients.

The heart weight was similar in IDC controls and IHD cases (446±151 g vs. 428±75 g, P=ns).

3.2 MBF, fibrosis, Formula retention and norepinephrine content
The MBF and fibrosis values in the IHD and IDC cases approached those obtained in the previously published group of eight patients included in this study. The data relating to MBF, fibrosis, Formula uptake and norepinephrine content are summarized in Table 1.


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  		Table 1 Table 1 and MBF distribution and biochemical indices in the 11 excised hearts

 
Mean transmural Formula retention was less homogeneous in the IHD cases than in the IDC controls (coefficient of variation: 37% vs. 13%). The endocardial to epicardial uptake ratio in the left ventricle was higher in the IDC than in the IHD cases (1.14±0.20 vs. 1.03±0.5, P=0.003), but it was similar in the right ventricle. Formula retention was not significantly different in the wedges supplied by <70% (n=40) or ≥70% (n=56) stenosed vessels, being 83±30% vs. 72±27% (P=ns) in the endocardium and 84±19% vs. 82±34% (P=ns) in the epicardium; the endocardial to epicardial uptake ratio averaged 1.00±0.36 vs. 1.04±0.65 (P=ns).

There was no difference between the IHD and IDC patients in terms of the norepinephrine content of either the left (P=ns) or right ventricle (P=ns). In the IDC cases, norepinephrine tissue content was slightly affected by differences in perfusion, being 736±523 pg/mg in the segments with an MBF of ≤0.6 ml·min–1·g–1, and 907±446 pg/mg in those with an MBF of >0.6 ml·min–1·g–1 (P=0.056), but definite flow-related dishomogeneities in norepinephrine concentrations were found in the IHD cases (684±433 vs. 1531±982 pg/mg, P=0.0015), regardless of the degree of fibrosis.

When the MBF values were plotted against Formula retention, no correlation was found in either the IHD (r=0.05) or the IDC patients (r=0.28). Biochemical fibrosis and Formula retention inversely correlated (r=–0.62, P=0.0001) (Fig. 1); as expected, the correlation was looser in specimens with <20% fibrosis (r=–0.34, P=ns) both in the IHD group and in IDC controls, due to the relatively large scatter of Formula retention at low fibrosis values.


Figure 1
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  		Fig. 1 Correlation between Figure 1 retention and biochemically determined myocardial fibrosis in IDC controls (closed circles) and IHD patients (open circles). See text for details.

 
3.3 Characterization of flow/Formula retention patterns in IHD
When the Formula retention and microsphere MBF values of the IHD patients were individually plotted, their behaviour was different in a number of regions characterized by disproportionately high Formula activity, as shown in the example given in Fig. 2. In order to identify these areas in each patient, flow was expressed as a percent of its maximum, and the region with the highest flow and least fibrosis was considered to be the ‘most normal’. Formula activity was normalized (100%) to this region, and the activity in the other regions was expressed as a percent of that in this segment. All of the regions with the most normal flow (mean 0.94±0.6 ml·min–1·g–1) showed a low degree of biochemical fibrosis (mean 11±8%).


Figure 2
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  		Fig. 2 Circumferential endocardial (upper panel) and epicardial (lower panel) profiles of myocardial blood flow (MBF, open squares), Figure 2 retention (closed circles) and fibrosis (crosses) in an IHD patient (patient 7). Figure 2 retention closely matches MBF in the anterior (segments 1–2), lateral (segments 3–4) and anterior septal walls (segment 8) of the left ventricle. In the posterior (segments 5–6) and posterior septal walls (segment 7), there is discordance between flow and tracer retention values in both layers, with a disproportionately high Figure 2 retention in relation to flow, leading to a flow/viability mismatch pattern (arrows). Figure 2 retention in these segments is greater than that observed in the segments (epicardium 1–2) with the most normal flow (>0.8 ml.min–1·g–1). The degree of fibrosis matches Figure 2 retention and MBF at the points with concordant cation and flow behaviour, but is consistently low in the areas with discordant behaviour.

 
Subsequently, some of the segments were arbitrarily defined flow/Formula mismatches when there was a >25% difference between the percent increase in Formula retention and the percent decrease in the corresponding flow value (see Fig. 2). This flow/viability mismatch pattern was found in 27/93 segments (29%): in the mismatched segments, the range of Formula retention (normalized to MBF and fibrosis as described above) was 87–160%.

Three segments with normal MBF values (>0.6 ml·min–1·g–1) but a low (<50%) Formula retention (‘reverse mismatch’ pattern) showed a high degree of fibrosis (mean 32±18%; range 21–53%), and were considered to represent reperfused myocardium within scar areas.

To separate segments with concordantly low or high Formula and MBF, the flow values were divided into four categories of impairment: severe (≤0.4 ml·min–1·g–1), moderate (0.41–0.60 ml·min–1·g–1), mild (0.61–0.80 ml·min–1·g–1), and normal (up to 0.80 ml·min–1·g–1). Concordant segments falling in the severely or moderately impaired flow categories were defined low match points (n=46); those falling in the mildly impaired or normal flow categories were considered high match points (n=20). We assumed that, in a continuum of tissue damage (myocardial fibrosis ranging from 1% to 99%), low match segments would represent the most damaged tissue, and high match segments the ‘most normal’ regions. Mismatching and matching points were similarly distributed between endocardial and epicardial wedges.

MBF, Formula retention, fibrosis, norepinephrine content, and the distribution of critically stenosed coronary vessels according to the different flow/Formula patterns are shown in Table 2. In comparison with low match segments, the mismatch points had a similar MBF and frequency of critically stenosed vessels; however, their norepinephrine content was higher and the percentage of fibrosis lower. Percent fibrosis did not exceed 10% in 21 out of 27 mismatch points; in 4 of the remaining 6 points, it was less than 20%. In comparison with high match segments, the mismatch points had a higher Formula retention, and similar fibrosis and norepinephrine content despite their markedly lower MBF. The individual data points of fibrosis and Formula retention in the matching and mismatching segments are shown in Fig. 3.


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  		Table 2 Characterization of flow/Table 2 patterns in ischemic patients

 

Figure 3
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  		Fig. 3 Individual data points of biochemical fibrosis (top panel) and Figure 3 retention (lower panel) in segments characterized as low match, high match and mismatch. When compared with low match segments, the mismatch points show less fibrosis despite similarly low flow values (see text); when compared with high match segments, they show a higher tracer retention despite a similar degree of fibrosis.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusions
 References
 
The main finding of this study, which examined the relationships between MBF, delayed Formula distribution, myocardial fibrosis and catecholamine content in chronically dysfunctioning hearts during transplant surgery, is the disproportionately high Formula retention found in 29% of the IHD segments with respect to microsphere flow estimates.

4.1 Formula uptake and retention in ischemic myocardium
The initial distribution of Formula in the myocardium is proportional to regional blood flow at the time of injection after coronary occlusion [1]and during the increase in metabolic demand [2], but it underestimates MBF during reactive hyperemia after transient ischemia [1]. In an experimental model of normal, ischemic and infarcted myocardium, a straight correspondence has been observed between initial Formula distribution and MBF [3], thus indicating that local metabolic conditions have a non-consistent effect on initial tracer deposition.

Early tracer redistribution to ischemic territories after flow restoration has been described in animal models [28], as well as in the clinical setting [5, 6], which suggests that Formula retention may not accurately reflect perfusion in normal, ischemic or acutely necrotic myocardium. Viable myocells in regions of flow restriction, which are exposed to a smaller amount of Formula in the early post-injection phase, continue to extract the recirculating tracer from the blood, and thus show a delayed peak uptake: in both animal models [13, 16, 28]and human disease [5, 6, 29], perfusion defects at initial rest imaging eventually decrease or disappear. Experimental data indicate that, when the levels of coronary blood flow and recirculating tracer are reduced, the intrinsic washout rate is decreased in the hypoperfused region [13–16, 30].

Formula redistribution is not simply flow dependent, but reflects intrinsic cell mechanisms that tend to re-establish an equilibrium within the cell potassium pool. In the acutely ischemic experimental model, it has been found that near-equalization of tracer activity between normal and acutely ischemic zones occurs within 4 h of tracer administration [12], thus indicating that the net loss of Formula from the hypoperfused myocardium proceeds more slowly and is less than in normoperfused areas. However, no experimental study has yet examined the behaviour of late Formula distribution in chronically hypoperfused, dysfunctioning but viable myocardium, mainly because of the lack of appropriate animal models of long-term hibernation. In our human model of chronic post-ischemic dysfunction, the late intracellular Formula concentrations in viable hypoperfused regions tended to normalize and even to exceed the accumulation in normoperfused areas; this led to super-normal tracer retention in mismatching points, a pattern that has never been clearly described in the experimental setting.

The rate of Formula presentation to the myocardial site is the product of arterial blood concentration and regional MBF, and so the time to peak Formula activity should be inversely related to blood flow: i.e. a progressive reduction in MBF should lead to a progressive lengthening in the time to peak activity. However, the higher level of tracer activity observed by us cannot be entirely explained by this mechanism: in fact, as the average MBF values in mismatching segments were less than 50% of those observed in normal segments (0.43 vs. 0.97 ml·min–1·g–1), a >50% reduction (in comparison with normoperfused areas) would be expected in the rate of Formula delivery to hypoperfused viable regions. And even assuming 100% tracer extraction at lower perfusion rates, the amount of Formula taken up by mismatching segments would still be expected to be much less than that taken up by normally perfused areas. Super-normal Formula retention at the mismatching points indicates that intramyocyte tracer transport and retention are greater in hypoperfused than normoperfused segments during the 4 h after tracer administration.

Yipintsoi et al. [31]and Becker et al. [32]found disproportionately high Formula concentrations in comparison with microspheres in severely ischemic regions whose microsphere content was less than 20% that of the control regions; the latter authors also reported that tracer content in peri-ischemic myocardium (which had 80–90% of the flows of the control segments) was significantly higher than in myocardium supplied by the patent coronary artery [32]. DiCola et al. [33]observed that Formula uptake in very low flow endocardial regions tends to overestimate the relative regional flow assessed by means of the microsphere technique. Although these authors suggested that the disproportionately high Formula or Formula content at very low flows was very probably due to the inverse relationship between flow and extraction, they did not provide any explanation for the higher tracer uptake in peri-ischemic segments with only a slightly reduced flow [32, 33].

In our study, several of the mismatching segments within the same myocardial slice, and with an equivalently low degree of fibrosis, showed a level of Formula activity that was more than 30% greater than that observed in regions with a flow rate that was three times higher (Fig. 3).

4.2 Mechanisms of Formula uptake and retention in hibernating myocardium
Many mechanisms have been proposed to explain functional down-resetting in viable but dysfunctioning (i.e. hibernating) myocardium [34]. Increased sympathetic stimulation and neurotransmitter spillover from cardiac adrenergic nerve endings, leading to a depletion of catecholamine stores [20, 21, 35], have been previously demonstrated in patients with heart failure. Adrenergic hyperstimulation may in turn activate membrane-bound Na+,K+-ATPase [36]. The transient K+ outward current [37]is also reduced in failing ventricles, and the intramyocardial activity of Formula (a potassium analogue) is likely to be influenced by changes in potassium influx and efflux into myocytes: Formula enters the cell via membrane-bound Na+,K+-ATPase, whereas its efflux is mainly linked to voltage-dependent K+ channels. Gradients of tracer content occur between areas with greater chronic exposure to cathecolamines and areas with a lower adrenergic drive. In line with the results of previous studies, we found that the levels of norepinephrine stores were similar in the failing hearts of our IDC and IHD patients; however, within the ischemic group, the norepinephrine content in the mismatching segments was higher than in the low match segments, but lower than in the high match segments (Table 2), thus indicating differences in the exposure of viable hypoperfused regions to adrenergic hyperstimulation. This latter finding may at least partially account for the increased Formula accumulation observed in the mismatching areas. Activation of Na+,K+-ATPase may increase K+ influx and reduce intracellular Na+ concentrations, and this reduction in intracellular Na+ could activate the Na+, Ca2+ exchanger, and consequently reduce the availability of calcium ions in intracellular stores. The net result would be a decline in regional inotropism in hypoperfused but viable myocardium. The accumulation of Formula in chronically hypoperfused viable segments may therefore reflect cell adaptation to chronic hypoperfusion.

As Formula cellular accumulation and retention depend on cell membrane integrity, its concentration into the myocyte does not mirror the myocellular utilization of 18F-fluorodeoxyglucose, a viability tracer which requires the presence of metabolically active myocytes. Therefore, possible discordancies obtained with these two radionuclides in the assessment of myocardial viability may be explained by their different uptake mechanisms and find a structural basis in clinico-morphologic studies [23, 38–40].

4.3 Limitations of the study
For ethical reasons, it was not possible to obtain the Formula input function during the 4-h interval between the injection of the tracer and transplant surgery: consequently, no absolute Formula uptake values for direct inter-patient comparison are available.

An inherent limitation of the design of this study is the lack of information regarding initial Formula uptake. However, as initial Formula activity is a generally accepted measure of relative blood flow, microsphere values are an acceptable surrogate for initial Formula distribution. In fact, although peri-operatively assessed MBF, which was determined under anaesthesia, might not precisely mirror myocardial perfusion at the time of Formula injection 4 h before surgery, previous experimental findings indicate that much higher pressure and rate changes from baseline than the average 20% difference observed in this patient series are required to obtain only mild flow changes with anaesthesia [41]. In addition, despite the absence of ischemic changes at ECG monitoring, the possibility of ischemia occurring during anesthesia and the first hour of surgery cannot be ruled out. However, the time interval between Formula injection and the induction of anesthesia was in any case greater than 3 h, so that tracer redistribution should have been almost complete.

Norepinephrine tissue concentrations provide only an indirect estimate of cardiac adrenergic function; whether catecholamine depletion in mismatching regions represents functional denervation of these areas, or indicates the exhaustion of cardiac stores due to excessive adrenergic nerve firing, cannot be determined from our data.

At the time of transplant surgery, the majority of our patients with severe refractory heart failure were assuming digitalis. Previous experimental work [42]has shown that Formula uptake is not altered by ouabain-induced inhibition of the Na+,K+-ATPase. The impact of treatment on our findings should therefore be minimal.


   5 Conclusions
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
References
 
Our data indicate that many viable hypoperfused regions in the chronically dysfunctioning hearts of IHD patients are characterized by a disproportionately high Formula retention, leading to a mismatching flow/viability pattern. This super-normal Formula retention may be a fingerprint of cell adaptive mechanisms to chronic hypoperfusion not previously reported in the experimental setting.

Time for primary review 42 days.


   Acknowledgements
 
This study was supported in part by the CNR-Targeted Project "Prevention and Control of Disease Factors", Subproject "Control of Cardiovascular Disease", from the National Research Council, Rome, Italy. The authors are grateful to the personnel of the Anesthesiology Service, the Cardiac Surgery, and the First Division of Cardiology for their skilful cooperation during the surgical procedure. We also thank Rosanna Vigorelli for her technical assistance, Marina Parolini for the statistical analysis of data and Elisabetta Spagnolo for her secretarial assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 

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