Molecular Imaging for Efficacy of Pharmacologic Intervention in Myocardial Remodeling
Susanne W.M. van den Borne, MD*, ,
Satoshi Isobe, MD, PhD*,
H. Reinier Zandbergen, MD,
Peng Li, MD, PhD*,
Artiom Petrov, PhD*,
Nathan D. Wong, PhD, FACC*,
Shinichiro Fujimoto, MD, PhD*,
Ai Fujimoto, MD, PhD*,
Dagfinn Lovhaug, PhD ,
Jos F.M. Smits, PhD,
Mat J.A.P. Daemen, MD, PhD,
W. Matthijs Blankesteijn, PhD,
Chris Reutelingsperger, PhD,
Faiez Zannad, MD, PhD, FACC ,
Navneet Narula, MD*,
Mani A. Vannan, MD, FACC*,
Bertram Pitt, MD, FACC¶,
Leonard Hofstra, MD, PhD,
Jagat Narula, MD, PhD, FACC*,*
* Division of Cardiology and Department of Pathology, University of California, Irvine School of Medicine, Irvine, California
Department of Cardiology, Biochemistry, Pathology and Pharmacology, University Hospital Maastricht, Maastricht, the Netherlands
GE Healthcare, AS, Oslo, Norway
University Henri Poincaré, Nancy, France
¶ Division of Cardiology, University of Michigan, Ann Arbor, Michigan
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Abstract
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Objectives: Using molecular imaging techniques, we examined interstitial alterations during postmyocardial infarction (MI) remodeling and assessed the efficacy of antiangiotensin and antimineralocorticoid intervention, alone and in combination.
Background: The antagonists of the renin-angiotensin-aldosterone axis restrict myocardial fibrosis and cardiac remodeling after MI and contribute to improved survival. Radionuclide imaging with technetium-99m–labeled Cy5.5 RGD imaging peptide (CRIP) targets myofibroblasts and indirectly allows monitoring of the extent of collagen deposition post-MI.
Methods: CRIP was intravenously administered for gamma imaging after 4 weeks of MI in 63 Swiss-Webster mice and in 6 unmanipulated mice. Of 63 animals, 50 were treated with captopril (C), losartan (L), spironolactone (S) alone, or in combination (CL, SC, SL, and SCL), 8 mice received no treatment. Echocardiography was performed for assessment of cardiac remodeling. Hearts were characterized histopathologically for the presence of myofibroblasts and thick and thin collagen fiber deposition.
Results: Acute MI size was similar in all groups. The quantitative CRIP percent injected dose per gram uptake was greatest in the infarct area of untreated control mice (2.30 ± 0.14%) and decreased significantly in animals treated with 1 agent (C, L, or S; 1.71 ± 0.35%; p = 0.0002). The addition of 2 (CL, SC, or SL 1.31 ± 0.40%; p < 0.0001) or 3 agents (SCL; 1.16 ± 0.26%; p < 0.0001) demonstrated further reduction in tracer uptake. The decrease in echocardiographic left ventricular function, strain and rotation parameters, as well as histologically verified deposition of thin collagen fibers, was significantly reduced in treatment groups and correlated with CRIP uptake.
Conclusions: Radiolabeled CRIP allows for the evaluation of the efficacy of neurohumoral antagonists after MI and reconfirms superiority of combination therapy. If proven clinically, molecular imaging of the myocardial healing process may help plan an optimal treatment for patients susceptible to heart failure.
Key Words: collagen • remodeling • radionuclide imaging • angiotensin receptors • angiotensin-converting enzyme • mineralocorticoids • heart failure
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Abbreviations and Acronyms
| | ACE-I = angiotensin-converting enzyme inhibitor | | ARB = angiotensin receptor blocker | | ASMA = alpha smooth muscle actin | | C = captopril | | CRIP = Cy5.5-RGD imaging peptide | | L = losartan | | LV = left ventricular | | MI = myocardial infarction | | RGD = Arg-Gly-Asp. | | S = spironolactone | | SARA = selective aldosterone receptor antagonist | | sCRIP = scrambled Cy5.5-RGD imaging peptide | | 99mTc = technetium-99m |
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Up regulation of the renin-angiotensin-aldosterone axis after myocardial infarction (MI) contributes to the process of cardiac remodeling and hence the evolution of heart failure (1–4). Although this remodeling process is initiated by myocyte loss, interstitial and myocytic alterations continue to occur inexorably even after the initial injury has abated (5–7). Antiangiotensin (8–10) and antialdosterone (11) treatments effectively control this process and delay or prevent the development of heart failure (12–15). These therapeutic interventions reduce the extent of interstitial collagen deposition, which is considered to be the basis of their efficacy (1,2). The combination of these agents has been shown to be more effective than the solitary agents (16,17).
The efficacy of pharmacologic intervention in a clinical milieu is variable, and it is desirable to define optimal use of antiremodeling agents for individual patients. Although pharmacogenomics has been proposed as an important determinant of such a strategy (18,19), the role of phenotypic characterization by novel imaging methods is also being explored (4). It has recently been reported that the extent of new collagen deposition can be assessed by molecular imaging employing technetium-99m-labeled Cy5.5-RGD-imaging peptide (99mTc-CRIP) (20). Through Arg-Gly-Asp (RGD)-based targeting, 99mTc-CRIP binds to the myofibroblasts in the healing infarct and correlates with the extent of new collagen deposition after experimental MI (20). In our preliminary experiment we proposed that such imaging strategy may have a role in identifying the impact of a therapeutic intervention on collagen deposition and hence the efficacy of therapy. In the present study, we evaluated the effect of an angiotensin-converting enzyme inhibitor (ACE-I), an angiotensin receptor blocker (ARB), and a selective aldosterone receptor antagonist (SARA), individually and in combination, on infarct healing by using 99mTc-CRIP imaging.
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Methods
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Study protocol.
The present study was performed in 69 Swiss-Webster mice. Of these, MI was produced in 63 mice by occlusion of the left coronary artery. Echocardiograms were obtained before and after coronary occlusion to demonstrate the basal left ventricular (LV) dimensions and function in addition to the infarct size. Of the 63 infarcted mice, 20 mice received a single agent intervention (1Rx); 8, 6, and 6 animals were started on spironolactone (S), captopril (C), and losartan (L), respectively. A total of 22 of 63 animals received dual therapy (2Rx); 6, 8, and 8 animals were started on a combination of captopril + losartan (CL), spironolactone + captopril (SC), or spironolactone + losartan (SL), respectively, and 8 of 63 post-MI animals received a combination of 3 neurohumoral antagonists (3Rx, spironolactone + captopril + losartan [SCL]) (Table 1). Eight post-MI animals did not receive any pharmacologic agent and served as treatment controls (No Rx), and 5 post-MI animals received a scrambled version of the imaging peptide (sCRIP) to serve as tracer controls. In addition to 63 infarcted mice, 6 unmanipulated animals were included in the imaging protocol as disease control animals. After 4 weeks, all animals again underwent echocardiographic examination for the characterization of the remodeling process and received 99mTc-labeled CRIP intravenously. After 3.5 h of CRIP administration, hearts were explanted and ex vivo imaging was performed with a microSPECT camera. The hearts were then sectioned into 3 cross-sectional bread-loaf slices to represent apical (predominantly infarct), middle, and the basal (predominantly remote) areas. All samples were gamma-counted for quantitative 99mTc-CRIP (and sCRIP) uptake. Subsequently, all myocardial specimens were processed for histopathological characterization, particularly the extent of myofibroblast proliferation and thick and thin collagen fiber deposition.
Experimental MI in mice.
This experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of California Irvine School of Medicine. In 63 adult Swiss Webster male mice (age, 3 months; body weight,  50 g), the lateral branch of the left coronary artery was ligated under pentobarbital (75 mg/kg intraperitoneally) and gas anesthesia (2.0 to 3.0% isoflurane) to induce MI, as per previous description (21). Successful ligation was verified by visual inspection of myocardial blanching and akinesis or dyskinesis of the apical segment. The chest was closed, and animals were gradually weaned from the respirator. Six of the 69 mice were left unmanipulated as disease control mice for comparison with the post-MI mice.
Treatment protocols after MI in mice.
Captopril (Sigma, St. Louis, Missouri) and losartan (kind gift of Merck, Rahway, New Jersey) were administered in the drinking water, 60 and 20 mg/kg/day, respectively. Spironolactone (Innovative Research of America, Sarasota, Florida) was administered via a subcutaneous pellet, releasing 1 mg/day, placed under the skin on the back immediately after MI surgery. The animals received the intervention for 4 weeks beginning immediately after surgery (Table 1).
Echocardiography.
Echocardiographic studies were performed in all mice for the evaluation of left ventricular dimensions and function, as reported previously (20). All animals underwent 3 echocardiographic studies; one performed at baseline level, one immediately after MI, and one 4 weeks after MI, just before CRIP imaging. Mice were anesthetized with 2% isoflurane, and echocardiograms were made (Sequoia, Siemens, Mountain View, California) with a 14-MHz linear probe (15L8, Acuson). An advanced high frame rate imaging technique (Paragon, Siemens) was adopted to increase temporal resolution at a frame rate of 120 fps. B-mode images of LV parasternal long-axis, parasternal short-axis, and apical views were digitally acquired at 2 to 3 cardiac cycle lengths. Images of LV short-axis were standardized at 3 levels: base, mid, and apex. M-mode imaging was unified according to the American Society of Echocardiography guidelines for measurements of wall thickness, chamber dimensions, and functional parameters (22). Infarction area and LV area were measured by tracing relative endocardial borders respectively on the long-axis images. Myocardial infarction percentage was calculated as: infarction area/LV area x 100. Left ventricular cavity dimensions, ejection fraction, strain, and rotation were calculated (23).
Radionuclide imaging with 99mTc-CRIP.
The technetium-99m-labeled Cy5.5-RGD imaging peptide (AH110863, kind gift of GE Healthcare, Oslo, Norway) is a 2.5-kDa peptide conjugated to both a fluorescent cyanine dye Cy5.5 and the chelating agent cPN216, which is linked for radiolabeling of the peptide with 99mTc. The peptide comprises 10 amino acids, contains an RGD motif, and has a bicyclic structure formed by a disulfide and a thioether bridge. The dye moiety and the chelating agent are conjugated to the peptide by the side-chains of lysine residues at the N- and C-terminal ends, respectively. The technetium-99m–labeled Cy5.5-RGD imaging peptide, by RGD-based targeting, binds to myofibroblasts (20) and is labeled with 99mTc for radionuclide imaging. For radiolabeling, 50 µg of 99mTc-CRIP was dissolved in 50 µl of methanol and then added to the freeze-dried kit. A total of 1.0 ml of 99mTcO4
- was added to the compound and left at room temperature for 20 to 30 min. Instant thin-layer chromatography confirmed radiopurity of more than 90%. Then, 3.7 ± 0.2 mCi (137 ± 7.4 MBq) of 99mTc-CRIP was injected intravenously through the tail vein. At 3.5 h after 99mTc-CRIP administration, mice were sacrificed with an overdose of pentobarbital (150 mg/kg intraperitoneally). Hearts were carefully dissected and planar images of ex vivo hearts were acquired for 15 min in 128 x 128 matrix with the use of a low-energy, high-resolution pinhole collimator, mounted in a dual-head micro single-photon emission computed tomography gamma camera (X-SPECT, Gamma Medica, Inc., Northridge, California). Thereafter, hearts were cut into apical, middle, and basal slices. The quantitative CRIP uptake was determined with a gamma scintillation counter (1480 Wizard 3'', Wallace & Co., Monrovia, California). We also determined CRIP biodistribution and radiation burden to lung, liver, spleen, and kidney.
Histological evaluation of myocardial specimens.
After completion of the nuclear studies, the 3 bread-loaf slices were washed in phosphate-buffered saline, fixed in 4% paraformaldehyde in phosphate-buffered saline for 24 h, processed, and embedded in paraffin; then, 4-µm sections were prepared. After deparaffinization and rehydration, sections were stained with hematoxylin & eosin, Masson's trichrome, and picro-Sirius red, as reported previously (20). In picro-Sirius–stained sections, the relative collagen areas in the infarcted, and remote areas were determined under a microscope (x200) coupled to a computerized morphometry system (Quantimet, Leica, Cambridge, United Kingdom). The quality of collagen fibers was investigated with the use of Sirius red polarization microscopy, allowing quantitation of the thick, tightly packed, more mature orange/red fibers and the newly formed, thin, loosely assembled yellow/green fibers (24,25). Approximately 4 to 5 fields per section were analyzed in the myocardium. Finally, alpha smooth muscle actin (ASMA) was used to determine myofibroblasts in the infarct area, as reported earlier (20). Alpha smooth muscle actin area was quantified by Quantimet analysis in 4 to 5 fields per section; ASMA uptake was only counted for myofibroblasts, and care was taken not to include ASMA uptake observed in vasculature.
Statistical analysis.
Quantitative radiotracer uptake was calculated as percent total injected dose per gram (%ID/g) of the tissue. Radiotracer uptake and echocardiographic measurements were expressed as mean ± standard deviation. In our previous study (20), CRIP uptake was reduced by >25% in the captopril group compared with the untreated group and in combination treatment (losartan plus captopril) compared with single therapy (captopril alone) group; as such, we propose that having at least 6 mice per group is sufficient to detect, with at least 90% power and alpha = 0.01, reductions in the uptake of CRIP of at least 25% in treated compared with untreated animals, or 2 versus 1 agent groups. This calculation confirms that our current study is well-powered. To determine the statistical significance of differences among treatment groups, we used 1-way analysis of variance followed by post-hoc analysis for multiple comparisons. Because we had a limited number of pre-specified pairwise comparisons, namely comparison of control and untreated animals with uptake among 1, 2, or 3 Rx-treated animals, we used the Bonferroni's method for post-hoc analysis. The correlation between the quantitative tracer uptake and echocardiographic parameters or histological characteristics was evaluated by linear regression analysis. p Values < 0.05 were considered as statistically significant.
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Results
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Uptake of 99mTc-CRIP and pharmacologic intervention.
Maximum CRIP uptake was observed in the infarct zone of the untreated post-MI mice; CRIP uptake was substantially reduced in the treated animals (Table 1, Fig. 1A); no CRIP uptake was observed in the unmanipulated hearts. Scrambled CRIP, which has a deranged RGD sequence, did not show uptake in the infarcted myocardium and demonstrated that CRIP uptake is RGD dependent and specific.
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Figure 1 Radiolabeled CRIP Uptake After Neurohumoral Antagonist Treatment in Post-MI Animals
(A) Disease control (unmanipulated) heart with RGD probe shows no radiotracer uptake in ex vivo image of the heart. However, intense 99mTc-CRIP uptake is found in 4-week untreated (no Rx) post-MI animals (upper panel, right). The uptake in the infarcted area is reduced after neurohumoral treatment with solitary agents and combination therapy (middle and lower panels). Losartan (L) images are displayed twice (middle panel) for the convenience of comparison. C = captopril; S = spironolactone. (B) Quantitative 99mTc-CRIP uptake in the infarct (apex, above) and remote (base, below) areas. The %ID/g uptake in the infarct area is greatest in untreated mice, followed by treatment with 1 (1Rx), 2 (2Rx), and 3 (3Rx) agents, respectively. On the other hand, the uptake in the remote area shows no significant differences between treated and untreated animals. Quantitative data confirmed the findings of ex vivo images. Data are presented as mean ± SEM. 99mTc-CRIP = technetium-99m-labeled Cy5.5-RGD imaging peptide; MI = myocardial infarction; %ID/g = percent of injected CRIP dose per gram; RGD = Arg-Gly-Asp.
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The quantitative CRIP uptake in the myocardial tissue specimens was calculated and represented as percent of injected CRIP dose per gram (%ID/g) of myocardial tissue. We observed CRIP uptake in the infarcted area to be the greatest in the untreated MI group (No Rx; 2.30 ± 0.14%). There was a significant reduction in CRIP uptake in the infarct area in all treated animals (1.46 ± 0.42%) compared with the untreated animals (p < 0.0001). The reduction of uptake was significant even when the animals were treated with a single neurohumoral antagonist (1Rx, 1.71 ± 0.35%, p = 0.0002) (Fig 1B). Each agent was independently effective; spironolactone (1.80 ± 0.44%; p = 0.0011), captopril (1.72 ± 0.30%; p = 0.0059), and losartan (1.58 ± 0.28%; p < 0.0001) (Table 1).
The use of 2 neurohumoral antagonists (2Rx, 1.31 ± 0.40%) influenced the CRIP uptake to a greater extent than a single agent (1Rx, 1.71 ± 0.35%) treatment (p = 0.0005) (Fig 1B). Uptake with various combinations was as follows; CL (1.37 ± 0.55%; p < 0.0001), SC (1.28 ± 0.41%; p < 0.0001), and SL (1.29 ± 0.32%; p<0.0001) (Table 1). The combination of all 3 agents further reduced the CRIP uptake (3Rx, 1.16 ± 0.26%) in the infarct area (Fig. 1B). The decrease in CRIP uptake in triple-treated animals was significantly lower than 1-agent treatment (p = 0.0001) but was statistically insignificantly reduced compared with 2-agent therapy (p = 0.33) (Fig. 1).
We found that CRIP uptake in the remote myocardium tended to be increased, although not statistically significant, in the untreated post-MI animals (0.92 ± 0.22) compared with the unmanipulated control animals with no infarcts (0.68 ± 0.16, p = 0.077) (Fig. 1B).
Echocardiographic examination and remodeling characteristics.
As shown in Table 2, initial MI size was similar in all animal groups. During the 4-week follow-up period, there was a reduction in LV ejection fraction in untreated post-MI (30 ± 8%) animals compared with unmanipulated disease control animals (64 ± 4%, p < 0.0001). Neurohumoral antagonists significantly prevented the functional loss (ejection fraction 44 ± 10%), compared with untreated animals (30 ± 8%; p = 0.0016). Similar to LV ejection fraction, there was a significant decrease in the myocardial strain development in untreated animals (–4.59 ± 1.85 U %) compared with unmanipulated (–12.00 ± 2.31 U %) animals (p < 0.0001); loss of myocardial strain was significantly prevented by treatment with neurohumoral agents (–8.04 ± 2.4 U %, p = 0.0005). The strain development in 1Rx (–7.17 ± 2.28 U %, p = 0.0126), 2Rx (–8.28 ± 2.51 U %, p = 0.0011), and 3Rx (–9.33 ± 1.7 U %, p < 0.0001) groups was better preserved compared with the untreated control group. However, the preservation of strain development did not reach statistical significance on comparison of 1Rx versus 2Rx (p = 0.13) or 2Rx versus 3Rx (p = 0.25) agents; 3Rx was statistically significantly better than the 1Rx intervention (p = 0.0168). The results of loss of apical rotation or counterclockwise twist of the LV were similar to the loss of strain development. The rotation in all (0.55 ± 0.18°, p < 0.0001), 1Rx (0.49 ± 0.17°, p = 0.0006), 2Rx (0.57 ± 0.19°, p < 0.0001), and 3Rx (0.63 ± 0.17°, p < 0.0001) groups was preserved compared with the untreated control group. However, once again, the prevention of rotational loss did not reach statistical significance on comparison of 1Rx versus 2Rx (p = 0.17) or 2Rx versus 3Rx (p = 0.36) agents; 3Rx was statistically significantly better than the 1Rx intervention (p = 0.048). The LV ejection fraction, strain development, and rotation correlated with %ID/g CRIP uptake (r2 = 0.309, = 0.397, and = 0.469, and p <0.0001, <0.0001, and <0.0001, respectively) (Fig. 2).
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Figure 2 Correlation of Radiolabeled 99mTc-CRIP Uptake and Echocardiographic Parameters of Remodeling an All Animals
A significant correlation between CRIP uptake and left ventricular ejection fraction (A), myocardial strain (B), and apical counterclockwise rotation (C) is observed. Abbreviations as in Figure 1.
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Histologic characterization of infarct and remote myocardium and correlation with 99mTc-CRIP uptake.
Masson's trichrome and picro Sirius red staining (Fig. 3) of the myocardial slices allowed the assessment of total collagen content and the composition in thick and thin collagen fibers. The percent collagen content in the infarcted area in untreated animals was 79 ± 4%, which reduced substantially in the treated animals (66 ± 8%, p < 0.0001) (Fig. 4A, Table 3). The collagen content decreased in animals treated with 1Rx (71 ± 7%, p = 0.006), 2Rx (64 ± 9%, p < 0.0001), or 3Rx (65 ± 4%, p = 0.0008) agents (Fig 4A). The results were similar for the yellow-green collagen fiber deposition in the infarcted myocardium, suggesting a decrease in new or thin collagen fiber synthesis (Fig. 4C). The new, thin collagen fibers correlated with CRIP uptake in the infarct (r2 = 0.5475, p < 0.0001) (Fig. 4E). In the remote region, the overall collagen deposition was markedly lower compared with the infarct region. The collagen content of the untreated remote myocardium (No Rx, 2.1 ± 0.6%) was significantly greater than the unmanipulated hearts (0.73 ± 0.2%, p < 0.0001) (Fig 4B). A marked decrease in collagen content and yellow-green collagen fiber deposition was observed in the remote region after treatment (Fig. 4D, Table 3); collagen content in treated animals was not different from unmanipulated animals. There was a direct correlation between tracer uptake and thin collagen fiber deposition in the remote region (r2 = 0.347; p < 0.0001) (Fig. 4F).
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Figure 3 Histopathological Characterization of the Treated and Untreated Animals
Masson's trichrome staining was performed for infarct localization in control, untreated, and treated animals (A). In addition, histological staining was performed with picro Sirius red to demonstrate collagen deposition, which on polarized light allowed identification of the quality of collagen, in the infarct (B and C) and remote regions (D and E). The left column presents control animals, the middle column shows untreated animals (no Rx), and the right column shows animals treated with 3Rx neurohumoral antagonists (SCL). The remote region shows minimal fibrosis and collagen deposition (D). On the other hand, infarct region shows significant wall thinning (A) and fibrosis with evidence of substantial collagen deposition in untreated animals (B, No Rx). 3Rx-treated animals show reduced fibrosis (A, SCL) and collagen deposition (B, SCL) in the infarcted area. The Sirius staining under polarized light provides distinction of thick red-orange and thin yellow-green collagen fibers in the infarct region (C). SCL = spironolactone, captopril, and losartan.
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Figure 4 Collagen Fiber Deposition in Infarct and Remote Regions
The total collagen content decreases with treatment in the infarcted (A), as well as the remote (B) areas. Further characterization of the collagen fibers by polarization reveals that the thin or new yellow-green collagen fibers decreased both in the infarct (C) and remote (D) regions after treatment. This indicates cessation of new collagen production and maturation of the collagen fibers after treatment. The prevalence of new collagen fibers paralleled the CRIP uptake and demonstrated a significant direct correlation, both in infarct (E) and remote (F) regions.
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We performed ASMA staining for quantitation of the myofibroblasts (Fig. 5A). The ASMA-positive area was substantially larger in the untreated animals (No Rx, 6.11 ± 5.17µm2) compared with the unmanipulated animals (0.007 ± 0.01µm2, p = 0.013). Treatment with 1Rx (4.91 ± 2.12 µm2, p = 0.51), 2Rx (2.22 ± 3.30 µm2, p = 0.037), or 3Rx (1.54 ± 0.92 µm2, p = 0.039) interventions substantially restricted the myofibroblast prevalence (Fig. 5B, Table 3). There was a directly proportional relationship between CRIP uptake and ASMA-positive area (r2 = 0.582, p < 0.0001) (Fig. 5C).
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Figure 5 Radiolabeled CRIP Uptake and the Prevalence of ASMA-Verified Myofibroblasts
Immuohistochemical staining with ASMA antibody allowed quantitative assessment of myofibroblasts infiltration in the infarct and remote regions (A). The left column presents control animals, the middle column shows untreated animals (no Rx), and the right column shows 3Rx-animals (SCL). There is a marked decrease in number of myofibroblasts in the infarct region in 2Rx and 3Rx groups (B). There is a significant direct correlation between the extent of myofibroblasts and the CRIP uptake in infarct region (C). ASMA = alpha smooth muscle actin; other abbreviations as in Figures 1 and 3.
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Discussion
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Molecular imaging in myocardial remodeling.
The present study used molecular imaging of myofibroblasts during infarct healing. The radiotracer, CRIP, contains an RGD sequence and binds to myofibroblasts through activated  vβ3/5 integrins with an affinity of 1 to 3 nmol/l (20); RGD is also known to commonly target the integrin expression associated with neoangiogenesis early after MI and macrophage influx during infarct healing. We have observed that after 2 weeks of infarct healing, myofibroblasts comprise a predominant target for RGD probes (20). In addition, a specific noncovalent interaction of CRIP also was observed with custom-made DDX-sequence contained within pro-collagen I. The previous study using 99mTc-CRIP had demonstrated the feasibility of noninvasively imaging the extent of myofibroblasts prevalence in a post-MI mouse model (20). The CRIP uptake receded over time in the infarct region with substantial resolution over the course of 3 months after the myocardial injury. This previous report also demonstrated that the uptake was significantly reduced at 4 weeks upon treatment with captopril alone or in combination with losartan and proposed that CRIP uptake could monitor the efficacy of neurohumoral antagonists and help identify the optimum level of therapeutic intervention (20). A simultaneously conducted clinical study, using RGD-seeking peptide demonstrated the feasibility of molecular imaging early after MI (26). The radiotracer uptake after MI predicted the extent of eventual magnetic resonance imaging-verified fibrosis after 1 year. On the basis of these results, the present study investigated the effect of various neurohumoral antagonists, alone or in combination, on the extent of myocardial interstitial alterations in a 4-week post-MI mouse model.
This study confirmed the efficacy of antiangiotensin and antialdosterone intervention on interstitial alterations and demonstrated that combination of 2 neurohumoral antagonists was superior to an individual agent; 3 agents decreased the radiotracer uptake further, albeit not significantly. The radiotracer uptake correlated closely with parameters of depressed myocardial mechanics, the quantitative prevalence of myofibroblasts, and thin fiber collagen deposition, suggesting that treatment with neurohumoral antagonists results in decreased new collagen production. The data obtained in the present study corroborate with information provided by the estimation of collagen synthesis and fragmentation markers in the serum (27–29). Procollagen type III amino-terminal peptide is decreased in chronic heart failure patients using antiangiotensin and anti-aldosterone treatment (30,31).
What is the optimum therapy?.
The clinical role of ACE-I, ARB, and SARA has been conclusively demonstrated in trials confirming the reduction in morbidity and mortality in patients with overt heart failure (8–11,17,32). These agents also decrease the likelihood of the development of heart failure in patients with asymptomatic decrease in LV ejection fraction (13,14), as well as those predisposed to development of heart failure but normal LV ejection fraction (12,33). It is also established that antiangiotensin agents in combination are superior to any agent used alone. The combination of ACE-I with ARB in the presence of beta-blockers ± aldosterone inhibitors reduces the combined end point of mortality (16) and hospitalization for worsening of heart failure (17). The experimental data from the present study confirm the superior effect of combination of 2 agents. However, the addition of the third agent did not decrease the uptake significantly. It is encouraging to observe the efficacy of an imaging agent to identify difference in infarct healing, which could be of translational clinical benefit.
Is lack of collagen deposition favorable?.
The present study raises an important question about the relevance of decrease in collagen deposition in the post-infarct patients. Total abolition of collagen deposition, which has been evaluated in clodronate liposome-treated animals (wherein infarct healing becomes defective with minimal collagen deposition and inefficient necrotic myocyte removal) (34), leads to greater tendency for aneurysmal formation and infarct rupture. On the other hand, direct myocardial injection of collagen in experimental infarcts leads to dense collagen deposition and prevents myocardial remodeling (35). Such experiments suggest that the collagen deposition may not necessarily be harmful. However, the use of ACE-I or SARA allows the reduction of collagen deposition as demonstrated by circulating collagen degradation products (30). The present study demonstrated a decrease in collagen deposition with suppression of angiotensin-aldosterone axis. Because neurohumoral antagonists lead to favorable clinical outcomes, conceivably reduced collagen deposition should be of benefit. Although the present study is not capable of explaining these paradoxical observations, it is possible that predominant decrease of collagen content observed in the remote myocardium in the present study, and the collagen fiber maturation (to thick fibers) observed in the infarct region may contribute to a more balanced healing.
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Conclusions
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The present study demonstrates that 99mTc-CRIP imaging allows for the evaluation of the efficacy of antiremodeling therapy. It also reconfirms the superiority of combination therapy over a solitary use of neurohumoral antagonists. If proven clinically, molecular imaging of the remodeling processes could facilitate individualizing the treatment for patients susceptible to heart failure.
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Acknowledgments
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The imaging agents (99mTc-CRIP and sCRIP) were kindly provided by GE Healthcare, and losartan was a kind gift of Merck & Co.
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Footnotes
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Dr. van den Borne was partially supported by a grant from the Netherlands Heart Foundation (2006R013). Dr. Lovhaug is an employee of GE Healthcare, involved in tracer preparation for the imaging studies. Dr. Pitt is a consultant to Pfizer, Merck, Takeda, Astra Zeneca, Synvista, Novartis, and Nile therapeutics but has no conflicts directly with the project. H. William Strauss, MD, acted as Guest Editor for this paper.
* Reprint requests and correspondence: Dr. Jagat Narula, Division of Cardiology, University of California, Irvine School of Medicine, UCI Main Campus, Medical-Science Building I, Room C 112, Irvine, California 92697 (Email: narula{at}uci.edu).
Manuscript received June 9, 2008;
revised manuscript received October 31, 2008,
accepted November 6, 2008.
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