Evolving role of molecular imaging for new understanding: targeting myofibroblasts to predict remodeling.

Containment of the process of cardiac remodeling is a prerequisite for prevention of development of heart failure (HF) after myocardial infarction. For personalization of therapeutic intervention strategy, it may be of benefit to identify the subset of patients who are at higher risk for development of HF. One such strategy may involve targeted imaging of various components involved in the remodeling process and interstitial fibrosis, including the myofibroblast. This cell type combines characteristics of fibroblasts and smooth muscle cells, and plays a crucial role in infarct healing and scar contraction. We define molecular targets on myofibroblasts and discuss the feasibility of molecular imaging of these cells for early detection and treatment of patients at risk for development of HF after myocardial infarction.

Myocardial remodeling after infarction: the role of myofibroblasts.

Myofibroblasts have characteristics of fibroblasts and smooth muscle cells: they produce extracellular matrix and are able to contract. In so doing, they can contribute to tissue replacement and interstitial fibrosis following cardiac injury. The scar formed after myocardial injury is no longer considered to be passive tissue; it is an active playground where myofibroblasts play a role in collagen turnover and scar contraction. Maintaining the extracellular matrix in the scar is essential and can prevent dilatation of the infarct area leading to heart failure. On the other hand, extracellular matrix deposition at sites remote from the infarct area can lead to cardiac stiffness, an inevitable process of myocardial remodeling that occurs in the aftermath of myocardial infarction and constitutes the basis of the development of heart failure. Defining molecular targets on myofibroblasts in conjunction with establishing the feasibility of molecular imaging of these cells might facilitate the early detection and treatment of patients who are at risk of developing heart failure after myocardial infarction.

Mouse strain determines the outcome of wound healing after myocardial infarction.

AIMS: Our objective was to study the effect of the genetic background on the wound healing process after myocardial infarction (MI) in mice. METHODS AND RESULTS: MI was induced in five different mouse strains (BalbC, C57Bl6, FVB, 129S6, and Swiss). At 3, 14, and 28 days after MI, cardiac dimensions were monitored by echocardiography and histology, whereas cardiac function was determined by direct intraventricular pressure measurements (dP/dt). Furthermore, matrix metalloproteinases were measured by zymography, and mRNA expression by quantitative PCR. Infarct rupture, which typically occurred at 3-6 days post-MI, was most frequent in 129S6 mice (62%), followed by C57Bl6 (36%), FVB (29%), Swiss (23%), and BalbC (5%). The high incidence of infarct rupture in 129S6 mice was associated with high systolic blood pressure and increased influx of inflammatory cells. Cardiac dilatation was most marked in Swiss mice and least prominent in 129S6 mice. The degree of dilatation was associated with a reduced ejection fraction and decreased dP/dt values at 14 and 28 days post-MI. At day 14 and 28 post-MI, secondary thinning of the infarct area was marked in BalbC, FVB, and Swiss, but absent in C57Bl6 and 129S6 mice. In the latter two groups, this was paralleled by the highest number of myofibroblasts at day 14 post-MI. CONCLUSION: The outcome of infarct healing in mice strongly depends on genetic background. On the basis of our results, we suggest that for studies on infarct rupture, the 129S6 mouse is the background of choice, whereas BalbC and Swiss mice are the preferred models to study infarct thinning post-MI.

Molecular imaging for efficacy of pharmacologic intervention in myocardial remodeling.

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.

Increased matrix metalloproteinase-8 and -9 activity in patients with infarct rupture after myocardial infarction.

BACKGROUND: Infarct rupture is a usually fatal complication of myocardial infarction (MI), for which no molecular mechanism has been described in humans. Experimental evidence in mouse models suggests that the degradation of the extracellular matrix by matrix metalloproteinases (MMPs) plays an important role in infarct rupture. The present study was designed to study the role of MMP-2, MMP-8, and MMP-9 in human infarct rupture. METHODS: Heart samples were obtained from patients who died from infarct rupture and control MI patients. The MMP activity was determined by zymography and quantitative immunocapture activity assay. TIMP-1 levels were measured and immunohistochemistry for MMP-2 and MMP-9 was performed. RESULTS: The amounts of both total and active MMP-8 and MMP-9 were significantly higher in ruptured infarct tissue than in control MI tissue, but no differences in MMP-2 activity were observed. Furthermore, the number of inflammatory cells was significantly higher in the ruptured infarcts than in control infarcts. CONCLUSIONS: These data suggest that increased MMP-8 and MMP-9 activity in the infarct area, caused by a more prominent infiltration of inflammatory cells, contribute to infarct rupture in humans.

Molecular imaging of interstitial alterations in remodeling myocardium after myocardial infarction.

OBJECTIVES: The purpose of this study was to evaluate interstitial alterations in myocardial remodeling using a radiolabeled Cy5.5-RGD imaging peptide (CRIP) that targets myofibroblasts. BACKGROUND: Collagen deposition and interstitial fibrosis contribute to cardiac remodeling and heart failure after myocardial infarction (MI). Evaluation of myofibroblastic proliferation should provide indirect evidence of the extent of fibrosis. METHODS: Of 46 Swiss-Webster mice, MI was induced in 41 by coronary artery occlusion, and 5 were unmanipulated. Of the 41 mice, 6, 6, and 5 received intravenous technetium-99m labeled CRIP for micro-single-photon emission computed tomography imaging 2, 4, and 12 weeks after MI, respectively; 8 received captopril or captopril with losartan up to 4 weeks after MI. Scrambled CRIP was used 4 weeks after MI in 6 mice; the remaining 10 of 46 mice received unradiolabeled CRIP for histologic characterization. RESULTS: Maximum CRIP uptake was observed in the infarct area; quantitative uptake (percent injected dose/g) was highest at 2 weeks (2.75 +/- 0.46%), followed by 4 (2.26 +/- 0.09%) and 12 (1.74 +/- 0.24%) weeks compared with that in unmanipulated mice (0.59 +/- 0.19%). Uptake was higher at 12 weeks in the remote areas. CRIP uptake was histologically traced to myofibroblasts. Captopril alone (1.78 +/- 0.31%) and with losartan (1.13 +/- 0.28%) significantly reduced tracer uptake; scrambled CRIP uptake in infarct area (0.74 +/- 0.17%) was similar to CRIP uptake in normal myocardium. CONCLUSIONS: Radiolabeled CRIP allows for noninvasive visualization of interstitial alterations during cardiac remodeling, and is responsive to antiangiotensin treatment. If proven clinically feasible, such a strategy would help identify post-MI patients likely to develop heart failure.

Defective intercellular adhesion complex in myocardium predisposes to infarct rupture in humans.

OBJECTIVES: Our goal was to evaluate intercellular adhesion complex proteins in myocardium in human infarct rupture. BACKGROUND: Infarct rupture, a fatal complication of myocardial infarction (MI), has been attributed to a defective cell adhesion complex in a transgenic mouse model. METHODS: Heart samples were collected from autopsies from infarct rupture and control (nonrupture) MI patients. Both infarcted and remote areas were included. Cell adhesion proteins including alphaE-catenin, beta-catenin, gamma-catenin, and N-cadherin were characterized by immunohistochemistry and immunoblotting. Genetic analysis was undertaken to evaluate mutations and polymorphisms in the alphaE-catenin gene. In addition, infarct rupture was studied in transgenic mice heterozygous for alphaE-catenin C-terminal deficiency, mimicking the situation in human infarct rupture patients. RESULTS: No alphaE-catenin was detected in 70% of remote samples of infarct rupture hearts compared with 20% in control MI by immunohistochemistry. The immunoblot analysis confirmed a significant reduction in remote areas, and complete absence of alphaE-catenin in infarct areas from infarct rupture patients. No mutation or polymorphism of the alphaE-catenin gene was discovered. Other cell adhesion proteins were not significantly affected in remote areas of infarct rupture hearts. Three-fourths of the heterozygous alphaE-catenin C-terminal truncated mice died of infarct rupture, compared with one-fourth of the wild-type littermates. CONCLUSIONS: The data show a reduced expression and defective localization of alphaE-catenin in the intercalated disc region in patients dying of infarct rupture. The mechanism of lower expression of alphaE-catenin remains to be elucidated.

Transglutaminase activity in acute infarcts predicts healing outcome and left ventricular remodelling: implications for FXIII therapy and antithrombin use in myocardial infarction.

AIMS: The transglutaminase factor XIII (FXIII) emerges as a key enzyme in healing after myocardial infarction (MI). Here we assess the impact of transglutaminase-modulating therapies on healing and evolution of heart failure using a novel, non-invasive molecular imaging technique. METHODS AND RESULTS: Immunoblotting revealed lower FXIII levels in the myocardium of nine patients with infarct rupture when compared to MI patients without rupture (P < 0.0045). In a murine model of MI, we assessed healing while modulating local FXIII activity. Infarct tissue activity was monitored with molecular in vivo single photon emission computed tomography-computed tomography (SPECT-CT) imaging, and activity was found to be increased by 80% in FXIII-treated mice (400 IU FXIII/kg iv.), and decreased by 65% in dalteparin (DP)-treated mice (600 IU/kg DP sc., P < 0.05). DP-treated mice exhibited increased mortality due to infarct rupture (64% by day 7, P < 0.018). Serial Magnetic Resonance Imaging (MRI) showed that left ventricular dilation post-MI was attenuated by FXIII treatment when compared to saline control-treated mice with MI (P = 0.04). Quantitative histological and reverse transcription-polymerase chain reaction analyses revealed that FXIII treatment induced a faster resolution of the neutrophil response, enhanced macrophage recruitment, increased collagen content and augmented angiogenesis in the healing infarct (P < 0.05 vs. control-treated mice with MI). CONCLUSION: FXIII tissue levels are decreased in patients with insufficient healing. Therapeutic strategies that modulate FXIII activity impact murine myocardial healing. Molecular imaging of FXIII activity predicts prognosis in mice with experimental MI.

Interruption of Wnt signaling attenuates the onset of pressure overload-induced cardiac hypertrophy.

The hypertrophic response of the heart has been recognized recently as the net result of activation of prohypertrophic and antihypertrophic pathways. Here we report the involvement of the Wnt/Frizzled pathway in the onset of cardiac hypertrophy development. Stimulation of the Wnt/Frizzled pathway activates the disheveled (Dvl) protein. Disheveled subsequently can inhibit glycogen synthase kinase-3beta, a protein with potent antihypertrophic actions through diverse molecular mechanisms. In the Wnt/Frizzled pathway, inhibition of glycogen synthase kinase-3beta leads to an increased amount of beta-catenin, which can act as a transcription factor for several hypertrophy-associated target genes. In this study we subjected mice lacking the Dvl-1 gene and their wild-type littermates to thoracic aortic constriction for 7, 14, and 35 days. In mice lacking the Dvl-1 gene, 7 days of pressure overload-induced increases in left ventricular posterior wall thickness and expression of atrial natriuretic factor and brain natriuretic protein were attenuated compared with their wild-type littermates. Beta-catenin protein amount was reduced in the group lacking the Dvl-1 gene, and an increased glycogen synthase kinase-3beta activity was observed. Moreover, the increase in the amount of Ser(473)-phosphorylated Akt, a stimulator of cardiac hypertrophy, was lower in the group lacking the Dvl-1 gene. In conclusion, we have demonstrated that interruption of Wnt signaling in the mice lacking the Dvl-1 gene attenuates the onset of pressure overload-induced cardiac hypertrophy through mechanisms involving glycogen synthase kinase-3beta and Akt. Therefore, the Wnt/Frizzled pathway may provide novel therapeutic targets for antihypertrophic therapy.