W. Matthijs Blankesteijn

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.

The inner nuclear membrane protein Emerin regulates β‐catenin activity by restricting its accumulation in the nucleus

Emerin is a type II inner nuclear membrane (INM) protein of unknown function. Emerin function is likely to be important because, when it is mutated, emerin promotes both skeletal muscle and heart defects. Here we show that one function of Emerin is to regulate the flux of β‐catenin, an important transcription coactivator, into the nucleus. Emerin interacts with β‐catenin through a conserved adenomatous polyposis coli (APC)‐like domain. When GFP‐emerin is expressed in HEK293 cells, β‐catenin is restricted to the cytoplasm and β‐catenin activity is inhibited. In contrast, expression of an emerin mutant, lacking its APC‐like domain (GFP‐emerinΔ), dominantly stimulates β‐catenin activity and increases nuclear accumulation of β‐catenin. Human fibroblasts that are null for emerin have an autostimulatory growth phenotype. This unusual growth phenotype arises through enhanced nuclear accumulation and activity of β‐catenin and can be replicated in wild‐type fibroblasts by transfection with constitutively active β‐catenin. Our results support recent findings that suggest that INM proteins can influence signalling pathways by restricting access of transcription coactivators to the nucleus.

Oxidation of CaMKII determines the cardiotoxic effects of aldosterone

Excessive activation of the β-adrenergic, angiotensin II (Ang II) and aldosterone signaling pathways promotes mortality after myocardial infarction, and antagonists targeting these pathways are core therapies for treating this condition. Catecholamines and Ang II activate the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII), the inhibition of which prevents isoproterenol-mediated and Ang II–mediated cardiomyopathy. Here we show that aldosterone exerts direct toxic actions on myocardium by oxidative activation of CaMKII, causing cardiac rupture and increased mortality in mice after myocardial infarction. Aldosterone induces CaMKII oxidation by recruiting NADPH oxidase, and this oxidized and activated CaMKII promotes matrix metalloproteinase 9 (MMP9) expression in cardiomyocytes. Myocardial CaMKII inhibition, overexpression of methionine sulfoxide reductase A (an enzyme that reduces oxidized CaMKII) or NADPH oxidase deficiency prevented aldosterone-enhanced cardiac rupture after myocardial infarction. These findings show that oxidized myocardial CaMKII mediates the cardiotoxic effects of aldosterone on the cardiac matrix and establish CaMKII as a nodal signal for the neurohumoral pathways associated with poor outcomes after myocardial infarction.

Thrombospondin-2 Is Essential for Myocardial Matrix Integrity: Increased Expression Identifies Failure-Prone Cardiac Hypertrophy

Cardiac hypertrophy can lead to heart failure (HF), but it is unpredictable which hypertrophied myocardium will progress to HF. We surmised that apart from hypertrophy-related genes, failure-related genes are expressed before the onset of failure, permitting molecular prediction of HF. Hearts from hypertensive homozygous renin-overexpressing (Ren-2) rats that had progressed to early HF were compared by microarray analysis to Ren-2 rats that had remained compensated. To identify which HF-related genes preceded failure, cardiac biopsy specimens were taken during compensated hypertrophy and we then monitored whether the rat progressed to HF or remained compensated. Among 48 genes overexpressed in failing hearts, we focused on thrombospondin-2 (TSP2). TSP2 was selectively overexpressed only in biopsy specimens from rats that later progressed to HF. Moreover, expression of TSP2 was increased in human hypertrophied hearts with decreased (0.19±0.01) versus normal ejection fraction (0.11±0.03 [arbitrary units]; P<0.05). Angiotensin II induced fatal cardiac rupture in 70% of TSP2 knockout mice, with cardiac failure in the surviving mice; this was not seen in wild-type mice. In TSP2 knockout mice, angiotensin II increased matrix metalloproteinase (MMP)-2 and MMP-9 activity by 120% and 390% compared with wild-type mice (P<0.05). In conclusion, we identify TSP2 as a crucial regulator of the integrity of the cardiac matrix that is necessary for the myocardium to cope with increased loading and that may function by its regulation of MMP activity. This suggests that expression of TSP2 marks an early-stage molecular program that is activated uniquely in hypertrophied hearts that are prone to fail.

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.

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.

β-Catenin, an Inducer of Uncontrolled Cell Proliferation and Migration in Malignancies, Is Localized in the Cytoplasm of Vascular Endothelium during Neovascularization after Myocardial Infarction

β-catenin is a protein involved in cell-cell adhesion and proliferation. In neoplastic diseases, defects in the regulation of the cellular β-catenin content and cytoplasmic accumulation of the protein contribute to the uncontrolled cell proliferation and migration. Whether β-catenin plays a role in the controlled proliferative and migratory responses to injury, eg, of vascular endothelial cells during neovascularization after myocardial infarction (MI), is not known. In the present study, we examined the localization of β-catenin in the infarcted rat heart at different time points after MI. Cytoplasmic β-catenin was observed in the endothelial cells of the newly formed and pre-existing blood vessels in the infarct area in the first week after MI, but not in the uninjured parts of the heart and not at later time points. Adenomatous polyposis coli (APC) protein was also detected; interaction of APC with β-catenin has been reported to be critical in epithelial tube formation in vitro. Moreover, the expression of dishevelled-1, an upstream regulatory molecule of the cellular β-catenin content, was observed in vascular endothelial cells in the infarct area. These findings suggest a role for the β-catenin-APC complex in the proliferation and migration of vascular endothelial cells during neovascularization of the infarct area.

Diffusion tensor imaging of left ventricular remodeling in response to myocardial infarction in the mouse

The cardiac muscle architecture lies at the basis of the mechanical and electrical properties of the heart, and dynamic alterations in fiber structure are known to be of prime importance in healing and remodeling after myocardial infarction. In this study, left ventricular remodeling was characterized using diffusion tensor imaging (DTI) in a mouse model of myocardial infarction. Myocardial infarction was induced in mice by permanent ligation of the left anterior descending coronary artery. Serial ex vivo DTI measurements were performed 7, 14, 28, and 60 days after ligation. Apparent diffusion coefficient, fractional anisotropy, the three eigenvalues of the diffusion tensor, and the myofiber disarray served as readout parameters. After myocardial infarction, the mouse hearts displayed extreme wall thinning in the infarcted area, which covered large parts of the apex and extended into the free wall up to the equator. Average heart mass increased by 70% 7–60 days after infarction. Histological analysis showed that the infarct at 7 days consisted of unstructured tissue with residual necrosis and infiltration of macrophages and myofibroblasts. At 14 days after infarction, the necrotic tissue had disappeared and collagen fibers were starting to appear. From 28 to 60 days, the infarct had fully developed into a mature scar. DTI parameters showed dynamic changes as a function of time after infarction. The apparent diffusion coefficient in the infarcted region was lower than in remote regions and increased as a function of time after infarction. The fractional anisotropy was higher in the infarcted region and was maximum at 28 days, which was attributed to the development of structured collagen fibers. Myofiber disarray, which was analyzed by considering the alignment of fibers in neighboring voxels, was significantly higher in infarcted regions. DTI provides a valuable non‐destructive tool for characterizing structural remodeling in diseased myocardium. Copyright

The wnt-frizzled cascade in cardiovascular disease

Time for primary review 31 days. Wnt-proteins constitute a family of secreted cystein-rich glycosylated proteins, involved in a variety of modeling and remodeling processes including cell proliferation, differentiation, apoptosis and the control of cell orientation [1–3]. Malfunctioning of the wnt-frizzled pathway has been implied in diseases as divergent as cancer and Alzheimers disease [1,4]. The wnt-frizzled signal transduction pathway plays an important role during non-vertebrate and vertebrate development [5]. Several studies have shown the importance of wnts in the control of processes such as patterning of the body axis and development of the central nervous system and the limbs [6,7]. Moreover, interventions in wnt signaling have been described to affect cardiac morphogenesis [8,9] and several members of the wnt-frizzled signal transduction pathway were found to be expressed during cardiac development in vertebrates [10–14]. In cardiovascular pathology, re-expression of a fetal gene expression pattern is a generally observed phenomenon [15]. The study of gene expression during development may therefore provide clues about the expression profile during cardiovascular pathology. Recently, considerable progress has been made concerning the role of the wnt-frizzled signal transduction pathway in the development and progression of cardiovascular pathology. This review will focus on the possible role of the wnt-frizzled signal transduction pathway in cardiovascular diseases. The family of wnt proteins consists of 16 members [5]. These proteins are extremely difficult to purify because they tend to bind to the extracellular matrix [16], which hampers the study of their characteristics. An overview of the wnt proteins and their proposed function during development, derived from the study of null mutants, is provided in Table 1. Based on functional differences, wnt proteins can be divided into two classes, the wnt1 class and the wnt5a class. Members of the wnt1 class are able … * Corresponding author. Tel.: +31-43-388-1417; fax: +31-43-388-4149 wm.blankesteijn{at}farmaco.unimaas.nl

Blocking of Frizzled Signaling With a Homologous Peptide Fragment of Wnt3a/Wnt5a Reduces Infarct Expansion and Prevents the Development of Heart Failure After Myocardial Infarction

Background— The molecular pathways that control the wound healing after myocardial infarction (MI) are not completely elucidated. One of these pathways is the Wnt/Frizzled pathway. In this study, we evaluated Frizzled as a novel therapeutic target for MI. These Frizzled proteins act as receptors for Wnt proteins and were previously shown to be expressed in the healing infarct. Methods and Results— Wnt/Frizzled signaling has been studied for decades, but synthetic ligands that interfere with the interaction between Wnts and Frizzled have not been described to date. Here we report the selection of 3 peptides derived from regions of high homology between Wnt3a and Wnt5a that act as antagonists for Frizzled proteins. UM206, the peptide with the highest affinity, antagonized the effect of Wnt3a and Wnt5a in different in vitro assays. Administration of UM206 to mice for 5 weeks, starting immediately after the induction of MI, reduced infarct expansion and increased the numbers of capillaries and myofibroblasts in the infarct area. Moreover, heart failure development was inhibited by this therapy. Conclusions— Blocking of Frizzled signaling reduces infarct expansion and preserves cardiac function after MI. Our findings underscore the potential of Frizzled receptors as a target for pharmacotherapy of cardiac remodeling after MI.