Morphological Representation of C1q in the Aging Central Nervous System.

INTRODUCTION: The complement protein C1q is essential for the innate immune system and neurophysiological and neuropathological processes. To gain more insight into these functions in the CNS, a comprehensive understanding of the morphological representation, especially of its cellular and subcellular target structures, is of great importance. METHODS: For a free-floating preparation, the brains of wild-type and ArcAß mice were cut into 100 µm slices. Living slices were incubated in Ringer's solution and then fixed in 4% paraformaldehyde (PFA) and stained with different primary and secondary antibodies or methoxy-X04. RESULTS: C1q was abundant in the entire brain. Interestingly, C1q accumulated around cell nuclei, with a perineuronal localization around neuronal somata and a paraneuronal accumulation around non-neuronal cells, e. g., microglia. Moreover, dendritic-like, linear, branched C1q signals were observed in the area between the dentate gyrus and the CA1 region of the hippocampus. Complementary staining revealed an overlap with ß-amyloid accumulation reflected by the deposition of C1q within plaques and modified basal C1q levels in the brains of transgenic ArcAß animals. DISCUSSION: The applied free-floating approach is suitable for C1q immunofluorescence imaging. The consistent colocalization of the complement protein C1q with ß-amyloid plaques may reflect an activated immune response, whereas the accumulation of C1q around neuronal structures such as somata and dendrites is still a matter of debate. Intriguingly, C1q surrounds those structures in older brains of both wild-type and ArcAß mice. Our results also indicate an involvement of C1q in neurophysiological and neurodegenerative processes.

CARM1 regulates senescence during airway epithelial cell injury in COPD pathogenesis.

Chronic obstructive pulmonary disease (COPD) is a life-threatening lung disease. Although cigarette smoke was considered the main cause of development, the heterogeneous nature of the disease leaves it unclear whether other factors contribute to the predisposition or impaired regeneration response observed. Recently, epigenetic modification has emerged to be a key player in the pathogenesis of COPD. The addition of methyl groups to arginine residues in both histone and nonhistone proteins by protein arginine methyltransferases (PRMTs) is an important posttranslational epigenetic modification event regulating cellular proliferation, differentiation, apoptosis, and senescence. Here, we hypothesize that coactivator-associated arginine methyltransferase-1 (CARM1) regulates airway epithelial cell injury in COPD pathogenesis by controlling cellular senescence. Using the naphthalene (NA)-induced mouse model of airway epithelial damage, we demonstrate that loss of CC10-positive club cells is accompanied by a reduction in CARM1-expressing cells of the airway epithelium. Furthermore, Carm1 haploinsuffficent mice showed perturbed club cell regeneration following NA treatment. In addition, CARM1 reduction led to decreased numbers of antisenescent sirtuin 1-expressing cells accompanied by higher p21, p16, and ß-galactosidase-positive senescent cells in the mouse airway following NA treatment. Importantly, CARM1-silenced human bronchial epithelial cells showed impaired wound healing and higher ß-galactosidase activity. These results demonstrate that CARM1 contributes to airway repair and regeneration by regulating airway epithelial cell senescence.

Coactivator-Associated Arginine Methyltransferase-1 Function in Alveolar Epithelial Senescence and Elastase-Induced Emphysema Susceptibility.

Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible loss of lung function and is one of the most prevalent and severe diseases worldwide. A major feature of COPD is emphysema, which is the progressive loss of alveolar tissue. Coactivator-associated arginine methyltransferase-1 (CARM1) regulates histone methylation and the transcription of genes involved in senescence, proliferation, and differentiation. Complete loss of CARM1 leads to disrupted differentiation and maturation of alveolar epithelial type II (ATII) cells. We thus hypothesized that CARM1 regulates the development and progression of emphysema. To address this, we investigated the contribution of CARM1 to alveolar rarefication using the mouse model of elastase-induced emphysema in vivo and small interfering (si)RNA-mediated knockdown in ATII-like LA4 cells in vitro. We demonstrate that emphysema progression in vivo is associated with a time-dependent down-regulation of CARM1. Importantly, elastase-treated CARM1 haploinsufficient mice show significantly increased airspace enlargement (52.5 ± 9.6 µm versus 38.8 ± 5.5 µm; P < 0.01) and lung compliance (2.8 ± 0.32 µl/cm H2O versus 2.4 ± 0.4 µl/cm H2O; P < 0.04) compared with controls. The knockdown of CARM1 in LA4 cells led to decreased sirtuin 1 expression (0.034 ± 0.003 versus 0.022 ± 0.001; P < 0.05) but increased expression of p16 (0.27 ± 0.013 versus 0.31 ± 0.010; P < 0.5) and p21 (0.81 ± 0.088 versus 1.28 ± 0.063; P < 0.01) and higher ß-galactosidase-positive senescent cells (50.57 ± 7.36% versus 2.21 ± 0.34%; P < 0.001) compared with scrambled siRNA. We further demonstrated that CARM1 haploinsufficiency impairs transdifferentiation and wound healing (32.18 ± 0.9512% versus 8.769 ± 1.967%; P < 0.001) of alveolar epithelial cells. Overall, these results reveal a novel function of CARM1 in regulating emphysema development and premature lung aging via alveolar senescence as well as impaired regeneration, repair, and differentiation of ATII cells.

Multiplexed imaging and automated signal quantification in formalin-fixed paraffin-embedded tissues by ChipCytometry.

Deciphering the spatial composition of cells in tissues is essential for detailed understanding of biological processes in health and disease. Recent technological advances enabled the assessment of the enormous complexity of tissue-derived parameters by highly multiplexed tissue imaging (HMTI), but elaborate machinery and data analyses are required. This severely limits broad applicability of HMTI. Here we demonstrate for the first time the application of ChipCytometry technology, which has unique features for widespread use, on formalin-fixed paraffin-embedded samples, the most commonly used storage technique of clinically relevant patient specimens worldwide. The excellent staining quality permits workflows for automated quantification of signal intensities, which we further optimized to compensate signal spillover from neighboring cells. In combination with the high number of validated markers, the reported platform can be used from unbiased analyses of tissue composition to detection of phenotypically complex rare cells, and can be easily implemented in both routine research and clinical pathology.

Integrin αvß3-dependent thyroid hormone effects on tumour proliferation and vascularisation.

Thyroid hormones are emerging as critical regulators of tumour growth and progression. To assess the contribution of thyroid hormone signalling via integrin αvß3, expressed on many tumour cells, endothelial cells, and stromal cells, to tumour growth, we compared the effects of thyroid hormones vs tetrac, a specific inhibitor of thyroid hormone action at integrin αvß3, in two murine xenograft tumour models with and without integrin αvß3 expression. Integrin αvß3-positive human anaplastic thyroid cancer cells SW1736 and integrin αvß3-negative human hepatocellular carcinoma cells HuH7 were injected into the flanks of nude mice. Tumour growth was monitored in euthyroid, hyperthyroid, hypothyroid, and euthyroid tetrac-treated mice. In SW1736 xenografts, hyperthyroidism led to a significantly increased tumour growth resulting in a decreased survival compared to euthyroid mice, while tumour growth was significantly reduced and, hence, survival prolonged in hypothyroid and tetrac-treated mice. Both proliferation and vascularisation, as determined by Ki67 and CD31 immunofluorescence staining, respectively, were significantly increased in tumours from hyperthyroid mice as compared to hypothyroid and tetrac-treated mice. No differences in tumour growth, survival, or Ki67 staining were observed between the different groups in integrin αvß3-negative HuH7 xenografts. Vascularisation, however, was significantly decreased in hypothyroid and tetrac-treated mice compared to euthyroid and hyperthyroid mice. Apoptosis was not affected in either tumour model, nor were cell proliferation or apoptosis in vitro. Tumour growth regulation by thyroid hormones in αvß3-positive tumours has important implications for cancer patients, especially those with thyroid dysfunctions and thyroid cancer patients treated with thyrotropin-suppressive L-thyroxine doses.

Decrease in coronary blood flow reserve during hyperlipidemia is secondary to an increase in blood viscosity.

BACKGROUND: During maximal hyperemia, capillaries provide the greatest resistance to flow. A major determinant of capillary resistance is viscosity. We, therefore, hypothesized that abnormal coronary blood flow (CBF) reserve observed during hyperlipidemia is secondary to increased blood viscosity and not abnormal coronary vasomotion. METHODS AND RESULTS: Maximal hyperemia was induced in 9 dogs using adenosine. Serum triglyceride levels were increased by incremental doses of Intralipid. A good correlation was noted between serum triglyceride levels and blood viscosity (r=0.82). Neither total coronary blood volume nor myocardial blood volume changed with increasing serum triglyceride levels, indicating lack of vasomotion. Myocardial vascular resistance (MVR) increased with increasing triglyceride levels (r=0.84), while hyperemic myocardial blood flow (MBF) decreased (r=-0.64). The decrease in hyperemic MBF was associated with a decrease in blood velocity (r=-0.56). These findings were confirmed with direct intravital microscopic observations in the mice cremaster muscle. CONCLUSIONS: Increasing lipid levels in a fully dilated normal coronary bed causes no change in large or small vessel dimensions. Instead, the increase in blood viscosity causes capillary resistance to rise, which attenuates hyperemic CBF. Therefore, the abnormal CBF reserve associated with hyperlipidemia is due to increase blood viscosity and not abnormal vascular function.

Quantification of cerebral perfusion with "Real-Time" contrast-enhanced ultrasound.

BACKGROUND: No noninvasive technique is currently capable of "real-time" assessment and monitoring of cerebral blood flow (CBF). We hypothesized that cerebral perfusion could be accurately measured and monitored in "real time" with contrast-enhanced ultrasound (CEU). METHODS AND RESULTS: Cerebral perfusion was assessed in 9 dogs through a craniotomy with CEU at baseline and during hypercapnia and hypocapnia while normoxia was maintained. Cerebral microvascular blood volume (A), microbubble velocity (beta), and blood flow (Axbeta) were calculated from time-versus-acoustic intensity relations. Compared with baseline, hypercapnia and hypocapnia significantly increased and decreased CBF, respectively, as measured by CEU. These changes in blood flow were mediated by changes in both A and beta. A good correlation was found between Axbeta derived from CEU and CBF measured by radiolabeled microspheres (y=0.67x-0.04, r=0.91, P<0.001). CONCLUSIONS: Changes in both cerebral microvascular blood volume and red blood cell velocity can be accurately assessed with CEU. Thus, CEU has the potential for bedside measurement and monitoring of cerebral perfusion in real time in patients with craniotomies or burr holes.

Is the left atrial v. wave the determinant of peak pulmonary artery pressure in patients with pure mitral stenosis?

A large left atrial (LA) v. wave can be observed in patients with pure mitral stenosis (MS) because of decreased LA compliance. Few data are available regarding the relation between the magnitude of the LA v wave and pulmonary artery pressure in MS. We hypothesized that pulmonary artery pressure may be higher in patients with decreased LA compliance and thus a large v. wave. We analyzed the right-sided cardiac and transseptal catheterization data in 113 patients (16 men and 97 women, mean age 39 years) with pure rheumatic MS in sinus rhythm. Peak systolic, diastolic, and mean pulmonary artery pressures were measured with right-sided cardiac catheterization. The magnitude of LA a and v. waves were measured through transseptal catheterization. Two-dimensional and Doppler echocardiography were also performed to measure LA size, mitral valve area, mean mitral gradient, and valvular regurgitation. Multiple regression analysis was performed to identify the most important factor in the determination of pulmonary artery pressure. A large v wave, defined if peak v wave height exceeded the mean LA pressure by > or =10 mm Hg, was observed in 43 of 113 patients (38%). Increased pulmonary artery systolic pressure (> or =50 mm Hg) was observed in 38 patients (34%). Univariate analysis showed that the factors associated with increased pulmonary artery systolic pressure were smaller mitral valve area, higher mean mitral gradient, higher mean LA pressure, and higher LA v. wave; among them, LA v wave (p <0. 001) and mean mitral gradient (p<0.001) were significant independent factors for pulmonary artery systolic pressure in multivariate analysis. In conclusion, in patients with pure MS and sinus rhythm, the magnitude of the LA v. wave is strongly associated with pulmonary artery pressure. This finding may suggest that LA compliance is a major contributing factor determining pulmonary artery pressure in pure MS.

Echocardiographic and morphologic characteristics of left atrial myxoma and their relation to systemic embolism.

We examined the relation between the echocardiographic morphology of cardiac myxoma and systemic embolism in 25 patients. Two distinct types of myxoma could be identified by echocardiography: round type characterized by solid and round shape with nonmobile surface (n = 13, 52%), and polypoid type characterized by soft and irregular shape with mobile surface (n = 12, 48%); multiple regression analysis revealed the polypoid type of tumor was the only independent predictor of systemic embolism (p = 0.0029).

Enhanced detection of right-to-left shunt through patent foramen ovale by transthoracic contrast echocardiography using harmonic imaging.

When the results of transesophageal echocardiography was regarded as the gold standard for detecting a patent foramen ovale (PFO) in 136 consecutive patients referred for evaluation of cardiac source of embolism, transthoracic harmonic imaging using saline contrast was superior to fundamental imaging in accuracy for detecting a PFO (sensitivity, 22.5%; specificity [p < 0.05] and sensitivity, 100%; specificity 100%, respectively.)

Quantification of myocardial perfusion and determination of coronary stenosis severity during hyperemia using real-time myocardial contrast echocardiography.

Although regional myocardial perfusion can be currently quantified with myocardial contrast echocardiography (MCE) by using intermittent harmonic imaging (IHI), the method is tedious and time-consuming in the clinical setting. We hypothesized that regional myocardial perfusion can be quantified and the severity of coronary stenosis determined during hyperemia with MCE using real-time imaging (RTI) where microbubbles are not destroyed. Six open-chest dogs were studied during maximal hyperemia induced by adenosine in the absence or presence of coronary stenoses varying from mild to severe. Myocardial blood flow (MBF) was measured at each stage by using radiolabeled microspheres. MCE was performed using both IHI and RTI. Data for the latter were acquired in both end-systole and end-diastole. No differences were found between myocardial flow velocity (MFV) derived from IHI and RTI when end-systolic frames were used for the latter. MFV was consistently higher for RTI (P <.01) when end-diastolic frames were used. A linear relation was noted between MFV and radiolabeled microsphere-derived MBF ratios from the stenosed and the normal beds when end-systolic frames were used for RTI (r = 0.78, P <.001), whereas no relation was found when end-diastolic frames were used (r = 0.08, P =.78). The scatter for assessing MBF (A.beta) was minimal for IHI and RTI (9%-10%) with end-systolic frames, whereas that for RTI with end-diastolic frames was large (30%). Furthermore the correlation with radiolabeled microsphere-derived MBF was significantly (P <.01) weaker with RTI when end-diastolic frames were used (r = 0.53) than when end-systolic frames (r = 0.94) or IHI was used (r = 0.99). Data acquisition for IHI was 10 minutes, whereas it was 8 seconds for RTI. Thus, RTI can be used to quantify regional myocardial perfusion and stenosis severity during MCE. Only end-systolic frames, however, provide accurate data. RTI offers a rapid and easy means of assessing regional myocardial perfusion with MCE.

Assessment of left atrial appendage filling pattern by using intravenous administration of microbubbles: comparison between mitral stenosis and mitral regurgitation.

Mitral stenosis (MS) and mitral regurgitation (MR) are the most frequent conditions that cause a dilation and dysfunction of the left atrial appendage (LAA). Despite similarly dilated LAA in patients with MS and MR, the incidence of LAA thrombi and the risk of thromboembolism is different between these patients. The purpose of this study was to characterize the filling pattern of LAA by using intravenous administration of perfluorocarbon-exposed dextrose albumin (PESDA) during transesophageal echocardiographic examination in patients with MS and MR. Twenty-four patients with moderate to severe MS, 12 patients with severe MR, and a control group including 30 patients with conditions other than mitral valve disease underwent transesophageal echocardiographic examination with an intravenous bolus injection of PESDA. LAA emptying and filling velocities and maximal and minimal areas of LAA and LAA ejection fraction were measured. Digital gray-scale intensity (GSI) of the left atrial (LA) and LAA cavity after PESDA injection was measured by off-line analysis. Compared with control patients, patients with MS or MR had larger maximal and minimal areas of LAA and reduced LAA ejection fraction. LAA peak emptying flow velocity was significantly lower in patients with MS compared with those of MR or control patients. LAA peak filling velocity was significantly lower in patients with MS compared with that of control patients. However, there was no significant difference of LAA peak filling velocity between the patients with MS and MR. There was no significant difference of GSI ratio of LAA and LA between patients with MR and control patients; however, GSI ratio of LAA and LA was significantly lower in patients with MS compared with that of MR. The incidence of LAA spontaneous echo contrast and LAA thrombi in patients with MS was significantly higher than that of the patients with MR and control subjects (P <.005). Despite similarly dilated LAA area and depressed contractile function of LAA in patients with MS and MR compared with control patients, profoundly impaired LAA filling with resultant flow stasis was demonstrated by contrast echocardiography in patients with MS. These findings may explain the higher incidence of LAA spontaneous echo contrast and thrombus in patients with MS.

Enhanced detection of left atrial spontaneous echo contrast by transthoracic harmonic imaging in mitral stenosis.

BACKGROUND: Spontaneous echo contrast (SEC) of the left atrium is associated with increased risk of thromboembolism in patients with mitral stenosis (MS). The determination of the presence and severity of left atrial (LA) SEC is of prognostic importance in these patients. Harmonic imaging (HI), a novel echocardiographic technique that differs from conventional fundamental imaging (FI) in that it involves transmitting ultrasound at one frequency and receiving at twice the transmitted frequency, produces better endocardial border definition and myocardial opacification. However, there are no data about its value for the detection of LA SEC. The purpose of this study was to investigate the utility of transthoracic noncontrast tissue HI in the detection of LA SEC in patients with MS. METHODS: Seventy-four consecutive patients with MS (49 women, mean age 51 years) underwent standard transthoracic echocardiography (TTE) in both HI and FI modes and transesophageal echocardiography (TEE) to determine the presence and severity of LA SEC. Left atrial SEC was graded by TEE as either mild (only seen at high gain) or severe (visible in the entire left atrium at normal gain control of the equipment). The control group comprised 30 patients randomly selected from patients who did not have LA SEC at the TEE examination. RESULTS: Atrial fibrillation was found in 46 patients (62.2%). The mean mitral valve area and mean mitral gradient were 1.0+/-0.3 cm(2) and 8.2+/-4.1 mm Hg, respectively. Nine patients (12.2%) had episodes of systemic embolism; 8 had stroke, and 1 had peripheral embolism. Left atrial thrombus was found in 11 patients (14.9%) by TEE. Left atrial SEC was present in all but one patient by TEE (mild in 35 patients, severe in 38). Fundamental imaging with TTE, however, revealed LA SEC in only 5 (6.8%) of the 73 patients. In contrast, with HI, LA SEC could be detected in 63 (86.3%) patients. In the detection of severe LA SEC, the sensitivities of FI and HI were 13.2% (5/38) and 100% (38/38), respectively. Left atrial SEC was not observed in control subjects by either FI or HI. CONCLUSIONS: Transthoracic HI significantly enhances the detection of LA SEC in patients with MS.

Transesophageal echocardiographic identification of left upper pulmonary venous obstruction caused by compression by spontaneous pseudoaneurysm of the descending thoracic aorta.

Pseudoaneurysm of the aorta usually occurs as a complication of nonpenetrating trauma or deceleration injuries. Spontaneous pseudoaneurysm of the aorta is, however, extremely rare. Pulmonary veins can be affected in this situation because of the anatomic proximity. However, it is often overlooked during clinical examination, during routine echocardiography, and even at invasive angiography. This report describes the importance of transesophageal echocardiography in the detection of pulmonary vein compression, which is not suspected during other noninvasive and invasive diagnostic tests, in a patient with spontaneous pseudoaneurysm of the descending thoracic aorta.

Index of myocardial performance using Doppler-derived parameters in the evaluation of left ventricular function in patients with essential hypertension.

We analyzed Doppler echocardiographic data in 120 subjects with normal sinus rhythm; normals (NL, n = 60, ages 54.1 +/- 15.1) and essential hypertensive patients (HT, n = 60, ages 57.3 +/- 10.2). The IMP was calculated as follows: IMP = (ICT + IRT)/ET, ICT; isovolumic contraction time, IRT; isovolumic relaxation time, ET; ejection time. There were no significant differences in ejection fraction (EF), stroke volume index (SVI), cardiac index (CI), ET and ICT between NL and HT. There were, however, significant differences in deceleration time (DT), E/A ratio, IRT and the IMP between the two groups (199.5 +/- 45.6 msec vs 222.3 +/- 54.3 msec, p < 0.01; 1.4 +/- 0.7 vs 0.9 +/- 0.2, p < 0.01; 113.6 +/- 30.2 msec vs 134.2 +/- 29.6 msec, p < 0.01; 0.6 +/- 0.1 vs 0.8 +/- 0.3, p < 0.05). In HT, there were no differences in EF, SVI, CI, E/A ratio and DT between the NYHA I (Gp I, n = 36) and II (Gp II, n = 24) groups. However, ET of Gp II was significantly shorter than that of Gp I (259.4 +/- 43.5 msec vs 297.8 +/- 33.6 msec, p < 0.01). ICT, IRT and the IMP were significantly increased in Gp II, compared to those of Gp I (64.4 +/- 23.9 msec vs 89.4 +/- 46.2 msec, p < 0.05; 120.3 +/- 21.0 msec vs 155.2 +/- 28.5 msec, p < 0.001; 0.6 +/- 0.2 vs 1.0 +/- 0.4, p < 0.001). There were no differences in heart rate and mean blood pressure between Gp I and Gp II (70.9 +/- 11.4/min vs 66.3 +/- 11.4/min, p > 0.05; 138.4 +/- 21.2 mmHg vs 131.3 +/- 19.9 mmHg, p > 0.05). These data suggest that the IMP may be a useful parameter and an early indicator of left ventricular dysfunction in essential hypertensive patients with normal systolic function.

Assessment of myocardial metaiodobenzylguanidine uptake and its relation to left ventricular systolic and diastolic functional parameters in dilated cardiomyopathy.

The purpose of this study was to assess the relation between myocardial metaiodobenzylguanidine (MIBG) uptake and left ventricular systolic and diastolic functional parameters, both of which are known as predictors of prognosis in patients with dilated cardiomyopathy. Echocardiography and iodine-123-MIBG myocardial scintigraphy were performed in 35 patients of dilated cardiomyopathy with normal sinus rhythm. Mean myocardial MIBG uptake in the patient group at early and delayed images were significantly lower than those in normal control subjects (10.6 +/- 1.1, 9.8 +/- 1.2 vs 12.4 +/- 1.0, 12.1 +/- 1.0, p < 0.01). There were, however, no significant differences of mean MIBG uptake in the lung and mediastinum between the two groups (p > 0.05). There were no significant correlations between myocardial MIBG uptake, expressed as the ratio of heart/mediastinum MIBG activity at delayed image, and left ventricular systolic and diastolic functional parameters [left ventricular ejection fraction, left ventricular end-diastolic dimension, peak velocity of early diastolic filling (E velocity), deceleration time of E wave, cardiac output, left ventricular end-diastolic pressure]. In conclusion, the myocardial uptake of MIBG is decreased in patients with dilated cardiomyopathy assessed by iodine-123-MIBG myocardial scintigraphy. There were, however, no significant correlations between myocardial MIBG uptake and left ventricular systolic and diastolic functional parameters derived from echocardiography.

Real-time determination of left ventricular ejection fraction by automatic boundary detection in patients with dilated cardiomyopathy: a comparison with radionuclide ventriculography.

Echocardiographic automatic boundary detection (ABD) is a new on-line technique which automatically outlines the left ventricular endocardial border and instantly calculates the left ventricular area and volume from two dimensional echocardiographic images. To determine if left ventricular ejection fraction (LVEF) can be derived using the ABD method, 25 consecutive patients with dilated cardiomyopathy, aged 52.1 +/- 15.2 (range 14 approximately 75), underwent complete echocardiographic examination with both the ABD method and radionuclide ventriculography (RVG). End-diastolic and end-systolic left ventricular areas were obtained on-line from the apical four chamber view. Left ventricular length was also measured from an apical view. Left ventricular volumes and ejection fraction were calculated using the single plane area-length method. ABD measurements could be obtained in all patients. Linear regression analysis compared ejection fraction derived by ABD and RVG. The mean radionuclide LVEF was 20.9 +/- 6.8% and mean ABD-derived LVEF was 22.7 +/- 5.8%. Linear regression analysis revealed that the ABD-derived LVEF is closely correlated with the RVG-derived LVEF (r = 0.87, p < 0.001). In conclusion, ABD echocardiography is a new on-line technique which may be used to accurately calculate LVEF in patients with dilated cardiomyopathy.