Replacement Fibrosis Research Articles

A lethal perinatal cardiac phenotype resulting from altered integrin function in cardiomyocytes

Background: Integrins are heterodimeric receptors that couple the extracellular matrix to intracellular signaling pathways and the cyoskeleton. Integrins are strain transducers and candidates for modulators or effectors of cardiac hypertrophy. Methods: To begin to probe this function, we have transgenically expressed a chimeric protein that alters integrin function in cardiomyocytes. The transgene (Tac-β1D) consists of the biologically inert extracellular and transmembrane domain of the interleukin-2 receptor α subunit (Tac) fused to the cytoplasmic tail of the human β1D integrin driven by the cardiac α-myosin heavy chain promoter. Transgene expression results in a severe, usually fatal, perinatal cardiac phenotype, characterized by initial electrocardiographic abnormalities followed by extensive myocyte loss, macrophage infiltration, and replacement fibrosis. Results: Expression of Tac-β1D resulted in displacement of endogenous β1D integrin from Z-lines and T-tubules, decreased expression of endogenous β1D, and disrupted the fibronectin pericellular matrix. These results are consistent with an essential role for β1 integrins in maintenance of cardiomyocyte viability and interaction with extracellular matrix. Conclusion: The appearance of conduction abnormalities before morphologic changes suggests that integrins are important in the development or maintenance of the conducting system of the heart.

Cardiocytic apoptosis and capillary endothelial swelling as morphological evidence of myocardial ischemia in ventricular biopsies from patients with angina and normal coronary arteriograms.

The cause of chest pain in patients with normal coronary arteriograms (CAG) remains poorly understood. Left ventricular endomyocardial biopsies from 11 anginal patients with normal CAG and normal left ventriculograms and from seven anginal patients with coronary stenosis were studied by light and electron microscopy. Biopsies from seven non-anginal patients (non-ischemic electrocardiogram abnormalities but no evident heart or systemic diseases) served as controls. In anginal patients with normal CAG, both cardiocytic diameter (17.2 +/- 5.5 microm) and interstitial space percentage area (37.6 +/- 14.9%) were significantly larger than those (13.7 +/- 0.9 microm, 14.9 +/- 2.9%) in control participants. Some cardiocytic nuclei (1.9% of 2000 randomly selected nuclei) exhibited DNA degradation by in-situ nick-end labeling. Electron microscopy revealed cardiocytic nuclei with distinct apoptotic ultrastructures (2.8% of 200 nuclei), phagocytic degradation of cardiocytic cytoplasm, and capillary endothelial swelling (7.1% of 200 capillary transverse sections). No significant infiltration of inflammatory cells was seen. In anginal patients with coronary narrowing (cardiocytic diameter, 16.8 +/- 1.1 microm; interstitial space, 20.1 +/- 5.8%; DNA degraded nuclei, 1.3%), there were however no apoptotic cardiocytic nuclei or cytoplasm and less capillary endothelial swelling (1.6%) in ultrastructure. In biopsies from anginal patients with normal CAG, the presence of cardiocytic hypertrophy and replacement fibrosis are both abnormal. Cardiocytic apoptosis and capillary endothelial swelling, found by others as characteristic of experimental myocardial reperfusion injury, are evident. This supports the possibility of myocardial transient ischemia and reperfusion injury in patients with angina and normal CAG.

Radiation-induced changes in gene expression and distribution of atrial natriuretic peptide (ANP) in different anatomical regions of the rat heart

Purpose : To examine the effects of whole-heart irradiation on gene expression and distribution of atrial natriuretic peptide (ANP) in atrial appendages and left ventricles of the rat heart. Material and methods : Female Sprague-Dawley rats were irradiated with a single dose of 0, 15 or 20 Gy locally to the heart. At intervals up to 16 months post-irradiation, the localization of ANP was examined using immunohistochemical techniques. Absolute mRNA concentrations were determined using the competitive PCR assay. Results : Histological and immunohistochemical studies demonstrated that whole-heart irradiation caused a reduction of atrial ANP, which is due to a substantial loss of ANP-producing atrial myocytes and accumulation of collagen (replacement fibrosis). On the other hand, ANP became detectable in the subendocardium of the irradiated left ventricles. Positive staining of ANP was mainly found in the proximity of injured areas consisting of degeneration of myocytes and fibrosis. At the transcriptional level, reduction of atrial ANP expression at 1 month post-irradiation was followed by recovery at 3 months. Thereafter, ANP mRNA concentrations followed the mRNA pattern of controls and even appeared to increase at 16 months. In the left ventricle, dose-dependent and progressive elevation of ANP gene expression could be observed during the observation period and reached a 20-fold increase as compared with sham-irradiated age-matched controls. Conclusions : The distribution of ANP after local irradiation of the rat heart depends on the severity of the pathological/structural changes (i.e. myocyte degeneration and fibrosis). In radiation-induced heart disease, elevated ANP expression in the left ventricle is most probably involved in the observed chronic elevation of plasma ANP levels.

Cardiac troponin I in feline hypertrophic cardiomyopathy.

Measurement of plasma cardiac troponin I concentration ([cTnI]) is a sensitive and specific means for detecting myocardial damage in many mammalian species. Studies have shown that [cTnI] increases rapidly after cardiomyocyte injury. The molecular structure of cTnl is highly conserved across species, and current assays developed for its detection in humans have been validated in many species. In this study, [cTnI] was quantified using a 2-site sandwich assay in plasma of healthy control cats (n = 33) and cats with moderate to severe hypertrophic cardiomyopathy (HCM) (n = 20). [cTnI] was significantly higher in cats with HCM (median, 0.66 ng/mL; range, 0.05-10.93 ng/mL) as compared with normal cats (median, <0.03 ng/mL; range, <0.03-0.16 ng/mL) (P < .0001). An increase in [cTnI] was also highly sensitive (sensitivity = 85%) and specific (specificity = 97%) for differentiating cats with moderate to severe HCM from normal cats. [cTnI] was weakly correlated with diastolic thickness of the left ventricular free wall (r2 = .354; P = .009) but not with the diastolic thickness of the interventricular septum (P = .8467) or the left atrium: aorta ratio (P = .0652). Furthermore, cats with congestive heart failure at the time of cTnI analysis had a significantly higher [cTnI] than did cats that had never had heart failure and those whose heart failure was controlled at the time of analysis (P = .0095 and P = .0201, respectively). These data indicate that cats with HCM have ongoing myocardial damage. Although the origin of this damage is unknown, it most likely explains the replacement fibrosis that is consistently identified in cats with moderate to severe HCM.

Relation of thallium uptake to morphologic features of chronic ischemic heart disease: evidence for myocardial remodeling in noninfarcted myocardium

OBJECTIVESThe aim of this study was to investigate the disparity between the extent of myocardial injury as assessed by thallium and the severity of left ventricular (LV) dysfunction in chronic ischemic heart disease.BACKGROUNDAlthough it is believed that thallium differentiates between viable and nonviable myocardium, in some patients with chronic ischemic heart disease, viable regions by thallium may fail to improve function after revascularization.METHODSThirteen transplant candidates with chronic ischemic heart disease (LV ejection fraction = 14 ± 6% at rest) were studied prospectively with stress-redistribution-reinjection thallium single-photon emission computed tomography. We examined pretransplantation quantitative thallium uptake and post-transplantation extent and the histological distribution of collagen replacement in infarcted and noninfarcted myocardium and in 13 age-matched control hearts.RESULTSThe volume fraction of collagen varied inversely with wall thickness (r = −0.70, p < 0.001) and was higher in irreversible (30.9 ± 15.8%) compared with reversible (20.2 ± 12.6%, p < 0.001) or normal thallium segments (15.0 ± 8.7%, p < 0.001). The irreversible thallium segments had lower wall thickness and more severe coronary artery narrowing (9.7 ± 2.8 mm and 95 ± 8%) compared with reversible (11.7 ± 2.7 mm and 87 ± 13%, p < 0.001) and normal thallium segments (12.8 ± 2.6 mm and 80 ± 14%, p < 0.001). Mean volume fraction of collagen was significantly lower in noninfarcted than it was in infarcted segments (13 ± 6% vs. 36 ± 13%, p < 0.001) but exceeded that in the control hearts (4 ± 2%, p < 0.001). Noninfarcted segments had predominantly interstitial fibrosis with either microscopic or patchy areas of replacement fibrosis.CONCLUSIONSIn chronic ischemic heart disease with severe LV dysfunction, patterns of normal, reversible and irreversible thallium uptake correlated with the magnitude of collagen replacement, segmental wall thickness and severity of coronary artery narrowing. The finding of scattered areas of replacement fibrosis in noninfarcted myocardium may explain the observed disparity between LV contractile dysfunction and the extent of myocardial injury assessed by thallium.

Disruption of Integrin Function in the Murine Myocardium Leads to Perinatal Lethality, Fibrosis, and Abnormal Cardiac Performance

The molecular mechanisms that regulate the cardiac hypertrophic response and the progression from compensated hypertrophy to decompensated heart failure have not been thoroughly defined. Alteration in cardiac extracellular matrix is a distinguishing characteristic of these pathological processes. Integrins, cell surface receptors that mediate cellular adhesion to the extracellular matrix, are signaling molecules that possess mechanotransduction properties. Therefore, we hypothesized that integrins are likely candidates to play an important role in cardiac function. To test this hypothesis, transgenic mice were constructed in which normal integrin function was disrupted by expression of a chimeric molecule encoding the transmembrane and extracellular domains of the Tac subunit of the IL-2 receptor, fused to the cytoplasmic domain of beta(1A) integrin (Tacbeta(1A)). Using the alpha myosin heavy chain promoter to target expression of this chimera to the cardiac myocyte, transgenic mice were generated that had varied levels of transgene expression. Multiple transgenic founders that expressed the transgene at high levels, died perinatally and exhibited replacement fibrosis. Lines that survived showed 1) hypertrophic changes concordant with reduction in endogenous beta(1) integrin levels, or 2) reduced basal contractility and relaxation as well as alterations in components of integrin signaling pathways. These data support an important role for beta(1) integrin in normal cardiac function.

Model of cardiovascular injury in magnesium deficiency

Magnesium deficiency is known to produce cardiovascular lesions. It is, however, not clear as to what constitutes magnesium deficiency – reduced serum levels, reduced tissue levels or reduced intracellular levels of the ionic form of the element. This article cites evidence in support of a hypothesis that a fall in serum magnesium levels may trigger a temporal sequence of events involving vasoconstriction, hemodynamic alterations and vascular endothelial injury to produce pro-inflammatory, pro-oxidant and pro-fibrogenic effects, resulting in initial perivascular myocardial fibrosis which, in turn, would cause myocardial damage and replacement fibrosis. Further, angiotensin II may be the prime mover of the pathogenetic cascade in magnesium deficiency. Importantly, such a mechanism of cardiovascular injury would be independent of a reduction in myocardial or vascular tissue levels of magnesium.

Histological remodeling in an ovine heart failure model resembles human ischemic cardiomyopathy

Staged coronary embolization, causing myocardial microinfarctions, has been shown in dogs and sheep to cause chronic ischemic heart failure (HF) that resembles the hemodynamics of the human condition. However, its histopathological basis remains unclear. We examined the hypothesis that the ventricular remodeling seen in such sheep resembles the histopathology of human ischemic cardiomyopathy (ICM). Understanding the pathophysiology of this model will determine its place in the development of treatment strategies for HF. Global left ventricular (LV) damage resulting in HF was induced by staged coronary embolization in 11 sheep. Six others served as controls (normal control, NC). In HF sheep, the heart was harvested 6 months after LV ejection fraction (EF) had stabilized at <35%. Histopathological profiles were compared in biventricular transverse sections at midpapillary level using computed image analysis. LV end-diastolic volume increased in the HF group from 84.9±29 to 122.4±30.3 ml ( n=11, P<.05), but myocytes across the LV wall in noninfarcted zones decreased (435.7±38.2 NC; 297.8±48.4/unit area HF; n=11, P<.0001) as did myocyte nuclear density (990.5±51.5 NC; 677.5±121.1/mm 2 HF, n=11, P<.0001). In contrast, LV replacement and interstitial fibrosis increased as did myocyte diameter in noninfarcted zones: 0.1±0.1 to 6.2±4.5% ( P=.0049); 2.0±1.0 to 7.6±4.9% ( P=.0149); and 10.0±0.5 to 15.9±2.2 μm ( P<.0001), respectively. Although LV myocyte nuclear length increased (10.2±1.0 NC; 12.2±0.9 μm HF, n=11, P=.0006), right ventricular (RV) myocyte nuclear density and length did not alter. In this ovine chronic HF model, LV dilation and interstitial and myocyte remodeling resemble human ICM.

Alterations in Cardiac Function and Gene Expression during Autoimmune Myocarditis in Mice

L. B. Stull, N. A. DiIulio, M. Yu, C. F. McTiernan, N. B. Ratliff, V. K. Tuohy and C. Schomisch Moravec. Alterations in Cardiac Function and Gene Expression during Autoimmune Myocarditis in Mice. Journal of Molecular and Cellular Cardiology (2000) 32, 2035–2049. Although myocarditis has been implicated in the pathogenesis of heart failure, a definitive relationship between myocardial inflammation, cardiac dysfunction, and changes in myocyte gene expression has not been established. In this study, we examined the hypothesis that myocardial inflammation and replacement fibrosis following an autoimmune response can progress to cardiac dysfunction and may result in progression to the heart failure phenotype. SWXJ mice were immunized with cardiac myosin on day 0 and day 7, in order to induce an autoimmune response to the myosin protein. Cardiac catheterization via the right carotid artery was performed on days 14, 21, 28, 35, and 42, using a 1.4F Millar transducer-tipped catheter. Hearts were weighed, and cross-sections were cut and stained with either haematoxylin and eosin or Masson's trichrome, in order to identify areas of inflammation and/or fibrosis. Myocardial gene expression was determined by Northern blot analysis. In mice with histological evidence of myocarditis, the heart weight/body weight ratio increased beginning on day 14, and cardiac function decreased beginning on day 21. Myocardial inflammation was accompanied by significant fibrosis beginning on day 21. Quantitation of mRNA showed expression of ventricular atrial naturietic factor, as well as a decrease in myosin heavy chain α, beginning on day 21. These data demonstrate that autoimmune inflammation of the heart results in significant cardiac dysfunction, leading to phenotypic alterations similar to those demonstrated in human heart failure and animal models of heart failure.

Myocardial-Directed Overexpression of the Human β1-Adrenergic Receptor in Transgenic Mice

The β1-adrenergic receptor (AR) is the dominant subtype in non-failing and failing myocardium. β1-AR signaling, by the endogenous neurotransmitter norepinephrine, is central to the regulation of myocardial contractility. In heart failure, the β1-AR undergoes subtype-selective downregulation which may protect against the increased cardiac adrenergic drive associated with this pathophysiological state. To examine the hypothesis that chronically increased β1-AR mediated signaling has adverse myocardial effects, transgenic mice overexpressing the human β1-AR in a cardiac-selective context were produced, utilizing an α-myosin heavy chain (MHC) promoter. In these mice, β1-AR protein abundance was ≈ 24–46-fold (1–2 pmol/mg protein) that of wild-type mice. Histopathological examination of young (4 months old) and old (≈ 9 months old) transgenic mouse hearts consistently demonstrated large areas of interstitial replacement fibrosis, marked myocyte hypertrophy and myofibrilar disarray. In addition, increased expression of the pre-apoptotic marker, Bax, was observed coincident with regions of fibrosis accompanied by an increased apoptotic index, as measured by TUNEL assay. Older non-transgenic mice exhibited a slight tendency towards a decreased fractional shortening, whereas older β1-AR transgenic mice had a marked reduction in fractional shortening (%FS ≈ 30) as determined by echocardiography. Additionally, older β1-AR transgenic mice had an increased left ventricular chamber size. In summary, cardiac-directed overexpression of the human β1-AR in transgenic mice leads to a significant histopathological phenotype with no apparent functional consequence in younger mice and a variable degree of cardiac dysfunction in older animals. This model system may ultimately prove useful for investigating the biological basis of adrenergically-mediated myocardial damage in humans.

Impact of Delayed Reperfusion of Myocardial Hibernation on Myocardial Ultrastructure and Function and Their Recoveries After Reperfusion in a Pig Model of Myocardial Hibernation

This study examined the effect of delayed reperfusion of myocardial hibernation from 24 hours to 7 days on myocardial ultrastructural and functional changes and their recoveries after reperfusion. Background: We have previously shown in pigs that after reperfusion the functional and structural alterations in short-term myocardial hibernation which was reperfused in 24 hours can recover in 7 days. The effect of delayed reperfusion of hibernating myocardium on the extent and severity of cellular and extracellular structural changes of hibernating myocardium, and their recoveries after reperfusion is not known. Methods and Results: A severe LAD stenosis was created in 27 pigs, reducing resting flow by 30–40% immediately after placement of the stenosis and producing acute ischemia as evidenced by regional lactate production, a decrease in regional coronary venous pH, reduced regional wall thickening (from 38.5 ± 5.1% to 10.4 ± 8.0%) and a 33% reduction of regional oxygen consumption. The stenosis was maintained either for 24 hours in 9 pigs (group 1) with LAD flow of 0.65 ± 0.13 ml/min/g (38% reduction), or for 7 days in 17 pigs (group 2) with LAD flow of 0.67 ± 0.14 ml/min/g (36% reduction). There were no differences (p = NS) in the reduction of wall thickening, rate-pressure product, lactate production, or regional oxygen consumption between group 1 and group 2. Quantitative morphometric evaluation of the ultrastructure on electromicrographs revealed a greater decrease in sarcomere volume and a higher incidence of myocytes with reduced sarcomere volume in 7-day than in 24-hour hibernating regions (53 ± 19% versus 33 ± 14%, p < 0.05). Patchy myocardial necrosis with replacement fibrosis was common, but 6 of the 18 pigs had no myocardial necrosis or replacement fibrosis in the 7-day hibernating group, and 4 of 9 pigs had no patchy myocyte necrosis in the 24 hour hibernating group. In 6 pigs in group 1 in which the stenosis was then released and hibernating myocardium reperfused in 24 hours, regional wall thickening recovered to 30 ± 6% (p = NS compared to baseline) after one week of reperfusion. In 12 pigs in group 2 in which the stenosis was released and hibernating myocardium reperfused in 7 days, regional wall thickening recovered slowly, from 10.1 ± 7.2% to 18.1 ± 8.3% at one week ( n = 5) and to 28.0 ± 3.6% at 3–4 weeks of reperfusion ( n = 7, p < 0.05 compared to baseline). Similarly, the sarcomere volume or myofilament recovered significantly (p < 0.01) and was not different compared to the normal region (p = NS) in the 24-hour hibernating region of group 1, but the recovery was much slower and was incomplete at 4 weeks (p < 0.01) compared to baseline in the 7-day hibernating region of group 2. Recovery of regional wall thickening correlated with ultrstructural recovery (p < 0.01). By multivariate stepwise regression analysis, the degree of LAD flow reduction, the extent of fibrosis, and myofilament loss were independent predictors of the extent of functional recovery. Conclusions: In a porcine model of myocardial hibernation with myocardial hypoperfusion, systolic dysfunction, and metabolic adaptations, a longer period of myocardial hibernation with delayed reperfusion was associated with more severe abnormalities of myocytes. an increasing interstitial fibrosis, and more protracted myofibrillar and functional recoveries after reperfusion. The extent of functional recovery is related to the degree of coronary flow reduction, the severity of the ultrastructural changes, and the extent of interstitial fibrosis.

Apoptotic cell death in heart failure.

Time for primary review 29 days. Progressive deterioration of left ventricular (LV) function is a characteristic feature of the failing heart. This hemodynamic deterioration, often accompanied by worsening of clinical symptoms, occurs despite the absence of clinically apparent intercurrent adverse events [1,2] and, invariably, culminates in the syndrome of congestive heart failure. The exact mechanisms that drive this process are not known. For years, a so-called ‘vicious circle’ postulate was adopted to explain this phenomenon whereby compensatory mechanisms, elicited to maintain homeostasis, themselves become factors that exacerbate the heart failure state. Such compensatory mechanisms included LV hypertrophy, LV chamber dilation, and enhanced and sustained activity of the sympathetic nervous system and renin–angiotensin system [3–6]. In recent years, we and others have put forward a working hypothesis that progressive LV dysfunction in heart failure may result, in part, from ongoing loss of cardiomyocytes. The notion that progression of heart failure may be due, in part, to ongoing loss of functional cardiac units was based on the presence of ultrastructural degenerative changes of cardiomyocytes in the failed human heart as well as in hearts of animals with experimentally-induced heart failure [7–10]. Structural abnormalities included myofibrillar disruption and disarray [8], abnormalities of mitochondria characterized by disruption of the internal and external membranes, hyperplasia and reduced organelle size [7], and abnormalities of the cardiac interstitium characterized by accumulation of collagen [9]. These observations provided some support, albeit indirect, to the concept that ongoing myocyte degeneration and loss may occur in the failing heart. Objective evidence in support of this concept, however, was only recently put forward [11]. Studies in dogs with heart failure produced by intracoronary microembolizations showed, for the first time, that progressive LV dysfunction was associated with increased volume fraction of replacement fibrosis [11] or, in … * Tel.: +1-313-876-7360; fax: +1-313-876-3001 HSABBAH1{at}hfhs.org

Structural Changes of Atrial Myocardium During Chronic Atrial Fibrillation

Of all known arrhythmia's, atrial fibrillation (AF) is the most often met in the clinical setting and it is associated with an increase in mortality risk. Several risk factors for AF have been described and several mechanisms of induction and maintenance have been proposed. Studies in patients with AF have shown that structural changes occur in the atria, but the relationship between the structural remodelling and the chronicity of the arrhythmia are not well understood. The changes mainly concern adaptive (dedifferentiation of cardiomyocytes) and maladaptive (degeneration of cells with replacement fibrosis) features. In order to characterise the time course of the structural remodelling the need for animal models which adequately mimic chronic atrial fibrillation in humans is felt essential. In this review, the structural changes that are observed during prolonged sustained AF in patients and animal models, are described. Furthermore, the time course and potential mechanisms of structural remodelling are discussed and methods for elucidation of the underlying molecular mechanisms are presented.

A self-perpetuating vicious cycle of tissue damage in human hibernating myocardium.

Recently, we proposed the hypothesis that a vicious cycle exists in human hibernating myocardium (HM) between the progression of myocyte degeneration and the development of fibrosis. We now investigated the pathomechanism of this cycle in more detail and established a correlation between the severity of the morphological changes and the degree of postoperative functional recovery of HM. HM was diagnosed by dobutamine echocardiography, thallium-201 scintigraphy and radionuclide ventriculography. Functional recovery was present at 3 months after coronary bypass surgery but remained unchanged at 15 months. Forty patients were subdivided into 2 groups: A with complete and B with incomplete recovery. Biopsies taken during surgery and studied by electron microscopy, immunocytochemistry, rt-PCR, and morphometry revealed myocyte degeneration and inflammatory and fibrinogenic changes in a widened interstitial space. We report here for the first time an upregulation of TGF-beta1 evident by a 5-fold increase of fibroblasts and macrophages exhibiting a TGF-beta1 content 3-fold larger than in control, and a > 3-fold increase in TGF-beta1 mRNAby rt-PCR. The number of angiotensin converting enzyme (ACE) containing structures was increased (n/mrm2: control-11.4, A-17.6, B-19.2, control vs. A and B, p < 0.05). Fibrosis was more severe in group B than A or control (%: C-10.1; A-21.2; B-40.6; p < 0.05). Capillary density was significantly reduced (n/mm2: C-1152; A-782; B-579, p < 0.05) and intercapillary distance was widened (microm: C-29.5, A-36.1, B-43.3, p < 0.05). The number of CD 3 (n/mm2: C-5.0; A-9.6; B-9.4, ns) and CD 68 positive cells (n/mm2: C-37.2; A-80.7; B-55.0, C vs. A p < 0.05) was elevated in HM as compared to control indicating an inflammatory reaction. Cut-off points for functional recovery are fibrosis > 32%, capillary density < 660/mm2 and intercapillary distance > 39.0 microm. In HM a self-perpetuating vicious cycle of tissue alterations leads to progressive replacement fibrosis and continuous intracellular degeneration which should be interrupted by early revascularization.

Profound apoptosis-mediated regional myocyte loss and compensatory hypertrophy in pigs with hibernating myocardium.

Myocyte apoptosis is seen in ischemic heart disease, but whether it can occur after reversible ischemia or independent of necrosis and replacement fibrosis is unknown. Pigs were instrumented with a stenosis of the left anterior descending coronary artery to chronically reduce coronary flow reserve over a period of 3 months. At this time, there was viable dysfunctional myocardium having the physiological features of hibernating myocardium. Resting subendocardial perfusion was reduced to 0.65+/-0.08 (mean+/-SEM) mL. min(-1). g(-1) in hibernating myocardium of instrumented pigs compared with 0.98+/-0.14 mL. min(-1). g(-1) in myocardium of sham-operated pigs (P<0.05). There was a critical limitation in subendocardial flow during vasodilation to 0.78+/-0.20 mL. min(-1). g(-1) in instrumented pigs versus 3. 24+/-0.50 mL. min(-1). g(-1) in sham-operated pigs (P<0.001). Histology revealed a regional reduction in myocyte nuclear density to 995+/-100 nuclei/mm(2) in hibernating myocardium from the instrumented group versus 1534+/-65 nuclei/mm(2) in myocardium from the sham-operated group (P<0.05), regional myocyte hypertrophy (myocyte volume per nucleus, 14 183+/-2594 in the instrumented group versus 9130+/-1301 microm(3) in the sham group; P<0.05), and minimal increases in connective tissue (5.8+/-0.9% in the instrumented group versus 3.0+/-0.2% in the sham group, P<0.05). Necrosis was not identified, but apoptosis was increased from 30+/-9 myocytes per 10(6) myocyte nuclei in myocardium from the sham group to 220+/-77 myocytes per 10(6) myocyte nuclei in hibernating myocardium (P<0.05). These findings indicate that reversible ischemia in an area of chronically reduced coronary flow reserve induces regional myocyte loss via an apoptotic mechanism. This may contribute to the progression of chronic coronary disease to heart failure and explain the lack of complete functional recovery after revascularization in hibernating myocardium.

Insulin-like growth factor-1 attenuates the detrimental impact of nonocclusive coronary artery constriction on the heart.

Coronary artery narrowing (CAN) induces tissue injury, which may involve myocyte necrosis and apoptosis. Insulin-like growth factor (IGF)-1 may counteract cell death, modifying the detrimental effects of myocardial ischemia. On this basis, CAN was produced in female FVB.Igf+/- mice and nontransgenic littermates, and the animals were euthanized 7 days later. CAN consisted of an 82% reduction in the vessel luminal cross-sectional area in both groups of mice. Severe left ventricular dysfunction was present in CAN nontransgenic and transgenic mice, but heart and left ventricular weights increased more in littermates than in FVB.Igf+/- mice. Similarly, the changes in chamber volume and diastolic wall stress were greater in nontransgenic mice. Subacute tissue injury, represented by foci of replacement fibrosis, was 2.6-fold higher in CAN littermates than in FVB.Igf+/- mice. Ongoing myocyte necrosis was 5-fold greater in nontransgenic mice, whereas apoptosis was low and did not differ in the 2 groups of mice. In CAN nontransgenic mice, myocyte necrosis was 12-fold more frequent than apoptosis but, in CAN transgenic mice, these 2 types of cell death were comparable. alpha-Myosin and beta-myosin isoform mRNAs were affected by CAN, but alpha-myosin mRNA was reduced more in nontransgenic mice. In conclusion, myocyte necrosis and replacement fibrosis are the prevailing forms of myocardial damage induced by CAN. Constitutive overexpression of IGF-1 attenuates myocyte necrosis and tissue injury, having no effect on cell apoptosis. These factors limit ventricular dilation, myocardial loading, cardiac hypertrophy, and alterations in alpha- and beta-myosin isoform expression.

Upregulated expression of cardiac endothelin-1 participates in myocardial cell growth in Bio14.6 Syrian cardiomyopathic hamsters

ObjectivesThe purpose of this study is to investigate the role of endogenous endothelin-1 (ET-1) in myocardial growth in Bio 14.6 Syrian cardiomyopathic hamsters (Bio).BackgroundWhile ET-1, as a growth-promoting peptide, has been implicated in the development of secondary cardiac hypertrophy, the role of endogenous ET-1 in cardiac growth in primary myocardial disease is unknown.MethodsWe measured left ventricular ET-1 levels by a specific sandwich enzyme-linked immunosorbent assay. Furthermore, we examined the chronic effect of T0201, an ET type A receptor-specific antagonist.ResultsThe ET-1 levels in the left ventricles were 1.8-fold higher (p < 0.0005) at 20 weeks and 6.4-fold higher (p < 0.0001) at 35 weeks in the Bio compared to age-matched control F1B hamsters (F1B). The Bio ET-1 levels in the lungs exhibited an only 1.3-fold elevation at 35 weeks. Immunohistochemistry demonstrated the localization of ET-1 mainly in the cardiac myocytes. The treatment with T0201 significantly reduced the heart weight/body weight ratio in the Bio, but did not affect the heart weight/body weight ratio in the F1B. Histologically, T0201 reduced the myocyte diameter of Bio to a level similar with that of F1B. However, T0201 did not affect the extent of fibrosis in Bio or F1B.ConclusionsThe ET-1 level in the heart of cardiomyopathic hamsters increases in stage-dependent and organ-specific manners. Though myocyte degeneration and subsequent replacement fibrosis do not require an ET-1 pathway, the accelerated synthesis of ET-1 in the heart may contribute to the pathological growth of remaining myocytes in this animal model.

Myocardial dna strand breaks are detected in biopsy tissues from patients with dilated cardiomyopathy

Progressive damage of cardiomyocytes with interstitial and replacement fibrosis accompanied by less inflammatory cell infiltration is observed in patients with dilated cardiomyopathy (DCM), suggesting some other mechanisms rather than necrotic cell death. The aim of this study was to assess the possible involvement of apoptotic process in the pathogenesis of DCM and myocarditis. Endomyocardial biopsy was performed in patients with DCM (n = 9), myocarditis (n = 4), or atypical chest pain syndrome (as controls; n = 5). The TUNEL method was used for in situ detection of oligonucleosomal DNA strand breaks. The TUNEL-positive cells were observed in three of nine patients with DCM and in all four with myocarditis, but in none of the controls. The TUNEL-positive nuclei were observed exclusively in cardiomyocytes in DCM, whereas in myocarditis they were detected mainly in interstitial cells and in a few myocytes. In DCM, interstitial fibrosis was greater in the TUNEL-positive than in TUNEL-negative patients (p < 0.05). In either DCM or myocarditis, electron microscopic examination could not reveal morphologic features of apoptosis of cardiomyocytes. The DNA strand breaks were detected in cardiomyocytes in patients with DCM and mainly in interstitial cells in myocarditis. It is possible that the DNA strand breaks can be involved in mechanisms of progressive loss of functional cardiac units in these myocardial diseases.

Proliferating Cell Nuclear Antigen (PCNA), DNA Synthesis and Mitosis in Myocytes Following Cardiac Transplantation in Man

Cardiac transplantation is characterized by rejection, myocyte loss, interstitial and replacement fibrosis, and loading abnormalities. These modifications contribute to enhance mural and muscle cell stress, activating reactive growth processes in myocytes and interstitial cells. However, it is unknown whether cell cycle related gene product, such as PCNA, and DNA synthesis are stimulated under these conditions. Therefore, 62 endomyocardial biopsies obtained from 17 patients who underwent cardiac transplantation were examined for the immunocytochemical detection of PCNA protein in myocyte and non-myocyte nuclei. In addition, tissue samples were labeledin vitrowith bromodeoxyuridine (BrdU) to document ongoing DNA synthesis. The presence of mitotic images in myocytes and non myocytes were also examined. Biopsies were collected from 1–768 days after surgery. Histologic examination of tissue sections documented that PCNA labeling involved nearly 30% of myocyte nuclei in all patients. Similar percentages of PCNA labeling were detected in interstitial cells, lymphocytes and mononuclear infiltrates. DNA synthesis in myocytes and connective tissue cells was observed in nine and 14 subjects, respectively. BrdU positive lymphocytes and mononuclear infiltrates in 13 cases. Three mitotic figures in myocyte nuclei were identified. PCNA, BrdU labeling and mitosis were not detected in eight myocardial samples obtained from control hearts. These results suggest that the evolution of the transplanted heart involves the expression of a gene which is implicated in DNA replication. The presence of ongoing DNA synthesis and mitosis support the notion that proliferation of myocytes and non muscle cells may be a component of ventricular remodeling after cardiac transplantation.

Clinical Features of Hypertrophic Cardiomyopathy Caused by Mutation of a “Hot Spot” in the Alpha-Tropomyosin Gene

Objectives. We studied the clinical and genetic features of familial hypertrophic cardiomyopathy (FHC) caused by an Asp175Asn mutation in the alpha-tropomyosin gene in affected subjects from three unrelated families.Background. Correlation of genotype and phenotype has provided important information in FHC caused by beta-cardiac myosin and cardiac troponin T mutations. Comparable analyses of hypertrophic cardiomyopathy caused by alpha-tropomyosin mutations have been hampered by the rarity of these genetic defects.Methods. The haplotypes of three kindreds with FHC due to an alpha-tropomyosin gene mutation, Asp175Asn, were analyzed. The cardiac histopathologic findings of this mutation are reported. Distribution of left ventricular hypertrophy in affected members was assessed by two-dimensional echocardiography, and patient survival rates were compared.Results. Genetic studies defined unique haplotypes in the three families, demonstrating that independent mutations caused the disease in each. The Asp175Asn mutation caused cardiac histopathologic findings of myocyte hypertrophy, disarray and replacement fibrosis. The severity and distribution of left ventricular hypertrophy varied considerably in affected members from the three families (mean maximal wall thickness ± SD: 24 ± 4.5 mm in anterior septum of Family DT; 15 ± 2.7 mm in anterior septum and free wall of Family DB; 18 ± 2.1 mm in posterior septum of Family MI), but survival was comparable and favorable.Conclusions. Nucleotide residue 579 in the alpha-tropomyosin gene may have increased susceptibility to mutation. On cardiac histopathologic study, defects in this sarcomere thin filament component are indistinguishable from other genetic etiologies of hypertrophic cardiomyopathy. The Asp175Asn mutation can elicit different morphologic responses, suggesting that the hypertrophic phenotype is modulated not by genetic etiologic factors alone. In contrast, prognosis reflected genotype; near normal life expectancy is found in hypertrophic cardiomyopathy caused by the alpha-tropomyosin mutation Asp175Asn.(J Am Coll Cardiol 1997;29:635–40)