We examined the prevalence, genotype-phenotype correlation, and natural history of lamin A/C gene (LMNA) mutations in subjects with dilated cardiomyopathy (DCM).
Mutations in LMNAhave been found in ...patients with DCM with familial conduction defects and muscular dystrophy, but the clinical spectrum, prognosis, and clinical relevance of laminopathiesin DCM are unknown.
A cohort of 49 nuclear families, 40 with familial DCM and 9 with sporadic DCM (269 subjects, 105 affected), was screened for mutations in LMNAusing denaturing high-performance liquid chromatography and sequence analysis. Bivariate analysis of clinical predictors of LMNAmutation carrier status and Kaplan-Meier survival analysis were performed.
Mutations in LMNAwere detected in four families (8%), three with familial (R89L, 959delT, R377H) and one with sporadic DCM (S573L). There was significant phenotypic variability, but the presence of skeletal muscle involvement (p < 0.001), supraventricular arrhythmia (p = 0.003), conduction defects (p = 0.01), and “mildly” DCM (p = 0.006) were predictors of LMNAmutations. The LMNAmutation carriers had a significantly poorer cumulative survival compared with non-carrier DCM patients: event-free survival at the age of 45 years was 31% versus 75% in non-carriers.
Mutations in LMNAcause a severe and progressive DCM in a relevant proportion of patients. Mutation screening should be considered in patients with DCM, in particular when clinical predictors of LMNAmutation are present, regardless of family history.
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.
The regulation of angiotensin II receptors and the two major subtypes (AT1 and AT2) in chronically failing human ventricular myocardium has not been previously examined.
Angiotensin II receptors were ...measured by saturation binding of 125I-Sar1,Ile8angiotensin II in crude membranes from nonfailing (n = 19) and failing human left ventricles with idiopathic dilated cardiomyopathy (IDC; n = 31) or ischemic cardiomyopathy (ISC; n = 21) and membranes from a limited number of right ventricles in each category. The AT1 and AT2 fractions were determined by use of an AT1-selective antagonist, losartan. beta-Adrenergic receptors were also measured by binding of 125I-iodocyanopindolol with the beta 1 and beta 2 fractions determined by use of a beta 1-selective antagonist, CGP20712A, AT1 but not AT2 density was significantly decreased in the combined (IDC + ISC) failing left ventricles (nonfailing: AT1 4.66 +/- 0.48, AT2 2.73 +/- 0.39; failing: AT1 3.20 +/- 0.29, AT2 2.70 +/- 0.33 fmol/mg protein; mean +/- SE). The decrease in AT1 density was greater in the IDC than in the ISC left ventricles (IDC: 2.73 +/- 0.40, P < .01; ISC: 3.89 +/- 0.39 fmol/mg protein, P = NS versus nonfailing). beta 1 but not beta 2 density was decreased in the failing left ventricles. AT1 density was correlated with beta 1 density in all left ventricles (r = .43). AT1 density was also decreased in IDC right ventricles. In situ reverse transcription-polymerase chain reaction in sections of nonfailing and failing ventricles indicated that AT1 mRNA was present in both myocytes and nonmyocytes.
AT1 receptors are selectively downregulated in failing human ventricles, similar to the selective downregulation of beta 1 receptors. The relative lack of AT1 downregulation in ISC hearts may be related to differences in the degree of ventricular dysfunction.
Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or ...idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of beta1-receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of beta2-receptor gene expression in PPH but not IDC. The major new findings were that (a) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of alpha-myosin heavy chain mRNA (alpha-MHC, 23-34% of total), and (b) in heart failure alpha-MHC was downregulated (by 67-84%) and beta-MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial alpha-MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction.
K. Asano, T. J. Bohlmeyer, J. Y. Westcott, L. S Zisman, K. Kinugawa, M. Good, W. A. Minobe, R. L. Roden, E. E. Wolfel, J. Lindenfeld, J. D. Port, M. B. Perryman, J. Cleveland, B. D. Lowes and M. R. ...Bristow. Altered Expression of Endothelin Receptors in Failing Human Left Ventricles. Journal of Molecular and Cellular Cardiology (2002) 34, 833–846. Background: Endothelin signaling is activated in failing human hearts, and may contribute to progressive myocardial dysfunction and remodeling. However, the behavior of endothelin receptor systems (ETA and ETB) in failing human hearts is not well understood.
Methods and Results:125I-endothelin-1 binding assays conducted in the presence of a non-hydrolyzable guanine nucleotide to uncouple agonist binding demonstrated that membranes prepared from nonfailing left ventricles (LVs) exhibit a mixed pattern of ETA (∼60%) and ETB (∼40%) receptor protein expression. Chronic LV failure from either idiopathic dilated (IDC) or ischemic (ISC) cardiomyopathy was accompanied by a significant (P<0.001) increase in ETA receptor density, to ∼80% of the total population, and a significant (P<0.02) decrease in ETB receptor density. Ribonuclease protection assays demonstrated an increase in ETA mRNA abundance in IDC and ISC LVs, and a significant (P<0.04) increase in ETB mRNA abundance in ISC LVs. Enzyme-linked immunoabsorbent assays demonstrated a significant increase in tissue immunoreactive endothelin-1 concentration in IDC (P=0.01) and in IDC+ISC LVs (P=0.02), but receptor subtype protein or mRNA level was not significantly correlated with tissue ET-1 across all LVs. In situ reverse-transcription polymerase chain reaction in LV sections demonstrated that in both failing and nonfailing LVs the ETA gene is expressed in cardiac myocytes, vascular smooth muscle and endothelium; the ETB gene is expressed in cardiac myocytes, fibroblasts and endothelium; and the prepro-endothelin-1 gene is expressed in myocytes and interstitial cells. Conclusions: In chronically failing human LVs, ETA receptor density is increased to become the dominant subtype while ETB receptor density is decreased. The ETA, but not the ETB density change is accompanied by cognate regulation of mRNA abundance. Both receptor genes and prepro-endothelin-1 are expressed in cardiac myocytes. Finally, based on a lack of correlation with endothelin-1 tissue levels, it is unlikely that the failure-related changes in ETA and ETB receptor protein and mRNA expression result from homologous regulation by agonist exposure.
Introduction: Hypoplastic left heart syndrome (HLHS) is the term used to describe a group of congenital malformations characterized by marked underdevelopment of the left side of the heart. HLHS ...accounts for nearly 25% of cardiac deaths in the first year of life. Although much has been reported regarding diagnosis, gross morphology and surgical treatment, no information on gene expression in HLHS myocytes is available.
Methods: We examined heart tissue from patients with HLHS using routine histology, immunohistochemistry, quantitative polymerase chain reaction (PCR), two-dimensional (2-D) gel electrophoresis and protein identification by mass spectrometry.
Results: Histologic examination of right and left ventricles from HLHS patients revealed characteristic features of myocyte differentiation, including striations and intercalated disc formation. Immunohistochemical staining using antibody to
N-cadherin demonstrated clear development of intercalated discs between myocytes. However, many of the myocytes contained scant cytoplasm and were grouped in small, disorganized bundles separated by abundant connective tissue and dilated, thin-walled vessels. Quantitative PCR analysis demonstrated that both left and right ventricular tissue from HLHS hearts expressed the fetal or “heart failure” gene expression pattern. Two-dimensional gel electrophoresis and protein identification by mass spectrometry also confirmed that myocytes from HLHS ventricles were differentiated but expressed the fetal isoform of some cardiac specific proteins. However, HLHS myocytes in all of the heart samples (
n=21) were inappropriately expressing platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), a member of the cell adhesion molecule (CAM) family that has a primary role in the regulation of tissue morphogenesis. These findings indicate that myocytes from HLHS syndrome patients, while differentiated, have a unique gene expression pattern.
Background: Excessive lengthening of cardiac myocytes attributed to series addition of sarcomeres is a consistent feature of left ventricular dilation in chronic heart failure. Currently, it is not ...feasible to assess myocyte dimensions, particularly myocyte length, in a manner that is of potential diagnostic usefulness.
Methods and Results: Isolated myocytes from three groups of normal rats (100, 200, and 300 g) were obtained by using two different methods: (1) digestion of formalin-fixed myocardial tissue using potassium hydroxide (KOH) and (2) retrograde aortic perfusion of fresh hearts with collagenase. There was no difference in mean cell length between the two methods. The KOH method was also used to isolate intact, rod-shaped myocytes from formalin-fixed human cadaver left ventricles (control, n = 3; heart failure, n = 3) and from human right ventricle biopsy specimens (n = 6). Confirming our previous work using collagenase-isolated myocytes from fresh human explants, left ventricular myocytes from failing hearts showed longer mean cell length compared with control hearts. Data from human right ventricle biopsy specimens confirmed our previous finding in rats that myocyte lengthening is less pronounced in this chamber in heart failure.
Conclusions: The KOH method can be used to obtain reliable measurements of myocyte length and other cellular parameters from myocardial biopsies and autopsy material. Such data may be useful in the diagnostic assessment of remodeling associated with heart failure.
The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative ...strategies, including beta-adrenergic receptor (betaAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in betaAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the beta(2)AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the betaAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.
Plasma CK concentrations have been widely used as the primary muscle enzyme marker for diagnosis and progression of myositis. Recently, total CK and CK-MB serum concentrations have been compared to, ...and used in conjunction with, serum concentrations of aspartate aminotransferase in diagnosis of myositis. The algorithmic use of CK, AST, and aldolase plasma concentrations to diagnose and categorize patients with myopathy may be a useful method of diagnosing specific muscle disease without invasive procedures. CAIII, as a specific marker for skeletal muscle damage, may replace CK as the enzyme of choice in diagnosis and progression of myositis and other muscle disease. Additional studies are required to determine the usefulness of carbonic anhydrase for the diagnosis and assessment of myositis.
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