Myosin heavy chain gene expression in human heart failure.

Two isoforms of myosin heavy chain (MyHC), alpha and beta, exist in the mammalian ventricular myocardium, and their relative expression is correlated with the contractile velocity of cardiac muscle. Several pathologic stimuli can cause a shift in the MyHC composition of the rodent ventricle from alpha- to beta-MyHC. Given the potential physiological consequences of cardiac MyHC isoform shifts, we determined MyHC gene expression in human heart failure where cardiac contractility is impaired significantly. In this study, we quantitated the relative amounts of alpha- and beta-MyHC mRNA in the left ventricular free walls (LVs) of 14 heart donor candidates with no history of cardiovascular disease or structural cardiovascular abnormalities. This group consisted of seven patients with nonfailing (NF) hearts and seven patients with hearts that exhibited donor heart dysfunction (DHD). These were compared with 19 patients undergoing cardiac transplantation for chronic end-stage heart failure (F). The relative amounts of alpha-MyHC mRNA to total (i.e., alpha + beta) MyHC mRNA in the NF- and DHD-LVs were surprisingly high compared with previous reports (33.3+/-18.9 and 35.4+/-16.5%, respectively), and were significantly higher than those in the F-LVs, regardless of the cause of heart failure (2.2+/-3.5%, P < 0.0001). There was no significant difference in the ratios in NF- and DHD-LVs. Our results demonstrate that a considerable amount of alpha-MyHC mRNA is expressed in the normal heart, and is decreased significantly in chronic end-stage heart failure. If protein and enzymatic activity correlate with mRNA expression, this molecular alteration may be sufficient to explain systolic dysfunction in F-LVs, and therapeutics oriented towards increasing alpha-MyHC gene expression may be feasible.     

Augmented expression of atrial natriuretic polypeptide gene in ventricle of human failing heart.

To elucidate the expression of the atrial natriuretic polypeptide (ANP) gene in the ventricle of the human failing heart, we have measured ANP and ANP messenger RNA (ANPmRNA) levels in left ventricular aneurysm obtained at operation, biopsy specimens of left ventricles from dilated cardiomyopathy (DCM) and autopsy samples of old myocardial infarction (OMI) and DCM hearts, and compared the levels with those in the normal ventricle. The ANP level (mean +/- SE) was 17.5 +/- 6.9 ng/g in the normal ventricle, and increased to 660.3 +/- 122.2 ng/g in the left ventricular aneurysm tissues and to 3,138.6 +/- 1,642.1 ng/g in the biopsy specimens of the DCM ventricle. These levels were approximately 40 and 200 times higher than in the normal ventricle. The increase of ANP levels was observed in both infarcted and noninfarcted regions of the OMI heart, and in the entire ventricle of the DCM heart. A significant positive correlation was found between the ANP level in aneurysm tissues and pulmonary capillary wedge pressure (r = 0.85). The ANPmRNA level in the left ventricular aneurysm showed about a 10-fold increase compared with that in the normal heart and reached 23% of that in the atrium of the same heart. A similar increase in the ANPmRNA level was observed in the entire ventricle of DCM. These data clearly indicate that the expression of the ANP gene in the ventricle is augmented in the failing heart in accordance with the severity of heart failure. In the atrium of the failing heart, ANP and ANPmRNA levels were only two times higher than those in the normal atrium. Thus, the augmentation in the expression of the ANP gene was more prominent in the ventricle than in the atrium. Taking tissue weight into account, the total content of ANPmRNA in the ventricle of the failing heart is much the same as that in the normal atrium. The ratio of the ANP level to the ANPmRNA level in the ventricle is much smaller than that in the atrium. These results suggest more rapid secretion of ANP after synthesis in the ventricle. These findings demonstrate that the expression of the ANP gene is augmented in the human ventricle of the failing heart and suggest that the ventricle becomes a substantial source of circulating ANP in congestive heart failure.     

Electrophysiologic effects of ketanserin on canine Purkinje fibers, ventricular myocardium and the intact heart

We studied the actions of ketanserin (KT) on transmembrane action potentials (AP) of canine Purkinje fibers (PF) and ventricular muscle (VM) and on rhythm in vivo. PF AP duration (APD) was increased by KT (10(-8) to 10(-6) M) and shortened at 10(-5) M. KT effect on APD was greater during stimulation at longer cycle lengths and KT induced early afterdepolarizations in two of six PF at [K+]0 = 2.7 mM. Maximum diastolic potential, AP amplitude and Vmax were not changed by KT. In VM, KT (10(-8) to 10(-6) M) prolonged APD; but 10(-5) M KT did not shorten APD, reducing the difference in APD between VM and PF. KT had no effect on slow response Vmax or amplitude but prolonged APD. To analyze whether changes in Na plateau current or transient outward current contributed to KT effects on APD, we used tetrodotoxin (TTX) and 4-aminopyridine. TTX shortened APD and in its presence, KT (10(-5) M) induced no further shortening. In contrast, the effect of KT persisted in the presence of 4-aminopyridine. In six anesthetized, open chest dogs, KT prolonged the QT interval, but did not modify QRS duration and epicardial conduction time or induce arrhythmias. KT facilitated the onset of torsades de pointes during epicardial aconitine application.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased negative inotropic effect of calcium-channel blockers in hypertrophied and failing rabbit heart.

The effects on ventricular function of calcium channel blockers and isoproterenol were studied in isovolumically beating perfused control rabbit hearts and in hearts subjected to a double pressure plus volume overload studied at the early phase of heart failure. In control hearts, isoproterenol produced an increase of systolic ventricular function and relaxation that was maximal at 10(-7) M. In failing hearts, inotropic state increase in response to isoproterenol was significantly smaller (P less than .01) with no observed lusitropic effect. In control hearts, verapamil and diltiazem produced dose-dependent decreases of ventricular function which were larger with verapamil than with diltiazem (median drug concentration50 of developed pressure was, respectively, 1163 +/- 131 nM and 4524 +/- 451 nM, P less than .001). In failing hearts, contractility decrease was larger than in control hearts (median drug concentration50 of developed pressure was 604 +/- 69 nM and 2691 +/- 580 nM with verapamil and diltiazem, respectively). In contrast, Ro 40-5967, a new calcium-channel blocker, did not produce reductions of inotropic state with concentrations up to 10(-5) M. All three calcium-channel blockers produced a 2-fold increase of coronary flow at 10(-6) M. We conclude that the deleterious effect of verapamil and diltiazem in heart failure is due, at least in part, to a direct depressant effect of these drugs on contractility, which is larger than in control hearts. Additionally, the in vivo sympathetic compensation is probably reduced, as indicated by the decreased ventricular responsiveness to isoproterenol.     

Left ventricular stiffness and chamber geometry in the pressure-overloaded hypertrophied heart.

Pressure-overloaded hypertrophy of the left ventricle (LV) was produced by coarctation of the ascending aorta in 7 dogs. The overall mean weight of the left ventricle (LVW) was 7.86 +/- 1.49 (S.D.) g/kg body weight; (normal, 5.99 +/- 0.70 g/kg: p less than 0.05). After potassium arrest, pressure-volume (P-V) relationships were examined with the left ventricles isolated from the normals and from the dogs of left ventricular hypertrophy (LVH-dogs). In both groups, the P-V relationships could be expressed by an equation deltaV=a-be-cP throughout the range of filling pressure of 2.5 to 35 cmH2O, where deltav was the actual volume change of LV, P intraventricular pressure, and a, b and c constants. A sensitive index of LV stiffness, the half-inflation pressure (h), was defined as 1n (2b/a)/c. In hypertrophied hearts, h was 10.5 +/- 0.7 cmH2O; (normal 8.0 +/- 0.4 cmH2O; P less than 0.001). The ratio of LVW to LVVp=h (the left ventricular volume at h) in hypertrophy, which was related to the LV chamber geometry, was 3.1 +/- 0.6 in contrast with the normal value of 2.0 +/- 0.3. The development of concentric hypertrophy was thus demonstrated. Moreover, h was closely correlated with LVW/LVVp=h in both the normals and the LVH-dogs (r=0.83; p less than 0.01). On the other hand, an index of LV wall stiffness h/LVW/LVVP=h was relatively constant. Therefore, the increase of LV stiffness in the LVH-dogs was attributed to the change in chamber geometry.     

Immunoglobulin heavy chain gene expression in peripheral blood B lymphocytes.

cDNA libraries for IgM heavy chain variable regions were prepared from unmanipulated peripheral blood lymphocytes of two healthy people. Partial sequencing of 103 clones revealed VH gene family use and complete CDR3 and JH sequences. The libraries differed in the two subjects. In one person's cDNA the VH5 family was overexpressed and the VH3 family underexpressed relative to genomic complexity. In the second person's cDNA, VH3 was most frequently expressed. In both libraries, JH4 was most frequent. VH segments of several clones were closely related to those in fetal repertoires. However, there was also evidence of mutation in many cDNAs. Three clones differed from the single nonpolymorphic VH6 germline gene by 7-13 bases. Clones with several differences from VH5 germline gene VH251 were identified. CDR3 segments were highly diverse. JH portions of several CDR3's differed from germline JH sequences. 44% of the clones had DH genes related to the DLR and DXP families, most with differences from germline sequences. In 11 DLR2-related sequences, several base substitutions could not be accounted for by polymorphism. Thus, circulating IgM-producing B cell populations include selected clones, some of which are encoded by variable region gene segments that have mutated from the germline form.     

Positive inotropic effects in isolated ventricular myocardium from non-failing and terminally failing human hearts

The positive inotropic responses to isoprenaline, dobutamine, histamine, forskolin, isobutyl-methylxanthine (IBMX), dibutyryl-cyclic adenosine monophosphate (db-cAMP), ouabain and calcium were studied in isolated, electrically driven papillary muscle strips from either terminally failing human hearts or non-failing donor myocardium. The positive inotropic effect of calcium has been taken to evaluate the maximal force of contraction of each individual muscle strip ('contractile reserve'). In the non-failing heart, the maximal positive inotropic effect of isoprenaline, dobutamine, IBMX, ouabain and calcium were not significantly different, but were significantly greater than histamine. In terminally failing hearts, the positive inotropic effects of agents stimulating the adenylate cyclase by a receptor-dependent mechanism (isoprenaline, dobutamine and histamine) and the phosphodiesterase inhibitor IBMX are less than in the normal heart. Furthermore, these compounds gave a markedly reduced inotropic effect compared with forskolin, db-cAMP and ouabain, which gave maximal responses similar to calcium in the failing hearts. The data did not differ when the increase of force of contraction was related to the diameter of each preparation. These results indicate that a defect in adenylate cyclase occurs in the failing human heart, presumably located at the regulatory stimulatory subunit (Gs) of the adenylate cyclase since effects through stimulatory receptors were reduced. Responses from activation of the catalytic subunit or through cAMP-dependent protein kinases were less affected. Since the positive inotropic effect of IBMX is also impaired, it is suggested that the basal rate of cAMP production is also reduced in heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient liposome-mediated gene transfection and expression in the intact human uterus.

Although gene therapy has been used for correction of metabolic defects in diseases such as cystic fibrosis, as adjuvant treatment in cancer, and in the treatment of infectious diseases, there has been no report of gene transfer to the intact female reproductive tract. We assessed the ability to transfect the human uterus ex vivo and thereby evaluate the applicability of gene therapy to gynecology. The uterine lumen was accessed transcervically, using an intrauterine insemination catheter. pcDNA3.1 plasmid containing the Escherichia coli lacZ reporter gene was delivered to each uterus via liposome-mediated transfection. Control uteri were transfected with empty pcDNA3.1. Immunohistochemical analysis revealed beta-galactosidase expression in the lacZ-treated uteri in endometrial epithelial cells, endometrial stromal cells, and myometrium to a depth of 1.75 cm from the endometrial-myometrial junction. Highest expression was seen in endometrial glandular epithelial cells, with significant expression in the stroma and adjacent myometrium. Each of these cell types in the control uteri showed no beta-galactosidase expression. Successful gene transfection and expression in the intact human uterus can be accomplished easily, rapidly, and efficiently. Gene therapy may have wide applicability in the treatment and study of gynecologic disease.     

Myostatin expression in ventricular myocardium in a rat model of volume-overload heart failure

BACKGROUND: Mechanical stress increases myocardial myostatin expression. However, the expression of myostatin in chronic heart failure resulting from volume-overload and after treatment with beta-blockers is little known. The authors hypothesize that myostatin plays a role in the failing myocardium because of volume-overload. MATERIALS AND METHODS: Aorto-caval shunt was created over a 4-week period in adult Sprague-Dawley rats to induce volume-overload heart failure. RESULTS: Heart weight and body weight ratio significantly increased after shunting. The left ventricular end-diastolic dimension also significantly increased. Treatment with carvedilol in the shunt group reversed the increase in heart weight and ventricular dimension to the baseline values. Myocardial and skeletal myostatin proteins were up-regulated in the shunt group. The mRNA of myocardial myostatin also increased in the shunt group. Treatment with carvedilol reversed both protein and mRNA of myocardial myostatin to the baseline values. Treatment with N-acetylcysteine and doxazosin partially decreased myostatin mRNA and protein expression as compared with the shunt group. Carvedilol normalized the increased immunohistochemical labelling of myocardial myostatin in the shunt group. CONCLUSION: Myocardial myostatin mRNA and protein expression were up-regulated in the rat model of volume-overload heart failure. Treatment with carvedilol is associated with a limitation of increased myostatin expression in the failing ventricular myocardium.     

Histometrical estimation of scar tissue in hypertrophied human heart muscle.

Histometrical estimation of scar tissue was made on 28 hypertrophied human hearts obtained at autopsy in order to know the significance of scar tissue in the process of cardiac hypertrophy and in the development of cardiac failure. Estimation was made on histological specimens of the anterior wall of the left ventricle and the posteromedial papillary muscle, according to Chalkley's point counting method, and the amount of scar tissue was expressed by percentages. The mean percentages were 4.6, 8.5 and 18.3 in the epicardial, endocardial part of the left ventricle, and the papillary muscle. There were very significant differences between these 3 mean percentages. There was a very significant correlation between the percentages of scar tissue in the endocardial part of the left ventricle and heart weight. There were no definite correlations between the percentages of scar tissue in the epicardial part or in the papillary muscle and heart weight. From these results, possible causes of scar formation and significance of scar tissue to the development of cardiac failure were discussed.     

Transcription, storage and release of atrial natriuretic factor in the failing human heart.

1. In this study the relationship between the synthesis of atrial natriuretic factor at the level of atrial natriuretic factor mRNA and the atrial storage and circulating plasma levels of atrial natriuretic factor were investigated in 15 patients with heart failure. The patients underwent right and left heart catheterization before cardiac surgery for valve replacement or coronary artery bypass grafting. 2. Plasma concentrations of atrial natriuretic factor were correlated to atrial levels of atrial natriuretic factor mRNA. Atrial levels of atrial natriuretic factor mRNA and plasma concentrations of atrial natriuretic factor exhibited a close correlation to both pulmonary artery pressure and left atrial pressure. No relationship, however, could be found between the right atrial content of atrial natriuretic factor and both the expression of atrial natriuretic factor mRNA in the atria and the plasma levels of atrial natriuretic factor. 3. From these data it may be concluded that increased plasma levels of atrial natriuretic factor in the pressure- and/or volume-overloaded heart are associated with an elevated level of atrial natriuretic factor mRNA. We suggest that not only plasma levels of atrial natriuretic factor but also the expression of atrial natriuretic factor in the atrial are related to left ventricular filling pressures in the failing human heart.     

Adenylylcyclase gene transfer increases function of the failing heart

A persistent question in cardiovascular gene transfer concerns whether an exogenously delivered gene can increase function of the failing heart. Here we test the hypothesis that intracoronary delivery of adenovirus encoding adenylylcyclase type VI (Ad.ACVI) in the setting of active heart failure will increase function of the failing heart. As a model of heart failure, we used transgenic mice with dilated and poorly functioning hearts resulting from cardiac-directed expression of Galphaq.Galphaq mice with equivalent pretreatment impairment in left ventricular (LV) function (echocardiography) received 2.5x1010 viral particles of Ad.ACVI or Ad.EGFP (enhanced green fluorescent protein), or saline, by indirect intracoronary delivery. Serial echocardiograms obtained before and 14 days after gene transfer showed that Ad.ACVI increased LV ejection fraction (p<0.01) and velocity of circumferential fiber shortening (p<0.03). Detailed measurements in isolated hearts showed that ACVI gene transfer increased LV positive dP/dt (p=0.02) and LV negative dP/dt (p=0.01). Gene transfer was confirmed by polymerase chain reaction. These data show that, in an animal model that mimics key aspects of clinical congestive heart failure, cardiac gene transfer of ACVI increases function of the failing heart.     

Mild metabolic acidosis impairs the beta-adrenergic response in isolated human failing myocardium

ABSTRACT: INTRODUCTION: Pronounced extracellular acidosis reduces both cardiac contractility and the beta-adrenergic response. In the past this was shown in some studies using animal models. However, only few data exist regarding how the human end-stage failing myocardium, in which compensatory mechanisms are exhausted, reacts to acute mild metabolic acidosis. The aim of this study was to investigate the effect of mild metabolic acidosis on contractility and betaadrenergic response of isolated trabeculae from human end-stage failing hearts. METHODS: Intact isometrically twitching trabeculae isolated from patients with end-stage heart failure were exposed to mild metabolic acidosis (pH=7.20). Trabeculae were stimulated at increasing frequencies and finally exposed to increasing concentrations of isoproterenol (0 to 1*10-6 mol/L). RESULTS: A mild metabolic acidosis caused a depression in twitch force amplitude of 26% (12.1+/-1.9 to 9.0+/-1.5 mN/mm2, n=12, P<0.01) as compared to pH 7.40. Force-frequency relation measurements yielded no further significant differences of twitch force. At the maximal isoproterenol concentration, the force amplitude was comparable in each of the two groups (pH 7.40 vs. pH 7.20). However, the half maximal effective concentration (EC50) was significantly increased in the acidosis group, with an EC50 of 5.834x10-8 mol/L (confidence interval [CI] 3.48x10-8 - 9.779x10-8, n=9), compared with the control group, which had an EC50 of 1.056x10-8 mol/L (CI 2.626x10-9 - 4.243x10-8, n=10, P<0.05), indicating an impaired beta-adrenergic force response. CONCLUSIONS: Our data show that mild metabolic acidosis reduces cardiac contractility and significantly impairs the beta-adrenergic force response in human failing myocardium. Thus, our results could contribute to the still controversial discussion about the therapy regimen of acidosis in patients with critical heart failure.     

Reduced beta 1 receptor messenger RNA abundance in the failing human heart.

Heart failure in humans is characterized by alterations in myocardial adrenergic signal transduction, the most prominent of which is down-regulation of beta 1-adrenergic receptors. We tested the hypothesis that down-regulation of beta 1-adrenergic receptors in the failing human heart is related to decreased steady-state levels of beta 1 receptor mRNA. Due to the extremely low abundance of beta 1 receptor mRNA, measurements were possible only by quantitative polymerase chain reaction (QPCR) or by RNase protection methods. Because the beta 1 receptor gene is intronless and beta 1 receptor mRNA abundance is low, QPCR yielded genomic amplification in total RNA, and mRNA measurements had to be performed in poly (A)(+)-enriched RNA. By QPCR the concentration of beta 1 receptor mRNA varied from 0.34 to 7.8 x 10(7) molecules/microgram poly(A)(+)-enriched RNA, and the assay was sensitive to 16.7 zeptomol. Using 100-mg aliquots of left ventricular myocardium obtained from organ donors (nonfailing ventricles, n = 12) or heart transplant recipients (failing ventricles, n = 13), the respective beta 1 mRNA levels measured by QPCR were 4.2 +/- 0.7 x 10(7)/micrograms vs. 2.10 +/- 0.3 x 10(7)/micrograms (P = 0.006). In these same nonfailing and failing left ventricles the respective beta 1-adrenergic receptor densities were 67.9 +/- 6.9 fmol/mg vs. 29.6 +/- 3.5 fmol/mg (P = 0.0001). Decreased mRNA abundance in the failing ventricles was confirmed by RNase protection assays in total RNA, which also demonstrated a 50% reduction in beta 1 message abundance. We conclude that down-regulation of beta 1 receptor mRNA contributes to down-regulation of beta 1 adrenergic receptors in the failing human heart.     

Noncompaction of the ventricular myocardium.

Noncompaction of the ventricular myocardium is a rare congenital cardiomyopathy resulting from an arrest in normal endomyocardial embryogenesis. The characteristic echocardiographic findings consist of multiple, prominent myocardial trabeculations and deep intertrabecular recesses communicating with the left ventricular cavity. The disease uniformly affects the left ventricle, with or without concomitant right ventricular involvement, and results in systolic and diastolic ventricular dysfunction and clinical heart failure. Noncompaction was initially described in children. However, recent studies have characterized this disease in the adult population, in whom this process may be more prevalent than currently appreciated. We describe an illustrative case of isolated noncompaction of the ventricular myocardium in a 57-year-old woman with the typical clinical and echocardiographic features of the disease. The literature on the topic is reviewed.