L. Dalla Libera

Apoptosis in the skeletal muscle of patients with heart failure: investigation of clinical and biochemical changes

OBJECTIVE To investigate the contribution of apoptosis in the development of the skeletal myopathy in chronic heart failure. DESIGN The electrophoretic pattern of myosin heavy chains (MHC), fibre cross sectional area, number of in situ nick end labelling (TUNEL) positive apoptotic myocyte nuclei, and the tissue levels of caspase-3, Bcl-2, and ubiquitin were determined in biopsies taken from the vastus lateralis muscle. The study involved nine patients with severe chronic heart failure caused by ischaemic heart disease and hibernating myocardium and five controls. RESULTS In chronic heart failure patients the vastus lateralis showed a significant increase of MHC2a and MHC2b and a greater degree of fibre atrophy, as demonstrated by the decreased cross sectional area. There was also an increased number of TUNEL positive apoptotic myocyte nuclei. Tissue concentrations of Bcl-2 were decreased, while those of caspase-3 and ubiquitin were increased. Peak oxygen consumption (Vo 2) was negatively correlated with the number of TUNEL positive nuclei and the fibre cross sectional area. There was a correlation between the number of apoptotic nuclei and the fibre cross sectional area, but no correlation between myosin heavy chains and number of apoptotic nuclei. CONCLUSIONS Myocyte apoptosis occurs in the skeletal muscle of patients with chronic heart failure, and its magnitude is associated with the severity of exercise capacity limitation and the degree of muscle atrophy. Muscle atrophy contributes to the limitation of exercise capacity, together with the increased synthesis of fast, more fatiguable myosin heavy chains.

Specific changes in skeletal muscle myosin heavy chain composition in cardiac failure: differences compared with disuse atrophy as assessed on microbiopsies by high resolution electrophoresis.

OBJECTIVE: In congestive heart failure (CHF) the skeletal muscle of the lower limbs develops a myopathy with atrophy and shift from the slow type to the fast type fibres. The aim was to test the hypothesis that this myopathy is specific and not simply related to detraining, by comparing patients with different degrees of CHF with patients with severe muscle atrophy due to disuse. DESIGN: Case-control study involving 50-150 micrograms needle biopsies of the gastrocnemius muscle. By an electrophoretic micromethod, the three isoforms of myosin heavy chains (MHC) were separated. PATIENTS: Five patients restricted to bed for more than one year because of stroke with disuse atrophy and normal ventricular function, and 19 with CHF were studied. There were seven age matched controls. MAIN OUTCOME MEASURES: The percentage of MHC1 (slow isoform), MHC2a (fast oxidative), and MHC2b (fast glycolytic) was determined by densitometric scan and correlated with indices of severity of cardiac failure. RESULTS: Ejection fraction was 42.5 (SD 15.2)% in CHF, 59.5 (1.0)% in disuse atrophy and 60.3 (1.4)% in controls (P < 0.001 v both). The degree of muscle atrophy as calculated by the body mass index/gastrocnemius cross sectional area, showed a profound degree of atrophy in patients with muscle disuse [0.94 (0.39)]. This was worse than in the controls [4.27 (0.16), P < 0.0005] and the CHF patients [2.60 (1.10), P < 0.005]. Atrophy in CHF patients was also greater than in controls (P < 0.005). MHC1 was lower in CHF than in disuse atrophy [51.83 (15.04) v 84.5 (17.04), P < 0.01] while MHC2b was higher [23.5 (7.4) v 7.25 (7.92), P < 0.001]. There was a similar trend for MHC2a [24.83 (15.01) v 8.25 (9.12), P < 0.05]. Within the CHF group there was a positive correlation between NYHA class and MHC2a (r = 0.47, P < 0.05) and MHC2b (r = 0.55, P < 0.01) and a negative correlation between NYHA class and MHC1 (r = -0.74, P < 0.001). Similarly, significant correlations were found for ejection fraction, diuretic consumption score, exercise test tolerance, and degree of muscle atrophy. CONCLUSIONS: The CHF myopathy appears to be specific and not related to detraining. The magnitude of MCH redistribution correlates with the severity of the disease. The electrophoretic micromethod used is very sensitive and reproducible. Biopsies are so well tolerated that can be repeated frequently, allowing thorough follow up.

Cardiac autoantibodies in dilated cardiomyopathy become undetectable with disease progression.

OBJECTIVE: To determine the relation of cardiac autoantibody and disease status in a consecutive series of patients with dilated cardiomyopathy by prospective antibody testing at diagnosis and at follow up. METHODS: Antibody status was assessed by indirect immunofluorescence in 110 patients with dilated cardiomyopathy (85 male, mean (SD) age 44 (13) years) at diagnosis and at follow up (mean (SD) 14 (12) months); in 57 of them cardiac specific anti-alpha myosin antibody titres were also measured by an enzyme-linked immunosorbent assay (ELISA). Patients underwent complete evaluation at diagnosis and clinical and non-invasive assessment at follow up, including exercise testing with maximal oxygen consumption measurements. RESULTS: The frequency of cardiac specific antibodies by immunofluorescence was lower at follow up than at diagnosis (28 (25%) v 11 (10%), P = 0.002). Mean (SEM) anti-alpha myosin antibody titres at follow up were also lower than at diagnosis (0.24 (0.02) v 0.30 (0.02), P = 0.038); 24% of patients at diagnosis and 14% at follow up had an abnormal ELISA result. None of the patients who were negative by immunofluorescence or ELISA at diagnosis became positive at follow up. Presence of antibody at diagnosis was associated with milder symptoms and greater exercise capacity at follow up and persistence of antibody at follow up was associated with stable disease and milder symptoms at diagnosis. CONCLUSIONS: Cardiac specific autoantibodies in dilated cardiomyopathy become undetectable with disease progression; this is a recognised feature of other autoimmune conditions, such as type 1 diabetes. Detection of these antibodies at diagnosis and at follow up may provide a non-invasive marker of early dilated cardiomyopathy.

Autoimmunity to alpha myosin in a subset of patients with idiopathic dilated cardiomyopathy.

OBJECTIVE--To use an enzyme linked immunoassay (ELISA) technique to assess frequency and disease specificity of anti-alpha-myosin antibodies in patients with dilated cardiomyopathy and their relatives. METHODS--Evaluation was performed on sera (dilution 1/320) from 123 consecutive patients with dilated cardiomyopathy (WHO criteria) (age 42 (SD 14) years), 252 of their relatives (35 (17) years), 203 healthy controls (45 (16) years), and 92 patients with ischaemic heart disease (63 (11) years). RESULTS--Abnormal antibody levels were commoner in patients with dilated cardiomyopathy (25, 20%) than in ischaemic heart disease (4, 4%), or normal controls (4, 2%, P = 0.001). Forty one (16%) of the relatives had abnormal results compared to the controls (4, 2%, P < 0.001) and antibodies were detected in 20 (38%) of pedigrees. Relatives from non-familial kindreds had higher antibody levels than those with familial disease (P << 0.001), and higher antibody levels were identified in 53 relatives of probands who had abnormal results compared to 116 relatives for whom the proband had a normal result (0.37 (SEM 0.02) v 0.22 (0.01); P < 0.001). CONCLUSIONS--The finding of anti-alpha-myosin antibodies in 20% of patients with dilated cardiomyopathy, in 16% of their asymptomatic relatives, and in 38% of families (particularly those with non-familial disease and where proband also had an abnormal result) provides additional evidence for autoimmunity against alpha myosin in a subset of patients.

Comparative analysis of chicken atrial and ventricular myosins

1. Structural and enzymic properties of myosins from atrial and ventricular cardiac muscle of the chicken were investigated and compared with myosins from the fast skeletal pectoralis and the slow skeletal anterior latissimus dorsi muscle. 2. The Ca2+-ATPase activity, both in function of pH and [K+], of atrial myosin closely resembled that of the fast pectoralis myosin, whereas the enzymic properties of ventricular myosin were similar to those of slow skeletal myosin. 3. By sodium dodecyl sulphate polyacrylamide gel electrophoresis on gradient gel and two-dimensional electrophoresis, involving isoelectric focusing in the first dimension and SDS gel electrophoresis in the second dimension, no difference could be demonstrated in the light-chain pattern of atrial and ventricular myosin. Complete identity was also found between anterior latissimus dorsi and cardiac light chains. 4. Electrophoretic analysis of soluble peptides released by tryptic digestion of myosin and electron microscopic study of light meromyosin paracrystals showed significant differences between the heavy chains of atrial and ventricular myosins, as well as between the heavy chains of cardiac and skeletal myosins. 5. The results confirm previous immunochemical findings and provide direct biochemical evidence for the existence of a new, unique type of myosin in the chicken atrial tissue.

Fractionation of rabbit ventricular myosins by affinity chromatography with insolubilized antimyosin antibodies

Immunofluorescence studies with specific antimyosin antibodies have demonstrated myosin polymorphism in avian [l] and mammalian heart (in preparation). In addition to the existence of distinct types of myosin in atria1 and ventricular tissue, myosin heterogeneity has been established in muscle cells of both tissues. For instance, when fluorescein-labelled anti-beef atria1 myosin antibodies were applied to sections of the rabbit heart, ventricular muscle cells with varying degree of reactivity were seen, suggesting the presence of multiple forms of ventricular myosin. Electrophoretic evidence for the existence of different myosin isoenzymes in the rat ventricles has been reported [2,3]. We show here that an immunoadsorbent made of specific antibodies against beef atrial myosin permits the fractionation of two myosin populations from rabbit ventricles with the same light chain composition but different heavy chain structure.

Ventricular myosin and creatine-kinase isoenzymes in hypertensive rats treated with captopril.

In the myocardium, myosin and creatine kinase isoforms possess different capacities for using O2 and energy-rich phosphates. We studied electrophoretically the distribution of these isoforms in 19 hypertensive rats (two-kidney, one clip model of hypertension) and in age-matched controls. After 6 weeks of hypertension, seven rats were treated with captopril (2 mg/kg daily) for 4 weeks, six were left hypertensive for another 4 weeks, and the remaining rats were killed under ether anesthesia. In the latter, ventricular mass was significantly higher than in controls; V3 isomyosin was 32.3 ±6.8% versus 0%, and both creatine kinase-MB and -BB were increased at the expense of creatine kinase-MM (creatine kinase-MB=29±2.8% vs. 14.7±1.8%, p < 0.001; creatine kinase-BB=3.1±0.6% vs. 1.7±0.8%, p < 0.001). After 10 weeks of hypertension, ventricular mass, V3 isomyosin, and both creatine kinase-MB and -BB isoforms were found to be persistently higher than in controls. At the same time, captopril-treated rats showed reduced but not normalized blood pressure levels, normalized ventricular mass, and prevalence of the V1 isomyosin (56.9±22% vs. 47.9±23.8% in normotensive controls, p < =NS). However, higher levels of creatine kinase-MB and -BB were still found in these rats in comparison with the normotensive controls (creatine kinase-MB=22.4±5.4% vs. 15.8±2.8%, p < 0.025; creatine kinase-BB=2.3±0.1% vs. 1.8±0.3%, p < 0.02). Therefore, despite the normalization in ventricular mass and isomyosin pattern, captopril-treated rats partly maintain the adaptive changes in creatine kinase isoenzymes that lead to a better use of energy-rich phosphates.

Changes in rat ventricular isomyosins with regression of cardiac hypertrophy.

The changes in ventricular isomyosin composition and Ca2+-activated ATPase activity occurring with regression of both hypertension and cardiac hypertrophy were investigated by using polyacrylamide gel electrophoresis under nondenaturing conditions, heavy chain peptide mapping, and an enzymatic assay. Eight control male Wistar rats and 14 two-kidney, one clip (Goldblatt II) hypertensive rats were studied from the fifth week of age. At 10 weeks of age, five Goldblatt II rats and four normotensive controls were killed. Five other Goldblatt II rats underwent nephrectomy of the ischemic kidney, which resulted in subsequent normalization of blood pressure. The remaining four control, four Goldblatt II rats, and five nephrectomized rats were killed at 15 weeks of age. Both the 10- and 15-week-old hypertensive rats had a significantly higher (p less than 0.001) biventricular weight to body weight ratio than the age-matched controls (3.84 +/- 0.76 X 10(-2) vs 2.75 +/- 0.25 X 10(-2); 5.93 +/- 2.26 X 10(-2) vs 2.65 +/- 0.17 X 10(-2]. The 15-week-old nephrectomized rats had a biventricular weight to body weight ratio (2.90 +/- 0.25 X 10(-2] close to that of age-matched controls and significantly lower (p less than 0.05) than that of age-matched hypertensive rats. In both the 10- and 15-week-old hypertensive rats left ventricular myosin Ca2+-activated ATPase activity was significantly lower (p less than 0.001) than in the age-matched controls (0.44 +/- 0.03 vs 0.59 +/- 0.06; 0.24 +/- 0.05 vs 0.48 +/- 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Light and heavy chains of myosin from atrial and ventricular myocardium of turkey and rat

SummarySubunit composition of myosins from atrial and ventricular cardiac muscle of turkey and rat was examined by two-dimensional polyacrylamide electrophoresis of light chains and by peptide mapping of electrophoretically purified heavy chains.In regard to light subunits in two-dimensional electrophoresis, the corresponding light chains from atrial and ventricular myocardium comigrate in the turkey. By contrast the atrial light chains LC1 and LC2 are easily distinguishable from the corresponding subunits of the ventricles in the rat.The peptide patterns of myosin heavy chains from atrial and ventricular cells were different both in turkey and rat, indicating differences in their primary structures. Therefore atrial and ventricular myosins differ in heavy chains composition in all the avian and mammalian species so far studied, suggesting that this heterogeneity represents a general feature related to the different physiological properties of atrial and ventricular cardiac cells.

Comparison between isomyosin pattern and contractility of right ventricular myocytes isolated from rats with right cardiac hypertrophy.

SummaryThe contractile properties of single rat cardiac cells isolated from normal and hypertrophied right ventricles have been investigated. These have been correlated with the isoenzyme composition of the whole ventricle. Right cardiac hypertrophy was induced by injecting rats with monocrotaline, an alkaloid which induces severe pulmonary hypertension. Ca2+ ATPase activity and myosin alpha-chain percentage were decreased in the hypertrophied right ventricle as compared with that of control rats. The contraction amplitude and speed of shortening of the isolated cells were measured using an inverted microscope, video camera, and edge detection device. Cells from the hypertrophied ventricle showed a significantly decreased contraction amplitude and speed of shortening in maximally activating concentrations of isoprenaline. A statistically significant correlation existed between myosin alpha-chain percentage and both contraction amplitude and speed of shortening in maximum isoprenaline. This was truc when all cells studied were included, as well as within the hypertrophy group. A similar, although not always statistically significant, correlation was observed when cells were maximally activated with calcium. These results suggest that changes in isomyosin pattern that occur in cardiac hypertrophy produce alterations in contraction amplitude and speed of shortening which can be detected in single cells isolated from the hypertrophied ventricles. Isolated cells appear to give responses representative of the function of the whole heart.