High-fat feeding stimulates endocrine, glucose-dependent insulinotropic polypeptide (GIP)-expressing cell hyperplasia in the duodenum of Wistar rats.

AIMS/HYPOTHESIS: Incretins are hormones released by enteroendocrine cells in response to meals, depending upon absorption of nutrients. The present study aimed to elucidate the mechanisms through which a high-fat diet (HFD) induces insulin resistance and insulin hypersecretion by focusing on the effects on enteroendocrine cells, especially those secreting glucose-dependent insulinotropic polypeptide (GIP). METHODS: Forty male Wistar rats, 4 months old, were randomised into two groups; one group received a chow diet and the other one received a purified tripalmitin-based HFD ad libitum. An OGTT was performed every 10 days and histological and immunofluorescence evaluations of the duodenum were obtained at 60 days from the beginning of the diets. Plasma glucose, insulin, GIP and glucagon-like peptide-1 (GLP-1) levels were measured. Immunofluorescence analysis of duodenal sections for pancreatic duodenal homeobox-1 (PDX-1), KI67, GLP-1, GIP and insulin were performed. RESULTS: Compared with chow diet, HFD induced a progressive significant increase of the glucose, insulin and GIP responses to OGTT, whereas GLP-1 circulating levels were reduced over time. After 60 days of HFD, cellular agglomerates of KI67 and PDX-1 positive cells, negative for insulin and GLP-1 but positive for GIP staining, were found inside the duodenal mucosa, and apoptosis was significantly increased. CONCLUSIONS/INTERPRETATION: With the limitation that we could not establish a causal relationship between events, our study shows that HFD stimulates duodenal proliferation of endocrine cells differentiating towards K cells and oversecreting GIP. The progressive increment of GIP levels might represent the stimulus for insulin hypersecretion and insulin resistance.

High saturated-fat diet induces apoptosis in rat enterocytes and blunts GIP and insulin-secretive response to oral glucose load.

Lipoapoptosis has been described in many organs and tissues, but never in enterocytes. We hypothesized that a high saturated-fat diet can induce duodenal enterocyte apoptosis and impair gastric inhibitory polypeptide (GIP) secretion. Forty male Wistar rats, approximately 4 months old, were randomized on standard laboratory or purified tripalmitin-based high-fat diet (59% calories). An oral-glucose tolerance test was performed after 30 and 90 days of diet to measure plasma glucose, insulin and GIP. Duodena were processed for histology and immunohistochemistry by transferase-mediated dUTP nick end-labeling (TUNEL) method. Apoptosis was confirmed by enzyme-linked immunosorbent assay. Glycemic response was significantly higher (P < 0.01 vs controls) in rats after 90 days. Insulin curve was markedly increased at 30 days, while it was blunted at 90 days. GIP area under the curve was 425.6 +/- 67.6 ng ml(-1) at 30 days vs 150.2 +/- 33.4 ng ml(-1) in controls (P < 0.001) and dropped to 53.8 +/- 25.8 ng ml(-1) at 90 days (P < 0.0001). TUNEL-positive nuclei were 66.08+/-26.19 at 30 days 57 (34.58+/-17 in controls, P < 0.05) and 216.99 +/- 129.42 nuclei per mm(3) at 90 days (38.75 +/- 18.36 in controls, P < 0.0001). A high saturated-fat diet stimulates GIP secretion but with time induces apoptosis of duodenal villi epithelium, showing for the first time that enterocytes are also prone to lipoapoptosis. The reduction of circulating GIP levels might contribute to hypoinsulinemia and hyperglycemia.

Apoptosis in the skeletal muscle of rats with heart failure is associated with increased serum levels of TNF-alpha and sphingosine.

Skeletal muscle in congestive heart failure (CHF) is responsible for increased fatigability, decreased endurance and exercise capacity. A specific myopathy with increased expression of fast myosin heavy chains (MHCs), myocyte atrophy, secondary to myocyte apoptosis, that is triggered by high levels of circulating tumor necrosis factor (TNF-alpha) has been described. However, a direct effect of TNF-alpha on skeletal muscle has not been described yet. In this paper we put forward the hypothesis that TNF-alpha plays an indirect effect on skeletal myocytes. In an animal model of CHF, the monocrotaline-treated rat, we have observed a significant (P<0.001) increase of circulating TNF-alpha that is paralleled by increased serum levels of the endogenous second messenger, sphingosine (SPH), (from 0.71+/-0.15 to 1.32+/-0.39 nmoles/ml, P<0.01). In the tibialis anterior (TA) muscle we found a marked increase of myocyte apoptosis (from 1.4+/-2.4 to 40.1+/-39.5 nuclei/mm(3), P<0.04). We correlated plasma levels of TNF-alpha with those of SPH and in turn with the magnitude of apoptosis. Linear regression showed a significant correlation between TNF-alpha, SPH, and apoptosis (r(2)=0.74, P=0.004 and r(2)=0.87, P=0.001 respectively). Analysis of covariance showed that TNF-alpha and SPH were independently correlated with the number of apoptotic nuclei (P=0.0001). In parallel in vitro experiments, where increasing concentrations of SPH were applied to skeletal muscle cells in culture, we observed a dose-dependent increase in apoptosis. These results suggest that TNF-alpha-induced SPH production may be responsible for skeletal muscle apoptosis. The link between TNF-alpha and skeletal muscle apoptosis could be represented by the second messenger SPH, which can directly induce apoptosis in these cells.

Apoptosis and changes in contractile protein pattern in the skeletal muscle in heart failure.

Chronic heart failure is characterized as a clinical disorder by exercise intolerance. There are two factors that are independently responsible for the reduced exercise capacity: (a) a shift from myosin heavy chain 1 (MHC1) to MHC2a and MHC2b and (b) muscle atrophy. We have demonstrated, both in experimental models of heart failure and in man, that the more severe the heart failure, the greater the magnitude of skeletal muscle apoptosis. In the monocrotaline treated rat, that develops a severe right-sided heart failure, the increased number of apoptotic nuclei was paralleled by increasing levels of circulating TNFalpha. In agreement with some recent observations showing that sphingolipids can mediate programmed cell death, we found that in animals with heart failure and high number of apoptotic nuclei, circulating levels of sphingosine were significantly increased. In a study conducted in patients with heart failure we found a correlation between exercise capacity limitation and skeletal myocytes apoptosis. There was also a correlation between degree of muscle atrophy and magnitude of apoptosis. The shift in MHCs, although with a different mechanism, is also responsible for the reduced exercise capacity in these patients. In fact there is a strong correlation between indices of severity of CHF and MHC composition. Muscle fatigue, appears earlier in patients that have a greater skeletal muscle expression of 'fast' MHCs. We have also demonstrated that MHCs shift and apoptosis can be prevented by using angiotensin II converting enzyme inhibitors and angiotensin II receptor blockers.

Beneficial effects on skeletal muscle of the angiotensin II type 1 receptor blocker irbesartan in experimental heart failure.

BACKGROUND: In congestive heart failure (CHF), skeletal muscle shows increased expression of fast myosin heavy chains (MHC) and fibers, muscle atrophy, increased fatigability, and decreased endurance. Atrophy is secondary to myocyte apoptosis, which is probably triggered by tumor necrosis factor-alpha (TNFalpha). Angiotensin II receptors are thought to play a role in controlling apoptosis. We tested the hypothesis that angiotensin II receptor blockade could prevent skeletal muscle apoptosis in rats with CHF. METHODS AND RESULTS: CHF was induced by injecting 36 rats with 30 mg/kg monocrotaline. Ten additional animals were injected with saline and acted as controls. After 2 weeks, 18 of the 36 rats with CHF were treated with 7 mg. kg(-1). d(-1) irbesartan through osmotic minipumps, and 10 of the 36 rats were treated with 2 mg. kg(-1). d(-1) nifedipine in drinking water. After 2 additional weeks, rats were killed. Tibialis anterior cross-sectional area, MHC composition, myocyte apoptosis, Bcl-2, pro-caspase 3, and activated caspases 3 and 9 were determined, as were plasma levels of TNFalpha and angiotensin II. Myocyte apoptosis and muscle atrophy were significantly decreased with irbesartan compared with untreated CHF rats. Irbesartan-treated rats had fewer cells labeled positively with terminal deoxynucleotidal transferase-mediated dUTP nick-end labeling and fewer caspases; however, they also had increased Bcl-2 levels and muscle fiber cross-sectional areas. The MHC pattern in irbesartan-treated animals was similar to that in controls. Nifedipine animals behaved like the untreated CHF animals. Angiotensin II was increased 3- to 4-fold in all CHF rats (treated and untreated). TNFalpha levels were decreased in irbesartan-treated rats but not in nifedipine-treated rats. CONCLUSIONS: Angiotensin II receptor blockade can protect from the development of apoptosis-dependent atrophy and from changes in MHCS: The reduction of TNFalpha may play a role in this process.

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 MHC(2a) and MHC(2b) 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.

Improved exercise tolerance after losartan and enalapril in heart failure: correlation with changes in skeletal muscle myosin heavy chain composition.

BACKGROUND: In congestive heart failure, fatigue-resistant, oxidative, slow type I fibers are decreased in leg skeletal muscle, contributing to exercise capacity (EC) limitation. The mechanisms by which ACE inhibitors and AII antagonists improve EC is still unclear. We tested the hypothesis that improvement in EC is related to changes in skeletal muscle composition toward type I fibers. METHODS AND RESULTS: Eight patients with congestive heart failure, NYHA classes I through IV, were treated for 6 months with enalapril (E) 20 mg/d, and another 8 with losartan (L) 50 mg/d. EC was assessed with maximal cardiopulmonary exercise testing at baseline and after treatment. Myosin heavy chain (MHC) composition of the gastrocnemius was studied after electrophoretic separation of slow MHC1, fast oxidative MHC2a, and fast glycolytic MHC2b isoforms from needle microbiopsies obtained at baseline and after 6 months. EC improved in both groups. Peak V(O2) increased from 21.0+/-4.7 to 27.6+/-4.3 mL . kg-1 . min -1 (P=0.011) in the L group and from 17.5+/-5.0 to 25.0+/-5.5 mL . kg-1 . min -1 (P=0.014) in the E group. Similarly, ventilatory threshold changed from 15.0+/-4.0 to 19.9+/-4.9 mL (P=0. 049) with L and from 12.0+/-1.9 to 15.4+/-3.5 mL (P=0.039) with E. MCH1 increased from 61.2+/-11.2% to 75.4+/-7.6% with L (P=0.012) and from 60.6+/-13.1% to 80.1+/-10.9% (P=0.006) with E. Similarly, MHC2a decreased from 21.20+/-9.5% to 12.9+/-4.4% (P=0.05) with L and from 19.9+/-7.8% to 11.8+/-7.9% (P=0.06) with E. MHC2b changed from 17. 5+/-6.5% to 11.7+/-5.2% (P=0.07) with L and from 19.5+/-6.4% to 8. 1+/-4.6% (P=0.0015) with E. There was a significant correlation between net changes in MHC1 and absolute changes in peak V(O2) (r2=0.29, P=0.029) and a trend to significance for MHC2a and 2b. CONCLUSIONS: Six months' treatment with L and with E produces an improvement in EC of similar magnitude. These changes are accompanied by a reshift of MHCs of leg skeletal muscle toward the slow, more fatigue-resistant isoforms. Magnitude of MHC1 changes correlates with the net peak V(O2) gain, which suggests that improved EC may be caused by favorable biochemical changes occurring in the skeletal muscle.

Skeletal muscle myosin heavy chains in heart failure: correlation between magnitude of the isozyme shift, exercise capacity, and gas exchange measurements.

BACKGROUND: Patients with congestive heart failure (CHF) have a reduced exercise capacity because of the early appearance of fatigue and dyspnea. Qualitative changes in the skeletal muscle composition and metabolism can be responsible for the origin of symptoms METHODS: We correlated the myosin heavy chain (MHC) composition of the gastrocnemius in 20 patients with different degrees of CHF to NYHA class, diuretic consumption, echocardiographic parameters, and expiratory gases measured during cardiopulmonary exercise testing. MHC composition was determined electrophoretically in skeletal muscle needle microbiopsies and the percent distribution was calculated by densitometry. Maximal cardiopulmonary exercise testing was performed on a treadmill with a modified Naughton protocol. A capnograph was used. RESULTS: There was no correlation between ejection fraction, left ventricular end systolic diameter, left ventricular end diastolic diameter, and MHC composition. We found a significant positive correlation between the percentage of MHC 1 (slow aerobic isoform) and NYHA class (r2 = 0.62, p < 0.0001), peak VO2 (r2 = 0.5, p < 0.0004), ventilatory threshold (VT) (r2 = 0.33, p = 0.008) and O2 pulse (peak VO2/HR) (r2 = 0.40, p = 0.003). There was a negative correlation between both MHC2a (fast oxidative) and MHC2b (fast glycolytic) with peak VO2 (r2 = 0.38, p = 0.004 and r2 = 0.37, p = 0.004, respectively), VT (r2 = 0.2, p = 0.046 and r2 = 0.34, p = 0.007, respectively), and O2 pulse (peak VO2/HR) (r2 = 0.39, p = 0.003 and r2 = 0.23, p = 0.03). NYHA class was also correlated positively with MHC2a and MHC2b (r2 = 0.46, p = 0.001 and r2 = 0.41, p < 0.006, respectively) and negatively with the same clinical and functional parameters. CONCLUSIONS: The correlation between the magnitude of the MHC shift from the slow aerobic to the fast glycolytic and fast oxidative with both functional and objective measurements of exercise capacity (peak VO2, VT, O2 pulse) seem to suggest that changes in skeletal muscle composition may play a determining role in exercise tolerance in patients with CHF.

Apoptosis of skeletal muscle myofibers and interstitial cells in experimental heart failure.

UNLABELLED: Congestive heart failure (CHF) is characterized by a limb skeletal muscle myopathy with shift from the slow aerobic, fatigue resistant fibers, to the fast, anaerobic ones, and muscle bulk loss. Apoptosis (A) has been recently demonstrated to play a role in several cardiovascular diseases. AIM OF THE STUDY: we have investigated the role of A in the skeletal muscle of the hindlimbs in an experimental model of CHF. ANIMALS AND METHODS: CHF was induced in 7 males 80-100 g Sprague-Dawley rats with 30 mg/kg monocrotaline. Five age and diet matched controls were also studied. The time course of A was also studied in additional animals at day 0, 17, 24 and 30 days. RESULTS: At day 27 the electrophoretic analysis of myosin heavy chains (MHCs) demonstrated in the CHF rats the occurrence of a myopathy, with disappearance of slow MHC1 in the Tibialis Anterior (TA), and a significant shift from the slow to the fast isoforms in the soleus and EDL. With in situ DNA nick-end labelling (TUNEL) we found in the TA of CHF animals a significantly higher number of TUNEL positive nuclei (0.43 +/- 0.24 v 0.08 +/- 0.02, P<0.02 and TUNEL positive myonuclei (0.031 +/- 0.012 v 0.0025 +/- 0.005, P<0.02). The time course of A showed a progressive rise in interstitial and myocyte A, accompanied by a drop in fibers cross-sectional area and muscle weight/body weight, that came out to be significant at 30 days. Western blot showed a lower expression of Bcl-2 at 27 days and a further drop at 30 days in the CHF rats. Double staining for TUNEL and antibody against anti-MHC2a and anti MHC2b + 2x showed that A occurs non-selectively in all the myofiber types. BetaANP and Right Ventricle Mass/Volume (RVM/V) correlated significantly with total apoptotic nuclei. CONCLUSIONS: In CHF myofibers A can lead to muscle atrophy. Endothelial cells A may produce an imbalance in myofibres nutrition with relative ischemia that triggers the preferential synthesis of fast anaerobic myosin as an adaptive mechanism or alternatively induce myofibres death.

[Myosin isoforms in skeletal muscle in patients with chronic heart decompensation: distribution and correlation with with exercise tolerance]. / Le isoforme miosiniche nel muscolo scheletrico del paziente con scompenso cronico di cuore: distribuzione e correlazione con la tolleranza allo sforzo.

Chronic heart failure (CHF) is accompanied by a reduced exercise capacity, and the symptoms can be at least in part explained by qualitative and quantitative changes in the skeletal muscle composition and metabolism. We have correlated the myosin heavy chain (MHC) composition of the gastrocnemius in 20 patients with different degrees of CHF to expiratory gases measured during maximal cardiopulmonary exercise testing, NYHA functional class and echocardiographic parameters. MHC composition was determined electrophoretically in skeletal muscle needle microbiopsies and the percent distribution calculated by laser densitometry. There was no correlation between ejection fraction, left ventricular end-diastolic and end-systolic diameters and MHC composition. The percentage of MHC 1 (slow aerobic isoform) was positively correlated with peak VO2 (r2 = 0.5, p = 0.0004), ventilatory threshold (VT, r2 = 0.33, p = 0.008), and O2 pulse (peak VO2/HR, r2 = 0.40, p = 0.003). There was a negative correlation between MHC 2a and 2b (fast isoforms) and peak VO2 (r2 = 0.38 and 0.37, p = 0.004, respectively), VT (r2 = 0.2, p = 0.05; r2 = 0.34, p = 0.007, respectively) and O2 pulse (r2 = 0.39, p = 0.003; r2 = 0.23, p = 0.03, respectively). NYHA functional class was also negatively correlated with the same parameters (r2 = 0.2, p = 0.01; r2 = 0.4, p = 0.001; r2 = 0.34, p = 0.006, respectively) as well as with MHC 1 (r2 = 0.62, p = 0.0001). A positive correlation was found between NYHA functional class and MHC 2a and 2b (r2 = 0.46, p = 0.001; r2 = 0.41, p = 0.002, respectively). The severity of heart failure is paralleled by a shift of the MHC pattern toward the fast MHC 2b. The correlation between the magnitude of the MHCs shift, from the slow aerobic to the fast type, with both clinical parameters (NYHA functional class) and functional measurements (peak VO2, VT, O2 pulse) of exercise capacity seem to suggest that changes in skeletal muscle composition may play a key role in exercise tolerance in patients with CHF.

Smooth muscle myosin light chain kinase is transiently expressed in skeletal muscle during embryogenesis and muscle regeneration both in vivo and in vitro.

By using a polyclonal antibody raised against smooth muscle Myosin Light Chain Kinase of adult chicken we show that the 135 kDa smooth muscle Myosin Light Chain Kinase isoform is present in neonatal and regenerating rat skeletal muscle, as well as in adult atrial myocardium. No reaction was evident in adult skeletal muscle fibres. In neonatal and in early regenerating muscle smooth muscle Myosin Light Chain Kinase is associated with embryonic myosin as revealed by their co-presence in muscle fibres. Experiments in vitro show the same results in myotubes. In atrial myocardium there is a patchy positivity in certain group of myocytes. Immunoblotting experiments show in muscle cell cultures, in neonatal and in regenerating skeletal muscle a protein band with electrophoretic mobility corresponding to that of smooth muscle Myosin Light Chain Kinase. These results suggest that the expression of smooth muscle Myosin Light Chain Kinase is not fully tissue-specific and that regulation of the contractile machinery could be different during myogenesis and in adulthood, in relation to the peculiar dynamic characteristics of developing muscles.

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.

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.

Angiotensin II and contraction of isolated myocytes from human, guinea pig, and infarcted rat hearts.

The effects of angiotensin II on myocardial contractility were assessed in isolated cardiac myocyte preparations, using video microscopy with a computerized edge-detection system. Angiotensin II (1 nM-10 microM) did not affect the contraction of rat (n = 10), guinea pig (n = 11), or human ventricular myocytes (n = 8) or of human atrial myocytes (n = 12). Isoproterenol or raised extracellular calcium increased the contraction amplitude of the cardiac myocytes to a maximum of between 150 and 560% above basal, and there were corresponding increases in the velocities of contraction and relaxation. In rat and guinea pig ventricular myocytes 1 microM angiotensin II did not affect the inotropic response to isoproterenol. Seven days after left coronary artery ligation in seven rats, the basal contraction amplitude was reduced in myocytes from the infarcted region (4.0 +/- 1.9%) compared with the noninfarcted region (5.0 +/- 2.8%, P = 0.03) and with myocytes from six sham-operated hearts (5.0 +/- 2.8%, P = 0.03). There was a switch in myosin isoform expression from the V1 to the V3 isoform in myocytes from both the infarcted and noninfarcted regions. Angiotensin II (1 nM-10 microM) had no significant effect on the contraction characteristics of myocytes from the infarcted rat hearts. In conclusion, angiotensin II had no significant inotropic effect on isolated cardiac myocyte preparations from guinea pig ventricle, normal and infarcted rat ventricle, human ventricle, and human atrium.

Contractile dysfunction of isolated ventricular myocytes in experimental uraemia.

In order to clarify the mechanism underlying impaired cardiac performance in uraemia, the contractile function of isolated cardiac myocytes from chronically uraemic and control rats has been compared. Rats were made uraemic by sub-total nephrectomy in a two-stage surgical procedure, and left for 4 weeks. Sham-operated controls were prepared at the same time. Animals were pairfed, and final body weights were not significantly different between the groups. Ventricular myocytes were isolated and their contraction amplitude and velocity were measured using a video-based edge-detection system. Contraction was depressed in myocytes from uraemic animals, with contraction amplitude in maximum Ca2+ reduced from 16.3 +/- 0.6% shortening, to 13.0 +/- 0.8% (p < 0.01, n = 10 animals for each group). There was a concomitant decrease in the velocity of shortening (5.6 +/- 0.4 vs. 3.9 +/- 0.5 micron s-1 change in sarcomere length, p < 0.02) and of relaxation (4.6 +/- 0.4 vs. 3.2 +/- 0.4 micron s-1 p < 0.02). Similar depression was seen at lower perfusate Ca2+ concentrations (1-2 mM) and the EC50 for Ca2+ was unchanged. The response to beta-adrenoceptor stimulation was decreased by the same magnitude as that to Ca2+, with no change in the EC50 for isoproterenol or the ratio of maximum response to isoproterenol or to Ca2+ in the same cell (isoproterenol/Ca2+ ratio). There was no shift in the myosin isozyme composition in uraemic cells, with both groups showing a heterogeneous V1/V2/V3 pattern. We conclude that chronic uraemia is associated with a depression of contractile function in the isolated myocyte but no shift in myosin isoforms or specific beta-adrenoceptor desensitisation.

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.

Human satellite cell proliferation in vitro is regulated by autocrine secretion of IL-6 stimulated by a soluble factor(s) released by activated monocytes.

We previously showed that macrophages, besides their scavenger role, selectively induce rat myoblast proliferation in vitro by releasing soluble factors. In this paper we demonstrate a relationship between human-activated monocytes and increased human myoblast proliferation due to IL-6 autocrine secretion by satellite cells. Indeed in the supernatants of muscle cultures treated with activated monocyte-conditioned medium we show by means of an ELISA quantitation a higher autocrine secretion of IL-6 associated with increased myoblast proliferation. This suggests that a growth factor(s) secreted by activated monocytes stimulates IL-6 production by myoblasts and then regulates proliferation of satellite cells.

Effects of beta 1-integrin antisense phosphorothioate-modified oligonucleotide on myoblast behaviour in vitro.

Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.