Gianluigi Scannapieco

Myosin isoforms and cell heterogeneity in vascular smooth muscle: I. Developing and adult bovine aorta

Monoclonal anti-smooth muscle (SM-E7, SM-F11, and BF-48) and anti-nonmuscle (NM-A9 and NM-G2) myosin antibodies, Western blotting, and immunocytochemical procedures were used to study myosin isoform composition and distribution in the smooth muscle (SM) cells of bovine aorta differentiating in vivo and in vitro. Two myosin heavy chain (MHC) isoforms were identified by SM-E7 in adult aorta: SM-MHC-1 (Mr = 205 kDa) and SM-MHC-2 (Mr = 200 kDa), respectively. When tested with the SM-F11 antibody, SM-MHC-2 isoform showed distinct antigenic properties compared to SM-MHC-1. Two bands of 205 and 200 kDa were also present in the aortic SM tissue from 3-month-old fetus and were equally recognized by the BF-48 antibody. The 200-kDa SM myosin isoform was labeled by SM-F11 but not by SM-E7, thus indicating the existence of a fetal-specific SM-MHC-2 isoform. At the cellular level, both developing and adult bovine aortic tissues showed the existence of distinct patterns of myosin isoform expression. Three or even more aortic cell populations are differently distributed in areas which appear as (1) a network of interconnecting sheet-like or compact tissue (early fetus) and (2) enriched of collagenous-elastic or muscular tissue (adult animal). In addition, the SM-MHC-2 isoform of the fetal type appears to be uniquely distributed in cultured SM cells grown in vitro from adult bovine aortic explants. Our data indicate that in bovine aorta (1) MHC isoform expression is developmentally regulated and (2) the distribution of myosin isoforms is heterogenous both among and within aortic cells. These findings may be related to the distinct physiological properties displayed by SM during vascular myogenesis.

Nonmuscle and smooth muscle myosin isoforms in bovine endothelial cells

A panel of monoclonal antibodies, specific for human platelet (NM-A9, NM-F6, and NM-G2) and for bovine smooth muscle (SM-E7) myosin heavy chains (MHC), were used to study the composition and the distribution of myosin isoforms in bovine endothelial cells (EC), in vivo and in vitro. Using indirect and double immunofluorescence techniques, we have found that in the intact aortic endothelium there is expression of nonmuscle MHC (NM-MHC), exclusively. By contrast, hepatic sinusoidal endothelium as well as cultured bovine aortic EC (BAEC) in the subconfluent phase of growth show coexistence of NM- and smooth muscle MHC (SM-MHC) isoforms. SM myosin immunoreactivity disappears when cultured BAEC become confluent. In this phase of cell growth, NM-MHC isoforms are localized differently within the cells, i.e., in the cytoplasm around the nucleus or in the cortical, submembranous region of EC cytoplasm. A third type of intracellular distribution of NM-MHC immunoreactivity was evident in the cell periphery of binucleated, confluent BAEC. These data indicate that (1) several myosin isoforms are differently distributed in bovine endothelia; and (2) SM myosin expression and the specific subcellular localization of NM myosin isoforms within EC might be regulated by cell-cell interactions.

Propranolol-induced changes in ventricular isomyosin composition in the rat

The ventricular isomyosin composition in the rat is characterized by three isoenzymes, V1, V2, and V3, with high, intermediate, and low Ca++-activated ATPase activity, speed of muscle shortening, and contractile economy. In this study, we examined the effects of propranolol on ventricular isomyosin composition in the rat. Eight 4-week-old male Wistar rats were treated from 4 to 12 weeks of age with daily 10 mg/kg subcutaneous doses of propranolol; four control rats were given subcutaneous distilled water. At the end of the treatment period, the efficacy of beta blockade was confirmed by isoproterenol test in some rats from each group. After the rats were killed left ventricular myosin from both control and propranolol-treated animals was purified and tested for Ca++-activated ATPase activity. Ventricular isomyosin composition was studied by gel electrophoresis in non-denaturing conditions. Heart rate was significantly lower in the propranolol group, while no differences in blood pressure, body weight, or ventricular weight were found between the two groups. Lower Ca++-activated ATPase activity values and a higher expression of myosin isoenzymes V2 and V3 were found in propranolol-treated rats. Possible links between the observed shift in ventricular isomyosin composition and the well-known modifications in myocardial contractility and oxygen consumption occurring after chronic propranolol administration remain to be established.

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)

The idiopathic dilated cardiomyopathy in man. A biochemical and molecular study on myosin

SummaryWe studied subunit composition and Ca++-activated ATPase activity of myosin isolated from atria and ventricles of hearts explanted from patients suffering from idiopathic dilated cardiomyopathy. At variance with previously published data, we have been unable to detect in the ventricular subendocardial layers a significant amount of myosin atrial-like light chain 1 (ALC1), which has been reported to be related to some hemodynamic features of the hypertrophied and failing heart. Such a subunit was not visible in the septum and in the subepicardial layers either. On the contrary, in both atria a ventricular-like light chain 2 (VLC2) was found. The nature of this additional light chain was confirmed on the basis of two-dimensional electrophoresis and immunoblotting techniques with polyclonal antibodies reacting with VLC2. In these patients we also observed a depressed Ca++-activated ATPase activity, both in atrial and ventricular myosin. The explanation for this finding in ventricles still remains obscure since neither myosin light chains, nor myosin heavy chains showed any difference between patients with dilated cardiomyopathy and controls. On the contrary, in atria we clearly identified changes consistent with the expression of myosin heavy chains of ventricular type and VLC2, which can account for the depressed Ca++-activated ATPase activity.

Low CD34(+) cells, high neutrophils and the metabolic syndrome are associated with an increased risk of venous thromboembolism.

The relationship between MetS (metabolic syndrome), levels of circulating progenitor/immune cells and the risk of VTE (venous thromboembolism) has not yet been investigated. We studied 240 patients with previous VTE and 240 controls. The presence of MetS was identified according to NCEP ATP III guidelines and flow cytometry was used to quantify circulating CD34(+) cells. VTE patients showed higher BMI (body mass index), waist circumference, triacylglycerol (triglyceride) levels, blood glucose, hs-CRP (high-sensitivity C-reactive protein) and lower HDL-C (high-density lipoprotein cholesterol) levels. The prevalence of MetS was significantly higher in VTE (38.3%) than in control individuals (21.3%) with an adjusted OR (odds ratio) for VTE of 1.96 (P=0.002). VTE patients had higher circulating neutrophils (P<0.0001), while the CD34(+) cell count was significantly lower among patients with unprovoked VTE compared with both provoked VTE (P=0.004) and controls (P=0.003). Subjects were also grouped according to the presence/absence of MetS (MetS(+) or MetS(-)) and the level (high/low) of both CD34(+) cells and neutrophils. Very high adjusted ORs for VTE were observed among neutrophils_high/MetS(+) (OR, 3.58; P<0.0001) and CD34(+)_low/MetS(+) (OR, 3.98; P<0.0001) subjects as compared with the neutrophils_low/MetS(-) and CD34(+)_high/MetS(-) groups respectively. In conclusion, low CD34(+) blood cell count and high circulating neutrophils interplay with MetS in raising the risk for venous thromboembolic events.

Left ventricular hypertrophy in hypertension: Changes in isomyosins and creatine-kinase isoenzymes

In hypertension, the heart of small mammals can express different isoenzymic forms of proteins under the influence of overload and other modulating factors. The increase in ventricular mass is generally paralleled by progressive changes in the isoforms of at least two proteins that are involved in the contraction process, namely, myosin and creatine-kinase. This review summarizes the biochemical and molecular changes occurring during progression and with regression of cardiac hypertrophy in rats, humans, and other animals, and focuses on the role played by antihypertensive drugs in modulation of ventricular isomyosins. The implications of these observations for humans remain to be fully determined.

Hematoporphyrin binding to cultured aortic smooth muscle cells from spontaneously atherosclerotic turkey

Porphyrins are known to be accumulated in vivo by tumors and atherosclerotic plaques. We studied the interaction of cultured aortic smooth muscle cells (SMC) from spontaneously atherosclerotic Broad Breasted White Turkeys (BBWT) with free hematoporphyrin (Hp) and low density lipoprotein (LDL)-Hp complexes. A significantly higher binding of LDL-Hp to SMC as compared to free Hp was observed. These data indicate that porphyrin binding to vascular SMC represents a possible mechanism for porphyrin accumulation by atherosclerotic plaques. This process is mediated, at least in part, by LDL.

Biochemical characterization of ventricular myosin from spontaneously hypertensive turkeys

Several stimuli are able to alter the synthesis of cardiac myosin isoenzymes. Particularly in the rat a shift toward a low-ATPase isomyosin is generally observed during development and in cardiac hypertrophy due to pressure overload. On the contrary in spontaneously hypertensive turkeys both ageing and the increasing degree of cardiac hypertrophy are accompanied by a different behaviour of ventricular myosin. In fact in a previous study we have shown that the Ca2+-activated ATPase activity of ventricular myosin increases about three folds from young normotensive to old hypertensive animals. Accordingly the peptide pattern obtained after chymotryptic digestion of myosin showed that some peptides, which are not evident or barely discernible in young animals, are present in the adult ones. In this study we compare the ventricular myosin from young normotensive and adult hypertensive turkeys with atrial myosin. The results obtained suggest that in the ventricles of hypertensive turkeys the synthesis of an isomyosin with biochemical properties close to those of atrial myosin occurs.