Redox regulation of signal transduction in cardiac and smooth muscle

In addition to the well-known property of reactive oxygen species (ROS) to cause non-specific cellular damage, the potential role of ROS in regulation of signal transduction has been recognized. Studies of vascular smooth muscle cells strongly suggest that ROS are required for cell growth signaling. The IP3-induced Ca2+ release from vascular smooth muscle can be selectively stimulated by ROS which may enhance signal transduction for muscle contraction and gene expression. The subunit-subunit contact within the ryanodine receptor complex, as well as intermolecular interactions between the ryanodine receptor and triadin, are redox sensitive, suggesting that ROS may regulate cardiac muscle Ca(2+)-signaling events. The biochemistry of ROS and thiol regulation may allow for specific interactions between ROS and target molecules during redox regulation.     

Modulation of human aorta smooth muscle cell phenotype: a study of muscle-specific variants of vinculin, caldesmon, and actin expression.

Vinculin- and caldesmon-immunoreactive forms and actin isoform patterns were studied in samples of normal and atherosclerotic human aorta. After removal of adventitia and endothelium, the remaining tissue was divided into three layers: media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. In media of normal aorta, meta-vinculin accounted for 41.0 +/- 0.9% (mean +/- SEM) of total immunoreactive vinculin (meta-vinculin + vinculin); 150-kDa caldesmon accounted for 78.2 +/- 5.1% of immunoreactive caldesmon (150-kDa + 70-kDa); the fractional contents of alpha-smooth muscle actin, beta-nonmuscle, and gamma-isoactins were 49.0 +/- 0.6%, 30.4 +/- 0.6%, and 20.8 +/- 0.8%, respectively. Muscular-elastic intima was very similar to media by these criteria. In subendothelial intima, the fractional content of meta-vinculin and 150-kDa caldesmon was significantly lower (6.9 +/- 1.5% and 32.7 +/- 7.0%, respectively) than in muscular-elastic intima and media, whereas the isoactin pattern was identical to that in adjacent layers, demonstrating the smooth muscle origin of subendothelial intima cells. In atherosclerotic fibrous plaque, the fractional content of alpha-actin was decreased in subendothelial intima, rather than in media and muscular-elastic intima. Additionally, the proportion of subendothelial intima cells [i.e., the cells that express low amounts of smooth muscle phenotype markers (meta-vinculin, 150-kDa caldesmon, and alpha-actin)] in the total intima cell population increased dramatically in atherosclerotic fibrous plaque. The results suggest that changes in the relative content of meta-vinculin and 150-kDa caldesmon as well as alpha-actin in human aortic intima are associated with atherosclerosis although, in subendothelial intima of normal aorta, a certain smooth muscle cell population exists that expresses reduced amounts of "contractile" phenotype markers, even in the absence of the disease.     

Comparison of net fluxes of Li and Na in vascular smooth muscle.

The rat tail artery enriched by incubation in a lithium-substituted medium loses Li at three distinct rates upon return to a normal medium at 37 degrees C. The first is very rapid with a single exponential rate constant of the order of 1 min-1, while the second and third are considerably slower with rate constants of the order of 3h-1 and 0.6h-1, respectively. Both slow components are cellular since they combine into a single component when active Na transport is inhibited by the absence of external K, by ouabain, or by low temperature. None of these procedures affects the rapid loss of Li from extracellular locations. It follows that cell Li can be measured simply by washing out extracellular Li at 2 degrees C, a procedure which removes only a small definable amount from cells. The influx of Li into cells during incubation in Li-substituted medium follows 1st order kinetics and exchanges 1:1 with cell K after a delay of about 20 min at 37 degrees C. This delay in influx is associated with active Na transport since it is abolished by the absence of external K, by low temperature, and by ouabain. The activation energy required for the influx of Li and of Na in K-free media is consistent, in the main, the passive diffusion through a restricted area although there is a suggestion in the case of Na that an additional mechanism (e.g. carrier) may be involved.     

Functional and autoradiographic evidence for endothelin 1 receptors on human and rat cardiac myocytes. Comparison with single smooth muscle cells

This study aimed to determine whether receptors for endothelin were present on the cardiac myocyte as well as on vascular smooth muscle cells. Low- and high-resolution autoradiography was performed using 125I-endothelin 1 on intact rat myocardium and samples of human ventricle obtained from explanted hearts at the time of transplant. In addition to specific binding to the smooth muscle of the blood vessel lumen, there was considerable binding associated with cardiac myocytes. To discover whether there was any functional correlate for this binding, muscle cells were isolated enzymatically from human and rat ventricle and from rat femoral artery, and their contractile characteristics were studied. Single cardiac cells were superfused with physiological saline at 32 degrees C, and their length change was displayed continuously on a chart recorder. Endothelin 1 had a pronounced effect on shortening in both rat and human myocytes. The contraction amplitude was approximately doubled in both cases, from 4.1 +/- 0.8% cell length to 8.1 +/- 1.3% for rat (mean +/- SEM, n = 9, p less than 0.001), and from 2.1 +/- 0.5% to 4.0 +/- 0.5% in human (n = 10, p less than 0.001). In rat, the magnitude of the effect was comparable to that of the alpha-adrenoceptor agonist phenylephrine. The maximum contraction amplitude of the human cells, produced by raising extracellular calcium to greater than 10 mM, was 11.4 +/- 1.1% cell length (n = 9), significantly greater than that produced by endothelin (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in cardiac muscle fructose content in streptozotocin-induced diabetic rats.

To evaluate the activation of the sorbitol pathway in cardiac muscle in diabetic rats, we measured sorbitol, fructose, and myo-inositol content in cardiac tissue obtained from control and streptozotocin-diabetic rats, with or without an 8-week insulin treatment, using gas chromatography-mass spectrometry (GC-MS). Cardiac fructose and sorbitol content in 10-week diabetic rats increased by 60-fold and 3.9-fold of those of control rats, respectively (P less than .001). In contrast, cardiac myo-inositol content in 10-week diabetic rats decreased to 56% (P less than .025) of the control value. The abnormalities in cardiac fructose, sorbitol, and myo-inositol content were completely normalized by the 8-week insulin treatment, which was initiated 2 weeks after the induction of diabetes. There was no difference in cardiac aldose reductase activity between control and diabetic rats. However, cardiac sorbitol dehydrogenase activity in diabetic rats was 151% (P less than .005) higher than that of control rats, although hepatic sorbitol dehydrogenase activity was not different between the two groups. These results indicate that the sorbitol pathway is significantly activated in cardiac tissue obtained from streptozotocin-induced diabetic rats, which results in the marked cardiac accumulation of fructose.     

Product inhibition of the actomyosin subfragment-1 ATPase in skeletal, cardiac, and smooth muscle.

We studied product inhibition of the actin-activated ATPase of myosin subfragment-1 (S-1) from the three types of muscle tissue: skeletal, cardiac, and smooth. Increasing levels of [MgADP] in the 0-1-mM range caused significant inhibition of the actin-activated MgATPase activity of cardiac and gizzard but not skeletal muscle S-1. When total nucleotide concentration ([ATP] + [ADP]) was kept constant at 1 mM, ATPase activity was inhibited by 50% at an ADP/ATP ratio of 6:1 for cardiac S-1 and 3:1 for gizzard S-1. For skeletal S-1, however, even a 19:1 ratio did not cause 50% inhibition of ATPase activity. The observed effect was not due to changes in pH or inorganic phosphate concentration, nor could it be explained by substrate (ATP) depletion. In the absence of actin, ADP had little or no inhibitory effect on the ATPase activity of S-1, and these observations imply that ADP is competing directly for the ATP binding site of the actin-S1 complexes of cardiac and smooth muscle S-1. ADP has previously been shown to be a weak competitive inhibitor of the ATPase activity in skeletal muscle. The current data imply that ADP is a very effective competitive inhibitor for the actin-activated ATPase activity of cardiac and gizzard S-1 and, therefore, that ADP may be a physiologically important modulator of contractile activity in cardiac and smooth muscle.     

Contraction of detergent-treated smooth muscle.

After exposure of segments of rabbit taenia coli to the nonionic detergent Triton X-100, tension could be induced by increasing the [Ca2+] in the micromolar range. In the presence of a saturating [Ca2+], this preparation developed nearly 100% of the control tetanus tension recorded from the intact muscle prior to the detergent treatment. In addition, tension could be induced by increasing the [Mg2+], in the virtual absence of Ca2+. Mg2+ seems to inhibit the Ca2+-induced tension in a predominantly competitive manner.     

Molecular biology of smooth muscle.

Several myosin isoforms are expressed in smooth muscle, based upon both heavy and light chain polymorphisms. These myosin isoenzymes reveal distinct detachment rate constants, thus regulating the responsiveness of the VSMC contractile apparatus to Ca2+, as well as its energetical and mechanical behavior.     

An elastic, biodegradable cardiac patch induces contractile smooth muscle and improves cardiac remodeling and function in subacute myocardial infarction.

OBJECTIVES: Our objective in this study was to apply an elastic, biodegradable polyester urethane urea (PEUU) cardiac patch onto subacute infarcts and to examine the resulting cardiac ventricular remodeling and performance. BACKGROUND: Myocardial infarction induces loss of contractile mass and scar formation resulting in adverse left ventricular (LV) remodeling and subsequent severe dysfunction. METHODS: Lewis rats underwent proximal left coronary ligation. Two weeks after coronary ligation, a 6-mm diameter microporous PEUU patch was implanted directly on the infarcted LV wall surface (PEUU patch group, n = 14). Sham surgery was performed as an infarction control (n = 12). The LV contractile function, regional myocardial wall compliance, and tissue histology were assessed 8 weeks after patch implantation. RESULTS: The end-diastolic LV cavity area (EDA) did not change, and the fractional area change (FAC) increased in the PEUU patch group (p < 0.05 vs. week 0), while EDA increased and FAC decreased in the infarction control group (p < 0.05). The PEUU patch was largely resorbed 8 weeks after implantation and the LV wall was thicker than infarction control (p < 0.05 vs. control group). Abundant smooth muscle bundles with mature contractile phenotype were found in the infarcted myocardium of the PEUU group. The myocardial compliance of the PEUU group was distributed between normal myocardium and infarction control (p < 0.001). CONCLUSIONS: Implantation of a novel biodegradable PEUU patch onto a subacute myocardial infarction promoted contractile phenotype smooth muscle tissue formation and improved cardiac remodeling and contractile function at the chronic stage. Our findings suggest a new therapeutic option against post-infarct cardiac failure.     

Measurement of collagen and smooth muscle cell content in atherosclerotic plaques using polarization-sensitive optical coherence tomography.

OBJECTIVES: The purpose of this study was to investigate the measurement of collagen and smooth muscle cell (SMC) content in atherosclerotic plaques using polarization-sensitive optical coherence tomography (PSOCT). BACKGROUND: A method capable of evaluating plaque collagen content and SMC density can provide a measure of the mechanical fidelity of the fibrous cap and can enable the identification of high-risk lesions. Optical coherence tomography has been demonstrated to provide cross-sectional images of tissue microstructure with a resolution of 10 mum. A recently developed technique, PSOCT measures birefringence, a material property that is elevated in tissues such as collagen and SMCs. METHODS: We acquired PSOCT images of 87 aortic plaques obtained from 20 human cadavers. Spatially averaged PSOCT birefringence, Phi, was measured and compared with plaque collagen and SMC content, quantified morphometrically by picrosirius red and smooth muscle actin staining at the corresponding locations. RESULTS: There was a high positive correlation between PSOCT measurements of Phi and total collagen content in all plaques (r = 0.67, p < 0.001) and in fibrous caps of necrotic core fibroatheromas (r = 0.68, p < 0.001). Polarization-sensitive optical coherence tomography measurements of Phi demonstrated a strong positive correlation with thick collagen fiber content (r = 0.76, p < 0.001) and SMC density (r = 0.74, p < 0.01). CONCLUSIONS: Our results demonstrate that PSOCT enables the measurement of birefringence in plaques and in fibrous caps of necrotic core fibroatheromas. Given its potential to evaluate collagen content, collagen fiber thickness, and SMC density, we anticipate that PSOCT will significantly improve our ability to evaluate plaque stability in patients.     

Similarities and differences in smooth muscle alpha-actin induction by TGF-beta in smooth muscle versus non-smooth muscle cells.

Transforming growth factor-beta (TGF-beta) has been shown to stimulate smooth muscle (SM) alpha-actin expression in smooth muscle cells (SMCs) and non-SMCs. We previously demonstrated that the 2 CArG boxes A and B and a novel TGF-beta control element (TCE) located within the first 125 bp of the SM alpha-actin promoter were required for TGF-beta inducibility of SM alpha-actin in SMCs. The aims of the present study were (1) to determine whether the TCE exhibits SMC specificity or contributes to TGF-beta induction of SM alpha-actin expression in non-SMCs (ie, endothelial cells and fibroblasts) and (2) to determine whether TGF-beta can induce expression of multiple TCE-containing SMC differentiation marker genes, such as SM22alpha, h(1) calponin, and SM myosin heavy chain (SM MHC) in non-SMCs. Results of transient transfection assays demonstrated that mutation of CArG A, CArG B, or the TCE within a 125-bp promoter context completely abolished TGF-beta inducibility of SM alpha-actin in endothelial cells and fibroblasts. However, in contrast to observations in SMCs, inclusion of regions upstream from (-155) completely repressed TGF-beta responsiveness in non-SMCs. Electrophoretic mobility shift assays showed that TGF-beta enhanced binding of a serum response factor to the CArG elements and the binding of an as-yet-unidentified factor to the TCE in endothelial cells and fibroblasts, but to a much lesser extent compared with SMCs. TGF-beta also stimulated expression of the SMC differentiation marker SM22alpha in non-SMCs. However, in contrast to SMCs, TGF-beta did not induce expression of h(1) calponin and SM MHC in non-SMCs. In summary, these results suggest a conserved role for CArG A, CArG B, and the TCE in TGF-beta-induced expression of SM alpha-actin in SMCs and non-SMCs that is modified by a complex interplay of positive- and negative-acting cis elements in a cell-specific manner. Furthermore, observations that TGF-beta stimulated expression of several early but not late differentiation markers in non-SMCs indicate that TGF-beta alone is not sufficient to induce transdifferentiation of non-SMCs into SMCs.     

Interaction between propranolol and calcium channel blockers in cardiac and vascular smooth muscle

The ability of the beta-receptor antagonist propranolol to influence the response of isolated cardiac and vascular smooth muscle to several classes of calcium channel blockers was examined. For comparison, the interactions between propranolol and other classes of negative inotropic and vasorelaxant agents was also evaluated. The results of these studies demonstrate that propranolol pre-treatment significantly enhances the in vitro response to the dihydropyridine calcium channel blocker nifedipine, but not the thiazapine calcium channel blocker diltiazem. This enhancement was unrelated to the negative inotropic or vasorelaxant properties of these agents. In addition, propranolol pre-treatment of rat cortical membranes also enhanced the affinity of nifedipine for the 3H-nitrendipine binding site, but did not alter the effect of diltiazem on 3H-nitrendipine binding. These observations suggest that a direct interaction may exist between beta-receptor antagonists and dihydropyrine-type calcium channel blockers. This interaction may be an important factor in selecting drug therapy for conditions such as hypertension and angina.     

Undernournishment and Yersinia enterocolitica enterocolitis alter intestinal contractility in the rabbit: role of smooth muscle contractile protein content.

Previous studies have demonstrated that the longitudinal smooth muscle of rabbits infected with Yersinia enterocolitica and undernourished because of reduced food intake exhibit a significantly reduced ability to develop tension in response to carbachol compared with pair-fed animals, which are uninfected but equivalently undernourished. To determine whether the alteration in smooth muscle contractility results from changes in cell number (hypo- or hyperplasia), or in contractile protein content or isoform distribution, New Zealand White rabbits (600 to 1000 g) were randomly assigned to one of three treatment groups: infected, pair-fed or control. Tissue contractility was measured, morphometric studies were performed and immunoassays were developed for the measurement of total actin, gamma-enteric and alpha-vascular isoactins, and myosin heavy chain. Consistent with what was found in previous reports, the contractility of longitudinal smooth muscle in response to carbachol was found to increase in pair-fed animals and to decrease in Y enterocolitica-infected animals. There was no significant change in the proportional thickness of the ileal longitudinal smooth muscle coat, and the number of cross-sectioned longitudinal smooth muscle cells/mm2 was not significantly different in infected, pair-fed or control tissues. Immunoassay indicated that the proportion of each specific contractile protein, relative to total protein content in the muscularis propria, was unaffected by Y enterocolitica infection or by pair-feeding. Thus, the alterations in intestinal longitudinal smooth muscle function observed after Y enterocolitica infection were concluded not to be associated with tissue hypo- or hyperplasia, or changes in the total content or isoform distribution of contractile proteins in the muscularis propria.     

Mechanisms of angiotensin II- and arginine vasopressin-induced increases in protein synthesis and content in cultured rat aortic smooth muscle cells. Evidence for selective increases in smooth muscle isoactin expression

Previous studies from this laboratory have demonstrated that angiotensin II (Ang II) and arginine vasopressin (AVP) are potent hypertrophic agents in cultured rat aortic smooth muscle cells. The present study identified major proteins that accumulate in Ang II-induced and AVP-induced hypertrophic cells and initiated studies of the mechanisms that contribute to their accumulation. Smooth muscle cell hypertrophy induced by Ang II and/or AVP (1 microM each) was associated with widespread increases in the content of many cellular proteins that were resolved by one- and two-dimensional gel electrophoresis. However, increases were also selective in nature, with increases in certain individual proteins, including actin (twofold to threefold), vimentin (2.5-fold to sevenfold), tropomyosin (threefold to sixfold), and myosin heavy chain, far exceeding overall increases in cellular protein content (20-40%). Increases in actin content were due largely to increased expression of smooth muscle alpha-actin (3.6- to 7.5-fold), as opposed to nonmuscle beta-actin (1.7- to 2.5-fold). Increases in smooth muscle alpha-actin were accompanied by a fivefold to eightfold increases in smooth muscle alpha-actin mRNA, indicating that these changes were not due exclusively to translational controls. Results demonstrate that contractile agonist-induced hypertrophy in cultured smooth muscle cells is due, in part, to increased expression of smooth muscle contractile proteins. Furthermore, the fact that Ang II and AVP induced selective increases in smooth muscle alpha-actin suggests that these agonists may not only regulate growth of vascular smooth muscle but may also promote expression of smooth muscle-specific contractile proteins during differentiation of vascular smooth muscle.     

Pharmacology of smooth muscle cell replication.

The suggestion that smooth muscle cell proliferation contributes to hypertension, atherosclerosis, and restenosis after angioplasty has led to a growing interest in the use of drugs to inhibit this process. This review summarizes pharmacological studies of smooth muscle cell proliferation in vitro and in vivo and identifies specific mediators of proliferation that are implicated by drugs binding with high affinity to enzymes or receptors.     

Protein kinase C of smooth muscle.

The primary mechanism of regulation of smooth muscle contraction involves the phosphorylation of myosin catalyzed by Ca2+/calmodulin-dependent myosin light chain kinase. However, additional mechanisms, both Ca(2+)-dependent and Ca(2+)-independent, can modulate the contractile state of smooth muscle. Protein kinase C was first implicated in the regulation of smooth muscle contraction with the observation that phorbol esters induce slowly developing, sustained contractions. Protein kinase C occurs in at least four Ca(2+)-dependent (alpha, beta I, beta II, and gamma) and four Ca(2+)-independent (delta, epsilon, zeta, and eta) isoenzymes. Only the alpha, beta, epsilon, and zeta isoenzymes have been identified in smooth muscle. Both classes of isoenzymes have been implicated in the regulation of smooth muscle contraction. However, the physiologically important protein substrates of protein kinase C have not yet been identified. Specific isoenzymes may be activated by different contractile agonists, and individual isoenzymes exhibit some degree of substrate specificity. Prolonged activation of protein kinase C can result in its proteolysis to the constitutively active catalytic fragment protein kinase M, which would dissociate from the sarcolemma and phosphorylate proteins such as myosin that are inaccessible to membrane-bound protein kinase C. Protein kinase M induces relaxation of demembranated smooth muscle fibers contracted at submaximal Ca2+ concentrations. We suggest that protein kinase C plays two distinct roles in regulating smooth muscle contractility. Stimuli triggering phosphoinositide turnover or phosphatidylcholine hydrolysis induce translocation of protein kinase C (probably specific isoenzymes) to the sarcolemma, phosphorylation of protein, and a slow contraction. Prolonged association of the kinase with the membrane may lead to proteolysis and release into the cytosol of protein kinase M, resulting in myosin phosphorylation and relaxation.