Cellular Contraction Research Articles

The cardiac contraction cycle: is Ca2+ going local?

the myriad of intracellular signaling pathways involving intracellular Ca2+ means Ca2+ plays a role in everything from cellular metabolism to cell death. In heart muscle Ca2+ is primarily known for its role in cellular contraction. Regulation of cardiac muscle contractility is achieved through

NADPH Oxidases: Functions and Pathologies in the Vasculature

Reactive oxygen species are ubiquitous signaling molecules in biological systems. Four members of the NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species in the vasculature: Nox1, Nox2, Nox4, and Nox5. Signaling cascades triggered by stresses, hormones, vasoactive agents, and cytokines control the expression and activity of these enzymes and of their regulatory subunits, among which p22phox, p47phox, Noxa1, and p67phox are present in blood vessels. Vascular Nox enzymes are also regulated by Rac, ClC-3, Poldip2, and protein disulfide isomerase. Multiple Nox subtypes, simultaneously present in different subcellular compartments, produce specific amounts of superoxide, some of which is rapidly converted to hydrogen peroxide. The identity and location of these reactive oxygen species, and of the enzymes that degrade them, determine their downstream signaling pathways. Nox enzymes participate in a broad array of cellular functions, including differentiation, fibrosis, growth, proliferation, apoptosis, cytoskeletal regulation, migration, and contraction. They are involved in vascular pathologies such as hypertension, restenosis, inflammation, atherosclerosis, and diabetes. As our understanding of the regulation of these oxidases progresses, so will our ability to alter their functions and associated pathologies.

Coming Full Circle: Will 'New Economics' Require Old Solutions in Cellular Market Regulation? (Hebrew)

Regulation of the Israeli cellular market is an ongoing project, in which several governmental institutions are involved. This study focuses on the dual regulation of the Ministry of Communication and the Antitrust Authority, who joined hands in mandating the reduction of access fees (a.k.a. interchange fees) charged when a subscriber to one network calls the subscriber of another.While studies of platform competition show us the anti-competitive nature of high access fees, regulation aiming to achieve lower consumer prices by mandating lower access fees is overly optimistic, as well as simplistic. The argument made here is that the focus on access prices is a method of 'minimalistic regulation' - the attempt by government agencies to promote consumer welfare and competition while avoiding the pitfalls of traditional regulatory oversight. Minimalistic regulation is a noble goal, though one easier achieved in theory than in practice. The paper outlines the methods by which cellular firms are able to circumvent access fee regulation, as well as highlighting the need for a difficult choice stemming from technological evolution of cellphones and consumer perception of cellular contracts: regulate extensively or forgo regulation altogether. I focus on the literature on two-sided markets to develop the former point, and on behavioral economics to develop the latter. Minimalistic regulation of the type assessed here, is presented as an attempt to hold the stick at both ends, with similar chances of success.

Identification of a mechanochemical checkpoint and negative feedback loop regulating branching morphogenesis.

Cleft formation is the initial step in submandibular salivary gland (SMG) branching morphogenesis, and may result from localized actomyosin-mediated cellular contraction. Since ROCK regulates cytoskeletal contraction, we investigated the effects of ROCK inhibition on mouse SMG ex vivo organ cultures. Pharmacological inhibitors of ROCK, isoform-specific ROCK I but not ROCK II siRNAs, as well as inhibitors of myosin II activity stalled clefts at initiation. This finding implies the existence of a mechanochemical checkpoint regulating the transition of initiated clefts into progression-competent clefts. Downstream of the checkpoint, clefts are rendered competent through localized assembly of fibronectin promoted by ROCK I/myosin II. Cleft progression is primarily mediated by ROCK I/myosin II-stimulated cell proliferation with a contribution from cellular contraction. Furthermore, we demonstrate that FN assembly itself promotes epithelial proliferation and cleft progression in a ROCK-dependent manner. ROCK also stimulates a proliferation-independent negative feedback loop to prevent further cleft initiations. These results reveal that cleft initiation and progression are two physically and biochemically distinct processes.

Immune System: Not So Superior

In his news focus story “on the origin of the immune system” (1 May, p. 580), J. Travis nicely lays out some of the issues surrounding the evolution of the adaptive immune system, particularly the biologically novel VDJ recombination process. The selective advantage of the adaptive immune system is, however, not at all clear. Citing the view of “many immunologists,” Travis describes the notion that the adaptive immune system allowed more complex organisms to deal with more complex threats. He also cites Craig Thompson’s suggestion that the adaptive immune system conserves resources, implying that the innate immune system is more resource intensive. These arguments fail by simple inspection. Whatever organismal complexity means, there is no evidence that invertebrates are less complex than gnathosomes or agnatha (which has its own version of adaptive immunity). On the issue of resource intensity, as T. Pradeu points out in his reply (“Immune system: ‘Big Bang’ in question,” Letters, 24 July, p. 393), the adaptive immune system never works on its own; it is activated first by an intensive innate response. In addition, adaptive immunity further amplifies innate reactions. An equally likely possibility is thus the adaptive immune response, with its high rate of cellular expansion and contraction, is actually more resource intensive. In any event, neither of these conjectures has been supported by experimentation. The underlying theme of the Travis news story as well as the reply by A. M. Silverstein (“Immune system: Promethean evolution,” Letters, 24 July, p. 393) is that the adaptive immune system is somehow a great advance (“Big Bang”) in the evolution of free-living organisms. Proponents of this view would thus be obliged to show that gnathosomes have a reduced incidence of mortality associated with disease, but I know of no such evidence. In fact, invertebrates are plagued by many of the same families of viruses, bacteria, and protists as vertebrates, and yet they do not exhibit high mortality rates due to infection nor do their populations collapse under the weight of parasitism. In fact, the immune system may be the cause of its own necessity (1). Incremental immune evolution surely confers a selective advantage, but such an advantage is quickly countered by the more facile evolution of thousands of parasitic agents. Over eons, there has grown an immune appendage that is nonetheless unable to decrease the overall burden of parasitism. This principle was described by van Valen as “The Red Queen Hypothesis” (2). The notion that the immune system plays the bouncer, raising the velvet rope for beneficial bacteria and giving attitude to their less desirable brethren, was taken one step further by Margaret McFall-Ngai. She proposed that the adaptive immune system evolved, in part, to recognize and manage complex communities of beneficial microbes living on or in vertebrates (3). In summary, comparative genomics has shown various components of the immune system to be under continuous positive selection, and yet we cannot assume we know the basis for this selective pressure (4).

Endogenous regulation of cardiovascular function by apelin-APJ

Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demonstrate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ.

Portal hypertensive response to kinin

Portal hypertension is the most common complication of chronic liver diseases, such as cirrhosis. The increased intrahepatic vascular resistance seen in hepatic disease is due to changes in cellular architecture and active contraction of stellate cells. In this article, we review the historical aspects of the kallikrein-kinin system, the role of bradykinin in the development of disease, and our main findings regarding the role of this nonapeptide in normal and experimental models of hepatic injury using the isolated rat liver perfusion model (mono and bivascular) and isolated liver cells. We demonstrated that: 1) the increase in intrahepatic vascular resistance induced by bradykinin is mediated by B2 receptors, involving sinusoidal endothelial and stellate cells, and is preserved in the presence of inflammation, fibrosis, and cirrhosis; 2) the hepatic arterial hypertensive response to bradykinin is calcium-independent and mediated by eicosanoids; 3) bradykinin does not have vasodilating effect on the pre-constricted perfused rat liver; and, 4) after exertion of its hypertensive effect, bradykinin is degraded by angiotensin converting enzyme. In conclusion, the hypertensive response to BK is mediated by the B2 receptor in normal and pathological situations. The B1 receptor is expressed more strongly in regenerating and cirrhotic livers, and its role is currently under investigation.

Characterization of the chondrocyte actin cytoskeleton in living three-dimensional culture: response to anabolic and catabolic stimuli.

The actin cytoskeleton is a dynamic network required for intracellular transport, signal transduction, movement, attachment to the extracellular matrix, cellular stiffness and cell shape. Cell shape and the actin cytoskeletal configuration are linked to chondrocyte phenotype with regard to gene expression and matrix synthesis. Historically, the chondrocyte actin cytoskeleton has been studied after formaldehyde fixation--precluding real-time measurements of actin dynamics, or in monolayer cultured cells. Here we characterize the actin cytoskeleton of living low-passage human chondrocytes grown in three-dimensional culture using a stably expressed actin-GFP construct. GFP-actin expression does not substantially alter the production of endogenous actin at the protein level. GFP-actin incorporates into all actin structures stained by fluorescent phalloidin, and does not affect the actin cytoskeleton as seen by fluorescence microscopy. GFP-actin expression does not significantly change the chondrocyte cytosolic stiffness. GFP-actin does not alter the gene expression response to cytokines and growth factors such as IL-1beta and TGF-beta. Finally, GFP-actin does not alter production of extracellular matrix as measured by radiosulfate incorporation. Having established that GFP-actin does not measurably affect the chondrocyte phenotype, we tested the hypothesis that IL-1beta and TGF-beta differentially alter the actin cytoskeleton using time-lapse microscopy. TGF-beta increases actin extensions, and lamellar ruffling indicative of Rac/CDC42 activation, while IL-1beta causes cellular contraction indicative of RhoA activation. The ability to visualize GFP-actin in living chondrocytes in 3D culture without disrupting the organization or function of the cytoskeleton is an advance in chondrocyte cell biology and provides a powerful tool for future studies in actin-dependent chondrocyte differentiation and mechanotransduction pathways.

Cellular Contractility Requires Ubiquitin Mediated Proteolysis

BackgroundCellular contractility, essential for cell movement and proliferation, is regulated by microtubules, RhoA and actomyosin. The RhoA dependent kinase ROCK ensures the phosphorylation of the regulatory Myosin II Light Chain (MLC) Ser19, thereby activating actomyosin contractions. Microtubules are upstream inhibitors of contractility and their depolymerization or depletion cause cells to contract by activating RhoA. How microtubule dynamics regulates RhoA remains, a major missing link in understanding contractility.Principal FindingsWe observed that contractility is inhibited by microtubules not only, as previously reported, in adherent cells, but also in non-adhering interphase and mitotic cells. Strikingly we observed that contractility requires ubiquitin mediated proteolysis by a Cullin-RING ubiquitin ligase. Inhibition of proteolysis, ubiquitination and neddylation all led to complete cessation of contractility and considerably reduced MLC Ser19 phosphorylation.ConclusionsOur results imply that cells express a contractility inhibitor that is degraded by ubiquitin mediated proteolysis, either constitutively or in response to microtubule depolymerization. This degradation seems to depend on a Cullin-RING ubiquitin ligase and is required for cellular contractions.

Breviscapine ameliorates hypertrophy of cardiomyocytes induced by high glucose in diabetic rats via the PKC signaling pathway

To investigate the influence of breviscapine on high glucose-induced hypertrophy of cardiomyocytes and the relevant mechanism in vitro and in vivo. Cultured neonatal cardiomyocytes were divided into i) control; ii) high glucose concentrations; iii) high glucose+PKC inhibitor Ro-31-8220; iv) high glucose+breviscapine; or v) high glucose+NF-kappaB inhibitor BAY11-7082. Cellular contraction frequency and volumes were measured; the expression of protein kinase C (PKC), NF-kappaB, TNF-alpha, and c-fos were assessed by Western blot or reverse transcription-polymerase chain reaction (RT-PCR). Diabetic rats were induced by a single intraperitoneal injection of streptozotocin, and randomly divided into i) control rats; ii) diabetic rats; or iii) diabetic rats administered with breviscapine (10 or 25 mg x kg(-1) x d(-1)). After treatment with breviscapine for six weeks, the echocardiographic parameters were measured. All rats were then sacrificed and heart tissue was obtained for microscopy. The expression patterns of PKC, NF-kappaB, TNF-alpha, and c-fos were measured by Western blot or RT-PCR. Cardiomyocytes cultured in a high concentration of glucose showed an increased pulsatile frequency and cellular volume, as well as a higher expression of PKC, NF-kappaB, TNF-alpha, and c-fos compared with the control group. Breviscapine could partly prevent these changes. Diabetic rats showed relative cardiac hypertrophy and a higher expression of PKC, NF-kappaB, TNF-alpha, and c-fos; treatment with breviscapine could ameliorate these changes in diabetic cardiomyopathy. Breviscapine prevented cardiac hypertrophy in diabetic rats by inhibiting the expression of PKC, which may have a protective effect in the pathogenesis of diabetic cardiomyopathy via the PKC/NF-kappaB/c-fos signal transduction pathway.

Cell volume variation under different concentrations of saline solution (NaCL)

It is very important to know the volumetric relation between the cellular contraction and the concentration of saline solution (NaCl) used in clinical practice. Blood samples of healthy volunteers were exposed to different concentrations of NaCl ranging from 0.42% to 11.70% and the resultant variation in the hematocrit was observed. The variation of the cellular volume as a function of the corresponding concentration of NaCl was studied. An equation theoretically predicting the observed behavior was obtained We studied 53 blood samples of 12 healthy volunteers. The relationship between cellular contraction and the concentration of NaCl shows that the maximum contraction of cellular volume is obtained when NaCl is about 5.85%. The mathematical model derived is summarized in the equation. Δ V = 1 C 4 Δ V means the cellular variation to the corresponding NaCl concentration. This model has a linear regression coefficient of 0.996 and applying chi-square goodness of fit test is observed a level of confidence superior to 99.5%. It was also noted that concentrations equal to or higher than 7.02% of NaCl provokes some degree of hemolysis. There is no reason to use concentrations of NaCl higher than 5.85%. Higher concentrations do not induce more contraction of cellular volume, but they do provoke cellular damage. Dentro de las prácticas clínicas, es de suma importancia conocer la relación volumétrica entre la contracción celular y la concentración de solución salina (NaCl). Muestras de sangre por parte de voluntarios que gozan de buena salud fueron expuestas a diferentes concentraciones de NaCl, las cuales oscilan entre 0.42% a 11.70%, observándose la variación resultante en el hematocrito. Se estudió la variación del volumen celular como función de la correspondiente concentración de NaCl. Se llegó a establecer una ecuación que predice teóricamente el comportamiento observado. Examinamos 53 muestras de sangre provenientes de 12 voluntarios sanos. La relación entre la contracción celular y la concentración de NaCl muestra que la máxima contracción del volumen celular se obtiene cuando el NaCl está en alrededor de 5.85%. Se resume el modelo matemático resultante en la ecuación. ΔV denota la variación celular a la correspondiente concentración de NaCl. Este modelo presenta un coeficiente de regresión lineal de 0.996 y aplicando el Chi-square goodness of fit test [es una prueba estadística] se observa un nivel de confianza superior a 99.5%. Igualmente, es menester mencionar que las concentraciones de NaCl iguales o superiores a 7.02% provoca cierto grado de hemólisis. No hay razón para utilizar concentraciones de NaCl superiores a 5.85%. Concentraciones más altas no inducen una mayor contracción del volumen celular, pero sí provocan lesión celular.41CV=Δ

Endothelin receptor selectivity: evidence from in vitro and pre‐clinical models of scleroderma

Scleroderma [systemic sclerosis (SSc)] is a spectrum of connective tissue diseases characterized by micro- and macro-vasculopathy, inflammation and autoimmunity and tissue remodelling that often leads to excessive scarring and fibrosis in both interstitial and vascular compartments. Pre-clinical investigations and gene association studies have led to improved understanding of the cell and molecular mechanisms underlying disease pathogenesis and to the identification of key molecular candidates that may represent potentially useful disease biomarkers and effective therapeutic targets. Studies on the endothelin (ET) system, pre-dominantly ET-1 and the cell surface receptors [type A (ET(A))] and type B (ET(B))], have provided evidence for an important role of this system in the vascular and fibrotic pathologies in SSc. To date, promising clinical results, utilizing dual/mixed ET receptor antagonism have been obtained in two of the vascular complications associated with SSc, ischaemic digital ulceration and pulmonary arterial hypertension. Evidence suggests that ET-1 is able to activate and re-program the functional phenotypes of vascular smooth muscle cells, microvascular pericytes and tissue fibroblasts into pro-fibrogenic cell populations with myofibroblasts-like properties. The impact of receptor-selective, over mixed-receptor, antagonism has also been studied in vitro with respect to cell differentiation and proliferation, extracellular matrix synthesis, production and deposition and in pathological cellular contraction. However, the complexity of the ET system, potential for receptor cross-talk, interactions with down-stream signal transduction cascades, as well as the potent inter-relationships with other important ligand-receptor pathways have made in vivo studies difficult to unravel. Moreover, little information is available on the role of the ET system and receptor selectivity in the recruitment and activation of mesenchymal progenitor cells in tissue remodelling and fibrosis or on the early inflammatory response. Here, we discuss the available pre-clinical evidence for the role of the ET system in tissue repair, scarring and fibrosis, using the connective tissue diseases SSc and model systems of fibrogenesis.

Plasma membrane Ca2+-ATPase isoform 4 antagonizes cardiac hypertrophy in association with calcineurin inhibition in rodents

How Ca2+-dependent signaling effectors are regulated in cardiomyocytes, given the extreme cytoplasmic Ca2+ concentration changes that underlie contraction, remains unknown. Cardiomyocyte plasma membrane Ca2+-ATPase (PMCA) extrudes Ca2+ but has little effect on excitation-contraction coupling, suggesting its potential role in controlling Ca2+-dependent signaling effectors such as calcineurin. We generated cardiac-specific inducible PMCA4b transgenic mice that displayed normal global Ca2+ transient and cellular contraction levels and reduced cardiac hypertrophy following transverse aortic constriction (TAC) or phenylephrine/Ang II infusion, but showed no reduction in exercise-induced hypertrophy. Transgenic mice were protected from decompensation and fibrosis following long-term TAC. The PMCA4b transgene reduced the hypertrophic augmentation associated with transient receptor potential canonical 3 channel overexpression, but not that associated with activated calcineurin. Furthermore, Pmca4 gene-targeted mice showed increased cardiac hypertrophy and heart failure events after TAC. Physical associations between PMCA4b and calcineurin were enhanced by TAC and by agonist stimulation of cultured neonatal cardiomyocytes. PMCA4b reduced calcineurin nuclear factor of activated T cell-luciferase activity after TAC and in cultured neonatal cardiomyocytes after agonist stimulation. PMCA4b overexpression inhibited cultured cardiomyocyte hypertrophy following agonist stimulation, but much less so in a Ca2+ pumping-deficient PMCA4b mutant. Thus, Pmca4b likely reduces the local Ca2+ signals involved in reactive cardiomyocyte hypertrophy via calcineurin regulation.

Biomechanics of the Sclera in Myopia: Extracellular and Cellular Factors

Excessive axial elongation of the eye is the principal structural cause of myopia. The increase in eye size results from active remodelling of the sclera, producing a weakened scleral matrix. The present study will detail the biomechanics of the sclera and highlight the matrix and cellular factors important in the control of eye size. Scleral elasticity (load vs. tissue extension) and creep rate (tissue extension vs. time) have been measured postmortem in human eyes. Animal models of myopia have allowed the direct relevance of scleral biomechanics to be investigated during myopia development. Recently, data on tissue matrices incorporating scleral fibroblasts have highlighted the role of cellular contraction in scleral biomechanics. Scleral elasticity is increased in eyes developing myopia, with a reduction in the failure load of the tissue. Scleral creep rate is increased in the sclera from eyes developing myopia, and reduced in eyes recovering from myopia. These changes in biomechanical properties of the sclera occur early in the development of myopia (within 24 h). Alterations in scleral biomechanics during myopia development have been attributed to changes in matrix constituents, principally reduced collagen content. Although the biochemical structure of the sclera plays a critical role in defining the mechanical properties, recent studies investigating the cellular mechanics of the sclera, implicate myofibroblasts in scleral biomechanics. Scleral myofibroblasts have the capacity to contract the matrix and are regulated by tissue stress and growth factors such as transforming growth factor-beta. Changes in these regulatory factors have been observed during myopia development, implicating cellular factors in the resultant weakened sclera. Changes in the biomechanical properties of the sclera are important in facilitating the increase in axial length that results in myopia. Understanding the matrix and cellular factors contributing to the weakened sclera may aid in the development of a clinically appropriate treatment for myopia.

An experimental model for assessing fibroblast migration in 3-D collagen matrices.

The purpose of this study was to develop and test a novel culture model for studying fibroblast migration in 3-D collagen matrices. Human corneal fibroblasts were seeded within dense, randomly oriented compressed collagen matrices. A 6 mm diameter button of this cell-seeded matrix was placed in the middle of an acellular, less dense outer collagen matrix. These constructs were cultured for 1, 3, 5 or 7 days in serum-free media, 10% fetal bovine serum (FBS), or 50 ng/ml PDGF. Constructs were then fixed and labeled with AlexaFluor 546 phalloidin (for f-actin) and TOTO-3 (for nuclei). Cell-matrix interactions were assessed using a combination of fluorescent and reflected light confocal imaging. Human corneal fibroblasts in serum-free media showed minimal migration into the outer (non-compressed) matrix. In contrast, culture in serum or PDGF stimulated cell migration. Cell-induced collagen matrix reorganization in the outer matrix could be directly visualized using reflected light imaging, and was highest following culture in 10% FBS. Cellular contraction in 10% FBS often led to alignment of cells parallel to the outer edge of the inner matrix, similar to the pattern observed during corneal wound healing following incisional surgery. Overall, this 3-D model allows the effects of different culture conditions on cell migration and matrix remodeling to be assessed simultaneously. In addition, the design allows for ECM density, geometry and mechanical constraints to be varied in a controlled fashion. These initial results demonstrate differences in cell and matrix patterning during migration in response to serum and PDGF.

Cytoskeletal-Mediated Tension Modulates the Directed Self-Assembly of Microtissues

The ability of cells to self-assemble into a microtissue such as a spheroid has been attributed mainly to intercellular cohesiveness achieved by the binding of surface membrane proteins such as cadherins. However, highly dynamic and complex cytoskeletal rearrangements are coordinated with these binding events and therefore are likely to participate in self-assembly. By inhibiting such cytoskeletal activity, Y-27632 has been used to prevent and treat fibrotic disease. Here, we used the Rho kinase inhibitor to investigate the role that cellular contraction plays in self-assembly. Normal human fibroblasts (NHFs), Reuber-H35 hepatoma cells (H35s), and mixes of the two (hybrid) were treated with drug during directed self-assembly of microtissues in nonadhesive, micromolded hydrogels. The kinetics of self-assembly of both constrained and unconstrained NHF microtissues were dramatically slowed by the drug, and inhibition was dose responsive and reversible. Although sorting of NHFs and H35s occurred normally in the presence of drug, Y-27632-treated NHFs sorted to the outside of a spheroid when mixed with untreated NHFs. When mixed with H35s in trough micromolds, NHFs could drive spheroid formation from the core of the hybrid microtissues even in small numbers relative to H35s (1:19). These findings demonstrate that cellular contraction controls the kinetics of self-assembly and suggests that NHFs within the core of a microtissue can transmit contractile forces through heterotypic bonds with H35s. The control of directed self-assembly using fibroblasts and contraction inhibitors may be useful for in vitro tissue engineering as well as represent an in vitro model for fibrotic disease.

Myocardial lesions after long-term administration of methamphetamine in rats

To demonstrate the myocardial lesion associated with long-term administration of methamphetamine in rats. The experimental models of intoxication of methamphetamine were established in Sprague-Dawley rats. Methamphetamine hydrochloride (3 mg x kg(-1) x d(-1)) was subcutaneously injected to rats in methamphetamine-treated group (n = 16), and normal saline at the same dose was injected to rats in control group (n = 16). After 1 week and 8 weeks of injection, 8 rats in each group were sacrificed and their hearts were examined with light microscopy and electron microscopy, respectively. After 1 week of methamphetamine exposure, foci of contraction band and cellular degeneration were present in subendocardial myocardium. Cellular degeneration, myocytolysis, and contraction band necrosis became prominent and extensive in methamphetamine-treated rats after 8 weeks. Hypertrophy, intracellular vacuolization, and fibrosis were also observed. The ultrastructural feature showed marked swelling and degeneration of mitochondria, enlargement of sarcoplasmic reticulum, and dissolution of myofilaments. No obvious cardiac myocyte lesions were observed in rats of control group. Methamphetamine abuse daily for a long time may result in an increased risk of cardiovascular lesions similar to cardiomyopathy.

IGSF4: A Novel Candidate Gene for Venous Thrombosis

Through a combination of genotyping and resequencing we have identified 3 SNPs (rs17564430, rs3802858 and rs6589488) in a venous thrombosis (VT) susceptibility gene encoding Immunoglobulin Superfamily 4 (IGSF4), that interacts with protein C (PC) deficiency (3363InsC) in a large thrombophilic kindred (n=516). The IGSF4 gene is located on chromosome 11q23. To verify the association with VT and to estimate the joint effect with PC deficiency, we genotyped the 3 SNPs in the 450 available kindred members, of whom 40 (32 PC deficient) had experienced VT. For each SNP we estimated the odds ratio (OR) and 95% confidence interval (CI95) for VT, adjusting for age and sex. Because earlier linkage analyses and SNP genotyping had suggested a dominant effect of the gene, we calculated the OR for carriers compared to non-carriers. Two of 3 SNPs in IGSF4 remained associated with VT. Carriers of the rs6589488 minor allele had a more than 10-fold increased risk of VT (OR 10.2, CI95 7.8–13.4), compared to those homozygous for the major allele. The effect was more pronounced in PC deficient family members (OR 17.0, CI95 13.5–21.4). PC deficient carriers of the rs3802858 minor allele had about 5-fold increased risk of VT (OR 4.7 CI95 2.2–10.0). We designed a replication study of these findings in the Leiden Thrombophilia Study (LETS) comprised of 471 VT patients and 471 control subjects. The 2 original and 11 additional SNPs were selected based on linkage disequilibrium (LD) in the locus surrounding the 2 SNPs associated with VT in the family. None of the SNPs was associated with VT across the whole LETS population. However, we observed a potential synergistic effect between some of these SNPs and the factor V Leiden (FVL) mutation, suggesting interaction with the PC system. For example, rs658948 major allele homozygotes who carry FVL have an 11–fold increased risk of VT (OR 11.2, CI95 5.1–24.3) compared to minor allele carriers who do not carry FVL. This is a 3-fold increase (CI95 0.9–9.9) over the VT risk associated with FVL alone. In LETS we took rs6589488 minor allele carriers as reference since the major allele was the higher risk allele. This is opposite of what we observed in the family. Apart from refuting associations, reverse associations in different populations might be true but caused by variation in LD patterns between the rs6589488 and other or true causal variants (Lin et al, Am. J. Hum. Genet. 80:531–8, 2007). Potential interactions between IGSF4 and the PC system in VT seem likely based on their functions. Activated protein C (APC) has important anticoagulant, anti-inflammatory and cytoprotective effects. The APC cytoprotective effect is characterized by reduction in endothelial permeability through stabilization of the endothelial cytoskeleton. This effect is mediated by protease activated receptor-1 and the sphingosine 1 phosphate receptor, S1P1, activation by APC bound to the endothelial cell PC receptor (APC-EPCR). The endothelial barrier protective effect likely improves endothelial thromboresistance. For example, exposure of endothelial cells to thrombin leads to transcellular actin stress fiber formation and cellular contraction with the formation of paracellular gaps. These gaps potentially expose plasma to subcellular tissue factor. This effect can be reversed by APC-EPCR which attenuates stress fiber formation and augments the cortical actin ring. (Finigan et al. J Biol Chem. 280:17286–93, 2005; Bae et al. Thromb Haemost. 100:101–9, 2008). IGSF4 is an immunoglobulin superfamily cell adhesion molecule with a well described role in basolateral cell-cell adhesion of pulmonary epithelial cells. We have demonstrated that IGSF4 is expressed in endothelial cells and has somewhat diminished expression in endothelial cells cultured from PC deficient kindred members compared to controls. It is likely that IGSF4 supports basolateral endothelial cell-cell adhesion. We hypothesize that a variant form or decreased expression of IGSF4, interacting with PC deficiency, impairs APC mediated endothelial barrier protection in response to thrombogenic stimuli and thus increases the risk of VT in this thrombophilic kindred.

Localized application of mechanical and biochemical stimuli in 3‐D culture

The goal of this study was to investigate the responses of isolated cells in 3-D culture to localized application of mechanical and biochemical signals. Corneal fibroblasts were plated inside collagen matrices for 24 hours, then imaged using time-lapse DIC. For mechanical perturbation, a microinjection needle (Femtotip) was inserted axially into the ECM, then displaced laterally to alter local ECM stress. For biochemical stimulation, PDGF or vehicle control solution was microinjected into the matrix. Compressing the ECM perpendicular to the cell axis had no appreciable effect on cell behavior. However, pushing the ECM parallel to the cell axis induced rapid cellular contraction, followed by secondary cell spreading and tractional force generation. Injection of PDGF induced a similar cell spreading response. Cells in 3-D matrices showed remarkable plasticity, and extension of pseudopodia could be induced at both the leading and trailing edges of migrating cells.

Ca 2+-Mobility in the Sarcoplasmic Reticulum of Ventricular Myocytes Is Low

The sarcoplasmic reticulum (SR) in ventricular myocytes contains releasable Ca 2+ for activating cellular contraction. Recent measurements of intra-SR (luminal) Ca 2+ suggest a high diffusive Ca 2+-mobility constant ( D CaSR). This could help spatially to unify SR Ca 2+-content ([Ca 2+] SRT) and standardize Ca 2+-release throughout the cell. But measurements of localized depletions of luminal Ca 2+ (Ca 2+-blinks), associated with local Ca 2+-release (Ca 2+-sparks), suggest D CaSR may actually be low. Here we describe a novel method for measuring D CaSR. Using a cytoplasmic Ca 2+-fluorophore, we estimate regional [Ca 2+] SRT from localized, caffeine-induced SR Ca 2+-release. Caffeine microperfusion of one end of a guinea pig or rat myocyte diffusively empties the whole SR at a rate indicating D CaSR is 8–9 μm 2/s, up to tenfold lower than previous estimates. Ignoring background SR Ca 2+-leakage in our measurement protocol produces an artifactually high D CaSR (>40 μm 2/s), which may also explain the previous high values. Diffusion-reaction modeling suggests that a low D CaSR would be sufficient to support local SR Ca 2+-signaling within sarcomeres during excitation-contraction coupling. Low D CaSR also implies that [Ca 2+] SRT may readily become spatially nonuniform, particularly under pathological conditions of spatially nonuniform Ca 2+-release. Local control of luminal Ca 2+, imposed by low D CaSR, may complement the well-established local control of SR Ca 2+-release by Ca 2+-channel/ryanodine receptor couplons.