Posts Tagged ‘cell migration’

Lymphocytes and Innate Immune Response

Curators: Larry H. Bernstein, MD, FCAP,  and Aviva Lev-Ari, PhD, RN



Scientists Shed Light on Key Role of Innate Lymphoid Cells in the Immune Response

Researchers at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) have discovered that innate lymphoid cells, early responders of the immune system, are primed at the DNA level for rapid action. [National Institutes of Health]


Scientists at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) have found that the development of innate lymphoid cells (ILCs) gradually prepares these cells for rapid response to infection. This work (“Developmental Acquisition of Regulomes Underlies Innate Lymphoid Cell Functionality”), which appears in Cell, sheds light on the development and function of a cell type that is increasingly recognized as having an important role in the body’s immune defense.

“Up until now, researchers have focused on T cells, another type of immune cell,” said John J. O’Shea, M.D., scientific director of NIAMS and senior author of the paper. “ILCs are coming into the spotlight because they appear to have a critical role in defending the body’s barrier regions, such as the skin, lungs, and gut, where microbes must first pass to make their way into the body.”

The importance of T cells became apparent during the 1980s with the emergence of the human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS). HIV attacks a certain class of T cells, destroying a person’s immune defenses and leaving him or her susceptible to infection and cancer. Since that time, investigators around the world have identified the distinct functions of T cell subclasses, providing new insights into their roles in host defense and opportunities for novel therapeutic strategies.

ILCs have received less attention, despite their critical role in mounting the innate immune response. Recent work has revealed that ILCs and T cells mirror each other in their subclasses, which are defined by the kinds of cytokines they produce. However, the relationships between the two types of cells have been unclear.

To determine what sets ILCs apart from T cells, Dr. O’Shea’s team looked to the foundation of a cell’s identity—its genetic information, which provides detailed instructions for how a cell functions. Part of what makes each cell type unique is its distinctive pattern of DNA structure and regulatory factors. The combination of a stretch of DNA and a set of regulatory factors can be thought of as a switch; it helps determine whether a gene is turned off or on.

Inactive regions of the DNA molecule are twisted into tight coils, whereas active regions are open and accessible to the cellular machinery that reads the genetic information. The open portions of the genome include genes themselves, as well as many regions that contribute to the regulation of their activities (the switches). The areas of the genome and the factors that control whether or not the information is read, in total, are referred to as the cell’s regulome.

Working in mice, the NIAMS researchers analyzed regions of the genome that control the cytokine genes produced by both ILCs and T cells. They found that each subclass of ILCs is associated with a distinct pattern of accessible regions. These patterns can be viewed as a type of barcode for each subclass. Further experiments showed that ILCs acquire their barcodes in a stepwise manner over the course of cellular development.

Importantly, the analysis showed that the barcodes are in place in ILCs before they encounter infection. This open, accessible configuration surrounding the switches that control cytokine genes may be instrumental in enabling ILCs to launch an assault rapidly upon infection.

In contrast, the researchers found that many of the DNA regions controlling cytokine genes in the mice’s T cells are inaccessible and silenced prior to exposure to a pathogen. But upon infection, T cells adopted barcodes similar to those of their ILC counterparts. This result reflected earlier findings that ILC and T cell subclasses produce similar sets of cytokines, but also revealed differences in how the two cell types control the activities of these key immune response genes.

While the regulatory landscapes of ILCs are primed for a quick defense upon infection, those of T cells are minimally prepared when the pathogen invades. Only following infection are modifications in the landscape made that enable T cells to launch their attack.

“ILCs and T cells appear very different, but in the end, the way they control key responses is amazingly similar,” said Han-Yu Shih, Ph.D., a postdoctoral fellow at NIAMS and first author of the paper. “ILCs were discovered less than a decade ago, but the parallels between them and T cells will enable us to more quickly understand how they work and to develop ways to enhance or inhibit their function in treating a variety of immune and inflammatory diseases.”



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How Cancer Cells “Grab” Neighbors and “Reel them in”

Larry H. Bernstein, MD, FCAP, Curator



How cancer cells form tumors by reaching out with ‘cables’ and grabbing cells

A cancer riddle solved; counters “cancer stem cell” explanation
January 27, 2016

University of Iowa | Cancer cells’ motion and accretion into tumors

Two University of Iowa studies have recorded the movements of cancerous human breast tissue cells in real time and in 3D — the first time cancer cells’ motion and accretion into tumors has been continuously tracked, the researchers believe.

The team discovered that cancerous cells, moving at move at 92 micrometers per hour (about twice the speed of healthy cells), actively recruit healthy cells into tumors by extending a kind of cable to grab their neighbors — both cancerous and healthy — and reel them in. Surprisingly, as little as five percent of cancerous cells are needed to form the tumors, a ratio previously unknown.

“It’s not like things sticking to each other,” said David Soll, biology professor at the UI and corresponding author on the open-access paper, published in the American Journal of Cancer Research. “It’s that these cells go out and actively recruit. It’s complicated stuff, and it’s not passive. No one had a clue that there were specialized cells in this process, and that it’s a small number that pulls all the rest in.”

The findings could lead to a more precise identification of tumorigenic cells (those that form tumors) and to testing which antibodies would be best equipped to eliminate them.*

How cancer cells “know” what to do

The question is: how do these cells know what to do. Soll hypothesizes they’re reaching back to a primitive past, when these cells were programmed to form embryos. If true, perhaps the cancerous cells — masquerading as embryo-forming cells — recruit other cells to make tissue that then forms the layered, self-sustaining architecture needed for a tumor to form and thrive. “It’s as if it’s building its own defenses against the body’s efforts to defeat them.”

University of Iowa researchers have documented how cancerous tumors form by tracking in real time the movement of individual cells in 3-D. They report that just 5 percent of cancer cells are needed to form tumors, a ratio that heretofore had been unknown. (credit: Soll Laboratory)

In the AJCR paper, the researchers found support for their previous observation that tumorigenic cell lines and fresh tumor cells possess the unique capacity to form tumors by the active formation of cellular cables.

The finding lends more weight to the idea that tumors are created concurrently, in multiple locations, by individual clusters of cells that employ the cancer-cell cables to draw in more cells and enlarge themselves. Some have argued that tumors come about more by cellular changes within the masses, known as the “cancer stem cell theory.”

The Developmental Studies Hybridoma Bank funded the study.

* Soll’s Monoclonal Antibody Research Institute and the Developmental Studies Hybridoma Bank, created by the National Institutes of Health as a national resource, directed by Soll and housed at the UI, together contain one of the world’s largest collections of antibodies that could be used for the anti-cancer testing, based on the new findings.

Abstract of Mediated coalescence: a possible mechanism for tumor cellular heterogeneity

Recently, we demonstrated that tumorigenic cell lines and fresh tumor cells seeded in a 3D Matrigel model, first grow as clonal islands (primary aggregates), then coalesce through the formation and contraction of cellular cables. Non-tumorigenic cell lines and cells from normal tissue form clonal islands, but do not form cables or coalesce. Here we show that as little as 5% tumorigenic cells will actively mediate coalescence between primary aggregates of majority non-tumorigenic or non-cancerous cells, by forming cellular cables between them. We suggest that this newly discovered, specialized characteristic of tumorigenic cells may explain, at least in part, why tumors contain primarily non-tumorigenic cells.



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Integrins, Cadherins, Signaling and the Cytoskeleton

Curator: Larry H. Bernstein, MD, FCAP 


We have reviewed the cytoskeleton, cytoskeleton pores and ionic translocation under lipids. We shall now look at this again, with specific attention to proteins, transporters and signaling.

Integrins and extracellular matrix in mechanotransduction

Lindsay Ramage
Queen’s Medical Research Institute, University of Edinburgh,

Edinburgh, UK
Cell Health and Cytoskeleton 2012; 4: 1–9

Integrins are a family of cell surface receptors which

  • mediate cell–matrix and cell–cell adhesions.

Among other functions they provide an important

  • mechanical link between the cells external and intracellular environments while
  • the adhesions that they form also have critical roles in cellular signal-transduction.

Cell–matrix contacts occur at zones in the cell surface where

  • adhesion receptors cluster and when activated
  • the receptors bind to ligands in the extracellular matrix.

The extracellular matrix surrounds the cells of tissues and forms the

  • structural support of tissue which is particularly important in connective tissues.

Cells attach to the extracellular matrix through

  • specific cell-surface receptors and molecules
  • including integrins and transmembrane proteoglycans.

Integrins work alongside other proteins such as

  • cadherins,
  • immunoglobulin superfamily
  • cell adhesion molecules,
  • selectins, and
  • syndecans

to mediate

  • cell–cell and
  • cell–matrix interactions and communication.

Activation of adhesion receptors triggers the formation of matrix contacts in which

  • bound matrix components,
  • adhesion receptors,
  • and associated intracellular cytoskeletal and signaling molecules

form large functional, localized multiprotein complexes.

Cell–matrix contacts are important in a variety of different cell and

tissue properties including

  1. embryonic development,
  2. inflammatory responses,
  3. wound healing,
  4. and adult tissue homeostasis.

This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.

Integrins are a family of αβ heterodimeric receptors which act as

  • cell adhesion molecules
  • connecting the ECM to the actin cytoskeleton.

The actin cytoskeleton is involved in the regulation of

  1. cell motility,
  2. cell polarity,
  3. cell growth, and
  4. cell survival.

The integrin family consists of around 25 members which are composed of differing

  • combinations of α and β subunits.

The combination of αβ subunits determines

  • binding specificity and
  • signaling properties.

In mammals around 19 α and eight β subunits have been characterized.

Both α and β integrin subunits contain two separate tails, which

  • penetrate the plasma membrane and possess small cytoplasmic domains which facilitate
  • the signaling functions of the receptor.

There is some evidence that the β subunit is the principal

site for

  • binding of cytoskeletal and signaling molecules,

whereas the α subunit has a regulatory role. The integrin


  • link the ECM to the actin cytoskeleton within the cell and with cytoplasmic proteins,

such as talin, tensin, and filamin. The extracellular domains of integrin receptors bind the ECM ligands.

The ECM is a complex mixture of matrix molecules, including -glycoproteins, collagens, laminins, glycosaminoglycans, proteoglycans,
and nonmatrix proteins, – including growth factors.
These can be categorized as insoluble molecules within the ECM, soluble molecules, and/or matrix-associated biochemicals, such as systemic hormones or growth factors and cytokines that act locally.

The integrin receptor formed from the binding of α and β subunits is shaped like a globular head supported by two rod-like legs (Figure 1). Most of the contact between the two subunits occurs in the head region, with the intracellular tails of the subunits forming the legs of the receptor.6 Integrin recognition of ligands is not constitutive but is regulated by alteration of integrin affinity for ligand binding. For integrin binding to ligands to occur the integrin must be primed and activated, both of which involve conformational changes to the receptor.

The integrins are composed of well-defined domains used for protein–protein interactions. The α-I domains of α integrin subunits comprise the ligand binding sites. X-ray crystallography has identified an α-I domain within the β subunit and a β propeller domain within the α subunit which complex to form the ligand-binding head of the integrin.

The use of activating and conformation-specific antibodies also suggests that the β chain is extended in the active integrin. It has since been identified that the hybrid domain in the β chain is critical for integrin activation, and a swing-out movement of this leg activates integrins.

DBP6: Integrin





Linking integrin conformation to function

Figure  Integrin binding to extracellular matrix (ECM). Conformational changes to integrin structure and clustering of subunits which allow enhanced function of the receptor.

integrin coupled to F-actin via linker

integrin coupled to F-actin via linker

Integrin extracellular binding activity is regulated from inside the cell and binding to the ECM induces signals that are transmitted into the cell.15 This bidirectional signaling requires

  • dynamic,
  • spatially, and
  • temporally regulated formation and
  • disassembly of multiprotein complexes that
    form around the short cytoplasmic tails of integrins.

Ligand binding to integrin family members leads to clustering of integrin molecules in the plasma membrane and recruitment of actin filaments and intracellular signaling molecules to the cytoplasmic domain of the integrins. This forms focal adhesion complexes which are able to maintain

  • not only adhesion to the ECM
  • but are involved in complex signaling pathways

which include establishing

  1. cell polarity,
  2. directed cell migration, and
  3. maintaining cell growth and survival.

Initial activation through integrin adhesion to matrix recruits up to around 50 diverse signaling molecules

  • to assemble the focal adhesion complex
  • which is capable of responding to environmental stimuli efficiently.

Mapping of the integrin

  • adhesome binding and signaling interactions

identified a network of 156 components linked together which can be modified by 690 interactions.

The binding of the adaptor protein talin to the β subunit cytoplasmic tail is known to have a key role in integrin activation. This is thought to occur through the disruption of

  • inhibitory interactions between α and β subunit cytoplasmic tails.

Talin also binds

  • to actin and to cytoskeletal and signaling proteins.

This allows talin to directly link activated integrins

to signaling events and the cytoskeleton.
Genetic programming occurs with the binding of integrins to the ECM

Signal transduction pathway activation arising from integrin-

ECM binding results in changes in gene expression of cells

and leads to alterations in cell and tissue function. Various

different effects can arise depending on the

  1. cell type,
  2. matrix composition, and
  3. integrins activated.

One way in which integrin expression is important in genetic programming is in the fate and differentiation of stem cells.
Osteoblast differentiation occurs through ECM interactions

with specific integrins

  • to initiate intracellular signaling pathways leading to osteoblast-specific gene expression
  • disruption of interactions between integrins and collagen;
  • fibronectin blocks osteoblast differentiation and

Disruption of α2 integrin prevents osteoblast differentiation, and activation of the transcription factor

  • osteoblast-specific factor 2/core-binding factor α1.

It was found that the ECM-integrin interaction induces osteoblast-specific factor 2/core-binding factor α1 to

  • increase its activity as a transcriptional enhancer
  • rather than increasing protein levels.

It was also found that modification of α2 integrin alters

  • induction of the osteocalcin promoter;
  • inhibition of α2 prevents activation of the osteocalcin promoter,
  • overexpression enhanced osteocalcin promoter activity.

It has been suggested that integrin-type I collagen interaction is necessary for the phosphorylation and activation of osteoblast-specific transcription factors present in committed osteoprogenitor cells.

A variety of growth factors and cytokines have been shown to be important in the regulation of integrin expression and function in chondrocytes. Mechanotransduction in chondrocytes occurs through several different receptors and ion channels including integrins. During osteoarthritis the expression of integrins by chondrocytes is altered, resulting in different cellular transduction pathways which contribute to tissue pathology.

In normal adult cartilage, chondrocytes express α1β1, α10β1 (collagen receptors), α5β1, and αvβ5 (fibronectin) receptors. During mechanical loading/stimulation of chondrocytes there is an influx of ions across the cell membrane resulting from activation of mechanosensitive ion channels which can be inhibited by subunit-specific anti-integrin blocking antibodies or RGD peptides. Using these strategies it was identified that α5β1 integrin is a major mechanoreceptor in articular chondrocyte responses to mechanical loading/stimulation.

Osteoarthritic chondrocytes show a depolarization response to 0.33 Hz stimulation in contrast to the hyperpolarization response of normal chondrocytes. The mechanotransduction pathway in chondrocytes derived from normal and osteoarthritic cartilage both involve recognition of the mechanical stimulus by integrin receptors resulting in the activation of integrin signaling pathways leading to the generation of a cytokine loop. Normal and osteoarthritic chondrocytes show differences at multiple stages of the mechanotransduction cascade (Figure 3). Early events are similar; α5β1 integrin and stretch activated ion channels are activated and result in rapid tyrosine phosphorylation events. The actin cytoskeleton is required for the integrin-dependent Mechanotransduction leading to changes in membrane potential in normal but not osteoarthritic chondrocytes.

Cell–matrix interactions are essential for maintaining the integrity of tissues. An intact matrix is essential for cell survival and proliferation and to allow efficient mechanotransduction and tissue homeostasis. Cell–matrix interactions have been extensively studied in many tissues and this knowledge is being used to develop strategies to treat pathology. This is particularly important in tissues subject to abnormal mechanical loading, such as musculoskeletal tissues. Integrin-ECM interactions are being used to enhance tissue repair mechanisms in these tissues through differentiation of progenitor cells for in vitro and in vivo use. Knowledge of how signaling cascades are differentially regulated in response to physiological and pathological external stimuli (including ECM availability and mechanical loading/stimulation) will enable future strategies to be developed to prevent and treat the progression of pathology associated with integrin-ECM interactions.

Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels

  1. Matthews, DR. Overby, R Mannix and DE. Ingber
    1Vascular Biology Program, Departments of Pathology and Surgery, Children’s Hospital, and 2Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, Boston, MA J Cell Sci 2006; 119: 508-518.

To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead isplacement with sub-micrometer resolution using optical microscopy. Cells exhibited four types of mechanical responses: (1) an immediate viscoelastic response;

(2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces;

(3) later adaptive cell stiffening with sustained (>15 second) static stresses; and

(4) a large-scale repositioning response with prolonged (>1 minute) stress.

Importantly, these adaptation responses differed biochemically. The immediate and early responses were affected by

  • chemically dissipating cytoskeletal prestress (isometric tension), whereas
  • the later adaptive response was not.

The repositioning response was prevented by

  • inhibiting tension through interference with Rho signaling,

similar to the case of the immediate and early responses, but it was also prevented by

  • blocking mechanosensitive ion channels or
  • by inhibiting Src tyrosine kinases.

All adaptive responses were suppressed by cooling cells to 4°C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.

Microtubule-Stimulated ADP Release, ATP Binding, and Force Generation In Transport Kinesins

J Atherton, I Farabella, I-Mei Yu, SS Rosenfeld, A Houdusse, M Topf, CA Moores

1Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, University of London, London, United Kingdom; 2Structural Motility, Institut Curie, Centre National de la Recherche Scientifique, Paris, France; 3Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States
eLife 2014;3:e03680.

Kinesins are a large family of microtubule (MT)-based motors that play important roles in many cellular activities including

  • mitosis,
  • motility, and
  • intracellular transport

Their involvement in a range of pathological processes also highlights their significance as therapeutic targets and the importance of understanding the molecular basis of their function They are defined by their motor domains that contain both the microtubule (MT) and ATP binding sites. Three ATP binding motifs—the P-loop, switch I, switch II–are highly conserved among kinesins, myosin motors, and small GTPases. They share a conserved mode of MT binding such that MT binding, ATP binding, and hydrolysis are functionally coupled for efficient MT-based work.

The interior of a cell is a hive of activity, filled with proteins and other items moving from one location to another. A network of filaments called microtubules forms tracks along which so-called motor proteins carry these items. Kinesins are one group of motor proteins, and a typical kinesin protein has one end (called the ‘motor domain’) that can attach itself to the microtubules.

The other end links to the cargo being carried, and a ‘neck’ connects the two. When two of these proteins work together, flexible regions of the neck allow the two motor domains to move past one another, which enable the kinesin to essentially walk along a microtubule in a stepwise manner.

Atherton et al. use a technique called cryo-electron microscopy to study—in more detail than previously seen—the structure of the motor domains of two types of kinesin called kinesin-1 and kinesin-3. Images were taken at different stages of the cycle used by the motor domains to extract the energy from ATP molecules. Although the two kinesins have been thought to move along the microtubule tracks in different ways, Atherton et al. find that the core mechanism used by their motor domains is the same.

When a motor domain binds to the microtubule, its shape changes, first stimulating release of the breakdown products of ATP from the previous cycle. This release makes room for a new ATP molecule to bind. The structural changes caused by ATP binding are relatively small but produce larger changes in the flexible neck region that enable individual motor domains within a kinesin pair to co-ordinate their movement and move in a consistent direction. This mechanism involves tight coupling between track binding and fuel usage and makes kinesins highly efficient motors.

A number of kinesins drive long distance transport of cellular cargo with dimerisation allowing them to take multiple 8 nm ATP-driven steps toward MT plus ends. Their processivity depends on communication between the two motor domains, which is achieved via the neck linker that connects each motor domain to the dimer-forming coiled-coil

Kinesins are a superfamily of microtubule-based

  • ATP-powered motors, important for multiple, essential cellular functions.

How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy.

All our reconstructions have, as their asymmetric unit, a triangle-shaped motor domain bound to an αβ-tubulin dimer within the MT lattice (Figure 1). The structural comparisons below are made with respect to the MT surface, which, at the resolution of our structures (∼7 Å, Table 1), is the same (CCC > 0.98 for all). As is well established across the superfamily, the major and largely invariant point of contact between kinesin motor domains and the MT is helix-α4, which lies at the tubulin intradimer interface (Figure 1C, Kikkawa et al., 2001).

However, multiple conformational changes are seen throughout the rest of each domain in response to bound nucleotide (Figure 1D). Below, we describe the conformational changes in functionally important regions of each motor domain starting with the nucleotide-binding site, from which all other conformational changes emanate.

The nucleotide-binding site (Figure 2) has three major elements: (1) the P-loop (brown) is visible in all our reconstructions;

(2) loop9 (yellow, contains switch I) undergoes major conformational changes through the ATPase cycle; and

(3) loop11 (red, contains switch II) that connects strand-β7 to helix-α4,

the conformation and flexibility of which is determined by MT binding and motor nucleotide state.

Movement and extension of helix-α6 controls neck linker docking

the N-terminus of helix-α6 is closely associated with elements of the nucleotide binding site suggesting that its conformation alters in response to different nucleotide states. In addition, because the orientation of helix-α6 with respect to helix-α4 controls neck linker docking and because helix-α4 is held against the MT during the ATPase cycle,

  • conformational changes in helix-α6 control movement of the neck linker.

Mechanical amplification and force generation involves conformational changes across the motor domain

A key conformational change in the motor domain following Mg-ATP binding is peeling of the central β-sheet from the C-terminus of helix-α4 increasing their separation (Figure 3—figure supplement 2); this is required to accommodate rotation of helix-α6 and consequent neck linker docking (Figure 3B–E).

Peeling of the central β-sheet has previously been proposed to arise from tilting of the entire motor domain relative to static MT contacts, pivoting around helix-α4 (the so-called ‘seesaw’ model; Sindelar, 2011). Specifically, this model predicts that the major difference in the motor before and after Mg-ATP binding would be the orientation of the motor domain with respect to helix-α4.

Kinesin mechanochemistry and the extent of mechanistic conservation within the motor superfamily are open questions, critical to explain how MT binding, and ATP binding and hydrolysis drive motor activity. Our structural characterisation of two transport motors now allows us to propose a model that describes the roles of mechanochemical elements that together drive conserved MT-based motor function.

Model of conserved MT-bound kinesin mechanochemistry. Loop11/N-terminus of helix-α4 is flexible in ADP-bound kinesin in solution, the neck linker is also flexible while loop9 chelates ADP. MT binding is sensed by loop11/helix-α4 N-terminus, biasing them towards more ordered conformations.

We propose that this favours crosstalk between loop11 and loop9, stimulating ADP release. In the NN conformation, both loop11 and loop9 are well ordered and primed to favour ATP binding, while helix-α6—which is required for mechanical amplification–is closely associated with the MT on the other side of the motor domain. ATP binding draws loop11 and loop9 closer together; causing

(1) tilting of most of the motor domain not contacting the MT towards the nucleotide-binding site,

(2) rotation, translation, and extension of helix-α6 which we propose contributes to force generation, and

(3) allows neck linker docking and biases movement of the 2nd head towards the MT plus end.

In both motors, microtubule binding promotes

  • ordered conformations of conserved loops that
  • stimulate ADP release,
  • enhance microtubule affinity and
  • prime the catalytic site for ATP binding.

ATP binding causes only small shifts of these nucleotide-coordinating loops but induces

  • large conformational changes elsewhere that
  • allow force generation and
  • neck linker docking towards the microtubule plus end.

Family-specific differences across the kinesin–microtubule interface account for the

  • distinctive properties of each motor.

Our data thus provide evidence for a

conserved ATP-driven

  • mechanism for kinesins and
  • reveal the critical mechanistic contribution of the microtubule interface.

Phosphorylation at endothelial cell–cell junctions: Implications for VE-cadherin function

I Timmerman, PL Hordijk, JD van Buul

Cell Health and Cytoskeleton 2010; 2: 23–31
Endothelial cell–cell junctions are strictly regulated in order to

  • control the barrier function of endothelium.

Vascular endothelial (VE)-cadherin is one of the proteins that is crucial in this process. It has been reported that

  • phosphorylation events control the function of VE-cadherin.

This review summarizes the role of VE-cadherin phosphorylation in the regulation of endothelial cell–cell junctions and highlights how this affects vascular permeability and leukocyte extravasation.

The vascular endothelium is the inner lining of blood vessels and

  • forms a physical barrier between the vessel lumen and surrounding tissue;
  • controlling the extravasation of fluids,
  • plasma proteins and leukocytes.

Changes in the permeability of the endothelium are tightly regulated. Under basal physiological conditions, there is a continuous transfer of substances across the capillary beds. In addition the endothelium can mediate inducible,

  • transient hyperpermeability
  • in response to stimulation with inflammatory mediators,
  • which takes place primarily in postcapillary venules.

However, when severe, inflammation may result in dysfunction of the endothelial barrier in various parts of the vascular tree, including large veins, arterioles and capillaries. Dysregulated permeability is observed in various pathological conditions, such as tumor-induced angiogenesis, cerebrovascular accident and atherosclerosis.

Two fundamentally different pathways regulate endothelial permeability,

  • the transcellular and paracellular pathways.

Solutes and cells can pass through the body of endothelial cells via the transcellular pathway, which includes

  • vesicular transport systems, fenestrae, and biochemical transporters.

The paracellular route is controlled by

  • the coordinated opening and closing of endothelial junctions and
  • thereby regulates traffic across the intercellular spaces between endothelial cells.

Endothelial cells are connected by

  • tight, gap and
  • adherens junctions,

of which the latter, and particularly the adherens junction component,

  • vascular endothelial (VE)-cadherin,
  • are of central importance for the initiation and stabilization of cell–cell contacts.

Although multiple adhesion molecules are localized at endothelial junctions, blocking the adhesive function of VE-cadherin using antibodies is sufficient to disrupt endothelial junctions and to increase endothelial monolayer permeability both in vitro and in vivo. Like other cadherins, VE-cadherin mediates adhesion via homophilic, calcium-dependent interactions.

This cell–cell adhesion

  • is strengthened by binding of cytoplasmic proteins, the catenins,
  • to the C-terminus of VE-cadherin.

VE-cadherin can directly bind β-catenin and plakoglobin, which

  • both associate with the actin binding protein α-catenin.

Initially, α-catenin was thought to directly anchor cadherins to the actin cytoskeleton, but recently it became clear that

  • α-catenin cannot bind to both β-catenin and actin simultaneously.

Data using purified proteins show that

  • monomeric α-catenin binds strongly to cadherin-bound β-catenin;
  • in contrast to the dimer which has a higher affinity for actin filaments,
  • indicating that α-catenin might function as a molecular switch regulating cadherin-mediated cell–cell adhesion and actin assembly.

Thus, interactions between the cadherin complex and the actin cytoskeleton are more complex than previously thought. Recently, Takeichi and colleagues reported that

  • the actin binding protein EPLIN (epithelial protein lost in neoplasm)
  • can associate with α-catenin and thereby
  • link the E-cadherin–catenin complex to the actin cytoskeleton.

Although this study was performed in epithelial cells,

  • an EPLIN-like molecule might serve as
  • a bridge between the cadherin–catenin complex and
  • the actin cytoskeleton in endothelial cells.

Next to β-catenin and plakoglobin, p120-catenin also binds directly to the intracellular tail of VE-cadherin.

Numerous lines of evidence indicate that

  • p120-catenin promotes VE-cadherin surface expression and stability at the plasma membrane.

Different models are proposed that describe how p120-catenin regulates cadherin membrane dynamics, including the hypothesis

  • that p120-catenin functions as a ‘cap’ that prevents the interaction of VE-cadherin
  • with the endocytic membrane trafficking machinery.

In addition, p120-catenin might regulate VE-cadherin internalization through interactions with small GTPases. Cytoplasmic p120-catenin, which is not bound to VE-cadherin, has been shown to

  • decrease RhoA activity,
  • elevate active Rac1 and Cdc42, and thereby is thought
  • to regulate actin cytoskeleton organization and membrane trafficking.

The intact cadherin-catenin complex is required for proper functioning of the adherens junction. Mutant forms of VE-cadherin which

  • lack either the β-catenin, plakoglobin or p120 binding regions reduce the strength of cell–cell adhesion.

Moreover, our own results showed that

  • interfering with the interaction between α-catenin and β-catenin,
  • using a cell-permeable peptide which encodes the binding site in α-catenin for β-catenin,
  • resulted in an increased permeability of the endothelial monolayer.

Several mechanisms may be involved in the regulation of the organization and function of the cadherin–catenin complex, including endocytosis of the complex, VE-cadherin cleavage and actin cytoskeleton reorganization. The remainder of this review primarily focuses on the

  • role of tyrosine phosphorylation in the control of VE-cadherin-mediated cell–cell adhesion.

Regulation of the adhesive function of VE-cadherin by tyrosine phosphorylation

It is a widely accepted concept that tyrosine phosphorylation of components of the VE–cadherin-catenin complex

  • Correlates with the weakening of cell–cell adhesion.

One of the first reports that supported this idea showed that the level of phosphorylation of VE-cadherin was

  • high in loosely confluent endothelial cells, but
  • low in tightly confluent monolayers,

when intercellular junctions are stabilized.

In addition, several conditions that induce tyrosine phosphorylation

of adherens junction components, like

  • v-Src transformation
  • and inhibition of phosphatase activity by pervanadate,

have been shown to shift cell–cell adhesion from a strong to a weak state. More physiologically relevant;

permeability-increasing agents such as

  • histamine,
  • tumor necrosis factor-α (TNF-α),
  • thrombin,
  • platelet-activating factor (PAF) and
  • vascular endothelial growth factor (VEGF)

increase tyrosine phosphorylation of various components of the cadherin–catenin complex.

A general idea has emerged that

  • tyrosine phosphorylation of the VE-cadherin complex
  • leads to the uncoupling of VE-cadherin from the actin cytoskeleton
  • through dissociation of catenins from the cadherin.

However, tyrosine phosphorylation of VE-cadherin is required for efficient transmigration of leukocytes.

This suggests that VE-cadherin-mediated cell–cell contacts

  1. are not just pushed open by the migrating leukocytes, but play
  2. a more active role in the transmigration process.

A schematic overview of leukocyte adhesion-induced signals leading to VE-cadherin phosphorylation

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin.

Notes: A) Permeability-inducing agents such as thrombin, histamine and VEGF, induce tyrosine phosphorylation (pY) of VE-cadherin and the associated catenins. Although the specific consequences of catenin tyrosine phosphorylation in endothelial cells are still unknown, VE-cadherin tyrosine phosphorylation results in opening of the cell–cell junctions (indicated by arrows) and enhanced vascular permeability. How tyrosine phosphorylation affects VE-cadherin adhesiveness is not yet well understood; disrupted binding of catenins, which link the cadherin to the actin cytoskeleton, may be involved. VEGF induces phosphorylation of VE-cadherin at specific residues, Y658 and Y731, which have been reported to regulate p120-catenin and β-catenin binding, respectively. Moreover, VEGF stimulation results in serine phosphorylation (pSer) of VE-cadherin, specifically at residue S665, which leads to its endocytosis. B) Adhesion of leukocytes to endothelial cells via ICAM-1 increases endothelial permeability by inducing phosphorylation of VE-cadherin on tyrosine residues. Essential mediators, such as the kinases Pyk2 and Src, and signaling routes involving reactive oxygen species (ROS) and Rho, have been shown to act downstream of ICAM-1. Different tyrosine residues within the cytoplasmic domain of VE-cadherin are involved in the extravasation of neutrophils and lymphocytes, including Y658 and Y731. (β: β-catenin, α: α-catenin, γ: γ-catenin/plakoglobin).

N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs

BT Jamal, MN Nita-Lazar, Z Gao, B Amin, J Walker, MA Kukuruzinska
Cell Health and Cytoskeleton 2009; 1: 67–80

N-glycosylation of E-cadherin has been shown to inhibit cell–cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/ threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how

  • N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions.

Using the hypoglycosylated E-cadherin variant, V13, we show that

  • V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein.

This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that

  • increased association of PP2A with V13-containing AJs promoted their tethering to microtubules.

On the other hand, V13/γ-catenin complexes associated more with vinculin, suggesting that they

  • mediated the interaction of AJs with the actin cytoskeleton.
  • N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin.

These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through

  • the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components.

Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle

Matteo Vatta, and Georgine Faulkner,

1 Departments of Pediatrics (Cardiology), Baylor College of Medicine, Houston, TX 2 Department of Reproductive and Developmental Sciences, University of Trieste, Trieste, Italy
3 Muscular Molecular Biology Unit, International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy

Future Cardiol. 2006 July 1; 2(4): 467–476.

The heart is a force-generating organ that responds to

  • self-generated electrical stimuli from specialized cardiomyocytes.

This function is modulated

  • by sympathetic and parasympathetic activity.

In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes

  • depend on their highly evolved and specialized cytoskeletal apparatus.

Defects in components of the cytoskeleton, in the long term,

  • affect the ability of the cell to compensate at both functional and structural levels.

In addition to the structural remodeling,

  • the myocardium becomes increasingly susceptible to altered electrical activity leading to arrhythmogenesis.

The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive. Here I will review

  • the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels

and, I will discuss the future impact of new data on molecular cardiology research and clinical practice.

Myocardial dysfunction in the end-stage failing heart is very often associated with increasing

  • susceptibility to ventricular tachycardia (VT) and ventricular fibrillation (VF),

both of which are common causes of sudden cardiac death (SCD).

Among the various forms of HF,

myocardial remodeling due to ischemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM)

  • is characterized by alterations in baseline ECG,

which includes the

  • prolongation of the QT interval,
  • as well as QT dispersion,
  • ST-segment elevation, and
  • T-wave abnormalities,

especially during exercise. In particular, subjects with

severe left ventricular chamber dilation such as in DCM can have left bundle branch block (LBBB), while right bundle branch block (RBBB) is more characteristic of right ventricular failure.  LBBB and RBBB have both been repeatedly associated with AV block in heart failure.

The impact of volume overload on structural and electro-cardiographic alterations has been noted in cardiomyopathy patients treated with left ventricular assist device (LVAD) therapy, which puts the heart at mechanical rest. In LVAD-treated subjects,

  • QRS- and both QT- and QTc duration decreased,
  • suggesting that QRS- and QT-duration are significantly influenced by mechanical load and
  • that the shortening of the action potential duration contributes to the improved contractile performance after LVAD support.

Despite the increasing use of LVAD supporting either continuous or pulsatile blood flow in patients with severe HF, the benefit of this treatment in dealing with the risk of arrhythmias is still controversial.

Large epidemiological studies, such as the REMATCH study, demonstrated that the

  • employment of LVAD significantly improved survival rate and the quality of life, in comparison to optimal medical management.

An early postoperative period study after cardiac unloading therapy in 17 HF patients showed that in the first two weeks after LVAD implantation,

  • HF was associated with a relatively high incidence of ventricular arrhythmias associated with QTc interval prolongation.

In addition, a recent retrospective study of 100 adult patients with advanced HF, treated with an axial-flow HeartMate LVAD suggested that

  • the rate of new-onset monomorphic ventricular tachycardia (MVT) was increased in LVAD treated patients compared to patients given only medical treatment,

while no effect was observed on the development of polymorphic ventricular tachycardia (PVT)/ventricular fibrillation (VF).

The sarcomere

The myocardium is exposed to severe and continuous biomechanical stress during each contraction-relaxation cycle. When fiber tension remains uncompensated or simply unbalanced,

  • it may represent a trigger for arrhythmogenesis caused by cytoskeletal stretching,
  • which ultimately leads to altered ion channel localization, and subsequent action potential and conduction alterations.

Cytoskeletal proteins not only provide the backbone of the cellular structure, but they also

  • maintain the shape and flexibility of the different sub-cellular compartments, including the
  1. plasma membrane,
  2. the double lipid layer, which defines the boundaries of the cell and where
  • ion channels are mainly localized.

The interaction between the sarcomere, which is the basic for the passive force during diastole and for the restoring force during systole. Titin connects

  • the Z-line to the M-line of the sarcomeric structure
    (Figure 1).

In addition to the strategic

  • localization and mechanical spring function,
  • titin is a length-dependent sensor during
  • stretch and promotes actin-myosin interaction

Titin is stabilized by the cross-linking protein

  • telethonin (T-Cap), which localizes at the Z-line and is also part of titin sensor machinery (Figure 1).

The complex protein interactions in the sarcomere entwine telethonin to other

  • Z-line components through the family of the telethonin-binding proteins of the Z-disc, FATZ, also known as calsarcin and myozenin.

FATZ binds to

  1. calcineurin,
  2. γ-filamin as well as the
  3. spectrin-like repeats (R3–R4) of α-actinin-2,

the major component of the Z-line and a pivotal

  • F-actin cross-linker (Figure 1).contractile unit of striated muscles, and
  • the sarcolemma,

the plasma membrane surrendering the muscle fibers in skeletal muscle and the muscle cell of the cardiomyocyte,

  • determines the mechanical plasticity of the cell, enabling it to complete and re-initiate each contraction-relaxation cycle.

At the level of the sarcomere,

  • actin (thin) and myosin (thick) filaments generate the contractile force,

while other components such as titin, the largest protein known to date, are responsible for

  • the passive force during diastole and for the restoring force during systole, and (titin).
  • the Z-line to the M-line of the sarcomeric structure
    (Figure 1).

In addition to the strategic

  • localization and mechanical spring function,
  • it acts as a length-dependent sensor during stretch and
  • promotes actin-myosin interaction.

Stabilized by the cross-linking protein telethonin (T-Cap),

  • titin localizes at the Z-line and is
  • part of titin sensor machinery

Another cross-linker of α-actinin-2 in the complex Z-line scaffold is

  • the Z-band alternatively spliced PDZ motif protein (ZASP),
  • which has an important role in maintaining Z-disc stability

in skeletal and cardiac muscle (Figure 1).

ZASP contains a PDZ motif at its N-terminus,

  • which interacts with C-terminus of α-actinin-2,
  • and a conserved sequence called the ZASP like motif (ZM)
  • found in the alternatively spliced exons 4 and 6.

It has also been reported

  • to bind to the FATZ (calsarcin) family of Z-disc proteins (Figure 1).

The complex protein interactions in the sarcomere entwine telethonin to other Z-line components through the family of the telethonin-binding proteins of the

  1. Z-disc,
  2. FATZ, also known as calsarcin and
  3. myozenin

FATZ binds to calcineurin,

  1. γ-filamin as well as the
  2. spectrin-like repeats (R3–R4) of α-actinin-2, the major component of the Z-line and a pivotal F-actin cross-linker (Figure 1).
sarcomere structure

sarcomere structure

Figure 1. Sarcomere structure

The diagram illustrates the sarcomeric structure. The Z-line determines the boundaries of the contractile unit, while Titin connects the Z-line to the M-line and acts as a functional spring during contraction/relaxation cycles.

Sarcomeric Proteins and Ion Channels

In addition to systolic dysfunction characteristic of dilated cardiomyopathy (DCM) and diastolic dysfunction featuring hypertrophic cardiomyopathy (HCM), the clinical phenotype of patients with severe cardiomyopathy is very often associated with a high incidence of cardiac arrhythmias. Therefore, besides fiber stretch associated with mechanical and hemodynamic impairment, cytoskeletal alterations due to primary genetic defects or indirectly to alterations in response to cellular injury can potentially

  1. affect ion channel anchoring, and trafficking, as well as
  2. functional regulation by second messenger pathways,
  3. causing an imbalance in cardiac ionic homeostasis that will trigger arrhythmogenesis.

Intense investigation of

  • the sarcomeric actin network,
  • the Z-line structure, and
  • chaperone molecules docking in the plasma membrane,

has shed new light on the molecular basis of

  • cytoskeletal interactions in regulating ion channels.

In 1991, Cantiello et al., demonstrated that

  • although the epithelial sodium channel and F-actin are in close proximity,
  • they do not co-localize.

Actin disruption using cytochalasin D, an agent that interferes with actin polymerization, increased Na+ channel activity in 90% of excised patches tested within 2 min, which indicated that

  • the integrity of the filamentous actin (F-actin) network was essential
  • for the maintenance of normal Na+ channel function.

Later, the group of Dr. Jonathan Makielski demonstrated that

  • actin disruption induced a dramatic reduction in Na+ peak current and
  • slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation.

These data were the first to support a role for the cytoskeleton in cardiac arrhythmias.

F-actin is intertwined in a multi-protein complex that includes

  • the composite Z-line structure.

Further, there is a direct binding between

  • the major protein of the Z-line, α-actinin-2 and
  • the voltage-gated K+ channel 1.5 (Kv1.5), (Figure 2).

The latter is expressed in human cardiomyocytes and localizes to

  • the intercalated disk of the cardiomyocyte
  • in association with connexin and N-cadherin.

Maruoka et al. treated HEK293 cells stably expressing Kv1.5 with cytochalasin D, which led to

  • a massive increase in ionic and gating IK+ currents.

This was prevented by pre-incubation with phalloidin, an F-actin stabilizing agent. In addition, the Z-line protein telethonin binds to the cytoplasmic domain of minK, the beta subunit of the potassium channel KCNQ1 (Figure 2).

Molecular interactions between the cytoskeleton and ion channels

Molecular interactions between the cytoskeleton and ion channels

Figure 2. Molecular interactions between the cytoskeleton and ion channels

The figure illustrates the interactions between the ion channels on the sarcolemma, and the sarcomere in cardiac myocytes. Note that the Z-line is connected to the cardiac T-tubules. The diagram illustrates the complex protein-protein interactions that occur between structural components of the cytoskeleton and ion channels. The cytoskeleton is involved in regulating the metabolism of ion channels, modifying their expression, localization, and electrical properties. The cardiac sodium channel Nav1.5 associates with the DGC, while potassium channels such as Kv1.5, associate with the Z-line.

Ion Channel Subunits and Trafficking

Correct localization is essential for ion channel function and this is dependent upon the ability of auxiliary proteins to

  • shuttle ion channels from the cytoplasm to their final destination such as
  • the plasma membrane or other sub-cellular compartments.

In this regard, Kvβ-subunits are

  • cytoplasmic components known to assemble with the α-subunits of voltage-dependent K+ (Kv) channels
  • at their N-terminus to form stable Kvα/β hetero-oligomeric channels.

When Kvβ is co-expressed with Kv1.4 or Kv1.5, it enhances Kv1.x channel trafficking to the cell membrane without changing the overall protein channel content. The regulatory Kvβ subunits, which are also expressed in cardiomyocytes, directly decrease K+ current by

  • accelerating Kv1.x channel inactivation.

Therefore, altered expression or mutations in Kvβ subunits could cause abnormal ion channel transport to the cell surface, thereby increasing the risk of cardiac arrhythmias.

Ion Channel Protein Motifs and Trafficking

Cell membrane trafficking in the Kv1.x family may occur in a Kvβ subunit-independent manner through specific motifs in their C-terminus. Mutagenesis of the final asparagine (N) in the Kv1.2 motif restores the leucine (L) of the Kv1.4 motif

  • re-establishing high expression levels at the plasma membrane in a Kvβ-independent manner

Cytoskeletal Proteins and Ion Channel Trafficking

Until recently, primary arrhythmias such as LQTS have been almost exclusively regarded as ion channelopathies. Other mutations have been identified with regard to channelopathies. However, the conviction that primary mutations in ion channels were solely responsible for

  • the electrical defects associated with arrhythmias

has been shaken by the identification of mutations in the

  • ANK2 gene encoding the cytoskeletal protein ankyrin-B

that is associated with LQTS in animal models and humans.

Ankyrin-B acts as a chaperone protein, which shuttles the cardiac sodium channel from the cytoplasm to the membrane. Immunohistochemical analysis has localized ankyrin-B to the Zlines/T-tubules on the plasma membrane in the myocardium. Mutations in ankyrin-B associated with LQTS

  • alter sodium channel trafficking due to loss of ankyrin-B localization at the Z-line/transverse (T)-tubules.

Reduced levels of ankyrin-B at cardiac Z-lines/T-tubules were associated with the deficiency of ankyrin-B-associated proteins such as Na/K-ATPase, Na/Ca exchanger (NCX) and inositol-1, 4, 5-trisphosphate receptors (InsP3R).

Dystrophin component of the Dystrophin Glycoprotien Complex (DGC)

Synchronized contraction is essential for cardiomyocytes, which are connected to each other via the extracellular matrix (ECM) through the DGC. The N-terminus domain of dystrophin

  • binds F-actin, and connects it to the sarcomere, while
  • the cysteine-rich (CR) C-terminus domain ensures its connection to the sarcolemma (Figure 2).

The central portion of dystrophin, the rod domain, is composed of

  • rigid spectrin-like repeats and four hinge portions (H1–H4) that determine the flexibility of the protein.

Dystrophin possesses another F-actin binding domain in the Rod domain region, between the basic repeats 11- 17 (DysN-R17).

Dystrophin, originally identified as the gene responsible for Duchenne and Becker muscular dystrophies (DMD/BMD), and later for the X-linked form of dilated cardiomyopathy (XLCM), exerts a major function in physical force transmission in striated muscle. In addition to its structural significance, dystrophin and other DGC proteins such as syntrophins are required for the

  • correct localization,
  • clustering and
  • regulation of ion channel function.

Syntrophins have been implicated in ion channel regulation.  Syntrophins contain two pleckstrin homology (PH) domains, a PDZ domain, and a syntrophin-unique (SU) C-terminal region. The interaction between syntrophins and dystrophin occurs at the PH domain distal to the syntrophin N-terminus and through the highly conserved SU domain. Conversely, the PH domain proximal to the N-terminal portion of the protein and the PDZ domain interact with other membrane components such as

  1. phosphatidyl inositol-4, 5-bisphosphate,
  2. neuronal NOS (nNOS),
  3. aquaporin-4,
  4. stress-activated protein kinase-3, and
  5. 5,

thereby linking all these molecules to the dystrophin complex (Figure 2).

Among the five known isoforms of syntrophin, the 59 KDa α1-syntrophin isoform is the most highly represented in human heart, whereas in skeletal muscle it is only present on the

  • sarcolemma of fast type II fibers.

In addition, the skeletal muscle γ2-syntrophin was found at high levels only at the

  • postsynaptic membrane of the neuromuscular junctions.

In addition to syntrophin, other scaffolding proteins such as caveolin-3 (CAV3), which is present in the caveolae, flask-shaped plasma membrane microdomains, are involved

  • in signal transduction and vesicle trafficking in myocytes,
  • modulating cardiac remodeling during heart failure.

CAV3 and α1-syntrophin, localizes at the T-tubule and are part of the DGC. In addition, α1-syntrophin binds Nav1.5, while

  • caveolin-3 binds the Na+/Ca2+ exchanger, Nav1.5 and the L-type Ca2+ channel as well as nNOS and the DGC (Figure 2).

Although ankyrin-B is the only protein found mutated in patients with primary arrhythmias, other proteins such as caveolin-3 and the syntrophins if mutated may alter ion channel function.


It is important to be aware of the enormous variety of clinical presentations that derive from distinct variants in the same pool of genetic factors. Knowledge of these variants could facilitate tailoring the therapy of choice for each patient. In particular, the recent findings of structural and functional links between

  • the cytoskeleton and ion channels

could expand the therapeutic interventions in

  • arrhythmia management in structurally abnormal myocardium, where aberrant binding
  • between cytoskeletal proteins can directly or indirectly alter ion channel function.

Executive Summary

Arrhythmogenesis and myocardial structure

  • Rhythm alterations can develop as a secondary consequence of myocardial structural abnormalities or as a result of a primary defect in the cardiac electric machinery.
  • Until recently, no molecular mechanism has been able to fully explain the occurrence of arrhythmogenesis in heart failure, however genetic defects that are found almost exclusively in ion channel genes account for the majority of primary arrhythmias such as long QT syndromes and Brugada syndrome. The contractile apparatus is linked to ion channels
  • The sarcomere, which represents the contractile unit of the myocardium not only generates the mechanical force necessary to exert the pump function, but also provides localization and anchorage to ion channels.
  • Alpha-actinin-2, and telethonin, two members of the Z-line scaffolding protein complex in the striated muscle associate with the potassium voltage-gated channel alpha subunit Kv1.5 and the beta subunit KCNE1 respectively.
  • Mutations in KCNE1 have previously been associated with the development of arrhythmias in LQTS subjects.
  • Mutations in both alpha-actinin-2, and telethonin were identified in individuals with cardiomyopathy. The primary defect is structural leading to ventricular dysfunction, but the secondary consequence is arrhythmia.

Ion channel trafficking and sub-cellular compartments

  • Ion channel trafficking from the endoplasmic reticulum (ER) to the Golgi complex is an important check-point for regulating the functional channel molecules on the plasma membrane. Several molecules acting as chaperones bind to and shuttle the channel proteins to their final localization on the cell surface
  • Ion channel subunits such as Kvβ enhance Kv1.x ion channel presentation on the sarcolemma. The α subunits of the Kv1.x potassium channels can be shuttled in a Kvβ-independent manner through specific sequence motif at Kv1.x protein level.
  • In addition, cytoskeletal proteins such as ankyrin-G bind Nav1.5 and are involved in the sodium channel trafficking. Another member of the ankyrin family, ankyrin-B was found mutated in patients with LQTS but the pathological mechanism of ankyrin-B mutations is still obscure, although the sodium current intensity is dramatically reduced.

The sarcolemma and ion channels

  • The sarcolemma contains a wide range of ion channels, which are responsible for the electrical propagating force in the myocardium.
  • The DGC is a protein complex, which forms a scaffold for cytoskeletal components and ion channels.
  • Dystrophin is the major component of the DGC and mutations in dystrophin and DGC cause muscular dystrophies and X-linked cardiomyopathies (XLCM) in humans. Cardiomyopathies are associated with arrhythmias
  • Caveolin-3 and syntrophins associate with Nav1.5, and are part of the DGC. Syntrophins can directly modulate Nav1.5 channel function.


  • The role of the cytoskeleton in ion channel function has been hypothesized in the past, but only recently the mechanism underlying the development of arrhythmias in structurally impaired myocardium has become clearer.
  • The recently acknowledged role of the cytoskeleton in ion channel function suggests that genes encoding cytoskeletal proteins should be regarded as potential candidates for variants involved in the susceptibility to arrhythmias, as well as the primary target of genetic mutations in patients with arrhythmogenic syndromes such as LQTS and Brugada syndrome.
  • Studies of genotype-phenotype correlation and and patient risk stratification for mutations in cytoskeletal proteins will help to tailor the therapy and management of patients with arrhythmias.

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Progenitor Cell Transplant for MI and Cardiogenesis  (Part 1

Author and Curator: Larry H. Bernstein, MD, FCAP
Curator: Aviva Lev-Ari, PhD, RN
This article is Part I of a review of three perspectives on stem cell transplantation onto a substantial size of infarcted myocardium to generate cardiogenesis in tissue that is composed of both repair fibroblasts and cardiomyocytes, after essentially nontransmural myocardial infarct.

Progenitor Cell Transplant for MI and Cardiogenesis (Part 1)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Source of Stem Cells to Ameliorate Damage Myocardium (Part 2)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN Damaged_Myocardium/

An Acellular 3-Dimensional Collagen Scaffold Induces Neo-angiogenesis
 (Part 3)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN _Induces_Neo-angiogenesis/

The same approach is considered for stroke in one of these studies.  These are issues that need to be considered
  1. Adult stem cells
  2. Umbilical cord tissue sourced cells
  3. Sheets of stem cells
  4. Available arterial supply at the margins
  5. Infarct diameter
  6. Depth of ischemic necrosis
  7. Distribution of stroke pressure
  8. Stroke volume
  9. Mean Arterial Pressure (MAP)
  10. Location of infarct
  11. Ratio of myocytes to fibrocytes
  12. Coexisting heart disease and, or
  13. Comorbidities predisposing to cardiovascular disease, hypertension
  14. Inflammatory reaction against the graft

Transplantation of cardiac progenitor cell sheet onto infarcted heart promotes cardiogenesis and improves function

L Zakharova1, D Mastroeni1, N Mutlu1, M Molina1, S Goldman2,3, E Diethrich4, and MA Gaballa1*
1Center for Cardiovascular Research, Banner Sun Health Research Institute, Sun City, AZ; 2Cardiology Section, Southern Arizona VA Health Care System, and 3Department of Internal Medicine, The University of Arizona, Tucson, AZ; and 4Arizona Heart Institute, Phoenix, AZ
Cardiovascular Research (2010) 87, 40–49



Cell-based therapy for myocardial infarction (MI) holds great promise; however, the ideal cell type and delivery system have not been established. Obstacles in the field are the massive cell death after direct injection and the small percentage of surviving cells differentiating into cardiomyocytes. To overcome these challenges we designed a novel study to deliver cardiac progenitor cells as a cell sheet.

Methods and results

Cell sheets composed of rat or human cardiac progenitor cells (cardiospheres), and cardiac stromal cells were transplanted onto the infarcted myocardium after coronary artery ligation in rats. Three weeks later, transplanted cells survived, proliferated, and differentiated into cardiomyocytes (14.6 ± 4.7%). Cell sheet transplantation suppressed cardiac wall thinning and increased capillary density (194 ± 20 vs. 97 ± 24 per mm2, P < 0.05) compared with the untreated MI. Cell migration from the sheet was observed along the necrotic trails within the infarcted area. The migrated cells were located in the vicinity of stromal-derived factor (SDF-1) released from the injured myocardium, and about 20% of these cells expressed CXCR4, suggesting that the SDF-1/CXCR4 axis plays, at least, a role in cell migration. Transplantation of cell sheets resulted in a preservation of cardiac contractile function after MI, as was shown by a greater ejection fraction and lower left ventricular end diastolic pressure compared with untreated MI.


The scaffold-free cardiosphere-derived cell sheet approach seeks to efficiently deliver cells and increase cell survival.These transplanted cells effectively rescue myocardium function after infarction by promoting not only neovascular-ization but also inducing a significant level of cardiomyogenesis
Keywords  Myocardial infarction • Cardiac progenitor cells • Cardiospheres • Cardiac regeneration • Contractility


Despite advances in cardiac treatment after myocardial infarction (MI), congestive heart failure remains the number one killer world-wide. MI results in an irreversible loss of functional cardiomyocytes followed by scar tissue formation. To date, heart transplant remains the gold standard for treatment of end-stage heart failure, a procedure which will always be limited by the availability of a donor heart. Hence, developing a new form of therapy is vital.
A number of adult non-cardiac progenitor cells have been tested for myocardial regeneration, including skeletal myoblasts,1 bone-marrow2, and endothelial progenitor cells.3,4 In addition, several cardiac resident stem cell populations have been characterized based on the expression of stem cell marker proteins.5–8 Among these, the c-Kit+ population has been reported to promote myocardial repair.5,9 Recently, an ex vivo method to expand cardiac-derived progenitor cells from human myocardial biopsies and murine hearts was developed.10 Using this approach, undifferentiated cells (or cardiospheres) grow as self-adherent clusters from postnatal atrium or ventricular biopsy specimens.11
To date, the most common technique for cell delivery is direct injection into the infarcted myocardium.12 This approach is inefficient because more than 90% of the delivered cells die by apoptosis and only a small number of the survived cells differentiated into cardiomyocytes.13 An alternative approach to cell delivery is a biodegradable scaffold-based engineered tissue.14,15 This approach has the clear advantage in creating tissue patches of different shapes and sizes and in creating a beating heart by decellularization technology.16 Advances are being made to overcome the issue of small patch thickness and to minimize possible toxicity of the degraded substances from the scaffold.15 Recently, scaffold-free cell sheets were created from fibroblasts, mesenchymal cells, or neonatal myocytes.17,18 Transplantation of these sheets resulted in a limited improvement in cardiac function due to induced neovascularization and angiogenesis through secretion of angiogenic factors.17–19 However, few of those progenitor cells have differentiated into cardiomyocytes.17 The need to improve cardiac contractile function suggests focusing on cells with higher potential to differentiate to cardiomyocytes with an improved delivery method.
In the present study, we report a cell-based therapeutic strategy that surpasses limitation inherent in previously used methodologies. We have created a scaffold-free sheet composed of cardiac progenitor cells (cardiospheres) incorporated into a layer of cardiac stromal cells. The progenitor cells survived when transplanted as a cell sheet onto the infarcted area, improved cardiac contractile functions, and supported recovery of damaged myocardium by promoting not only vascularization but also a significant level of cardiomyogenesis. We also showed that cells from a sheet can be recruited to the site of injury driven, at least partially, by the stromal-derived factor (SDF-1) gradient.


Detailed methods are provided in the Supplementary Methods


Three-month-old Sprague Dawley male rats were used. Rats were randomly placed into four groups:
(1) sham-operated rats, n = 12;
(2) MI, n = 12;
(3) MI treated with rat sheet, n = 10; and
(4) MI treated with human sheet, n = 10.

Myocardial infarction

MI was created by the ligation of the left coronary artery.20 Animals were intubated and ventilated using a small animal ventilator (Harvard Apparatus). A left thoracotomy was performed via the third intercostal rib, and the left coronary artery was ligated. The extent of infarct was verified by measuring the area at risk: heart was perfused with PBS containing 4 mg/mL Evans Blue as previously described by our laboratory.20 The area at risk was estimated by recording the size of the under-perfused (pale-colored) area of myocardium (see Supplementary material online, Figure S1). Only animals with an area at risk >30% were used in the present study. Post-mortem infarct size was measured using triphenyl tetrazolium chloride staining as previously described by our laboratory.20

Isolation of cardiosphere-forming cells

Cardiospheres were generated as described10 from atrial tissues obtained from:
(1) human atrial resection samples obtained from patients (aged from 53 to 73 years old) undergoing cardiac bypass surgery at Arizonam Heart Hospital (Phoenix, AZ) in compliance with Institutional Review Board protocol (n = 10),
(2) 3-month-old SD rats (n = 10). Briefly, tissues were cut into 1–2 mm3 pieces and tissue fragments were cultured ‘as explants’ in a complete explants medium for 4 weeks (Supplementary Methods).
Cell sheet preparation, labelling, handling, and transplantation
Cardiosphere-forming cells (CFCs) combined with cardiac stromal cells were seeded on double-coated plates (poly-L-lysine and collagen type IV from human placenta) in cardiosphere growing medium (Supplementary Methods). The sheets created from the same cell donors were divided into two groups,
one for transplantation and the other for characterization by immunostaining and RT–PCR (Supplementary Methods).
Prior to transplantation, rat cell sheets were labelled with 2 mM 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine, DiI, for tracking transplanted cells in rat host myocardium (Molecular Probes, Eugene, OR). Sheets created using human cells were transplanted unlabelled. Sheets were gently peeled off the collagen-coated plate and folded twice to form four layers. The entire sheet with 200 ml of media was
  • gently aspirated into the pipette tip,
  • transferred to the supporting polycarbonate filter (Costar) and
  • spread off by adding media drops on the sheet (Figure 2A).
Polycarbonate filter was used as a flexible mechanical support for cell sheet to facilitate handling during the transplantation. Immediately after LAD occlusion, the cell sheet was transplanted onto the infarcted area, allowed to adhere to the ventricle for 5–7 min, and the filter was removed before closing the chest (Figure 2A).

Cardiac function

Three weeks after MI, closed-chest in vivo cardiac function was measured using a Millar pressure conductance catheter system (Millar Instruments, Houston, TX) (Supplementary Methods).

Cell sheet survival, engraftment, and cell migration

Rat host myocardium and cell sheet composition after transplantation were characterized by immunostaining (Supplementary Methods). Rat-originated cells were traced by DiI, while human-originated cells were identified by immunostaining with anti-human nuclei or human lamin antibodies.
  1. To assess sheet-originated cardiomyocytes within the host myocardium, the number of cells positive for both human nuclei and myosin heavy chain (MHC) (human sheet); or both DiI and MHC (rat sheet) were counted.
  2. To assess sheet-originated capillaries within the rat host myocardium, the number of cells positive for both human nuclei and von Willebrand factor (vWf) (human sheet); or both DiI and vWf (rat sheet) were counted. Cells were counted in five microscopic fields within cell sheet and area of infarct (n = 5). The number of cells expressing specific markers was normalized to the total number of cells determined by 40,6-diamidino-2-phenylindole staining of the nuclei DNA.
  3. To assess the survival of transplanted cells, sections were stained with Ki-67 antibody followed by fluorescent detection and caspase 3 primary antibodies followed by DAB detection (Supplementary Methods).
  4. To evaluate human sheet engraftment, sections were stained with human lamin antibody followed by fluorescent detection (Supplementary Methods).
  5. Rat host inflammatory response to the transplanted human cell sheet 21 days after transplantation was evaluated by counting tissue mononuclear phagocytes and neutrophils (Supplementary Methods).


Images were captured using Olympus IX70 confocal microscope (Olympus Corp, Tokyo, Japan) equipped with argon and krypton lasers or Olympus IX-51 epifluorescence microscope using excitation/emission maximum filters: 490/520 nm, 570 /595 nm, and 355 /465 nm. Images were processed using DP2-BSW software (Olympus Corp).


All data are represented as mean ± SE Significance (P < 0.05) was deter-mined using ANOVA (StatView).


Generation of cardiospheres

Cardiospheres were generated from atrial tissue explants. After 7–14 days in culture, a layer of stromal cells arose from the attached explants (Supplementary material online, Figure S2a). CFCs, small phase-bright single cells, emerged from explants and bedded down on the stromal cell layer (Supplementary material online, Figure S2b).
  • After 4 weeks, single CFCs, as well as cardiospheres (spherical colonies generated from CFCs) were observed (Supplementary material online, Figure S2c).
Cellular characteristics of cardiospheres in vitro
Immunocytochemical analysis of dissociated cardiospheres revealed that
  • 30% of cells were c-Kitþ indicating that the CFCs maintain multi-potency. About
  • 22 and 28% of cells expressed a, b-MHC and cardiac troponin I, respectively.
These cells represent an immature cardiomyocyte population because they were smaller (10–15 pm in length vs. 60–80 pm for mature cardiomyocytes) and no organized structure of MHC was detected. Furthermore
  • 17% of the cells expressed a-smooth muscle actin (SMA) and
  • 6% were positive for vimentin,
    • both are mesenchymal cell markers (Supplementary material online, Figure S3a and b).
  • Less then 5% of cells were positive for endothelial cell marker; vWf.
Cell characteristics of human cardiospheres are similar to those from rat tissues (Supplementary material online, Figure S3c).
Cardiospheres were further characterized based on the expression of c-Kit antigen. RT–PCR analysis was performed on both c-Kitþ and c-Kit2 subsets isolated from re-suspended cardiospheres. KDR, kinase domain protein receptor, was recently identified as a marker for cardiovascular lineage progenitors in differentiating embryonic stem cells.21 Here, we found that
  • the c-Kitþ cells were also Nkx2.5 and GATA4-positive, but were low or negative for KDR (Supplementary material online, Figure S3d). In contrast,
  • c-Kit2 cells strongly expressed KDR and GATA4, but were negative for Nkx2.5.
  • Both c-Kitþ and c-Kit2 subsets did not express Isl1, a marker for multipotent secondary heart field progenitors.22
Characteristics of cell sheet prior to transplantation
The cell sheet is a layer of cardiac stromal cells in which the cardiospheres were incorporated at a frequency of 21 ± 0.5 spheres per 100,000 viable cells (Figure 1A). The average diameter of cardiospheres within a sheet was 0.13 ± 0.02 mm and their average area was 0.2 ± 0.06 mm2 (Figure 1A). After sheets were peeled off the plate, it exhibited a heterogeneous thickness ranging from 0.05– 0.1 mm (n 1/4 10), H&E staining (Figure1B) and Masson’s Trichrome staining (Figure 1C) of the sheet sections revealed tissue-like organized structures composed of muscle tissue intertwined with streaks of collagen with no necrotic core. Based on the immunostaining results, sheet compiled of several cell types including
  • SMAþ cardiac stromal cells (50%),
  • MHCþ cardiomyocytes (20%), and
  • vWfþ endothelial cells (10%) (Figure 1D and E).
  • 15% of the sheet-forming cells were c-Kitþ suggesting the cells multipotency (Figure 1E).
  • Cells within the sheet expressed gap-junction protein C43, an indicator of electromechanical coupling between cells (Figure 1D).
  • 40% of cells were positive for the proliferation marker Ki-67 suggesting an active cell cycle state (Figure 1D, middle panel).
Human sheet expressed genes
  1. known to be upregulated in undifferentiated cardiovascular progenitors such as c-Kit and KDR;
  2. cardiac transcription factors Nkx2.5 and GATA4; genes related to adhesion, cell homing, and
  3. migration such as ICAM (intercellular adhesion molecule), CXCR4 (receptor for SDF-1), and
  4. matrix metalloprotease 2 (MMP2).
No expression of Isl1 was detected in human sheet (Figure 1F).
sheet transplant on MI_Image_2
Figure 1 Cell sheet characteristics. (A) Fully formed cell sheet. Arrow indicates integrated cardiosphere. (B) H&E staining; pink colour (arrowhead) indicates cytosol and blue (arrows) indicates nuclear stain. Note that there is no necrotic core within the cell sheet. (C) Masson’s Trichrome staining of sheet section. Arrowhead indicates collagen deposition within the sheet. (D and E) Sheet sections were labelled with antibodies against following markers: (D) vWf (green), Ki-67 (green), C43 (green); (E) c-Kit (green), MHC (red), SMA (red) as indicated on top of each panel. Nuclei were labelled with blue fluorescence of 40,6-diamidino-2-phenylindole (DAPI). (F) Gene expression analysis of the cell sheet. Scale bars, 200 pm (A) or 50 pm (B–E).

Cell sheet survival and proliferation

Two approaches were used to track transplanted cells in the host myocardium.
  • rat cell sheets were labelled with red fluorescent dye, DiI, prior to the transplantation.
  • the sheet created from human cells (human sheet) were identified in rat host myocardium by immunostaining with human nuclei antibodies.
DiI-labelling together with trichrome staining showed engraftment of the cardiosphere-derived cell sheet to the infarcted myocardium (Figure 2B–D). In vivo sheets grew into a stratum with heterogeneous thickness ranging from 0.1–0.5 mm over native tissue. The percentage of Ki-67þ cells within the sheet was 37.5 ± 6.5 (Figure 2F) whereas host tissue was mostly negative (except for the vasculature).
To assess the viability of transplanted cells, the heart sections were stained with the apoptosis marker, caspase 3. A low level of caspase 3 was detected within the sheet, suggesting that the majority of transplanted cells survived after transplantation (Figure 2G).
sheet transplant on MI_Image_3
Figure 2 Transplantation and growth of cell sheet after transplantation.
(A) Sheet transplantation onto infarcted heart. Detached cell sheet on six-well plate (left); cell sheet folded on filter (middle); and transplanted onto left ventricle (right). Scale bar 2 mm. DiI-labelled cell sheets grafted above MI area at day 3
(B) and day 21
(C) after transplantation.
(D) LV section of untreated MI rat at day 21 showing no significant red fluorescence background.
Bottom row (B–D) demonstrates the enlargement of box-selected area of corresponding top panels.
(E) Similar sections stained with Masson’s Trichrome. Section of rat (F) or human (G) sheet treated rat at day 21 after MI.
(F) Section was stained with antibody against Ki-67 (green). Cell sheet was pre-labelled with DiI (red). Nuclei stained with blue fluorescence of DAPI.
(G) Section was double stained with human nuclei (blue) and caspase 3 (brown, arrows) antibodies and counterstained with eosin.
Asterisks (**) indicate cell sheet area. Scale bars 200 mm (B–D, top row), 100 mm (B–D, bottom row, and E) or 50 mm (F, G).
Identification of inflammatory response
Twenty-one days after transplantation of human cell sheet, inflammatory response of rat host was examined. Transplantation of human sheet on infarcted rats reduced the number of mononuclear phagocytes (ED1-like positive cells) compared with untreated MI control (Supplementary material online, Figure S4a–e and l). In addition, the number of neutrophils was similar in both control untreated MI and sheet-treated sections (Supplementary material online, Figure S4f–k and m). These data suggest that at 21 days post transplantation, human cell sheet was not associated with significant infiltration of host immune cells.

Cell sheet engraftment and migration

Development of new vasculature was determined in cardiac tissue sections by co-localization of DiI labelling and vWf staining (Figure 3C). Three weeks after transplantation, the capillary density of ischaemic myocardium in the sheet-treated group significantly increased compared with MI animals (194 ± 20 vs. 97 ± 24 per mm2, P < 0.05, Figure 3A and B). The capillaries originated from the sheet ranged in diameter from 10 to 40 jim (n 1/4 30). A gradient in capillary density was observed with higher density in the sheet area which was decreased towards underlying infarcted myocardium. Mature blood vessels were identified within the sheet area and in the underlying myocardium in close proximity to the sheet evident by vWf and SMA double staining (Figure 3D).
sheet transplant on MI_Image_4
Figure 3 Neovascularization of infarcted wall. (A) Frozen tissue sections stained with vWf antibody (green). LV section of control (sham), infarcted (MI), and MI treated with cell sheet (sheet) rats. Scale bar, 100 jim. (B) Capillary density decreased in the MI compared with sham (*P < 0.05) and improved after cell sheet treatment (#P < 0.05). (C) Neovascularization within cell sheet area was recognized by co-localization of DiI- (red) and vWf (green) staining. Scale bar 100 jim. (D) Mature blood vessels (arrows) were identified by co-localization of SMA (red) and vWf (green) staining. Scale bar 50 jim.
Furthermore, 3 weeks after transplantation, a large number of labelled human nuclei positive or DiI-labelled cells were detected deep within the infarcted area indicating cell migration from the epicardial surface to the infarct (Figure 4A, B, and D). Minor or no migration was detected when the cell sheet was transplanted onto non-infarcted myocardium, sham control (Figure 4C). To evaluate engraftment of sheet-originated cells, sections were labelled with anti-human nuclear lamin antibody. Quantification of engraftment was performed using two approaches: fluorescence intensity and cell counting. Fluorescence intensity of the signal was analysed and compared for different areas of myocardium (Figure 4E–J). Since the transplanted sheets are created by human cells and are stained with human nuclear lamin-labelled with green fluorescence, the signal intensity of the sheet is set to 100% (100% of cells are lamin-positive). Myocardial area with no or limited number of labelled cells had the lowest level of fluorescence signal (13%, or 3.2 ± 1.4% of total number of cells), while
  1. the area where the cell migrated from the sheet to the infarcted myocardium had higher signal intensity (47%, or 11.9 ± 1.7% of total number of cells), indicating a higher number of sheet-originated cells are engrafted in the infarcted area.) (Figure 4K and L).
  2. Migrated cells were positive for KDR (Supplementary material online, Figure S5).
sheet transplant on MI_Image_5
Figure 4 Engraftment quantification of cells migrated from the sheet into the infarcted area of MI. Animals were treated with rat (A) or human (B–F) sheets. Cardiomyocytes were labelled with MHC antibody (A, green or B, red). Rat sheet-originated cells were identified with DiI-labelling, red (A). Arrows indicate the track of migrating cells. Human sheet-originated cells were identified by immunostaining with human nuclei antibody followed by secondary antibodies conjugated with either Alexa 488 (B, E and F, green) or AP (C, D, blue). No migration was detected when the cell sheet was transplanted onto non-infarcted myocardium (C). Heart sections were counterstained with eosin, pink (C–D). Higher magnification of area selected in the box is presented (D, right). Immunofluorescence of sheet (green) grafted to the myocardium surface (E) or cells migrated to the infarction area (F). Fluorescence profiles acrossthe cell sheet itself(G, box 1), area underlying cell sheet (I, box 2) and infarction areawith migrated cells (F, box 3). Mean fluorescence intensityofthe grafted human (K) cells was determined by outlining the region of interest (ROI) and subtracting the background fluorescence for the same region. Fluorescence intensity was normalized to the area of ROI (ii 1/4 6). (L) Percent engraftment was defined as number of lamin-positive cells divided by total number of cells per ROI. ‘M’, myocardium,’S’ sheet, ‘I’ infarction. Scale bars 100 mm (A–C, D, left, E and F), or 50 mm (D, right).
To elucidate a possible mechanism of cell migration, sections were stained to detect SDF1 and its unique receptor CXCR4. The migration patterns of cells from the sheet coincided with SDF-1 expression. Within 3 days after MI, SDF-1 was expressed in the injured myocardium (Figure 5A). At 3 weeks after MI and sheet transplantation, SDF-1 was co-localized with the migrated labelled cells (Figure 5B). PCR analysis revealed CXCR4 expression in cell sheet before transplantation (Figure 1F). However, after transplantation only a fraction of migrated cells expressed CXCR4 (Figure 5C).
sheet transplant on MI_Image_6
Figure 5 Migration of sheet-originated cells into the infarcted area. Confocal images of MI animals treated with sheets from rats (A and B) or human (C). SDF1 (green) was detected at border zone of the infarct at day 3 (A) and day 21 (B). Rat sheet-originated cells were identified with DiI-labelling (red). Note co-localization of DiI-positive sheet-originated cells with SDF1 at 21 days after MI (B). Human cells were identified by immunostaining with human nuclei antibody, red, (C). Note human cells that migrated to the area of infarct express CXCR4 (green) (C). Scale bar, 200 mm (A, B) or 50 mm (C). ‘M’, myocardium, ‘S’ sheet, ‘I’ infarct.

3.7 Cardiac regeneration

The differentiation of migrating cells into cardiomyocytes was evident by the co-localization of MHC staining with either human nuclei (Figure 6A) or DiI (Figure 6B and C). In contrast to the immature cardiomyocyte-like cells within the pre-transplanted cell sheet, the migrated and newly differentiated cells within the myocardium were about 30–50 mm in size and co-expressed C43 (see Supplementary material online, Figure S6). Cardiomyogenesis within the infarcted myocardium was observed in the sheets created from either rat or human cells.
sheet transplant on MI_Image_6
Figure 6 Cardiac regeneration. Sections of MI animals treated with human (A) or rat (B, C) sheets. Human sheet was identified by immunostaining with human nuclei antibody (green). Section was double-stained with MHC (red) antibody. Newly formed cardiomyocytes was identified by co-localization of human nuclei and MHC (yellow, arrow). (B) Rat sheet-originated cells were identified by DiI labelling (red). Section was double-stained with MHC (green) antibody. Newly formed cardiomyocytes were detected by co-localization of DiI with MHC (yellow, arrows). (C) Higher magnification of area selected in the boxes (B). Scale bars 200 mm (B), or 20 mm (A, C). ‘M’, myocardium, ‘S’ sheet, ‘I’ infarct.

Cell sheet improved cardiac contractile function and retarded LV remodelling after MI

Closed-chest in vivo cardiac function was derived from left ventricle (LV) pressure–volume loops (PV loops), which were measured using a solid-state Millar conductance catheter system. MI resulted in a characteristic decline in LV systolic parameters and an increase in diastolic parameters (Table 1). Cell sheet treatment improved both systolic and diastolic parameters (Table 1). Specifically, load-dependent parameters of systolic function: ejection fraction (EF), dP/dTmax, and cardiac index (CI) were decreased in MI rats and increased towards sham control with the cell sheet treatment (Table 1). Diastolic function parameters, dP/dTmin, relaxation constant (Tau), EDV, and EDP were increased in the MI rats and returned towards sham control parameters after sheet treatment (Table 1). However, load-independent systolic function, Emax, was decreased after MI. Treatment with human sheet improved Emax, while treatment with rat sheet had no effect (Table 1). Treatment with either rat or human sheets retarded LV remodelling; as such that it increased the ratio of anteriolateral wall thickness/LV inner diameter (t/Di) and wall thickness/LV outer diameter (t/Do) (see Supplementary material online, Table S3). However, human sheets appear to further improve LV remodelling compared with rat sheets as indicated by increased ratio of wall thickness to ventricular diameter and decreased both EDV and EDP (Table 1 and see Supplementary material online, Table S3).
Table 1 Hemodynamic parameters
Table 1. hemodynamic parameters


The majority of the cardiac progenitor cells delivered using our scaffold-free cell sheet survived after transplantation onto the infarcted heart. A significant percentage of transplanted cells migrated from the cell sheet to the site of infarction and differentiated into car-diomyocytes and vasculature leading to improving cardiac contractile function and retarding LV remodelling. Thus, delivery of cardiac progenitor cells together with cardiac mesenchymal cells in a form of scaffold-free cell sheet is an effective approach for cardiac regeneration after MI.
Consistent with previous studies,5,11 here we showed that cardio-spheres are composed of multipotent precursors, which have the capacity to differentiate to cardiomyocytes and other cardiac cell types. When we fractioned cardiospheres based on c-Kit expression, we identified two subsets: Kitþ /KDR2/low/Nkx2.5þ and Kit2/KDRþ/ Nkx2.52(Supplementary material online, Figure S3d), which are likely reflecting cardiac and vascular progenitors.20
In the present study, delivery of cardiac progenitor cells as a cell sheet facilitates cell survival after transplantation. Necrotic cores, commonly observed in tissue engineered patches,23,24 are absent in cardiosphere sheets prior to transplantation (Figure 1B and C). Poor cell survival is caused by multiple processes such as: ischemia from the lack of vasculature and anoikis due to cell detachment from sub-strate.25 A possible mechanism of cell survival within the sheet is the induction of neo-vessels soon after transplantation due to the presence of endothelial cells within the sheet before transplantation (Figure 10). The cell sheet continued to grow in vivo (Figure 2B and C), suppressed cardiac wall thinning, and prevented LV remodelling at 21 days after transplantation (see Supplementary material online, Table S3). This maybe due to the induction of neovascularization (Figure 3), which may prevents ischemia-induced cell death (Figure 2G). Another likely mechanism of cell survival is that the cells within the scaffold-free sheet maintained cell-to-cell adhesion16 as shown by ICAM expression (Figure 1F). The cells also exhibit C43-positive junctions (Figure 10, see Supplementary material online, Figure S6), which may facilitate electromechanical coupling between the transplanted cells and the native myocardium.
We observed cell migration from the sheet to the infarcted myocardium (Figure 4A and B, E and F), which may be facilitated by the strong expression of MMP2 in the cell sheet (Figure 1F). Although, the mechanism of cardiac progenitor cell migration remains unclear, previous observations showed that SDF-1 is upregulated after MI and plays a role in bone-marrow and cardiac stem cell migration.26,27 Our data suggest that SDF-1-CXCR4 axis plays, at least in part, a role in cardiac progenitor cell migration from cell sheet to the infarcted myocardium. This conclusion is based on the following observations: (1) cell sheet expresses CXCR4 prior to transplantation (Figure 1F), (2) migrated cells are located in the vicinity of SDF-1 release (Figure 5A and B), and (3) about 20% of migrated cells expressed CXCR4. Note, not all the migrated cells expressed CXCR4 suggesting other mechanisms are involved in cell migration (Figure 5C).
Here we report that implanting cardiosphere-generated cell sheet onto infarcted myocardium not only improved vascularization but also promoted cardiogenesis within the infarcted area (Figure 6). A larger number of newly formed cardiomyocytes were found deep within the infarct compared with the cell sheet periphery. Notably the transplantation of the cell sheet resulted in a significant improvement of the cardiac contractile function after MI, as was shown by an increase of EF and decrease of LV end diastolic pressure (Table 1).
The beneficial effect of cell sheet is, in part, due to the presence of a large number of activated cardiac mesenchymal stromal cells (myofibroblasts) within the sheet. Myofibroblasts are known to provide a mechanical support for grafted cells, facilitating contraction28 and to induce neovascularization through the release of cytokines.17 In addition, mesenchymal cells are uniquely immunotolerant. In xenograft models unmatched mesenchymal cells transplanted to the heart of immunocompetent rats were shown to suppress host immune response29 presumably due to inhibition of T-cell activation.30 Consistently with previous study from our laboratory,31 here, we demonstrated host tolerance to the cell sheet 21 days after MI. Finally, phase II and III clinical trials are currently undergoing in which allogeneic MSCs are used to treat MI in patients (Osiris Therapeutic, Inc.).
In summary, our results show that cardiac progenitor cells can be delivered as a cell sheet, composed of a layer of cardiac stromal cells impregnated with cardiospheres. After transplantation, cells from the cell sheet migrated to the infarct, partially driven by SDF-1 gradient, and differentiated into cardiomyocytes and vasculature. Transplantation of cell sheet was associated with prevention of LV remodelling, reconstitution of cardiac mass, reversal of wall thinning, and significant improvement in cardiac contractile function after MI. Our data also suggest that strategies, which utilize undigested cells, intact cell–cell interactions, and combined cell types such as our scaffold-free cell sheet should be considered in designing effective cell therapy.


Fuchs JR, Nasseri BA, Vacanti JP, Fauza DO. Postnatal myocardial augmentation with skeletal myoblast-based fetal tissue engineering. Surgery 2006;140:100–107.
Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P. Bone marrow stem cells regenerate infarcted myocardium. Pediatr Transplant 2003;7(Suppl. 3):86–88.
Kawamoto A, Tkebuchava T, Yamaguchi J, Nishimura H, Yoon YS, Milliken C et al. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003;107:461–468.
Iwasaki H, Kawamoto A, Ishikawa M, Oyamada A, Nakamori S, Nishimura H et al. Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction. Circulation 2006;113:1311–1325.
Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114: 763–776.
Oh H, Bradfute SB, Gallardo TD, Nakamura T, Gaussin V, Mishina Y et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci USA 2003;100:12313–12318.
Laugwitz KL, Moretti A, Lam J, Gruber P, Chen Y, Woodard S et al. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 2005;433: 647–653.
Pfister O, Mouquet F, Jain M, Summer R, Helmes M, Fine A et al. CD31- but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res 2005;97:52–61.
Dawn B, Stein AB, Urbanek K, Rota M, Whang B, Rastaldo R et al. Cardiac stem cells delivered intravascularly traverse the vessel barrier, regenerate infarcted myocardium, and improve cardiac function. Proc Natl Acad Sci USA 2005;102:3766–3771.


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Author and Reporter: Ritu Saxena, Ph.D.  

Mitochondria is an important cell organelle that is associated with several key cellular functions as energy production, anabolism, calcium homeostasis and cell programmed death, and any abnormalities occurring in mitochondria would lead to alteration of normal cellular function.

Role of mitochondria in cancer has long been implicated. Post published on September 1, 2012 ( presents a brief overview of the mechanisms by which mitochondrial defects could be associated with cancer. Different studies on various types of Cancers have tried to determine the mtDNA mutations and the mechanisms involved. An important aspect of cancerous progression is the cancer cell migration and it has been observed that mitochondrial dysfunction is involved in cancer cell migration. However, the molecular mechanism still needs to be deciphered.

A group from Taiwan recently published their findings in the Biochimica et Biophysica Acta journal stating that enhanced β5-integrin expression was involved in promoting cell migration in human gastric cancer cell line as a result of mitochondrial dysfunction.

The authors used human gastric cancer cell line, SC-M1 cells for their studies. The methodology followed was to first create mitochondrial dysfunction in the SC-M1 cells by the use of oxidative phosphorylation inhibitors: oligomycin (Complex V inhibitor) and antimycin A (Complex III inhibitor) thereby inhibiting mitochondrial function. The results indicated that impaired oxidative phosphorylation caused an increase in the intracellular Reactive Oxygen Species (ROS) that lead to an increased cell migration in SC-M1 cells.

Different types of integrin molecules have been implicated in cell migration. Hung et al extracted RNA and protein from SC-M1 cells in order to study the different types of integrins, and observed that the levels of β5-integrin were significantly upregulated in SC-M1 cells.  Simultaneously, the surface expression of the dimer- β5-integrin and αv-integrin, was studied in cancer cells with using FACS. The analysis revealed a higher surface expression of the dimer corresponding to the higher levels of the protein and RNA results of  β5-integrin expression in SC-M1 cells with mitochondrial dysfunction. Infact, a subpopulation of SC-M1 cells that showed higher migration capability (SC-M1-3rd) was observed to harbor a higher lever of β5-integrin expression, correlating β5-integrin expression with cell migration ability. The experiments supported the role of β5-integrin in cell migration in gastric cancer cells.

Finally, authors confirmed the in vitro results in the human gastric cancer samples. Immunohistochemical analysis revealed that β5-integrin was stained positive in around 73% of the cancer samples. Additionally, the higher expression levels of β5-integrin could be correlated with the invasive ability and more aggressive behavior of gastric cancer cells.

Authors stated “our study pinpoints another aspect that links the induction of intracellular ROS level in mitochondrial dysfunction gastric cancer cells with the activation of αvβ5-integrin. Taken together, the induction of β5-integrin is important to gastric cancer metastasis, especially in cancer cells that exhibit mitochondrial dysfunction.”

Thus, blockage of αvβ5-integrin function by antibodies might be tested as a potential therapy for preventing or delaying gastric cancer metastasis, especially in gastric cancers harboring mitochondrial dysfunction.


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