Binding of αvß1 and αvß6 integrins to tenascin-C induces epithelial-mesenchymal transition-like change of breast cancer cells.

Tenascin-C (TNC), a large hexameric extracellular glycoprotein, is a pleiotropic molecule with multiple domains binding to a variety of receptors mediating a wide range of cellular functions. We earlier reported that TNC induces epithelial-mesenchymal transition (EMT)-like change in breast cancer cells. In the present study, we clarified TNC receptor involvement in this process. Among integrins previously reported as TNC receptors, substantial expression of αv, α2, ß1 and ß6 subunits was detected by quantitative PCR and immunoblotting in MCF-7 cells. Integrin ß6 mRNA was remarkably upregulated by transforming growth factor (TGF)-ß1 treatment, and protein expression was prominently increased by additional exposure to TNC. Immunofluorescent labeling demonstrated integrin αvß6 accumulation in focal adhesions after TNC treatment, especially in combination with TGF-ß1. The α2 and ß1 subunits were mainly localized at cell-cell contacts, αv being found near cell cluster surfaces. Immunoprecipitation showed increase in αvß1 heterodimers, but not α2ß1, after TNC treatment. Activated ß1 subunits detected by an antibody against the Ca(2+)-dependent epitope colocalized with αv in focal adhesion complexes, associated with FAK phosphorylation at tyrosine 925. Neutralizing antibodies against αv and ß1 blocked EMT-like change caused by TNC alone. In addition, anti-αv and combined treatment with anti-ß1 and anti-αvß6 inhibited TGF-ß1/TNC-induced EMT, whereas either of these alone did not. Integrin subunits αv, ß1 and ß6, but not α2, bound to TNC immobilized on agarose beads in a divalent cation-dependent manner. Treatments with neutralizing antibodies against ß1 and αvß6 reduced αv subunit bound to the beads. Immunohistochemistry of these receptors in human breast cancer tissues demonstrated frequent expression of ß6 subunits in cancer cells forming scattered nests localized in TNC-rich stroma. These findings provide direct evidence that binding of αvß6 and αvß1 integrins to TNC as their essential ligand induces EMT-like change in breast cancer cells.

Deficiency of tenascin-C delays articular cartilage repair in mice.

OBJECTIVE: In human articular cartilage, tenascin-C (TN-C) expression decreases during maturation of chondrocytes, and almost disappears in adults; however, it reappears in damaged cartilage. To examine the effects of TN-C on cartilage degeneration and repair, we compared articular cartilage degeneration between wild-type (WT) and tenascin-C knockout mouse (TNKO) mice using a spontaneous osteoarthritis (OA) in aged joints and surgical OA model. In addition, we made full-thickness cartilage defects and compared the cartilage repair process between the two groups. METHODS: The surgical procedure to create degenerative OA model was performed by transecting the anterior cruciate ligament and medial collateral ligament. Full-thickness defects were created in the center of the femoral trochlea to evaluate cartilage repair. Sections of cartilage were stained with hematoxylin and eosin or safranin-O, and immunostaining for TN-C. The degrees of degeneration and repair were graded. RESULTS: In the WT surgical OA model, the articular cartilage was almost normal at 2 weeks, but safranin-O decreased staining at 4 weeks. In TNKO mice, safranin-O decreased staining at 2 weeks, and cartilage was injured intensely at 4 weeks. In the cartilage repair model, TN-C was expressed after 1 week, was strongly expressed in the upper layer of regenerated tissue after 3 weeks, and disappeared after 6 weeks. The defects were restored until 6 weeks in WT mice; however, defects in TNKO mice were filled with fibrous tissue with no cartilage-like tissue. CONCLUSIONS: This study revealed that cartilage repair in TNKO mice was significantly slower than that in WT mice and that the deficiency of TN-C progressed during cartilage degeneration.

Deficiency of tenascin-C attenuates liver fibrosis in immune-mediated chronic hepatitis in mice.

Tenascin-C (TNC), an extracellular matrix glycoprotein, is upregulated in chronic liver disease. Here, we investigated the contribution of TNC to liver fibrogenesis by comparing immune-mediated hepatitis in wild-type (WT) and TNC-null (TNKO) mice. Eight-week-old BALB/c mice received weekly intravenous injections of concanavalin A to induce hepatitis, and were sacrificed one week after the 3rd, 6th, 9th, and 12th injections. In WT livers, immunohistochemical staining revealed a gradual increase in TNC deposition. TNC mRNA levels also increased sequentially and peaked after the 9th injection. Collagen deposition, stained with picrosirius red, was significantly less intense in TNKO mice than in WT mice, and procollagen I and III transcripts were significantly upregulated in WT mice compared with TNKO mice. Inflammatory infiltrates were most prominent after the 3rd-6th injections in both groups and were less intense in TNKO mice than in WT mice. Interferon-gamma, tumour necrosis factor-alpha, and interleukin-4 mRNA levels were significantly higher in WT mice than in TNKO mice, while activated hepatic stellate cells (HSCs) and myofibroblasts, a cellular source of TNC and procollagens, were more common in WT livers. Transforming growth factor (TGF)-beta1 mRNA expression was significantly upregulated in WT mice, but not in TNKO mice. In conclusion, TNC can promote liver fibrogenesis through enhancement of inflammatory response with cytokine upregulation, HSC recruitment, and TGF-beta expression during progression of hepatitis to fibrosis.

Cooperation of oncogenic K-ras and p53 deficiency in pleomorphic rhabdomyosarcoma development in adult mice.

Human rhabdomyosarcomas (RMSs) frequently demonstrate genetic alterations in ras and p53. To investigate their possible involvement in the tumorigenesis, we generated a knock-in mouse line with oncogenic K-ras, conditionally expressed by Cre/LoxP system on a background of p53 alteration. Electroporation of Cre expression vector in skeletal muscle tissues resulted in the generation of tumor in adults with tumor incidences of 100% at 10 weeks and 40% at 15 weeks, in p53(-/-) and p53(-/+) backgrounds, respectively. The tumor histology was pleomorphic RMS with characteristic bizarre giant cells, positive for desmin and alpha-sarcomeric actin and exhibiting remarkable increase in total and phosphorylated extracellular signal-regulated protein kinase (ERK)1 and ERK2. Loss of the wild-type p53 was detected in K-rasG12V-expressed tumors of p53(-/+) mice. Early lesions 3 weeks after electroporation consisted of proliferating populations of myogenic progenitors, including stem cells positive for ScaI antigen, immature cells positive for desmin and neural cell adhesion molecule-positive myotubes. Thus, cooperation of oncogenic K-ras and p53 deficiency resulted in the development of pleomorphic RMS in adult mice, providing a useful mouse model for further detailed studies.

Involvement of tenascin-C and PG-M/versican in flexor tenosynovial pathology of idiopathic carpal tunnel syndrome.

Increased intra-carpal-tunnel pressure due to swelling of the flexor tenosynovium is the most probable pathological mechanism of idiopathic carpal tunnel syndrome (CTS). To clarify the role of tenascin-C and PG-M/versican, which have often been found to be involved in tissue remodeling and vascular stenosis in the pathogenesis of CTS, we histologically and biochemically examined the production of extracellular matrix in the flexor tenosynovium from 40 idiopathic CTS patients. Tenascin-C was temporarily expressed in the vessel wall, synovial lining and fibrous tissue, with expression regulated differently in each tissue. Tenascin-C expression by vessels correlated with disease duration and appeared to be involved in vascular lesion pathology. Morphometric analysis showed that tenascin-C expression by small arteries is correlated with PG-M/versican expression in surrounding connective tissue. PG-M/versican was also present at the neointima of severely narrowed vessels. Although tenascin-C expression by synovial lining and connective tissue shows marked regional variation and seems inconsistent, in vitro examination suggested that tenascin-C production by these tissues is regulated in response to mechanical strain on the flexor tenosynovium.

Interaction between cell and extracellular matrix in heart disease: multiple roles of tenascin-C in tissue remodeling.

The heart remodels myocardial tissue in physiological and pathological response. The cell-extracellular matrix (ECM) interaction provides not only structural and mechanical support but also important biological signaling during tissue remodeling. Among various ECM molecules, tenascin-C (TNC) is well known as a regulator of multiple cellular functions during embryogenesis, wound healing or cancer progression. In the heart, TNC appears in several important steps of embryonic development such as the initial differentiation of cardiomyocytes or coronary vasculo/angiogenesis, but it is not detected in a normal adult myocardium. However, TNC is found to re-express after myocardial injury and may regulate cellular behavior during tissue remodeling by modulating the attachment of cardiomyocytes to connective tissue, by enhancing migration and differentiation of myofibroblasts, and by inducing matrix metallo-proteinases. TNC also interacts with other ECM molecules and may modulate progression of fibrosis. Furthermore, transient and site specific expression of TNC closely associated with myocardial injury and inflammation suggests not only its key roles during tissue remodeling but also that TNC can be a marker for myocardial disease activity.

A single administration of interleukin-4 antagonistic mutant DNA inhibits allergic airway inflammation in a mouse model of asthma.

Interleukin 4 (IL-4) is essential for the switching of B cells to IgE antibody production and for the maturation of T helper (Th) cells toward the Th2 phenotype. These mechanisms are thought to play a crucial role in the pathogenesis of the allergic airway inflammation observed in asthma. In the present study, we examined the anti-inflammatory effects of DNA administration of murine IL-4 mutant Q116D/Y119D (IL-4 double mutant, IL-4DM), which binds to the IL-4 receptor alpha and is an antagonist for IL-4. Immunization of BALB/c mice with alum-adsorbed ovalbumin (OVA) followed by aspiration with aerosolized OVA resulted in the development of allergic airway inflammation. A single administration of IL-4DM DNA before the aerosolized OVA challenge protected the mice from the subsequent induction of allergic airway inflammation. Serum IgE level and extent of eosinophil infiltration in bronchoalveolar lavage (BAL) from IL-4DM DNA-administered mice were significantly lower than those in BAL from control plasmid-immunized mice. In our study, IL-4 or IL-4 mutants were not detected in sera from mice that had received a single administration of IL-4DM DNA. The results of this study provide evidence for the potential utility of IL-4 mutant antagonist DNA inoculation as an approach to gene therapy for asthma.

Serial extracellular matrix changes in neointimal lesions of human coronary artery after percutaneous transluminal coronary angioplasty: clinical significance of early tenascin-C expression.

It has become clear that deposition of extracellular matrix(ECM) proteins is a major cause of human restenosis after percutaneous coronary angioplasty (PTCA). To define the composition and organization of the involved ECM in human restenotic tissue, we morphologically and semiquantitatively analyzed specimens obtained by means of directional coronary atherectomy at various stages after PTCA with anti-fibronectin, tenascin-C, collagens I and III, and PG-M/versican antibodies. Tenascin-C deposition transiently increased within 1 month after PTCA, when smooth muscle cell migration and proliferation was active. Following the disappearance of tenascin-C, PG-M/versican accumulation increased and peaked between 1 month and 3 months when clinical restenosis was most actively progressing. At later stages, the PG-M/versican was replaced by a more mature ECM consisting of collagens I and III. The volume ratio of elastin remained at a low level throughout. Our results demonstrate that the matrix proteins of human restenotic lesions sequentially change after angioplasty and that tenascin-C could be a key molecule in the early stages.

Rho kinases play an obligatory role in vertebrate embryonic organogenesis.

Rho-associated kinases (Rho kinases), which are downstream effectors of RhoA GTPase, regulate diverse cellular functions including actin cytoskeletal organization. We have demonstrated that Rho kinases also direct the early stages of chick and mouse embryonic morphogenesis. We observed that Rho kinase transcripts were enriched in cardiac mesoderm, lateral plate mesoderm and the neural plate. Treatment of neurulating embryos with Y27632, a specific inhibitor of Rho kinases, blocked migration and fusion of the bilateral heart primordia, formation of the brain and neural tube, caudalward movement of Hensen's node, and establishment of normal left-right asymmetry. Moreover, Y27632 induced precocious expression of cardiac alpha-actin, an early marker of cardiomyocyte differentiation, coincident with the upregulated expression of serum response factor and GATA4. In addition, specific antisense oligonucleotides significantly diminished Rho kinase mRNA levels and replicated many of the teratologies induced by Y27632. Thus, our study reveals new biological functions for Rho kinases in regulating major morphogenetic events during early chick and mouse development.

Tenascin-C modulates adhesion of cardiomyocytes to extracellular matrix during tissue remodeling after myocardial infarction.

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays important roles in tissue remodeling. TNC is not normally expressed in adults but reappears under pathologic conditions. The present study was designed to clarify the contribution of TNC to ventricular remodeling after myocardial infarction. We examined the expression of TNC after experimental myocardial infarction in the rat by immunohistochemistry and in situ hybridization. Within 24 hours of permanent coronary ligation, interstitial fibroblasts in the border zone started to express TNC mRNA. The expression of TNC was down-regulated on Day 7 and was no longer apparent by Day 14 after infarction. During the healing process, TNC protein and TNC-producing cells were found at the edges of the residual myocardium. Some of the TNC-producing cells were immunoreactive for alpha-smooth muscle actin. In culture, TNC increased the number of cardiomyocytes attached to laminin but inhibited the formation of focal contacts at costameres. The results indicate that during the acute phase after myocardial infarction, interstitial cells in the border zone synthesize TNC, which may loosen the strong adhesion of surviving cardiomyocytes to connective tissue and thereby facilitate tissue reorganization.

beta1 integrin and organized actin filaments facilitate cardiomyocyte-specific RhoA-dependent activation of the skeletal alpha-actin promoter.

Activation of RhoA GTPase causes actin filament bundling into stress fibers, integrin clustering, and focal adhesion formation through its action on actin cytoskeleton organization. RhoA also regulates transcriptional activity of serum response factor (SRF). Recent studies in NIH 3T3 fibroblasts have shown that SRF activation by RhoA does not require an organized cytoskeleton and may be regulated by G-actin level. In cardiac myocytes, the organization of actin fibers into myofibrils is one of the primary characteristics of cardiac differentiation and hypertrophy. The primary purpose of this study was to examine if RhoA regulates SRF-dependent gene expression in neonatal cardiomyocytes in a manner different from that observed in fibroblasts. Our results show that RhoA-dependent skeletal alpha-actin promoter activation requires beta1 integrin and a functional cytoskeleton in cardiomyocytes but not in NIH 3T3 fibroblasts. Activation of the alpha-actin promoter by RhoA is greatly potentiated (up to 15-fold) by co-expression of the integrin beta1A or beta1D isoform but is significantly reduced by 70% with a co-expressed dominant negative mutant of beta1 integrin. Furthermore, clustering of beta1 integrin with anti-beta1 integrin antibodies potentiates synergistic RhoA and beta1 integrin activation of the alpha-actin promoter. Cytochalasin D and latrunculin B, inhibitors of actin polymerization, significantly reduced RhoA-induced activation of the alpha-actin promoter. Jasplakinolide, an actin polymerizing agent, mimics the synergistic effect of RhoA and beta1 integrin on the actin promoter. These observations support the concept that RhoA regulates SRF-dependent cardiac gene expression through cross-talk with beta1 integrin signal pathway via an organized actin cytoskeleton.

Expression of tenascin-C in stromal cells of the murine uterus during early pregnancy: induction by interleukin-1 alpha, prostaglandin E(2), and prostaglandin F(2 alpha).

Tenascin-C (TN-C), an extracellular matrix glycoprotein, is known to be expressed in uterine stroma in the peri-implantation period. Examination of the spatiotemporal pattern during early pregnancy using immunohistochemistry and in situ hybridization revealed TN-C expression in the stroma beneath the luminal epithelia of the murine endometrium on Days 0 and 1 of pregnancy, subsequent disappearance, and reappearance on Day 4. After decidualization, tissue around the deciduoma was positive. In situ hybridization demonstrated TN-C production by the stromal cells adjacent to the epithelia. To investigate the regulation of TN-C expression in vitro, murine uterine stromal and epithelial cells were isolated and cultured. Addition of interleukin-1 alpha (IL-1 alpha) and prostaglandin E(2) (PGE(2)) and F(2 alpha) (PGF(2 alpha)) induced TN-C expression in the stromal cells at both protein and mRNA levels, while the sex steroid hormones, progesterone and ss-estradiol, exerted little effect. Immunohistochemistry using anti-IL-1 alpha antibody showed epithelial cells to be positive on Days 2-4 of pregnancy, and addition of progesterone but not ss-estradiol enhanced IL-1 alpha expression in epithelial cells in vitro. In a culture insert system, TN-C expression by stromal cells cocultured with epithelial cells was induced by addition of progesterone alone that was blocked by additions of anti-IL-1 alpha antibody. Collectively, these findings indicate that TN-C expression in the preimplantation period is under the control of progesterone, but not directly, possibly by the paracrine and autocrine intervention of IL-1 alpha secreted by epithelial cells and PGE(2) and PGF(2 alpha) secreted by stromal cells.

Immunohistochemical localization of type I collagen, fibronectin and tenascin C during embryonic osteogenesis in the dentary of mandibles and tibias in rats.

Type I collagen, fibronectin and tenascin C play an important role in regulating early osteoblast differentiation, but the temporal and spatial relationship of their localization during embryonic osteogenesis in vivo is notknown. The present study was designed to localize these three molecules in the dentary of mandibles and tibias in rat embryos using immunohistochemistry. Serial paraffin sections were cut and adjacent sections were processed for von Kossa staining or immunohistochemistry for type I collagen, fibronectin and tenascin C. In the dentary, tenascin C was localized within and around the mesenchymal cell condensation in embryos at 14 days in utero. The bone matrix at 15 days showed immunoreactivity for both type I collagen and fibronectin. The immunoreactivity of type I collagen was persistent, whereas that of fibronectin decreased with age of embryos. In tibias, tenascin C was localized in the perichondral mesenchymal tissue at 17 days. Immunoreactivity for type I collagen was persistent in the bone matrix, whereas the tibial bone showed little immunoreactivity for fibronectin at any embryonic age examined. The present study demonstrated characteristic localization of type I collagen, fibronectin and tenascin C during embryonic osteogenesis in the dentary of mandibles and tibias.

Chronic colchicine administration attenuates cardiac hypertrophy in spontaneously hypertensive rats.

To determine whether the long-term inhibition of microtubule integrity in vivo by colchicine could attenuate the development of cardiac hypertrophy, we studied five groups of rats: Wistar-Kyoto rats receiving saline for 4 weeks (WKYsaline); WKY receiving colchicine, which depolymerizes microtubules (WKYcolchicine); spontaneously hypertensive rats receiving saline (SHRsaline); SHRs receiving colchicine (SHRcolchicine); and SHRs receiving lumicolchicine, an inactive stereoisomer of colchicine (SHRlumicolchicine). Seven-week-old animals were administered drugs or control substances via alternate day intraperitoneal injection for a period of 4 weeks. Dosage was gradually increased from 0.6 to 1.0 mg/kg to avoid drug toxicity. Depolymerization of myocardial microtubules by the in vivo administration of colchicine into the rats was confirmed by both Western blot analysis and immunofluorescence of tubulin protein in the hearts. Body weight (BW) was lower, while systolic blood pressure was significantly elevated in SHRs vs the WKY rats. No significant difference was found in either of these parameters between the control or treatment groups of each strain. Left ventricular (LV) weight-to-BW ratio was elevated and showed significant increases in the SHRs as compared to WKY animals, indicative of cardiac hypertrophy. When the SHRs were treated with colchicine but not vehicle or lumicolchicine, LV/BW was similar to the WKY. Changes of myocyte cross-sectional area determined using LV mid-free wall specimens were concordant with the LV/BW data. No significant changes were found in collagen volume fraction between groups. Thus the inhibition of microtubule polymerization abolished the progression of cardiac myocyte hypertrophy in SHRs independently of blood pressure.

Low cytoplasmic pH causes fragmentation and dispersal of the Golgi apparatus in human hepatoma cells.

The centrosomal localization of the Golgi apparatus in interphase cells is thought to be maintained by retrograde microtubule-based motility. It is well established that, when intracellular pH is lowered, lysosomes and endosomes, also showing pericentrosomal localization, translocate towards the plus ends of microtubules within 15 min. In this study, we found that prolonged incubation in low pH medium (pH 6.6) with 20 mM Na acetate induced the fragmentation and dispersal of the Golgi apparatus in the human hepatoma cell line PLC/PRF/5. The fraction of Golgi-dispersed cells increased in a time-dependent manner, and reached over 60% after the 16-h incubation. The cytoplasmic pH was dropped to approximately 7.10. Replacement with normal pH medium restored the structure and localization of the apparatus within 30 min. In the low pH condition, the microtubular network and endoplasmic reticulum appeared normal, and cytoplasmic dynein was still bound to the fragmented Golgi membranes. These findings suggest that low cytoplasmic pH suppresses the retrograde movement of the Golgi apparatus as well as that of lysosomes and endosomes.

Vinculin, Talin, Integrin alpha6beta1 and laminin can serve as components of attachment complex mediating contraction force transmission from cardiomyocytes to extracellular matrix.

Recently, we reported that cardiomyocytes adhere to extracellular matrix at costameres, the striated distribution of vinculin between Z-lines and the sarcolemma, where transmission of contraction forces from myofibrils to the extracellular matrix occurs. To identify other molecules involved in force transmission at costameres, we examined adult rat and embryonic chick cardiomyocytes cultured on coverslips or flexible thin silicone rubber substrata. Immunolocalization of talin showed a costameric, striated distribution, which corresponded to dark contacts with interference reflection microscopy. The molecules involved in substrate adhesion were cross-linked with the non-penetrating cross-linking agent Bis(sulfosuccinimidyl)-suberate and detected by immunohistochemical staining with anti-alpha6, alpha3, alphav, or beta1 integrin antibodies. Both alpha6 and beta1 showed costameric distributions, but alpha3 and alpha(v) did not. The distribution of laminin after cross-linking and extraction also showed a costameric distribution. When anti-integrin beta1 antibody was added to live cardiomyocytes grown on the silicone rubber substratum, the transmission of contraction forces was inhibited. These findings suggest that vinculin, talin, integrin alpha6beta1 and laminin system can be involved in transmission of contraction force to the extracellular matrix.

Role of microtubules in the viscoelastic properties of isolated cardiac muscle.

Myocardial viscoelastic properties are determined by both interstitial collagens and intramocyte structures, including sarcolemma, contractile proteins and the cytoskeleton. It is not known whether myocyte microtubules are significant constituents that contribute to the viscoelastic properties of cardiac muscle. We examined the passive properties of isolated right-ventricular papillary muscles before and after altering the polymerization states of microtubules. The muscles were subjected to sinusoidal changes in length (strain) and the resultant changes in resting tension (stress) were measured. The elastic constant was determined by the slope of the stress-strain relation during the slow increase in muscle length (duration 60 s). The viscous constant was determined by the loop area between the stress-strain relation obtained during the rapid increase and decrease in muscle length (duration 1 s). Colchicine (1 micromol/l, 1 h), which depolymerized microtubules, had little effect on either the elastic constant or viscous constant. In contrast, taxol (10 micromol/l), which hyperpolymerized and stabilized microtubules, exerted a time-dependent increase in the viscous constant (133+/-9% of control; n=9, P<0.05), but did not affect the elastic constant (18. 9+/-2.2 to 17.7+/-2.1; n=7, P=n.s.). The increase of viscosity by taxol closely paralleled the increase in the strain rate. The specificity of each pharmacological intervention for the microtubule polymerization state was confirmed by both a Western blot analysis and the immunofluorescence micrographs of myocyte tubulin. Like other cytoskeleton and extracellular collagens, the increase in the myocyte microtubule density was able to modify the viscous component of the passive properties of the isolated cardiac muscle.

Microtubules are involved in early hypertrophic responses of myocardium during pressure overload.

Mechanical overloading to cardiac muscle causes fetal contractile protein gene expression and acceleration of protein synthesis. Myocyte microtubules might be involved in these pressure overload-induced hypertrophic responses. We assessed c-fos and fetal contractile protein genes such as beta-myosin heavy chain (MHC) and alpha-skeletal actin using Northern blot analysis and quantified total cardiac protein, DNA, and RNA content in the left ventricular myocardium obtained from four groups of rats: sham-operated rats; sham-operated rats treated with colchicine, which depolymerized microtubules; rats in which acute pressure overload was imposed by abdominal aortic constriction for 3 days (AoC); and AoC rats treated with colchicine (AoC + colchicine). Systolic arterial pressure was elevated to a similar degree in AoC and AoC + colchicine rats. c-fos and beta-MHC mRNA levels were significantly upregulated in AoC rats, which was attenuated by microtubule inhibition. Both RNA content and RNA-to-DNA ratio, the index of the protein synthesis capacity, were increased in AoC rats, which effect was also abolished by colchicine. Furthermore, induction of nonfunctioning microtubules by taxol or deuterium oxide exerted the same inhibitory effects. Thus the hypertrophic responses of the myocardium during pressure overload might depend on the integrity of myocyte microtubules.

Terminal warm blood cardioplegia improves cardiac function through microtubule repolymerization.

BACKGROUND: To elucidate the mechanisms responsible for the beneficial effects of terminal warm blood cardioplegia, we studied dynamic change in microtubules induced by cold cardioplegia followed by rewarming. Further, we investigated the relationship between cardiac function and morphologic changes in microtubules caused by hyperkalemic, hypocalcemic warm cardioplegia during initial reperfusion. METHODS: In protocol 1 isolated rat hearts were perfused at 37 degrees C with Krebs-Henseleit buffer (KHB). After 3 hours of hypothermic cardiac arrest at 10 degrees C, hearts were reperfused at 37 degrees C with one of two buffers: group C, 60-minute reperfusion with KHB (K+, 5.9 mmol/L; Ca2+, 2.5 mmol/L); and group TC, 10-minute initial reperfusion with modified KHB (K+, 15 mmol/L; Ca2+, 0.25 mmol/L), followed by 50 minutes of reperfusion with KHB. Cardiac function after reperfusion was determined as a percentage of the prearrest value. In protocol 2 hearts were perfused at 37 degrees C with KHB containing colchicine (10(-5) mol/L) for 60 minutes. RESULTS: There was spontaneous contractile recovery after 10 minutes of initial reperfusion in hearts from group TC as well as improved cardiac function after 15, 30, and 60 minutes of reperfusion compared with that in group C. Immunohistochemical staining and immunoblot analysis demonstrated microtubule depolymerization during hypothermic cardiac arrest and complete repolymerization after 10 minutes of reperfusion with warm buffers in both groups. Colchicine-induced microtubule depolymerization is associated with deterioration of cardiac function. CONCLUSIONS: One mechanism responsible for improved cardiac function mediated by terminal warm blood cardioplegia is the restart of contraction after complete microtubule repolymerization.