A new hemostatic agent composed of Zn2+-enriched Ca2+ alginate activates vascular endothelial cells in vitro and promotes tissue repair in vivo.

To control capillary bleeding, surgeons may use absorbable hemostatic agents, such as Surgicel® and TachoSil®. Due to their slow resorption, their persistence in situ can have a negative impact on tissue repair in the resected organ. To avoid complications and obtain a hemostatic agent that promotes tissue repair, a zinc-supplemented calcium alginate compress was developed: HEMO-IONIC®. This compress is non-absorbable and is therefore removed once hemostasis has been achieved. After demonstrating the hemostatic efficacy and stability of the blood clot obtained with HEMO-IONIC, the impact of Surgicel, TachoSil, and HEMO-IONIC on cell activation and tissue repair were compared (i) in vitro on endothelial cells, which are essential to tissue repair, and (ii) in vivo in a mouse skin excision model. In vitro, only HEMO-IONIC maintained the phenotypic and functional properties of endothelial cells and induced their migration. In comparison, Surgicel was found to be highly cytotoxic, and TachoSil inhibited endothelial cell migration. In vivo, only HEMO-IONIC increased angiogenesis, the recruitment of cells essential to tissue repair (macrophages, fibroblasts, and epithelial cells), and accelerated maturation of the extracellular matrix. These results demonstrate that a zinc-supplemented calcium alginate, HEMO-IONIC, applied for 10 min at the end of surgery and then removed has a long-term positive effect on all phases of tissue repair.

Effect of CFTR correctors on the traffic and the function of intracellularly retained ABCB4 variants.

BACKGROUND & AIM: ABCB4 is expressed at the canalicular membrane of hepatocytes. This ATP-binding cassette (ABC) transporter is responsible for the secretion of phosphatidylcholine into bile canaliculi. Missense genetic variations of ABCB4 are correlated with several rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). In a repurposing strategy to correct intracellularly retained ABCB4 variants, we tested 16 compounds previously validated as cystic fibrosis transmembrane conductance regulator (CFTR) correctors. METHODS: The maturation, intracellular localization and activity of intracellularly retained ABCB4 variants were analyzed in cell models after treatment with CFTR correctors. In addition, in silico molecular docking calculations were performed to test the potential interaction of CFTR correctors with ABCB4. RESULTS: We observed that the correctors C10, C13, and C17, as well as the combinations of C3 + C18 and C4 + C18, allowed the rescue of maturation and canalicular localization of four distinct traffic-defective ABCB4 variants. However, such treatments did not permit a rescue of the phosphatidylcholine secretion activity of these defective variants and were also inhibitory of the activity of wild type ABCB4. In silico molecular docking analyses suggest that these CFTR correctors might directly interact with transmembrane domains and/or ATP-binding sites of the transporter. CONCLUSION: Our results illustrate the uncoupling between the traffic and the activity of ABCB4 because the same molecules can rescue the traffic of defective variants while they inhibit the secretion activity of the transporter. We expect that this study will help to design new pharmacological tools with potential clinical interest.

TGR5 controls bile acid composition and gallbladder function to protect the liver from bile acid overload.

BACKGROUND & AIMS: As the composition of the bile acid (BA) pool has a major impact on liver pathophysiology, we studied its regulation by the BA receptor Takeda G protein coupled receptor (TGR5), which promotes hepatoprotection against BA overload. METHODS: Wild-type, total and hepatocyte-specific TGR5-knockout, and TGR5-overexpressing mice were used in: partial (66%) and 89% extended hepatectomies (EHs) upon normal, ursodeoxycholic acid (UDCA)- or cholestyramine (CT)-enriched diet, bile duct ligation (BDL), cholic acid (CA)-enriched diet, and TGR5 agonist (RO) treatments. We thereby studied the impact of TGR5 on: BA composition, liver injury, regeneration and survival. We also performed analyses on the gut microbiota (GM) and gallbladder (GB). Liver BA composition was analysed in patients undergoing major hepatectomy. RESULTS: The TGR5-KO hyperhydrophobic BA composition was not directly related to altered BA synthesis, nor to TGR5-KO GM dysbiosis, as supported by hepatocyte-specific KO mice and co-housing experiments, respectively. The TGR5-dependent control of GB dilatation was crucial for BA composition, as determined by experiments including RO treatment and/or cholecystectomy. The poor TGR5-KO post-EH survival rate, related to exacerbated peribiliary necrosis and BA overload, was improved by shifting BAs toward a less toxic composition (CT treatment). After either BDL or a CA-enriched diet with or without cholecystectomy, we found that GB dilatation had strong TGR5-dependent hepatoprotective properties. In patients, a more hydrophobic liver BA composition was correlated with an unfavourable outcome after hepatectomy. CONCLUSIONS: BA composition is crucial for hepatoprotection in mice and humans. We indicate TGR5 as a key regulator of BA profile and thereby as a potential hepatoprotective target under BA overload conditions. LAY SUMMARY: Through multiple in vivo experimental approaches in mice, together with a patient study, this work brings some new light on the relationships between biliary homeostasis, gallbladder function, and liver protection. We showed that hepatic bile acid composition is crucial for optimal liver repair, not only in mice, but also in human patients undergoing major hepatectomy.

Hepatoprotective impact of the bile acid receptor TGR5.

During liver repair after injury, bile secretion has to be tightly modulated in order to preserve liver parenchyma from bile acid (BA)-induced injury. The mechanisms allowing the liver to maintain biliary homeostasis during repair after injury are not completely understood. Besides their historical role in lipid digestion, bile acids (BA) and their receptors constitute a signalling network with multiple impacts on liver repair, both stimulating regeneration and protecting the liver from BA overload. BA signal through nuclear (mainly Farnesoid X Receptor, FXR) and membrane (mainly G Protein-coupled BA Receptor 1, GPBAR-1 or TGR5) receptors to elicit a wide array of biological responses. While a great number of studies have been dedicated to the hepato-protective impact of FXR signalling, TGR5 is by far less explored in this context. Because the liver has to face massive and potentially harmful BA overload after partial ablation or destruction, BA-induced protective responses crucially contribute to spare liver repair capacities. Based on the available literature, the TGR5 BA receptor protects the remnant liver and maintains biliary homeostasis, mainly through the control of inflammation, biliary epithelial barrier permeability, BA pool hydrophobicity and sinusoidal blood flow. Mouse experimental models of liver injury reveal that in the lack of TGR5, excessive inflammation, leaky biliary epithelium and hydrophobic BA overload result in parenchymal insult and compromise optimal restoration of a functional liver mass. Translational perspectives are thus opened to target TGR5 with the aim of protecting the liver in the context of injury and BA overload.

TGR5-dependent hepatoprotection through the regulation of biliary epithelium barrier function.

OBJECTIVE: We explored the hypothesis that TGR5, the bile acid (BA) G-protein-coupled receptor highly expressed in biliary epithelial cells, protects the liver against BA overload through the regulation of biliary epithelium permeability. DESIGN: Experiments were performed under basal and TGR5 agonist treatment. In vitro transepithelial electric resistance (TER) and FITC-dextran diffusion were measured in different cell lines. In vivo FITC-dextran was injected in the gallbladder (GB) lumen and traced in plasma. Tight junction proteins and TGR5-induced signalling were investigated in vitro and in vivo (wild-type [WT] and TGR5-KO livers and GB). WT and TGR5-KO mice were submitted to bile duct ligation or alpha-naphtylisothiocyanate intoxication under vehicle or TGR5 agonist treatment, and liver injury was studied. RESULTS: In vitro TGR5 stimulation increased TER and reduced paracellular permeability for dextran. In vivo dextran diffusion after GB injection was increased in TGR5-knock-out (KO) as compared with WT mice and decreased on TGR5 stimulation. In TGR5-KO bile ducts and GB, junctional adhesion molecule A (JAM-A) was hypophosphorylated and selectively downregulated among TJP analysed. TGR5 stimulation induced JAM-A phosphorylation and stabilisation both in vitro and in vivo, associated with protein kinase C-ζ activation. TGR5 agonist-induced TER increase as well as JAM-A protein stabilisation was dependent on JAM-A Ser285 phosphorylation. TGR5 agonist-treated mice were protected from cholestasis-induced liver injury, and this protection was significantly impaired in JAM-A-KO mice. CONCLUSION: The BA receptor TGR5 regulates biliary epithelial barrier function in vitro and in vivo through an impact on JAM-A expression and phosphorylation, thereby protecting liver parenchyma against bile leakage.

The P2X4 purinergic receptor regulates hepatic myofibroblast activation during liver fibrogenesis.

BACKGROUND & AIMS: Liver fibrosis is characterized by the accumulation of extracellular matrix produced by hepatic myofibroblasts (hMF), the activation of which is critical to the fibrogenic process. Extracellular ATP, released by dying or stressed cells, and its purinergic receptors, constitute a powerful signaling network after injury. Although the purinergic receptor P2X4 (P2RX4) is highly expressed in the liver, its functions in hMF had never been investigated during liver fibrogenesis. METHODS: In vivo, bile duct ligation was performed and methionine- and choline-deficient diet administered in wild-type and P2x4 knock-out (P2x4-KO) mice. In vitro, hMF were isolated from mouse (wild-type and P2x4-KO) and human liver. P2X4 pharmacological inhibition (in vitro and in vivo) and P2X4 siRNAs (in vitro) were used. Histological, biochemical and cell culture analysis allowed us to study P2X4 expression and its involvement in the regulation of fibrogenic and fibrolytic factors, as well as of hMF activation markers and properties. RESULTS: P2X4 genetic invalidation or pharmacological inhibition protected mice from liver fibrosis and hMF accumulation after bile duct ligation or methionine- and choline-deficient diet. Human and mouse hMFs expressed P2X4, mainly in lysosomes. Invalidation of P2X4 in human and mouse hMFs blunted their activation marker expression and their fibrogenic properties. Finally, we showed that P2X4 regulates calcium entry and lysosomal exocytosis in hMF, impacting on ATP release, profibrogenic secretory profile, and transcription factor activation. CONCLUSION: P2X4 expression and activation is critical for hMF to sustain their activated and fibrogenic phenotype. Therefore, the inactivation of P2X4 may be of therapeutic interest during liver fibrotic diseases. LAY SUMMARY: During chronic injury, the liver often repairs with fibrotic tissue, which impairs liver function, and for which there is currently no treatment. We found that a previously unexplored pathway involving the purinergic receptor P2X4, can modulate fibrotic liver repair. Therefore, this receptor could be of interest in the development of novel therapies for fibrotic liver diseases.

The P2X4 purinergic receptor impacts liver regeneration after partial hepatectomy in mice through the regulation of biliary homeostasis.

UNLABELLED: Many regulatory pathways are involved in liver regeneration after partial hepatectomy (PH), to initiate growth, protect liver cells, and sustain remnant liver functions. Extracellular adenosine triphosphate rises in blood and bile after PH and contributes to liver regeneration, although purinergic receptors and mechanisms remain to be precisely explored. In this work we analyzed during regeneration after PH the involvement of P2X4 purinergic receptors, highly expressed in the liver. P2X4 receptor expression in the liver, liver histology, hepatocyte proliferation, plasma bile acid concentration, bile flow and composition, and lysosome distribution in hepatocytes were studied in wild-type and P2X4 knockout (KO) mice, before and after PH. P2X4 receptors were expressed in hepatocytes and Kupffer cells; in hepatocytes, P2X4 was concentrated in subcanalicular areas closely costained with lysosomal markers. After PH, delayed regeneration, hepatocyte necrosis, and cholestasis were observed in P2X4-KO mice. In P2X4-KO mice, post-PH biliary adaptation was impaired with a smaller increase in bile flow and HCO3 (-) biliary output, as well as altered biliary composition with reduced adenosine triphosphate and lysosomal enzyme release. In line with these data, lysosome distribution and biogenesis were altered in P2X4-KO compared with wild-type mice. CONCLUSION: During liver regeneration after PH, P2X4 contributes to the complex control of biliary homeostasis through mechanisms involving pericanalicular lysosomes, with a resulting impact on hepatocyte protection and proliferation. (Hepatology 2016;64:941-953).

The receptor TGR5 protects the liver from bile acid overload during liver regeneration in mice.

UNLABELLED: Many regulatory pathways are involved in liver regeneration after partial hepatectomy (PH) to initiate growth, protect liver cells, and sustain functions of the remnant liver. Bile acids (BAs), whose levels rise in the blood early after PH, stimulate both hepatocyte proliferation and protection, in part through their binding to the nuclear farnesoid X receptor (FXR). However, the effect of the BA receptor, TGR5 (G-protein-coupled BA receptor 1) after PH remains to be studied. Liver histology, hepatocyte proliferation, BA concentrations (plasma, bile, liver, urine, and feces), bile flow and composition, and cytokine production were studied in wild-type (WT) and TGR5 KO (knockout) mice before and after PH. BA composition (plasma, bile, liver, urine, and feces) was more hydrophobic in TGR5 KO than in WT mice. After PH, severe hepatocyte necrosis, prolonged cholestasis, exacerbated inflammatory response, and delayed regeneration were observed in TGR5 KO mice. Although hepatocyte adaptive response to post-PH BA overload was similar in WT and TGR5 KO mice, kidney and biliary adaptive responses were strongly impaired in TGR5 KO mice. Cholestyramine treatment, as well as Kupffer cell depletion, significantly improved the post-PH TGR5 KO mice phenotype. After bile duct ligation or upon a cholic acid-enriched diet, TGR5 KO mice exhibited more severe liver injury than WT as well as impaired BA elimination in urine. CONCLUSION: TGR5 is crucial for liver protection against BA overload after PH, primarily through the control of bile hydrophobicity and cytokine secretion. In the absence of TGR5, intrahepatic stasis of abnormally hydrophobic bile and excessive inflammation, in association with impaired bile flow adaptation and deficient urinary BA efflux, lead to BA overload-induced liver injury and delayed regeneration.

Calcium signalling and liver regeneration.

After partial hepatectomy (PH) the initial mass of the organ is restored through a complex network of cellular interactions that orchestrate both proliferative and hepatoprotective signalling cascades. Among agonists involved in this network many of them drive Ca(2+) movements. During liver regeneration in the rat, hepatocyte cytosolic Ca(2+) signalling has been shown on the one hand to be deeply remodelled and on the other hand to enhance progression of hepatocytes through the cell cycle. Mechanisms through which cytosolic Ca(2+) signals impact on hepatocyte cell cycle early after PH are not completely understood, but at least they include regulation of immediate early gene transcription and ERK and CREB phosphorylation. In addition to cytosolic Ca(2+), there is also evidence that mitochondrial Ca(2+) and also nuclear Ca(2+) may be critical for the regulation of liver regeneration. Finally, Ca(2+) movements in hepatocytes, and possibly in other liver cells, not only impact hepatocyte progression in the cell cycle but more generally may regulate cellular homeostasis after PH.

Immediate neuroendocrine signaling after partial hepatectomy through acute portal hyperpressure and cholestasis.

BACKGROUND & AIMS: Early neuroendocrine pathways contribute to liver regeneration after partial hepatectomy (PH). We investigated one of these pathways involving acute cholestasis, immediate portal hyperpressure, and arginine vasopressin (AVP) secretion. METHODS: Surgical procedure (PH, Portal vein stenosis (PVS), bile duct ligation (BDL), spinal cord lesion (SCL)) and treatments (capsaicin, bile acids (BA), oleanolic acid (OA)) were performed on rats and/or wild type or TGR5 (GPBAR1) knock-out mice. In these models, the activation of AVP-secreting supraoptic nuclei (SON) was analyzed, as well as plasma BA, AVP, and portal vein pressure (PVP). Plasma BA, AVP, and PVP were also determined in human living donors for liver transplantation. RESULTS: Acute cholestasis (mimicked by BDL or BA injection) as well as portal hyperpressure (mimicked by PVS) independently activated SON and AVP secretion. BA accumulated in the brain after PH or BDL, and TGR5 was expressed in SON. SON activation was mimicked by the TGR5 agonist OA and inhibited in TGR5 KO mice after BDL. An afferent nerve pathway also contributed to post-PH AVP secretion, as capsaicin treatment or SCL resulted in a weaker SON activation after PH. CONCLUSIONS: After PH in rodents, acute cholestasis and portal hypertension, via the nervous and endocrine routes, stimulate the secretion of AVP that may protect the liver against shear stress and bile acids overload. Data in living donors suggest that this pathway may also operate in humans.

Cytosolic calcium regulates liver regeneration in the rat.

UNLABELLED: Liver regeneration is regulated by growth factors, cytokines, and other endocrine and metabolic factors. Calcium is important for cell division, but its role in liver regeneration is not known. The purpose of this study was to understand the effects of cytosolic calcium signals in liver growth after partial hepatectomy (PH). The gene encoding the calcium-binding protein parvalbumin (PV) targeted to the cytosol using a nuclear export sequence (NES), and using a discosoma red fluorescent protein (DsR) marker, was transfected into rat livers by injecting it, in recombinant adenovirus (Ad), into the portal vein. We performed two-thirds PH 4 days after Ad-PV-NES-DsR or Ad-DsR injection, and liver regeneration was analyzed. Calcium signals were analyzed with fura-2-acetoxymethyl ester in hepatocytes isolated from Ad-infected rats and in Ad-infected Hela cells. Also, isolated hepatocytes were infected with Ad-DsR or Ad-PV-NES-DsR and assayed for bromodeoxyuridine incorporation. Ad-PV-NES-DsR injection resulted in PV expression in the hepatocyte cytosol. Agonist-induced cytosolic calcium oscillations were attenuated in both PV-NES-expressing Hela cells and hepatocytes, as compared to DsR-expressing cells. Bromodeoxyuridine incorporation (S phase), phosphorylated histone 3 immunostaining (mitosis), and liver mass restoration after PH were all significantly delayed in PV-NES rats. Reduced cyclin expression and retinoblastoma protein phosphorylation confirmed this observation. PV-NES rats exhibited reduced c-fos induction and delayed extracellular signal-regulated kinase 1/2 phosphorylation after PH. Finally, primary PV-NES-expressing hepatocytes exhibited less proliferation and agonist-induced cyclic adenosine monophosphate responsive element binding and extracellular signal-regulated kinase 1/2 phosphorylation, as compared with control cells. CONCLUSION: Cytosolic calcium signals promote liver regeneration by enhancing progression of hepatocytes through the cell cycle.

ATP release after partial hepatectomy regulates liver regeneration in the rat.

BACKGROUND & AIMS: Paracrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger, remains unclear. The physiological release of ATP into extracellular milieu and its impact on regeneration after partial hepatectomy were investigated in this study. METHODS: Hepatic ATP release after hepatectomy was examined in the rat and in human living donors for liver transplantation. Quinacrine was used for in vivo staining of ATP-enriched compartments in rat liver sections and isolated hepatocytes. Rats were treated with an antagonist for purinergic receptors (Phosphate-6-azo(benzene-2,4-disulfonic acid), PPADS), and liver regeneration after hepatectomy was analyzed. RESULTS: A robust and transient ATP release due to acute portal hyperpressure was observed immediately after hepatectomy in rats and humans. Clodronate liposomal pre-treatment partly inhibited ATP release in rats. Quinacrine-stained vesicles, co-labeled with a lysosomal marker in liver sections and isolated hepatocytes, were predominantly detected in periportal areas. These vesicles significantly disappeared after hepatectomy, in parallel with a decrease in liver ATP content. PPADS treatment inhibited hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in bromodeoxyuridine incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early gene induction. CONCLUSION: Extracellular ATP is released immediately after hepatectomy from hepatocytes and Kupffer cells under mechanical stress and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP is released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration.

Rapid procoagulant phosphatidylserine exposure relies on high cytosolic calcium rather than on mitochondrial depolarization.

OBJECTIVE: Relationships between intracellular Ca(2+) concentration ([Ca(2+)](cyt)) and apoptotic events, such as mitochondrial depolarization (DeltaPsim loss) and Bcl-2 and Bad phosphorylation, were analyzed in platelets and Jurkat cells in relation to rapid procoagulant phosphatidylserine (PS) exposure. METHODS AND RESULTS: Platelets were stimulated with A23187, thapsigargin (TG) and thrombin plus convulxin (Thr/Cvx), and Jurkat cells with ionomycin, in the presence or absence of cyclosporin A (CsA), a mitochondrial permeability transition pore inhibitor. DeltaPsim loss occurred when platelets were stimulated in Ca(2+) medium in conditions exposing PS, but also in EGTA medium. CsA inhibited PS exposure, [Ca(2+)](cyt) increase, and DeltaPsim loss in platelets stimulated with TG and Thr/Cvx, but had no inhibitory effect on A23187 stimulation. CsA reduced TG-induced Ca(2+) release from the endoplasmic reticulum and, consequently, external Ca(2+) influx. In ionomycin-stimulated Jurkat cells, rapid PS exposure was evidenced but not DeltaPsim loss, and CsA did not inhibit the process. The status of phosphorylated Bad and Bcl-2 in both cell types remained unchanged on stimulation. CONCLUSIONS: Whether DeltaPsim loss occurs or not, PS exposure is triggered by a high [Ca(2+)](cyt) increase. Data further demonstrate that CsA prevents membrane scrambling by inhibiting the high [Ca(2+)](cyt) increase, independently of its effect on mitochondrial permeability transition pore.

Can cold or heat shock improve skeletal myoblast engraftment in infarcted myocardium?

OBJECTIVE: Cell death remains a major limitation of skeletal myoblast (SM) transplantation but the patterns of cell survival and proliferation in heart and their potential modulation by thermic stresses like heat shock (HS) and cryopreservation (Cryo) are still incompletely characterized. METHODS: To track SMs in situ, we developed a dual-marker system based on the semiconservative expression of the foreign soluble protein, beta-Galactosidase (beta-Gal) and the constitutive expression of the Y chromosome in a myocardial infarction model. Control medium or Lewis male rat SMs (fresh or subjected to Cryo or HS) were injected in Lewis female rats. RESULTS: There was a massive cell loss early after transplantation in the fresh group, which was only partially compensated for by a subsequent proliferation. Conversely, both Cryo and HS significantly improved early cell survival but blunted subsequent proliferation so that, at 15 days posttransplantation, the total number of engrafted donor-derived Y-positive cells did not differ significantly between the three groups. Most of them expressed a skeletal muscle phenotype. CONCLUSIONS: These data confirm the high death rate of in-scar transplanted myoblasts, demonstrate the ability of those that survive to proliferate and differentiate along the myogenic pathway but do not support the efficacy of either Cryo or HS for increasing the ultimate magnitude of myoblast engraftment.

Myoblasts transplanted into rat infarcted myocardium are functionally isolated from their host.

Survival and differentiation of myogenic cells grafted into infarcted myocardium have raised the hope that cell transplantation becomes a new therapy for cardiovascular diseases. The approach was further supported by transplantation of skeletal myoblasts, which was shown to improve cardiac performance in several animal species. Despite the success of myoblast transplantation and its recent trial in human, the mechanism responsible for the functional improvement remains unclear. Here, we used intracellular recordings coupled to video and fluorescence microscopy to establish whether myoblasts, genetically labeled with enhanced GFP and transplanted into rat infarcted myocardium, retain excitable and contractile properties, and participate actively to cardiac function. Our results indicate that grafted myoblasts differentiate into peculiar hyperexcitable myotubes with a contractile activity fully independent of neighboring cardiomyocytes. We conclude that mechanisms other than electromechanical coupling between grafted and host cells are involved in the improvement of cardiac function.

Long-term (1 year) functional and histological results of autologous skeletal muscle cells transplantation in rat.

BACKGROUND: Several studies have demonstrated the short-term benefits of autologous skeletal muscle cell transplantation on postinfarction left ventricular function. The present experiments were designed to assess the long-term effects of the procedure. METHODS AND RESULTS: Thirteen Wistar rats that had undergone skeletal muscle cell transplantation (n=6) or injection of control culture medium (n=7) in isoforms areas after myocardial infarction created by coronary artery ligation and survived for 1 year were functionally assessed by combining echocardiography and pressure-volume loops. At 1 year after transplantation, both contractile and relaxation indices were significantly improved in the skeletal muscle cell-grafted group compared with controls. One-year echocardiographic measurements of ejection fraction were similar to those recorded 2 months after the procedure. The stability of the functional outcome contrasted with a decrease in the number of histologically detectable skeletal myotubes over time. However, the proportion of the slow and composite (fast and slow) myosin isoforms expressed by skeletal muscle fibers still present after 1 year was greater than that found in animals sacrificed after 2 months. CONCLUSION: The functional benefits of autologous skeletal muscle cell transplantation are sustained over time and are associated with either selection, preservation or an increased expression of slow myosin heavy chain isoforms. The discrepancy between maintenance of this improvement and the decay in the engrafted myotubes suggests protective mechanisms operative from the early post-transplantation stage and possibly involving modulation of extracellular matrix remodelling or paracrinally induced maturation of putative cardiac resident stem cells.

The transplant of cardiac cells and myoblast skeletal cells in myocardial infarction

OBJETIVO:Comparar o resultado funcional e anátomo-patológico entre o transplante de células mioblásticas e cardíacas no infarto do miocárdio. MÉTODO: Realizado infarto da parede ântero-lateral do ventrículo esquerdo em 26 ratos Wistar, com ligadura da artéria coronária esquerda. Após cinco dias, os animais foram submetidos a ecocardiografia transtorácica para cálculo dos volumes sistólico (VSFVE) e diastólico (VDFVE) finais e da fraçäo de ejeçäo do ventrículo esquerdo (FEVE). Os animais foram divididos em três grupos: 1) controle (n=10), 2) células cardíacas adultas (n=8) e 3) células musculares esqueléticas adultas (n=8). Sete dias após o infarto do miocárdio, os animais foram reoperados por esternotomia mediana, sendo identificada a regiäo de fibrose no ventrículo esquerdo e nela, injetado 0.15ml de meio de cultura no grupo I, 8.5x106/0.15ml de células cardíacas heterólogas no grupo II e 8.5x106/0.15ml de células musculares esqueléticas heterólogas no grupo III. Todos os animais receberam ciclosporina (15mg/kg/dia). Após dois meses do transplante, realizou-se nova ecocardiografia avaliando os mesmos parâmetros. RESULTADOS: Após dois meses do transplante celular, o grupo I apresentou um decréscimo da FEVE (48.18 porcento vs. 33.25 porcento p=0.0003), sendo que houve um acréscimo dos VSFVE e VDFVE (0.308ml vs. 0.536ml p=0.026 e 0.597ml vs. 0.776ml p=0.054, respectivamente). No grupo II houve uma estabilizaçäo da FEVE (42.48 porcento vs. 41.31 porcento p=0.4968, respectivamente) e um discreto aumento do VDFVE (0.602ml vs. 0.771ml p=0.0711). O VSFVE variou de 0.358ml a 0.450ml p=0.0400. O grupo III apresentou um acréscimo da FEVE, VDFVE e VSFVE (40 porcento vs. 47.35 porcento p=0.0142, 0.643ml vs. 0.931ml p=0.0026 e 0.388ml vs. 0.491ml p=0.0557 (sem significância), respectivamente. O GIII apresentou um maior valor, considerado estatisticamente significativo, da fraçäo de ejeçäo do ventrículo esquerdo, em comparaçäo ao GI e ao GII (47.35 porcento + 6.89 porcento vs. 41.31 porcento + 8.46 porcento vs. 33.25 porcento + 12.41 porcento p=0.0200, respectivamente). Identificou-se uma diferença estatisticamente significativa da fraçäo de ejeçäo do ventrículo esquerdo entre o GIII e o GI, após dois meses do transplante (47.35 porcento + 6.891 porcento vs. 33.25 porcento + 12.41 porcento p=0.0213, respectivamente)...