Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.

INTRODUCTION: Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism. METHODS AND RESULTS: We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy). CONCLUSION: This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux.

Toward 3-D Echocardiographic Determination of Regional Myofiber Structure.

As a step toward the goal of relating changes in underlying myocardial structure to observed altered cardiac function in the hearts of individual patients, this study addresses the feasibility of creating echocardiography-derived maps of regional myocardial fiber structure for entire, intact, excised sheep hearts. Backscatter data were obtained from apical echocardiographic images acquired with a clinical ultrasonic imaging system and used to determine local fiber orientations in each of seven hearts. Systematic acquisition across the entire heart volume provided information sufficient to give a complete map for each heart. Results from the echocardiography-derived fiber maps compare favorably with corresponding results derived from diffusion tensor magnetic resonance imaging. The results of this study provide evidence of the feasibility of using echocardiographic methods to generate individualized whole heart fiber maps for patients.

Autophagy Portends the Level of Cardiac Hypertrophy in Experimental Hypertensive Swine Model.

BACKGROUND: Left ventricular (LV) hypertrophy (LVH) plays an important role in hypertensive heart disease, and may be accompanied by myocardial autophagy. However, the pattern of autophagy during evolution of LVH is unclear. We hypothesized that autophagy activation indicates advancing cardiac LVH with tissue remodeling. METHODS: Ten domestic pigs with a 10-week unilateral renovascular hypertension (HTN) were classified as mild or moderate HTN (n = 5 each group) based on the degree of renal artery stenosis (above or below 75%). Seven normal pigs served as controls. Left ventricular remodeling, function, and microvascular density were assessed using multi-detector- and micro-computed tomography and histology. Markers of myocardial autophagic and endoplasmic reticulum (ER) stress-related unfolded protein response (UPR), apoptosis, and fibrosis were examined ex vivo. RESULTS: Both HTN groups had increased myocyte cross-sectional area, but it was greater in moderate HTN, accompanied by elevated LV muscle-mass. Moderate, but not mild HTN, also showed impaired microvascular density and impaired myocardial perfusion. Autophagy mediators were unaltered in mild HTN but UPR markers were increased, while in moderate HTN they were all upregulated, whereas UPR markers were suppressed. Myocardial apoptosis and fibrosis were also greater in moderate HTN. Autophagic proteins were correlated with LVH and fibrosis. CONCLUSIONS: Autophagic activity is stimulated during the exacerbation of LVH, following a transient early increase in ER stress, and may be involved in the progression of cardiac remodeling in renovascular hypertensive heart disease.

Blueberry Anthocyanins-Enriched Extracts Attenuate Cyclophosphamide-Induced Cardiac Injury.

We sought to explore the effect of blueberry anthocyanins-enriched extracts (BAE) on cyclophosphamide (CTX)-induced cardiac injury. The rats were divided randomly into five groups including normal control, CTX 100 mg/kg, BAE 80mg/kg, CTX+BAE 20mg/kg and CTX+BAE 80mg/kg groups. The rats in the three BAE-treated groups were administered BAE for four weeks. Seven days after BAE administration, rats in CTX group and two BAE-treated groups were intraperitoneally injected with a single dose of 100 mg/kg CTX. Cardiac injury was assessed using physiological parameters, Echo, morphological staining, real-time PCR and western blot. In addition, cardiotoxicity indices, inflammatory cytokines expression and oxidative stress markers were also detected. Four weeks 20mg/kg and 80mg/kg dose of BAE treatment following CTX exposure attenuated mean arterial blood pressure, heart rate and activities of heart enzymes, improved cardiac dysfunction, left ventricular hypertrophy and fibrosis. Importantly, BAE also attenuated CTX-induced LV leukocyte infiltration and inflammatory cytokines expression, ameliorated oxidative stress as well as cardiomyocyte apoptosis. In conclusion, BAE attenuated the CTX-induced cardiac injury and the protective mechanisms were related closely to the anti-inflammatory, antioxidant and anti-inflammatory characteristics of BAE.

(18)F-labeled rhodamines as potential myocardial perfusion agents: comparison of pharmacokinetic properties of several rhodamines.

INTRODUCTION: We recently reported the development of the [(18)F]fluorodiethylene glycol ester of rhodamine B as a potential positron emission tomography (PET) tracer for myocardial perfusion imaging (MPI). This compound was developed by optimizing the ester moiety on the rhodamine B core, and its pharmacokinetic properties were found to be superior to those of the prototype ethyl ester. The goal of the present study was to optimize the rhodamine core while retaining the fluorodiethyleneglycol ester prosthetic group. METHODS: A series of different rhodamine cores (rhodamine 6G, rhodamine 101, and tetramethylrhodamine) were labeled with (18)F using the corresponding rhodamine lactones as the precursors and [(18)F]fluorodiethylene glycol ester as the prosthetic group. The compounds were purified by semipreparative HPLC, and their biodistribution was measured in rats. Additionally, the uptake of the compounds was evaluated in isolated rat cardiomyocytes. RESULTS: As was the case with the different prosthetic groups, we found that the rhodamine core has a significant effect on the in vitro and in vivo properties of this series of compounds. Of the rhodamines evaluated to date, the pharmacologic properties of the (18)F-labeled diethylene glycol ester of rhodamine 6G are superior to those of the (18)F-labeled diethylene glycol esters of rhodamine B, rhodamine 101, and tetramethylrhodamine. As with (18)F-labeled rhodamine B, [(18)F]rhodamine 6G was observed to localize in the mitochondria of isolated rat cardiomyocytes. CONCLUSIONS: Based on these results, the (18)F-labeled diethylene glycol ester of rhodamine 6G is the most promising potential PET MPI radiopharmaceutical of those that have evaluated to date, and we are now preparing to carry out first-in-human clinical studies with this compound.

Preclinical characterization of a novel radiolabeled analog of practolol for the molecular imaging of myocardial ß-adrenoceptor density.

BACKGROUND: The great clinical potential of myocardial ß-AR imaging has been shown by recent studies evaluating the ß-AR-specific, non-selective agent [(11)C]-CGP12177 in the setting of idiopathic-dilated cardiomyopathy, and myocardial infarction. However, the short half-life of (11)C hampers the potential of [(11)C]-CGP12177 for routine clinical use. AMI9 is an analog of the ß-adrenoceptor ligand practolol that can readily be labeled using radioactive isotopes of iodine. The present study was aimed at characterizing the in vitro, ex vivo, and in vivo ß-AR binding properties of [(125)I]-AMI9. METHODS AND RESULTS: Newborn rat cardiomyocytes were used for saturation and kinetic binding assays as well as for displacement and competition experiments. Isolated perfused rat hearts were used to evaluate the pharmacological activity of AMI9. The in vivo kinetics of [(125)I]-AMI9 were studied using biodistribution experiments in mice. [1(25)I]-AMI9 displayed high specific affinity for ß-AR with no ß-AR subtype selectivity (K D, 5.6 ± 0.3 nM; B max, 231 ± 7 fmol·(mg protein)(-1)). AMI9 potently inhibited the inotropic effects of isoproterenol. The early in vivo cardiac and lung activities of [(125)I]-AMI9 compared favorably with those of the clinically validated tracer CGP12177. CONCLUSION: Iodine-labeled AMI9 is a promising agent for the molecular imaging of myocardial ß-AR density.

Enhanced delivery of microRNA mimics to cardiomyocytes using ultrasound responsive microbubbles reverses hypertrophy in an in-vitro model.

BACKGROUND: Cardiovascular diseases (CVD) account for 36% of deaths in Europe and the United States. Gene therapy can act as a therapeutic modality for the treatment of CVD. The use of microRNA mimetics may be advantageous as they regulate important processes in health and pathology. A major hurdle for using miRNA therapies relates to site specific delivery and sufficient cellular uptake of material to achieve efficacy OBJECTIVE: To assess the feasibility of ultrasound responsive microbubble mediated delivery of miR mimics to cardiomyocytes. METHODS: Liposome/microbubble formulations were added to HL-1 cardiomyocytes in the presence/absence of ultrasound (US). Transfection efficacy and functionality was assessed using epifluorescent microscopy, flow cytometry and qRT-PCR. DNA Quantification post-ultrasound mediated transfection of HL-1s using microbubbles was quantified. The capability of miR-133 microbubble formulations to suppress hypertrophy were measured by quantifying changes in cell size. RESULTS: Ultrasound mediated microbubble formulations enhanced intracellular delivery of miR mimics in cardiomyocytes. Both complexed/encapsulated miR-microbubble formulations delivered functional miR mimics and showed no adverse effect on cardiomyocyte viability. Furthermore, ultrasound mediated microbubble transfection of miR-133 mimics reversed cardiomyocyte hypertrophy in an in-vitro model. CONCLUSIONS: This novel delivery method has the potential for further development as a targeted delivery strategy for miR therapeutics to the heart.

Cellular bioenergetics is an important determinant of the molecular imaging signal derived from luciferase and the sodium-iodide symporter.

RATIONALE: Molecular imaging is useful for longitudinal assessment of engraftment. However, it is not known which factors, other than cell number, can influence the molecular imaging signal obtained from reporter genes. OBJECTIVE: The effects of cell dissociation/suspension on cellular bioenergetics and the signal obtained by firefly luciferase and human sodium-iodide symporter labeling of cardiosphere-derived cells were investigated. METHODS AND RESULTS: (18)Fluorodeoxyglucose uptake, ATP levels, (99m)Tc-pertechnetate uptake, and bioluminescence were measured in vitro in adherent and suspended cardiosphere-derived cells. In vivo dual-isotope single-photon emission computed tomography/computed tomography imaging or bioluminescence imaging (BLI) was performed 1 hour and 24 hours after cardiosphere-derived cell transplantation. Single-photon emission computed tomography quantification was performed using a phantom for signal calibration. Cell loss between 1 hour and 24 hours after transplantation was quantified by quantitative polymerase chain reaction and ex vivo luciferase assay. Cell dissociation followed by suspension for 1 hour resulted in decreased glucose uptake, cellular ATP, (99m)Tc uptake, and BLI signal by 82%, 43%, 42%, and 44%, respectively, compared with adherent cells, in vitro. In vivo (99m)Tc uptake was significantly lower at 1 hour compared with 24 hours after cell transplantation in the noninfarct (P<0.001; n=3) and infarct (P<0.001; n=4) models, despite significant cell loss during this period. The in vivo BLI signal was significantly higher at 1 hour than at 24 hours (P<0.01), with the BLI signal being higher when cardiosphere-derived cells were suspended in glucose-containing medium compared with saline (PBS). CONCLUSIONS: Adhesion is an important determinant of cellular bioenergetics, (99m)Tc-pertechnetate uptake, and BLI signal. BLI and sodium-iodide symporter imaging may be useful for in vivo optimization of bioenergetics in transplanted cells.

Increased passive stiffness of cardiomyocytes in the transverse direction and residual actin and myosin cross-bridge formation in hypertrophied rat hearts induced by chronic ß-adrenergic stimulation.

BACKGROUND: Left ventricular (LV) hypertrophy is often present in patients with diastolic heart failure. However, stiffness of hypertrophied cardiomyocytes in the transverse direction has not been fully elucidated. The aim of this study was to assess passive cardiomyocyte stiffness of hypertrophied hearts in the transverse direction and the influence of actin-myosin cross-bridge formation on the stiffness. METHODS AND RESULTS: Wistar rats received a vehicle (control) or isoproterenol (ISO) subcutaneously. After 7 days, compared with the controls, ISO administration had significantly increased heart weight and LV wall thickness and had decreased peak early annular relaxation velocity (e') assessed by echocardiography. Elastic modulus of living cardiomyocytes in the transverse direction assessed by an atomic force microscope was significantly higher in the ISO group than in controls. We added butanedione monoxime (BDM), an inhibitor of actin-myosin interaction, and blebbistatin, a specific myosin II inhibitor, to the medium. BDM and blebbistatin significantly reduced the elastic modulus of cardiomyocytes in the ISO group. X-ray diffraction analysis showed that the reflection intensity ratio (I((1,0))/I((1,1))) at diastole was not different before and after treatment with BDM, which induces complete relaxation, in control hearts, but that I((1,0))/I((1,1)) was significantly increased after BDM treatment in the ISO group, indicating residual cross-bridge formation in hypertrophied hearts. CONCLUSIONS: Passive cardiomyocyte stiffness in the transverse direction is increased in hearts with ISO-induced hypertrophy and this is caused by residual actin-myosin cross-bridge formation.

Functional and morphological preservation of adult ventricular myocytes in culture by sub-micromolar cytochalasin D supplement.

In cardiac myocytes, cytochalasin D (CytoD) was reported to act as an actin disruptor and mechanical uncoupler. Using confocal and super-resolution STED microscopy, we show that CytoD preserves the actin filament architecture of adult rat ventricular myocytes in culture. Five hundred nanomolar CytoD was the optimal concentration to achieve both preservation of the T-tubular structure during culture periods of 3 days and conservation of major functional characteristics such as action potentials, calcium transients and, importantly, the contractile properties of single myocytes. Therefore, we conclude that the addition of CytoD to the culture of adult cardiac myocytes can indeed be used to generate a solid single-cell model that preserves both morphology and function of freshly isolated cells. Moreover, we reveal a putative link between cytoskeletal and T-tubular remodeling. In the absence of CytoD, we observed a loss of T-tubules that led to significant dyssynchronous Ca(2+)-induced Ca(2+) release (CICR), while in the presence of 0.5 µM CytoD, T-tubules and homogeneous CICR were majorly preserved. Such data suggested a possible link between the actin cytoskeleton, T-tubules and synchronous, reliable excitation-contraction-coupling. Thus, T-tubular re-organization in cell culture sheds some additional light onto similar processes found during many cardiac diseases and might link cytoskeletal alterations to changes in subcellular Ca(2+) signaling revealed under such pathophysiological conditions.

A mouse model of reverse cardiac remodelling following banding-debanding of the ascending aorta.

AIM: Myocardial remodelling during pressure overload might contribute to development of heart failure. Reverse remodelling normally occurs following aortic valve replacement for aortic stenosis; however, the details and regulatory mechanisms of reverse remodelling remain unknown. Thus, an experimental model of reverse remodelling would allow for studies of this process. Although models of aortic banding are widely used, only few reports of debanding models exist. The aim of this study was to establish a banding-debanding model in the mouse with repetitive careful haemodynamic evaluation by high-resolution echocardiography. METHODS: C57Bl/6 mice were subjected to ascending aortic banding and subsequent debanding. Cardiac geometry and function were evaluated by echocardiography, and left ventricular myocardium was analysed by histology and quantitative real-time polymerase chain reaction. RESULTS: The degree of aortic banding was controlled by non-invasive estimation of the gradient, and we found a close correlation between left ventricular mass estimated by echocardiography and weight at the time of killing. Aortic banding led to left ventricular hypertrophy, fibrosis and expression of foetal genes, indicating myocardial remodelling. Echocardiography revealed concentric left ventricular remodelling and myocardial dysfunction. Following debanding, performed via a different incision, there was rapid regression of left ventricular weight and normalization of both cardiac geometry and function by 14 days. CONCLUSIONS: We have established a reproducible and carefully characterized mouse model of reverse remodelling by banding and debanding of the ascending aorta. Such a model might contribute to increased understanding of the reversibility of cardiac pathology, which in turn might give rise to new strategies in heart failure treatment.

Mechanism of pressure-overload right ventricular hypertrophy in infant rabbits.

Although pressure-overload right ventricular hypertrophy is a long-term risk in some congenital heart diseases such as tetralogy of Fallot, how it develops is unclear. The aim of this study was to investigate the mechanism of development of this right ventricular heart failure.Pulmonary artery banding in 10-day-old rabbits induced pressure-overload right ventricular hypertrophy as they grew. Comparisons were made with age-matched sham controls (n = 24 per group). In weekly serial echocardiography, the right ventricular contraction and diastolic function decreased from 3 weeks after surgery (P < 0.01), and the right ventricle became hypertrophic from 4 weeks after (P < 0.05). Pressure-overload increased cardiomyocyte apoptosis from 4 weeks postoperatively (TUNEL staining and Western blotting analysis, P < 0.05); and fibrosis occurred in the right ventricular cardiomyocytes at 8 weeks after operation (Masson's trichrome stain, P < 0.01). In our model, pressure-overload to the right ventricle caused the right ventricular disorder, hypertrophy, and fibrosis. Apoptosis of right ventricular cardiomyocytes was involved in progression. We have shown for the first time the mechanism whereby pressure-overload right ventricular hypertrophy develops in an infant rabbit model.

Are ECG premature complexes induced by ultrasonic cavitation electrophysiological responses to irreversible cardiomyocyte injury?

The objective of this study was to explore the relationship between premature complexes (PCs) in the electrocardiogram (ECG) and lethal injury of cardiomyocytes induced by ultrasound exposure of the heart with contrast-agent gas bodies in the circulation. Anesthetized rats were exposed in a heated water bath to 1.55 MHz focused ultrasound with bursts triggered at end systole during contrast agent infusion. PCs were detected in ECG recordings and cardiomyocyte necrosis was scored by identifying Evans blue-stained cells in multiple frozen sections. With 0.1 µL/kg/min infusion of contrast agent for 5 min, both effects increased strongly for 2-ms bursts with increasing peak rarefactional pressure amplitude >1 MPa. At 8 MPa, statistically significant effects were found even for no agent infusion relative to sham tests. For 2-ms bursts at 2 MPa, the highly significant bioeffects seen for 10-, 1- and 0.1-µL/kg/min infusion became marginally significant for 0.01 µL/kg/min, which indicated a lower probability of cavitation nucleation. Burst duration variation from 0.2-20 ms produced no substantial trends in the results. Overall, the two effects were well correlated (r(2) = 0.88). The PCs occurring during contrast-enhanced ultrasound therefore appear to be electrophysiological responses to irreversible cardiomyocyte injury induced by ultrasonic cavitation.

Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover.

Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously ¹4C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that ¹4C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans.

Myocardial fiber shortening in the circumferential direction produces left ventricular wall thickening during contraction.

When one bends the elbow by shortening of the biceps, a knot of muscle is observed in his or her upper arm, indicating that muscle shortening is converted to muscle standing in the perpendicular direction due to the incompressibility of skeletal muscle. A similar mechanism may work in the thickening process of the left ventricular (LV) wall. Although myocardial fibers of the left ventricle shorten by about 20% along the fiber direction when they contract, thickening of the LV wall during contraction often exceeds 50%. Thus, the aim of the present study was to clarify the mechanism by which myocardial fiber shortening produces such remarkable thickening of the LV wall. We hypothesized that myocardial fiber shortening in the circumferential direction causes myocardial transformation perpendicular to the fiber direction, thereby producing LV wall thickening. We evaluated this hypothesis using an incompressible model of the LV wall. In 15 healthy male volunteers (38±13 years), we calculated theoretical peak thickening values of the inner and outer LV wall layers and compared them with directly measured peak thickening values using Doppler strain imaging at the corresponding areas. The theoretical peak thickening and directly measured peak thickening were >60% in the LV inner layer. The theoretical peak thickening was correlated with the directly measured peak thickening in the inner (r=0.75, p<0.05) and outer (r=0.61, p<0.05) layers. We conclude that shortening of LV circumferential myocardial fiber and incompressibility of myocardium produce LV wall thickening during contraction.

Annexin A5 uptake in ischemic myocardium: demonstration of reversible phosphatidylserine externalization and feasibility of radionuclide imaging.

UNLABELLED: Ischemic insult to the myocardium is associated with cardiomyocyte apoptosis. Because apoptotic cell death is characterized by phosphatidylserine externalization on cell membrane and annexin-A5 (AA5) avidly binds to phosphatidylserine, we hypothesized that radiolabeled AA5 should be able to identify the regions of myocardial ischemia. METHODS: Models of brief myocardial ischemia by the occlusion of the coronary artery for 10 min (I-10) and reperfusion for 180 min (R-180) for the detection of phosphatidylserine exteriorization using (99m)Tc-labeled AA5 and gamma-imaging were produced in rabbits. (99m)Tc-AA5 uptake after brief ischemia was compared with an I-40/R-180 infarct model. Histologic characterization of both myocardial necrosis and apoptosis was performed in ischemia and infarct models. Phosphatidylserine exteriorization was also studied in a mouse model, and the dynamics and kinetics of phosphatidylserine exposure were assessed using unlabeled recombinant AA5 and AA5 labeled with biotin, Oregon Green, or Alexa 568. Appropriate controls were established. RESULTS: Phosphatidylserine exposure after ischemia in the rabbit heart could be detected by radionuclide imaging with (99m)Tc-AA5. Pathologic characterization of the explanted rabbit hearts did not show apoptosis or necrosis. Homogenization and ultracentrifugation of the ischemic myocardial tissue from rabbit hearts recovered two thirds of the radiolabeled AA5 from the cytoplasmic compartment. Murine experiments demonstrated that the cardiomyocytes expressed phosphatidylserine on their cell surface after an ischemic insult of 5 min. Phosphatidylserine exposure occurred continuously for at least 6 h after solitary ischemic insult. AA5 targeted the exposed phosphatidylserine on cardiomyocytes; AA5 was internalized into cytoplasmic vesicles within 10-30 min. Twenty-four hours after ischemia, cardiomyocytes with internalized AA5 had restored phosphatidylserine asymmetry of the sarcolemma, and no detectable phosphatidylserine remained on the cell surface. The preadministration of a pan-caspase inhibitor, zVAD-fmk, prevented phosphatidylserine exposure after ischemia. CONCLUSIONS: After a single episode of ischemia, cardiomyocytes express phosphatidylserine, which is amenable to targeting by AA5, for at least 6 h. Phosphatidylserine exposure is transient and internalized in cytoplasmic vesicles after AA5 binding, indicating the reversibility of the apoptotic process.

Weaving hypothesis of cardiomyocyte sarcomeres: discovery of periodic broadening and narrowing of intercalated disk during volume-load change.

To investigate how cardiomyocytes change their length, echocardiographic and morphological studies were performed on rabbit hearts that were subjected to volume overload, overload removal, and repeated cycles of overload and overload removal. These conditions were created by arterio-venous fistula between the carotid artery and jugular vein, closure of the fistula, and cycles of repeatedly forming and closing fistula, respectively. After overload, hearts dilated and myocytes elongated. Intercalated disks repeatedly broadened and narrowed with a 2-day cycle, which continued for 8 weeks in many animals. The cycle consisted of shifts between five modes characterized by two interdigitation elongation-and-shortenings as follows: (I) flat with short ( approximately 1/4 to approximately 1/3 sarcomere long) interdigitations; (II) flat with long (one sarcomere long) interdigitations; (III) grooved with short interdigitations; (IV) grooved with long interdigitations; (V) flat with short interdigitations intermingled by sporadic long interdigitations; and return to (I). After overload removal, hearts contracted and myocytes shortened with similar 2-day broadening and narrowing cycle of intercalated disks, in which the five modes were reversed. Repeated overload and overload removal resulted in the repetition of myocyte elongation and shortening. We hypothesize that a single elongation-and-shortening event creates or disposes one sarcomere layer, and the two consecutive elongation-and-shortenings occur complementarily to each other so that the disks return to their original state after each cycle. Our hypothesis predicts that intercalated disks weave and unravel one sarcomere per myocyte per day.

Omentopexy enhances graft function in myocardial cell sheet transplantation.

Myocardial cell sheets (MCS) are a potentially valuable tool for tissue engineering aimed at heart regeneration. Several methods have recently been established for the fabrication of MCS. However, the lack of a sufficient blood supply has inhibited functional recovery of the MCS. To address this challenge, we combined MCS transplantation with omentopexy (OP), which utilizes omental tissue as a surgical flap. Rats were divided into five groups: sham, myocardial infarction (MI), MCS transplantation, OP, and MCS+OP. Histologic analysis revealed that MCS+OP drastically reversed MI-induced cardiac remodeling. Echocardiography revealed that MCS increased cardiac function, while OP had a synergistic beneficial effect with MCS transplantation. Immunofluorescence imaging showed that OP increased the survival of transplanted cardiomyocytes, and increased the blood supply through enhancement of angiogenesis and migration of small arteries into the MCS. Taken together, we concluded that OP is a promising strategy for the enhancement of graft function in MCS transplantation.