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The need to regenerate tissue is paramount, especially for the heart that lacks the ability to regenerate after injury. The urinary bladder extracellular matrix (ECM), when used to repair a right ventricular defect, successfully regenerated some mechanical function. The objective of the current study was to determine whether the regenerative effect of ECM could be improved by seeding the patch with human mesenchymal stem cells (hMSCs) enhanced to differentiate down a cardiac linage, hMSCs were used to form three-dimensional spheroids. The expression of cardiac proteins was determined in cells exposed to the spheroid formation and compared with nonmanipulated hMSCs. To determine whether functional calcium channels were present, the cells were patch clamped. To evaluate the ability of these cells to regenerate mechanical function, the spheroids were seeded on ECM and then implanted into the canine heart to repair a full-thickness right ventricular defect. As a result, many of the cells spreading from the spheroids expressed cardiac-specific proteins, including sarcomeric [alpha]-actinin, cardiotin, and atrial natriuretic peptide, as well as the cell cycle markers cyclin D1 and proliferating cell nuclear antigen. A calcium current similar in amplitude to that of ventricular myocytes was present in 16% of the cells. The cardiogenic cell-seeded scaffolds increased the regional mechanical function in the canine heart compared with the unmanipulated hMSC-seeded scaffolds. In addition, the cells prelabeled with fluorescent markers demonstrated myocyte-specific actinin staining with sarcomere spacing similar to that of normal myocytes. In conclusion, the spheroid-derived cells express cardiac-specific proteins and demonstrate a calcium current similar to adult ventricular myocytes. When these cells are implanted into the canine heart, some of these cells appear striated and mechanical function is improved compared with the unmanipulated hMSCs. Further investigation will be required to determine whether the increased mechanical function is due to a differentiation of the cardiogenic cells to myocytes or to other effects. human mesenchymal stem cells; myocyte structure; cardiac contractile function; myogenesis; cardiac regeneration; myocardium; heart failure

Background Surgically induced ischemia in the arrested heart can result in changes in the mechanical properties of the myocardium. Regions of ischemia may be characterized based on the amount of epicardial deformation for a given load. Computer aided speckle interferometrv (CASI), which tracks the movement of clusters of particles, is developed as a technique for measuring epicardial deformation, thereby determining the perfusion status of the passive heart. Materials and Methods Silicone carbide particles and retroreflective beads were dispersed randomly onto the epicardial surface of 11 isolated rabbit hearts to form speckle images. The hearts were arrested with hyperkalemic Krebs-Henseleit buffered solution. Each heart was then exposed to a series of intracavitary pressures, and at each pressure speckle images were acquired with a charge-coupled device (CCD) camera. Nine hearts were exposed to global ischemia, and two hearts were exposed to regional ischemia by occluding the second diagonal branch of the left anterior descending artery (LAD). The hearts were again loaded and the speckle images were acquired. CASI was used to determine the distribution of deformation field. Results CASI was able to determine displacements with a spatial resolution of about 50 microns. Global ischemia resulted in a significant increase in the maximum principle strain and the first invariant of the 2-D strain tensor. In the regionally ischemic heart, a large difference in deformation between the ischemic and perfused regions was clearly observed. Conclusion Based on epicardial deformation, CASI is able to distinguish between perfused and ischemic myocardium, with a spatial resolution of 50 [micro]m. [DOI: 10.1115/1.1645524]

It is well-known that micromolar to millimolar concentrations of cardiac glycosides inhibit Na/K pump activity, however, some early reports suggested nanomolar concentrations of these glycosides stimulate activity. These early reports were based on indirect measurements in multicellular preparations, hence, there was some uncertainty whether ion accumulation/depletion rather than pump stimulation caused the observations. Here, we utilize the whole-cell patch-clamp technique on isolated cardiac myocytes to directly measure Na/K pump current ([I.sub.p]) in conditions that minimize the possibility of ion accumulation/depletion causing the observed effects. In guinea pig ventricular myocytes, nanomolar concentrations of dihydro-ouabain (DHO) caused an outward current that appeared to be due to stimulation of [I.sub.p] because of the following: (1) it was absent in 0 mM [[[K.sup.+]].sub.o], as was [I.sub.p]; (2) it was absent in 0 mM [[[Na.sup.+]].sub.i], as was [I.sub.p]; (3) at reduced [[[Na.sup.+]].sub.i], the outward current was reduced in proportion to the reduction in [I.sub.p]; (4) it was eliminated by intracellular vanadate, as was [I.sub.p]. Our previous work suggested guinea pig ventricular myocytes coexpress the [[alpha].sub.1]- and [[alpha].sub.2]-isoforms of the Na/K pumps. The stimulation of [I.sub.p] appears to be through stimulation of the high glycoside affinity [[alpha].sub.2]-isoform and not the [[alpha].sub.1]-isoform because of the following: (1) regulatory signals that specifically increased activity of the [[alpha].sub.2]-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the [[alpha].sub.1]-isoform did not affect the stimulation; (3) changes in [[[K.sup.+]].sub.o] that affected activity of the [[alpha].sub.1]-isoform, but not the [[alpha].sub.2]-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the [[alpha].sub.1]-isoform but not the [[alpha].sub.2]-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total [I.sub.p] increased by 35 [+ or -] 10% (mean [+ or -] SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the [[alpha].sub.2]-isoform, then activity of the [[alpha].sub.2]-isoform increased by 107 [+ or -] 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the [[alpha].sub.2]-isoform, but both the stimulatory and inhibitory concentrations of ouabain were ~10-fold lower than those for DHO. Stimulation of [I.sub.p] by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the [[alpha].sub.1]- and [[alpha].sub.3]-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the [[alpha].sub.3]-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of [I.sub.p] that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of [I.sub.p] and where the contributions of the high glycoside affinity [[alpha].sub.2]-and [[alpha].sub.3]-isoforms could be separated from that of the [[alpha].sub.1]-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of [I.sub.p] in heart by nanomolar concentrations of endogenous ouabain-like molecules. KEY WORDS: cardiac electrophysiology * Na/K ATPase * cardiac glycosides