ABSTRACT

Regenerative medicine approaches for the treatment of damaged or missing myocardial tissue include cell-based therapies, scaffold-based therapies, and/or the use of specific growth factors and cytokines. The present study evaluated the ability of extracellular matrix (ECM) derived from porcine urinary bladder to serve as an inductive scaffold for myocardial repair. ECM scaffolds have been shown to support constructive remodeling of other tissue types including the lower urinary tract, the dermis, the esophagus, and dura mater by mechanisms that include the recruitment of bone marrow-derived progenitor cells, angiogenesis, and the generation of bioactive molecules that result from degradation of the ECM. ECM derived from the urinary bladder matrix, identified as UBM, was configured as a single layer sheet and used as a biologic scaffold for a surgically created 2 cm2 full-thickness defect in the right ventricular free wall. Sixteen dogs were divided into two equal groups of eight each. The defect in one group was repaired with a UBM scaffold and the defect in the second group was repaired with a Dacron patch. Each group was divided into two equal subgroups (n = 4), one of which was sacrificed 15 min after surgical repair and the other of which was sacrificed after 8 weeks. Global right ventricular contractility was similar in all four subgroups groups at the time of sacrifice. However, 8 weeks after implantation the UBM-treated defect area showed significantly greater (p < 0.05) regional systolic contraction compared to the myocardial defects repaired with by Dacron (3.3 +/- 1.3% vs. -1.8 +/- 1.1%; respectively). Unlike the Dacron-repaired region, the UBM-repaired region showed an increase in systolic contraction over the 8-week implantation period (-4.2 +/- 1.7% at the time of implantation vs. 3.3 +/- 1.3% at 8 weeks). Histological analysis showed the expected fibrotic reaction surrounding the embedded Dacron material with no evidence for myocardial regeneration. Histologic examination of the UBM scaffold site showed cardiomyocytes accounting for approximately 30% of the remodeled tissue. The cardiomyocytes were arranged in an apparently randomly dispersed pattern throughout the entire tissue specimen and stained positive for alpha- sarcomeric actinin and Connexin 43. The thickness of the UBM graft site increased greatly from the time of implantation to the 8-week sacrifice time point when it was approximately the thickness of the normal right ventricular wall. Histologic examination suggested complete degradation of the originally implanted ECM scaffold and replacement by host tissues. We conclude that UBM facilitates a constructive remodeling of myocardial tissue when used as replacement scaffold for excisional defects.

Receptors coupled to G␣ q play a key role in the development of heart failure. Studies using genetically modified mice suggest that G␣ q mediates a hypertrophic response in cardiac myocytes. G␣ q signaling in these models is modified during early growth and development, whereas most heart failure in humans occurs after cardiac damage sustained during adulthood. To determine the phenotype of animals that express increased G␣ q signaling only as adults, we generated transgenic mice that express a silent G␣ q protein (G␣ q Q209L-hbER) in cardiac myocytes that can be activated by tamoxifen. Following drug treatment to activate G␣ q Q209L-hbER, these mice rapidly develop a dilated cardiomyopathy and heart failure. This phenotype does not appear to involve myocyte hypertrophy but is associated with dephosphorylation of phospholamban (PLB), decreased sarcoplasmic reticulum Ca 2؉ -ATPase activity, and a decrease in L-type Ca 2؉ current density. Changes in Ca 2؉handling and decreased cardiac contractility are apparent 1 week after G␣ q Q209L-hbER activation. In contrast, transgenic mice that express an inducible G␣ q mutant that cannot activate phospholipase C␤ (PLC␤) do not develop heart failure or changes in PLB phosphorylation, but do show decreased L-type Ca 2؉ current density. These results demonstrate that activation of G␣ q in cardiac myocytes of adult mice causes a dilated cardiomyopathy that requires the activation of PLC␤. However, increased PLC␤ signaling is not required for all of the G␣ q -induced cardiac abnormalities.G␣ q is a member of the heterotrimeric G protein superfamily. Heterotrimeric G proteins are composed of three subunits (␣, ␤, and ␥). Receptor activation leads to the exchange of GTP for GDP on the ␣ subunit, which causes G␣ to dissociate from the tightly bound ␤␥ complex. Both G␣ and ␤␥ can then interact with specific effector proteins, resulting in changes in cellular function. Phospholipase C␤ (PLC␤) 3 is the best known effector of the G␣ q subfamily of G proteins (1). Activated G␣ q binds to PLC␤ and increases its enzymatic activity to hydrolyze phosphatidylinositol (4,5)-bisphosphate to form inositol (1,4,5)-trisphosphate and diacylglycerol. Although less well characterized, other signaling effectors of G␣ q include the p110␣/p85␣ phosphatidylinositol 3-kinase (PI3K) complex, which is inhibited by G␣ q ⅐GTP binding 4 (2), and Bruton's tyrosine kinase, which is activated by G␣ q (3). Following cardiac injury, up-regulation of angiotensin II and catecholamines leads to activation of G␣ q and progression to heart failure. The signaling pathway that mediates G␣ q -induced cardiomyopathy is unclear. The presumption has been that activation of PLC␤ by G␣ q is responsible for the cardiac pathology, but this hypothesis has not been directly tested. Activation of PLC␤ leads to the release of Ca 2ϩ from inositol (1,4,5)-trisphosphate-sensitive stores, but the role of this signaling event in cardiac myocytes is unknown. Activation of PLC␤ also leads to the diacylglycerol-dependent activation of so...

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.