Cellular cardiomyoplasty for cardiac repair.

Hans Reinecke, Ph.D. Department of Pathology, University of Washington, Seattle, WA.

Several strategies for using cell grafts to repair the heart may be envisioned. The most intuitive is placing new muscle tissue into the infarct to restore systolic wall motion. A second strategy is the use of cellular grafts to induce angiogenesis. This strategy might be used after an acute infarction or to treat chronic ischemic heart disease. Finally, cellular grafting may be used to change the passive mechanical properties of the infarcted wall (e.g., wall thickness or compliance) to attenuate ventricular dilation and other adverse consequences of remodeling. This review will focus on the use of committed cardiac and skeletal muscle cells for myocardial repair.

Cardiomyocyte Grafting

It seems logical that cardiac myocytes would be the best cell type to repair a myocardial infarct. Initial studies generated significant excitement after they demonstrated that cardiomyocytes from fetal mice formed viable grafts after injection into normal myocardium of syngeneic hosts. Electron microscopic analysis showed formation of intercalated disks, complete with gap junctions, between graft and host cardiomyocytes.

Although the behavior of cardiomyocyte grafts in normal hearts appeared straightforward, their biology in the injured heart remains controversial. Several groups reported that rat and fetal human cardiomyocytes could be grafted into infarcted or cryoinjured hearts, whereas others reported that fetal and neonatal pig cardiomyocytes died after implantation. Furthermore, even the studies reporting survival disagreed as to whether the graft cells differentiated normally. We systematically surveyed how the developmental stage of implanted cardiomyocytes and the status of the recipient heart tissue (normal myocardium, acute necrosis, healing granulation tissue) influenced the success of grafting. We found that both fetal and neonatal rat cardiomyocytes formed viable grafts in normal myocardium, acutely necrotic myocardium, or 1-week-old granulation tissue. In contrast, adult cardiomyocytes died irrespective of the type of tissue into which they were grafted. A detailed time course analysis of neonatal cells implanted into acutely cryoinjured hearts revealed that the grafted cells underwent a normal differentiation program, including hypertrophy and formation of intercalated disks (Fig. 1, A-D). At early times after grafting (6 days) it was possible to demonstrate gap junctions between graft and host cardiomyocytes, suggesting electromechanical coupling. At later times, however, the grafted cardiomyocytes were more commonly separated from host myocardium by scar tissue. These studies showed that cardiomyocyte grafting could generate new, normal-appearing myocardium in injured hearts.

Larger grafts, however, proved to be difficult to achieve due to the fact that the fraction of dying cells increases with increasing graft cell number (Fig. 1, E-G). This suggests that the cells are competing for limited resources (e.g., oxygen, survival factors). Current evidence points to ischemic injury as the principal culprit in causing cell death. Other paths to death, including inflammation, loss of matrix attachments (anoikis), or other apoptotic stimuli are also possible contributors to the poor survival of grafted cardiomyocytes.

Skeletal Muscle Grafting

Because of some of the limitations of cardiomyocytes, our group and others have studied skeletal muscle as a repair cell for the infarcted heart. Before describing these studies, it is worthwhile to review a few points of basic skeletal muscle biology. Mature skeletal muscle fibers originate from undifferentiated, mononucleated progenitor cells, which are termed myoblasts. Myoblasts proliferate in response to local mitogens, such as fibroblast growth factor (FGF) family members. When local grwth factors are depleted, myoblasts withdraw irreversibly from the cell cycle, activate expression of muscle-specific genes (e.g., actins, myosins, creatine kinase) and fuse to form multinucleated cells called myotubes. Myotubes undergo progressive maturation and hypertrophy to form differentiated myofibers characteristic of adult skeletal muscle. Not all myoblasts fuse into myotubes, however. Rather, some become quiescent stem cells, or satellite cells, residing in close apposition to the muscle fiber. Satellite cells can reenter the cell cycle in response to muscle injury and are responsible for the ability of skeletal muscle to regenerate. No comparable cell population has been found in the heart. Several clinical trials are under way to determine whether autologous satellite cell/myoblast grafts in the heart are an effective strategy for myocardialinfarct repair.

Differentiation, “Transdifferentiation,� and Electromechanical Coupling

In contrast to cardiac myocytes, skeletal muscle cells are among the most ischemia-tolerant in the body and, consequently, are capable of forming large grafts in the injured heart (Fig 2A). When injected into acutely cryoinjured myocardium, skeletal myoblasts proliferate for up to 3 days and then differentiate to form multinucleated myotubes. Despite their ectopic location, the skeletal muscle cells undergo a normal maturation process, eventually forming hypertrophic cells with peripherally located nuclei. Several investigators have proposed that skeletal muscle cells will transdifferentiate into cardiomyocytes after cardiac engraftment. Our group has looked carefully at this question in several studies, most recently using BrdU prelabeling of the skeletal muscle cells to follow their lineage, coupled with highly specific cardiac-or skeletal muscle-specific anti-bodies. These studies have shown unambiguously that these grafts express skeletal muscle myosin heavy chains and fail to express cardiac markers such as α-myosin heavy chain (Fig. 2B), cardiac troponin-I and atrial natriuretic factor. Thus the skeletal myoblasts appear firmly committed to their fate and form only skeletal muscle in the heart. Studies with myocardial wound strips showed that the skeletal muscle grafts would contract when exogenously stimulated. The grafts showed the ability to undergo tetanic contraction under high-frequency stimulation, a property not shared by myocardium because of its refractory period after depolarization. As the electrical field stimulation was increased, the skeletal muscle grafts showed increasing twitch tension, indicating recruitment of additional fibers. Fiber recruitment implied that the skeletal muscle grafts were electrically insulated from one another, unlike cardiomyocytes, which are electrically coupled by gap junctions. These observations led us to explore expression of the intercalated disk proteins N-cadherin (mechanical junctions) and connexin43 (gap junctions) in skeletal muscle.27 Cell culture experiments showed that proliferating skeletal myoblasts expressed abundant amounts of N-cadherin and connexin43. When cells differentiated into myotubes, however, both proteins were markedly downregulated. Immunostaining revealed that skeletal muscle grafts in the heart had undetectable levels of N-cadherin and connexin43, indicating that the grafts were not electromechanically coupled with one another or with host myocardium (Fig. 2C). These findings make it unlikely that skeletal muscle grafts in the heart are beating synchronously with the host myocardium. Much to our surprise, however, when skeletal and cardiac muscle cells were placed in coculture, the cells formed a synchronously beating network. The α-adrenergic agonist isoproterenol increased synchronous beating rates, suggesting cardiomyocytes were the pacemakers. Conversely, the gap junction blocker, heptanol, stopped skeletal muscle contractions and restricted individual cardiomyocytes to their intrinsic pacemaker frequency, suggesting the two cell types were coupled with gap junctions. Fluorescent calcium imaging studies showed that cardiomyocytes and skeletal muscle cells had synchronous calcium transients, indicating tight coupling between the cell types (Fig. 2F). Microinjection studies showed that the gap junction permeant dye, Lucifer yellow, could pass from skeletal muscle cells to cardiomyocytes. Finally, confocal microscopy revealed the presence of N-cadherin–mediated adherens junctions and connexin43-mediated gap junctions between skeletal muscle cells and cardiomyocytes (Fig. 2D, E).

Taken together, these experiments indicate that cardiomyocytes have the capacity to form electromechanical junctions with skeletal muscle cells and to use these junctions to induce synchronous beating in the skeletal muscle. Why does this coupling not occur in vivo after grafting? Skeletal muscle cells in culture are less differentiated than in vivo graft cells, and in culture the cells still have low levels of N-cadherin and connexin43. It appears that this low-level expression is sufficient to permit physiologic coupling. As the graft cells mature in vivo, however, N-cadherin and connexin43 appear to be downregulated to undetectable levels, thereby precluding coupling. Studies are currently directed at inducing expression of these two genes in grafted skeletal muscle cells to determine if this permits coupling between skeletal and cardiac muscle in vivo.

Fig. 1. Cardiomyocyte grafting for myocardial repair. Adult rat hearts were injured by a cryoprobe and 5 million syngeneic neonatal cardiomyocytes injected into the lesion immediately thereafter. (A) At 1 day after grafting the graft cardiomyocytes were round, undifferentiated-looking cells contained within the necrotic host myocardium. Note that some of the cells have plump nuclei while others have condensed, shrunken nuclei. (B) At 2 months the graft cardiomyocytes had rod-shaped morphology and formed interconnecting fibers, similar to normal host myocardium. (C, D) At 2 months the graft cardiomyocytes were coupled with one another by intercalated disks, containing N-cadherin and connexin43. (E, F) To study graft cell death, cells were prelabeled with Orange Cell Tracker (Molecular Probes) and a fluorescent green TUNEL reaction performed to quantify DNA fragmentation. The TUNEL reaction appears yellow in the merged, confocal image. At 30 minutes after grafting, the cells had no DNA fragmentation, whereas by 18 hours there was extensive DNA fragmentation. (G) Time course of TUNEL staining. Cell counts were performed to quantify the amount of cell death after grafting. At 1 day 32% of graft cells were TUNEL-positive. The incidence of cell death declined to 10% at day 4 and to 1% at day 7. Data are means±SEM.

Fig. 2. Skeletal muscle grafting and coculture. (A) Seven-week-old syngeneic skeletal muscle graft in a cryoinjured rat heart, immunostained for embryonic skeletal myosin heavy chain. Skeletal muscle cells formed a large mass of muscle in the injured heart. Grafts were typically separated from underlying myocardium by scar (wound) tissue. Note that the underlying myocardium does not express embryonic skeletalmyosin. (B) A serialsection immunostained for the cardiac marker -myosin heavy chain. Note that the graft does not stain, indicating no transdifferentiation to cardiac muscle has occurred. (C) Skeletal muscle graft stained for the intercalated disk adherens junction protein, N-cadherin. Although the intercalated disks of the host myocardium stain vigorously for N-cadherin (arrows), the skeletal muscle graft does not express this protein. Similar results were obtained after immunostaining for the gap junction protein, connexin43 (not shown). (D, E) Skeletal myotubes (MT) and cardiomyocytes in coculture, stained for F-actin (red) and N-cadherin or connexin43 (green). In contrast to the grafting results, in coculture the two cell types formed intercalated disk-like junctions containing both N-cadherin and connexin43. (F) Calcium imaging in cocultures using Oregon Green. Synchronous calcium transients were observed in the cardiomyocyte and the coupled skeletal MT. Note that the pause at 3 seconds and the synchronous resumption of calcium fluxes.