Two clinical trials since 2002 using transplanted adult stem cells successfully led to tissue regrowth in damaged hearts, but 11 of 18 patients later developed potentially fatal heart rhythm disturbances, including one who required cardiac resuscitation. "It was a potential case of the cure being worse than the disease," says senior researcher and cardiovascular physiologist Eduardo Marbán, M.D., Ph.D., professor and chief of cardiology at The Johns Hopkins University School of Medicine and its Heart Institute. "It was very discouraging to know that these patients developed arrhythmias, yet not know if it was the muscle stem cells at fault or simply a progression of the patients' heart disease."
Marbán is also editor in chief of the journal Circulation Research, in which the findings will be published online June 23.
Marbán's team says it has discovered the source of the arrhythmias to be transplantation of myoblasts, which are adult stem cells taken from patients' own healthy skeletal muscle. In patients, these myoblasts are injected directly into damaged heart muscle to regrow new tissue. In petri dish studies using these cells, the transplantation process caused an immediate disruption in heart muscle tissue's regular electrical rhythm, or conductivity, which is necessary to stimulate a regular heart beat.
Moreover, the Hopkins group was able to minimize arrhythmias dramatically by using gene therapy to replace a key protein, called connexin 43, missing in heart muscle fibers that regrew as a result of the stem cell injections. Connexin 43 makes up the gap junctions between muscle cells, allowing cells to communicate with each other to regularly contract and expand.
"We believe that by combining gene therapy with adult stem cell transplantation, we can go a long way to prevent the development of potentially fatal arrhythmias in patients who will have these myoblast transplants," says Marbán. According to the latest statistics from the American Heart Association, in 2002, there were an estimated 565,000 new cases of heart attack in the United States, plus an additional 300,000 cases of recurrent heart attack. More than 3 million Americans suffer from congestive heart failure, a common target of the myoblast stem cell therapy.
In the study, Marbán and his team created a simulation of stem cell transplantation using healthy muscle cells taken from rats' hearts and mixing them with myoblasts from healthy human skeletal muscle. A liquid suspension kept both cell types alive, and their electrical interactions were mapped with a voltage-sensitive dye. The team found that mixing the two cell types slowed normal conduction rates among heart muscle cells by two-thirds. Computer printouts of the optical maps showed a spiral-wave pattern, the most common kind of arrhythmia.
Four different mixtures of muscle stem cells were used, with the highest concentrations of stem cells, 20 percent and 50 percent, producing spiral-wave electrical patterns in all cultured heart cells. Lower stem cell concentrations of 1 percent resulted in no arrhythmia, and a 10 percent concentration led to only half of the cultured heart cells showing signs of arrhythmia.
"Our results confirmed that myoblast transplantation was responsible for the arrhythmias and that higher doses of stem cells aggravate the problem," says cardiologist M. Roselle Abraham, M.B.B.S., an assistant professor at Hopkins who led the study. "But we were not exactly sure how this was related to the subsequent cell regrowth occurring in the heart, or if it could be treated using gene therapy."
Previous research from animal transplants showed that heart tissue regrowth produced a mix of skeletal and heart muscle, resulting in long strands of muscle fibers. In the Hopkins experiment, for example, muscle fibers ranged in length from 500 microns to 1.5 millimeters when viewed under the microscope.
Though otherwise healthy, these muscle fibers are known to lack gap junctions, or protein connections between cell membranes that allow neighboring cells to communicate with each other through the exchange of ions and other electrical signals, the researchers say.
In the gene therapy experiments, the Hopkins team increased production of connexin 43 by injecting a virus carrying the gene that codes for the gap-junction protein into the cultured cells. The researchers found that the addition of connexin 43 dramatically increased conductivity between cells to normal levels, with only two of nine cultures developing signs of arrhythmia. Meanwhile, a majority of cultures, 13 of 14, which did not receive connexin gene therapy, went on to develop irregular heart-cell signals.
"We think we can now explain some of the underlying problems associated with adult stem cell therapies for heart failure and show how combining them with ex vivo therapy can possibly be more effective," says Marbán.
While their precise biological action is not known, adult stem cells are a special type of body cell found in skeletal muscle, heart, bone marrow and other tissues that gives rise to other types of specialized cells, including bone, cartilage, fat, and muscle, such as the heart. Because they can be extracted and reinjected into the same person, use of adult stem cells avoids the potiential for rejection by the body's immune system.
Marbán is also the Michel Mirowski, M.D., Professor of Medicine at Hopkins and director of its Institute of Molecular Cardiobiology.
The study was funded by the Donald W. Reynolds Foundation, the American College of Cardiology and the National Institutes of Health.
Other researchers who participated in this research, conducted solely at Hopkins, were Charles Henrikson, M.D., M.P.H.; Leslie Tung, Ph.D.; Marvin Chang, B.S.; Miguel Aon, Ph.D.; Ronald Li, Ph.D.; and Brian O'Rourke, Ph.D.
Note to Editors: Marban's paper was reviewed externally, by peers not affiliated with the journal Circulation Research, before its acceptance for publication, and the manuscript's disposition was handled by an external consulting editor.