Cardiovascular disease remains the leading cause of death worldwide. Because of the heart's limited ability to regenerate, injuries such as myocardial infarction (heart attack) heal by scar formation rather than muscle regeneration. As a result, the heart pumps less efficiently, leading to the burgeoning epidemic of heart failure seen today. Current medical therapies support the heart with its reduced function, but scientists and clinicians are eager to learn how to regenerate damaged heart muscle. On pages 593 and 599 of this issue, Qian et al.1 and Song et al.2 describe how, in an effort to improve cardiac function, they have induced scar-forming cells (fibroblasts) to become muscle cells (cardiomyocytes) in the injured hearts of live mice.
The reprogramming of cells from one fate to another moved from the realm of alchemy to biochemistry after the discovery of MYOD1, a transcription factor that regulates the expression of genes involved in the development of skeletal muscle. When experimentally expressed, MYOD1 can convert many cell types into skeletal muscle in vitro3, as well as cells in the injured hearts of live rats4. More recently, it was found5 that somatic (non-germline) cells from adult mammals could be reprogrammed to become pluripotent stem cells — which can differentiate into any cell type — by expressing 'cocktails' of transcription factors. Researchers have recently used this approach to convert differentiated cells directly into other differentiated cell types such as cardiomyocytes6,7,8,9,10.
Qian et al.1 and Song et al.2 built on previous work6 which showed that fibroblasts could be reprogrammed into cardiomyocytes in vitro by the introduction of genes coding for three transcription factors that regulate heart development (GATA4, MEF2C and TBX5). Qian et al. used only these three genes, whereas Song et al. observed better in vitro reprogramming efficiency by adding a fourth one, which encodes the transcription factor HAND2. In both studies, the authors induced myocardial infarction in mice by occluding a coronary artery (a blood vessel that supplies blood to heart muscle), and used retroviruses to deliver the transcription-factor genes to the injured heart. These viruses can insert genes into the chromosomes of actively dividing cells, such as scar-forming fibroblasts, but not into those of non-dividing cells such as cardiomyocytes. One month after treatment, reprogrammed cardiomyocyte-like cells comprised 2.4–6.5% of the cardiomyocytes in the region bordering the injured area (the infarct border zone) in the study by Song et al. and, remarkably, up to 35% in Qian and colleagues' experiments. Moreover, in both studies, the hearts of treated mice showed improved function compared with those of control mice.
A key challenge for the authors was how to distinguish pre-existing cardiomyocytes from those derived from reprogrammed fibroblasts. To address this, both groups used mice that had been genetically manipulated so that a fluorescent protein was permanently produced only in fibroblasts and their descendants (including those that became cardiomyocytes). The specificity of this lineage-tracing technique depended on the use of certain regulatory sequences, or promoters, that had been taken from genes encoding either periostin or FSP1 — two proteins that are typically produced by fibroblasts, but not cardiomyocytes. In cells in which the promoter was active, a genetic rearrangement led to permanent activation of the gene encoding the fluorescent protein.
Such lineage-tracing approaches are state of the art, but they are not perfect. The biggest pitfall would be activation of the fibroblast promoter in pre-existing cardiomyocytes, so that these would then be mistaken for reprogrammed cells. Neither periostin nor FSP1 is specific to fibroblasts11,12 (although we know of no evidence for their expression in cardiomyocytes). For these reasons, Song et al. carried out further experiments in which they controlled the timing of the fibroblast-marking event using a 'genetic pulse–chase' technique. They report that no cardiomyocytes were marked unless they expressed the transcription-factor cocktail. This finding enhances confidence that true reprogramming had occurred.
Interestingly, both studies found that, although some of the cells had been only partially reprogrammed, others were morphologically and functionally indistinguishable from normal cardiomyocytes. In particular, fibroblast-derived cardiomyocytes in short-term culture were able to contract when stimulated electrically and had electrochemical activities typical of this cell type, including action potentials and electrical coupling. Both research groups used non-invasive diagnostic procedures (echocardiography and magnetic resonance imaging) to identify the improved functional performance and reduced scar area of the treated mice when compared with untreated animals.
The finding of enhanced heart function is certainly important, but how is this happening, and can it be improved on? Although the authors' results suggest that the treatment generated new, functional cardiomyocytes that directly improved pump performance, it is important to remember that the reprogrammed cells constituted only a fraction of the cardiomyocytes in the infarct border zone, which is by nature ill-defined and forms only a fraction of the injured area. Can such a small number of cells directly account for a global increase in heart function? Researchers in stem-cell therapy have encountered similarly disproportionate benefits of cellular grafts in the heart. This therefore raises the possibility that grafted or reprogrammed cells may produce growth factors, cytokines or other signalling molecules that improve the performance of pre-existing cells by enhancing blood flow or cell survival13.
Going forward, it will be necessary to validate the authors' results in independent labs using different lineage-tracing approaches, and the efficiency of cell reprogramming must be increased. Also, for clinical applications, reprogramming must be achieved without inserting the transcription-factor genes into the fibroblasts' chromosomes, to prevent complications such as malignant transformation. Moreover, are cardiomyocytes the best choice of outcome for reprogramming, or would immature progenitors of cardiomyocytes (which have greater proliferative ability) be better?
Although clinical trials are probably far off, the studies by Qian et al. and Song et al. open up a new line of investigation in cardiovascular translational medicine. If we can understand the reprogramming mechanisms correctly, regenerative therapy might simply involve inducing the heart to reprogram its own cells after injury.