With few exceptions, stem cell injections into the heart produce short-term improvement in cardiac function but a notable lack of integration and differentiation. This temporal improvement in cardiac function may be due to paracrine factors, but the process is not well understood. Recently, mitochondria have been observed to be transferred through tunneling nanotubes (TNTs) into myocytes, which might explain the transient effects of stem cell injections on improved cellular function. An understanding of mitochondrial transfer between stem cells and myocytes might enable rescue of failing cardiomyocytes by directed transfer of functional mitochondrial replacements. Significantly, there are pediatric genetic mitochondrial cardiomyopathies that in which such transfer could be particularly useful as a therapy. TNT formation has been observed in many settings; however, difficulty in capturing such a small structure using currently available tissue-sectioning techniques has made current knowledge of TNT formation and function heavily dependent on cell co-culture models. As there has been little systematic exploration of TNT formation and function, all that is known about TNTs is that they are widely observed and that they appear to transport both organelles and cytoplasmic molecules. Systematic study is difficult because in conventional cell co-culture, multiple TNTs of various lengths form between randomly distributed cells. To overcome this limitation, here it is proposed to develop a microfabricated coculture model, in which cardiomyocytes and stem cells are deposited on respective sides of a chamber with a perforated barrier between them. Due to geometric confinement, TNT formation and mitochondrial transfer between specific cell pairs can be defined in length and orientation. Using such a model, the proposed studies will explore the process of TNT communication between stem cells and myocytes and test model systems in which delivery of mitochondria will provide measurable improvement in outcomes. Of potential significance is that TNT-mitochondria transfer-based cell rescue is an intrinsic targeting process as opposed to conventional paracrine-based rescue mechanisms. An understanding of the underlying principles might lead to the formulation of strategies to develop a targeting therapy to rescue mitochondrial cardiomyopathies or to provide additional energy to failing hearts.
The specific aims are 1) Determine ontogeny of mitochondria-transferring TNTs in a microfabricated, compartmental coculture model; 2) Determine whether formation of a nanotube or transfer of mitochondria through the nanotube mediates a myocyte-survival function in an in vitro survival model; 3) Determine the rescue effect of mitochondrial transfer through TNTs on mitochondrial genetic cardiomyopathy; and 4) Determine the rescue effect on cardiac infarct of mitochondrial or other materials transfer through TNTs. Achievement of these aims will provide answers to the following questions: 1) Do all TNTs transmit mitochondria or is there a subset, discernible by size or structure, that facilitates this transfer? 2) Is TNT formation a response to myocyte stress? 3) Is the transfer of mitochondria, other molecules, or organelles the mediator of enhanced myocyte survival in coculture? 4) Do transferred mitochondria remain distinct or do they fuse with host mitochondria? 6) Does mitochondrial transfer utilize microtubular motor molecules? 7) Can normal mitochondria rescue myocyte function in a genetic model of mitochondrial cardiomyopathy? 8) In an in vivo infarct model, can discernible mitochondrial transfer with corresponding measurable changes in cardiac function be seen? These answers will have translational significance for stem cell therapies and will be informative as to design and success of specific approaches. These studies will also impact pediatric mitochondrial myopathies that are rare but fatal. If it is established that there is a functional advantage to TN formation, future studies can address the mechanisms of TNT formation and the optimization of this process for clinical utilization.
Understanding long-range mitochondrial transfer from stem cells to cardiomyocytes and the associated rescue effects should lead to development of therapies for heart diseases such as pediatric cardiomyopathies that are produced by mutations in mitochondria genes. Data obtained during the proposed research will enable formulation of novel targeting therapy at the cellular level.
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