Cell transfer to the myocardium

Heart failure frequently occurs as the result of ischemia and necrosis developing after the occlusion of a coronary vessel. The myocardium cannot (er only to a very limited extent) regenerate. Myocardial infarcts heal, therefore, by scaring. The inevitable loss in contractile cells alter a myocardial infarction results consequently often in cardiac insufficiency. Autologous transfer of skeletal muscle myoblasts (MB) and transfer (er cytocine-activation) of bone marrow derived stem cells (BMC) have been shown to promote tissue repair after myocardial infarcts. There is, however, an ongoing debate about the efficacy of each of these therapeutic approaches. A significant part of the project will, therefore, focus an a comparison of the efficacy and optimal use of MB vs. BMC transfer in the therapy of heart failure in an animal model (Fisher rat). A second aim of the research proposal is to evaluate the arrhythmogenic potential of skeletal myoblast- and BMC-grafts and the intracellular calcium handling of the cells that have been transplanted to the myocardium. A major part of the experiments will be performed an enzymatically isolated muscle cells (identified by marker genes) from the myocardium (confocal microscopy, patch clamp analysis). We will evaluate the expression of the different sodium and calcium channel subtypes in the MB or BMC grafts. The project will also focus an the clinical potential of BMC vs. MB vs. MB/fibroblast therapy (in in vivo studies to be performed in collaboration with Prof. Laufers group [Cardiac Surgery] and Prof Gastls group [Hematology] an the ischemic heart failure model). Here we will perform telemetric monitoring of ECG and blood pressure after cell transfer and investigate changes in functional parameters of the myocardium an isolated perfused, working hearts in ex vivo studies. Functional parameters (e.g. ejection fraction) will be estimated by echocardiography in conscious rats. Part of these experiments will be performed to evaluate the optimal time point and the optimal amount of cell material for transfer (MB and BMC) into the necrotic area of the infarct zone. The expertise of the participating groups (Departments of Cardiac Surgery [infarct model, in vivo and ex vivo studies], Hematology [stem cell research] and Biochemical Pharmacology [studies an enzymatically isolated single cells] all University of Innsbruck) and the shared use of the heart failure model, tissue culture facilities (ie. established GFP-positive cell lineages of MB and fibroblasts of Fisher rats) and their specific research capabilities provide optimal conditions for a successfull realisation of the proposal.

Transplantation of adult stem cells (myogenic or marrow-derived) is a potential new means of improving the prognosis of patients with cardiac failure. It is assumed that the loss of cardiomyocytes after a myocardial infarction can be partly reversed by implantation of new contractile cells into (or around) the scar area. In proof of concept studies transfer of skeletal muscle myoblasts (SM) and bone marrow derived stem cells (BMC) have been shown to promote tissue repair after a myocardial infarction. A significant part of the project focused on the question whether intramyocardial transplantation of combined SM and mononuclear BMC is superior to the isolated transplantation of these cell types. Our studies clearly demonstrated that the concept of combining SMs with BMC may be of clinical relevance by merging the beneficial effects of each cell line and potentially reducing the required cell quantity (Ott et al. 2004). In more recent studies we could demonstrate that transplantation of SM or angiopoietic progenitor BMC leads to improvement of left ventricular function, and reduction of scar size and myocardial apoptosis by means of neoangiogenesis in chronic ischemia. During the project we developed a novel cell delivery technique for intramyocardial multisite pressure injection. This technique enabled a safer and more reliable transplantation of multiple myoblast microdepots into an infarcted myocardium. The compared to the standard technique the efficacy of myoblast transplantation was increased, target tissue damage was reduced and cell engraftment increased as well. A major aim of our research project was to evaluate the electrophysiological properties of SM that have been transplanted into the myocardium. For this purpose we studied the voltage-dependent sodium, calcium and potassium channels of SM after transplantation into the infarcted myocardium of syngenic rats. We observed a down-regulation of all three types of ion channels after engraftment. Before injection myoblasts expressed predominantly transient outward potassium channels whereas after isolation from the myocardium exclusively rapid delayed rectifier channels. After transplantation into the myocardium SM seem to dedifferentiate towards an unexcitable "hibernating" cell type. Under optimal growth conditions ionic currents of the previously unexcitable cells recover completely between 1 and 6 weeks. The cells remain fusion competent and differentiate into multinucleated myotubes. Further studies will show if these mononucleated myogenic cells have common properties with skeletal muscle satellite cells. More recent studies in in our lab suggest that the dedifferentiation is not caused by the "aggressive environment" of the infarct region but occur also human myoblasts that were implanted into non infracted heart muscle tissue (Berjukow et al. in preparation).