The promise of myocardial repair – towards a better understanding

Moshe Y. Flugelman* and Basil S. Lewis1

Cardiovascular Research Center, Department of Cardiovascular Medicine, Lady Davis Carmel Medical Center and the Bruce Rappaport School of Medicine, Technion IIT, Haifa, Israel

* Correspondence to: Moshe Y. Flugelman, Cardiovascular Research Center, Department of Cardiovascular Medicine, Lady Davis Carmel Medical Center and the Bruce Rappaport School of Medicine, Technion IIT, Haifa, Israel. Tel.: +972-4-8250812/972-4-8250575; fax: +972-4-8250841/972-4-8250936
myf{at}tx.technion.ac.il

Despite major advances in the management of acute myocardial infarction, including effective and early urgent revascularisation, myocardial damage is common and may be severe. In many patients myocardial infarction is followed by myocardial scarring, ventricular remodelling, dilatation and reduced cardiac function followed over a period of months to years by an inexorable downhill course culminating in heart failure and death regardless of best current treatment modalities.

Myocyte regeneration – a paradigm shift

In the new millennium, we are witnessing a paradigm shift in our approach to myocardial damage, following the landmark observation that the adult heart is not necessarily a post-mitotic organ and that regeneration of cardiac myocytes may occur in the circumstances of the appropriate signalling processes.1 Within a relatively short time period, the findings from the basic science and animal laboratory have been applied in the clinical arena. Intracoronary injection of mesenchymal stromal cells (MSCs) of bone-marrow origin produced encouraging results in humans recovering from acute infarction,2 with an improvement in myocardial perfusion by thallium-201 scintigraphy, and higher ejection fraction compared with patients not treated. The intracoronary injection of progenitor cells originating in marrow or peripheral blood (PBSCs) was followed by enhanced regional contractile function, a significant increase at 4 months in coronary flow reserve in the infarct artery and a significant increase in myocardial viability by quantitative F-18-fluorodeoxyglucose-positron emission tomography in the infarct zone.3 Ejection fraction increased significantly post-infarction.3 There were no differences for any measured parameter between blood-derived or bone-marrow-derived progenitor cells. Britten et al.4 showed that the migratory capacity of the infused cells is a major determinant of infarct remodelling, disclosing a causal effect of progenitor cell therapy on regeneration enhancement.

None of the published studies regarding injection of MSCs or PBSCs have been randomised or placebo-controlled and a large number of questions remain unanswered as we apply with enthusiasm our relatively frugal basic knowledge to the clinical setting. Experience from recent studies has highlighted our relative lack of understanding concerning many of the basic processes underlying myocardial repair. These studies have indeed introduced a note of caution regarding possible unwanted effects of stem cell mobilisation by granulocyte colony-stimulating factor (g-CSF) regarding aggravated vasculopathy5 or the possible deleterious effects of infusion involving inappropriate cell size6 in an already compromised peripheral coronary circulation, at least in an animal model. The unravelling of the signalling processes in cellular regeneration and a better understanding of the cellular possibilities are mandatory if we are to apply cell therapy as a routine clinical modality in patients with acute or chronic myocardial damage. In particular, we need to elucidate the mechanisms by which transplanted stem cells can home in and differentiate into endothelial and vascular cells and/or into cardiac myocytes.

What do we know about the mechanisms of cellular regeneration?

Three major issues currently under debate relate to: (1) the optimal source of cells to promote myocardial repair (bone-marrow, peripheral blood autologous cells or indeed the possibility of "off the shelf" immune tolerant stem cells), (2) the relative importance in the repair process of vascular-oriented cell processes (enhanced angiogenesis with indirect improvement in myocardial function) or haematopoietic inflammatory processes (also indirect stimulation of cellular growth), versus direct regeneration of cardiac myocytes by mitosis and true transdifferentiation of migratory and/or implanted cells, and (3) the importance of the mobilised or transplanted cells themselves or the paracrine effects of the activated cells on resident progenitors in or around areas of myocardial damage.

Hocht and colleagues7 recently studied these issues using a model based on sex-mismatched male heart transplant recipients who underwent post-transplant myocardial infarction 1–5 years later. Such patients provide the opportunity for clear identification and tracking of cells involved in the inflammatory and repair process, whether these be of local cardiac (female, Y-chromosome negative cells) or extracardiac host (male, Y-chromosome positive cells) origin. Using fluorescence in situ hybridisation (FISH) for detection of the Y-chromosome and 3D-confocal imaging techniques, combined with immunofluorescence staining for CD45 and CD68 to distinguish host-derived inflammatory cells, the authors focused on the issue of repopulation of the infarcted heart with myocytes originating from the (male) bone-marrow cells. The study, although based on a small number of patients, showed that host-derived non-inflammatory progenitor and endothelial cells were significantly increased compared to nine non-infarcted patients. Yet, by using this novel multi-step approach, they were able to show that only 0.02% of all cells were estimated to be male cardiomyocytes, and their increase in infarcted regions to 0.07% was not significant, implying that myocardial infarction enhanced invasion of extra-cardiac progenitor cells and regeneration of endothelial cells, but a significant differentiation into cardiomyocytes did not occur. The low number of bone-marrow-derived myocytes in the study of Höcht et al.7 can be related to several characteristics of the patients they studied – their patients were immuno-suppressed, they died of heart failure (which is associated with multiple organ, including bone marrow, failure), and the time from infarct to death was relatively short. In particular, new growth of vascular tissue is well-known in transplant patients, with extracardiac host cells playing an important role in transplant vasculopathy, both in the heart and in other organs such as following renal transplantation.

Laflamme et al.8 showed that the transplanted heart contained a minute but readily detectable fraction of Y chromosome-positive cardiomyocytes. In their study, the mean percentage of cardiomyocytes arising from the host was estimated to be 0.04% with a median of 0.016%. Most Y-positive cardiomyocytes were associated with regions of acute rejection, suggesting such chimerism involves an injury event. Furthermore, the sole patient whose immediate cause of death was allograft rejection showed a higher percentage of host-derived cardiomyocytes, up to 29% in local, 1 mm "hot spots" of inflammation. Two recent studies in mice cast doubt on the ability of bone-marrow-derived cells to differentiate into cardiomyocytes.9,10 The difference between the findings reported by Beltrami et al.,1 and Thiele et al.11 may be based on a different model, different cause of death and different methodology. Muller et al.12 detected cardiomyocytes of recipient origin in 8 of 13 male recipients of female hearts and were connected by gap junctions with adjacent myocytes.

To further identify the mechanisms and durability of bone-marrow-derived reconstitution of cardiomyocytes, Nygren et al.13 studied mouse marrow cells with transgenic green fluorescent protein (GFP) and expressing lacZ in host tissue. Disappointingly, they found no increase in peripheral blood cellularity or progenitor stem cell activity following coronary ligation (unlike following cytokine stimulation). In agreement with Orlic et al.14 they did show engraftment of marrow-derived cells in damaged myocardium on day 9, but most engraftment was not maintained and indeed lost, at 28 days. By haematopoietic cell markers (CD45) it was haematopoietic cells rather than cardiomyocytes which accounted for this transient engraftment. Importantly, however, the authors did find bone-marrow-derived cardiomyocytes outside or bordering the infarcted zone, and demonstrated that the bone-marrow-derived cardiomyocytes were a result of cell fusion with existing myocytes.13 This suggested that cell fusion rather than cell transdifferentiation may be the basis of the changes operative in myocardial regeneration. Further studies are clearly needed in this regard.

Temporal relations and chronic damage

The signalling processes triggered by an acute event are of major importance in determining the course of events leading to myocardial repair. There is almost certainly a "natural" window of opportunity where cellular intervention will produce an optimal effect. This area has yet to be unravelled and a different approach may be needed in the patient with chronic myocardial damage. Bel et al.15 found that the benefits of extemporaneous transplantation of fresh (unfractionated) autologous bone marrow were of limited value for regenerating chronically infarcted myocardium in a sheep model.

An alternative approach to chronic damage focused on the use of autologous skeletal myoblast transplantation in patients with severe ischaemic cardiomyopathy. Menasche et al.16 implanted, at the time of bypass surgery, skeletal myoblasts grown from a biopsy taken at the thigh in 10 patients with severe left ventricular dysfunction and the post-infarction presence non-viable scar by dobutamine echocardiography and 18-fluorodeoxyglucose positron emission tomography. Blinded echocardiographic analysis showed that 63% of the cell-implanted scars (14 of 22) demonstrated improved systolic thickening, but arrhythmias were not uncommon.16 In a 12-month animal study from the same group,17 transplantation of skeletal muscle limited post-infarction deterioration and improved systolic scar function through colonisation of fibrosis by skeletal muscle cells with expression of both MHC isoforms, which may confer to the graft the ability to withstand a cardiac-type workload.17

Are we headed in the right direction?

Undoubtedly, cells of extracardiac origin may play an important role in myocardial repair, may reduce apoptosis and prevent remodelling in infarcted hearts. There is clearly, then, the rationale for the current intense research activity revolving around the application of cell therapy to the treatment of patients with myocardial damage. At this stage, however, the magnitude and importance of direct myocyte regeneration from extracardiac progenitors appears to be limited. Bone-marrow-derived cells found in the heart appear to be mainly inflammatory or part of blood vessels. They can promote proliferation of resident progenitors that can differentiate into myocytes. New cardiomyocytes may be generated in the adult heart from non-myocyte precursors, but there is still debate regarding the mechanisms and relative importance of the various processes. We need to understand how to activate signalling processes and perhaps use a different approach in the chronic situation. Many studies are based on a transplant model and we need to know whether the findings would be the same in a non-transplant population. Above all we must take care to ensure the parallel expansion of clinical and basic molecular knowledge to properly define the best approach to myocardial repair in this very large and very challenging group of patients.

Footnotes

1 Louis Edelstein Professor of Medicine and Medical Research. Back

References

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