Bernardo Nadal-Ginard

Bone marrow cells regenerate infarcted myocardium

Myocardial infarction leads to loss of tissue and impairment of cardiac performance. The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time. Injury to a target organ is sensed by distant stem cells, which migrate to the site of damage and undergo alternate stem cell differentiation; these events promote structural and functional repair. This high degree of stem cell plasticity prompted us to test whether dead myocardium could be restored by transplanting bone marrow cells in infarcted mice. We sorted lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein by fluorescence-activated cell sorting on the basis of c-kit expression. Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct. Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells. The developing tissue comprised proliferating myocytes and vascular structures. Our studies indicate that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease.

Adult Cardiac Stem Cells Are Multipotent and Support Myocardial Regeneration

The notion of the adult heart as terminally differentiated organ without self-renewal potential has been undermined by the existence of a subpopulation of replicating myocytes in normal and pathological states. The origin and significance of these cells has remained obscure for lack of a proper biological context. We report the existence of Lin(-) c-kit(POS) cells with the properties of cardiac stem cells. They are self-renewing, clonogenic, and multipotent, giving rise to myocytes, smooth muscle, and endothelial cells. When injected into an ischemic heart, these cells or their clonal progeny reconstitute well-differentiated myocardium, formed by blood-carrying new vessels and myocytes with the characteristics of young cells, encompassing approximately 70% of the ventricle. Thus, the adult heart, like the brain, is mainly composed of terminally differentiated cells, but is not a terminally differentiated organ because it contains stem cells supporting its regeneration. The existence of these cells opens new opportunities for myocardial repair.

Mobilized bone marrow cells repair the infarcted heart, improving function and survival

Attempts to repair myocardial infarcts by transplanting cardiomyocytes or skeletal myoblasts have failed to reconstitute healthy myocardium and coronary vessels integrated structurally and functionally with the remaining viable portion of the ventricular wall. The recently discovered growth and transdifferentiation potential of primitive bone marrow cells (BMC) prompted us, in an earlier study, to inject in the border zone of acute infarcts Lin− c-kitPOS BMC from syngeneic animals. These BMC differentiated into myocytes and vascular structures, ameliorating the function of the infarcted heart. Two critical determinants seem to be required for the transdifferentiation of primitive BMC: tissue damage and a high level of pluripotent cells. On this basis, we hypothesized here that BMC, mobilized by stem cell factor and granulocyte-colony stimulating factor, would home to the infarcted region, replicate, differentiate, and ultimately promote myocardial repair. We report that, in the presence of an acute myocardial infarct, cytokine-mediated translocation of BMC resulted in a significant degree of tissue regeneration 27 days later. Cytokine-induced cardiac repair decreased mortality by 68%, infarct size by 40%, cavitary dilation by 26%, and diastolic stress by 70%. Ejection fraction progressively increased and hemodynamics significantly improved as a consequence of the formation of 15 × 106 new myocytes with arterioles and capillaries connected with the circulation of the unaffected ventricle. In conclusion, mobilization of primitive BMC by cytokines might offer a noninvasive therapeutic strategy for the regeneration of the myocardium lost as a result of ischemic heart disease and, perhaps, other forms of cardiac pathology.

Evidence That Human Cardiac Myocytes Divide after Myocardial Infarction

BACKGROUND The scarring of the heart that results from myocardial infarction has been interpreted as evidence that the heart is composed of myocytes that are unable to divide. However, recent observations have provided evidence of proliferation of myocytes in the adult heart. Therefore, we studied the extent of mitosis among myocytes after myocardial infarction in humans. METHODS Samples from the border of the infarct and from areas of the myocardium distant from the infarct were obtained from 13 patients who had died 4 to 12 days after infarction. Ten normal hearts were used as controls. Myocytes that had entered the cell cycle in preparation for cell division were measured by labeling of the nuclear antigen Ki-67, which is associated with cell division. The fraction of myocyte nuclei that were undergoing mitosis was determined, and the mitotic index (the ratio of the number of nuclei undergoing mitosis to the number not undergoing mitosis) was calculated. The presence of mitotic spindles, contractile rings, karyokinesis, and cytokinesis was also recorded. RESULTS In the infarcted hearts, Ki-67 expression was detected in 4 percent of myocyte nuclei in the regions adjacent to the infarcts and in 1 percent of those in regions distant from the infarcts. The reentry of myocytes into the cell cycle resulted in mitotic indexes of 0.08 percent and 0.03 percent, respectively, in the zones adjacent to and distant from the infarcts. Events characteristic of cell division--the formation of the mitotic spindles, the formation of contractile rings, karyokinesis, and cytokinesis--were identified; these features demonstrated that there was myocyte proliferation after myocardial infarction. CONCLUSIONS Our results challenge the dogma that the adult heart is a postmitotic organ and indicate that the regeneration of myocytes may be a critical component of the increase in muscle mass of the myocardium.

Myocardial Cell Death in Human Diabetes

Abstract— The renin-angiotensin system is upregulated with diabetes, and this may contribute to the development of a dilated myopathy. Angiotensin II (Ang II) locally may lead to oxidative damage, activating cardiac cell death. Moreover, diabetes and hypertension could synergistically impair myocardial structure and function. Therefore, apoptosis and necrosis were measured in ventricular myocardial biopsies obtained from diabetic and diabetic-hypertensive patients. Accumulation of a marker of oxidative stress, nitrotyrosine, and Ang II labeling were evaluated quantitatively. The diabetic heart showed cardiac hypertrophy, cavitary dilation, and depressed ventricular performance. These alterations were more severe with diabetes and hypertension. Diabetes was characterized by an 85-fold, 61-fold, and 26-fold increase in apoptosis of myocytes, endothelial cells, and fibroblasts, respectively. Apoptosis in cardiac cells did not increase additionally with diabetes and hypertension. Diabetes increased necrosis by 4-fold in myocytes, 9-fold in endothelial cells, and 6-fold in fibroblasts. However, diabetes and hypertension increased necrosis by 7-fold in myocytes and 18-fold in endothelial cells. Similarly, Ang II labeling in myocytes and endothelial cells increased more with diabetes and hypertension than with diabetes alone. Nitrotyrosine localization in cardiac cells followed a comparable pattern. In spite of the difference in the number of nitrotyrosine-positive cells with diabetes and with diabetes and hypertension, apoptosis and necrosis of myocytes, endothelial cells, and fibroblasts were detected only in cells containing this modified amino acid. In conclusion, local increases in Ang II with diabetes and with diabetes and hypertension may enhance oxidative damage, activating cardiac cell apoptosis and necrosis.

Interaction of myogenic factors and the retinoblastoma protein mediates muscle cell commitment and differentiation

The experiments reported here document that the tumor suppressor retinoblastoma protein (pRB) plays an important role in the production and maintenance of the terminally differentiated phenotype of muscle cells. We show that pRB inactivation, through either phosphorylation, binding to T antigen, or genetic alteration, inhibits myogenesis. Moreover, inactivation of pRB in terminally differentiated cells allows them to reenter the cell cycle. In addition to its involvement in the myogenic activities of MyoD, pRB is also required for the cell growth-inhibitory activity of this myogenic factor. We also show that pRB and MyoD directly bind to each other, both in vivo and in vitro, through a region that involves the pocket and the basic-helix-loop-helix domains, respectively. All the results obtained are consistent with the proposal that the effects of MyoD on the cell cycle and of pRB on the myogenic pathway result from the direct binding of the two molecules.

Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy.

It is generally believed that increase in adult contractile cardiac mass can be accomplished only by hypertrophy of existing myocytes. Documentation of myocardial regeneration in acute stress has challenged this dogma and led to the proposition that myocyte renewal is fundamental to cardiac homeostasis. Here we report that in human aortic stenosis, increased cardiac mass results from a combination of myocyte hypertrophy and hyperplasia. Intense new myocyte formation results from the differentiation of stem-like cells committed to the myocyte lineage. These cells express stem cell markers and telomerase. Their number increased >13-fold in aortic stenosis. The finding of cell clusters with stem cells making the transition to cardiogenic and myocyte precursors, as well as very primitive myocytes that turn into terminally differentiated myocytes, provides a link between cardiac stem cells and myocyte differentiation. Growth and differentiation of these primitive cells was markedly enhanced in hypertrophy, consistent with activation of a restricted number of stem cells that, through symmetrical cell division, generate asynchronously differentiating progeny. These clusters strongly support the existence of cardiac stem cells that amplify and commit to the myocyte lineage in response to increased workload. Their presence is consistent with the notion that myocyte hyperplasia significantly contributes to cardiac hypertrophy and accounts for the subpopulation of cycling myocytes.

Bone Marrow Cells Differentiate in Cardiac Cell Lineages After Infarction Independently of Cell Fusion

Recent studies in mice have challenged the ability of bone marrow cells (BMCs) to differentiate into myocytes and coronary vessels. The claim has also been made that BMCs acquire a cell phenotype different from the blood lineages only by fusing with resident cells. Technical problems exist in the induction of myocardial infarction and the successful injection of BMCs in the mouse heart. Similarly, the accurate analysis of the cell populations implicated in the regeneration of the dead tissue is complex and these factors together may account for the negative findings. In this study, we have implemented a simple protocol that can easily be reproduced and have reevaluated whether injection of BMCs restores the infarcted myocardium in mice and whether cell fusion is involved in tissue reconstitution. For this purpose, c-kit–positive BMCs were obtained from male transgenic mice expressing enhanced green fluorescence protein (EGFP). EGFP and the Y-chromosome were used as markers of the progeny of the transplanted cells in the recipient heart. By this approach, we have demonstrated that BMCs, when properly administrated in the infarcted heart, efficiently differentiate into myocytes and coronary vessels with no detectable differentiation into hemopoietic lineages. However, BMCs have no apparent paracrine effect on the growth behavior of the surviving myocardium. Within the infarct, in 10 days, nearly 4.5 million biochemically and morphologically differentiated myocytes together with coronary arterioles and capillary structures were generated independently of cell fusion. In conclusion, BMCs adopt the cardiac cell lineages and have an important therapeutic impact on ischemic heart failure.

Myocyte Death, Growth, and Regeneration in Cardiac Hypertrophy and Failure

The accepted paradigm considers the adult mammalian heart as a postmitotic organ, which possesses a relatively constant number of myocytes from shortly after birth to adulthood and senescence. This notion is questioned by the demonstration that although most adult myocytes are terminally differentiated, there is a small and continuously renewed subpopulation of cycling myocytes produced by the differentiation of cardiac stem-like cells. Myocyte death and myocyte regeneration are introduced as major determinants of cardiac homeostasis and alterations of ventricular anatomy and function in physiological and pathological states. The possibility of reconstituting dead myocardium by stem-like cells is advanced and proposed as a major area of future research.

Myocyte Renewal and Ventricular Remodelling

Remaining young at heart is a desirable but elusive goal. Unbeknown to us, however, myocyte regeneration may accomplish just that. Continuous cell renewal in the adult myocardium was thought to be impossible, but multipotent cardiac stem cells may be able to renew the myocardium and, under certain circumstances, can be coaxed to repair the broken heart after infarction.