Functional and developmental properties of human embryonic stem cells–derived cardiomyocytes

Abstract

Cardiovascular diseases are the most frequent cause of death in the industrialized world, with the main contributor being myocardial infarction. Given the high morbidity and mortality rates associated with congestive heart failure, the shortage of donor hearts for transplantation, complications resulting from immunosuppression, and long-term failure of transplanted organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. Because it is desired that the transplanted cells fully integrate within the diseased myocardium, contribute to its contractile performance, and respond appropriately to various physiological stimuli (eg, β-adrenergic stimulation), our major long-term goal is to investigate the developmental changes in functional properties and hormonal responsiveness of human embryonic stem cells–derived cardiomyocytes (hESC-CM). Furthermore, because one of the key obstacles in advancing cardiac cell therapy is the low differentiation rate of hESC into cardiomyocytes, which reduces the clinical efficacy of cell transplantation, our second major goal is to develop efficient protocols for directing the cardiomyogenic differentiation of hESC in vitro.

To accomplish the first goal, we investigated the functional properties of hESC-CM (<90 days old), respecting the contractile function and the underlying intracellular Ca²⁺ handling. In addition, we performed Western blot analysis of the key Ca²⁺-handling proteins SERCA2, calsequestrin, phospholamban and the Na⁺/Ca²⁺ exchanger. Our major findings were the following: (1) In contrast to the mature myocardium, hESC-CM exhibit negative force-frequency relationships and do not present postrest potentiation. (2) Ryanodine and thapsigargin do not affect the [Ca²⁺]_i transient and contraction, suggesting that, at this developmental stage, the contraction does not depend on sarcoplasmic reticulum Ca²⁺ release. (3) In agreement with the finding that a voltage-dependent Ca²⁺ current is present in hESC-CM and contributes to the mechanical function, verapamil completely blocks contraction. (4) Although hESC-CM express SERCA2 and Na⁺/Ca²⁺ exchanger at levels comparable to those of the adult human myocardium, calsequestrin and phospholamban are not expressed. (4) In agreement with other reports, hESC-CM are responsive to β-adrenergic stimulation. These findings show that the mechanical function related to intracellular Ca²⁺ handling of hESC-CM differs from the adult myocardium, probably because of immature sarcoplasmic reticulum capacity.

Introduction

Cardiovascular diseases are the most frequent cause of death in the industrialized world. In the United States alone, congestive heart failure—the ineffective pumping of the heart caused by the loss or dysfunction of heart muscle cells, affects approximately 5 million patients, with 400 000 new cases each year.¹ The main contributor to the development of this condition is myocardial infarction, affecting about 1.1 million Americans each year. End-stage heart failure still has a poor prognosis, underlined by the fact that only 50% of these patients survive to the following year. The current pharmacotherapy for congestive heart failure, including neurohormonal inhibition with angiotensin-converting enzyme inhibitors and β-blockers, improves clinical outcomes. Nevertheless, other treatment options including various interventional and surgical therapeutic methods are limited in preventing ventricular remodeling because of their inability to repair or replace damaged myocardium. Given the high morbidity and mortality rates associated with congestive heart failure, shortage of donor hearts for transplantation, complications resulting from immunosuppression, and long-term failure of transplanted organs, novel therapeutic modalities for improving cardiac function and preventing heart failure are in critical demand.² Hence, regeneration or repair of infracted or ischemic myocardium may be accomplished by the use of cell therapy—the transplantation of healthy, functional, and propagating cells to restore the viability or function of deficient tissues.3, 4, 5

Stem cells are characterized by prolonged self-renewal and long-term potential to form differentiated cell types. In most adult tissues, stem or progenitor cells are mobilized in response to environmental stimuli. However, stem cells present in adult tissues form only a limited number of cell types. In early mammalian embryos at the blastocyst stage, the inner cell mass is pluripotent, and therefore, the identification, derivation and characterization of human embryonic stem cells may open the door to the rapidly progressing field of therapeutic cell transplantation. Human embryonic stem cells (hESC), which are derived from human preimplantation embryos at the blastocyst stage4, 6, 7 were demonstrated to fulfill all the criteria defining embryonic stem cells: immortality, capability to proliferate indefinitely in culture while maintaining the undifferentiated phenotype, and the capacity to form derivatives of all three germ layers. Hence, hESC can therefore serve as a source of numerous types of differentiated cells.

The most attractive application of hESC is cell replacement therapy aimed at substituting healthy cells for diseased, missing, or degenerated tissue. The use of cell replacement therapy is of particular significance in the field of regenerative cardiovascular medicine because massive loss of terminally differentiated adult cardiomyocytes, as what occurs, for example, during myocardial infarction, is irreversible. Recent experimental observations suggest that cell transplantation has a potential therapeutic value for treating heart diseases (eg, Refs.8, 9, 10). This notion is based on the fundamental concept that the transplanted donor cardiomyocytes will integrate with the host myocardium and contribute directly to the mechanical cardiac performance. Preferably, cell transplantation allows the replacement of nonfunctional myocytes and scar tissue with new fully functional contracting cells, improving cardiac function and relieving the symptoms of heart failure. Of the various potential sources of transplantable cardiomyocytes, our work is focused on hESC-derived cardiomyocytes (hESC-CM). In recent years, several studies have shown that improvement of myocardial function can been achieved in experimental animal models of heart failure and infarction by transplanting exogenous adult and embryonic cell types into the compromised myocardium. The transplanted cells include embryonic, fetal, and neonatal cardiomyocytes from rodents and pigs, as well as human adult and fetal cardiomyocytes, autologous adult atrial cells, and dermal fibroblasts (eg, Refs.10, 11). Independent of the cell type used, the improved myocardial function accompanying the transplantation may result from either the ability of the muscle cells to contract or from the passive contribution of mechanical support to the myocardial architecture. Thus, to improve the prospects of cardiac cell transplantation, it is widely realized that the functional properties and hormonal responsiveness of hESC-CM should be thoroughly investigated. In view of that, we (OB and JI-E) have embarked on a project aimed at establishing a comprehensive scientific infrastructure, which will significantly improve our abilities to use hESC-CM for cell therapy in heart diseases. Because it is desired that the transplanted cells fully integrate within the diseased myocardium, contribute to its contractile performance, and respond appropriately to various stimuli (eg, β-adrenergic stimulation), it is important to decipher their adaptability with the host myocardium. Therefore, our major goal is to investigate the functional properties and hormonal responsiveness of developing hESC-CM.