br Experimental Procedures br Author Contributions

Experimental Procedures
Author Contributions
Acknowledgments
Introduction Pluripotent stem RGDfK (PSCs) have the remarkable capacity to generate all cell types of the body (Thomson et al., 1998). Potential biomedical applications for the derivatives of PSCs are vast and diverse, including disease modeling, drug testing, tissue engineering, and cell therapies. However, to fully realize the potential of any of these applications, it is essential to understand more about their functional properties and to identify the factors that control their stability and maturation, since all differentiated derivatives of PSCs in vitro are immature, with fetal rather than adult characteristics (Murry and Keller, 2008). Here, we were interested in examining the properties of cardiomyocytes derived in vitro from human embryonic stem cells (hESCs). Electrically and contraction-competent cardiomyocytes can now be generated efficiently under defined conditions from hESCs and human induced pluripotent stem cells (hiPSCs) (Mummery et al., 2012). These cardiomyocytes have the potential to be used for all of the applications relevant to heart physiology and disease mentioned above. Now that the efficiency of differentiation is not rate limiting, a deeper study of the cardiomyocyte function is feasible and warranted. Of particular relevance to the heart’s function as a pump is the ability of the cardiomyocytes to supply themselves with the necessary energy for their work. During development in vivo, cardiomyocytes acquire a high density of mitochondria, which ultimately occupy 20%–30% of the cell volume in the adult (Schaper et al., 1980). This gives these cells a huge capacity for ATP synthesis, which is necessary to fund the high energy demands of ion pumping and contractility during strenuous activity. The importance of mitochondria for heart function is highlighted by the fact that functionally important mutations that affect mitochondria frequently cause cardiomyopathy (Bates et al., 2012; Hirano et al., 2001), and diminished mitochondrial function is an almost universal feature of cardiac disease (Ventura-Clapier et al., 2011). Heart disease remains a major cause of morbidity and mortality in the Western world and there is an urgent need for better models and treatment strategies. Surprisingly, though, investigation of mitochondrial involvement in heart disease has largely been limited to mice, which have a markedly different cardiac physiology compared with humans (Davis et al., 2011) and have not proved to be a highly predictable model for mitochondrial disease. The advent of human PSC research has created opportunities to probe the functional relationship between mitochondria and heart failure, and to study the specific cardiac pathogenic mechanisms of mitochondrial diseases using iPSCs generated from patients. However, little is known about how mitochondrial functions and bioenergetics change in the transition from a PSC to a cardiomyocyte, or how important these functions are. An analysis of these fundamental characteristics is thus warranted. Such an analysis would have practical implications for investigating the response to an energetic stress, such as a hypertrophic or chronotropic stimulus, and for studying disease phenotypes in which mitochondria are implicated, such as cardiomyopathy and cardiac hypertrophy. Another important consideration is that if cardiomyocytes acquire a high density of highly polarized mitochondria, one would also expect reactive oxygen species (ROS) production to be high. It is not known what impact this would have on cardiomyocyte function, stability, or maturation in this in vitro context, and therefore whether ROS levels should be controlled. ROS have been shown to affect a variety of important ion channels and pumps, so the benefit of having a large energy reserve could be offset by a greater burden on the cell as a consequence of oxidative modifications and damage (Goldhaber et al., 1989; Liu et al., 2010; Zima and Blatter, 2006).