Heart disease remains a major cause of death despite advances in medical technology. Heart-regenerative therapy that uses pluripotent stem cells (PSCs) is a potentially promising strategy for patients with heart disease, but the inability to generate highly purified cardiomyocytes in sufficient quantities has been a barrier to realizing this potential. Here, we report a nongenetic method for mass-producing cardiomyocytes from mouse and human PSC derivatives that is based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and noncardiomyocytes, including undifferentiated cells. We cultured PSC derivatives with glucose-depleted culture medium containing abundant lactate and found that only cardiomyocytes survived. Using this approach, we obtained cardiomyocytes of up to 99% purity that did not form tumors after transplantation. We believe that our technological method broadens the range of potential applications for purified PSC-derived cardiomyocytes and could facilitate progress toward PSC-based cardiac regenerative therapy.

Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.

Abstract-Mitochondrial ATP-sensitive potassium (mitoK ATP ) channels play a key role in ischemic preconditioning of the heart. However, the mechanism of cardioprotection remains controversial. We measured rhod-2 fluorescence in adult rabbit ventricular cardiomyocytes as an index of mitochondrial matrix Ca 2ϩ concentration ([Ca 2ϩ ] m ), using time-lapse confocal microscopy. To simulate ischemia and reperfusion (I/R), cells were exposed to metabolic inhibition (50 minutes) followed by washout with control solution. Rhod-2 fluorescence gradually increased during simulated ischemia and rose even further with reperfusion. The mitoK ATP channel opener diazoxide attenuated the accumulation of [Ca 2ϩ ] m during simulated I/R (EC 50 ϭ18 mol/L). These effects of diazoxide were blocked by the mitoK ATP channel antagonist 5-hydroxydecanoate (5HD). In contrast, inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A and bongkrekic acid, did not alter [Ca 2ϩ ] m accumulation during ischemia, but markedly suppressed the surge in rhod-2 fluorescence during reperfusion. Measurements of mitochondrial membrane potential, ⌬⌿ m , in permeabilized myocytes revealed that diazoxide depolarized ⌬⌿ m (by 12% at 10 mol/L, PϽ0.01) in a 5HD-inhibitable manner. Our data support the hypothesis that attenuation of mitochondrial Ca 2ϩ overload, as a consequence of partial mitochondrial membrane depolarization by mitoK ATP channels, underlies cardioprotection. Furthermore, mitoK ATP channels and the MPT differentially affect mitochondrial calcium homeostasis: mitoK ATP channels suppress calcium accumulation during I/R, while the MPT comes into play only upon reperfusion. Key Words: mitochondrial calcium overload Ⅲ cardioprotection Ⅲ ischemia I schemic preconditioning (IPC) 1 is the endogenous mechanism whereby brief periods of ischemia paradoxically protect the myocardium against the damaging effects of subsequent prolonged ischemia. IPC exists in all species examined, including humans. 2 Mitochondrial K ATP (mitoK ATP ) channels feature prominently in the mechanism of cardioprotection 3-9 ; however, their precise role remains controversial. A decrease in the extent of mitochondrial Ca 2ϩ overload during ischemia and reperfusion (I/R) has been proposed to prevent or delay cell death, 10,11 and activation of the mitochondrial permeability transition (MPT) may be involved in this process. MPT opening results in the collapse of oxidative phosphorylation and can be accelerated synergistically by Ca 2ϩ , oxidative stress, and ATP depletion during I/R. 12,13 These observations prompted us to investigate not only whether the cytoprotective effect of mitoK ATP channel activation is related to inhibition of mitochondrial Ca 2ϩ overload during I/R, but also whether mitoK ATP channel opening can depolarize ⌬⌿ m . 4 To test our hypothesis, we have monitored mitochondrial Ca 2ϩ concentration ([Ca 2ϩ ] m ) during simulated I/R and separately measured mitochondrial membrane potential in response to mitoK ATP channel mediators. Our...

SUMMARY Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked-down 7061 evolutionarily conserved genes under conditions of stress. We present a first global road-map of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and post-transcriptional regulatory mechanisms. Silencing of the CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of lethal arrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.

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This study demonstrated that iPSCs could be useful to characterize LQTS disease as well as drug responses in the LQTS patient with a novel mutation. Such analyses may in turn lead to future progress in personalized medicine.