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.

Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H 2 S formed by cystathionine β-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H 2 S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2-null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.gas biology | neurovascular unit | energy metabolism | gasotransmitter T he cerebral circulation is maintained by autoregulation, which prevents marked alterations in response to changes in blood pressure, whereas functional hyperemia links blood flow to neural activity (1). Blood flow regulation in the brain is modulated by O 2 (2), with increased cerebral blood flow in response to hypoxia critical for protecting the brain against diverse insults. Such regulation also participates in functional hyperemia, as demonstrated by functional MRI investigations indicating a transient decrease in O 2 levels preceding activation of blood flow in response to neuronal firing (3).Alterations in cerebral blood flow in response to hypoxia and neural activity are mediated via several neurotransmitter systems, with prominent involvement of the gaseous mediator nitric oxide (NO) (1, 2). In response to glutamate acting on NMDA receptors, neuronal NO synthase (nNOS) is activated by increases in intracellular calcium, with the generated NO stimulating soluble guanylyl cyclase, thereby increasing cGMP levels to dilate blood vessels (4). Functional hyperemia is decreased by ∼50% in rats in response to inhibition of nNOS (5). Another gaseous mediator, CO (6-8), is also vasoactive. In some blood vessel systems (e.g., liver sinusoids), CO causes vasodilation, and inhibition of its biosynthetic enzyme HO-2 leads to vasoconstriction (9-13). However, in the cerebral circulation, CO elicits vasoconstriction. Thus, HO inhibitors cause cerebral vasodilation, an effect reversed by CO (14). This action of CO cannot be readily explained by previously identified CO receptors, such as soluble guanylyl cyclase (6-12, 15) or potassium channels (13, 16), both of which mediate vasodilation. The CO and NO systems interface; thus, the vasodilatory actions of HO inhibitors are partially reversed by inhibitors of NOS (14). A third gaseous mediator, H 2 S, is also vasoactive, eliciting vasodilation in both the peripheral and cerebral circulation (17-21). H 2 S can be physiologically ...

Haem oxygenase (HO)-1/carbon monoxide (CO) protects cancer cells from oxidative stress, but the gas-responsive signalling mechanisms remain unknown. Here we show using metabolomics that CO-sensitive methylation of PFKFB3, an enzyme producing fructose 2,6-bisphosphate (F-2,6-BP), serves as a switch to activate phosphofructokinase-1, a rate-limiting glycolytic enzyme. In human leukaemia U937 cells, PFKFB3 is asymmetrically di-methylated at R131 and R134 through modification by protein arginine methyltransferase 1. HO-1 induction or CO results in reduced methylation of PFKFB3 in varied cancer cells to suppress F-2,6-BP, shifting glucose utilization from glycolysis toward the pentose phosphate pathway. Loss of PFKFB3 methylation depends on the inhibitory effects of CO on haem-containing cystathionine β-synthase (CBS). CBS modulates remethylation metabolism, and increases NADPH to supply reduced glutathione, protecting cells from oxidative stress and anti-cancer reagents. Once the methylation of PFKFB3 is reduced, the protein undergoes polyubiquitination and is degraded in the proteasome. These results suggest that the CO/CBS-dependent regulation of PFKFB3 methylation determines directional glucose utilization to ensure resistance against oxidative stress for cancer cell survival.