Background-Ex vivo expansion of resident cardiac stem cells, followed by delivery to the heart, may favor regeneration and functional improvement. Methods and Results-Percutaneous endomyocardial biopsy specimens grown in primary culture developed multicellular clusters known as cardiospheres, which were plated to yield cardiosphere-derived cells (CDCs). CDCs from human biopsy specimens and from comparable porcine samples were examined in vitro for biophysical and cytochemical evidence of cardiogenic differentiation. In addition, human CDCs were injected into the border zone of acute myocardial infarcts in immunodeficient mice. Biopsy specimens from 69 of 70 patients yielded cardiosphere-forming cells. Cardiospheres and CDCs expressed antigenic characteristics of stem cells at each stage of processing, as well as proteins vital for cardiac contractile and electrical function. Human and porcine CDCs cocultured with neonatal rat ventricular myocytes exhibited biophysical signatures characteristic of myocytes, including calcium transients synchronous with those of neighboring myocytes. Human CDCs injected into the border zone of myocardial infarcts engrafted and migrated into the infarct zone. After 20 days, the percentage of viable myocardium within the infarct zone was greater in the CDC-treated group than in the fibroblast-treated control group; likewise, left ventricular ejection fraction was higher in the CDC-treated group. Conclusions-A method is presented for the isolation of adult human stem cells from endomyocardial biopsy specimens.CDCs are cardiogenic in vitro; they promote cardiac regeneration and improve heart function in a mouse infarct model, which provides motivation for further development for therapeutic applications in patients. Key Words: cells Ⅲ biopsy Ⅲ electrophysiology Ⅲ myocardial infarction Ⅲ myocytes W e sought to develop a clinically applicable method for the isolation and expansion of adult stem cells capable of regenerating myocytes and vessels and improving function in the injured heart. Given recent evidence that the adult mammalian heart contains endogenous, cardiac-committed stem cells, 1-5 we began with cardiac tissue as our stem cell source, postulating that cardiac-derived cells might be particularly well-suited for myocardial regeneration. Percutaneous endomyocardial biopsy specimens were utilized as a convenient, minimally invasive tissue source. 6,7 We began with the observation that cardiac surgical biopsy specimens in culture yield spherical multicellular clusters dubbed "cardiospheres." 8 Cardiospheres resemble neurospheres 9 in that they are derived from primary tissue culture and contain many proliferative cells that express stem cell-related antigens, as well as other cells undergoing spontaneous cardiac differentiation. 8 We modified the original culture method to improve efficiency and added a postcardiosphere expansion step to obtain reasonable numbers of cells (cardiosphere-derived cells [CDCs]) for transplantation from the small specimens in a timely manner. Editori...

SUMMARY Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1β and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor.

Cardiomyocyte T-tubules are important for regulating ionic flux. Bridging Integrator 1 (BIN1) is a T-tubule protein associated with calcium channel trafficking that is down-regulated in failing hearts. Here we find that cardiac T-tubules normally contain dense protective inner membrane folds that are formed by a cardiac spliced isoform of BIN1. In mice with cardiac Bin1 deletion, T-tubule folding is decreased which does not change overall cardiomyocyte morphology, but frees diffusion of local extracellular calcium and potassium ions, prolonging action potential duration, and increasing susceptibility to ventricular arrhythmias. We also find that T-tubule inner folds are rescued only by the BIN1 isoform BIN1+13+17, which promotes N-WASP dependent actin polymerization to stabilize T-tubule membrane at cardiac Z-discs. In conclusion, BIN1+13+17 recruits actin to fold T-tubule membrane, creating a fuzzy space that protectively restricts ionic flux. When BIN1+13+17 is decreased, as occurs in acquired cardiomyopathy, T-tubule morphology is altered and arrhythmias can result.

The heartbeat originates within the sinoatrial node (SAN), a small highly-specialized structure containing <10,000 genuine pacemaker cells. The ~5 billion working cardiomyocytes downstream of the SAN remain quiescent when it fails, leading to circulatory collapse and fueling a $6B/year electronic pacemaker industry. To engineer faithful biological replicas of rare SAN cells as an alternative therapeutic strategy, we expressed a gene critical for early SAN specification in working cardiomyocytes in vitro, and in vivo in a model of bradycardia. Within days of transduction with Tbx18, ventricular cardiomyocytes in culture developed spontaneous electrical firing physiologically indistinguishable from that of SAN cells, along with morphological and epigenetic features characteristic of SAN cells. Focal Tbx18 gene transfer in the guinea-pig ventricle yielded ectopic pacemaker activity in vivo, correcting a bradycardic disease phenotype. Myocytes transduced in vivo acquired the cardinal tapering morphology and physiological automaticity of native SAN pacemaker cells, while controls remained rectangular and quiescent. The creation of induced SAN-like pacemaker (iSAN) cells by Tbx18 gene transfer opens new prospects for bioengineered pacemakers.

We conclude that electrically active, hESC-derived CMs are capable of actively pacing quiescent, recipient, ventricular CMs in vitro and ventricular myocardium in vivo. Our results may lead to an alternative or a supplemental method for correcting defects in cardiac impulse generation, such as cell-based pacemakers.