OF WORK This research area involves the study of embryonic stem and induced pluripotent stem cells prior to and during differentiation to cardiomyocytes. Past accomplishments include establishment of efficient in vitro systems to generation of cardiomyocytes from ES cells in vitro, and the analysis of ryanodine type 2 deficient ES cells and their effects on cardiomyocytes. Selection protocols (most recently with a cardiac-restricted portion of the Na/Ca exchanger promoter) have also permitted the isolation of purified cardiomyocytes from these heterogeneous cultures. Recently, we established an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CMs that can be employed that facilitate the analysis of cell cycle control mechanisms. These cells, which represent one promising source for cell replacement therapy in heart, are highly heterogeneous and show a variety of maturation states. In a recently published study, we employed an ESC clonal line that contains a cardiac-restricted ncx1 promoter driven puromycin resistance cassette together with a mass culture system to isolate ESC-CMs that display traits characteristic of very immature CMs. The cells display properties of proliferation, CM-restricted markers, reduced mitochondrial mass and hypoxia resistance. Following transplantation into rodent hearts, bioluminescence imaging revealed that immature cells, but not more mature CMs, survived for at least one month following injection. These data and comparisons with more mature cells lead us to conclude that immature hypoxia resistant ESC-CMs can be isolated in mass in vitro and, following injection into heart, form grafts that may mediate long-term recovery of global and regional myocardial contractile function following infarction. Separately, we collaborated to show that embryonic stem cell-derived cardiocytes (ESCs)often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca(2+)Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca(2+)current (I(CaL)) induces a global Ca(2+) release (CICR), depleting the Ca(2+) content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca(2+)release of multiple LCRs throughout the cell. The LCRs generate an inward Na(+)/Ca(2+)exchanger (NCX) current (absent in Na(+)-free solution) that ignites the next AP. Interfering with SR Ca(2+) cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na(+)-free solution), or I(CaL) (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca(2+) loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic """"""""pacemaker-like"""""""" cells. SR Ca(2+) loading and LCR activity could be also increased with a selective activation of SR Ca(2+) pumping by a phospholamban antibody. We conclude that SR Ca(2+) loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I(CaL) synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca(2+) release and NCX current. Rhythmic ESC automaticity can be achieved by boosting """"""""coupling"""""""" factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma. More recent data have been generated from targeted mouse ESCs which were used to generate inducible knockouts of the ryanodine receptor. These data have been recently published.

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Bround, Michael J; Asghari, Parisa; Wambolt, Rich B et al. (2012) Cardiac ryanodine receptors control heart rate and rhythmicity in adult mice. Cardiovasc Res 96:372-80
Qiao, Hui; Zhang, Hualei; Yamanaka, Satoshi et al. (2011) Long-term improvement in postinfarct left ventricular global and regional contractile function is mediated by embryonic stem cell-derived cardiomyocytes. Circ Cardiovasc Imaging 4:33-41
Boheler, Kenneth R; Joodi, Robert N; Qiao, Hui et al. (2011) Embryonic stem cell-derived cardiomyocyte heterogeneity and the isolation of immature and committed cells for cardiac remodeling and regeneration. Stem Cells Int 2011:214203
Zahanich, Ihor; Sirenko, Syevda G; Maltseva, Larissa A et al. (2011) Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling. J Mol Cell Cardiol 50:66-76
Kania, Gabriela; Boheler, Kenneth R; Landmesser, Ulf et al. (2011) Stem cells in heart failure. Stem Cells Int 2011:193918
Fu, Ji-Dong; Rushing, Stephanie N; Lieu, Deborah K et al. (2011) Distinct roles of microRNA-1 and -499 in ventricular specification and functional maturation of human embryonic stem cell-derived cardiomyocytes. PLoS One 6:e27417
Wiese, Cornelia; Nikolova, Teodora; Zahanich, Ihor et al. (2011) Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin. Int J Cardiol 147:95-111
Zhang, Zhuoli; Hancock, Brynne; Leen, Stephanie et al. (2010) Compatibility of superparamagnetic iron oxide nanoparticle labeling for ¹H MRI cell tracking with ³¹P MRS for bioenergetic measurements. NMR Biomed 23:1166-72
Boheler, Kenneth R (2009) The golden age of cardiomyogenic stem cells: avoiding a fool's fate. Expert Rev Cardiovasc Ther 7:1-4
Biehl, Jesse K; Yamanaka, Satoshi; Desai, Tejal A et al. (2009) Proliferation of mouse embryonic stem cell progeny and the spontaneous contractile activity of cardiomyocytes are affected by microtopography. Dev Dyn 238:1964-73

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