Miyako Takaki

In vitro functional gut-like organ formation from mouse embryonic stem cells.

Background and Aims. Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently found that ES cells can give rise to a functional gut‐like unit, which forms a three‐dimensional dome‐like structure with lumen and exhibits mechanical activity, such as spontaneous contraction and peristalsis. The aim of the present study was to investigate the electrophysiological and morphological properties of ES cell‐derived contracting clusters.

Properties of spontaneously active cells distributed in the submucosal layer of mouse proximal colon

Intracellular electrical activity was recorded from smooth muscle tissues of the mouse proximal colon, and the impaled cells were visualized by injection of neurobiotin. Slow potentials with initial fast and subsequent plateau components (plateau potentials), generated at a frequency of 14.8 min−1, were recorded from oval‐shaped cells with bipolar processes. Periodic bursts of spike potentials (4.6 min−1) and bursts of oscillatory potentials (4.3 min−1) were recorded in circular and longitudinal smooth muscle cells, respectively. Nifedipine (0.1 μm) abolished the bursts of spike and oscillatory potentials and reduced the duration of plateau potentials. The plateau potentials were abolished by 1 μm nifedipine. The plateau potentials were also abolished by cyclopiazonic acid (an inhibitor of Ca2+ uptake into internal stores) or 2‐aminoethoxydiphenyl borate (an inhibitor of inositol 1,4,5‐trisphosphate receptor‐mediated Ca2+ release), and were inhibited by bis‐(aminophenoxy) ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (a chelator of intracellular Ca2+). Carbonyl cyanide m‐chlorophenylhydrazone (a mitochondrial protonophore) abolished plateau potentials, and its action was not mimicked by oligomycin (an inhibitor of mitochondrial ATPase). It is concluded that in mouse proximal colon, submucosal c‐kit‐positive bipolar cells spontaneously generate plateau potentials with rhythms different from those generated by smooth muscle cells. The plateau potentials are generated through activation of voltage‐gated Ca2+ channels, which are coupled to the release of Ca2+ from the internal stores and the handling of Ca2+ in mitochondria.

In Vitro Formation of Enteric Neural Network Structure in a Gut‐Like Organ Differentiated from Mouse Embryonic Stem Cells

Using an embryoid body (EB) culture system, we developed a functional organ‐like cluster—a “gut”—from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited spontaneous contractions but did not exhibit distinct peristalsis‐like movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (c‐kit [a transmembrane receptor that has tyrosine kinase activity]‐positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified; however, enteric neural ganglia were absent in the spontaneously differentiated ES gut. By adding brain‐derived neurotrophic factor (BDNF) only during EB formation, we for the first time succeeded in in vitro formation of enteric neural ganglia with connecting nerve fiber tracts (enteric nervous system [ENS]) in the ES gut. The ES gut with ENS exhibited strong peristalsis‐like movements. During EB culture in BDNF+ medium, we detected each immunoreactivity associated with the trk proto‐oncogenes (trkB; BDNF receptors) and neural crest marker, proto‐oncogene tyrosine‐protein kinase receptor ret precursor (c‐ret), p75, or sox9. These results indicated that the present ENS is differentiated from enteric neural crest‐derived cells. Moreover, focal stimulation of ES guts with ENS elicited propagated increases in intracellular Ca2+ concentration ([Ca2+]i) at single or multiple sites that were attenuated by atropine or abolished by tetrodotoxin. These results suggest in vitro formation of physiologically functioning enteric cholinergic excitatory neurons. We for the first time succeeded in the differentiation of functional neurons in ENS by exogenously adding BDNF in the ES gut, resulting in generation of distinct peristalsis‐like movements.

Rescue of Ca2+ overload-induced left ventriclur dysfunction by targeted ablation of phospholamban

In failing hearts, a deficiency in sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA)2a results in abnormal Ca2+ handling and diminished contraction. In addition, a decrease in the phosphorylation of phospholamban (PLB) has been reported. Gene transfer of antisense PLB (asPLB) can improve contractile function in the failing human myocardium. Gene transfer of SERCA2a improves survival and the energy potential in failing hearts. The aim of present study was to evaluate whether enhancement of SERCA2a function prevents acute Ca2+ overload-induced left ventricular (LV) dysfunction in rat hearts. We ablated PLB using adenoviral gene transfer of asPLB by a new and less invasive gene delivery method, which involved a percutaneous technique. Experiments were performed on 13 excised cross-circulated rat hearts: 5 rats underwent sham operations, 4 rats underwent gene transfer of the reporter gene beta-galactosidase (Ad.beta-gal), and 4 rats underwent gene transfer of asPLB (Ad.asPLB). After clearance of high Ca2+ infused into the coronary, there was LV contractile dysfunction associated with the decreased myocardial O2 consumption per beat (Vo2) intercept (equal to decreased Vo2 for Ca2+ handling in excitation-contraction coupling) of the Vo2-systolic pressure-volume area (PVA; total mechanical energy per beat) linear relation in the hearts that underwent sham operation and had been infected with Ad.beta-gal. Hearts that had been infected with Ad.asPLB were rescued from LV contractile dysfunction associated with an unchanged Vo2 intercept of the Vo2-PVA linear relation. We conclude that SERCA2a function enhanced by adenoviral gene transfer of asPLB prevents Ca2+ overload-induced LV contractile dysfunction in terms of mechanical work and especially energetics.

A new integrative method to quantify total Ca2+ handling and futile Ca2+ cycling in failing hearts

Ca2+ handling in excitation-contraction coupling requires considerable O2 consumption (Vo 2) in cardiac contraction. We have developed an integrative method to quantify total Ca2+ handling in normal hearts. However, its direct application to failing hearts, where futile Ca2+ cycling via the Ca2+-leaky sarcoplasmic reticulum (SR) required an increased Ca2+handling Vo 2, was not legitimate. To quantify total Ca2+ handling even in such failing hearts, we combined futile Ca2+ cycling with Ca2+ handling Vo 2 and the internal Ca2+ recirculation fraction via the SR. We applied this method to the canine heart mechanoenergetics before and after intracoronary ryanodine at nanomolar concentrations. We found that total Ca2+ handling per beat was halved after the ryanodine treatment from ∼60 μmol/kg left ventricle before ryanodine. We also found that futile Ca2+ cycling via the SR increased to >1 cycle/beat after ryanodine from presumably zero before ryanodine. These results support the applicability of the present method to the failing hearts with futile Ca2+ cycling via the SR.

A 5-HT(4)-receptor activation-induced neural plasticity enhances in vivo reconstructs of enteric nerve circuit insult.

Background  It was recently reported that some 5‐HT4‐receptor agonists increased neuronal numbers and length of neurites in enteric neurons developing in vitro from immunoselected neural crest‐derived precursors. We aimed to explore a novel approach in vivo to reconstruct the enteric neural circuitry that mediates a fundamental distal gut reflex.

Isoproterenol-Induced Hypertrophied Rat Hearts: Does Short-Term Treatment Correspond to Long-Term Treatment?

In consideration of clinical implications, it is often complained that short-term experimental diseased heart models do not mimic long-term diseased hearts that are often clinically encountered. The aim of the present study was (i) to compare the left ventricular function between rat cardiac hypertrophy models treated with isoproterenol for 3 days (Iso 3d) and 7 days (Iso 7d) by pressure-volume measurements with a catheter method, and (ii) to follow up the left ventricular function in the same model treated with Iso up to 16 weeks with a less-invasive echocardiography. An infusion of either Iso (1.2 mg x kg(-1) x day(-1) for 3 days-16 weeks) or vehicle (saline 24 microl x day(-1) for 3 days-16 weeks; Sa group) was performed by subcutaneously implanting an osmotic minipump. There were no significant differences in the systolic pressure-volume area at midrange left ventricular volume (PVA(mLVV): a mechanical work capability index) between Iso 3d and 7d groups, though PVA(mLVV) in both groups was significantly reduced from that in the Sa group. From echocardiography, the left ventricular function of the hypertrophy models at 3 days, 1 week, and 2 weeks was unchanged, but the model at a term longer than 4 weeks resulted in prolonged systolic failure. The results indicated that (i) no marked differences in the left ventricular mechanical work capability were found between the Iso 3d and 7d groups, and that (ii) only a 3-day Iso infusion induced the hypertrophy model similar in shape and function to that induced by a 2-week Iso infusion. We concluded that the 3-day model was sufficient.

Pacemaker activity from submucosal interstitial cells of Cajal drives high‐frequency and low‐amplitude circular muscle contractions in the mouse proximal colon

The present study was aimed at elucidating how pacemaker activity (plateau potentials) (mean frequency: 15.9 ± 2.8 times min−1) from submucosal interstitial cells of Cajal (ICC‐SM) control spontaneous contractions in the mouse proximal colon. Mechanical activities in the circular muscle direction showed high‐frequency (mean frequency: 15.6 ± 2.7 times min−1) and low‐amplitude (mean amplitude: 0.01 ± 0.005 g) (HFLA) rhythmic contractions. Simultaneous recordings of circular muscle mechanical activity and electrical activity from ICC‐SM revealed that HFLA contractions were synchronized with plateau potentials (mean frequency: 15.9 ± 2.8 times min−1). Although low‐frequency (3.5 ± 2.1 times min−1) and high‐amplitude (0.12 ± 0.03 g) (LFHA) contractions in both longitudinal and circular muscle directions were synchronized with burst of action potentials in both muscle cells, these LFHA contractions were not synchronous with plateau potentials. Intracellular Ca2+ release from the internal stores through IP3 receptors is not a major factor to generate both action potentials differently from plateau potentials. Neither tetrodotoxin nor atropine affected the plateau potentials. The results reveal that the pacemaker activity from ICC‐SM drives only the spontaneous HFLA (one‐tenth amplitude of the LFHA circular and longitudinal muscle contractions) circular muscle contractions without control by enteric nerves.

Recent advances in studies of spontaneous activity in smooth muscle: Ubiquitous pacemaker cells

The general and specific properties of pacemaker cells, including Kit-negative cells, that are distributed in gastrointestinal, urethral and uterine smooth muscle tissues, are discussed herein. In intestinal tissues, interstitial cells of Cajal (ICC) are heterogeneous in both their forms and roles. ICC distributed in the myenteric layer (ICC-MY) act as primary pacemaker cells for intestinal mechanical and electrical activity. ICC distributed in muscle bundles play a role as mediators of signals from autonomic nerves to smooth muscle cells. A group of ICC also appears to act as a stretch sensor. Intracellular Ca2+ dynamics play a crucial role in ICC-MY pacemaking; intracellular Ca2+ ([Ca2+](i)) oscillations periodically activate plasmalemmal Ca2+-activated ion channels, such as Ca2+-activated Cl(-) channels and/or non-selective cation channels, although the relative contributions of these channels are not defined. With respect to gut motility, both the ICC network and enteric nervous system, including excitatory and inhibitory enteric neurons, play an essential role in producing highly coordinated peristalsis.

Cardioprotective effects of a novel calpain inhibitor SNJ-1945 for reperfusion injury after cardioplegic cardiac arrest

We have previously indicated that calpain inhibitor-1 prevents the heart from ischemia- reperfusion injury associated with the impairment of total Ca(2+) handling by inhibiting the proteolysis of alpha-fodrin. However, this inhibitor is insoluble with water and inappropriate for clinical application. The aim of the present study was to investigate the protective effect of a newly developed calpain inhibitor, SNJ-1945 (SNJ), with good aqueous solubility on left ventricular (LV) mechanical work and energetics in the cross-circulated rat hearts. SNJ (150 microM) was added to KCl (30 meq) cardioplegia (CP). Mean end-systolic pressure at midrange LV volume (ESP(mLVV)) and systolic pressure-volume area (PVA) at mLVV (PVA(mLVV); a total mechanical energy per beat) were hardly changed after CP plus SNJ arrest-reperfusion (post-CP + SNJ), whereas ESP(mLVV) and PVA(mLVV) in post-CP group were significantly (P < 0.01) decreased. Mean myocardial oxygen consumption for the total Ca(2+) handling in excitation-contraction coupling did not significantly decrease in post-CP + SNJ group, whereas it was significantly (P < 0.01) decreased in post-CP group. The mean amounts of 145- and 150-kDa fragments of alpha-fodrin in the post-CP group were significantly larger than those in normal and post-CP + SNJ groups. In contrast, the mean amounts of L-type Ca(2+) channel and sarcoplasmic reticulum Ca(2+)-ATPase were not significantly different among normal, post-CP, and post-CP + SNJ groups. Our results indicate that soluble SNJ attenuates cardiac dysfunction due to CP arrest-reperfusion injury associated with the impairment of the total Ca(2+) handling in excitation-contraction coupling by inhibiting the proteolysis of alpha-fodrin.