Embryonic germ cells as well as germline stem cells from neonatal mouse testis are pluripotent and have differentiation potential similar to embryonic stem cells, suggesting that the germline lineage may retain the ability to generate pluripotent cells. However, until now there has been no evidence for the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male. Here we show the isolation of SSCs from adult mouse testis using genetic selection, with a success rate of 27%. These isolated SSCs respond to culture conditions and acquire embryonic stem cell properties. We name these cells multipotent adult germline stem cells (maGSCs). They are able to spontaneously differentiate into derivatives of the three embryonic germ layers in vitro and generate teratomas in immunodeficient mice. When injected into an early blastocyst, SSCs contribute to the development of various organs and show germline transmission. Thus, the capacity to form multipotent cells persists in adult mouse testis. Establishment of human maGSCs from testicular biopsies may allow individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells. Furthermore, these cells may provide new opportunities to study genetic diseases in various cell lineages.

In heart failure (HF), Ca 2+ /calmodulin kinase II (CaMKII) expression is increased. Altered Na + channel gating is linked to and may promote ventricular tachyarrhythmias (VTs) in HF. Calmodulin regulates Na + channel gating, in part perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and Tg (chronic) overexpression of cytosolic CaMKIIδ C on Na + current (I Na ) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIδ C overexpression shifted voltage dependence of Na + channel availability by -6 mV (P < 0.05), and the shift was Ca 2+ dependent. CaMKII also enhanced intermediate inactivation and slowed recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2-related inhibitory peptide [AIP] or KN93). CaMKIIδ C markedly increased persistent (late) inward I Na and intracellular Na + concentration (as measured by the Na + indicator sodium-binding benzofuran isophthalate [SBFI]), which was prevented by CaMKII inhibition in the case of acute CaMKIIδ C overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na + channels. In vivo, transgenic CaMKIIδ C overexpression prolonged QRS duration and repolarization (QT intervals), decreased effective refractory periods, and increased the propensity to develop VT. We conclude that CaMKII associates with and phosphorylates cardiac Na + channels. This alters I Na gating to reduce availability at high heart rate, while enhancing late I Na (which could prolong action potential duration). In mice, enhanced CaMKIIδ C activity predisposed to VT. Thus, CaMKIIdependent regulation of Na + channel function may contribute to arrhythmogenesis in HF.

Induced pluripotent stem cells (iPSCs) may represent an ideal cell source for future regenerative therapies. A critical issue concerning the clinical use of patient-specific iPSCs is the accumulation of mutations in somatic (stem) cells over an organism's lifetime. Acquired somatic mutations are passed onto iPSCs during reprogramming and may be associated with loss of cellular functions and cancer formation. Here we report the generation of human iPSCs from cord blood (CB) as a juvenescent cell source. CBiPSCs show characteristics typical of embryonic stem cells and can be differentiated into derivatives of all three germ layers, including functional cardiomyocytes. For future therapeutic production of autologous and allogeneic iPSC derivatives, CB could be routinely harvested for public and commercial CB banks without any donor risk. CB could readily become available for pediatric patients and, in particular, for newborns with genetic diseases or congenital malformations.

Rationale In heart failure (HF), CaMKII expression and reactive oxygen species (ROS) are increased. Both ROS and CaMKII can increase late INa leading to intracellular Na accumulation and arrhythmias. It has been shown that ROS can activate CaMKII via oxidation. Objective We tested whether CaMKIIδ is required for ROS-dependent late INa regulation and if ROS-induced Ca released from the sarcoplasmic reticulum (SR) is involved. Methods and Results 40 µmol/L H2O2 significantly increased CaMKII oxidation and autophosphorylation in permeabilized rabbit cardiomyocytes. Without free [Ca]i (5 mmol/L BAPTA/1 mmol/L Br2-BAPTA) or after SR depletion (caffeine 10 mmol/L, thapsigargin 5 µmol/L) the H2O2-dependent CaMKII oxidation and autophosphorylation was abolished. H2O2 significantly increased SR Ca spark frequency (confocal microscopy) but reduced SR Ca load. In wildtype (WT) mouse myocytes, H2O2 increased late INa (whole cell patch-clamp). This increase was abolished in CaMKIIδ−/− myocytes. H2O2-induced [Na]i and [Ca]i accumulation (SBFI and Indo-1 epifluorescence) was significantly slowed in CaMKIIδ−/− myocytes (vs. WT). CaMKIIδ−/− myocytes developed significantly less H2O2-induced arrhythmias, and were more resistant to hypercontracture. Opposite results (increased late INa, [Na]i and [Ca]i accumulation) were obtained by overexpression of CaMKIIδ in rabbit myocytes (adenoviral gene transfer) reversible with CaMKII inhibition (10 µmol/L KN93 or 0.1 µmol/L AIP). Conclusion Free [Ca]i and a functional SR are required for ROS activation of CaMKII. ROS-activated CaMKIIδ enhances late INa, which may lead to cellular Na and Ca overload. This may be of relevance in HF, where enhanced ROS production meets increased CaMKII expression.

Reactive oxygen species (ROS), including H 2 O 2 , cause intracellular calcium overload and ischemia-reperfusion damage. The objective of this study was to examine the hypothesis that H 2 O 2 -induced arrhythmic activity and contractile dysfunction are the results of an effect of H 2 O 2 to increase the magnitude of the late sodium current (late I Na ). Guinea pig and rabbit isolated ventricular myocytes were exposed to 200 M H 2 O 2 . Transmembrane voltages and currents and twitch shortening were measured using the whole-cell patch-clamp technique and video edge detection, respectively.

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Abstract-The predominant cardiac Ca 2ϩ /calmodulin-dependent protein kinase (CaMK) is CaMKII␦. Here we acutely overexpress CaMKII␦ C using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKII␦ C transgenic mice. CaMKII␦ C protein expression and activation state (autophosphorylation) were increased 5-to 6-fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKII␦ C versus LacZ expressing myocytes. However, the contraction-frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced ( 0.5Hz / 3Hz ϭ2.14Ϯ0.10 versus 1.87Ϯ0.10), and peak Ca 2ϩ current (I Ca ) was increased by 31% (Ϫ7.1Ϯ0.

Background-Potassium currents contribute to action potential duration (APD) and arrhythmogenesis. In heart failure, Ca/calmodulin-dependent protein kinase II (CaMKII) is upregulated and can alter ion channel regulation and expression. Methods and Results-We examine the influence of overexpressing cytoplasmic CaMKII␦ C , both acutely in rabbit ventricular myocytes (24-hour adenoviral gene transfer) and chronically in CaMKII␦ C -transgenic mice, on transient outward potassium current (I to ), and inward rectifying current (I K1 ). Acute and chronic CaMKII overexpression increases I to,slow amplitude and expression of the underlying channel protein K V 1.4. Chronic but not acute CaMKII overexpression causes downregulation of I to,fast , as well as K V 4.2 and KChIP2, suggesting that K V 1.4 expression responds faster and oppositely to K V 4.2 on CaMKII activation. These amplitude changes were not reversed by CaMKII inhibition, consistent with CaMKII-dependent regulation of channel expression and/or trafficking. CaMKII (acute and chronic) greatly accelerated recovery from inactivation for both I to components, but these effects were acutely reversed by AIP (CaMKII inhibitor), suggesting that CaMKII activity directly accelerates I to recovery. Expression levels of I K1 and Kir2.1 mRNA were downregulated by CaMKII overexpression. CaMKII acutely increased I K1 , based on inhibition by AIP (in both models). CaMKII overexpression in mouse prolonged APD (consistent with reduced I to,fast and I K1 ), whereas CaMKII overexpression in rabbit shortened APD (consistent with enhanced I K1 and I to,slow and faster I to recovery). Computational models allowed discrimination of contributions of different channel effects on APD. Key Words: action potentials Ⅲ potassium Ⅲ arrhythmia Ⅲ electrophysiology Ⅲ heart failure H eart failure (HF) is accompanied by arrhythmogenic changes related to electric remodeling. This is associated with prolongation of action potential duration (APD) 1 and downregulation of transient outward K-current (I to ) and inward rectifying K-current (I K1 ). I K1 is responsible for stabilizing the diastolic membrane potential (E m ), such that decreased I K1 increases the propensity for triggered arrhythmias. 2 I to is important in early repolarization and influences the effects of other currents and transporters by affecting AP voltage-time trajectory. There are at least 2 components of I to generated by different K-channel isoforms, which can be distinguished according to their recovery and inactivation kinetics. 3,4 The fast component (I to,fast ) recovers and inactivates with time constants () of Ͻ100 ms, whereas the slow component (I to,slow ) recovers with of hundreds of milliseconds up to several seconds and inactivates with of Ϸ200 ms. 3-5 Downregulation of I to has been described in animal models of hypertrophy and human HF, 2,6,7 is associated with APD prolongation, 8 and predisposes to early afterdepolarizations. Conclusion-CaMKII Clinical Perspective on p 294In HF, expression and activity of Ca/cal...