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.

AimsEmpagliflozin, a clinically used oral antidiabetic drug that inhibits the sodium‐dependent glucose co‐transporter 2, has recently been evaluated for its cardiovascular safety. Surprisingly, empagliflozin reduced mortality and hospitalization for heart failure (HF) compared to placebo. However, the underlying mechanisms remain unclear. Therefore, our study aims to investigate whether empagliflozin may cause direct pleiotropic effects on the myocardium.Methods and resultsIn order to assess possible direct myocardial effects of empagliflozin, we performed contractility experiments with in toto‐isolated human systolic end‐stage HF ventricular trabeculae. Empagliflozin significantly reduced diastolic tension, whereas systolic force was not changed. These results were confirmed in murine myocardium from diabetic and non‐diabetic mice, suggesting independent effects from diabetic conditions. In human HF cardiomyocytes, empagliflozin did not influence calcium transient amplitude or diastolic calcium level. The mechanisms underlying the improved diastolic function were further elucidated by studying myocardial fibres from patients and rats with diastolic HF (HF with preserved ejection fraction, HFpEF). Empagliflozin beneficially reduced myofilament passive stiffness by enhancing phosphorylation levels of myofilament regulatory proteins. Intravenous injection of empagliflozin in anaesthetized HFpEF rats significantly improved diastolic function measured by echocardiography, while systolic contractility was unaffected.ConclusionEmpagliflozin causes direct pleiotropic effects on the myocardium by improving diastolic stiffness and hence diastolic function. These effects were independent of diabetic conditions. Since pharmacological therapy of diastolic dysfunction and HF is an unmet need, our results provide a rationale for new translational studies and might also contribute to the understanding of the EMPA‐REG OUTCOME trial.