Lars S. Maier

Pluripotency of spermatogonial stem cells from adult mouse testis

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.

Generation of Functional Murine Cardiac Myocytes From Induced Pluripotent Stem Cells

Background— The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease– and human patient–specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line. Methods and Results— With the use of a standard embryoid body–based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell–derived cardiomyocytes show typical features of ES cell–derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes typical for mesoderm, cardiac mesoderm, and cardiomyocytes including Brachyury, mesoderm posterior factor 1 (Mesp1), friend of GATA2 (FOG-2), GATA-binding protein 4 (GATA4), NK2 transcription factor related, locus 5 (Nkx2.5), T-box 5 (Tbx5), T-box 20 (Tbx20), atrial natriuretic factor (ANF), myosin light chain 2 atrial transcripts (MLC2a), myosin light chain 2 ventricular transcripts (MLC2v), &agr;-myosin heavy chain (&agr;-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric &agr;-actinin, titin, cardiac troponin T, MLC2v, and connexin 43. iPS cell cardiomyocytes displayed spontaneous rhythmic intracellular Ca2+ fluctuations with amplitudes of Ca2+ transients comparable to ES cell cardiomyocytes. Simultaneous Ca2+ release within clusters of iPS cell–derived cardiomyocytes indicated functional coupling of the cells. Electrophysiological studies with multielectrode arrays demonstrated functionality and presence of the &bgr;-adrenergic and muscarinic signaling cascade in these cells. Conclusions— iPS cells differentiate into functional cardiomyocytes. In contrast to ES cells, iPS cells allow derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.

The δC Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure

Abstract— Recent studies have demonstrated that transgenic (TG) expression of either Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKII&dgr;B, both of which localize to the nucleus, induces cardiac hypertrophy. However, CaMKIV is not present in heart, and cardiomyocytes express not only the nuclear CaMKII&dgr;B but also a cytoplasmic isoform, CaMKII&dgr;C. In the present study, we demonstrate that expression of the &dgr;C isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload. To determine whether enhanced activity of this cytoplasmic &dgr;C isoform of CaMKII can lead to phosphorylation of Ca2+ regulatory proteins and induce hypertrophy, we generated TG mice that expressed the &dgr;C isoform of CaMKII. Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca2+ handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and CaMKII is found associated with the RyR in immunoprecipitates from the CaMKII TG mice. Phosphorylation of phospholamban is also increased specifically at the CaMKII but not at the PKA phosphorylation site. These findings are the first to demonstrate that CaMKII&dgr;C can mediate phosphorylation of Ca2+ regulatory proteins in vivo and provide evidence for the involvement of CaMKII&dgr;C activation in the pathogenesis of dilated cardiomyopathy and heart failure.

Relationship Between Na+-Ca2+–Exchanger Protein Levels and Diastolic Function of Failing Human Myocardium

BACKGROUND In the failing human heart, sarcoplasmic reticulum (SR) calcium handling is impaired, and therefore, calcium elimination and diastolic function may depend on the expression of sarcolemmal Na+-Ca2+ exchanger. METHODS AND RESULTS Force-frequency relations were studied in ventricular muscle strip preparations from failing human hearts (n=29). Protein levels of Na+-Ca2+ exchanger and SR Ca2+-ATPase were measured in the same hearts. Hearts were divided into 3 groups by discriminant analysis according to the behavior of diastolic function when stimulation rate of muscle strips was increased from 30 to 180 min-1. At 180 compared with 30 min-1, diastolic force was increased by 160%, maximum rate of force decline was decreased by 46%, and relaxation time was unchanged in group III. In contrast, in group I, diastolic force and maximum rate of force decline did not change, and relaxation time decreased by 20%. Na+-Ca2+ exchanger was 66% higher in group I than in group III. Na+-Ca2+ exchanger was inversely correlated with the frequency-dependent rise of diastolic force when stimulation rate was increased (r=-0.74; P<0.001). Compared with nonfailing human hearts (n=6), SR Ca2+-ATPase was decreased and Na+-Ca2+ exchanger unchanged in group III, whereas Na+-Ca2+ exchanger was increased and SR Ca2+-ATPase unchanged in group I. Results with group II hearts were between those of group I and group III hearts. CONCLUSIONS By discriminating failing human hearts according to their diastolic function, we identified different phenotypes. Disturbed diastolic function occurs in hearts with decreased SR Ca2+-ATPase and unchanged Na+-Ca2+ exchanger, whereas increased expression of the Na+-Ca2+ exchanger is associated with preserved diastolic function.

Ca2+/calmodulin-dependent protein kinase II regulates cardiac Na+ channels.

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 CaMKIIdelta(C) on Na(+) current (I(Na)) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIdelta(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). CaMKIIdelta(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 CaMKIIdelta(C) overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na(+) channels. In vivo, transgenic CaMKIIdelta(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 CaMKIIdelta(C) activity predisposed to VT. Thus, CaMKII-dependent regulation of Na(+) channel function may contribute to arrhythmogenesis in HF.

Generation of Induced Pluripotent Stem Cells from Human Cord Blood

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 organisms 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.

Transgenic CaMKIIδC Overexpression Uniquely Alters Cardiac Myocyte Ca2+ Handling: Reduced SR Ca2+ Load and Activated SR Ca2+ Release

Abstract— Ca2+/calmodulin-dependent protein kinase II (CaMKII) &dgr; is the predominant cardiac isoform, and the &dgr;C splice variant is cytoplasmic. We overexpressed CaMKII&dgr;C in mouse heart and observed dilated heart failure and altered myocyte Ca2+ regulation in 3-month-old CaMKII&dgr;C transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca2+]i transient amplitude, and diastolic [Ca2+]i were all reduced by ≈50% in TG versus WT. This is explained by >50% reduction in SR Ca2+ content in TG versus WT. Peak Ca2+ current (ICa) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca2+ load and diastolic [Ca2+]i, fractional SR Ca2+ release was increased and resting spontaneous SR Ca2+ release events (Ca2+ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca2+ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca2+ spark frequency and ICa, consistent with direct CaMKII activation of ryanodine receptors (and ICa) in TG. The rate of [Ca2+]i decline during caffeine exposure was faster in TG, indicating enhanced Na+-Ca2+ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca2+ leak (via sparks), reduced SR Ca2+-ATPase expression, and increased Na+-Ca2+ exchanger explain the reduced diastolic [Ca2+]i and SR Ca2+ content in TG. We conclude that CaMKII&dgr;C overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure.

Ca2+ Handling and Sarcoplasmic Reticulum Ca2+ Content in Isolated Failing and Nonfailing Human Myocardium

Disturbed sarcoplasmic reticulum (SR) Ca2+ content may underlie the altered force-frequency and postrest contractile behavior in failing human myocardium. We used rapid cooling contractures (RCCs) to assess SR Ca2+ content in ventricular muscle strips isolated from nonfailing and end-stage failing human hearts. With an increase in rest intervals (1 to 240 s; 37 degrees C), nonfailing human myocardium (n=7) exhibited a parallel increase in postrest twitch force (at 240 s by 121+/-44%; P<0.05) and RCC amplitude (by 69+/-53%; P<0.05). In contrast, in failing myocardium (n=30), postrest twitch force decreased at long rest intervals and RCC amplitude declined monotonically with rest (by 25+/-9% and 53+/-9%, respectively; P<0.05). With an increase in stimulation frequencies (0.25 to 3 Hz), twitch force increased continuously in nonfailing human myocardium (n=7) by 71+/-17% (at 3 Hz; P<0.05) and RCC amplitude increased in parallel by 247+/-55% (P<0.05). In contrast, in failing myocardium (n=26), twitch force declined by 29+/-7% (P<0. 05) and RCC amplitude increased only slightly by 36+/-14% (P<0.05). Paired RCCs were evoked to investigate the relative contribution of SR Ca2+ uptake and Na+/Ca2+ exchange to cytosolic Ca2+ removal during relaxation. SR Ca2+ uptake (relative to the Na+/Ca2+ exchange) increased significantly in nonfailing but not in failing human myocardium as stimulation rates increased. We conclude that the negative force-frequency relation in failing human myocardium is due to an inability of SR Ca2+ content to increase sufficiently at high frequencies and thus cannot overcome the frequency-dependent refractoriness of SR Ca2+ release. The rest-dependent decay in twitch force in failing myocardium is due to rest-dependent decline in SR Ca2+ content. These alterations could be secondary to depressed SR Ca2+-ATPase combined with enhanced cytosolic Ca2+ extrusion via Na+/Ca2+ exchange.

The δ isoform of CaM kinase II is required for pathological cardiac hypertrophy and remodeling after pressure overload

Acute and chronic injuries to the heart result in perturbation of intracellular calcium signaling, which leads to pathological cardiac hypertrophy and remodeling. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the transduction of calcium signals in the heart, but the specific isoforms of CaMKII that mediate pathological cardiac signaling have not been fully defined. To investigate the potential involvement in heart disease of CaMKIIδ, the major CaMKII isoform expressed in the heart, we generated CaMKIIδ-null mice. These mice are viable and display no overt abnormalities in cardiac structure or function in the absence of stress. However, pathological cardiac hypertrophy and remodeling are attenuated in response to pressure overload in these animals. Cardiac extracts from CaMKIIδ-null mice showed diminished kinase activity toward histone deacetylase 4 (HDAC4), a substrate of stress-responsive protein kinases and suppressor of stress-dependent cardiac remodeling. In contrast, phosphorylation of the closely related HDAC5 was unaffected in hearts of CaMKIIδ-null mice, underscoring the specificity of the CaMKIIδ signaling pathway for HDAC4 phosphorylation. We conclude that CaMKIIδ functions as an important transducer of stress stimuli involved in pathological cardiac remodeling in vivo, which is mediated, at least in part, by the phosphorylation of HDAC4. These findings point to CaMKIIδ as a potential therapeutic target for the maintenance of cardiac function in the setting of pressure overload.

Influence of the Novel Inotropic Agent Levosimendan on Isometric Tension and Calcium Cycling in Failing Human Myocardium

BACKGROUND Levosimendan was shown to increase calcium sensitivity by a novel mechanism and to inhibit phosphodiesterase III activity in animal myocardium. METHODS AND RESULTS We investigated the influence of levosimendan on isometric contractions and calcium transients (aequorin method) in muscle strips from human hearts with end-stage failing dilated or ischemic cardiomyopathy (n=27). Data were compared with the effects of the phosphodiesterase inhibitor milrinone (n=9). The average maximum increase in twitch tension was 47+/-14% (range, 6% to 150%) at a levosimendan concentration of 0. 8+/-0.3 micromol/L (P<0.01). This was associated with significant increases in maximum rates of tension rise and fall and decreases in times to peak tension, to 50% relaxation, and to 95% relaxation. In aequorin-loaded muscles, levosimendan 10(-6) mol/L increased average tension by 50% (P<0.02), associated with a nonsignificant increase in aequorin light (16%). With milrinone 10(-5) mol/L, average tension increased by 58% and aequorin light by 49% (P<0.05). In those muscle strips with pronounced inotropic effects (>50% increase in tension), there was a comparable and pronounced increase in aequorin light with both agents. However, in muscle strips with weak inotropic responses (<50% increase in tension), the increase in light was significantly higher with milrinone than with levosimendan. CONCLUSIONS Levosimendan has inotropic and lusitropic actions in failing human myocardium. Comparison with the phosphodiesterase inhibitor milrinone indicates that in case of pronounced inotropic stimulation, the modes of action of the two agents may be similar (phosphodiesterase inhibition), whereas small inotropic effects of levosimendan may result predominantly from calcium sensitization.