Type-2 cells as link between glial and neuronal lineage in adult hippocampal neurogenesis.

In the course of adult hippocampal neurogenesis, new cells go through a series of stages associated with proliferative activity. The most highly proliferative cell type is an intermediate precursor cell, called type-2 cell. We here report that on the level of type-2 cells a transition takes place between features associated with the glial and the neuronal lineage. We show that stem-cell marker Sox2 and radial glia marker BLBP are expressed in type-2 cells but label only a small percentage of the proliferating cells. By and large, precursor cell marker Sox2 was found to be widely expressed in hippocampal astrocytes. Between 3 h and 1 week after a single injection of permanent S-phase marker bromodeoxyuridine (BrdU), the number of BrdU-labeled BLBP-positive cells did not change, consistent with the idea that both markers here are associated with the maintained precursor cell pool. Using reporter gene mice expressing the green fluorescent protein (GFP) under the promoter for nestin we found an overlap of GFP with markers of the neuronal lineage, doublecortin (DCX) and transcription factor NeuroD1 in type-2 cells, whereas in glial fibrillary acidic protein (GFAP)-GFP mice expression of GFP and NeuroD1 or DCX was mutually exclusive. Electrophysiologically, the group of type-2 cells fell into two subgroups: one with astrocytic properties and another with an early "complex" phenotype of neural progenitor cells. Our data further support the existence of proliferative precursor cells that mark the transition between glia-like states and neuronal differentiation.

Human atrial myosin light chain 1 expression attenuates heart failure.

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, replacing the ventricular essential light chains (VLC-1). VLC-1/ALC-1 isoform shift is correlated with increases in cardiac contractile parameters of a transgenic rat model overexpressing hALC-1 in the heart (TGR/hALC-1) compared to normal WKY rats. To investigate, whether the benefical effects of the hALC-1 on cardiac contractility could attenuate contractile failure of the overloaded heart, aortocaval shunt operations of 9-10 weeks old WKY and TGR/hALC-1 were performed. 5 weeks later, both animals groups were sacrificed for analysis of cardiac contraction and transgene expression. Control animals were operated but remained normal body and heart weights. The whole heart contractility parameters were evaluated using the Langendorff heart preparation. Shunt-operated TGR/hALC-1 and WKY rats developed comparable levels of cardiac hypertrophy which was associated with significant reduction of contractile parameters of the Langendorff hearts. However, the decline of cardiac contractility was less pronounced in shunt-operated TGR/hALC-1 compared to shunt-operated WKY. In fact, developed left ventricular pressure as well as maximal velocity of pressure development and relaxation were significantly higher in shunt-operated TGR/hALC-1 as compared to shunt-operated WKY. Expression of hALC-1 was 17 microg/mg whole SDS-protein in control (sham-operated) controls and declined significantly to 14 microg/mg whole SDS-protein in hypertrophied TGR/hALC-1. These results demonstrate that the expression of hALC-1 could have a beneficial effect on the overloaded hypertrophied heart.

Regulation of the human atrial myosin light chain 1 promoter by Ca2+-calmodulin-dependent signaling pathways.

We investigated expression regulation of the human atrial myosin light chain 1 (hALC-1) gene using a cardiomyocyte H9c2 cell line stably transfected with a construct consisting of the human ALC-1 promoter cloned in front of the luciferase gene (H9c2T1). H9c2T1 cells were stimulated with vasopressin, which is known to induce cardiomyocyte hypertrophy and to activate a panel of signaling pathways. Those pathways involved in hALC-1 promoter activity regulation were dissected by using pharmacological inhibitor substances. Stimulation with vasopressin was associated with nuclear NFAT translocation and significantly increased human ALC-1 promoter activity. Inhibition of calcineurin by cyclosporin A blocked the effects of vasopressin on ALC-1 promoter activity to approximately 50%. This suggests that the Ca2+-calmodulin-calcineurin-NFAT pathway is involved in human ALC-1 promoter activation. However, inhibition of multifunctional Ca2+-calmodulin-dependent protein kinases (CaMK) by KN-93 decreased human ALC-1 promoter activity to almost basal levels. CaMK regulation of ALC-1 promoter activity effect could well be mediated by CaMKIV, which accumulated in the nucleus upon vasopressin stimulation. Inhibition of protein kinase C (PKC) isoforms by bisindolylmaleimide had no significant influence on human ALC-1 promoter activity. Thus, our results demonstrate a dominant role of Ca2+-calmodulin-dependent signaling pathways in the regulation of human ALC-1 expression.

The carboxyl-terminal ahnak domain induces actin bundling and stabilizes muscle contraction.

Ahnak, a 700 kDa protein, is expressed in a variety of cells and has been implicated in different cell-type-specific functions. In the human heart, we observed an endogenous carboxyl-terminal 72 kDa ahnak fragment that copurified with myofibrillar proteins. Immunocytochemistry combined with confocal microscopy localized this fragment to the intercalated discs and close to the Z-line of cardiomyocytes. No endogenous myofibrillar ahnak fragment was observed in the skeletal muscle. We elucidated the role of the recombinant carboxyl-terminal ahnak fragment (ahnak-C2) in actin filament organization and in the function of muscle fibers. Addition of ahnak-C2 to actin filaments induced filament bundling into paracrystalline-like structures as revealed by electron microscopy. Incubation of demembranated skeletal muscle fibers with ahnak-C2 attenuated the decline in isometric force development upon repeated contraction-relaxation cycles. Our results suggest that the carboxyl-terminal ahnak domain exerts a stabilizing effect on muscle contractility via its interaction with actin of thin filaments.

Functional characterization of the human atrial essential myosin light chain (hALC-1) in a transgenic rat model.

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using skinned fibers from the hypertrophied ventricles of human hearts. To study the functional importance of hALC-1 in the intact perfused heart, we generated a transgenic rat model (TGR) overexpressing hALC-1 in the heart. Twelve-week-old TGR rats expressed 17 +/- 4 microg hALC-1 per mg of whole SDS-soluble protein. Their perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (P<0.001) in the contractile parameters of the hearts of the TGR compared to the age matched control (WKY) animals, represented by increases from 20.8 +/- 2.3 to 45.1 +/- 3.6 mmHg/g heart weight in the developed left ventricular pressure, 1,035.7 +/- 89.8 to 2,181 +/- 135.4 mmHg/s in the contraction rate, and 713 +/- 60.2 to 1,364 +/- 137.4 mmHg/s in the relaxation rate in the WKY and the TGR groups respectively. Characterizing the functional effects of hALC-1 at the whole organ level represents a step towards gene therapy of heart failure.

Analysis of the energetic state of heart cells after adenovirus-mediated expression of hALC-1.

Expression of the human atrial myosin light chain 1 (hALC-1) in the cardiac ventricle in vivo as well as in primary cultivated adult cardiomyocytes caused a pronounced positive inotropic effect. Therefore, it is one of the most promising candidate gene to treat congestive heart failure (CHF). In this work, we investigated, whether hALC-1 expression also modifies the energetic state of cardiomyocytes. Primary cultivated neonatal rat hearts cells (NRHC) were infected with adenoviral vectors (Ad vectors) containing a hALC-1 cDNA (AdCMV.hALC-1) or a control Ad vector. Infection efficiency of NRHC reached 100% at 50 multiplicity of infection (MOI). Interestingly and in contrast to primary cultures of liver cells, there were no cytotoxic side effects or induction of apoptosis up to MOI 50 in Ad vector infected NRHC. NRHC expressed large amounts of hALC-1 upon infection with AdCMV.hALC-1 which could easily been detected by protein staining and Western blot analysis. Analysis of intracellular hALC-1 localization by double-labeling immunofluorescence of AdCMV.hALC-1 infected cardiomyocytes revealed the typical myofibrillar striation pattern, as well as co-localization of hALC-1 with myosin heavy chains. There was no difference in the oxygen consumption between controls and AdCMV.hALC-1 infected NRHC. These data suggest that first: adenoviral vectors could be used as a safe and effective tool for gene transfer to cardiomyocytes, and second: that a positive inotropic effect of hALC-1 is not associated with enhanced oxygen consumption.