Hannelore Haase

From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model.

Vascular smooth muscle cell (VSMC) differentiation is important in understanding vascular disease; however, no in vitro model is available. Totipotent mouse embryonic stem (ES) cells were used to establish such a model. To test whether the ES cell‐derived smooth muscle cells expressed VSMC‐specific properties, the differentiated cells were characterized by 1) morphological analysis, 2) gene expression, 3) immunostaining for VSMC‐specific proteins, 4) expression of characteristic VSMC ion channels, and 5) formation of [Ca2+]i transients in response to VSMC‐specific agonists. Treatment of embryonic stem cell‐derived embryoid bodies with retinoic acid and dibutyryl‐cyclic adenosine monophosphate (db‐cAMP) induced differentiation of spontaneously contracting cell clusters in 67% of embryoid bodies compared with 10% of untreated controls. The highest differentiation rate was observed when retinoic acid and db‐cAMP were applied to the embryoid bodies between days 7 and 11 in combination with frequent changes of culture medium. Other protocols with retinoic acid and db‐cAMP, as well as single or combined treatment with VEGF, ECGF, bFGF, aFGF, fibronectin, matrigel, or hypoxia did not influence the differentiation rate. Single‐cell RT‐PCR and sequencing of the PCR products identified myosin heavy chain (MHC) splice variants distinguishing between gut and VSMC isoforms. RT‐PCR with VSMC‐specific MHC primers and immunostaining confirmed the presence of VSMC transcripts and MHC protein. Furthermore, VSMC expressing MHC had typical ion channels and responded to specific agonists with an increased [Ca2+]i. Here we present a retinoic acid + db‐cAMP‐inducible embryonic stem cell model of in vitro vasculogenesis. ES cell‐derived cells expressing VSMC‐specific MHC and functional VSMC properties may be a suitable system to study mechanisms of VSMC differentiation.—Drab, M., Haller, H., Bychkov, R., Erdmann, B., Lindschau, C., Haase, H., Morano, I., Luft, F. C., Wobus, A. M. From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db‐cAMP in vitro differentiation model. FASEB J. 11, 905–915 (1997)

Smooth-muscle contraction without smooth-muscle myosin

Here we have used gene-targeting to eliminate expression of smooth-muscle myosin heavy chain. Elimination of this gene does not affect expression of non-muscle myosin heavy chain, and knockout individuals typically survive for three days. Prolonged activation, by KCl depolarisation, of intact bladder preparations from wild-type neonatal mice produces an initial transient state (phase 1) of high force generation and maximal shortening velocity, which is followed by a sustained state (phase 2) characterized by low force generation and maximal shortening velocity. Similar preparations from knockout neonatal mice do not undergo phase 1, but exhibit a normal phase 2. We propose that, in neonatal smooth muscle phase 1 is generated by recruitment of smooth-muscle myosin heavy chain, whereas phase 2 can be generated by activation of non-muscle myosin heavy chain. We conclude that phase 1 becomes indispensable for survival and normal growth soon after birth, particularly for functions such as homeostasis and circulation.

L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells

Despite intensive interest in understanding the differentiation of vascular smooth muscle cells (VSMC), no information is available about differential regulation of ion channels in these cells. Since expression of the L‐type Ca2+ channel can be influenced by differentiation in other cell types, we tested the hypothesis that the L‐type (C class) channel is a specific differentiation marker of VSMC and that expression of these channels depends on the state of cell differentiation. We used rat aortic (A7r5) VSMC, which express functional L‐type Ca2+ channels, and induced dedifferentiation by cell culture in different media. Treatment with retinoic acid was used to redifferentiate the VSMC. We characterized the differentiated state of the cells by using immunohistochemistry and Western blot analysis for smooth muscle (SM) α‐actin and SM‐myosin heavy chain (MHC). The number of functional Ca2+ channels was significantly decreased in dedifferentiated VSMC and increased upon differentiation with retinoic acid. Ca2+ channel function was assessed by whole‐cell voltage clamp techniques. Using Western blot and dihydropyridine binding analysis, we found that the expression of the Ca2+ channel α1 subunit, and to a lesser extent the β2 subunit, was directly correlated with the expression of SM α‐actin and SM‐MHC. We conclude that expression of L‐type Ca2+ channel α1 subunits, and thus a functional Ca2+ channel, is highly coordinated with expression of the SM‐specific proteins required for specialized smooth muscle cell functions. Furthermore, our results demonstrate that the L‐type Ca2+ channel is a novel marker for differentiation of VSMC. The data suggest that regulation of ion channel expression during differentiation may have physiological importance for normal smooth muscle function and may influence VSMC behavior under pathophysiological conditions.

The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton

Ahnak is a ubiquitously expressed giant protein of 5643 amino acids implicated in cell differentiation and signal transduction. In a recent study, we demonstrated the association of ahnak with the regulatory β2 subunit of the cardiac L‐type Ca2+ channel. Here we identify the most carboxyl‐terminal ahnak region (aa 5262–5643) to interact with recombinant β2a as well as with β2 and β1a isoforms of native muscle Ca2+ channels using a panel of GST fusion proteins. Equilibrium sedimentation analysis revealed Kd values of 55 ± 11 nM and 328 ± 24 nM for carboxyl‐terminal (aa 195–606) and amino‐terminal (aa 1–200) truncates of the β2a subunit, respectively. The same carboxylterminal ahnak region (aa 5262–5643) bound to G‐actin and cosedimented with F‐actin. Confocal microscopy of human left ventricular tissue localized the carboxylterminal ahnak portion to the sarcolemma including the T‐tubular system and the intercalated disks of cardiomyocytes. These results suggest that ahnak provides a structural basis for the subsarcolemmal cytoarchitecture and confers the regulatory role of the actin‐based cytoskeleton to the L‐type Ca2+ channel.—Hohaus, A., Person, V., Behlke, J., Schaper, J., Morano, I., Haase, H. The carboxyl‐terminal region of ahnak provides a link between cardiac L‐type Ca2+ channels and the actin‐based cytoskeleton. FASEB J. 16, 1205–1216 (2002)

Molecular mechanisms of early electrical remodeling: transcriptional downregulation of ion channel subunits reduces ICa,Land Itoin rapid atrial pacing in rabbits ☆

OBJECTIVES The purpose of the study was to characterize the ionic and molecular mechanisms in the very early phases of electrical remodeling in a rabbit model of rapid atrial pacing (RAP). BACKGROUND Long-term atrial fibrillation reduces L-type Ca(2+) (I(Ca,L)) and transient outward K(+) (I(to)) currents by transcriptional downregulation of the underlying ionic channels. However, electrical remodeling starts early after the onset of rapid atrial rates. The time course of ion current and channel modulation in these early phases of remodeling is currently unknown. METHODS Rapid (600 beats/min) right atrial pacing was performed in rabbits. Animals were divided into five groups with pacing durations between 0 and 96 h. Ionic currents were measured by patch clamp techniques; messenger ribonucleic acid (mRNA) and protein expression were measured by reverse transcription-polymerase chain reaction and Western blot, respectively. RESULTS L-type calcium current started to be reduced (by 47%) after 12 h of RAP and continued to decline as pacing continued. Current changes were preceded or paralleled by decreased mRNA expression of the Ca(2+) channel beta subunits CaB2a, CaB2b, and CaB3, whereas significant reductions in the alpha(1) subunit mRNA and protein expression began 24 h after pacing onset. Transient outward potassium current densities were not altered within the first 12 h, but after 24 h, currents were reduced by 48%. Longer pacing periods did not further decrease I(to). Current changes were paralleled by reduced Kv4.3 mRNA expression. Kv4.2, Kv1.4, and the auxiliary subunit KChIP2 were not affected. CONCLUSIONS L-type calcium current and I(to) are reduced in early phases of electrical remodeling. A major mechanism appears to be transcriptional downregulation of underlying ion channels, which partially preceded ion current changes.

Phosphorylation of the L‐type calcium channel β subunit is involved in β‐adrenergic signal transduction in canine myocardium

Cyclic AMP‐mediated phosphorylation of calcium channel submits was studied in vitro and in vivo in preparations from dog heart. Calcium channels in native cardiac membranes were phosphorylated by cAMP‐dependent protein kinase (PKA) solubilized with digitonin and subsequently immunoprecipitated using a polyclonal antibody generated against the deduced carboxy‐terminal sequence of the cardiac β subunit. A 62 kDa protein was identified as the major PKA‐substrate in the immunoprecipitates. In the intact myocardium, this putative β subunit was found to be phosphorylated in response to cAMP elevating agents. In contrast, no phosphorylation of a protein with an electrophoretic mobility similar to the α1 subunit was detected, although 1,4‐dihydropyridine receptor sites were recovered in the immunoprecipitates. Thus, we suggest that PKA‐mediated phosphorylation of the β subunit is the major mechanism for β‐adrenergic regulation of cardiac L‐type calcium channel activity.

Angiotensin II type 1 receptor antibodies and increased angiotensin II sensitivity in pregnant rats.

Pregnant women who subsequently develop preeclampsia are highly sensitive to infused angiotensin (Ang) II; the sensitivity persists postpartum. Activating autoantibodies against the Ang II type 1 (AT1) receptor are present in preeclampsia. In vitro and in vivo data suggest that they could be involved in the disease process. We generated and purified activating antibodies against the AT1 receptor (AT1-AB) by immunizing rabbits against the AFHYESQ epitope of the second extracellular loop, which is the binding epitope of endogenous activating autoantibodies against AT1 from patients with preeclampsia. We then purified AT1-AB using affinity chromatography with the AFHYESQ peptide. We were able to detect AT1-AB both by ELISA and a functional bioassay. We then passively transferred AT1-AB into pregnant rats, alone or combined with Ang II. AT1-AB activated protein kinase C-&agr; and extracellular-related kinase 1/2. Passive transfer of AT1-AB alone or Ang II (435 ng/kg per minute) infused alone did not induce a preeclampsia-like syndrome in pregnant rats. However, the combination (AT1-AB plus Ang II) induced hypertension, proteinuria, intrauterine growth retardation, and arteriolosclerosis in the uteroplacental unit. We next performed gene-array profiling of the uteroplacental unit and found that hypoxia-inducible factor 1&agr; was upregulated by Ang II plus AT1-AB, which we then confirmed by Western blotting in villous explants. Furthermore, endothelin 1 was upregulated in endothelial cells by Ang II plus AT1-AB. We show that AT1-AB induces Ang II sensitivity. Our mechanistic study supports the existence of an “autoimmune-activating receptor” that could contribute to Ang II sensitivity and possible to preeclampsia.

Atrial Glutathione Content, Calcium Current, and Contractility

Atrial fibrillation (AF) is characterized by decreased L-type calcium current (ICa,L) in atrial myocytes and decreased atrial contractility. Oxidant stress and redox modulation of calcium channels are implicated in these pathologic changes. We evaluated the relationship between glutathione content (the primary cellular reducing moiety) and ICa,L in atrial specimens from AF patients undergoing cardiac surgery. Left atrial glutathione content was significantly lower in patients with either paroxysmal or persistent AF relative to control patients with no history of AF. Incubation of atrial myocytes from AF patients (but not controls) with the glutathione precursor N-acetylcysteine caused a marked increase in ICa,L. To test the hypothesis that glutathione levels were mechanistically linked with the reduction in ICa,L, dogs were treated for 48 h with buthionine sulfoximine, an inhibitor of glutathione synthesis. Buthionine sulfoximine treatment resulted in a 24% reduction in canine atrial glutathione content, a reduction in atrial contractility, and an attenuation of ICa,L in the canine atrial myocytes. Incubation of these myocytes with exogenous glutathione also restored ICa,L to normal or greater than normal levels. To probe the mechanism linking decreased glutathione levels to down-regulation of ICa, the biotin switch technique was used to evaluate S-nitrosylation of calcium channels. S-Nitrosylation was apparent in left atrial tissues from AF patients; the extent of S-nitrosylation was inversely related to tissue glutathione content. S-Nitrosylation was also detectable in HEK cells expressing recombinant human cardiac calcium channel subunits following exposure to nitrosoglutathione. S-Nitrosylation may contribute to the glutathione-sensitive attenuation of ICa,L observed in AF.

Potential Relevance of α1-Adrenergic Receptor Autoantibodies in Refractory Hypertension

Background Agonistic autoantibodies directed at the α1-adrenergic receptor (α1-AAB) have been described in patients with hypertension. We implied earlier that α1-AAB might have a mechanistic role and could represent a therapeutic target. Methodology/Principal Findings To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had α1-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate α1-adrenergic receptor antibodies (α1-AB). Patient α1-AAB and rabbit α1-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human α1A-adrenergic receptor were incubated with patient α1-AAB and rabbit α1-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient α1-AAB and rabbit α1-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca2+ in cardiomyocytes and induce mesentery artery segment contraction. Conclusions/Significance Patient α1-AAB and rabbit α1-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for α1-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.

Small Molecule AKAP-Protein Kinase A (PKA) Interaction Disruptors That Activate PKA Interfere with Compartmentalized cAMP Signaling in Cardiac Myocytes

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.