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H Haase

Publications and source records attributed to H Haase.

At least 55 records · Page 3Linked to original sources
PubMed1998

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) alpha-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 alpha1 subunit, and to a lesser extent the beta2 subunit, was directly correlated with the expression of SM alpha-actin and SM-MHC. We conclude that expression of L-type Ca2+ channel alpha1 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.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1998

Cardiomyopathic lentiginosis/LEOPARD syndrome presenting as sudden cardiac arrest.

A 26-year-old apparently healthy man with numerous pigmented skin lesions collapsed during an evening party and was resuscitated from ventricular fibrillation. Hypertrophic cardiomyopathy and subaortic tunnel were disclosed by angiocardiography. A diagnosis of cardiomyopathic lentiginosis/lentigines (multiple), electrocardiographic abnormalities, ocular hypertelorism, pulmonary stenosis, abnormalities of the genitalia, retardation of growth, and deafness (sensorineural) syndrome was made. The patient then underwent treatment with an implantable pacer-cardioverter-defibrillator device. Further evaluation revealed several well-established features of the disorder. This is the first reported case of survival from ventricular fibrillation associated with this rare and little known multifaceted syndrome. Disseminated lentiginosis must prompt clinicians to evaluate such cases further since underlying disorders may be associated with considerable morbidity and, apparently, sudden death.

Abnormalities, Multiple↗
PubMed1997

Beta1B subunit of voltage-dependent Ca2+ channels is predominant isoform expressed in human neuroblastoma cell line IMR32.

Human neuroblastoma cells (IMR32) respond to treatment with either dibutyryl-cAMP or nerve factor by acquiring a neuronal phenotype which is accompanied by a marked increase in the density of neuronal (N-type) VDCC currents. Using IMR32 cells as a model for neuronal differentiation, we were interested in examining possible changes in the level of expression of the alpha1B subunit of N-type calcium channels as well as beta subunit isoforms. Upon differentiation with dibutyryl-cAMP and 5-bromo-2-deoxyuridine for 16 days, we observed a dramatic increase in alpha1B protein which initiated between day 8 and 10. Day 10 evidenced maximal expression of alpha1B protein, which was followed by an interval of relatively constant expression of alpha1B (day 12 to day 16). Monitoring beta subunit expression using a pan specific anti-beta antibody (Ab CW20), we observed an increase in expression of a single 82 kDa beta subunit. The predominant 82 kDa beta subunit expressed throughout the course of differentiation was identified as the beta1b isoform using a panel of beta subunit specific antibodies. Of significance, neither the beta2 nor beta3 isoforms were detected in full differentiated IMR32 cells. Contrary to a previous report on the absence of neurotypic expression of VDCC beta subunits in a second model for in vitro differentiation, NGF-treated rat pheochromocytoma cells (PC12 cells) [1], we report the regulated expression of the beta1b protein in differentiated IMR32 cells suggesting a cell specific function for this beta subunit which parallels the acquisition of the neuronal phenotype. The restrictive expression of the beta1b in IMR32 cells may reflect a cell-type specific function that extends beyond its role as an auxiliary subunit of VDCC complexes.

Amino Acid Sequence↗
PubMed1997

Beta subunit heterogeneity in neuronal L-type Ca2+ channels.

Heterologous expression studies have shown that the activity of voltage-gated Ca2+ channels is regulated by their beta subunits in a beta subunit isoform-specific manner. In this study we therefore investigated if one or several beta subunit isoforms associate with L-type Ca2+ channels in different regions of mammalian brain. All four beta subunit isoforms (beta1b, beta2, beta3, and beta4) are expressed in cerebral cortex as shown in immunoblots. Immunoprecipitation of (+)-[3H]isradipine-labeled L-type channels revealed that the majority of beta subunit-associated L-type channels was associated with beta3 (42 +/- 8%) and beta4 (42 +/- 7%) subunits, whereas beta1b and beta2 were present in a smaller fraction of channel complexes. beta3 and beta4 were also the major L-type channel beta subunits in hippocampus. In cerebellum beta1b, beta2, and beta3 but not beta4 subunits were expressed at lower levels than in cortex. Accordingly, beta4 was the most prominent beta subunit in cerebellar L-type channels. This beta subunit composition was very similar to the one determined for 125I-omega-conotoxin-GVIA-labeled N-type and 125I-omega-conotoxin-MVIIC-labeled P/Q-type channel complexes in cerebral cortex and cerebellum. Our data show that all four beta subunit isoforms associate with L-type Ca2+ channels in mammalian brain. This beta subunit heterogeneity may play an important role for the fine tuning of L-type channel function and modulation in neurons.

Animals↗
PubMed1997

Different actin affinities of human cardiac essential myosin light chain isoforms.

The N terminus of myosin light chain 1 (MLC-1) of skeletal muscle bind to the C terminus of actin. We investigated whether the N termini of human cardiac MLC-1 isoforms likewise bind to actin. Furthermore, we investigated whether the N-terminal sequence 5-15 (P5-14) of MLC-1 of human atrium (ALC-1) and ventricle (VLC-1) bind with different affinities to actin. Affinity beads were produced by covalently coupling a synthetic peptide corresponding to the N-terminal sequence 4-14 of human VLC-1 to aminohexylagarose in order to bind G-actin. We found, that G-actin specifically binds to the affinity beads. Furthermore, preincubation of G-actin with P5-14 of both ALC-1 and VLC-1 decreased the amount of G-actin recovered from the affinity beads in a concentration-dependent manner. The half-maximal effective concentrations, however were significantly (p < 0.01) different being 0.32 +/- 0.02 microM and 0.71 +/- 0.02 microM for the VLC-1 and ALC-1 peptide, respectively. The appropriate scrambled peptides were without effect up to 3 microM. These results demonstrate the specific interaction between the N-terminal domains of human cardiac MLC-1 isoforms and actin and reveal different actin affinities of MLC-1 isoforms. Weak binding of ALC-1 to actin could explain the higher cycling kinetics of cross-bridges with ALC-1 compared to those with VLC-1.

Actins↗
PubMed1997

Influence of mechanical activity, adrenergic stimulation, and calcium on the expression of myosin heavy chains in cultivated neonatal cardiomyocytes.

It is generally accepted that mechanical stress of cardiomyocytes increases RNA and protein synthesis of myosin heavy chain (MHC) quantitatively but it is still a matter of debate whether MHC gene expression is also changed qualitatively. We investigated expression of MHC genes of spontaneously contracting neonatal cardiomyocytes experimentally arrested by permanent depolarization [potassium chloride (KCl)] as well as by electromechanical uncoupling [2,3 butanedione monoxime (BDM)]. Relative distribution of MHC mRNA isoforms (alpha and beta) was studied by quantitative polymerase chain reaction. Expression of MHC isoenzymes was the same in contracting (34.5% beta-MHC) and arrested (40.5% and 33.0% beta-MHC in KCl and BDM, respectively) cardiomyocytes. However, treatment with phenylephrine for the same period increased significantly beta-MHC expression to 55%. We conclude that hormonal factors rather than Ca2+ or mechanical stress regulate qualitatively MHC gene expression.

Adrenergic Agents↗
PubMed1997

Alternative splicing and cycling kinetics of myosin change during hypertrophy of human smooth muscle cells.

We investigated in vivo expression of myosin heavy chain (MHC) isoforms, 17 kDa myosin light chain (MLC17), and phosphorylation of the 20 kDa MLC (MLC20) as well as mechanical performance of chemically skinned fibers of normal and hypertrophied smooth muscle (SM) of human myometrium. According to their immunological reactivity, we identified three MHC isoenzymes in the human myometrium: two SM-MHC (SM1 with 204 kDa and SM2 with 200 kDa), and one non-muscle specific MHC (NM with 196 kDa). No cross-reactivity was detected with an antibody raised against a peptide corresponding to a seven amino acid insert at the 25K/50K junction of the myosin head (a-25K/50K) in both normal and hypertrophied myometrium. In contrast, SM-MHC of human myomatous tissue strongly reacted with a-25K/50K. Expression of SM1/SM2/NM (%) in normal myometrium was 31.7/34.7/33.6 and 35.1/40.9/24 in hypertrophied myometrium. The increased SM2 and decreased NM expression in the hypertrophied state was statistically significant (P < 0.05). MHC isoform distribution in myomatous tissue was similar to normal myometrium (36.3/35.3/29.4). In vivo expression of MLC17a increased from 25.5% in normal to 44.2% in hypertrophied (P < 0.001) myometrium. Phosphorylation levels of MLC20 upon maximal Ca(2+)-calmodulin activation of skinned myometrial fibers were the same in normal and hypertrophied myometrial fibers. Maximal force of isometric contraction of skinned fibers (pCa 4.5, slack-length) was 2.85 mN/mm2 and 5.6 mN/mm2 in the normal and hypertrophied state, respectively (P < 0.001). Apparent maximal shortening velocity (Vmax(appt), extrapolated from the force-velocity relation) of myometrium rose from 0.13 muscle length s-1 (ML/s) in normal to 0.24 ML/s in hypertrophied fibers (P < 0.001).

Adult↗
PubMed1997

Expression of sense and naturally occurring antisense mRNA of myosin heavy chain in rat heart tissue and cultivated cardiomyocytes.

We investigated the expression of sense- as well as naturally occurring antisense-mRNA of myosin heavy chains (MHC) in rat cardiomyocytes during development and cultivation. Relative distribution of the two MHC mRNA isoforms (alpha and beta) was studied by quantitative polymerase chain reaction. Primers were specific for the 3' untranslated region of alpha- and beta-MHC. Sense-alpha-MHC mRNA increased from 0% in the fetal heart to 45 +/- 8% in neonatal and to 75 +/- 5% in adult rat myocardium. In neonatal heart cell culture (10% bovine calf serum), alpha-MHC mRNA levels changed from 49 +/- 3% (day 0 = fresh isolated cells) to 61 +/- 6% (day 5 of cultivation). alpha-MHC mRNA of cultivated adult cardiomyocytes (serum-free) decreased from 90 +/- 7% (day 0) to 30 +/- 5% (day 5). Antisense-mRNA of both alpha- and beta-MHC was detected in rat cardiac tissue and cultivated cardiomyocytes. The expression of antisense-MHC mRNA did not change during development and cultivation. Antisense-alpha-MHC mRNA was the abundant isoform and was half of sense-alpha-MHC mRNA.

Animals↗
PubMed1997

Changes in essential myosin light chain isoform expression provide a molecular basis for isometric force regulation in the failing human heart.

We investigated the effects of the expression of myosin light chain (MLC) isoforms on the Ca2+ sensitivity of isometric force production of demembranated (skinned) fibers of papillary muscle from the left ventricle of three groups: patients with ischemic cardiomyopathy, patients with dilated cardiomyopathy (NYHA IV) and normal human hearts. Expression and phosphorylation of the phosphorylatable MLC isoforms (MLC-2) was equal within all three groups. However, 72% of the patients investigated in this study expressed the atrial essential MLC (ALC-1) in addition to the essential ventricular MLC (VLC-1) ranging between 2.4% and 10.3%. Using fibers from failing hearts, we observed a significant positive correlation between ALC-1 and Ca2+ sensitivity in that the higher the ALC-1 expression the higher the Ca2(+)-sensitivity: pCa50 (Ca2+ required for half-maximal force production) was 5.87 without ALC-1 and 6.08 with 10.3% ALC-1. Fibers from a normal heart (no ALC-1) revealed a pCa50 of 5.85. Isoform and phosphorylation patterns of tropomyosin and troponin I remained unchanged in the patients and normal hearts. Our results suggest that Ca2+ responsiveness and force development of the human heart is regulated by the expression of different MLC-1 isoforms.

Calcium↗
PubMed1997

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.

Animals↗
PubMed1997

Hypertrophy of colonic smooth muscle: contractile proteins, shortening velocity, and regulation.

Smooth muscle in megacolon was studied in the lethal spotted mouse and its normal sibling. In megacolon, structural remodeling and a very large increase in total protein content are associated with some changes in the contractile protein isoform composition. 1) There is a higher actin concentration in megacolon, primarily caused by a larger proportion of gamma-isoforms. 2) There was no difference in myosin concentration or in SM1/SM2 heavy chains in megacolon and normal muscle contractile proteins. 3) Only LC17a essential light chain is present in both normal and megacolon. 4) The 25- to 50-kDa 5'-insert occurs in 15-20% of the myosin in normal colon, compared with 5- to 10-fold lower amounts in megacolon. In permeabilized muscles there was no significant difference in unloaded shortening velocity (Vo) with maximal thiophosphorylation of the 20-kDa light chains, nor was there significant difference in the force vs. Ca2+ and force vs. myosin light chain phosphorylation relationships. At approximately 60% myosin light chain phosphorylation after Ca2+ activation, Vo of megacolon was approximately two times faster than Vo of normal muscle. These cellular changes largely account for the higher propulsive velocity of the colon in situ. The distribution of myosin and actin isoforms and the lack of a simple relationship between myosin light chain phosphorylation and Vo point to the operation of additional regulatory processes.

Actins↗
PubMed1997

L-type calcium channels in insulin-secreting cells: biochemical characterization and phosphorylation in RINm5F cells.

Opening of dihydropyridine-sensitive voltage-dependent L-type Ca2+-channels (LTCCs) represents the final common pathway for insulin secretion in pancreatic beta-cells and related cell lines. In insulin-secreting cells their exact subunit composition is unknown. We therefore investigated the subunit structure of (+)-[3H]isradipine-labeled LTCCs in insulin-secreting RINm5F cells. Using subunit-specific antibodies we demonstrate that alpha1C subunits (199 kDa, short form) contribute only a minor portion of the total alpha1 immunoreactivity in membranes and partially purified Ca2+-channel preparations. However, alpha1C forms a major constituent of (+)-[3H]isradipine-labeled LTCCs as 54% of solubilized (+)-[3H]isradipine-binding activity was specifically immunoprecipitated by alpha1C antibodies. Phosphorylation of immunopurified alpha1C with cAMP-dependent protein kinase revealed the existence of an additional 240-kDa species (long form), that remained undetected in Western blots. Fifty seven percent of labeled LTCCs were immunoprecipitated by an anti-beta-antibody directed against all known beta-subunits. Isoform-specific antibodies revealed that these mainly corresponded to beta1b- and beta3-subunits. We found beta2- and beta4-subunits to be major constituents of cardiac and brain L-type channels, respectively, but not part of L-type channels in RINm5F cells. We conclude that alpha1C is a major constituent of dihydropyridine-labeled LTCCs in RINm5F cells, its long form serving as a substrate for cAMP-dependent protein kinase. beta1b- and beta3-Subunits were also found to associate with L-type channels in these cells. These isoforms may therefore represent biochemical targets for the modulation of LTCC activity in RINm5F cells.

Calcium Channels↗
PubMed1997

Apical hypertrophic cardiomyopathy of the Japanese type coexistent with a coronary muscle bridge. A case report and review.

We describe the case of a 21-year-old Italian male who presented with giant negative T-waves and left ventricular hypertrophy on the electrocardiogram suggestive of apical hypertrophic cardiomyopathy. Clinically, he suffered from new onset of exertional angina, dyspnea and palpitations during soccer playing or heavy exercise beginning one week before admission. Echocardiography and cardiac catheterization conformed the rare combination of a nonobstructive apical hypertrophic cardiomyopathy of the "Japanese" type coexistent with an extensive muscular bridge involving almost the entire anterior interventricular branch of the left coronary artery. Although the patient complained of exertional angina pectoris, absence of objective evidence of myocardial ischemia by means of treadmill stress test, exercise thallium scan, dobutamine stress echocardiography as well as atrial pacing stress emphasized the benign nature of this complex anomaly.

Adult↗
PubMed1996

Regulation of human heart contractility by essential myosin light chain isoforms.

Most of the patients with congenital heart diseases express the atrial myosin light chain 1 (ALC-1) in the right ventricle. We investigated the functional consequences of ALC-1 expression on the myosin cycling kinetics in the intact sarcomeric structure using multicellular demembranated fibers ("skinned fibers") from the right ventricular infundibulum of patients with Tetralogy of Fallot (TOF), double outlet right ventricle (DORV), and infundibular pulmonary stenosis (IPS), Force-velocity relation was analyzed by the constant-load technique at maximal Ca2+ activation (pCa 4.5). Half-time of tension development (t1/2) was investigated by monitoring contraction initiation upon photolytic release of ATP from caged-ATP in rigor. The patients investigated here expressed between 0 and 27% ALC-1. There was a statistically significant correlation between ALC-l and maximal shortening velocity (Vmax) which rose 1.87-fold from 1.2 muscle length per second (ML/s) to 2.25 ML/s in a normal (0% ALC-1) and diseased (19.9% ALC-1) ventricle. Half-time of tension development decreased 1.85-fold with increasing ALC-1 expression (t1/2) was 0.252 s and 0.136 s at 2 and 18.4% ALC-1, respectively). We conclude that the expression of ALC-1 in the human heart modulates cross-bridge cycling kinetics accelerating shortening velocity and isometric tension production.

Adenosine Triphosphate↗
PubMed1996

Alternative splicing of smooth muscle myosin heavy chains and its functional consequences.

The aim of our study was to determine the relation between alternatively spliced myosin heavy chain (MHC) isoforms and the contractility of smooth muscle. The relative amount of MHC with an alternatively spliced insert in the 5' (amino terminal) domain was determined on the protein level using a peptide-directed antibody (a25K/50K) raised against the inserted sequence (QGPSFAY). Smooth muscle MHC isoforms of both bladder and myometrium but not nonmuscle MHC reacted with a25/50K. Using a quantitative Western-blot approach the amount of 5'-inserted MHC in rat bladder was detected to be about eightfold higher than in normal rat myometrium. The amount of heavy chain with insert was found to be decreased by about 50% in the myometrium of pregnant rats. Although bladder contained significantly more 5'-inserted MHC than myometrium, apparent maximal shortening velocities (Vmax) were comparable, being 0.138 +/- 0.012 and 0.114 +/- 0.023 muscle length per second of skinned bladder and normal myometrium fibers, respectively. Phosphorylation of myosin light chain 20 induced by maximal Ca2+/calmodulin activation was the same in bladder and myometrial fibers. These results suggest that the amount of 5'-inserted MHC is not necessarily associated with contractile properties of smooth muscle.

Alternative Splicing↗