Ingo Morano

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)

The failing human heart is unable to use the Frank-Starling mechanism.

There is evidence that the failing human left ventricle in vivo subjected to additional preload is unable to use the Frank-Starling mechanism. The present study compared the force-tension relation in human nonfailing and terminally failing (heart transplants required because of dilated cardiomyopathy) myocardium. Isometric force of contraction of electrically driven left ventricular papillary muscle strips was studied under various preload conditions (2 to 20 mN). To investigate the influence of inotropic stimulation, the force-tension relation was studied in the presence of the cardiac glycoside ouabain. In skinned-fiber preparations of the left ventricle, developed tension was measured after stretching the preparations to 150% of the resting length. To evaluate the length-dependent activation of cardiac myofibrils by Ca2+ in failing and nonfailing myocardium, the tension-Ca2+ relations were also measured. After an increase of preload, the force of contraction gradually increased in nonfailing myocardium but was unchanged in failing myocardium. There were no differences in resting tension, muscle length, or cross-sectional area of the muscles between both groups. Pretreatment with ouabain (0.02 mumol/L) restored the force-tension relation in failing myocardium and preserved the force-tension relation in nonfailing tissue. In skinned-fiber preparations of the same hearts, developed tension increased significantly after stretching only in preparations from nonfailing but not from failing myocardium. The Ca2+ sensitivity of skinned fibers was significantly higher in failing myocardium (EC50, 1.0; 95% confidence limit, 0.88 to 1.21 mumol/L) compared with nonfailing myocardium (EC50, 1.7; 95% confidence limit, 1.55 to 1.86 mumol/L). After increasing the fiber length by stretching, a significant increase in the sensitivity of the myofibrils to Ca2+ was observed in nonfailing but not in failing myocardium. These experiments provide evidence for an impaired force-tension relation in failing human myocardium. On the subcellular level, this phenomenon might be explained by a failure of the myofibrils to increase the Ca2+ sensitivity after an increase of the sarcomere length.

Adipocyte Fatty Acid-Binding Protein Suppresses Cardiomyocyte Contraction. A New Link Between Obesity and Heart Disease

Rationale: Adipocyte fatty acid–binding protein (FABP4) is a member of the intracellular lipid-binding protein family and is predominantly expressed in adipose tissue. Emerging evidence suggests that FABP4 plays a role in some aspects of the metabolic syndrome including the development of type 2 diabetes and atherosclerosis. We have recently reported that secretory products from human adipocytes directly and acutely depressed cardiac contractile function. Objective: The purpose of this study was to identify this adipocyte-derived cardiodepressant factor. Methods and Results: Through mass spectrometry and immunoblotting, we have identified this cardiodepressant factor as FABP4. FABP4 represents 1.8% to 8.1% of total protein secreted by adipocytes in extracellular medium. FABP4 acutely depressed shortening amplitude as well as intracellular systolic peak Ca2+ in a dose-dependent manner in isolated rat cardiomyocytes. Heart-specific FABP isoform (FABP3) revealed a similar cardiodepressant effect. The N-terminal amino acids 1 to 20 of FABP4 could be identified as the most effective cardiodepressive domain. We could exclude any effect of FABP4 on action potential duration and L-type Ca2+ current, suggesting a reduced excitation-contraction gain caused by FABP4 as the main inhibitory mechanism. Conclusion: We conclude that the release of FABP4 from adipocytes may be involved in the development of cardiac contractile dysfunction of obese subjects.

Counting target molecules by exponential polymerase chain reaction: copy number of mitochondrial DNA in rat tissues.

In this report, we show that the actual number of target molecules of the polymerase chain reaction can be determined by measuring the concentration of product accumulating in consecutive cycles. The equation describing product accumulation, log Nn = log eff x n + log N0, can be analyzed by linear regression and the molar concentration of target at cycle zero, N0, is obtained. Using this new approach, the actual content of mitochondrial DNA was determined in rat tissues and ranged from 116 x 10(9) molecules/g in fast-twitch skeletal muscle to 743 x 10(9) molecules/g in liver. Using morphometric data from the literature, mitochondria were found to contain 1 to 3 DNA molecules. There was no relation between the oxidative capacity of a tissue and its content of mitochondrial DNA, indicating that transcriptional and posttranscriptional mechanisms rather than gene dosage, as postulated by others, determine to what extent the mitochondrial genome is expressed.

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.

Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms

Cardiac hypertrophy is an adaptive response that normalizes wall stress and compensates for increased workload. It is accompanied by distinct qualitative and quantitative changes in the expression of protein isoforms concerning contractility, intracellular Ca(2+)-homeostasis and metabolism. Changes in the myosin subunit isoform expression improves contractility by an increase in force generation at a given Ca(2+)-concentration (increased Ca(2+)-sensitivity) and by improving the economy of the chemo-mechanical transduction process per amount of utilised ATP (increased duty ratio). In the human atrium this is achieved by partial replacement of the endogenous fast myosin by the ventricular slow-type heavy and light chains. In the hypertrophic human ventricle the slow-type beta-myosin heavy chains remain unchanged, but the ectopic expression of the atrial myosin essential light chain (ALC1) partially replaces the endogenous ventricular isoform (VLC1). The ventricular contractile apparatus with myosin containing ALC1 is characterised by faster cross-bridge kinetics, a higher Ca(2+)-sensitivity of force generation and an increased duty ratio. The mechanism for cross-bridge modulation relies on the extended Ala-Pro-rich N-terminus of the essential light chains of which the first eleven residues interact with the C-terminus of actin. A change in charge in this region between ALC1 and VLC1 explains their functional difference. The intracellular Ca(2+)-handling may be impaired in heart failure, resulting in either higher or lower cytosolic Ca(2+)-levels. Thus the state of the cardiomyocyte determines whether this hypertrophic adaptation remains beneficial or becomes detrimental during failure. Also discussed are the effects on contractility of long-term changes in isoform expression of other sarcomeric proteins. Positive and negative modulation of contractility by short-term phosphorylation reactions at multiple sites in the myosin regulatory light chain, troponin-I, troponin-T, alpha-tropomyosin and myosin binding protein-C are considered in detail.

Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides

In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration.

Connective tissue growth factor overexpression in cardiomyocytes promotes cardiac hypertrophy and protection against pressure overload.

Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca2+ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls. Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGFs profibrotic function in the heart.

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)