Heimo Ehmke

Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts

The concept of regenerating diseased myocardium by implantation of tissue-engineered heart muscle is intriguing, but convincing evidence is lacking that heart tissues can be generated at a size and with contractile properties that would lend considerable support to failing hearts. Here we created large (thickness/diameter, 1–4 mm/15 mm), force-generating engineered heart tissue from neonatal rat heart cells. Engineered heart tissue formed thick cardiac muscle layers when implanted on myocardial infarcts in immune-suppressed rats. When evaluated 28 d later, engineered heart tissue showed undelayed electrical coupling to the native myocardium without evidence of arrhythmia induction. Moreover, engineered heart tissue prevented further dilation, induced systolic wall thickening of infarcted myocardial segments and improved fractional area shortening of infarcted hearts compared to controls (sham operation and noncontractile constructs). Thus, our study provides evidence that large contractile cardiac tissue grafts can be constructed in vitro, can survive after implantation and can support contractile function of infarcted hearts.

Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice.

Fibroblast growth factor‐2 (FGF‐2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF‐2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF‐2‐deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin‐positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin‐positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF‐2‐deficient adult mice are hypotensive. They respond normally to angiotensin II‐induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF‐2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF‐2‐deficient adult mice uncovers more general roles in neural development and function.

Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold

K‐Cl co‐transporters are encoded by four homologous genes and may have roles in transepithelial transport and in the regulation of cell volume and cytoplasmic chloride. KCC3, an isoform mutated in the human Anderman syndrome, is expressed in brain, epithelia and other tissues. To investigate the physiological functions of KCC3, we disrupted its gene in mice. This severely impaired cell volume regulation as assessed in renal tubules and neurons, and moderately raised intraneuronal Cl− concentration. Kcc3−/− mice showed severe motor abnormalities correlating with a progressive neurodegeneration in the peripheral and CNS. Although no spontaneous seizures were observed, Kcc3−/− mice displayed reduced seizure threshold and spike‐wave complexes on electrocorticograms. These resembled EEG abnormalities in patients with Anderman syndrome. Kcc3−/− mice also displayed arterial hypertension and a slowly progressive deafness. KCC3 was expressed in many, but not all cells of the inner ear K+ recycling pathway. These cells slowly degenerated, as did sensory hair cells. The present mouse model has revealed important cellular and systemic functions of KCC3 and is highly relevant for Anderman syndrome.

Myeloperoxidase acts as a profibrotic mediator of atrial fibrillation

Observational clinical and ex vivo studies have established a strong association between atrial fibrillation and inflammation. However, whether inflammation is the cause or the consequence of atrial fibrillation and which specific inflammatory mediators may increase the atrias susceptibility to fibrillation remain elusive. Here we provide experimental and clinical evidence for the mechanistic involvement of myeloperoxidase (MPO), a heme enzyme abundantly expressed by neutrophils, in the pathophysiology of atrial fibrillation. MPO-deficient mice pretreated with angiotensin II (AngII) to provoke leukocyte activation showed lower atrial tissue abundance of the MPO product 3-chlorotyrosine, reduced activity of matrix metalloproteinases and blunted atrial fibrosis as compared to wild-type mice. Upon right atrial electrophysiological stimulation, MPO-deficient mice were protected from atrial fibrillation, which was reversed when MPO was restored. Humans with atrial fibrillation had higher plasma concentrations of MPO and a larger MPO burden in right atrial tissue as compared to individuals devoid of atrial fibrillation. In the atria, MPO colocalized with markedly increased formation of 3-chlorotyrosine. Our data demonstrate that MPO is a crucial prerequisite for structural remodeling of the myocardium, leading to an increased vulnerability to atrial fibrillation.

The Apolipoprotein E Knockout Mouse: A Model Documenting Accelerated Atherogenesis In Uremia

Rodents do not develop spontaneous atherosclerosis. Currently, there is no good animal model to study the effect of uremia on atherosclerosis. This study evaluated whether apolipoprotein E knockout (Apoe-/-) mice are useful to study the effect of renal dysfunction on cardiovascular risk. Apoe-/- mice have decreased serum apolipoprotein E and exhibit lipid abnormalities and atherosclerosis even on a low-cholesterol diet. Ten-wk-old Apoe-/- mice were subtotally nephrectomised (SNX Apoe-/-; n = 8), uninephrectomised (UNX Apoe-/-; n = 5), or sham-operated (sham Apoe-/-; n = 5) and compared with their genetic controls (SNX C57/BL6; UNX C57/BL6; sham C57/BL6). After 12 wk, BP was measured intraarterially, blood samples were taken, and the experiment was terminated by perfusion fixation. The heart weight was determined, and quantitative morphologic analysis of intramyocardial arteries and aortic changes was performed. At the end of the experiment, heart weight and relative left ventricular weight were comparable in all groups. Intraarterial BP was somewhat higher in Apoe-/- mice compared with controls. Baseline serum cholesterol and triglyceride levels were higher in Apoe-/- mice than in C57/BL6. Atherosclerotic plaques were not present in sham or UNX C57/BL6, but minor plaque formation was noted in some SNX control animals. In contrast, beginning plaques were seen even in untouched Apoe-/- mice, and strikingly increased plaque formation was noted in UNX and SNX Apoe-/- mice. Maximal plaque diameter (cross-section) was 37 +/- 74 micro m in SNX C57/BL6, 191 +/- 90 micro m in sham Apoe-/-, 323 +/- 66 micro m in UNX Apoe-/-, and 457 +/- 17 micro m in SNX Apoe-/-. The plaque morphology corresponded with that of early plaques characterized by foam cells and virtual absence of lymphocytes or smooth muscle cell infiltration. In conclusion, even mild renal dysfunction, i.e., after uninephrectomy, causes a dramatic increase in plaque size and aggressive morphology (foam cell rich soft plaques) in the animal model of the Apoe-/- mouse.

Gating, modulation and subunit composition of voltage‐gated K+ channels in dendritic inhibitory interneurones of rat hippocampus

GABAergic interneurones are diverse in their morphological and functional properties. Perisomatic inhibitory cells show fast spiking during sustained current injection, whereas dendritic inhibitory cells fire action potentials with lower frequency. We examined functional and molecular properties of K+ channels in interneurones with horizontal dendrites in stratum oriens‐alveus (OA) of the hippocampal CA1 region, which mainly comprise somatostatin‐positive dendritic inhibitory cells. Voltage‐gated K+ currents in nucleated patches isolated from OA interneurones consisted of three major components: a fast delayed rectifier K+ current component that was highly sensitive to external 4‐aminopyridine (4‐AP) and tetraethylammonium (TEA) (half‐maximal inhibitory concentrations < 0.1 mm for both blockers), a slow delayed rectifier K+ current component that was sensitive to high concentrations of TEA, but insensitive to 4‐AP, and a rapidly inactivating A‐type K+ current component that was blocked by high concentrations of 4‐AP, but resistant to TEA. The relative contributions of these components to the macroscopic K+ current were estimated as 57 ± 5, 25 ± 6, and 19 ± 2 %, respectively. Dendrotoxin, a selective blocker of Kv1 channels had only minimal effects on K+ currents in nucleated patches. Coapplication of the membrane‐permeant cAMP analogue 8‐(4‐chlorophenylthio)‐adenosine 3′:5′‐cyclic monophosphate (cpt‐cAMP) and the phosphodiesterase blocker isobutyl‐methylxanthine (IBMX) resulted in a selective inhibition of the fast delayed rectifier K+ current component. This inhibition was absent in the presence of the protein kinase A (PKA) inhibitor H‐89, implying the involvement of PKA‐mediated phosphorylation. Single‐cell reverse transcription‐polymerase chain reaction (RT‐PCR) analysis revealed a high abundance of Kv3.2 mRNA in OA interneurones, whereas the expression level of Kv3.1 mRNA was markedly lower. Similarly, RT‐PCR analysis showed a high abundance of Kv4.3 mRNA, whereas Kv4.2 mRNA was undetectable. This suggests that the fast delayed rectifier K+ current and the A‐type K+ current component are mediated predominantly by homomeric Kv3.2 and Kv4.3 channels. Selective modulation of Kv3.2 channels in OA interneurones by cAMP is likely to be an important factor regulating the activity of dendritic inhibitory cells in principal neurone‐interneurone microcircuits.

Myeloperoxidase attracts neutrophils by physical forces

Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocytes surface. In vivo, PMN recruitment was shown to be MPO-dependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPOs affinity to both the endothelial and the leukocytes surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces.

Autoregulation of renal blood flow, glomerular filtration rate and renin release in conscious dogs

The relationship between renal artery pressure (RAP), renal blood flow (RBF), glomerular filtration rate (GFR) and the renal venous-arterial plasma renin activity difference (PRAD) was studied in 22 chronically instrumented, conscious foxhounds with a daily sodium intake of 6.6 mmol/kg. RAP was reduced in steps and maintained constant for 5 min using an inflatable renal artery cuff and a pressure control system.Between 160 and 81 mm Hg we observed a concomitant autoregulation of GFR and RBF with a high precision. The “break off points” for GRF- and RBF-autoregulation were sharp and were significantly different from each other (GFR: 80.5±3.5 mm Hg; RBF: 65.6±1.3 mm Hg;P<0.01). In the subautoregulatory range GFR and RBF decreased in a linerar fashion and ceased at 40 and 19 mm Hg, respectively.Between 160 mm Hg and 95 mm Hg (threshold pressure for renin release) PRAD remained unchanged; below threshold pressure PRAD increased steeply (average slope: 0.34 ng AI·ml−1·h−1· mm Hg−1) indicating that resting renin release may be doubled by a fall of RAP by only 3 mm Hg. At the “break-off point” of RBF-autoregulation (66 mm Hg) renin release was 10-fold higher than the resting level.It is concluded that under physiological conditions (normal sodium diet) GFR and RBF are perfectly autoregulated over a wide pressure range. Renin release remains suppressed until RAP falls below a well defined threshold pressure slightly below the animals resting systemic pressure. RBF is maintained at significantly lower pressures than GFR, indicating that autoregulation of RBF also involves postglomerular vessels. Our data are in agreement with the myogenic hypothesis as a basic mechanism of autoregulation.

Autoregulation of renal blood flow in the conscious dog and the contribution of the tubuloglomerular feedback.

1 The aim of this study was to investigate the autoregulation of renal blood flow under physiological conditions, when challenged by the normal pressure fluctuations, and the contribution of the tubuloglomerular feedback (TGF). 2 The transfer function between 0.0018 and 0.5 Hz was calculated from the spontaneous fluctuations in renal arterial blood pressure (RABP) and renal blood flow (RBF) in conscious resting dogs. The response of RBF to stepwise artificially induced reductions in RABP was also studied (stepwise autoregulation). 3 Under control conditions (n= 12 dogs), the gain of the transfer function started to decrease, indicating improving autoregulation, below 0.06‐0.15 Hz (t= 7‐17 s). At 0.027 Hz a prominent peak of high gain was found. Below 0.01 Hz (t> 100 s), the gain reached a minimum (maximal autoregulation) of ‐6.3 ± 0.6 dB. The stepwise autoregulation (n= 4) was much stronger (‐19.5 dB). The time delay of the transfer function was remarkably constant from 0.03 to 0.08 Hz (high frequency (HF) range) at 1.7 s and from 0.0034 to 0.01 Hz (low frequency (LF) range) at 14.3 s, respectively. 4 Nifedipine, infused into the renal artery, abolished the stepwise autoregulation (‐2.0 ± 1.1 dB, n= 3). The gain of the transfer function (n= 4) remained high down to 0.0034 Hz; in the LF range it was higher than in the control (0.3 ± 1.0 dB, P< 0.05). The time delay in the HF range was reduced to 0.5 s (P < 0.05). 5 After ganglionic blockade (n= 7) no major changes in the transfer function were observed. 6 Under furosemide (frusemide) (40 mg + 10 mg h−1 or 300 mg + 300 mg h−1 i.v.) the stepwise autoregulation was impaired to ‐7.8 ± 0.3 or ‐6.7 ± 1.9 dB, respectively (n= 4). In the transfer function (n= 7 or n= 4) the peak at 0.027 Hz was abolished. The delay in the LF range was reduced to ‐1.1 or ‐1.6 s, respectively. The transfer gain in the LF range (‐5.5 ± 1.2 or ‐3.8 ± 0.8 dB, respectively) did not differ from the control but was smaller than that under nifedipine (P < 0.05). 7 It is concluded that the ample capacity for regulation of RBF is only partially employed under physiological conditions. The abolition by nifedipine and the negligible effect of ganglionic blockade show that above 0.0034 Hz it is almost exclusively due to autoregulation by the kidney itself. TGF contributes to the maximum autoregulatory capacity, but it is not required for the level of autoregulation expended under physiological conditions. Around 0.027 Hz, TGF even reduces the degree of autoregulation.

Regional alterations of repolarizing K+ currents among the left ventricular free wall of rats with ascending aortic stenosis

1 The effect of cardiac hypertrophy on electrocardiogram (ECG), action potential duration (APD) and repolarizing K+ currents was investigated in epicardial, midmyocardial and endocardial myocytes isolated from the rat left ventricular free wall. 2 Cardiac hypertrophy was induced by stenosis of the ascending aorta (AS), which led to an increased pressure load (+85 ± 10 mm) of the left ventricle; sham‐operated animals served as controls. 3 In ECG recordings from AS rats, the QTc interval was prolonged and the main vectors of the QRS complex and the T‐wave pointed in opposite directions, indicating an abnormal sequence of repolarization. 4 APD and K+ currents were recorded using the whole‐cell patch‐clamp technique. In the AS group, APD90 (90 % repolarization) was significantly prolonged in epicardial and midmyocardial, but not endocardial myocytes. 5 Corresponding to the increase in APD, the magnitude of the transient outward K+ current (Ito1) was significantly smaller (‐30 %) in epicardial and midmyocardial, but not endocardial myocytes. 6 Inactivation and steady‐state inactivation of Ito1 were not affected by hypertrophy. Recovery from inactivation was slightly prolonged in endocardial myocytes from AS rats. 7 No differences in delayed rectifier currents (IK) or inwardly rectifying K+ currents (IK1) were detected between myocytes of the three regions of sham‐operated or AS animals. However, both currents were reduced by AS. 8 The present data show that cardiac hypertrophy caused by pressure overload leads to an increase in APD and a decrease in Ito1 primarily in epicardial and midmyocardial myocytes, which implies a major role of alterations in Ito1 for the reduced gradient in APD. The effects of AS on IK1 and IK may slightly counteract the decrease in APD gradient. The observed changes in APD and underlying ionic currents could well explain the alterations in repolarization observed in the ECG induced by cardiac hypertrophy.