Joachim Günther

Cardiac fibroblasts and the mechano-electric feedback mechanism in healthy and diseased hearts.

Cardiac arrhythmia is a serious clinical condition, which is frequently associated with abnormalities of mechanical loading and changes in wall tension of the heart. Recent novel findings suggest that fibroblasts may function as mechano-electric transducers in healthy and diseased hearts. Cardiac fibroblasts are electrically non-excitable cells that respond to spontaneous contractions of the myocardium with rhythmical changes of their resting membrane potential. This phenomenon is referred to as mechanically induced potential (MIP) and has been implicated in the mechano-electric feedback mechanism of the heart. Mechano-electric feedback is thought to adjust the frequency of spontaneous myocardial contractions to changes in wall tension, which may result from variable filling pressure. Electrophysiological recordings of single atrial fibroblasts indicate that mechanical compression of the cells may activate a non-selective cation conductance leading to depolarisation of the membrane potential. Reduced amplitudes of MIPs due to pharmacological disruption of F-actin and tubulin suggest a role for the cytoskeleton in the mechano-electric signal transduction process. Enhanced sensitivity of the membrane potential of the fibroblasts to mechanical stretch after myocardial infarction correlates with depression of heart rates. It is assumed that altered electrical function of cardiac fibroblasts may contribute to the increased risk of post-infarct arrhythmia.

Mechanically Induced Potentials in Fibroblasts from Human Right Atrium

It has been shown that cardiac fibroblasts of the human heart are electrically non‐excitable and mechanosensitive. The resting membrane potential of these cells is ‐15.9 ± 2.1 mV and the membrane resistance is 4.1 ± 0.1 GΩ. Rhythmic contractions of the myocardium associated with stretch of the surrounding tissue produce reversible changes in the membrane potential of cardiac fibroblasts. These mechanically induced potentials (MIPs) follow the rhythm of myocardial contractions. Simultaneous recording of the action potential of cardiomyocytes and MIPs of cardiac fibroblasts demonstrates a delay of 40.0 ± 0.4 ms after the action potential before the appearance of the MIP. Contraction produces a MIP which is more positive or more negative than the reversal potential ‐ the membrane potential due to current injection at which the MIP reverses its direction. Regardless of the initial orientation of the MIP, intracellular polarization increases the amplitude towards the reversal potential if the background MIP had depolarized the membrane or away from the reversal potential if the initial background MIP had hyperpolarized the membrane. Artificial intracellular polarization changed the amplitude but not the frequency of the MIP. The pool of electrically non‐excitable mechanosensitive cells, which change their electrical activity during contraction and relaxation of the heart, may play a role in the mechano‐electrical feedback mechanism which has to be taken into account in the normal function of the heart as well as in pathological processes.

Mechanoelectric feedback after left ventricular infarction in rats

BACKGROUND Myocardial infarction can lead to electrical abnormalities and rhythm disturbances. However, there is limited data on the electrophysiological basis for these events. Since regional contraction abnormalities feature prominently in infarction, we investigated whether stretch of myocardium from the infarction borderzone can modulate the electrophysiological properties of cardiomyocytes via mechanoelectric feedback providing a mechanism for post-infarction arrhythmia. METHODS Five weeks after experimental myocardial infarction (MI) in rats due to ligation of the left coronary artery (n = 26) or after sham operation (SO, n = 16), action potentials (AP) were measured in left ventricular preparations from the infarction borderzone. Sustained stretch was applied via a micrometer. RESULTS Preparations from MI generated spontaneous electrical and contractile activity. Cardiomyocytes from MI had a comparable AP amplitude, a more negative resting membrane potential, and a prolonged AP duration (APD) when compared to SO. In SO, stretch of 150 microns increased the APD90. This was associated with stretch activated depolarizations near APD90 (SAD-90). In MI, significantly lower stretch, of only 20 microns, elicited SAD-90s, or SADs near APD50 (SAD-50). Stretch-induced events were suppressed by gadolinium, at a concentration (40 microM) normally used to inhibit stretch-activated channels. CONCLUSION After MI, SADs are generated in the infarction borderzone at lower degrees of stretch. Increased sensitivity of the membrane potential of cardiac myocytes to mechanical stimuli may contribute to the high risk of arrhythmia after infarction. These SADs may involve the opening of stretch-activated channels.

Myocardial contractility after infarction and carnitine palmitoyltransferase I inhibition in rats.

Inhibition of carnitine palmitoyltransferase I with etomoxir increases sarcoplasmic reticulum Ca(2+)-transport and V(1) isomyosin expression. To test whether etomoxir attenuates contractile dysfunction after myocardial infarction, we compared the contractility of papillary muscles from etomoxir- and placebo-treated rats 6 weeks after infarction. Etomoxir induced cardiac hypertrophy in animals with small infarctions, and enhanced compensatory heart growth at large infarct size. Contractile function of papillary muscles from etomoxir-treated rats was improved particularly in animals with small infarctions. Thus, induction of mild cardiac hypertrophy by etomoxir in rats with small infarctions may be beneficial for myocardial performance.

Alterations of cardiac contractile function are related to changes in membrane calcium transport in spontaneously hypertensive rats

Objective: Transport activities of cardiac sodium-calcium exchange and sarcoplasmic reticulum calcium ATPase were measured biochemically in spontaneously hypertensive rats (SHR) with hypertrophied myocardium and in normotensive Wistar-Kyoto (WKY) rats and it was tested whether possible differences have consequences for the contractile properties of papillary muscle. Methods: Sarcolemmal sodium-dependent calcium transport via sodium-calcium exchange and oxalate-supported sarcoplasmic reticulum calcium uptake were measured in left ventricular membranes of 22-week-old rats. Postextrastimulatory potentiated contractions, postrest potentiated contractions, the twitch-to-twitch decay of those potentiations and the response to increasing stimulation frequency of left ventricular papillary muscles were analysed. Results: Compared with WKY rats we found in SHR: a significant increase in sodium-calcium exchange (65%) and in sarcoplasmic reticulum calcium uptake (24%); a steeper twitch-to-twitch decay in postextrastimulatory potentiated contractions and postrest potentiated contractions, suggesting a lower calcium fraction recirculating between myofilaments and sarcoplasmic reticulum and, consequently, a relatively higher calcium efflux via sodium-calcium exchange; a stronger rest-dependent decrease in recirculating calcium fraction in postrest potentiated contractions accompanied by accelerated relaxation, suggesting an increasing driving force for calcium extrusion via sodium-calcium exchange, probably caused by decreasing intracellular sodium during rest; a greater transient decrease in peak force of subsequent twitches after postrest potentiated contractions below pre-interventional level, indicating higher cellular calcium loss; and a smaller negative inotropic effect in response to doubling of stimulation rate as a manoeuvre to increase the intracellular sodium level. Conclusion: In SHR, the contractile properties suggest an increased contribution of sodium-calcium exchange to cellular calcium removal, which is strongly supported by the enhanced sodium-calcium exchange activity in cardiac membrane vesicles.

Combined Treatment with Ramipril and Metoprolol Prevents Changes in the Creatine Kinase Isoenzyme System and Improves Hemodynamic Function in Rat Hearts after Myocardial Infarction

Beneficial effects of monotherapy with ACE inhibitors or beta-blockers on hemodynamic function after myocardial infarction are well known. Until now, the effects of combined treatment on cardiac function and energy metabolism have been poorly described. This study examines the effects of combined ramipril and metoprolol treatment on the creatine kinase (CK) system and hemodynamic function in rats after infarction. Wistar rats with experimental infarction were randomized for treatment with ramipril (R), metoprolol (M), combined treatment (MR), or placebo (P). Sham-operated (SO) animals served as controls. After 6 weeks, we assayed for CK isoenzymes and performed hemodynamic measurements. In P versus SO, left ventricular systolic pressures (dp/dtmax and dp/dtmin) diminished, whereas left ventricular end-diastolic pressure (LVEDP) increased. Decreased total CK activity and mitochondrial CK isoenzyme, increased CK-MB, and increased CK-BB isoenzymes were measured in P versus SO. With infarct size ≤45%, mitochondrial CK increased in M and R versus P. Combined treatment had an additional enhancing effect on mitochondrial CK isoenzyme level versus M and R, decreased LVEDP versus P, as well as increased dp/dtmax and dp/dtmin versus R. These results provide evidence of an interaction between normalization of energy metabolism and improvement in cardiac function due to a combination of ACE inhibition and beta blockade after myocardial infarction.

Contractile function of rat myocardium is less susceptible to hypoxia/reoxygenation after acute infarction

In this study we tested the hypothesis that induction of heat shock proteins (HSPs) and antioxidant enzymes is a compensatory mechanism, which preserves the contractility of the surviving myocardium after acute myocardial infarction. For this purpose, mechanical function of isolated rat papillary muscles was tested 15 h after experimental myocardial infarction and sham operation, respectively. Contractility of the preparations was compared to the expression of HSP25, HSP72, and glutathione peroxidase activity (GSH-Px) at normoxia and during hypoxia/reoxygenation. At normoxic conditions, rates of isometric contraction and, in particular, of relaxation were significantly higher after acute myocardial infarction than after sham operation. Improved relaxation rates were reflected in 2- to 3-fold higher heat shock protein levels in papillary muscles from rats with myocardial infarction compared to sham operated animals. During hypoxia/reoxygenation, the rates of contraction and relaxation were better preserved after myocardial infarction than after sham surgery. Recovery of relaxation rates during reoxygenation was associated with increased HSP25 levels and enhanced GSH-Px activity after myocardial infarction. In conclusion, heat shock proteins exert a beneficial effect on cardiac muscle relaxation after acute myocardial infarction. Enhanced heat shock protein expression and GSH-Px activity may protect the contractile function of the surviving myocardium against the damaging influence of hypoxia/reoxygenation during the early post-infarct period.

Effects of metoprolol and ramipril on action potentials after myocardial infarction in rats

The effects of chronic treatment with the beta-adrenoceptor antagonist metoprolol, the angiotensin converting enzyme inhibitor ramipril, their combination, or placebo on action potential configuration 6 weeks after myocardial infarction in rats were studied. Action potentials were measured in isolated left ventricular posterior papillary muscles and compared with action potentials from a sham operated group without infarction. After infarction, the action potential amplitude was reduced and this phenomenon was partially reversed by metoprolol- and ramipril-treatment. Prolonged repolarisation after infarction compared to sham operated animals was additionally delayed after metoprolol treatment. Thus, metoprolol extends the refractory period, which may counteract tachyarrhythmia.

Influence of Transgenic Expression of Sarcoplasmic Reticulum Ca2+ATPase on Reticular Ca2+ Transport in Rat Hearts

Cardiac relaxation partially depends on the expression of the sarcoplasmic reticulum (SR) Ca2+-ATPase SERCA2a. To evaluate the impact of SERCA2a overexpression on cardiac SR Ca2+ handling under normal and pathological conditions we generated a new transgenic rats model expressing a human cytomegalovirus enhancer/chicken β-actin promotor-controlled rat SERCA2a transgene. Characterization of a heterozygous transgenic rat line (L1167) showed that the steady-state SERCA2 mRNA and protein levels increased by +69% and +25%, respectively, relative to wild-type rats. The levels of mRNA encoded by some of the other genes involved in cardiac Ca2+ control, such as phospholamban and Na+/Ca2+ exchanger, remained unchanged. Functional analysis of SR Ca2+ handling in isolated membranes in the presence of the synthetic protein kinase A inhibitor peptide [PKI (6–22)amide] indicated that the rate of oxalate-supported Ca2+ uptake was increased in average by 49% at free Ca2+ concentrations ranging from 0.5 to 3.7 μM if compared to wild-type controls. The sensitivity of uptake to the specific SR Ca2+-ATPase inhibitor thapsigargin was similar in transgenic and wild-type animals (IC50:3.4 ± 0.7 vs. 3.8 ± 0.4 nM, respectively). Cardiac expression of the SERCA2a transgene also occurred in streptozotocin-induced diabetes mellitus and propylthiouracil-induced hypothyroidism and this rescued, at least partially, the compromised cardiac SR Ca2+ transport in these diseased conditions. At 3.7 μM free Ca2+, homogenate SR Ca2+ uptake of hypothyroid and diabetic transgenic animals was 42% and 33% higher than in respective diseased hearts of wild-type rats (p < 0.05, respectively). Our results suggest that transgenic rats overexpressing SERCA2a can serve as a valid model for further evaluation concerning the possible therapeutic impact of specifically targeting gene expression of the SR Ca2+-ATPase under pathological conditions with compro- mised cardiac SR Ca2+ transport.

Einfluß der Bindungskonstanten des Ca2+-TnC-Komplexes auf die Ca2+-Dynamik in der Herzmuskelzelle

EINLEITUNG Die Dynamik der intrazellulären Ca-Konzentration (Cai(t)) in der Herzmuskelzelle ergibt sich aus der Bilanz des Zusammenwirkens der Ca+-Flüsse (Jj(t)) zwischen den intraund extrazellulären Ca+-Speichern. In Abhängigkeit von Cai(t) aktivieren Ca-lonen die Aktin-Myosin-Querbrücken im Sarkomer und damit die mechanische Spannung, die als zellulärer Ca-lndikator in der Myokardzelle dient. Dabei werden Ca-lonen mit Troponin-C (TnC) komplexiert, so daß sie in der intrazellulären Bilanz fehlen und den zeitlichen Verlauf von Cai(t) verändern (1). Die Ca^-TnC-Komplexbildung wird u.a. durch die zugehörige Bindungskonstante bestimmt, die physiologisch und pharmakologisch verändert werden kann (2). Analysiert man den Verlauf von Cai(t) anhand gemessener Ca*-lndikator-Fluoreszenzsignale (Caf(t)) ist der Einfluß unterschiedlicher Bindungskonstanten des Ca^-TnC-Komplexes zu berücksichtigen. Mittels eines dafür modifizierten Ca*-Kompartiment-Modells wird der Einfluß der Bindungskonstante auf alle Ca*-Konzentrationen der Kompartimente Caj(t) simuliert (3). Neue Ca+Indikatoren, die sich z.B. hochselektiv in zelluläre Kompartimente einlagern, könnten eine genaue experimentelle Prüfung ermöglichen (4).