Biomedical subjects

Dirk J Beuckelmann

Publications and source records attributed to Dirk J Beuckelmann.

5 recordsLinked to original sources

PubMed2003

Dominant-negative suppression of HCN channels markedly reduces the native pacemaker current I(f) and undermines spontaneous beating of neonatal cardiomyocytes.

BACKGROUND: The pacemaker current I(f) contributes to spontaneous diastolic depolarization of cardiac autonomic cells. In heterologous expression, HCN channels exhibit a hyperpolarization-activated inward current similar to I(f). However, the links between HCN genes and native I(f) are largely inferential, and it remains unknown whether I(f) is essential for cardiac pacing. METHODS AND RESULTS: To clarify this situation, we generated a GYG(402-404)AYA pore mutation of HCN2, which rendered the channel nonfunctional and suppressed wild-type HCN2 in a dominant-negative manner in Chinese hamster ovary cells. In addition, HCN2-AYA suppressed I(HCN4) in a dominant-negative manner when coexpressed with wild-type HCN4, indicating that the 2 isoforms HCN2 and HCN4 are able to coassemble to form heteromultimeric complexes. Given that HCN2 and HCN4 are the dominant HCN mRNA transcripts in neonatal rat ventricle, we expressed HCN2-AYA in neonatal cardiocytes using adenoviral gene transfer to test the effect of HCN suppression on native I(f). I(f) density was indeed reduced markedly, from 7.8+/-1.6 pA/pF (n=13) in control cells to 0.3+/-0.2 pA/pF (n=11) in HCN2-AYA-infected cells when measured at -130 mV (P<0.001). To probe the effect of HCN on cardiac pacing, we infected spontaneously beating neonatal monolayers with adenoviral vectors expressing wild-type and mutant HCN channels. Infection with HCN2 and HCN4 accelerated the beating rate significantly, to 230.5+/-8.6 bpm (n=12) and 223.5+/-12.3 bpm (n=10), respectively, compared with control cultures (83.4+/-4.5 bpm, n=13, P<0.001). Conversely, HCN2-AYA completely undermined spontaneous pacing of neonatal cardiocytes. CONCLUSIONS: HCN channels are the major molecular component of native I(f) and are critical for spontaneous beating of neonatal cardiomyocytes.

Adenoviridae↗

PubMed2002

Identification of gating modes in single native Na+ channels from human atrium and ventricle.

The aim of the present study was to investigate the single-channel properties of different gating modes in the native human cardiac Na+ channel. Patch-clamp experiments were performed at low noise using ultrathick-walled pipettes. In 17 cell-attached patches containing only one channel, fast back and forth switching between five different Na+-channel gating modes (F-mode, M1-mode, M2-mode, S-mode, and P-mode) was identified, but no difference in the gating properties was found between normal and diseased cardiomyocytes from atrium or ventricle, respectively. Hodgkin-Huxley fits to the ensemble-averaged currents yielded the activation-time (tau(m)) and inactivation-time (tau(h)) constants. tau(m) was comparably fast in the F-mode, M1-mode, M2-mode, and S-mode (0.15 ms) and slow in the P-mode (0.3 ms). tau(h) ranged from 0.35 ms (F-mode) to 4.5 ms (S-mode and P-mode). The mean open-channel lifetime (tau(o)) was shortest in the F-mode and P-mode (0.15 ms) and longest in the S-mode (1.25 ms). The time before which half of the first channel openings occurred (t(0.5)) was comparably short in the F-mode, M1-mode, M2-mode, and S-mode (0.3 ms) and long in the P-mode (0.9 ms). It is concluded that (1) a single native human cardiac Na+ channel can be recorded at low noise, (2) this channel can change its gating properties at a time scale of milliseconds, (3) lifetimes of the observed gating modes are short ranging from milliseconds to seconds only, and (4) the gating modes are characterized by specific activation and inactivation kinetics and differ at least in their mean open time and first latency.

Atrial Function↗

PubMed2002

Role of the cardiac Na(+)/H(+)exchanger in [Ca(2+)](i)and [Na(+)](i)handling during intracellular acidosis. Effect of cariporide (Hoe 642).

Intracellular acidosis is one of the alterations occurring in cardiac ischemia and has been discussed to be important in altering excitation--contraction coupling. The aim of this study was to determine how intracellular acidosis may affect intracellular sodium and calcium handling. Cardiomyocytes were isolated from the hearts of adult male guinea-pigs by standard techniques and superfused with modified Tyrode's solution at room temperature, either HEPES buffered containing 10 mM NaHCO(3)or HEPES buffered without NaHCO(3), in order to examine a possible interaction with the sodium bicarbonate symport. The whole cell voltage clamp technique was used utilizing 3 M Omega pipettes filled with (mM): Cs aspartate 120, CsCl 20, MgCl(2)1, NaCl 5, Mg-ATP 2, HEPES 10 and either 100 microM Fura-2 or 100 microM SBFI. The pH of the pipette solution was either 7.2 or 6.5. Cells were kept at a holding potential of -80 mV and after a pre-pulse to -40 mV the membrane was continuously clamped to potentials from -30 to +80 mV in 10 mV steps. Intracellular Ca(2+)or Na(+)were estimated using the Fura-2 or SBFI technique (impermeable salt), respectively. The cardiac Na(+)/H(+)exchanger was inhibited using the Na(+)/H(+)- exchange inhibitor cariporide (Hoe 642) (1 microM), when indicated. In NaHCO(3)-free experiments we found an increase in intracellular sodium reflected by a rise in the SBFI ratio of 0.326 +/- 0.01 upon intracellular acidification, in contrast to cells perfused at pH = 7.2 (no significant increase in intracellular Na(+)) (P< 0.05). There was no difference in intracellular calcium handling between cells perfused with solutions of pH = 7.2 or 6.5 (Fura-2 Delta ratio: 0.79 +/- 0.10 vs 0.82 +/- 0.07, n.s.). The l -type calcium current also remained unchanged. Blockade of the Na(+)/H(+)exchanger by Hoe 642 had no influence on cells perfused at pH = 7.2 but inhibited the increase in intracellular Na(+)at pH = 6.5 (0.023 +/- 0.026 in the presence of Hoe 642 vs 0.326 +/- 0.01 without Hoe 642, P< 0.05) without affecting [Ca(2+)](i)or the L-type calcium current. In cells superfused with a Tyrode solution containing NaHCO(3), the increase in intracellular sodium concentration was even more pronounced. Under these conditions Hoe 642 also antagonized this increase in intracellular sodium but without reaching the control level. We conclude that under these experimental conditions intracellular acidification causes an increase in [Na(+)](i)without changing intracellular Ca(2+)or the L-type calcium current. In addition in bicarbonate-buffered systems the acidosis-induced increase in sodium is enhanced which may involve the Na(+)/HCO(3)(minus sign)symport. The effect of cariporide (Hoe 642) in intracellular acidosis seems to be based on antagonization of the rise in intracellular sodium rather than calcium in this model.

Animals↗

PubMed2002

Ca2+-handling in heart failure--a review focusing on Ca2+ sparks.

[Ca2+]i-transients have been shown to be altered in isolated ventricular myocytes from terminally failing human myocardium. It has been demonstrated that one reason for this alteration is a reduction in the Ca2+ content of the sarcoplasmic reticulum (SR). Further investigations were done to investigate, whether there may be an additional defect of the Ca2+-release mechanisms from the SR. These release mechanisms were investigated through the recording of Ca2+ sparks in single human myocytes. In cardiac myocytes, Ca2+ sparks are elementary units of Ca2+ release, which occur spontaneously, or which are triggered by Ca2+ influx through L-type Ca2+-channels (Ca2+-induced Ca2+ release). Ca2+ sparks have been investigated in various animal models of cardiac hypertrophy and cardiac failure and results were conflicting. Discrepancies may be explained by different species and also by the mechanisms underlying hypertrophy and heart failure. This review summarizes our current knowledge on Ca2+ sparks in heart failure.

Animals↗

PubMed2002

[Hormonal hyperactivity in heart failure. Differences in beta blockers].

PATHOPHYSIOLOGY: Heart failure is characterized by a disturbed contractility and activation of neurohumoral mechanisms. Activation of the adrenergic system and the beta-adrenergic signal transduction pathway leads to downregulation of beta 1-receptors of the heart muscle cell membrane. THERAPY: In addition to ACE inhibitors, diuretics and glycosides, beta blockers are an integral part in the combination therapy of patients with heart failure. Carvedilol, bisoprolol and metoprolol have been approved for use in patients with heart failure. Carvedilol is a nonselective beta-adrenoceptor antagonist with additional alpha 1-receptor-blocking properties. Furthermore, it has additional antioxidative and antiproliferative effects. Bisoprolol and metoprolol are beta 1-selective beta blockers without intrinsic sympathomimetic activity. Based on the results of the US carvedilol trials, the CIBIS-II trial, the MERIT-HF study and the COPERNICUS study, it has been shown that beta blocker therapy can improve the prognosis of patients with compensated heart failure (NYHA II and III) and carvedilol can improve prognosis in severe heart failure (stage IV). Induction of therapy must be performed using very low doses, that can be increased in 2- to 3-week intervals. Drug dosages should be increased as much as possible. Besides a better prognosis, clinical symptoms and left ventricular ejection fraction can be expected to improve. The final effect can be expected only after months up to 1 year. Therapy should continue lifelong. CONCLUSION: In summary, beta blocker therapy of congestive heart failure has been established as an integral part in the combination therapy of patients with all clinical stages of heart failure.

Adrenergic beta-Antagonists↗