Stephan Hirt

Increased Availability and Open Probability of Single L-Type Calcium Channels From Failing Compared With Nonfailing Human Ventricle

BACKGROUND The role of the L-type calcium channel in human heart failure is unclear, on the basis of previous whole-cell recordings. METHODS AND RESULTS We investigated the properties of L-type calcium channels in left ventricular myocytes isolated from nonfailing donor hearts (n= 16 cells) or failing hearts of transplant recipients with dilated (n=9) or ischemic (n=7) cardiomyopathy. The single-channel recording technique was used (70 mmol/L Ba2+). Peak average currents were significantly enhanced in heart failure (38.2+/-9.3 fA) versus nonfailing control hearts (13.2+/-4.5 fA, P=0.02) because of an elevation of channel availability (55.9+/-6.7% versus 26.4+/-5.3%, P=0.001) and open probability within active sweeps (7.36+/-1.51% versus 3.18+/-1.33%, P=0.04). These differences closely resembled the effects of a cAMP-dependent stimulation with 8-Br-cAMP (n= 11). Kinetic analysis of the slow gating shows that channels from failing hearts remain available for a longer time, suggesting a defect in the dephosphorylation. Indeed, the phosphatase inhibitor okadaic acid was unable to stimulate channel activity in myocytes from failing hearts (n=5). Expression of calcium channel subunits was measured by Northern blot analysis. Expression of alpha1c- and beta-subunits was unaltered. Whole-cell current measurements did not reveal an increase of current density in heart failure. CONCLUSIONS Individual L-type calcium channels are fundamentally affected in severe human heart failure. This is probably important for the impairment of cardiac excitation-contraction coupling.

Expression of ten RGS proteins in human myocardium: functional characterization of an upregulation of RGS4 in heart failure

OBJECTIVE RGS proteins (regulators of G protein signalling) negatively regulate G protein function as GTPase activating proteins. By controlling heterotrimeric G proteins they may regulate myocardial hypertrophy and contractility. We investigated the expression of RGS proteins in the human heart and whether they take part in the pathophysiological changes of heart failure. METHODS AND RESULTS Using RNase protection assays (RPAs) RGS2, 3L, 3S, 4, 5 and 6 were identified in the myocardium from terminally failing human hearts with dilated (DCM, n=22) or ischemic (ICM, n=18) cardiomyopathy and from nonfailing donor hearts (NF, n=9). With reverse transcriptase polymerase chain reaction in addition mRNA of RGS1, 9, 12, 14 and 16 were detectable. Compared to NF in failing LV myocardium RGS4 mRNA and protein was upregulated 2-3-fold (mRNA, 10(-21) mol/microg+/-S.E.M.: NF: 22+/-5, DCM: 51+/-10*, ICM: 37+/-8; P<0.05 vs. DCM+ICM, *P<0.05 vs. NF, P<0.05 vs. DCM+ICM; protein, % of NF+/-S.E.M.: NF: 100+/-35, DCM 266+/-60*, ICM: 205+/-64, n=5, *P<0.05 vs. NF). In contrast, RGS2, 3L, 3S, 5, 6, and 16 protein and mRNA levels did not vary between failing and NF hearts. In order to investigate the impact of RGS4 on Gq/11 mediated signalling, PLC activity was measured in human LV membranes. Recombinant RGS4 blunted the endothelin-1 (ET-1) stimulated PLC activity. When overexpressed by adenoviral mediated gene transfer in rabbit ventricular myocytes RGS4 abolished the inotropic effect of ET-1. CONCLUSION The upregulation of RGS4 in failing human myocardium diminishes Gq/11-mediated signalling and can be involved in the desensitization of Gq/11-mediated positive inotropic effects.

Influence of Bicaval Anastomoses on Late Occurrence of Atrial Arrhythmia After Heart Transplantation

BACKGROUND The standard technique for orthotopic heart transplantation includes right and left atrial anastomoses, which potentially disturb the integrity of the donor atria. Consequently, electrophysiologic abnormalities such as atrial flutter and fibrillation may occur even late after heart transplanation. METHODS Over a 3-year period, 39 heart transplantations were performed using a standard right atrial anastomosis (group A), and 40 were done using bicaval anastomoses (group B). In each group, data of 30 consecutive patients with a minimum follow-up of 9 months were reviewed retrospectively to assess the incidence of atrial arrhythmia after hospital discharge. RESULTS Early postoperatively, there was no difference in the duration of temporary pacemaker requirement and incidence of permanent pacemaker implantation (group A, 7%; group B, 7%; not significant) between the two groups. In 12 patients in group A (40%), 16 episodes of atrial flutter and fibrillation were detected 20 to 205 days after heart transplantation. In group B, 1 patient (4%) suffered from atrial fibrillation on day 116 after the operation (p < 0.001). CONCLUSIONS Preservation of the integrity of the right donor atrium by construction of bicaval anastomoses results in a significantly decreased incidence of atrial flutter and fibrillation after heart transplantation when compared with the standard technique.

Development of obliterative bronchiolitis after allogeneic rat lung transplantation: implication of acute rejection and the time point of treatment

BACKGROUND Chronic allograft failure represents the major cause of late morbidity and mortality after solid organ transplantation. Despite the pathological and clinical changes of this disease being well-described, the etiology and the causative factors are still under discussion. Several clinical, as well experimental studies, emphasize the significance of acute rejection. In rat model of left lung allo-transplantation (F344-to-WKY) the influence of acute rejection (AR) on the development of chronic rejection (CR) was studied. METHODS In Group I (n = 25) no immunosuppression was used, while methylprednisolone (MP) (10 mg/kg) was applied in Group II (n = 20) in the early phase of AR on postoperative Days 9, 10, 11 and in Group III (n = 20) during AR on Day 14, Day 15, Day 16. The rats were sacrificed on Day 5, Day 15/20, Day 30, Day 60, Day 100 and following HE-staining the extend of AR as well CR was graded according to the working formulation of The International Society of Heart and Lung Transplantation. RESULTS In Group I, AR was found at Day 15 and Day 30 which resolved spontaneously and resulted in CR on Day 60 and Day 100. In Group II, signs of AR were less evident on Day 20, while mild AR persisted on Day 30 and Day 60. On Day 100, normal lung structure was found in all rats. The recipients of Group III showed decreased signs of AR in the early course, however, severe CR was found on Day 60 and Day 100. Extensive airway inflammation with destruction of the subepithelial layer of the smaller airways resulted in severe early obliterative bronchiolitis. CONCLUSIONS Untreated severe AR in the early course after lung transplantation results in CR in the F344-to-WKY model. Preventive treatment with MP during the early phase of AR clearly diminishes the degree of AR and the graft recovers completely without any evidence of CR. Late application of steroids during the zenith of AR is successful to control the extent of AR, however, it fails to prevent CR.

Single-channel properties of L-type calcium channels from failing human ventricle

OBJECTIVE The aim of our study was to analyse the single-channel properties of L-type calcium channels from failing human heart and to compare them to the respective animal data. Furthermore, we intended to evaluate the feasibility of future single-channel studies on the role of calcium channels in the pathophysiology of heart failure. METHODS Single L-type calcium channels were recorded in ventricular myocytes from explanted failing human heart, using the cell-attached configuration of the patch-clamp technique. RESULTS One or more successful registrations of calcium channels could be obtained in 11 of 19 cell isolations. Determination of single-channel conductance yielded a mean value of 16.6 +/- 1.2 pS (70 mM Ba2+ as the charge carrier) under control conditions and 23.7 +/- 2.8 pS in presence of the calcium-channel agonist FPL 64176. The rapid gating process could be described by a C<-->C<-->O gating scheme. Slow gating analysis revealed a highly significant clustering of active and non-active sweeps. CONCLUSION Single-channel measurements of L-type calcium channels in human failing ventricle are feasible and reproducible despite the varying patient characteristics. Their channel properties are qualitatively comparable to those found in other mammals. Whether there are quantitative differences due to the underlying heart failure can be elucidated in further studies.

C1-esterase inhibitor in graft failure after lung transplantation

Abstract Graft failure after lung transplantation may occur immediately after transplantation due to reperfusion injury or later due to rejection and infection. Although the pathological mechanisms are not completely known, the clinical findings are similar to the adult respiratory distress syndrome. In this condition, the blood coagulation contact system and the complement system are activated, leading to a capillary leak syndrome. Activation of the contact as well as the complement system is regulated by a common inhibitor, C1-esterase inhibitor (C1-INH). We report on two patients who received high doses of C1-INH for 2 days during graft failure either due to reperfusion injury immediately after transplantation or due to an acute rejection 2 months after double-lung transplantation. In both cases of graft failure, a capillary leak syndrome occurred with pleural effusions of 7 l to more than 10 l per day. In case 1 disturbance of gas exchange during severe reperfusion injury could not be treated effectively with other treatment modalities like nitric oxide ventilation or surfactant administration. With the use of C1-INH, pleural effusions reduced within 12 h, leading to normal graft function within 4 days. In the second recipient, acute rejection forced the use of extracorporeal membrane oxygenation (ECMO) within 24 h despite immunosuppressive therapy. After administration of C1-INH, pleural effusions reduced from 19 l per day to 300 ml within 3 days of treatment. ECMO was discontinued after C1-INH treatment and the patient extubated 2 weeks later. This experience indicates that C1-INH may play a role in the management of capillary leak syndrome after lung transplantation.

TOPICAL AMPHOTERICIN B APPLICATION IN SEVERE BRONCHIAL ASPERGILLOSIS AFTER LUNG TRANSPLANTATION: REPORT OF EXPERIENCES IN 3 CASES

Ulcerative tracheobronchial aspergillosis after lung transplantation (ltx) may lead to bronchial-pulmonary artery fistula that results in fatal bleeding. We report our early experience with combined systemic, aerolized and topical application of amphotericin B in 3 cases of bronchial aspergillosis after ltx. Two patients are still alive, but 1 died of bleeding from a fistula between the left upper lobe bronchus and the pulmonary artery. Aspergillosis in the second patient resolved with minimal stenosis of the left main and the left upper lobe bronchus, and the third patient developed an anastomotic stenosis that was successfully dilated.

Therapy for lung failure using nitric oxide inhalation and surfactant replacement

Nitric oxide inhalation and surfactant replacement therapy are relatively new concepts in the treatment of respiratory failure due to hypoxia and reperfusion injury after lung transplantation. We report on a patient in whom reperfusion injury of the lung developed after resuscitation and implantation of a biventricular assist device for sudden cardiac arrest. Lung failure developed within 12 hours after implantation of the biventricular assist device. Lung function was reestablished using combined therapy of nitric oxide and surfactant. Heart transplantation was performed successfully thereafter. This case indicates the potential role of a combined therapy of nitric oxide and surfactant in acute hypoxic lung failure.

Detection of diastolic dysfunction: acoustic quantification (AQ) in comparison to Doppler echocardiography

Objectives. To evaluate the potential of acoustic quantification (AQ) in detection of diastolic dysfunction in comparison to Doppler analysis, we investigated, as a model of restrictive filling pattern, nonrejecting heart transplant recipients early postoperatively. Background. AQ, an ultrasonic backscatter imaging system, enables inst-antaneous calculation of cavity areas and thus provides a new approach to diastolic function. Methods. Of 27 pts who have undergone heart transplantation, echocardiography has been performed at the day of biopsy. During a time course of 8 weeks echocardiographic data have been analysed at 3 different time points (early, mid and late) in 16 nonrejecting pts. Indexes of the area-change waveform and its 1. derivative (dA/dt) obtained by AQ were opposed to usual Doppler indexes. Results. In comparing data of the early and late time point of investigation, significant changes of early diastolic filling were detectable by AQ as well as by Doppler: End-diastolic areas have increased (p<0.001), while peak filling rate (p<0.0001), slope of area change during rapid filling (p<0.001) and amount of relative area change during rapid filling (p<0.001) have decreased. Complementary, Doppler derived pressure half-time (p<0.0001) and isovolumic relaxation time (p<0.0001) have increased while the peak early filling velocity (p<0.0001) and its time velocity integral (p<0.001) have decreased. Conclusion. An initial restrictive filling pattern has improved 8 weeks postoperatively. Since multiple indexes, obtained from the area change waveforms, in particular the for end-diastolic area normalized peak filling rate, seem to be highly sensitive in detecting changes of diastolic function, AQ may play an important complementary role in non-invasive evaluation of restrictive filling pattern.

Dantrolene sodium improves the force–frequency relationship and β-adrenergic responsiveness in failing human myocardium

Failing human myocardium is characterized by a negative force–frequency relationship and impaired β‐adrenergic responsiveness which have been related to alterations of the intracellular Ca2 homeostasis. Dantrolene sodium is a clinically used drug that modulates myocardial Ca2 i handling in animal models. This study investigated the effects of dantrolene sodium on intracellular Ca2 handling and contractile function in failing human myocardium.