Rabea Verhaegh

Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca2+-flux and sarcoplasmic reticulum Ca2+-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca2+-concentration, trans-sarcolemmal Ca2+-flux via L-type Ca2+-channels and Na+-Ca2+-exchanger, and Ca2+-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca2+-channels by verapamil (1 μM) decreased force of contraction to 22±7% compared to baseline (n=4, p<0.05). Ni2+ was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73±3 ms vs. 101±8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca2+-release decreased force development to 54±3% in ventricular (n=13, p<0.05) and to 52±8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca2+-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169±7% force compared to baseline, n=8, p<0.05) and 10 s (198±9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca2+-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

Transient dilutional acidosis but no lactic acidosis upon cardiopulmonary bypass in patients undergoing coronary artery bypass grafting

Introduction Dilutional acidosis may result from the introduction of a large fluid volume into the patients’ systemic circulation, resulting in a considerable dilution of endogenous bicarbonate in the presence of a constant carbon dioxide partial pressure. Its significance or even existence, however, has been strongly questioned. Blood gas samples of patients operated on with standard cardiopulmonary bypass (CPB) were analyzed in order to provide further evidence for the existence of dilutional acidosis. Material and methods Between 07/2014 and 10/2014, a total of 25 consecutive patients scheduled for elective isolated coronary artery bypass grafting with CPB were enrolled in this prospective observational study. Blood gas samples taken regularly after CPB initiation were analyzed for dilutional effects and acid-base changes. Results After CPB initiation, hemoglobin concentration dropped from an average initial value of 12.8 g/dl to 8.8 g/dl. Before the beginning of CPB, the mean value of the patients’ pH and base excess (BE) value averaged 7.41 and 0.5 mEq/l, respectively. After the onset of CPB, pH and BE values significantly dropped to a mean value of 7.33 (p < 0.0001) and –3.3 mEq/l (p < 0.0001), respectively, within the first 20 min. In the following period during CPB they recovered to 7.38 and –0.5 mEq/l, respectively, on average. Patients did not show overt lactic acidosis. Conclusions The present data underline the general existence of dilutional acidosis, albeit very limited in its duration. In patients undergoing coronary artery bypass grafting it seems to be the only obvious disturbance in acid-base homeostasis during CPB.

Severe blunt muscle trauma in rats: only marginal hypoxia in the injured area.

Background After severe muscle trauma, hypoxia due to microvascular perfusion failure is generally believed to further increase local injury and to impair healing. However, detailed analysis of hypoxia at the cellular level is missing. Therefore, in the present work, spectroscopic measurements of microvascular blood flow and O2 supply were combined with immunological detection of hypoxic cells to estimate O2 conditions within the injured muscle area. Materials and Methods Severe blunt muscle trauma was induced in the right Musculus gastrocnemius of male Wistar rats by a standardized “weight-drop” device. Microvascular blood flow, relative hemoglobin amount, and hemoglobin O2 saturation were determined by laser Doppler and white-light spectroscopy. Hypoxic cells were detected by histologic evaluation of covalent binding of pimonidazole and expression of HIF-1α. Results Directly after trauma and until the end of experiment (480 minutes), microvascular blood flow and relative hemoglobin amount were clearly increased. In contrast to blood flow and relative hemoglobin amount, there was no immediate but a delayed increase of microvascular hemoglobin O2 saturation. Pimonidazole immunostaining revealed a hypoxic fraction (percentage area of pimonidazole-labelled muscle cells within the injured area) between 8 to 3%. There was almost no HIF-1α expression detectable in the muscle cells under each condition studied. Conclusions In the early phase (up to 8 hours) after severe blunt muscle trauma, the overall microvascular perfusion of the injured area and thus its O2 supply is clearly increased. This increased O2 supply is obviously sufficient to ensure normoxic (or even hyperoxic) conditions in the vast majority of the cells.

Impact of Acute Intestinal Ischemia and Reperfusion Injury on Hemodynamics and Remote Organs in a Rat Model

Background Acute mesenteric ischemia following cardiovascular surgery is a rare but fatal complication. We established a new rat model for hemodynamic monitoring during mesenteric ischemia/reperfusion (I/R) and evaluated the impact of mesenteric I/R on hemodynamics and remote organ injury. Methods Mesenteric I/R was induced in male Wistar rats by superior mesenteric artery occlusion for 90 minutes, followed by 120 minutes of reperfusion. Before I/R, ventilation and hemodynamic monitoring including mean arterial blood pressure (MAP) and cardiac output (CO) were established. During reperfusion Geloplasma (I/R + Geloplasma, N = 6) and Ringers solution (I/R + Ringer, N = 6) were titrated according to CO and compared with I/R without volume resuscitation (I/R only, N = 6) and a sham group (sham, N = 6). Blood samples were regularly taken for serum marker measurements. After reperfusion organs were harvested for histology studies. Results After acute mesenteric I/R, MAP and CO decreased (p < 0.01) while systemic and pulmonary vascular resistance increased (p < 0.01) continuously in the I/R group. Volume substitution according to CO initially stabilized hemodynamic parameters, but CO declined independently in the late stage. Compared with the I/R + Ringer group, the I/R + Geloplasma group required less volume for resuscitation (p < 0.01), experienced less metabolic acidosis. I/R groups had more organ injuries, more neutrophils sequestration, and higher creatine phosphokinase‐MB levels than sham group. Conclusion A new model for CO monitoring after mesenteric I/R injury demonstrated severe hypovolemic shock during reperfusion followed by remote myocardial and lung injury. Far less colloid volume is needed for hemodynamic stabilization after I/R compared with crystalloid volume.