Flexibility of the larval circulatory system

The circulatory system typically starts operating earlier than any other organ, but in these early stages blood flow is not yet linked to metabolic requirements of tissues. Furthermore, in these early stages control of blood flow is possible by blood born and/or local hormones, but not by the autonomic nervous system. While the formation of major blood vessels during early development appears to be driven be genetic information, in adults the vascular bed of various organs and tumors can be significantly modified by local signals. It is not known, to what extend local or environmental parameters are able to modify or remodel the vascular bed in larval animals. In order to understand how flexible and adaptable the cardiovascular system of developing larvae is we will subject zebrafish larvae to a prolonged training program in a swim tunnel in order to increase the oxygen demand of the muscle tissue. As already shown in our previous studies, this results in a significantly lower critical PO2 and an increased survival rate at low oxygen tensions. In this project we will test the hypothesis, that these observations are the result of an improved oxygen transport system, achieved either by an improved performance of the cardiac system or by an increased oxygen carrying capacity in the blood, i.e. by an increase in hematocrit. We will also test the idea that an improved capillarization of tissues contributes to the improved performance of the trained larvae at low oxygen tensions and analyze the capillarization of muscle tissue by using our digital motion analysis techniques. Pharmacological studies will demonstrate whether in trained animals an increased sensitivity or an increase in the expression of receptors inducing a vasodilation facilitates blood flow to the tissues and thus contributes to an improved oxygen supply to the tissue under hypoxic conditions. The experiments will provide insight in the functional development of the cardiovascular system and in its adaptability during larval devevlopment.

Skeletal muscle of vertebrates comprises red, intermediate and white muscle fibers, specialized for prolonged muscular work or for burst activity. For mammalian muscles it is known that appropriate training programs are able to modify the internal structure of a muscle fiber and to induce the transition from one fiber type to another. In this project we addressed the question how chronic muscular activity affects tissue differentiation during early development. Due to the transparency of the zebrafish larvae during early development cardiac activity as well as the formation of the vascular bed can be analyzed in vivo by optical methods, developed in our lab in a previous project, which makes the zebrafish a suitable model for these experiments. The results revealed that chronic swim training of zebrafish larvae resulted in a significantly improved oxygen transport system. Zebrafish larvae exposed to a water current (water velocity 5 body length/sec) for either 6 days or 11 days were able to tolerate higher water velocities than untrained larvae, and at any given water velocity had a lower oxygen consumption than untrained animals. In addition, the critical PO 2 was significantly reduced compared to untrained control larvae. At the end of the training period no adaptations in cardiac activity could be detected that could explain the improvements in the oxygen transport system present in trained fish. At the tissue level however, significant changes were observed. Especially in primarily aerobic muscle cells (i.e. red muscle fiber and intermediate muscle fiber) mitochondrial density was increased, and in animals trained between 21 days and 32 days post fertilization (dpf) capillarization of muscle tissue and of the tail fin was significantly improved. This clearly shows that chronic muscular activity induces quite complex adaptations, pertaining cell metabolism, cellularity of muscle fibers and also the vascular bed. Accordingly, even in these early developmental stages tissue differentiation is not only determined by the genetic background of the larvae, but shows some plasticity in response to changing environmental conditions. In additional experiments a method was developed to analyze hemoglobin oxygen saturation in vivo. The results showed that even at a time when convective oxygen transport by the circulatory system is not yet required for oxygen transport to the tissues hemoglobin can be partly deoxygenated. Especially at 12 dpf low saturation values were detected in the central circulatory system, which is in line with the conclusion from previous experiments that this is the time when convective oxygen transport becomes necessary to support cutaneous respiration. In the next project the molecular basis for the observed plasticity in tissue differentiation will be addressed.