Contrary to what has long been believed, the role of the sympathetic nervous system in muscle tissue goes far beyond controlling blood flow by contracting or relaxing blood vessels, according to studies conducted at the University of São Paulo (USP) in Brazil.
With support from FAPESP and the collaboration of researchers at Mannheim University and Heidelberg University in Germany, a group of Brazilian researchers led by Isis do Carmo Kettelhut and Luiz Carlos Carvalho Navegantes at the University of São Paulo’s Ribeirão Preto Medical School (FMRP–USP) have demonstrated the importance of sympathetic innervation for the growth and maintenance of muscle mass and also for the control of movement.
“The findings contribute to a better understanding of skeletal muscle physiology,” co-author Navegantes told Agência FAPESP. “They also have implications for the treatment of neuromuscular diseases such as myasthenic syndromes.”
The researchers set out to understand how the nervous system regulates the expression of proteins in skeletal muscle tissue 23 years ago, when Navegantes joined Kettelhut and Professor Renato Migliorini at FMRP-USP’s Metabolism Control Laboratory.
In a series of studies published between 2000 and 2014, the group unveiled the anabolic role played in muscle protein metabolism by this autonomic innervation, which also controls functions such as heartbeat, bronchial dilation and gut motility.
These results had no parallel in the literature and attracted the interest of Rüdiger Rudolf and his group in Germany. Through anagreement between FAPESP and DFG, the German Research Foundation, a collaborative project began in 2012.
“Thanks to our partnership with the German group, we were able to confirm our hypothesis that sympathetic innervation was present and fundamentally active in the motor endplate or neuromuscular junction, the tissue region that generates muscle contraction. We also showed that these nerves help motor innervation maintain endplate structure and control contractions,” Navegantes said.
“Our Brazilian colleagues contributed their vast experience in protein metabolism and experimental models of sympathetic function activation and blocking, as well as biochemical analysis of sympathetic activity in skeletal muscle. We contributed in vivo imaging know-how using biosensors and other techniques and our experience in the physiopathology of neuromuscular junctions. It’s been a perfect match, and the interaction has been highly enjoyable,” Rudolf told Agência FAPESP.
Action mechanism explained
The group first used immunohistochemistry to confirm the presence of sympathetic innervation in the motor endplate. Immunohistochemistry is a technique that consists of using antibodies against specific proteins to locate these molecules and view them under a microscope.
Next they used FRET (Förster resonance energy transfer) in living animals under a microscope to examine the presence and dynamics of the neurotransmitters and receptors involved in the transmission of signals from sympathetic nerves to muscles.
“We observed the presence of β2-adrenergic receptors in the motor endplate. These receptors are activated by noradrenaline, the main neurotransmitter released by sympathetic innervation. Previously, these receptors were thought to be present only in the blood vessels that irrigate muscles and in the membranes of muscle fibers,” Navegantes said.
Images produced with the aid of fluorescent biosensors showed that when the noradrenaline released by sympathetic nerves binds to β2-adrenergic receptors, a second messenger called cyclic adenosine monophosphate (cAMP) is released in the motor endplate. “The presence of cAMP proves that sympathetic innervation is not just present in the endplate but also active,” Navegantes explained.
Based on data in the scientific literature, the researchers defend the theory that an increase in the concentration of cAMP tends to enhance the stability of cholinergic receptors, which recognize the neurotransmitter acetylcholine released by motor innervation. Thus, sympathetic innervation indirectly helps motor innervation control muscle contraction.
“There are accounts in the literature that cholinergic receptors become more stable when drugs that increase the concentration of cAMP in the motor endplate are administered in vitro,” Navegantes said. “However, it was unclear how regulation occurred, i.e., what induced the increase in this second messenger in the motor endplate. We’re suggesting that this extracellular signal is noradrenaline released by sympathetic innervation.”
The next step was to investigate what happened to mice when sympathetic innervation was eliminated from muscle tissue via denervation. To this end, the researchers treated the mice with a neurotoxin (6-hydroxydopamine) that destroyed sympathetic neurons selectively, i.e., without interfering with motor innervation or other cells.
“After this intervention, we observed that the mice displayed deficient contractile activity. Using immunohistochemistry, we found that both the size and shape of the motor endplate were totally altered. The number of cholinergic receptors was reduced by 57%. These findings suggest sympathetic innervation is essential to motor endplate maintenance,” Navegantes said.
In an attempt to reverse the dysfunction caused by the neurotoxin, the researchers treated the mice with a sympathomimetic drug whose structure is very similar to that of the noradrenaline released by sympathetic innervation.
“This drug has an affinity with β2-adrenergic receptors, which we had already shown to be present in the motor endplate, so in this way, we simulated the natural release of noradrenaline by sympathetic innervation. We observed an improvement in endplate structure and morphology,” Navegantes said.
In the next experiment, the group tested the same treatment in an animal model of myasthenic syndrome, in which mice were genetically modified to develop a condition similar to the syndrome in human patients. In these cases, both motor endplate and cholinergic receptors are altered, leading to deficient muscle contraction.
“Before the treatment, motor endplates in the genetically modified group were 44% smaller than in the control group. After administration of the sympathomimetic, the difference fell to 6%, and locomotive activity improved,” Navegantes said.
Although the beneficial effects of sympathomimetic drugs in the treatment of myasthenic disorders are well known, he explained, the risk of adverse side effects limits their use. They are considered anabolics, and one of the possible side effects is cardiac hypertrophy, which can lead to heart failure.
However, now that the mechanism of action of these drugs has been elucidated, it is possible that novel substances to increase the concentration of cAMP in muscle cells without activating β2-adrenergic receptors could be tested.
“There are drugs approved for human use that do this and that have never been tested for the treatment of neuromuscular diseases or to combat the loss of muscle mass seen in patients with sepsis, diabetes, cancer or dystrophies. This will be our next step,” Navegantes said.