Sarcoplasmic reticulum Ca2+ dynamics in aging Drosophila and correlation with sarcopenia

Abstract

Aging still remains a mystery of biology and one of the most affected tissues in aging is skeletal muscle, whose loss of muscle mass and strength is called sarcopenia. Age-dependent sarcopenia is not restricted to mammals, as it affects other animal species including nematodes or flies. Cytosolic Ca2+ ion is the intracellular second messenger that triggers muscle contraction. The sarcoplasmic reticulum is the store of Ca2+ in the muscle cell, and it releases Ca2+ to the cytosol when muscle contracts. Sarcopenia has been linked to the loss of Ca2+ homeostasis that trigger muscle contraction, but mechanistic details remain unsolved. Here we explore the hypothesis that an alteration of the Ca2+ content within the sarcoplasmic reticulum (SR) is at the origin of this loss of Ca2+ homeostasis observed in sarcopenia. For investigating this hypothesis, we generated transgenic flies that express the ratiometric low affinity Ca2+ indicator GAP3 targeted to the muscle sarcoplasmic reticulum (erGAP3), and we developed a new method to calibrate erGAP3 fluorescent signals into SR/ER Ca2+ concentrations ([Ca2+]SR/ER). With these tools we measured resting [Ca2+]SR in vivo along the fly life, and found a progressive decrease with aging that results in a tenfold reduction in the [Ca2+]SR in the oldest flies. Then, to explore the molecular mechanisms involved in this decrease of [Ca2+]SR we studied the expression levels of the main proteins involved in [Ca2+]SR resting levels. In old muscle, we found a slight non-significant increase in the ryanodine receptors (RyR) and in the immunoglobulin protein (BiP) expression whereas the expression of the sarco/endoplasmic reticulum Ca2+- ATPase (SERCA) decreased by 35%. Moreover, the loss of function of the skeletal muscle was monitored by the well-characterized climbing assay, and found a strong correlation between the Ca2+ content of the sarcoplasmic reticulum and fly climbing ability with aging. Furthermore, to assess whether the reduction of [Ca2+]SR content in the aged flies also affected the [Ca2+]C transients, we studied the cytosolic Ca2+ dynamics during muscle contraction in transgenic flies expressing the cytosolic Ca2+ sensor GCaMP in the muscle tissue. This experiments showed that old flies released less Ca2+ to the cytosol in comparison to young flies and, thus, these results validated those obtained in the SR. In order to investigate whether the reduction of SR Ca2+ content observed in muscle was a universal phenomenon of aging that occurred also in other tissues we studied the progression of [Ca2+]ER in brain neurons and in the peripheral sensory wing neurons using the pan neuronal transgenic line, which expresses erGAP3 in all types of neurons. The [Ca2+]ER of the brain neurons did not change significantly with age, and remained stable along the whole fly life. However, the behaviour is different in other neurons as we can also appreciate a decrease in the [Ca2+]ER of the sensory wing neurons, similar to what occurs in the skeletal muscle. Regarding the key molecular players, in contrast to the muscle, SERCA levels remained unchanged in brain neurons whereas BiP and RyR levels are increased in the aging brain.