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

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.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

Caffeine chelates calcium in the lumen of the endoplasmic reticulum

Producción CientíficaCytosolic Ca2+ signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca2+ channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca2+ levels. Caffeine sensitizes RyR to Ca2+ and promotes ER Ca2+ release at basal cytosolic Ca2+ levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca2+ with the low-affinity genetic Ca2+ probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca2+ content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca2+ release does not go along with the expected cytosolic Ca2+ increase. These results can be explained by Ca2+ chelation by caffeine inside the ER. Ca2+-overloaded mitochondria also display a drop of the matrix Ca2+ concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca2+ concentration (10−7 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca2+ release) and in the cytosol (increase in Ca2+ peak) at low caffeine concentrations (0.3–1 mM) that have no effects in control cells. Our results illustrate the fact that simultaneous monitoring of both cytosolic- and ER Ca2+ are necessary to understand the action of caffeine and raise concerns against the use of high concentrations of caffeine as a readout of the presence of CICR.Ministerio de Economía, Industria y Competitividad (Project BFU2017-83066-P

Use of aequorin-based indicators for monitoring Ca2+ in acidic organelles

Producción CientíficaOver the last years, there is accumulating evidence that acidic organelles can accumulate and release Ca2+ upon cell activation. Hence, reliable recording of Ca2+ dynamics in these compartments is essential for understanding the physiopathological aspects of acidic organelles. Genetically encoded Ca2+ indicators (GECIs) are valuable tools to monitor Ca2+ in specific locations, although their use in acidic compartments is challenging due to the pH sensitivity of most available fluorescent GECIs. By contrast, bioluminescent GECIs have a combination of features (marginal pH sensitivity, low background, no phototoxicity, no photobleaching, high dynamic range and tunable affinity) that render them advantageous to achieve an enhanced signal-to-noise ratio in acidic compartments. This article reviews the use of bioluminescent aequorin-based GECIs targeted to acidic compartments. A need for more measurements in highly acidic compartments is identified.Ministerio de Ciencia e Innovación (PID2020-116086RB-I00 )Junta de Castilla y León (Ref. CLU- 2019-02)Biotechnology and Biological Sciences Research Council, Reino Unido (BB/T015853/1 y BB/W01551X/1

Sarcoplasmic reticulum Ca2+ decreases with age and correlates with the decline in muscle function in Drosophila

Producción CientíficaSarcopenia, the loss of muscle mass and strength associated with age, has been linked to impairment of the cytosolic Ca2+ peak that triggers muscle contraction, but mechanistic details remain unknown. Here we explore the hypothesis that a reduction in sarcoplasmic reticulum (SR) Ca2+ concentration ([Ca2+]SR) is at the origin of this loss of Ca2+ homeostasis. We engineered Drosophila melanogaster to express the Ca2+ indicator GAP3 targeted to muscle SR, and we developed a new method to calibrate the signal into [Ca2+]SR in vivo. [Ca2+]SR fell with age from ∼600 µM to 50 µM in close correlation with muscle function, which declined monotonically when [Ca2+]SR was <400 µM. [Ca2+]SR results from the pump-leak steady state at the SR membrane. However, changes in expression of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump and of the ryanodine receptor leak were too modest to explain the large changes seen in [Ca2+]SR. Instead, these changes are compatible with increased leakiness through the ryanodine receptor as the main determinant of the [Ca2+]SR decline in aging muscle. In contrast, there were no changes in endoplasmic reticulum [Ca2+] with age in brain neurons.Ministerio de Economía, Industria y Competitividad (grant BFU2017-83066-P)Junta de Castilla y León (project GR175

Contemporary use of cefazolin for MSSA infective endocarditis: analysis of a national prospective cohort

Objectives: This study aimed to assess the real use of cefazolin for methicillin-susceptible Staphylococcus aureus (MSSA) infective endocarditis (IE) in the Spanish National Endocarditis Database (GAMES) and to compare it with antistaphylococcal penicillin (ASP). Methods: Prospective cohort study with retrospective analysis of a cohort of MSSA IE treated with cloxacillin and/or cefazolin. Outcomes assessed were relapse; intra-hospital, overall, and endocarditis-related mortality; and adverse events. Risk of renal toxicity with each treatment was evaluated separately. Results: We included 631 IE episodes caused by MSSA treated with cloxacillin and/or cefazolin. Antibiotic treatment was cloxacillin, cefazolin, or both in 537 (85%), 57 (9%), and 37 (6%) episodes, respectively. Patients treated with cefazolin had significantly higher rates of comorbidities (median Charlson Index 7, P <0.01) and previous renal failure (57.9%, P <0.01). Patients treated with cloxacillin presented higher rates of septic shock (25%, P = 0.033) and new-onset or worsening renal failure (47.3%, P = 0.024) with significantly higher rates of in-hospital mortality (38.5%, P = 0.017). One-year IE-related mortality and rate of relapses were similar between treatment groups. None of the treatments were identified as risk or protective factors. Conclusion: Our results suggest that cefazolin is a valuable option for the treatment of MSSA IE, without differences in 1-year mortality or relapses compared with cloxacillin, and might be considered equally effective