How Important Is SERCA In Neuronal Calcium Homeostasis?

Abstract

Maintaining intracellular calcium homeostasis is critical for excitation‐contraction coupling in the heart, and is essentially dependent on primary and secondary active transport systems. However, upregulating sarcoendoplasmic reticulum calcium ATPase (SERCA) in cardiomyocytes from patients with heart failure has recently proved inconclusive (CUPID2 trial). Neuronal calcium impairment is also seen in cardiovascular diseases resulting in heightened sympathetic responsiveness that further exacerbate the disease phenotype. We tested whether overexpression of SERCA in cardiac sympathetic neurons could modulate intracellular calcium transients. Isolated neurons were incubated with either Ad‐MCherry‐SERCA2A or Ad‐MCherry‐Empty (control) for 24 hours immediately post isolation. Calcium imaging using fura‐2AM (2.5μM) was carried out three days post transfection. SERCA2A transgene neurons showed greater depolarisation induced intracellular calcium transients, compared to control (Δ 380/340 ratio (au): SERCA 0.75±0.03, n=68; Empty 0.64±0.03, n=57. P<0.01). SERCA2A expressing neurons also showed greater SR calcium storage compared to control, as demonstrated by significantly higher calcium responses to caffeine (10mM: Δ 380/340 ratio (au): SERCA 0.15±0.01, n=45; Empty 0.03±0.01, n=35. P<0.0001) and thapsigargin stimulation (1μM: Δ 380/340 ratio (au): SERCA 0.12±0.02, n=42; Empty 0.03±0.01, n=33. P<0.0001). Additionally mitochondrial calcium uncoupling with FCCP (1μM) resulted in a greater intracellular calcium change in the SERCA2A transgenic neurons (Δ 380/340 ratio (au): SERCA 0.13±0.01, n=22; Empty 0.05±0.01, n=22. P<0.0001), indicating that altering neuronal calcium handling with upregulation of SERCA has also effected mitochondrial calcium handling and potentially metabolic function in these neurons. Within this study we demonstrated that a SERCA2A gene construct can be transfected in isolated sympathetic neurons from the stellate ganglia. Moreover, this transduction caused an increase in intracellular calcium transients that would be expected to facilitate neurotransmission and be potentially deleterious for cardiac function.Support or Funding InformationBritish Heart Foundation