Calcium Deregulation: Novel Insights to Understand Friedreich's Ataxia Pathophysiology

Abstract

Friedreich's Ataxia (FRDA) is a neurodegenerative disorder, characterized by degeneration of dorsal root ganglia, cerebellum and cardiomyopathy. Heart failure is one of the most common causes of death for FRDA patients. Deficiency of frataxin, a small mitochondrial protein, is responsible for all clinical and morphological manifestations of FRDA. The focus of our study was to investigate the unexplored Ca²⁺ homeostasis in cerebellar granule neurons (CGNs) and in cardiomyocytes of FRDA cellular models to understand the pathogenesis of degeneration. Ca²⁺ homeostasis in neurons and cardiomyocytes is not only crucial for the cellular wellbeing but more importantly to generate action potential in both neurons and cardiomyocytes. By challenging Ca²⁺ homeostasis in CGNs, and in adult and neonatal cardiomyocytes of FRDA models, we have assessed the impact of frataxin decrease on both neuronal and cardiac physiopathology. Interestingly, we have found that Ca²⁺ homeostasis is altered both cell types. CGNs showed a Ca²⁺ mishandling under depolarizing conditions and this was also reflected in the endoplasmic reticulum (ER) content. In cardiomyocytes we found that the sarcoplasmic reticulum (SR) Ca²⁺ content was pathologically reduced, and that mitochondrial Ca²⁺ uptake was impaired. This phenomenon is due to the excess of oxidative stress under FRDA like conditions and the consequent aberrant modulation of key players at the SR/ER and mitochondrial level that usually restore the Ca²⁺ homeostasis. Our findings demonstrate that in both neurons and cardiomyocytes the decreased Ca²⁺ level within the stores has a comparable detrimental impact in their physiology. In cardiomyocytes, we found that ryanodine receptors (RyRs) may be leaking and expel more Ca²⁺ out from the SR. At the same time mitochondrial uptake was altered and we found that Vitamin E can restore this defect. Moreover, Vitamin E protects from cell death induced by hypoxia-reperfusion injury, revealing novel properties of Vitamin E as potential therapeutic tool for FRDA cardiomyopathy.

Keywords: CGNs; FRDA; calcium; cardiomyocytes; oxidative stress; ryanodine receptors and mitochondrial membrane potential; sarcoplasmic reticulum.

FIGURE 1

Oxidative stress in CGNs of FRDA mouse model. (A) The picture shows the loading of 1 μM CM-H₂Xros in CGNs. (B) The graph shows the kinetic curves normalized, for Control (black) and YG8R (red). (C) The histogram represents the rates of ArbU over minutes in percentage normalized to the Control. mROS were significantly increased in YG8R CGNs compared to Control (^∗p < 0.05). (D) The picture shows CGNs loaded with 10 μM C11 BODIPY (581/591), top left bright field (bf), at 0 minutes it is shown the oxidized form (top green), the un-oxidized form (top red) and the merge; at 9 min fluorescence it is shown the oxidized form increased (bottom green), the un-oxidized form decreased (bottom red) and the merge. (E,F) Show respectively the kinetic curves and the rates the lipid peroxidation (^∗∗∗p < 0.0005). Scale bar (20 μm).

FIGURE 2

Frataxin decrease causes oxidative stress in cardiomyocytes. (A,B) HL-1 cells, Scr and FxnKD, were loaded with 10 μM dihydroethydium (Het) and imaged over time. (A) The kinetic curve show an increase of Het, meaning an increase of cytosolic ROS in FxnKD cells (red). (B) Confirmed by the rate of the dye (^∗p < 0.05). (C) Here it is shown the loading of the dye (red) and the transfected cells (green in the merge). (D,E) The kinetic curves and the histogram represent the increase of mROS measured with 1 μM CM-H2Xros. Mitochondrial ROS were significantly increased in FxnKD (^∗∗∗p < 0.0005). (F,G) We report the results of cytosolic ROS in H9c2 cells, that also showed a relevant increase in FxnKD (^∗p < 0.05). (H–J) Here we report similar mROS measurements in H9c2 cells (^∗∗p < 0.005). Scale bar (20 mm).

FIGURE 3

Failure to restore Ca²⁺ homeostasis after depolarization in YG8R CGNs. (A–C) CGNs were loaded with Fluo4-AM (A) and challenged with 30 mM KCL (B), amplitudes were measured and showed a higher response in YG8R than Control (C; ^∗p < 0.05). (D,E) CGNs response to 1 μM Thapsigargin and 1mM CaCl₂, and relative amplitudes (^∗∗∗p < 0.0005). Scale bar (20 μm).

FIGURE 4

FxnKD induces a smaller caffeine response and RyRs potentiation. (A,B) HL-1 cells FxnKD shows a smaller caffeine response compared to Scr cells. (A) Shows the kinetic of an average of cells and (B) reports the full sets of experiments combined together showing the decreased amplitude in FxnKD cells (^∗∗p < 0.005). (C,D) Highlight phase II of the Ca²⁺ response in Scr cells and in FxnKD cells. Showing an early end of the Ca²⁺ response probably ascribed to energy failure. (E,F) In H9c2 cells the diminished caffeine response in FxnKD was even more marked than HL-1 cells, however, the second phase of Ca²⁺ response was flat and the receptors saturated (^∗∗p < 0.005).

FIGURE 5

Frataxin silencing causes SR Ca²⁺ store depletion in cardiomyocytes. (A) Loading of Fura2-AM. (B,D,F) FxnKD cells show a marked difference to 1 μM thapsigargin response compared to Scr. (C–G) The amplitude of the response in relative values % shows a significant decrease in FxnKD cells which reveals a decrease of Ca²⁺ content in the SR (^∗p < 0.05; ^∗∗p < 0.005). Scale bar (20 mm).

FIGURE 6

Dantrolene prevents SR Ca²⁺ store depletion in FxnKD cardiomyocytes. (A) HL-1 cells show an increased calcium response after pre-incubation with dantrolene, an inhibitor of RyRs. (B) Comparing the response between FxnKD and Dantrolene_FxnKD we found a significant increase of the thapsigargin–induced calcium response in dantrolene pre-treated FxnKD, demonstrating that the RyRs were not inhibited (^∗p < 0.05).

FIGURE 7

FxnKD causes an abnormal energetic response to caffeine in cardiomyocytes. (A) TMRM was loaded in de-quence mode, at high concentration, where a depolarization of ΔΨ_m is seen by an increase of fluorescence. The response to caffeine depolarises the potential as the mitochondria are active taking up Ca²⁺ to restore the Ca²⁺ homeostasis of the cell. (B,C) In FxnKD cells the response is reduced revealing that the mitochondria may not take up Ca²⁺ correctly (^∗p < 0.05). Scale bar (20 μm).

FIGURE 8

Mitochondrial Ca²⁺ is altered during Thapsigargin response. During Thapsigargin response mitochondrial Ca²⁺ uptake is reduced in FxnKD cells. By pre-incubation with Vitamin E the abnormal effect induced by FxnKD was recovered during the response. However, the decay of the response was not completely restored.

FIGURE 9

Vitamin E prevents cell death after hypoxia reperfusion. FxnKD cardiomyocytes exposed to H/R revealed an enounced vulnerability as the cell death increased significantly. However, cells pre-treated with Vitamin E seemed to be protected. The numbers of TUNEL-positive cells were calculated and the apoptotic cell death was expressed as percentage (^∗∗∗p < 0.0005).