Role of defective calcium regulation in cardiorespiratory dysfunction in Huntington’s disease

Huntington’s disease (HD) is a progressive, autosomal dominant neurodegenerative disorder affecting striatal neurons beginning in young adults with loss of muscle coordination and cognitive decline. Less appreciated is the fact that patients with HD also exhibit cardiac and respiratory dysfunction, including pulmonary insufficiency and cardiac arrhythmias. The underlying mechanism for these symptoms is poorly understood. In the present study we provide insight into the cause of cardiorespiratory dysfunction in HD and identify a potentially novel therapeutic target. We now show that intracellular calcium (Ca2+) leak via posttranslationally modified ryanodine receptor/intracellular calcium release (RyR) channels plays an important role in HD pathology. RyR channels were oxidized, PKA phosphorylated, and leaky in brain, heart, and diaphragm both in patients with HD and in a murine model of HD (Q175). HD mice (Q175) with endoplasmic reticulum Ca2+ leak exhibited cognitive dysfunction, decreased parasympathetic tone associated with cardiac arrhythmias, and reduced diaphragmatic contractile function resulting in impaired respiratory function. Defects in cognitive, motor, and respiratory functions were ameliorated by treatment with a novel Rycal small-molecule drug (S107) that fixes leaky RyR. Thus, leaky RyRs likely play a role in neuronal, cardiac, and diaphragmatic pathophysiology in HD, and RyRs are a potential novel therapeutic target.


WT
Q175 Q175+S107 Hind limb clasping (0-3)  immunoprecipitated from whole brain lysates (band intensity were normalized to total RyR2). The blots show RyR2 phosphorylation, oxidation, nitrosylation, and calstabin2/RyR2 association in WT (n=4), R6/1 (n=4) and R6/1 mice treated with S107 (n=4   Speckle Tracking Echocardiography analysis. Global and regional left ventricular dynamics were assessed by speckle tracking analysis from B-mode cine loops acquired during the echocardiographic sessions. Parasternal long axis views were used for the evaluation of LV longitudinal strain. Strain analysis was conducted by the same trained blinded investigator as previously described 3. ROS production: Mice brains were placed in isolation medium (containing 225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, pH 7.4) and homogenized using a glass-teflon homogenizer. The tissue homogenate was centrifuged 5minx1000g and 10minx10,000g.
Mitochondrial pellet was suspended in isolation medium and total mitochondrial protein was determined using Bradford method. Mitochondrial ROS production were measured using DCF-DA method as previously described 4 and according to manufacture instructions.
Mitochondria were incubated for 1hr at 37C with 20 μM H2DCF-DA (Thermo Fisher scientific) which is activated by ROS to generate a highly fluorescent 2′7′-dichlorofluorescein (DCF) molecule. DCF fluorescence was measured at 490/528 nm during 80min using a multimode plate reader.

Mass spectrometry analysis
Brain tissues collected from treated Q175 mice were extracted with 500ul MeOH/CAN/H2 2:2:1 (vol/vol/vol). The samples were then vortexed for 30s. For protein precipitation, the samples were sonicated for 15min and incubated 1h at -20C, then centrifuged 15min at 13,000 r.p.m and 4C. The resulting supernatant was removed and evaporated to dryness in a vaccum concentrator. Dry extracts were then reconstituted in 100 ul of CAN/H2O 1:1 (vol/vol), sonoicated 15min and centrifuged 15min at 13,000 r.p.m and 4C to remove insoluble debris.
Supernatants were transferred to HPLC vials and transferred to the mass spectrometry for small molecule analysis as previously described 5.