Altered Heart Rates Precede Death.

<p>A. DS mice exhibit significant QT prolongation (50 - 90%). B. Heart rates in DS mice decrease 100 min before death, followed by a sharp increase just prior to the terminal event, while the WT heart rates remains high and constant. (100 minutes = 10:16 PM in WT-1 and DS-1; 7:46 PM in WT-2 and DS-2). C. WT-3 and DS-3 HR cycling, followed by DS exhibiting sudden drops in heart rate in the 72 h preceding death. D and E. Increased R-R variability 60 min prior to SUDEP in DS-1 (blue) and DS-2 (red), respectively, with further increased variability immediately preceding the lethal arrhythmia, while 1 day prior at the same time the R-R interval was constant (black). F. Progressive bradycardia and increased R-R variability in DS-3 at several time points preceding an agonal state and euthanasia (denoted by colored arrows in C).</p

mScn5a and Nav1.5 levels are unchanged in DS mutant hearts.

<p>A. Heart RNA from biological replicates (DS mice, n = 4; WT mice, n = 5) were used to generate two independent cDNAs per animal. The cDNAs were assayed using qPCR in quadruplicate with two independent <i>Scn5a</i> TaqMan primer sets and normalized to 18s RNA. B. Western blots of membrane proteins isolated from DS and WT ventricular CMs. 50 µg of protein was loaded in each lane, and probed with anti-Na_v1.5 (Mohler 1:1000), and anti-α-actin (Sigma 1:500), which served as the loading control. C. Quantification of Na_v1.5 expression normalized to α-actin expression.</p

Isolation of TTX-R and TTX-S I_Na Biophysical Properties.

<p>A. Boltzman curves for the voltage dependence of I_Na availability and conductance for the total cardiac I_Na (TTX-S + TTX-R I_Na; reproduction of the curve-fits from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077843#pone-0077843-g001" target="_blank">Figure 1C</a>). In both WT and DS myocytes the V_½ values of TTX-R I_Na (closed circles, following blockade of TTX-S I_Na with 100 nM TTX) and TTX-S I_Na (open circles, defined as total I_Na minus TTX-R I_Na) are plotted. Pharmacological separation of TTX-S and TTX-R I_Na was confirmed by the loss of difference in the V_½ values between WT vs DS, and the development of a significant difference between the TTX-S vs. TTX-R V ½ values for I_Na availability and conductance. B. Zoom-in of the boxed region in A.</p

Cardiac Arrhythmias Precede SUDEP in DS.

<p>Lead II ECG traces illustrating cardiac arrhythmias preceding death. A-C. In mouse DS-2, muscle artifact consistent with convulsive seizures was preceded by idioventricular rhythms, including premature ventricular complexes (PVCs), bundle branch block (BBB), altered QRS morphology, and R-R variability. D and E. Initiation of high frequency electrical activity without any discernible sinus activity, consistent with VF. F. Low amplitude wide complex focal bradycardia with a BBB morphology, and eventual asystole. </p

Dominant Frequency Analysis.

<p><i>A. Sinus</i> rhythm 1 day prior to SUDEP, which is consistent with heart rate (728 bpm) analysis. B. Muscle artifact embedded in the sinus ECG (same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077843#pone-0077843-g009" target="_blank">Figure 9 C</a>) without any clear frequency peaks. C. High frequency electrical activity without any discernible sinus activity, consistent with VF (~25 Hz, same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077843#pone-0077843-g009" target="_blank">Figure 9, D and E</a>). D. PTZ induced seizures lead to a lower frequency electrical signal (~10 - 20 Hz). <i>Inset</i>: Representative snapshots of the ECG signal included in the fast-fourier transformation.</p

Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence

Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux and a massive enlargement of lysosomes. Collectively, this leads to various physiological defects, including embryonic lethality, neurodegeneration and overt inflammation. The reasons for such drastic lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF, enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition, and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.</p

DS Mice Have Altered Cardiac I_Na Properties.

<p>A. Current-voltage (I-V) relationship of transient I_Na. Peak transient I_Na density is increased 2-fold in the DS (N = 6, n = 14) vs WT cardiac myocytes (N = 8, n = 20, <i>p</i> < 0.0001). <i>Inset</i>: Representative traces from each group. B. I-V relationship for persistent I_Na (pre- minus post-30 µM TTX) also shows a 2-fold increase in peak persistent I_Na in the DS vs. WT groups. To further confirm these results we employed the P/4 method to measure the persistent I_Na, yielding similar results (-60 mV, WT, -1.72 ± 0.50; DS, -3.88 ± 0.72, N = 2, n = 5-9, <i>p</i> = 0.02). C. Leftward shift (V_½ of Boltzman fit, <i>p</i> = 0.04) in the voltage dependence of I_Na availability and conductance in the DS group. D. Similar percent change in peak transient I_Na density upon administration of 100 nM TTX in the WT and DS groups. Unpaired t-test with Welch’s correction.</p