Nanoscale organization of cardiac calcium channels is dependent on thyroid hormone status.

Thyroid hormone dysfunction is frequently observed in patients with chronic illnesses including heart failure which increases risk of adverse events. This study examined effects of thyroid hormones (TH) on cardiac T-tubule (TT) integrity, Ca2+ sparks, and nanoscale organization of ion channels in excitation-contraction (EC)-coupling, including L-type calcium channel (Cav1.2), ryanodine receptor-type 2 (RyR2), and junctophilin-2 (Jph2). TH deficiency was established in adult female rats by propyl-thiouracil (PTU) ingestion for 8 weeks; followed by randomization to continued PTU without or with oral triiodo-L-thyronine (T3; 10 ug/kg/d) for two additional weeks (PTU+T3). Confocal microscopy of isolated cardiomyocytes (CM) showed significant misalignment of TTs, and increased Ca2+ sparks in thyroid-deficient CMs. Density-Based Spatial Clustering of Applications with Noise (DBSCAN) analysis of STochastic Optical Reconstruction Microscopy (STORM) images showed decreased (p<0.0001) RyR2 cluster number per cell area in PTU CMs compared to euthyroid (EU) control myocytes, and this was normalized by T3-treatment. Cav1.2 channels and Jph2 localized within 210 nm radius of the RyR2 clusters were significantly reduced in PTU myocytes, and these values were increased with T3 treatment. A significant percentage of the RyR2 clusters in the PTU myocytes had neither Cav1.2 or Jph2, suggesting fewer functional clusters in EC-coupling. Nearest neighbor distances between RyR2 clusters were greater (p<0.001) in PTU cells compared to EU and T3-treated CMs that corresponds to disarray of TTs at the sarcomere z-discs. These results support a regulatory role of T3 in the nanoscale organization of RyR2 clusters and co-localization of Cav1.2 and Jph2 in optimizing EC-coupling.

Aberrant hippocampal Atp8a1 levels are associated with altered synaptic strength, electrical activity, and autistic-like behavior.

Type IV ATPases are putative aminophospholipid translocases (APLTs), more commonly known as flippases. A pronounced induction of the flippase Atp8a1 was observed in post-mortem tissue homogenates from the hippocampus and temporal lobe of juvenile autistic subjects compared to age-matched controls. In order to simulate the human data, C57BL/6 mice were allowed to develop after intra-hippocampal injection of recombinant lentivirus expressing Atp8a1 at the early developmental stage of postnatal day 6 (P6). Transmission electron microscopy (TEM) analysis of the lentivirus-Atp8a1 treated (Atp8a1+) mice in adulthood revealed fewer and weaker excitatory synapses in the hippocampal CA1 region compared to mice injected with empty virus. Significant inhibition of the Schaffer collateral pathway was observed in the Atp8a1+ mice in paired-pulse recording (PPR) at 20-ms inter-stimulus interval. In the three-chambered sociability test, the Atp8a1+ mice displayed no preference for an encaged stranger mouse over a novel object, which is a characteristic autistic-like behavior. In sharp contrast, Atp8a1 (-/-) mice displayed a preference for a stranger mouse over the novel object, which is characteristic of neurotypical mouse behavior. However, similar to the Atp8a1+ mice, the Atp8a1 (-/-) mice harbored fewer and weaker excitatory synapses in CA1 compared to wild-type controls, and displayed inhibition at 20-ms inter-stimulus interval in PPR. These findings suggest that both elevated and diminished levels of Atp8a1 during early development are detrimental to brain connectivity, but only elevated Atp8a1 is associated with aberrant social behavior. Mice with augmented levels of Atp8a1 may therefore serve as a potential model in autism research.

PKC epsilon as a neonatal target to correct FXS-linked AMPA receptor translocation in the hippocampus, boost PVN oxytocin expression, and normalize adult behavior in Fmr1 knockout mice.

Fragile X Syndrome (FXS) is an inherited developmental disorder caused by the non-expression of the Fmr1 gene. FXS is associated with abnormal social and anxiety behavior that is more prominent among males. Given that oxytocin (OXT) regulates both social and anxiety behavior, we studied the effect of FXS in the hypothalamic paraventricular nucleus (PVN), the major central source of OXT. We observed a significant suppression of protein kinase C epsilon (PKCε) (34%) in the ventral hippocampal CA1 region of postnatal day-18 (P18) male Fmr1 knockout (KO) mice, which displayed social behavior deficits and hyper-anxiety in adulthood. These mice also displayed a 39% increase in cell surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) at P18 (measured by the surface level of the AMPAR subunit GluR2), thereby indicating excitation of the CA1 neurons. It is known that neuronal activation at CA1 is linked to an inhibition of the PVN neurons. As expected, these mice also displayed a 25% suppression of oxytocin+ (OXT+) cells in the PVN at P20. Stimulating PKCε during postnatal days 6-,14 (P6-14) mice using a selective activator, dicyclopropyl-linoleic acid (DCP-LA), corrected AMPAR externalization in CA1 and suppression of OXT+ cell number in PVN in a PKCε dependent manner. Most notably, neonatal DCP-LA treatment rescued social behavior deficits and hyper-anxiety, displayed by adult (≥P60) male but not female KO mice. Thus, neonatal stimulation of PKCε could be a strategy to correct endophenotypic anomalies during brain development and aberrant adult behavior of the FXS males to the wild-type levels.