Fig 1: MiR-208a suppresses PDE4D in adult ventricular myocytes.(A) Two sites of PDE4D 3' UTR sequence alignment are shown across species highlighting the seed match sequences of PDE4D 3'UTR (red) complementary to the seed sequences of miR-208a. Expression of PDE4D as assessed by immunohistochemistry (B–D). Significant reduction of total PDE4D staining was observed using a PDE4D specific antibody both in the cytoplasm and nucleus of miR-208a transduced but not in control myocytes (B–D). Sarcomeric actinin (green) shows the presence of the myocytes that were also stained by PDE4D (D; small box green for actinin). The images were taken with 40x objective confocal microscopy and bar shows scale of 50 µm. Western blots show that PDE4D was reduced significantly in miR-208a transduced but not in untreated or miR-208a mutant myocytes (E,F). Effects of pharmacologic inhibition of miR-208a via miR-208a power inhibitor that increased PDE4D content (G,H). Note that the Western blot in G was run longer resulting in distinct band separation versus E. Acute suppression of miR-208a in adult ventricular cardiac myocytes by antimiR-208a (I,J). Data are shown as mean +/- SEM, n = 3 *P < 0.05.
Fig 2: PDE4D inhibition exacerbates PKA phosphorylation of tau in vitro. (A) Representative blot of three replicates from one cortical neuron preparation. Control (Cntrl) samples, treated with DMSO, are labeled in blue, GEBR-7b samples are in purple, FSK samples are in orange, FSK + GEBR samples are in red. pS214-tau and total tau (tau46) were blotted on the same membrane so no loading control was used. (B) Quantification of normalized pS214-tau across three independent neuronal preparations, four experimental replicates per preparation (N = 12) are shown for each condition. Means for each group were compared using a Tukey’s multiple comparison test. The significance based on adjusted p-value is shown: Cntrl vs. GEBR p = 0.1429, Cntrl vs. FSK ****p < 0.0001, Cntrl vs. GEBR + FSK ****p < 0.001, FSK vs. GEBR + FSK **p = 0.0017. Bar graph represents mean and SEM.
Fig 3: PDE4D inversely correlates with elevated pS214-tau in FC in an extreme aged cohort of rats. (A) Levels of pS214-tau normalized to total tau in total lysate were compared between young (blue dots) and aged (red diagonal lines) animals in the cognitively characterized cohort. No significant difference between young and aged animals was found using an unpaired t-test (p = 0.3069). (B) Levels of normalized pS214-tau were compared between young (blue horizontal lines) and aged (red vertical lines) animals in the extreme age cohort. A significant difference was detected by an unpaired t-test (*p = 0.0308). Bar graph represents mean and SEM. (C) Levels of normalized pS214-tau were plotted against levels of PDE4D in the extreme age cohort. The relationship between the two markers was fit by linear regression utilizing Pearson Correlation. The slope and correlation coefficient are shown in the box in the bottom right of the graph. The slope was significantly non-zero (*p = 0.019). The average age of each cohort is displayed beneath graphs (A,B). (D) Expression of pS214-tau in young rat mPFC layer II/III. Immunolabeling was virtually absent within neurons and the neuropil. (E) Expression of pS214-tau in aged rat mPFC layer II/III. There was a marked elevation in the density and intensity of pS214-tau immunopositive neurons. Scale bar, 100 µm.
Fig 4: Working model: miR-208a titration and contractile function.Cardiac expression of miR208a suppresses PDE4D through engagement of the seed sequence of miR-208a to the seed-match sequence of PDE4D 3' UTR. MiR-208a also up-regulates other miRs targeting PDE4D. Suppression of PDE4D by miR-208a and other miRs promotes increased phosphorylation of cTnI and PLN thereby enhancing cardiac function (A, Green). Baseline contractility is depicted in red (A). Suppression of regulatory subunit of PKA (PRKAR1a) by miR-208a leads to active catalytic subunit (PRKCA) and PKA activation (B). This concerted amplification of PKA signaling in turn increases phosphorylation of cTnI and PLN thereby enhancing cardiac function (A, green and B). The dotted line indicates indirect regulation.
Fig 5: Advanced age highlights loss of PDE4D particularly from total lysate in rat FC. (A) Schematic demonstrating the process from crude total lysate to triton-soluble lysate and pellet. (B) Levels of total PDE4D normalized to GAPDH were compared between young (blue, N = 10) and aged (red, N = 10) animals. An unpaired t-test showed a significant difference between the two age groups (****p < 0.0001). Bar graph represents mean and SEM (C) Quantification of triton-soluble PDE4D normalized to GAPDH in an extreme age cohort of young and aged animals. Young animals in blue (N = 10) were compared to aged animals in red (N = 10) via a Mann-Whitney test (*p = 0.0232). Bar graph represents mean and SEM (D) PDE4D was detected in the pellet created by the centrifugation step between total lysate and triton-soluble lysate. Comparison of PDE4D in the pellet between young and animals was conducted via an unpaired t-test and revealed no difference (p = 0.7026). Variation within groups is noted by the SEM depicted with each bar graph. The average age of each cohort is displayed beneath graphs (B–D). (E) Expression of PDE4D in young rat mPFC layer II/III. Pyramidal neurons in layer II/III were immunolabeled against PDE4D, especially with staining being found in the pyramidal somata and delicate labeling in the neuropil visualized in the higher-magnification panel (right). (F) Expression of PDE4D in aged rat mPFC layer II/III. There was a marked decrement in the density of PDE4D-immunopositive neurons in aged rat mPFC layer II/III observed in the higher-magnification panel (right). Scale bar, 100 µm.
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