AAV9-mediated heart-failure-inducible PP1βshRNA increased PLN phosphorylation at Ser16, reduced BNP expression, and ameliorated cardiac interstitial fibrosis.

<p><b>A</b>: Immunoblots of the key SR phosphoproteins and analysis of the phosphorylation levels by using phosphospecific antibodies in LV homogenates at 3 months after gene transfer. <b>B</b>: Summaries of the phospholylation levels of PLN at Ser16 and RyR at Ser2808. “*” indicates p<0.05 vs. the NCshRNA treated group. (n = 8 in PP1βshRNA treated group and n = 8 in NCshRNA treated group). <b>C</b>: Expression analysis of normalized BNP (nBNP) using real-time RT-PCR from the AAV9 shRNA transfected heart tissue. “*” indicates p<0.05 vs. the NCshRNA treated group. (n = 8 in the NCshRNA treated group, n = 8 in the PP1βshRNA treated group). <b>D</b>: Representative images of Heidenhain's trichrome staining in the AAV9-BNP-EmGFP-NCshRNA- andAAV9-BNP-EmGFP-PP1βshRNA treated hearts at 3 months after gene transfer. The lower graph shows the quantitative image analysis of percentage of the area of interstitial fibrosis. “*” indicates p<0.05 vs. the NCshRNA treated group. (n = 6 in NCshRNA treated group and n = 7 in PP1βshRNA treated group).</p

AAV9-mediated heart-failure-inducible PP1βshRNA improved systolic and diastolic function and inhibited LV remodeling.

<p><b>A</b>: Representative M-mode echocardiographic tracings 12 weeks after AAV9-BNP-PP1βshRNA treated MLP knockout mice compared with that of AAV9-BNP-NCshRNA treated mice. <b>B</b>: Serial cardiac function evaluated by echocardiogram after gene transfer. “*” indicates p<0.05 vs the NC group post GT after 12 weeks. “#” indicates p<0.05 vs. an age-matched wild type mouse. <b>C</b>: Representative tracing of LV pressure and dP/dt in the PP1βshRNA and NCshRNA treated groups. The scale bar indicates 200 msec. <b>D</b>: Summaries of the hemodynamic data analysis. “*” indicates p<0.05 vs. the NCshRNA treated group, (n = 7 in the PP1βshRNA treated group, n = 6 in NCshRNA treated group.).</p

Experimental designs.

<p><b>A</b>: The experiment confirming the effect of <i>in vivo</i> RNA polymerase II promoter-mediated shRNA in normal mice using AdV vectors. <b>B</b>: The experiment confirming AAV9-BNP promoter-mediated gene transfer transfection efficiency at 1 month after gene transfer in normal and MLPKO mice. <b>C</b>: The experiment assessing the effects of <i>in vivo</i> AAV9-BNP promoter-mediated heart-failure condition-specific shRNA in MLPKO mice cardiomyopathy.</p

<i>In vivo</i> adenoviral PP1βshRNA significantly augmented the Ca²⁺ transient and % sarcomere shortening in AdV-PP1βshRNA mice cardiomyocytes.

<p>Cardiomyocytes were isolated from the mouse hearts 7 days after gene transfer, followed by an analysis of sarcomere shortening and the Ca²⁺ transient using the Ionoptix system with fura-2 AM dye. Sarcomere length was calculated in a real-time manner using the SarcLen software of the Ionoptix system. <b>A</b>: Representative tracing of sarcomere shortening and Ca²⁺ transient in enzymatically isolated cardiomyocytes from the mouse heart 7 days after adenoviral transfection. <b>B</b>: Summaries of the analysis in sarcomere shortening and Ca²⁺ transients; namely, % sarcomere shortening (%SS), the maximum/minimum value of the first derivatives of sarcomere length (+dL/dt, −dL/dt, respectively), the time constant of the Ca²⁺ transient decay slope (Ca²⁺ transient Tau), and the amplitude of the Ca²⁺ transient (Ca²⁺ transient amplitude). “*” indicates p<0.01 vs. NCshRNA control group. (n = 38 in the NCshRNA group, n = 35 in the PP1βshRNA group). <b>C</b>: Immunoblots of the key SR phosphoproteins and the phospholylation levels 7 days after gene transfer. Note that the phosphorylation levels of PLN at Ser16 was augmented in the PP1βshRNA treated group compared with that of the NCshRNA treated group. <b>D</b>: Summaries of the phospholylation levels of PLN at Ser16 and RyR at 2808 in each group. “*” indicates p<0.05 vs. the NCshRNA treated group. (n = 6 in the NCshRNA group, n = 6 in the PP1βshRNA group).</p

<i>In vivo</i> and <i>in vitro</i> adenoviral mediated PP1βshRNA significantly reduced expression of PP1β in C2C12 cells and mice hearts.

<p><b>A</b>: Vector structure of the adenoviral-PP1βshRNA under the control of the CMV promoter and marker gene expression of EmGFP. The negative control sequence of miR was used as a shRNA control sequence (NCshRNA). <b>B</b> and <b>C</b>: Efficiency of AdV-CMV-EmGFP-PP1βshRNA assessed by real-time RT-PCR (left panel) and immunoblotting of PP1β (right panel) at increasing MOIs of transfections 5 days after adenoviral infection in differentiated C2C12 cells. <b>D</b>: Immunostaining of GFP in the transverse heart section at 7 days after direct adenoviral injection, showing the representative transfection efficiency. The lower panel shows the site of local inflammation (indicated by arrows) 7 days after adenoviral gene transfer. <b>E</b>: Immunoblotting of PP1β from LV homogenates and a summary of quantitative image analysis normalized by the amount of GAPDH expression. “*” indicates p<0.05 vs. control. (n = 8 in the PP1βshRNA group, n = 8 in the NCshRNA group).</p

AAV9- mediated heart-failure-inducible PP1βshRNA and partial reduction in PP1βexpression.

<p><b>A</b>: Vector structure of the adeno-associated virus 9 (AAV9) -PP1βshRNA flanked by 3′ and 5′ inverted terminal repeats (ITR) under the control of the brain natriuretic peptide (BNP) promoter and marker gene expressison of EmGFP. The negative control sequence of miR was used as a shRNA control sequence (NCshRNA). AAV9 vector expressing lacZ driven by the CMV promoter was used to assess the <i>in vivo</i> transfection efficiency through the tail vein injection of AAV9 gene transfer into the heart. <b>B</b>: The expression levels of BNP mRNA and EmGFP protein were dose-dependently increased with phenyrephrine (PE) in the pZAC2.1-BNP-EmGFP-PP1βshRNA (AAV9 vector plasmid) transfected HL-1 cells. “*” indicates p<0.001 vs PE(−),“#” indicates p<0.001 vs PE 1×10⁻⁴ M, “**” indicates p<0.05 vs PE 2×10⁻⁴ M, “##” indicates p<0.01 vs PE 2×10⁻⁴ M, “†” p<0.05 vs PE(−),“††” p<0.05 vs PE 2×10⁻⁴ M. (nBNP; normalized BNP expression, nGFP; normalized EmGFP expression). <b>C</b>: Comparison of EmGFP expression levels with BNP- versus CMV- promoter. The normalized EmGFP expression levels were estimated 48 hours after plasmid transfection of pDC316-CMV-EmGFP-PP1βshRNA and pZAC2.1-BNP- EmGFP-PP1βshRNA in HL-cells. “*” indicates p<0.01 vs the CMV promoter treated group. <b>D</b>: Bluo-gal staining of transverse heart sections at the papillary muscle level in AAV9-CMV-LacZ transfected MLPKO mice (left panel), and immunostaining of EmGFP proteins in the AAV9-BNP-PP1βshRNA transfected hearts in MLP knockout mice(middle panel) and wild type mice (right panel). The expression level of EmGFP was clearly detected by an anti-GFP antibody (Abcam) as a brown color in the GFP-immunostaining indicates positive cardiomyocytes. The scale bar indicates 50 µm. <b>E</b>: Immunoblotting of PP1β in LV homogenates from the transfected MLP knockout hearts with AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA. The graph under the immunoblot indicates quantitative analysis of PP1β expression normalized by GAPDH. PP1β expression levels were decreased by 25% in the PP1βshRNA treated group. “*” indicates p<0.05 vs. NCshRNA treated group. (n = 8 in each group). <b>F</b>: Bluo-gal staining of AAV-CMV-LacZ injected mice organs (left column) and immunostaining of GFP inAAV9-EmGFP-PP1βshRNA. There were no detectable levels of the GFP shRNA vector in organs other than the heart, whereas the AAV9-CMV-lacZ vector exhibited a trace expression of lacZ in skeletal muscle, kidney and pancreas. There was no detectable expression in liver, spleen and lung. The scale bar indicates either 50 µm or 100 µm, as indicated in each panel.</p

A hybrid-scale spatial model of myofbirils, mitochondria and RyR clusters.

<p>(A): Confocal image of a tissue section from the left ventricule of an adult male Wistar rat; numbered cells were processed for RyR cluster distribution analysis and development of a novel computational fusion algorithm (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004417#pcbi.1004417.s017" target="_blank">S1 Text</a>). (B): 3D rendering of cell number 1 in (A) showing green immuno-labeling of RyR clusters and red, phalloidin staining of myofibrillar actin. (C): Electron micrographs of a 240 nm tissue section from another left ventricular sample of a similar male Wistar rat were acquired at different tilts (left) to construct a 3D electron tomogram; the stack was manually segmented (right) for myofibrils and mitochondria. More details on the data acquisition are in the Materials and Methods. (D): One image slice from the electron tomogram with an overlay of the FE computational mesh (mitochondrial regions in green) and the simulated RyR clusters (red spheres) from the computational fusion algorithm; the mesh is partly removed for visualization. (E): A 3D view of the RyR clusters and the mitochondrial regions. (F): 3D view of the predicted Fluo-4-bound Ca²⁺ at the end of 30 ms; an isosurface of the solution field is partially in view at the mid-plane of the half-sarcomere model.</p

The impact of mitochondria and RyR cluster distribution on the [Ca²⁺]_i transient.

<p>(A): The F/F₀ signal from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004417#pcbi.1004417.g002" target="_blank">Fig 2C</a>. (B) F/F₀ signal with mitochondrial regions being assigned cytosolic properties. (C) same as (B) without mitochondria rendered. The red dots illustrate the RyR cluster distribution that matches experimental data. (D) Changed F/F₀ signal when the RyR cluster distribution is forced to have a fixed nearest-neighbor distance between all clusters.</p

[Ca²⁺]_i is robust to alterations in RyR cluster distribution.

<p>[Ca²⁺]_i (top panel) and [F4Ca]_i (bottom panel) are shown at t = 30 ms, at the z-disc transverse plane. Results on the left column were generated from a simulation using RyR cluster distribution properties of the rat. Results on the right column were generated from a simulation using RyR cluster distribution properties from human myocyte measurements.</p

RyR cluster simulation algorithm workflow.

<p>(1) segment RyR clusters from original data; (2) calculate the nearest-neighborhood distances for the RyR clusters; (3) Identify Z-disc planes from RyR cluster data; (4) Convert phalloidin stack into binary image stack; (5) Identify Z-plane positions on the binary myofibrils stack from (4) and set rest of the planes to background colour; (6) Calculate background distance transform for z-disc stack and decompose to radial and axial components; (7) Determine W, window of voxels available for RyR cluster simulation; (8) Use W, nearest-neighborhood distances and axial and radial z-disc distance statistics as input for RyR cluster simulation algorithm.</p