An inhibitor of the δPKC interaction with the d subunit of F1Fo ATP synthase reduces cardiac troponin I release from ischemic rat hearts: utility of a novel ammonium sulfate precipitation technique.

We have previously reported protection against hypoxic injury by a cell-permeable, mitochondrially-targeted δPKC-d subunit of F1Fo ATPase (dF1Fo) interaction inhibitor [NH2-YGRKKRRQRRRMLA TRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-COOH] in neonatal cardiac myo-cytes. In the present work we demonstrate the partitioning of this peptide to the inner membrane and matrix of mitochondria when it is perfused into isolated rat hearts. We also used ammonium sulfate ((NH4)2SO4) and chloroform/methanol precipitation of heart effluents to demonstrate reduced card-iac troponin I (cTnI) release from ischemic rat hearts perfused with this inhibitor. 50% (NH4)2SO4 saturation of perfusates collected from Langendorff rat heart preparations optimally precipitated cTnI, allowing its detection in Western blots. In hearts receiving 20 min of ischemia followed by 30, or 60 min of reperfusion, the Mean±S.E. (n=5) percentage of maximal cTnI release was 30 ± 7 and 60 ± 17, respectively, with additional cTnI release occurring after 150 min of reperfusion. Perfusion of hearts with the δPKC-dF1Fo interaction inhibitor, prior to 20 min of ischemia and 60-150 min of reperfusion, reduced cTnI release by 80%. Additionally, we found that when soybean trypsin inhibitor (SBTI), was added to rat heart effluents, it could also be precipitated using (NH4)2SO4 and detected in western blots. This provided a convenient method for normalizing protein recoveries between groups. Our results support the further development of the δPKC-dF1Fo inhibitor as a potential therapeutic for combating cardiac ischemic injury. In addition, we have developed an improved method for the detection of cTnI release from perfused rat hearts

Chloroform methanol precipitation of proteins improves cardiac troponin I (cTnI) detection in Western blots.

<p>Cardiac left ventricular tissue from adult rats was homogenized as described in Methods. Homogenate protein (10–50 µg) was (left side) or was not (right side) subjected to chloroform/methanol precipitation prior to its resolution on SDSPAGE. Western blot analyses with anti-cardiac troponin I (cTnI) antisera were then conducted. A typical autoradiograph is shown (top). Histograms represent Mean±S.E. densitometry values, expressed as the percentage of maximal densitometry, from 4 independent experiments. Asterisk shown in the figure indicates statistically significant difference (p<0.01) between 10 µg protein groups for chloroform/methanol precipitated (left) vs. not chloroform/methanol precipitated (right) proteins.</p

Commercially available soybean trypsin inhibitor (SBTI) preparations are devoid of cardiac troponin I (cTnI).

<p>Lane 1 contains 35 µg of rat left ventricle homogenate prepared as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070580#pone-0070580-g001" target="_blank">Figure 1</a>. Lanes 2–7 contain 0–60 µg of commercially available SBTI. All samples were subjected to SDSPAGE and proteins were electro-transferred onto nitrocellulose paper (NCP). The resulting blots were stained for total proteins using Ponceau S stain (Panel A) and then subjected to Western blot analyses for the detection of SBTI and cTnI (Panel B). Note the absence of cTnI immunoreactivity in the commercially available SBTI samples. Typical autoradiographs are shown in the top of Panel B with the histogram representing Mean±S.E. densitometry values from all autoradiographs (expressed as the % of maximal densitometry scores) from 5 independent experiments.</p

Detection of cardiac troponin I (cTnI) in effluents collected from Langendorff-perfused rat hearts following ischemia/reperfusion injury.

<p>Rat hearts were first given a 20 min equilibration (Eq) period, followed by a 1 hr perfusion (which will serve as the peptide loading phase (PepLoad), but no peptide was added for experiments in this figure). Next, hearts were given a 20 min global ischemia (I) exposure followed by 2.5 hr of perfusion with oxygenated Kreb’s buffer (Reperfusion). Effluents (50 ml) were collected from each of the protocol stages indicated by arrows in panel A. Proteins in each effluent were then precipitated by adding crystalline (NH₄)₂SO₄ to a level of 50% saturation and centrifuging them. Precipitated proteins were subjected to SDSPAGE and transferred onto nitrocellulose paper (NCP). A representative Ponceau S stain of a typical NCP blot is shown in Panel B and autoradiographs from Western blots with anti-cTnI and anti-SBTI antisera are shown in Panel C. Mean±S.E densitometry values for SBTI (Panel D) and cTnI (Panel E) are also shown. cTnI densitometry values were normalized to SBTI levels in the same blots and SBTI values were not statistically different between groups in which cTnI was released. Results shown are typical of the responses of effluents collected from 5 independent rat hearts and are represented in the histograms.</p

Optimization of ammonium sulfate (NH₄)₂SO₄ precipitation of cardiac troponin I (cTnI) and the utility of soybean trypsin inhibitor as a normalization tool.

<p>In each experiment homogenate protein (200 µg) from rat cardiac left ventricle was added to each of 5 different aliquots of Kreb’s buffer (50 ml) on ice. The protease inhibitors leupeptin, aprotinin, phenylmethylsulfonyl-fluoride and soybean trypsin inhibitor (SBTI) were then added to each aliquot. Different amounts of (NH₄)₂SO₄ were then added to each 50 ml sample to determine the optimal percentage of (NH₄)₂SO₄ (0–90% (NH₄)₂SO₄-saturated solutions) for precipitation of cTnI. Panel A is a Ponceau S. stain of a blot of the (NH₄)₂SO₄ precipitated proteins from each sample. The arrow in Panel A marks the position of the most prominent protein band in the gel, which we later determined to be SBTI. Panel B is a Western blot for exogenously added SBTI. Panel C is a similar blot, but anti-cTnI immunoreactivity was monitored instead of SBTI immunoreactivity. Data in the histogram of Panel D shows Mean±S.E. autoradiograph densitometry scores, expressed as the percentage of maximal densitometry, from 5 independent experiments. Note the optimal level of (NH₄)₂SO₄ saturation for the precipitation of SBTI was ≥80% whereas for cTnI it was 50%.</p

The δPKC-dF₁Fo interaction inhibitor reduces cardiac troponin I (cTnI) release when perfused into Langendorff hearts.

<p>Experimental details were as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070580#pone-0070580-g004" target="_blank">Figure 4</a> except that during the peptide loading phase 50 nM concentrations of the scrambled sequence (inactive) control peptide or the δPKC-dF₁Fo interaction inhibitor peptide were administered to hearts for 1 hr. Effluents were collected and Western blot analyses for cTnI were conducted. The left-most lane contains rat left ventricle homogenate used as a positive control for cTnI immunoreactivity. The remaining groups are described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070580#pone-0070580-g004" target="_blank">Figure 4</a>. Results shown are typical of the responses of 3 independent rat hearts in each peptide treatment group.</p

Sub-mitochondrial Localization of the δPKC-dF₁Fo peptide inhibitor after its perfusion into Langendorff rat hearts.

<p>Adult rat hearts were first perfused with 50 nM concentrations of the FLAG-epitope tagged δPKC-dF₁Fo inhibitor peptide for 1 hr. Hearts were then washed for 5 min with Krebs buffer that contained no peptide. In Panel A mitochondria were then isolated from the left ventricle and subfractionated into mitochondrial inner (IM) and matrix fractions as described in Methods. Sub-mitochondrial fractions were then probed in Western blots with monoclonal mouse anti-FLAG antisera at 1∶500 dilution. Results are expressed as the mean±S.E. percentage of the maximal densitometry scores from 3 independent experiments. Extensive FLAG immunoreactivity was observed in the matrix and IMM fractions where δPKC would be predicted to interact with the “d: subunit of F1Fo ATP synthase. Panels B–H refer to results from immuno-gold transmission electron microscopy studies. Results shown are typical analyses conducted in 3 independent experiments in which random fields were selected from embedded sections and a total of 364 mitochondria were analyzed.</p

Sex-Dependent Role of Adipose Tissue HDAC9 in Diet-Induced Obesity and Metabolic Dysfunction

Obesity is a major risk factor for both metabolic and cardiovascular disease. We reported that, in obese male mice, histone deacetylase 9 (HDAC9) is upregulated in adipose tissues, and global deletion of HDAC9 protected against high fat diet (HFD)-induced obesity and metabolic disease. Here, we investigated the impact of adipocyte-specific HDAC9 gene deletion on diet-induced obesity in male and female mice. The HDAC9 gene expression was increased in adipose tissues of obese male and female mice and HDAC9 expression correlated positively with body mass index in humans. Interestingly, female, but not male, adipocyte-specific HDAC9 KO mice on HFD exhibited reduced body weight and visceral adipose tissue mass, adipocyte hypertrophy, and improved insulin sensitivity, glucose tolerance and adipogenic differentiation gene expression. Furthermore, adipocyte-specific HDAC9 gene deletion in female mice improved metabolic health as assessed by whole body energy expenditure, oxygen consumption, and adaptive thermogenesis. Mechanistically, compared to female mice, HFD-fed male mice exhibited preferential HDAC9 expression in the stromovascular fraction, which may have offset the impact of adipocyte-specific HDAC9 gene deletion in male mice. These results suggest that HDAC9 expressed in adipocytes is detrimental to obesity in female mice and provides novel evidence of sex-related differences in HDAC9 cellular expression and contribution to obesity-related metabolic disease