Rheb prevents cardiac hypertrophy and interacts with Bnip3.

<p>Myocardial lysates from Bnip3-TG and wild type (WT) mice were prepared and processed for reciprocal Bnip3 and Rheb immunoprecipitation as described in Methods. Representative western blots showing immunoprecipitation (i.p.) and western blot (WB) antibodies are shown in (A). Quantification of western blot immunoprecipitation is shown in (B) (n = 3; * p < 0.002). (C) Representative plates of neonatal rat cardiac myocytes after transfection with random sequence (RS) or Rheb siRNA and exposure to serum as a hypertrophy stimulus as described in Methods. Top panels GFP; bottom phalloidin (red); arrows indicate hypertrophied myocytes in RS panels and reduced myoplasm area with Rheb-siRNA treatment. (D) Quantification of cell area from ~100 myocytes per group as described in Methods (n = 3; * p < 0.002). (E) Representative western blot shown efficient KD of Rheb by siRNA. All results are mean ± S.E.M.</p

Curcumin and GATA4 sensitive changes in myocyte morphology.

<p>In (A) cardiac myocytes were transfected with expression plasmids for GFP, Bnip3, or Bnip3ΔTM and the ratio of the cell length to width determined. Myocyte identity was confirmed by troponin (TNI) immunocytochemistry. Quantitative analysis of the major to minor axis ratio is shown below. The effect of curcumin (Curc) on the major to minor axis ratio is shown in (B) with quantitative analysis of the major to minor axis ratio again shown below. The effect of GATA4 and random (RS) siRNA on the major to minor axis ratio is shown in (C) with quantitative analysis of the major to minor axis ratio also shown below. Results are mean ± S.E.M., significantly different then controls (p < 0.05), n = 3.</p

Curcumin treatment improves Bnip3-TG cardiac performance.

<p>(A) Histone H3 acetylation (Ac-H3) levels in left ventricular lysates from wild type (WT) and Bnip3-TG mice were measured and quantified as described in Methods. (n = 3; * p < 0.05). Example images of short axis M-mode echo cardiograms of WT and Bnip3-TG mice treated with either vehicle or curcumin as described in Methods (B). The effect of vehicle (Veh) or curcumin (Cur) treatment on echocardiographically determined left ventricular ejection fraction (LVEF), left ventricular end diastolic diameter (LVEDD), and heart weight to body weight (HW/BW) ratio of WT and Bnip3-TG mice is shown in (C–E). In (F) p300 was immunoprecipitated from the hearts of wild type (WT), Bnip3-TG, and Bnip3-TG mice treated with vehicle (Veh) or curcumin (Cur) and the association of p300 with GATA4 determined by western blot analysis. Quantitation of p300 bound GATA4 is shown below (*p < 0.05, n = 5). Results are mean ± S.E.M.</p

Bnip3 regulates histone acetylation during hypoxia-acidosis.

<p>In (A and B), cardiac myocytes were treated with random sequence or Bnip3 specific siRNA as indicated and histone acetylation determined by western blot analysis as described in Methods (n = 4; * p < 0.05). In (C and D), cardiac myocytes were subjected to hypoxia (HX) or hypoxia-acidosis (HA) in the presence of Bnip3 or random (RS) siRNA treatments as in (A and B) and the level of acetylated histone H3 determined by immunocytochemistry. Nuclei were labeled with Hoechst. (n = 4; * p<0.05). All results are mean ± S.E.M.</p

Bnip3 increases p300 acetyltransferase activity.

<p>Histone deacetylase (HDAC) (A) and acetyltransferase (HAT) (B) activities were measured in cardiac myocytes exposed to aerobic, hypoxia (HX) and hypoxia-acidosis (HA) as described in Methods. P300-specific acetyltransferase activity is shown in (C). The effect of the p300 inhibitor, curcumin, on histone H3 acetylation (Ac-H3), and Bnip3 protein levels during hypoxia and HA is shown in (D). Western blot quantitation of histone H3 acetylation levels in the presence of curcumin (Cur) is shown in (E). In (F and G), cardiac myocytes were treated with random (RS) or Bnip3 specific siRNA and total cellular HAT (F) or p300 specific HAT (G) activities were determined during hypoxia-acidosis (HA). In (H and I) recombinant Bnip3 or transmembrane deletion mutant Bnip3 (Bnip3-ΔTM) protein were incubated with nuclear lysates from normoxic cardiac myocytes as described in Methods and the HAT activity determined. Results are mean ± S.E.M., * significantly different then controls (p < 0.05), n = 3.</p

Bnip3 binds GATA4 and p300.

<p>GATA4 and MEF2 were immunoprecipitated from cardiac myocytes exposed to aerobic (A), hypoxia (HX) or hypoxia-acidosis (HA) as indicated and the acetylation level of each protein determined by western blot analysis (A). Western blot quantitation of changes in GATA4 and MEF2 acetylation levels in hypoxic-acidotic cardiac myocytes is shown to the right. In (B-D), Bnip3, GATA4 and p300 were immunoprecipitated from cardiac myocytes exposed aerobic (A), hypoxia (HX), or hypoxia-acidosis (HA). Precipitated proteins were detected by western blot analysis using antibodies directed against the indicated proteins. In (E) p300 was immunoprecipitated from cardiac myocytes treated with either random sequence siRNA (RS RNA) or Bnip3 specific siRNA (siRNA) and the western blot probed for GATA4. Western blot quantitation of immunoprecipitated GATA4 is shown to the right (F). Results are mean ± S.E.M., * significantly different then controls (p < 0.05), n = 3.</p

Apoptosis in aged but not young Bnip3-TG mice.

<p>TUNEL staining was implemented as described in Methods. (A) The sensitivity of the TUNEL assay was demonstrated by treating heart sections with DNAase I to induce TUNEL positive DNA strain breaks; top panel TUNEL, bottom panel DAPI; >80% of nuclei are TUNEL-positive (data not shown). (B) Apoptotic indices of wild type (WT) and Bnip3-TG mice were determined by TUNEL at the indicated times as described in Methods. Examples of TUNEL staining in heart sections from 5-week old wild type (C) and Bnip3-TG mice (D). Nuclei were co-stained with DAPI. Yellow arrows point to TUNEL positive nuclei and the corresponding DAPI stained nuclei. Results in (B) are mean ± S.E.M.; * p < 0.05, comparing apoptotic indices with 4–6 weeks (n = 5).</p

Histone hyperacetylation is independent of cell death.

<p>In (A and B), cardiac myocytes were pretreated with mitochondrial permeability transition pore (mPTP) inhibitor TAT-BH4, before exposure to hypoxia or HA, and histone acetylation or DNA fragmentation measured as described in Methods. In (C and D), cardiac myocytes exposed to hypoxia (HX) or hypoxia-acidosis (HA) in the presence and absence of the mPTP inhibitor cyclosporine A (CsA), the caspase inhibitor BocD, or the calpain inhibitor calpeptin as indicated. Western blot quantitation of histone H3 acetylation in the presence of BocD and calpeptin (Cal) is shown in (E). In (F and G) cardiac myocytes were treated with vehicle (UT), or with staurosporine or hydrogen peroxide (H₂O₂) as indicated, to induce cell death, and the acetylation level of histone H3 was determined by western blot analysis. As a positive control for histone acetylation cardiac myocytes were also treated with the HDAC inhibitor TSA. The effect of staurosporine (ST) treatment on histone H3 and H4 acetylation is shown in (H). All results are mean ± S.E.M., n = 3.</p

Bnip3 associates with p300 and GATA4 leading to altered gene expression.

<p>In (A) GATA4, MEF2 and acetylated lysine were immunoprecipitated from total heart lysates generated from wild type (WT) and Bnip3-TG mice. Co-immunoprecipitated (IP) proteins were determined by western blot (WB) analysis as indicated. Quantitation of the changes in GATA4 and MEF-2 acetylation levels are shown in (B). In (C) nuclear extracts were made from heart lysates and reciprocal immunoprecipitation of p300 and GATA4 performed as indicated. Quantitation of p300 and GATA4 reciprocal immunoprecipitation is shown in (D). In (E) transcript levels of atrial natriuretic protein (ANP) in Bnip3-TG hearts ± curcumin relative to wild type hearts was determined by RT-PCR at the indicated ages. All results are mean ± S.E.M., (p < 0.05), n = 3.</p

Bnip3 dependent ventricular dilation and contractile dysfunction.

<p>(A) Bnip3 protein levels were determined in heart lysates from wild type (WT) and α-MHC-Bnip3 transgenic mice (Bnip3-TG) at the indicated ages. (B) Hematoxylin and eosin—stained coronal heart sections from 3 and 10 month old wild type (WT) and Bnip3-TG mice. Examples of short axis M-mode echocardiograms of WT and Bnip3-TG mice at the indicated ages are shown in (C). (D-F) echocardiographically determined left ventricular end diastolic diameter (LVEDD), left ventricular fractional shortening (FS) and left ventricular ejection fraction (LVEF) at the indicated ages. Results are mean ± S.E.M. (* p < 0.05); n = 5.</p