Postconditioning activates Akt and eNOS.

<p><b>A.</b> Western blot of total eNOS and GADPH in WT (C57BL6/J), S1176D mice (S1176ki), and eNOS ko mice. <b>B.</b> Western blot demonstrating phosphorylation (Ser1176) and total protein levels of eNOS in wild-type mice under conditions of control (CTL), MIR, and MIPc. <b>C.</b> Representative Western blot demonstrating phosphorylated (Ser473) and total protein levels of Akt. Densities (arbitrary units, AU) show that MIPc phosphorylates Akt in WT and eNOS ko mice. n = 5 per group. Data are expressed as the mean±SD. *P<0.05.</p

Temporal myocardial contrast echocardiography.

<p>Myocardial blood flow (Aβ) profiles in the apical region for <b>A.</b> C57BL/6J mice. <b>B.</b> eNOS knockout mice, and <b>C.</b> S1176D knockin mice. Levels of myocardial myocardial blood flow were normalized to baseline values and measured at 2, 10 and 30 minutes after reperfusion. <b>D.</b> Apical myocardial blood flow 30 minutes after reperfusion. MIR: Traditional myocardial ischemia with reperfusion, MIPc: Myocardial ischemia with postconditioning. n = 5–6 per group. Data are expressed as the mean±SD. *P<0.05.</p

eNOS S1176 phosphorylation protects against I/R injury in vivo.

<p>Wild-type, S1176D and eNOS knockout mice were subjected to 45 minutes of myocardial ischemia (LAD ligation) followed by traditional reperfusion (MIR) or postconditioned reperfusion (MIPc: 6 cycles of 10sec reperfusion, 10 sec ischemia). <b>A.</b> Percentage of left ventricle area at risk (AAR), (P = NS). <b>B.</b> Quantitative analysis of infarct size over AAR, *P<0.05 compared to wild-type control. <b>C.</b> Representative heart sections perfused with 1% Evans blue and stained with 2% TTC; infarct areas are outlined in black. MIR: Myocardial ischemia with reperfusion, MIPc: Myocardial ischemia with postconditioning, AAR: Area at Risk, LV: Left Ventricle. n = 6–9 mice per group. Data are expressed as the mean±SD.</p

Effect of postconditioning and S1176D mutation on no-reflow zones.

<p><b>A.</b> Representative images 30 minutes after reperfusion. Superimposed areas (blue) indicate regions with ≤20% residual blood flow. <b>B.</b> Composite graph showing areas of the myocardium with ≤20% (black) and ≤30% (white) residual blood flow compared to preischemic baseline. MBF: myocardial blood flow. n = 5–6 per group. Data are expressed as the mean±SD. *P<0.05.</p

BMP7 signaling is enhanced in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs.

<p>(A) Immunoblots of lysates of WT (Bmpr2^+/+) or <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs treated with BMP4 or BMP7 (10 ng/ml) for various times were reacted with antibodies directed against phosphorylated and total Smad1/5/8. Quantification of the ratio of phosphorylated Smad1/5/8 to total Smad1/5/8 (analysis of 3 independent experiments) demonstrated that BMP4 signaling is similar in <i>Bmpr2</i>^<i>+/+</i> or <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs, whereas BMP7 signaling is greater in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs. *P<0.05 compared to <i>Bmpr2</i>^<i>+/+</i> PaSMC group treated with BMP7. <i>Id1</i> (B) and <i>Smad6</i> (C) mRNA levels were measured by qPCR in <i>Bmpr2</i>^<i>+/+</i> or <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs treated with BMP4 or BMP7 (10 ng/ml) for various times. <i>Id1</i> and <i>Smad6</i> gene expression was normalized to <i>Gapdh</i> and expressed as fold-change relative to control <i>Bmpr2</i>^<i>+/+</i> PaSMC group. *P < 0.01 compared to <i>Bmpr2</i>^<i>+/+</i> PaSMC group treated with BMP7.</p

Bmpr2 expression in PaSMCs obtained from WT or <i>Bmpr2</i>^{<i>Δtd/+</i>} mice.

<p>(A) Levels of <i>Bmpr2</i> mRNA were measured in WT (Bmpr2^+/+) or <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs by qPCR using hydrolysis probes for <i>Bmpr2</i> exon junctions 6–7 and 12–13. <i>Bmpr2</i> mRNA levels were normalized to <i>Gapdh</i> and expressed as the fold-change relative to <i>Bmpr2</i>^<i>+/+</i> PaSMCs. *P < 0.01 compared to <i>Bmpr2</i>^<i>+/+</i> PaSMCs. (B) Immunoblots prepared from lysates of <i>Bmpr2</i>^<i>+/+</i> and <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were incubated with an antibody directed against the tail domain of Bmpr2 to detect Bmpr2‑WT or with an anti-GFP antibody to detect Bmpr2‑ΔTD. Immunoblots were subsequently incubated with an antibody directed against Gapdh as a control for protein loading. (C) Confocal microscopy image of a PaSMC transiently transfected with a plasmid directing expression of <i>Bmpr2</i>^<i>Δtd</i> and reacted with an anti-GFP antibody showing localization of Bmpr2‑ΔTD at the cell membrane.</p

Bmpr2‑ΔTD contributes to BMP7 signaling in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs.

<p>(A) <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were transfected with negative control siRNA (siNC), si<i>Bmpr2</i>‑ex12, or si<i>Egfp</i> (30 nM). After 48 h, the ability of BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> mRNA expression was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs transfected with siNC. *P < 0.01 compared to siNC group treated with BMP7, ^† P<0.01 compared to si<i>Bmpr2</i>‑ex12 group treated with BMP7. Efficiency of silencing <i>Bmpr2</i>^<i>+</i> (si<i>Bmpr2</i>‑ex12) and <i>Bmpr2</i>^<i>Δtd</i> (si<i>Egfp</i>) transcripts was measured by qPCR. (B) <i>Bmpr2</i>^{<i>Δtd/flox</i>} and <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs were treated with BMP4 or BMP7 (10 ng/ml) for 30 and 60 minutes, upon which the activation of Smad1/5/8 was evaluated by immunoblotting. Quantification of the Smad1/5/8 activation is plotted as the ratio of pSmad1/5/8 to total Smad1/5/8. (C) The ability of BMP4 or BMP7 to induce <i>Id1</i> and <i>Smad6</i> gene expression in <i>Bmpr2</i>^{<i>Δtd/flox</i>} and <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to untreated <i>Bmpr2</i>^{<i>Δtd/flox</i>} PaSMCs. *P < 0.01 compared to <i>Bmpr2</i>^{<i>Δtd/flox</i>} PaSMC group treated with BMP7.</p

Bmpr2‑TD attenuates Alk2‑mediated BMP7 signaling in PaSMCs.

<p>Alk3‑deficient <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were transfected with specific siRNA to silence <i>Bmpr2</i>^<i>+</i> (si<i>Bmpr2</i>‑ex12) or <i>Bmpr2</i>^<i>Δtd</i> (si<i>Egfp</i>) transcripts. After 48 h, the ability of BMP7 to induce <i>Id1</i> and <i>Smad6</i> gene expression was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>}; <i>Alk3</i>^{<i>del/del</i>} PaSMCs treated with siNC. *P < 0.01 compared to control cells (siNC) treated with BMP7. Silencing efficiency was quantified by qPCR.</p

BMP7 preferentially utilizes Alk2 in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs.

<p>(A) The ability of BMP4 or BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> gene expression, in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs deficient in Alk2 or expressing Alk2 was examined by qPCR. <i>Id1</i> and <i>Smad6</i> gene expression was normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>}<i>; Alk2</i>^{<i>flox/flox</i>} PaSMCs. *P < 0.01 compared to <i>Bmpr2</i>^{<i>Δtd/+</i>}<i>; Alk2</i>^{<i>flox/flox</i>} PaSMCs treated with BMP7. (B) The ability of BMP4 or BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> gene expression, in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs deficient in Alk3 or expressing Alk3 was measured by qPCR. <i>Id1</i> and <i>Smad6</i> gene expression was normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>}<i>; Alk3</i>^{<i>flox/flox</i>} PaSMCs. *P < 0.01 compared to <i>Bmpr2</i>^{<i>Δtd/+</i>}<i>; Alk3</i>^{<i>flox/flox</i>} PaSMC treated with BMP4.</p

BMP7 signaling in <i>Bmpr2</i>^{<i>Δtd/+</i>} and <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs does not depend on the presence of Acvr2a.

<p>(A) <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were treated with a siRNA specific for <i>Acvr2a</i> transcripts. The ability of BMP4 or BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> gene expression was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs treated with siNC. *P < 0.01 compared to siNC within BMP treatment. Silencing efficiency was quantified by measuring <i>Acvr2a</i> mRNA levels. (B) <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs were treated with si<i>Acvr2a</i>. The ability of BMP4 or BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> gene expression was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs treated with siNC. *P < 0.01 compared to siNC within BMP treatment. <i>Acvr2a</i> silencing efficiency was measured by qPCR.</p