IP3 receptor orchestrates maladaptive vascular responses in heart failure

Figures

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  Figure 1. IP3R1 remodeling in aortic tissues from patients with HF. (A) Representative immunoblots showing total IP3R1 expression, phosphorylation of serine/tyrosine kinases, and AKAP9 binding to the channels, as assessed by immunoprecipitation (IP: IP3R1); RyR2 phosphorylation levels of PKA-induced RyR2 phosphorylation on serine 2808 (pSer-2808); and p-MLC20 levels in aortic tissues from patients with HF (n = 5) and controls (n = 4). (B–G) Quantification of the immunoblots shown in A. Individual values with the mean ± SEM are shown. *P < 0.05 versus control, by 2-tailed Student’s t test.

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  Figure 2. Global evaluation of vascular responses in IP3R1VSMC–/– mice. (A and B) MAP was measured by radiotelemetry in IP3R1fl/fl and IP3R1VSMC–/– mice at baseline (days –20 to 0) and following administration of ATII (A) or NE (B) via miniosmotic pumps. n ≥6 mice/group. *P < 0.05 versus IP3R1fl/fl mice, by repeated-measures ANOVA. (C–F) Vascular reactivity of aortic segments (C and D) and third-order mesenteric arteries (E and F) in response to ATII (C and E) and NE (D and F). Data are presented as the mean ± SEM. mN, millinewton. n = 8–20 rings from at least 6 mice per group. *P < 0.05 versus IP3R1fl/fl mice, by repeated-measures ANOVA.

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  Figure 3. Functional role of VSMC IP3R1 in HF. (A) LVEF evaluated by serial echocardiography following surgical ligation of the LAD. (B–D) Neurohormonal activation in HF assessed by measuring blood levels of catecholamines and aldosterone. (E) Representative Masson’s trichrome–stained images showing interstitial cardiac fibrosis. Scale bars: 50 μm. (F) Quantification of interstitial cardiac fibrosis. (G) Measurement of cardiac afterload (ratio of end-systolic pressure and stroke volume); other hemodynamic parameters are reported in Supplemental Table 2. (H) Coronary flow reserve determined in vivo in IP3R1fl/fl and IP3R1VSMC–/– mice. (I) Representative immunoblots of denuded mesenteric arteries (n ≥8 mice/group) showing MLC protein phosphorylation levels in sham-operated and MI IP3R1fl/fl and IP3R1VSMC–/– mice. (J) Quantification of results in I. Data are shown as individual values with the mean ± SEM. *P < 0.05 versus sham, by 2-tailed Student’s t test; #P < 0.05 versus MI IP3R1fl/fl mice, by repeated-measures ANOVA.

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  Figure 4. VSMC IP3R1 deletion attenuates peripheral vascular constriction induced by HF. (A) Myography traces recorded in cannulated mesenteric arteries during stepwise incremental increases in pressure (namely 80, 100, and 120 mmHg) at a constant temperature of 37°C in regular buffer (green background) and a Ca2+-free buffer (light blue background). For clarity, only the sham curve for IP3R1fl/fl mice is shown (not significantly different from the responses in IP3R1VSMC–/– mice). (B) Individual myogenic tone values are shown as the mean ± SEM. n = 5/group. *P < 0.05 versus IP3R1fl/fl mice, by 2-tailed Student’s t test. Of note, the difference in the myogenic contractile response (*P < 0.05) cannot be attributed to modifications in wall thickness (see Supplemental Figure 6).

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  Figure 5. Increased IP3R1 phosphorylation by PKA in VSMCs during HF. (A) PKA activity in VSMCs from sham-operated and MI IP3R1fl/fl and IP3R1VSMC–/– mice. (B) PKA activity in VSMCs from MI and ML-7–treated MI mice (n = 6 mice per group). (C and D) Representative immunoblots (C) and quantification (D) showing increased IP3R1 phosphorylation by PKA in VSMCs from IP3R1fl/fl mice after MI compared with sham-operated mice (n = 6 mice per group). (E and F) Representative immunoblots (E) and quantification (F) showing reduced IP3R1 phosphorylation by PKA in VSMCs from HF-treated mice compared with untreated mice (n = 4 mice per group). Individual values are shown with the mean ± SEM. *P < 0.05 for IP3R1fl/fl sham versus IP3R1fl/fl MI mice, IP3R1VSMC–/– sham versus IP3R1VSMC–/– MI mice, and MI mice versus ML-7–treated MI mice; 2-tailed Student’s t test. #P < 0.05, for IP3R1fl/fl MI versus IP3R1VSMC–/– MI mice; repeated-measures ANOVA.

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  Figure 6. IP3R1 in vascular smooth muscle mediates increased vascular tone during HF. ATII binds to GPCRs (Gq–G11) and activates PLC, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2), resulting in 2 second messengers: IP3 and diacylglycerol (DAG). IP3 binds to its receptor IP3R1 on the SR, causing Ca2+ release into the cytosol. Furthermore, increased catecholamines during HF bind to adrenergic (AD) receptors and activate the Gs protein and AC, leading to increased levels of cAMP. cAMP activates PKA, which phosphorylate the IP3R1 channels, causing further Ca2+ release into the cytosol. IP3R1 binds AKAP9, which anchors a pool of PKA to the channel. Of note, PKA phosphorylates RyR2 and SR/ER Ca2+-ATPase type 2 (SERCA2a), which play a role in SR Ca2+ release and uptake processes, respectively. An increase in the cytosolic Ca2+ concentration activates the CAM protein. CAM activates MLCK, which in turn phosphorylates MLC20, leading to smooth muscle contraction and vasoconstriction. Chronic vasoconstriction increases cardiac afterload, thereby promoting decompensated HF. Both genetic depletion of VSMC IP3R1 and pharmacologic inhibition of MLCK with ML-7 attenuate MLCK activation and…