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. 2013 Oct 1;305(7):H1089-97.
doi: 10.1152/ajpheart.00067.2013. Epub 2013 Aug 2.

Cardiomyocyte-specific p65 NF-κB deletion protects the injured heart by preservation of calcium handling

Xiu Q Zhang  1 , Ruhang Tang, Ling Li, Amanda Szucsik, Hadi Javan, Noriko Saegusa, Ken W Spitzer, Craig H Selzman
Affiliations

Cardiomyocyte-specific p65 NF-κB deletion protects the injured heart by preservation of calcium handling

Xiu Q Zhang et al. Am J Physiol Heart Circ Physiol. .

Abstract

NF-κB is a well-known transcription factor that is intimately involved with inflammation and immunity. We have previously shown that NF-κB promotes inflammatory events and mediates adverse cardiac remodeling following ischemia reperfusion (I/R). Conversely, others have pointed to the beneficial influence of NF-κB in I/R injury related to its anti-apoptotic effects. Understanding the seemingly disparate influence of manipulating NF-κB is hindered, in part, by current approaches that only indirectly interfere with the function of its most transcriptionally active unit, p65 NF-κB. Mice were generated with cardiomyocyte-specific deletion of p65 NF-κB. Phenotypically, these mice and their hearts appeared normal. Basal and stimulated p65 expression were significantly reduced in whole hearts and completely ablated in isolated cardiomyocytes. When compared with wild-type mice, transgenic animals were protected from both global I/R by Langendorff as well as regional I/R by coronary ligation and release. The protected, transgenic hearts had less cytokine activity and decreased apoptosis. Furthermore, p65 ablation was associated with enhanced calcium reuptake by the sarcoplasmic reticulum. This influence on calcium handling was related to increased expression of phosphorylated phospholamban in conditional p65 null mice. In conclusion, cardiomyocyte-specific deletion of the most active, canonical NF-κB subunit affords cardioprotection to both global and regional I/R injury. The beneficial effects of NF-κB inhibition are related, in part, to modulation of intracellular calcium homeostasis.

Keywords: calcium; ischemia-reperfusion; nuclear factor-κB; phospholamban.

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Figures

Fig. 1.
Fig. 1.
Characterization of mice with cardiomyocyte-specific deletion of p65 NF-κB. A: X-gal staining of wild-type (WT; left) and α-MHC-Cre (right) animals crossed with ROSA26 mice. B: gross, cross-sectional, and microscopic histology of WT and p65 knockout (KO) mice. C: Western blot analysis of whole heart and spleen homogenates for p65 and phospho-p65 NF-κB with densitometry normalized to GADPH expression (spleen blots modified to align with the heart sequence at top). D: mice were treated with intraperitoneal LPS or saline. Hearts were procured 12 h later, and Western blots performed for p65 and phospo-p65. Western blots are representative of 4–6 mice/group. *P < 0.05.
Fig. 2.
Fig. 2.
Cardiomyocyte NF-κB activity in vitro. A: cardiomyocytes were cultured, and Western blots were performed for NF-κB-related proteins including proximal kinase, IKKα, IKKβ, and IκBα, as well as its sister subunits, p50 and p105. B: cardiomyocytes were stimulated with TNF-α (10 ng/ml) for 30 min, and Western blots performed for phospho-p65. Western blots are representative of 4–6 mice/group (*vs. vehicle; †vs. WT; P < 0.05).
Fig. 3.
Fig. 3.
Ex vivo perfusion of WT and CMC p65-deficient mouse hearts undergoing simulated global ischemia-reperfusion. A: pressure measurements during the experimental period as a percentage of contractile function. n = 7–9/group. LVDP, left ventricular (LV) developed pressure; RPP, rate pressure product; +dP/dt, rate of rise of LV pressure per unit time; −dP/dt, rate of fall of LV pressure. B: triphenyltetrazolium chloride (TTC) staining and infarct size depicted as a ratio of infarcted area to whole heart tissue (n = 5/group). C: myocardial enzyme release for lactate dehydrogenase (LDH) and creatine phosphokinase (LDH, CK). n = 7–9. Pre-isc, preischemic; post-isc, postischemic. For each experiment, *P < 0.05, vs. WT.
Fig. 4.
Fig. 4.
In vivo ischemia-reperfusion with LAD ligation and release in WT and CMC p65-deficient mice. A: twenty-four hours after reperfusion, hearts were analyzed after TTC staining in vivo (n = 7/group). Infarct area stains white, injured area stains red, and viable areas stain blue. B: quantitative analysis of infarct area, area at risk (AAR), and viable area (n = 7/group; *P < 0.05). LV, left ventricular.
Fig. 5.
Fig. 5.
In vivo ischemia-reperfusion 6 h after LAD ligation and release. A: terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining for apoptosis in area at risk of sham-operated, WT, and CMC p65-deficient mice. Included are macroscopic views demonstrating the area at risk as well as high-powered views with nuclear counterstaining. B: quantification of apoptotic cells. C and D: Western blot for active p65 NF-κB expression and its quantification. E: RT-PCR expression of IL-6 and TNF-α. *P < 0.05; n = 3–5.
Fig. 6.
Fig. 6.
NF-κB and calcium handling. A: calcium transients during simulated ischemia-reperfusion. Myocytes were field stimulated (cycle length = 1 s), and transients were recorded. Representative calcium transients during control (black), after 5 min of ischemia followed by 5 min of reperfusion (blue) (n = 7), are shown. Western blot analysis (B) and densitometry (C) of whole mouse hearts for sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), phospholamban (PLN), and phosphorylated PLN-Ser16 6 h after in vivo regional ischemia-reperfusion (n = 4 to 5). *P < 0.05 compared with WT.
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