Upstream necroptotic regulators signal to the mitochondrion through Mcl-1.

<p>A, Western blots for Bax, Bak, BID, Bcl-2, Mcl-1, MLKL, RIP3, cofilin-1, CypD, Complex V and III (mitochondrial loading control), and αtubulin (cytoplasmic loading control) from cytoplasmic and mitochondrial extracts from WT, <i>Ripk3</i> null, <i>Bax/Bak1</i> DKO, and <i>Ppif</i> null MEFs treated with TNFα and zVAD-FMK (TZ) or vehicle (Veh.) for 6 hours. B, Immunocytochemistry for MLKL (green) and mitochondrial protein SAMM50 (red) on WT MEFs treated with TNFα and zVAD-FMK (TZ) or vehicle (Veh.) for 3 hours. Nuclei are stained with Dapi (blue).</p

MPTP opening is required for necroptosis.

<p>A, Western blots for CypD, Bax, Bak, RIP1, RIP3, and gapdh (control) from extracts of WT and <i>Ppif</i>^<i>-/-</i> MEFs. B, Quantitation of dead cells in WT and <i>Ppif</i>^<i>-/-</i> MEFs treated with TNFα and zVAD-FMK with and without necrostatin. All assays were performed in duplicate and averaged from three independent experiments. *p<0.01 vs untreated; # p<0.01vs WT treated. C, Transmission electron microscopy in <i>Ppif</i>^<i>-/-</i> MEFs treated with and without TNFα and zVAD-FMK for 2 hours (Early) and 12 hours (Late) at 2 different magnifications. D, Images of pancreas stained with H&E from WT and <i>Ppif</i>^<i>-/-</i> mice treated with caerulein to induce necroptosis-mediated pancreatitis (200X magnification). E, Calcium uptake capacity assay with membrane impermeable calcium indicator dye Calcium Green-5N in purified mitochondria from WT liver pretreated with tBID (red) or vehicle (black). The calcium addition is labeled with an arrowhead. Fluorescence reduces as the mitochondria take up the calcium from the solution; three independent experiments were performed, although a representative trace is shown.</p

Cardiac-specific deletion of <i>Nlk</i> protects mice from stress stimulation.

<p>(A) Western blot assessment of NLK cardiac protein levels in 8 week-old <i>Nlk</i>^{<i>fl/fl-βMHC-Cre</i>} mice versus control <i>Nlk</i>^<i>fl/fl</i> mice. Gapdh was used as a processing and Western blot loading control. (B) LV FS (%) in hearts of the mice shown at 10 weeks of age as either sham operated or 2 weeks after a TAC surgical procedure. Number of mice used is shown in the bars. #p<0.05 versus sham; *p<0.05 versus <i>Nlk</i>^<i>fl/fl</i> TAC. (C) HW/BW ratio in the groups of mice shown at 10 weeks of age as either sham operated or 2 weeks after a TAC surgical procedure. Number of mice used is shown in the bars. #p<0.05 versus sham; *p<0.05 versus <i>Nlk</i>^<i>fl/fl</i> TAC. (D) Western blot assessment of NLK cardiac protein levels in 8 week-old <i>Nlk</i>^{<i>fl/fl-αMHC-MCM</i>} mice 2 weeks after TAC. Gapdh was used as a processing and Western blot loading control. Mice were given tamoxifen injection via intraperitoneal (IP) injection for 7 consecutive days (0.5 mg/day), then subjected to TAC. (E) LV FS (%) in hearts of the mice shown at 10 weeks of age as either sham-operated or 2 weeks after a TAC surgical procedure. Number of mice used is shown in the bars. #p<0.05 versus sham; *p<0.05 versus <i>Nlk</i>^<i>fl/fl</i> TAC. (F) HW/BW ratio of the groups of mice shown at 10 weeks of age as either sham operated or 2 weeks after a TAC surgical procedure. Number of mice used is shown in the bars. #p<0.05 versus sham; *p<0.05 versus <i>Nlk</i>^<i>fl/fl</i> TAC. (G) Quantification of the ischemic area versus the area at risk (IA/AAR) for the indicated groups of mice. *p<0.05 vs <i>Nlk</i>^<i>fl/fl</i>. Number of animals used is shown in the bars. (H) Quantification of the area at risk (AAR) to the total LV area from the hearts of mice show in G. Number of animals used is shown in the bars.</p

The necroptotic pathway and mitochondrial involvement.

<p>Schematic representation of the necroptotic pathway showing that upon treatment of TNFα and zVAD-FMK, upstream effectors RIP1 and RIP3 lead to MLKL translocation to the mitochondria where matrix Mcl-1 is depleted and the MOMP and MPTP are engaged, thus leading to mitochondrial dysfunction that is necessary for effective cell death through necroptosis.</p

NLK DTG mice develop cardiomyopathy.

<p>(A) Heart weight (HW) normalized to body weight (BW) in tTA and NLK DTG mice at the indicated ages. Number of mice used is shown in the bars. *p<0.05 vs tTA. (B) Cross-sectional area of cardiomyocytes from histological heart sections from tTA and DTG mice analyzed by ImageJ software. Number of mice used is shown in the bars. At least 600 cardiomyocytes were analyzed for each group across the number of hearts shown. *p<0.05 vs tTA. (C) Real-time PCR analysis of mRNA for the indicated genes from the hearts of tTA control and DTG mice at 2 months of age. At least 4 hearts were used for each group and all PCR samples were performed in duplicate. *p<0.05 vs tTA. (D) Representative Masson’s trichrome-stained histological heart sections from tTA and NLK DTG mice at 2 months of age. Magnification is 100x total. (E) Immunohistochemical assessment of cardiac myocytes positive for both TUNEL and α-actinin antibody staining in histological sections from hearts of tTA or DTG mice. *p< 0.05 vs tTA (at least 14,000 nuclei were counted across 4 hearts in each group).</p

Analysis of cardiac hypertrophy and cell death in NLK DTG after stress stimulation.

<p>(A and B) Echocardiographic assessment of FS% and LVIDd in tTA and DTG mice after 2 weeks of a sham or TAC surgical procedure. Number of mice analyzed is shown in the bars. *p<0.05 vs sham; #p<0.05 versus tTA TAC. (C) HW normalized to BW in tTA and NLK DTG mice 2 weeks after sham or a TAC surgical procedure. Number of mice used is shown in the bars. *p<0.05 vs tTA TAC. (D) Representative Masson’s trichrome-stained histological heart sections from tTA and NLK DTG mice after 2 weeks of TAC. Magnification is 100x total. (E) Representative images of transverse heart sections stained with triphenyltetrazolium chloride (red area) following I/R injury. Ischemic area is outlined in yellow. (F) Quantification of the ischemic area versus the area at risk (IA/AAR) for the indicated groups of mice. *p<0.05 vs tTA. Number of mice used is shown in the bars. (G) Quantification of the percent area at risk (AAR) normalized to the left ventricle (LV) area from hearts of tTA or DTG mice after I/R injury. Number of animals used is shown in the bars.</p

NLK interacts with Stat1.

<p>(A) Top, silver stained 10% SDS-PAGE gel of immunoprecipitated protein samples from HEK293 cells transfected with either an empty Flag containing expression plasmid or Flag-NLK expression plasmid. Bottom, Western blot for Flag-tagged NLK protein eluted from anti-Flag M2 magnetic beads. Two pools of interacting proteins were extracted and analyzed by mass spectrometry. IgG light chain protein is non-specific but shows the same loading of anti-Flag M2 beads in the 2 samples. (B) Western blot validation of the interaction between overexpressed NLK and endogenous Stat1 from HEK293 cells. (C) Immunoprecipitation followed by Western blotting to show that overexpressed NLK interacted with endogenous Stat1 in isolated neonatal cardiac myocytes. (D) Western blot analysis for Stat3, Stat1, and NLK in tTA and DTG hearts at 3 months of age with ~9 weeks of NLK induction after Dox removal. Gapdh was used as a processing and loading control. (E) Quantification of expression levels of Stat1 and Stat3 shown in D. These data were generated from at least two experiments, 3 mice each. *p<0.05 vs tTA.</p

Cardiac-specific over-expression of TGFBI.

<p>(A) Schematic representation of the double transgenic (DTG) system used to generate cardiac-specific, doxycycline inducible expression of TGFBI. (B) Western blot analysis of TGFBI in hearts of DTG mice. GAPDH was used as a control. (C) Immunohistochemistry of heart histological sections at 6 months of age from WT or TGFBI DTG mice. Sections were imaged for the proteins or markers shown. Scale = 10 μm. (D) Heart-weight (HW) normalized to body-weight (BW) in WT and DTG mice at 6 months of age. *p<0.05 vs WT using an unpaired Student’s T-test. (E) Percent fractional shortening (FS%) in WT and DTG mice at 6 months of age. *p<0.05 vs WT using an unpaired Student’s T-test. (F) Left ventricular dimension in diastole (LVED) in WT and DTG mice at 6 months of age as measured by echocardiography. (G) Percent fibrosis measured from Masson’s trichrome-stained cardiac histological sections from WT and DTG mice at 1 year of age. (H) Assessment of FS% in the indicated groups of mice over 12 weeks after sham or TAC surgery. (I) HW normalized to BW in the indicated groups of mice after 12 weeks after TAC. *p<0.05 vs corresponding sham group. (J) Assessment of lung-weight (LW) normalized to BW in the indicated groups of mice after 12 weeks of TAC. *p<0.05 vs corresponding sham group analyzed by parametric one way ANOVA with a Newman-Keuls post-hoc test.</p

Loss of <i>Tgfbi</i> does not alter disease progression after MI.

<p>(A) Percent survival in the groups of mice shown over 7 days following MI surgery. No mortality was observed in sham-operated WT or <i>Tgfbi</i>^<i>-/-</i> mice. No differences in survival between WT and <i>Tgfbi</i>^<i>-/-</i> after MI was detected using the log rank Mantel-Cox statistical test. (B) Western blot analysis for TGFBI and periostin from isolated infarct areas of the hearts of the indicated groups of mice 7 days after MI. GAPDH was used as a loading control. (C and D) Quantification of protein levels normalized to GAPDH using densitometry for periostin in WT and <i>Tgfbi</i>^<i>-/-</i>-operated hearts and for TGFBI in WT and <i>Postn</i>^<i>-/-</i>-operated hearts. *p<0.05 vs WT using an unpaired Student’s T-test. (E) Percent survival in the indicated groups of mice over 7 days after sham or MI surgery. p<0.05 vs WT MI-operated animals using the log rank Mantel-Cox test (F) Percent ventricular fractional shortening (FS%) as measured by echocardiography in the indicated genotypes of mice 8 weeks after MI. *p<0.05 vs sham. (G) Left ventricular end diastolic dimension (LVED) as measured by echocardiography in the indicated groups of mice 8 weeks after MI. *p<0.05 vs sham using a parametric one way ANOVA with a Newman-Keuls post-hoc test.</p

TGFBI and periostin are induced in the heart after injury.

<p>(A) Western blot analysis of periostin and TGFBI in isolated adult cardiomyocytes and fibroblasts. Sarcomeric α-actin was used as a control for cardiomyocyte purity and GAPDH was used as a loading control. (B) Quantitative real time PCR for <i>Postn</i> and <i>Tgfbi</i> from 1 week sham or MI-operated hearts. mRNA levels were normalized to 18s ribosomal RNA. *p<0.05 for both genes in MI-operated animals compared to sham animals using an unpaired Student’s T-test. n = 3 animals. (C) Western blot analysis for periostin and TGFBI in the infarcted areas isolated from hearts 24 hours, 7 and 14 days after MI surgery. GAPDH was used as a loading control. Each lane corresponds to protein from one mouse. (D and E) Quantification of TGFBI (D) and periostin (E) protein levels from the conditions shown in panel C except that 3 hearts were analyzed in total each. (F) Immunohistochemistry for the indicated markers/proteins on sham or MI-operated hearts after 1 week. Images were taken at 400x magnification. Scale = 20 μm.</p