Article Figures & Data
Figures
- FIG. 1.
In vivo IR and IGF-1R signaling deficiency in muscle tissue of MI2RKO mice. Phosphotyrosine (pY) immunoblot analysis of (a) IR or (b) IGF-1R immunoprecipitates of tissue protein extracts from mice injected with saline (−) or with insulin or IGF-1 (+).
- FIG. 2.
MI2RKO mice die within 4 weeks of birth. A group of 69 mice including all study group genotypes were monitored closely for 4 weeks after birth. All 15 MI2RKO mice died within this time period.
- FIG. 3.
MI2RKO mice have normal glucose homeostasis. (a) Blood glucose and (b) plasma insulin levels were measured in 2-week-old (2 wks), random-fed male mice (four to nine mice per group). The value that was significantly different (P < 0.05) from the value for DLox mice is indicated by an asterisk. (c) Glucose tolerance tests were performed on 2.5-week-old DLox and MI2RKO male mice (three to five mice per group). (d) Insulin tolerance tests were performed on 2.5-week-old DLox and MI2RKO male mice (three mice per group). (e) Phospho-Akt (pAkt) (phospho-Ser473) and total Akt immunoblot analysis of whole-skeletal-muscle protein extracts from mice stimulated with saline (−), insulin (Ins), or IGF-1 (IGF1).
- FIG. 4.
Mice with combinatorial knockouts have normal glucose homeostasis. (a) Blood glucose levels in random-fed and fasted 6-month-old male DLox, MIRKO, MIGF1RKO, MIRKO/+, and MIGF1RKO/+ mice (7 to 10 mice per group). (b and c) Plasma insulin levels were measured in 6-month-old random-fed mice (b) or mice fasted overnight (c) (six to nine mice per group). (d) Glucose tolerance tests were performed on 4- and 6-month-old DLox, MIRKO, MIGF1RKO, MIRKO/+, and MIGF1RKO/+ male mice (five to seven mice per group). (e) Insulin tolerance tests were performed on mice of the same genotypes and at the same ages as in panel d (seven to nine mice per group).
- FIG. 5.
MI2RKO mice develop dilated cardiomyopathy. (a) Phospho-Akt (pAkt) (phospho-Ser473) and total Akt immunoblot analysis of whole-heart protein extracts from mice stimulated with saline (—), insulin (Ins), or IGF-1 (IGF1). (b) Longitudinal sections of hearts removed from 20-day-old DLox, MIRKO, MIGF1RKO, and MI2RKO mice (left panels). Representative echocardiograms from 17-day-old male mice (right panels). (c) Transverse sections of hearts in 20-day-old DLox and MI2RKO mice. The right ventricle (RV) and left ventricle (LV) are indicated.
- FIG. 6.
Altered morphology of cardiac muscle of MI2RKO hearts at the ultrastructural level. The Z and M lines of MI2RKO hearts are disrupted, and the number of mitochondria is increased. Arrows indicate Z lines in DLox (Lox) and MI2RKO (DKO) myofibrils. Electron microscopic images were obtained from longitudinal sections of cardiac muscle from DLox and MI2RKO mice at postnatal day 20 and processed as described in Materials and Methods. Scale bars, 1 μm.
- FIG. 7.
Number of probe sets detecting altered gene expression (P < 0.01). Heart failure is accompanied by a large shift in gene expression patterns. This graph indicates the number of probe sets up- and down-regulated in the hearts of MIRKO, MIGF1RKO, and MI2RKO mice at postnatal days 8 and 20. The Affymetrix chip contained 22,602 probe sets. Between three and six hearts were analyzed, each with an individual RNA preparation and chip hybridization, for each genotype and time point.
Tables
- TABLE 1.
MI2RKO male mice have small hearts at postnatal days 8 and 20 and small cardiomyocytes at postnatal day 20
Timea Mice Heart wt (mg)b Body wt (g)b HW/BW (%)b Cell circumference (μm)c P8 DLox 27.6 ± 0.8 (13) 4.4 ± 0.2 (13) 0.64 ± 0.02 (13) 26.5 ± 1.1 (7) MI2RKO 23.5 ± 1.1d (10) 4.5 ± 0.2 (10) 0.54 ± 0.03d (10) 26.8 ± 1.2 (5) P20 DLox 68.6 ± 2.5 (14) 9.9 ± 0.3 (22) 0.67 ± 0.03 (9) 49.7 ± 0.6 (4) MIRKO 57.2 ± 2.6d,g (6) 10.3 ± 0.7 (12) 0.49 ± 0.01e,g (4) 49.4 ± 0.4 (4) MIGF1RKO 79.3 ± 2.7d (11) 10.4 ± 0.3 (15) 0.75 ± 0.03 (9) 49.0 ± 1.2 (4) MI2RKO 50.5 ± 2.3e,g (11) 8.3 ± 0.2e,g,h (15) 0.60 ± 0.04f,h (7) 39.7 ± 0.9e (4) ↵ a P8, postnatal day 8; P20, postnatal days 19 to 21.
↵ b Values are means ± standard errors of the means. The number of mice is shown in parentheses.
↵ c Values are means ± standard errors of the means. The number of sections is shown in parentheses. At least 100 cell outlines per heart were measured for volume estimation.
↵ d Significantly different from value obtained with DLox mice (P < 0.01).
↵ e Significantly different from value obtained with DLox mice (P < 0.001).
↵ f Significantly different from value obtained with MIGF1RKO mice (P < 0.01).
↵ g Significantly different from value obtained with MIGF1RKO mice (P < 0.001).
↵ h Significantly different from value obtained with MIRKO mice (P < 0.05).
- TABLE 2.
Echocardiographic analysis of in vivo heart function in 17-day-old male micea
Mice (n) IVS-D (mm) IVS-S (mm) LV-D (mm) LV-S (mm) PW-D (mm) PW-S (mm) LVFS (%) HR (no. of contractions min−1) DLox (8) 0.40 ± 0.00 0.75 ± 0.04 3.09 ± 0.06 1.26 ± 0.08 0.40 ± 0.00 0.76 ± 0.04 59.1 ± 2.3 728 ± 11 MIRKO (3) 0.37 ± 0.03 0.70 ± 0.06 3.07 ± 0.09 1.40 ± 0.15 0.40 ± 0.00 0.73 ± 0.03 54.8 ± 3.2 700 ± 20 MIGF1RKO (2) 0.40 ± 0.00 0.75 ± 0.05 3.20 ± 0.10 1.10 ± 0.20 0.40 ± 0.00 0.75 ± 0.05 66.0 ± 5.6 705 ± 15 MI2RKO (6) 0.28 ± 0.02b 0.48 ± 0.02b 3.30 ± 0.07b 1.98 ± 0.07b 0.30 ± 0.03b 0.50 ± 0.03b 39.4 ± 2.1b 645 ± 15b ↵ a Values are means ± standard errors of the means. IVS-D, diastolic interventricular septum wall thickness; IVS-S, systolic interventricular septum wall thickness; LV-D, diastolic left ventricle diameter; LV-S, systolic left ventricle diameter; PW-D, diastolic left ventricle posterior wall thickness; PW-S, systolic left ventricle posterior wall thickness; LVFS, left ventricular fractional shortening; HR, heart rate.
↵ b Significantly different from value obtained with DLox mice (P < 0.05).
- TABLE 3.
Genes of the contractile apparatus are up-regulated in MI2RKO hearts at postnatal day 20a
Contractile apparatus proteinb Mean ratio of expressionc at: P8 P20 MI2RKO MIRKO MIGF1RKO MI2RKO MIRKO MIGF1RKO β-MHC 4.32 ± 0.52 54.31 ± 5.20 {5.74 ± 0.92d 1.88 ± 0.53 0.99 ± 0.24 Tnni1 22.55 ± 5.52 Tpm2 1.44 ± 0.10 0.91 ± 0.04 1.50 ± 0.11 8.63 ± 1.16 2.26 ± 0.24 1.24 ± 0.15 CARP 0.96 ± 0.06 7.96 ± 0.31 2.12 ± 0.19 1.31 ± 0.12 Acta1 3.92 ± 0.71 2.65 ± 0.28 Flnc 2.81 ± 0.41 Myom2 0.68 ± 0.04 2.82 ± 0.24 2.16 ± 0.23 Actn4 1.75 ± 0.09 Smpx 1.55 ± 0.18 Des 0.77 ± 0.07 0.78 ± 0.05 1.57 ± 0.17 ↵ a Genes encoding proteins associated with the contractile apparatus were selected if the mean change in expression (n-fold change) by microarray for any pairwise comparison of MI2RKO, MIRKO, or MIGF1RKO mice versus the mean change in expression for DLox mice for all probe sets at either time point was >1.5.
↵ b Genes encoding proteins directly associated with the Z disk are shown in bold type. β-MHC, myosin heavy chain, beta isoform; Tnni1, troponin, slow skeletal, 1; Tpm2, tropomyosin 2; CARP, cardiac ankyrin repeat protein; Acta1, alpha-actin 1; Flnc, filamin C; Myom2, myomesin 2; Actn4, alpha-actinin 4; Smpx, small muscle protein, X-linked; Des, desmin.
↵ c The mean ratio of expression of the indicated mice versus that of DLox mice for all probe sets specific to the genes encoding the listed proteins is reported. Values are means ± standard errors of the means. Cells are blank when no probe sets met the significance criterion. The lower values for β-MHC at P8 indicated by the bracket represent quantitative real-time PCR data, for which mean ratios versus that of DLox mice are reported regardless of significance.
↵ d Significantly different from value obtained with DLox mice (P < 0.01).
- TABLE 4.
Genes of the electron transport chain are down-regulated in MI2RKO and MIRKO hearts
Electron transport chain proteina Mean ratio of expressionb MI2RKO MIRKO MIGF1RKO Cytochrome c Cycs 0.84 ± 0.04 {0.79 ± 0.07c 1.04 ± 0.04 1.07 ± 0.04 Cyct 0.55 ± 0.13 Complex I (NADH dehydrogenase [ubiquinone]) Ndufa3 0.72 ± 0.04 0.72 ± 0.04 {0.73 ± 0.03d 0.93 ± 0.07 0.99 ± 0.06 Ndufa6 0.84 ± 0.04 Ndufa7 0.83 ± 0.02 0.86 ± 0.03 Ndufb4 0.84 ± 0.04 0.90 ± 0.06 Ndufb9 0.80 ± 0.03 0.76 ± 0.03 0.80 ± 0.04 Ndufb2 0.82 ± 0.03 Ndufa4 0.73 ± 0.06 Ndufa1 0.73 ± 0.06 0.75 ± 0.04 0.68 ± 0.03 Ndufv2 0.79 ± 0.03 0.87 ± 0.03 Ndufa9 0.79 ± 0.03 0.81 ± 0.03 0.77 ± 0.05 Complex II (succinate dehydrogenase) Sdhc 1.13 ± 0.12 1.08 ± 0.16 Sdhd 1.25 ± 0.15 Complex III (ubiquinol-cytochrome c) reductase Uqcrc1 0.98 ± 0.06 1.00 ± 0.11 Uqcrc2 0.93 ± 0.07 1.00 ± 0.11 Uqcrb 0.87 ± 0.08 1500040F11Riken 0.71 ± 0.07 0.71 ± 0.05 0.69 ± 0.08 Complex IV (cytochrome c oxidase) Cox4i1 0.77 ± 0.05 0.73 ± 0.04 {0.82 ± 0.08e 0.94 ± 0.07 0.88 ± 0.09 Cox5a 1.02 ± 0.10 Cox5b 0.75 ± 0.04 0.77 ± 0.05 0.75 ± 0.04 Cox6a2 0.77 ± 0.04 0.60 ± 0.05 Cox7a2 0.86 ± 0.12 0.86 ± 0.04 Cox8b 0.75 ± 0.06 0.73 ± 0.06 Cox8a 0.71 ± 0.07 0.85 ± 0.17 Complex V (ATP synthase) Atp5a1 0.94 ± 0.08 Atp5b 0.83 ± 0.03 0.82 ± 0.05 {0.85 ± 0.05f 0.87 ± 0.05e 0.97 ± 0.08 Atp5c1 0.95 ± 0.07 Atp5d 0.83 ± 0.03 0.84 ± 0.06 0.90 ± 0.08 Atp5e 0.82 ± 0.04 Atp5j 0.83 ± 0.04 Atp5o 0.96 ± 0.06 1.16 ± 0.15 Atp5g1 0.85 ± 0.04 0.82 ± 0.06 Atp5g2 0.99 ± 0.10 Atp5g3 0.79 ± 0.04 0.77 ± 0.05 0.78 ± 0.05 Atp5h 0.80 ± 0.03 0.84 ± 0.02 0.80 ± 0.02 Atp5k 0.67 ± 0.06 Atp5j2 0.85 ± 0.03 0.85 ± 0.03 Atp5l 0.80 ± 0.04 Atp5s 0.82 ± 0.06 0.84 ± 0.04 Atpi (inhibitor) 1.37 ± 0.15 No. of genes significantly decreased (microarray data)/total no. of genes 27/27 26/30 13/17 ↵ a All genes encoding electron transport chain proteins for which at least one probe set had a P value of <0.05 for one of the pairwise comparisons of MI2RKO, MIRKO, or MIGF1RKO heart versus DLox heart are listed.
↵ b For each individual pairwise comparison, the mean ratio of expression of the indicated mice versus that of DLox mice for all probe sets specific to genes encoding the listed proteins is reported. Cells are blank when no probe set met the significance criterion. The lower set of values indicated by each set of brackets is quantitative real-time PCR data, for which mean ratios versus that of DLox are reported regardless of significance.
↵ c Significantly different from value obtained with DLox mice (P < 0.05).
↵ d Significantly different from value obtained with DLox mice (P < 0.0005).
↵ e Significantly different from value obtained with DLox mice (P = 0.08).
↵ f Significantly different from value obtained with DLox mice (P = 0.07).
- TABLE 5.
Genes of mitochondrial fatty acid beta-oxidation are down-regulated in MI2RKO hearts
Fatty acid beta-oxidation proteina Enzyme Mean ratio of expressionb MI2RKO MIRKO MIGF1RKO Facl2 Fatty acid-CoA ligase,g long chain, 2 0.90 ± 0.03 1.09 ± 0.05 1.05 ± 0.05 Cpt1a Carnitine palmitoyltransferase 1a 1.25 ± 0.14 Slc25a20 Carnitine/acylcarnitine translocase 0.85 ± 0.08 {0.80 ± 0.07f 1.06 ± 0.11 1.12 ± 0.10 Cpt2 Carnitine palmitoyltransferase 2 0.80 ± 0.12 1.31 ± 0.12 {0.60 ± 0.05e 0.84 ± 0.05c 1.03 ± 0.08 Acadm Acyl-CoA dehydrogenase, medium chain 0.77 ± 0.04 Acads Acyl-CoA dehydrogenase, short chain 0.65 ± 0.04 0.73 ± 0.05 Pecr Peroxisomal trans-2-enoyl-CoA reductase 0.65 ± 0.04 {0.99 ± 0.10 0.92 ± 0.09 1.05 ± 0.13 Hadha Trifunctional enzyme alpha subunit 0.80 ± 0.04 {0.77 ± 0.05d 0.96 ± 0.05 0.97 ± 0.05 Scp2 Sterol carrier protein 2 0.81 ± 0.06 0.85 ± 0.03 No. of genes significantly decreased (microarray data)/total no. of genes 8/8 0/1 2/5 ↵ a The genes encoding all proteins for which at least one probe set on the microarray had a P value of <0.05 for one of the pairwise comparisons of MI2RKO, MIRKO, or MIGF1RKO heart versus DLox heart are listed.
↵ b For each individual pairwise comparison, the mean ratio of expression of the indicated mice versus DLox mice for all probe sets specific to the genes encoding the listed proteins is reported. Cells are blank when no probe set met the significance criterion. The lower set of values in brackets are quantitative real-time PCR data, for which mean ratios versus DLox are reported regardless of significance.
↵ c Significantly different from value obtained with DLox mice (P < 0.05).
↵ d Significantly different from value obtained with DLox mice (P < 0.005).
↵ e Significantly different from value obtained with DLox mice (P < 0.0005).
↵ f Significantly different from value obtained with DLox mice (P = 0.08).
↵ g CoA, coenzyme A.
