Figure 1.
Lrrc10−/− mice develop dilated cardiomyopathy.
Echocardiography reveals cardiac functional deficits in Lrrc10−/− mice. Left ventricular inner diameter (LVID) measurements during (A) diastole and (B) systole and (C) fractional shortening (F.S.) in WT and Lrrc10−/− mice at the indicated age. (D) Representative M-mode images at 10 months of age. E, embryonic day; NB, newborn; m, months; d, diastole; s, systole. n = 4–13.
Table 1.
Echocardiographic assessment of cardiac structure and function in Lrrc10−/− mice.
Figure 2.
Characterization of Lrrc10−/− hearts.
(A) qRT-PCR analysis for ANF (Nppa), β-MHC (Myh7), and BNP (Nppb) demonstrates re-expression of the fetal genes in Lrrc10−/− hearts at 4 months of age (n = 7–8). (B) Representative immunoblot and (C) quantitation of ANF expression in WT and Lrrc10−/− hearts (n = 4). Expression level was normalized to GAPDH as a loading control. (D) Histology of WT and Lrrc10−/− mice at ten months of age. Representative (a) frontal midline heart sections (scale bar = 1 mm) and (b) 40X high magnification images (scale bar = 20 μm) of H&E and (c) Masson's trichrome staining. (E) Wet heart weight to body weight ratios indicate increased size of Lrrc10−/− hearts in adulthood (n = 6–12). (F) Isolated adult WT and Lrrc10−/− ventricular cardiomyocytes were micrographed (top left, scale bar = 10 μm) to measure cell length and width (n = 384 cells from 3 WT hearts and 435 cells from 3 Lrrc10−/− hearts). (G) Cardiomyocyte cross-sectional area (CSA) in WT and Lrrc10−/− hearts at 10 months (n = 4–6). (H) To measure relative left ventricle wall thickness as compared to left ventricle size, H/R ratio was calculated from echocardiography data. H/R ratio = (LVPWd+LVAWd)/LVIDd. m, months.
Figure 3.
LRRC10 interacts with actin and α-actinin.
(A) LRRC10 cofractionates with α-actin and α-actinin in the particulate fraction. Heart extracts from 10-week-old mice were fractionated into a nonparticulate fraction (Non) and a particulate fraction (Par) enriched in myofibril proteins. GAPDH and myosin heavy chain (MyHC) were used as nonparticulate and particulate controls, respectively. (B) Yeast-two hybrid screening identified α-actinin and γ-actin as binding partners of LRRC10. (C) α-actinin and α-actin endogenously interact with LRRC10 in the heart. WT or Lrrc10−/− mouse heart extracts were immunoprecipitated (IP) with preimmune serum (PreIM) or an LRRC10 antibody and immunblotted for α-actinin or α-actin. (D) LRRC10 interacts with α-, β-, and γ-actin in vitro. Purified α-actin or cytoskeletal actin was incubated with GST or a GST-LRRC10 fusion protein and pulled down proteins were immunoblotted for α-actin, γ-actin, or β-actin. (E) LRRC10 colocalizes with α-actin. Coimmunostaining for LRRC10 and α-actin in adult mouse ventricular cardiomyocytes. Enlargement of boxed area is shown at bottom. Scale bar = 10 μm.
Figure 4.
Gene expression profiling of embryonic Lrrc10−/− hearts.
Microarray analysis was performed on WT and Lrrc10−/− hearts at E15.5 to identify dysregulated pathways in the absence of Lrrc10. (A) Pathway analysis on genes upregulated 1.2-fold or greater (p<0.05, signal ≥20) at E15.5. Shown are the five most significantly upregulated KEGG pathways in Lrrc10−/− hearts using DAVID. The number at the end of the histograms represents the number of upregulated genes in the KEGG pathway. (B) Heat maps from expression profiling and (C) qRT-PCR analyses demonstrate upregulation of actin cytoskeloton transcripts in embryonic Lrrc10−/− hearts (n = 5–10). (D) Western blotting analysis of cytoskeletal protein expression in E15.5 hearts (n = 4–5). Actn2, actinin alpha 2; Actc1, actin alpha, cardiac muscle 1;Vcl, vinculin; Tln1, talin 1; ILK, integrin-linked kinase; Itgb1, integrin β1; Parva, parvin alpha; E; embryonic day.
Figure 5.
Gene expression profiling of adult Lrrc10−/− hearts.
Microarray analysis was performed on WT and Lrrc10−/− hearts at two months of age to identify dysregulated pathways in the absence of Lrrc10. (A) Pathway analysis on genes upregulated 1.2-fold or greater (p<0.05, signal ≥20) at two months of age. Shown are the five most significantly upregulated KEGG pathways in Lrrc10−/− hearts using DAVID. The number at the end of the histograms represents the number of upregulated genes in the KEGG pathway. (B, C) Heat maps from expression profiling (left) and qRT-PCR analyses (right, n = 6–12) demonstrate upregulation of many (B) cardiac muscle contraction and (C) oxidative phosphorylation transcripts in adult Lrrc10−/− hearts. Myh7, β-myosin heavy chain; Tnni3, troponin I, cardiac 3; Tnnt1, troponin T1, skeletal, slow; Tpm1, tropomyosin 1, alpha; Mybpc3, myosin binding protein C, cardiac; Mybpc2, myosin binding protein C, fast-type; Cytb, cytochrome b; Cytc, cytochrome c; Cox5a, cytochrome c oxidase subunit Va; Cox7a1, cytochrome c oxidase subunit VIIa1; Atp5c1, ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1; Atp5o, ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit; Atp5h, ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d; ATP6, ATP synthase F0 subunit 6; ND1, NADH dehydrogenase subunit 1; Ndufb9, NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9; Ndufa13, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13; Slc25a4, solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 4.
Figure 6.
Signal transduction in Lrrc10−/− hearts.
(A) Western blotting analysis for activation of Akt, p38, ERK, and STAT signaling pathways and PKC expression reveals no changes in phosphorylation of p38, ERK, or STAT or protein abundance of PKCα or PKCε. (B) Western blotting indicates hyperphosphorylation of Akt at Ser 473 in Lrrc10−/− hearts. (C) Quantitation of Western blotting confirms increased phosphorylation of Akt in Lrrc10−/− hearts (n = 7). Phosphorylated Akt was normalized to total Akt expression and GAPDH was used as a loading control. (D) Representative Western blotting of particulate and nonparticulate fractions of adult heart extracts in control (WT) and Lrrc10−/− mice. (E) Quantification of PKCε expression in the particulate fraction relative to the nonparticulate fraction. Myosin heavy chain (MyHC) and α-actinin were used as a particulate/myofilament loading control.
Figure 7.
Contractility of skinned myocardium and myofibril protein composition are not altered in Lrrc10−/− mice.
(A) SL-dependent increase in passive force is unaltered in Lrrc10−/− skinned trabeculae. The passive forces were measured in pCa 9.0 first at SL 2.00 μm then at 2.22 and finally at 2.35 μm in WT (n = 4; closed circles) and Lrrc10−/− (n = 4; open circles) skinned trabeculae. (B) Force-pCa relationships established at SL 2.20 μm yielded nearly identical sigmoidal plots in WT and Lrrc10−/− skinned trabeculae. (C–D) Deletion of Lrrc10 has no effect on the rate of force redevelopment. (C) ktr-pCa and (D) ktr-relative force relationships were established at SL 2.22 μm in skinned trabeculae. (E) Silver staining for myofibril protein composition in WT and Lrrc10−/− hearts.