Gene expression and β-adrenergic signaling are altered in hypoplastic left heart syndrome

Abstract

Background: The purpose of the current study was to define the myocellular changes and adaptation of the β-adrenergic receptor (β-AR) system that occur in the systemic right ventricle (RV) of children with hypoplastic left heart syndrome (HLHS).

Methods: Explanted hearts from children with HLHS and non-failing controls were used for this study. HLHS patients were divided into 2 groups: "compensated" (C-HLHS), infants listed for primary transplant with normal RV function and absence of heart failure symptoms, and "decompensated" (D-HLHS), patients listed for transplant after failed surgical palliation with RV failure and/or refractory protein-losing enteropathy or plastic bronchitis.

Results: Compared with non-failing control RVs, the HLHS RV demonstrated decreased sarcoplasmic reticulum calcium-adenosine triphosphatase 2a and α-myosin heavy chain (MHC) gene expression, decreased total β-AR due to down-regulation of β1-AR, preserved cyclic adenosine monophosphate levels, and increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity. There was increased atrial natriuretic peptide expression only in the C-HLHS group. Unique to those in the D-HLHS group was increased β-MHC and decreased α-MHC protein expression (MHC isoform switching), increased adenylyl cyclase 5 expression, and increased phosphorylation of the CaMK target site on phospholamban, threonine 17.

Conclusions: The HLHS RV has an abnormal myocardial gene expression pattern, downregulation of β1-AR, preserved cyclic adenosine monophosphate levels, and increased CaMKII activity compared with the non-failing control RV. There is MHC isoform switching, increased adenylyl cyclase 5, and increased phosphorylation of phospholamban threonine 17 only in the D-HLHS group. Although abnormal gene expression and changes in the β-AR system precede clinically evident ventricular failure in HLHS, additional unique adaptations occur in those with HLHS and failed surgical palliation.

Keywords: gene expression; hypoplastic left heart syndrome; right ventricle; β-adrenergic receptor system.

Figure 1

Recapitulation of the fetal gene program (FGP) occurs in the hypoplastic left heart syndrome (HLHS) right ventricle (RV). Messenger (m)RNA expression was determined in tissue from non-failing RV (NF-RV) controls (n = 11) and in RV tissue from compensated (C)-HLHS (n = 8) and decompensated (D)-HLHS patients (n = 11). There was a pathologic gene expression pattern in HLHS with increased expression of (A) atrial natriuretic peptide (ANP) in C-HLHS and (B) β-myosin heavy chain (MHC) in D-HLHS, along with decreased expression of (C) sarcoplasmic reticulum calcium-adenosine triphosphatase (SERCA) and (D) α-MHC in both HLHS groups compared with NF-RV. (E) B-type natriuretic peptide (BNP) was unchanged in HLHS compared with NF-RV. Results are presented as the mean with the standard error (error bars).

Figure 2

Western blot analysis demonstrates that protein expression of α-myosin heavy chain (MHC) is decreased in decompensated hypoplastic left heart syndrome (D-HLHS, n = 7) compared with non-failing right ventricle (NF-RV, n = 7) and compensated (C)-HLHS (n = 7). Results are presented as the mean with the standard error (error bars)

Figure 3

β-Adrenergic receptor (β-AR) is downregulated in compensated hypoplastic left heart syndrome (C-HLHS, n = 4) and decompensated (D)-HLHS (n = 7) compared with non-failing right ventricle (NF-RV, n = 6). (A) Total β-AR (Bmax) is decreased in HLHS as determined by [¹²⁵I]-iodocyanopindolol binding assay. The decrease of β-AR in both HLHS groups is due to (B) β₁-AR downregulation with (C) preserved β₂-AR expression. Results are presented as the mean with the standard error (error bars).

Figure 4

Cyclic adenosine monophosphate (cAMP) levels and adenylyl cyclase (AC) 6 (AC6) expression are unchanged in hypoplastic left heart syndrome (HLHS) compared with the non-failing right ventricle (NF-RV), whereas expression of AC5 is increased in decompensated (D)-HLHS. (A) cAMP levels determined by enzyme-linked immunosorbent assay are unchanged in HLHS (NF-RV, n = 8; compensated [C]-HLHS, n = 7; D-HLHS, n = 8). Messenger RNA levels of (B) AC5 and (C) AC6 were determined by real-time polymerase chain reaction (NF-RV, n = 8; C-HLHS, n = 8; D-HLHS, n = 8). Results are presented as the mean with the standard error (error bars).

Figure 5

Calcium/calmodulin-dependent protein kinase II (CaMKII) activity is increased in hypoplastic left heart syndrome (HLHS) compared with non-failing right ventricle (NF-RV, n = 9; compensated [C]-HLHS, n = 10; decompensated [D]-HLHS, n = 7). Results are presented as the mean with the standard error (error bars).

Figure 6

Phosphorylation of the calcium/calmodulin-dependent protein kinase (CaMK) target site on phospholamban (PLB), threonine 17 (PT17), is increased in decompensated hypoplastic left heart syndrome (D-HLHS) compared with the non-failing right ventricle (NF-RV). (A) By Western blot analysis, there was no difference in total PLB between HLHS (compensated [C]-HLHS, n = 9; D-HLHS, n = 9) and NF-RV (n = 9). (B) There was increased phosphorylation of PT17 in D-HLHS (n = 6) compared with NF-RV (n = 8) and C-HLHS (n = 10) (C) but no differences in phosphorylation of PLB at the protein kinase A (PKA) target site, Ser16, between groups (NF-RV, n = 9; C-HLHS, n = 10; and D-HLHS, n = 7). Results are presented as the mean with the standard error (error bars).