Abnormal response of costal chondrocytes to acidosis in patients with chest wall deformity

Abstract

Costal cartilage is much understudied compared to the load bearing cartilages. Abnormally grown costal cartilages are associated with the inherited chest wall deformities pectus excavatum and pectus carinatum resulting in sunken or pigeon chest respectively. A lack of understanding of the ultrastructural and molecular biology properties of costal cartilage is a major confounder in predicting causes and outcomes of these disorders. Due to the avascular nature of cartilage, chondrocytes metabolize glycolytically, producing an acidic environment. During physical activity hydrogen ions move within cartilage driven by compressive forces, thus at any one time, chondrocytes experience transient changes in pH. A variety of ion channels on chondrocytes plasma membrane equip them to function in the rapidly changing conditions they experience. In this paper we describe reduced expression of the ASIC2 gene encoding the acid sensing ion channel isoform 2 (previously referred to as ACCN1 or ACCN) in patients with chest wall deformities. We hypothesized that chondrocytes from these patients cannot respond normally to changes in pH that are an integral part of the biology of this tissue. Activation of ASICs indirectly creates a cascade ultimately dependent on intracellular calcium transients. The objective of this paper was to compare internal calcium signaling in response to external pH changes in costal chondrocytes from patients with chest wall deformities and healthy individuals. Although the molecular mechanism through which chondrocytes are regulated by acidosis remains unknown, we observed reduced amplitudes of calcium rise in patient chondrocytes exposed to low pH that become further impaired upon repeat exposure.

Keywords: Calcium; Cartilage; Chondrocytes; Ion channels; Pectus carinatum; Pectus excavatum.

Figure 1.

Median fold changes in non-selective ion channels in chondrocytes of CWD patients using qPCR. Pectus excavatum (PE; purple box plot) and pectus carinatum (PC; blue box plot) are shown relative to normal costal cartilage. Each sample was analyzed with an n = 3 and significant changes (p < 0.05) relative to normal costal cartilage are indicated (*). The green and red horizontal lines indicated a +2 or −2 fold change, respectively. Outlying data points in PC patients are indicated by unconnected orange dots. Genes analyzed are ASIC1, 2, and 3 Acid Sensing Ion Channel unit 1, 2, and 3; BCNG2 (HCN2) Hyperpolarization Activated Cyclic Nucleotide Gated Potassium and Sodium Channel 2; BEST1 Bestrophin; TRPC1 Transient Receptor Potential Cation Channel Subfamily C Member 1; TRPV1–4 Transient Receptor Potential Cation Channel Subfamily V Members 1–4.

Figure 2.

Costal chondrocytes from a control (C-08) and a PC patient (PC16) were exposed to change of pH and ratiometric measurements of calcium release made. pH was changed from 7.4 to 5.5 at 60 seconds and calcium release plotted against time.

Figure 3.

Graphical display of intracellular calcium transient time properties following pH challenge in C-08 and PC16 chondrocytes. Significant differences (p < 0.05, < 0.01, and < 0.001) are indicated by asterisks (*,**, and ***, respectively).

Figure 4.

Calcium release from patient and control cells following a second exposure to acidic pH. (A) The pH was reduced to pH 5.5 at 30 seconds and calcium release measured in PC34 and C-10 cells. (B) Cells were restored to pH 7.4 and then changed for the second time to pH 5.5. Calcium release from patient cells were significantly lower than the first exposure (p < 0001).

Figure 5.

Calcium is released from internal stores upon exposure to low pH. Calcium release was measured in C-08 cells when the pH was reduced to 5.5 at 30 seconds. External calcium was chelated with EGTA.

Figure 6.

(A) Ca²⁺-release from intracellular stores of three individual C-08 cells (black, red, and blue traces) upon perfusion with low pH (5.5) in zero Ca²⁺solution (B) Dynamic of Ca²⁺ release from internal stores in zero Ca²⁺ solution. To distinguish components of compound Ca²⁺ release traces from (A) were shifted to the same point in the moment of initial Ca²⁺-rise differentiated and averaged. (B) show two speed components (fast and slow) in the dynamic release of Ca²⁺ .

Figure 7.

Metabolic activity of costal chondrocytes were measured after being subject to low pH: (A) 6.5, (B) 6.0, and (C) 5.5, at several time points between control (blue), PC (green), and PE (red) cells. Metabolic activity levels displayed are compared to cells treated with normal media(y-axis) at each time point (x-axis). Significant differences (p < 0.05) between control and both PC and PE are indicated (*).