Spontaneous calcium signaling of cartilage cells: from spatiotemporal features to biophysical modeling

Abstract

Intracellular calcium ([Ca²⁺]_i) oscillation is a fundamental signaling response of cartilage cells under mechanical loading or osmotic stress. Chondrocytes are usually considered as nonexcitable cells with no spontaneous [Ca²⁺]_i signaling. This study proved that chondrocytes can exhibit robust spontaneous [Ca²⁺]_i signaling without explicit external stimuli. The intensity of [Ca²⁺]_i peaks from individual chondrocytes maintain a consistent spatiotemporal pattern, acting as a unique "fingerprint" for each cell. Statistical analysis revealed lognormal distributions of the temporal parameters of [Ca²⁺]_i peaks, as well as strong linear correlations between their means and sds. Based on these statistical findings, we hypothesized that the spontaneous [Ca²⁺]_i peaks may result from an autocatalytic process and that [Ca²⁺]_i oscillation is controlled by a threshold-regulating mechanism. To test these 2 mechanisms, we established a multistage biophysical model by assuming the spontaneous [Ca²⁺]_i signaling of chondrocytes as a combination of deterministic and stochastic processes. The theoretical model successfully explained the lognormal distribution of the temporal parameters and the fingerprint feature of [Ca²⁺]_i peaks. In addition, by using antagonists for 10 pathways, we revealed that the initiation of spontaneous [Ca²⁺]_i peaks in chondrocytes requires the presence of extracellular Ca²⁺, and that the PLC-inositol 1,4,5-trisphosphate pathway, which controls the release of calcium from the endoplasmic reticulum, can affect the initiation of spontaneous [Ca²⁺]_i peaks in chondrocytes. The purinoceptors and transient receptor potential vanilloid 4 channels on the plasma membrane also play key roles in the spontaneous [Ca²⁺]_i signaling of chondrocytes. In contrast, blocking the T-type or L-type voltage-gated calcium channel promoted the spontaneous calcium signaling. This study represents a systematic effort to understand the features and initiation mechanisms of spontaneous [Ca²⁺]_i signaling in chondrocytes, which are critical for chondrocyte mechanobiology.-Zhou, Y., Lv, M., Li, T., Zhang, T., Duncan, R., Wang, L., Lu, X. L. Spontaneous calcium signaling of cartilage cells: from spatiotemporal features to biophysical modeling.

Keywords: articular; autocatalytic; chondrocytes; fingerprint; threshold regulating.

Figure 1

Harvest of fresh cartilage explant and fluorescent calcium imaging of in situ chondrocytes. A) Fresh cartilage explants were harvested from the central region of femoral condyle heads of bovine knee joint. B) Half cylindrical cartilage explant was dyed with Fluo-8 AM, placed in an imaging chamber filled with DMEM medium, and imaged on a confocal microscope. Imaging area was at the central region of the cross-section area, which was 200 µm below the articular surface. Fluorescent images of the in situ chondrocytes located 50 µm inside the tissue were taken at 1.5 s per frame for 16 min (scale bar, 50 µm). C) A typical [Ca²⁺]_i intensity curve of chondrocyte with 2 spontaneous peaks. Spatiotemporal parameters of the [Ca²⁺]_i peaks include number of peaks, normalized maximum magnitude of a peak (m₁), time from baseline to the peak (t₁), and time from peak value to 50% relaxation (t₂).

Figure 2

Calcium signaling pathways and their effects on the calcium responsive percentage of chondrocytes. A) Ten treatment groups were designed to understand the roles of several essential pathways in the spontaneous calcium signaling of in situ chondrocytes. The pathways include extracellular Ca²⁺ source, intracellular ER calcium store, purinoceptors, mechanosensitive channel, TRPV4 channel, T-type VGCC (T-VGCC) and L-type VGCC (L-VGCC), and intercellular gap junctions on plasma membrane. The corresponding antagonist of each pathway is listed in black. Length scales of the components involved in calcium signaling are listed at the bottom for reference. B–D) Responsive percentage of in situ chondrocytes, defined as the number of cells with spontaneous [Ca²⁺]_i peaks divided by the total number of cells in the recorded fluorescent videos. Responsive percentages of the antagonist-treated samples and their corresponding controls are compared in each plot. Data are shown as means + sd with data points overlapped. ***P < 0.001.

Figure 3

Spatiotemporal parameters of spontaneous [Ca²⁺]_i peaks and the effects of pathway antagonists. Data from all treated groups were normalized to their corresponding control sample (Ctrl). A) Magnitude of [Ca²⁺]_i peaks. B) Number of [Ca²⁺]_i peaks in the responsive chondrocytes. C) Time to reach a peak from baseline. D) Peak relaxation time. Data are shown as means + sd. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Statistical analysis of the spatiotemporal parameters of the spontaneous [Ca²⁺]_i peaks. A, B) Statistical distribution of time to reach a peak plotted at a regular (A) and a logarithmic (B) scale. The distribution trends were plotted in the lognormal form (A) and gaussian normal form (B). Shapiro-Wilk normality test indicates that the distribution in logarithmic scale follows a gaussian normal form (P = 0.797). C) Mean and sd of spatiotemporal parameters of each explant were plotted together, and the correlation between the means and sd were tested. Pearson’s r value was calculated to confirm the linear relationship; the value r > 0.8 indicates a very strong linear correlation.

Figure 5

Fingerprint-like feature of [Ca²⁺]_i peaks for each individual cell. A) [Ca²⁺]_i peaks from a single cell at different times displayed almost identical spatiotemporal patterns. B) [Ca²⁺]_i peaks released by the same chondrocyte were stacked to illustrate the fingerprint-like feature. Each plot has 2 [Ca²⁺]_i peaks from an individual chondrocyte. One-dimensional (1D) cross-correlation coefficient was calculated to evaluate the similarity between the 2 peaks. Coefficient of 1 indicates a perfect match.

Figure 6

Curve-fitting of [Ca²⁺]_i peaks using a simplified multistage biophysical model. A) Typical curve fitting of [Ca²⁺]_i peaks using the new theoretical model in this study. s₁ is the exponential initiation stage, s₂ denotes the logistic rising stage, and s₃ is the reciprocal relaxation stage. R² values of the curve fitting in each stage are reported. B, C) Autocatalytic-related parameters of the spontaneous [Ca²⁺]_i peaks were calculated for all treated groups using the theoretical model. Parameters from all treated groups were normalized to their corresponding control. B) The calcium induced calcium influx constant, in Supplemental Eq. S1, indicates the reaction rate for current [Ca²⁺]_i concentration to induce Ca²⁺ influx in the autocatalytic process. C) The calcium induced calcium outflux constant, in Supplemental Eq. S3, indicates the reaction rate for current [Ca²⁺]_i concentration to induce Ca²⁺ outflux in the autocatalytic process. Data are shown as means + sd; **P < 0.01, ***P < 0.001.