Cardiac fibroblast activation and hyaluronan synthesis in response to hyperglycemia and diet-induced insulin resistance

Abstract

Diabetic patients are at a greater risk of heart failure due to diabetic cardiomyopathy and worsened outcome post-myocardial infarction. While the molecular mechanisms remain unclear, fibrosis and chronic inflammation are common characteristics of both conditions. Diabetes mellitus (types I and II) results in excessive hyaluronan (HA) deposition in vivo, and hyperglycemia stimulates HA synthesis for several cell types in vitro. HA-rich extracellular matrix contributes to fibrotic, hyperplastic and inflammatory disease progression. We hypothesized that excessive hyperglycemia-driven HA accumulation may contribute to pathological fibroblast activation and fibrotic remodelling in diabetic patients. Therefore, we analysed the impact of both hyperglycemia and diet-induced obesity and insulin resistance on HA matrix formation and cardiac fibroblast activation. Here we report that cardiac fibroblasts isolated from mice on a diabetogenic diet acquire pro-fibrotic gene expression without a concomitant increase in HA matrix deposition. Additionally, hyperglycemia alone does not stimulate HA synthesis or cardiac fibroblast activation in vitro, suggesting that the direct effect of hyperglycemia on fibroblasts is not the primary driver of fibrotic remodelling in cardiac diabetic maladaptation.

Figure 1

Hyperglycemia does not augment either HA matrix production or cardiac fibroblast activation in vitro. Cultures of primary cardiac fibroblasts were treated with media containing 5.5 or 25 mM glucose in 10% FBS for up to 72 hours. (a) Experimental design schematic. (b) Quantification of HA secretion into the media (n = 7) and deposition into the cell layer (n = 3). (c) Quantification of Has1, 2 and 3 mRNA expression, expressed as fold relative to 24 h 5.5 mM, 24 h (n = 6–9), 48 h (n = 5–8) and 72 h (n = 9–12). (d) Quantification of Acta2 mRNA expression after 72 h, expressed as fold of 5.5 mM (n = 7). (e) Quantification of Col1a1 mRNA expression after 72 h, expressed as fold of 5.5 mM (n = 7). For analysis of mRNA expression, Rn18S was used as an internal control. Data represent mean ± SEM; one-way ANOVA with Sidak’s multiple-comparison correction (b,c) and unpaired t-test (d,e).

Figure 2

Hyperglycemia does not enhance the HA matrix production or activation of cardiac fibroblasts stimulated with TGF-β1. Cultures of primary cardiac fibroblasts were treated with media containing 5.5 or 25 mM glucose in 1% FBS ± 10 ng/mL TGF-β1 for 72 hours. (a) Experimental design schematic. (b) Representative images of immunocytochemical staining of HA (red) and α-SMA (green) with quantification (c,e) (n = 7). (d) Quantification of HA secretion into the media (n = 8). (f) Quantification of Acta2 mRNA expression, expressed as fold of 5.5 mM without TGF-β1 (n = 4). (g) Quantification of Col1a1 mRNA expression, expressed as fold of 5.5 mM without TGF-β1 (n = 4). For analysis of mRNA expression, Rn18S was used as an internal control. Data represent mean ± SEM; one-way ANOVA with Sidak’s multiple-comparison correction (c,d,e,f,g). *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P < 0.0001.

Figure 3

Hyperglycemia does not augment the glucose uptake of cardiac fibroblasts in vitro. Cultures of primary cardiac fibroblasts were treated with media containing 5.5 or 25 mM glucose supplemented with radiolabeled 2-deoxyglucose in 10% FBS for up to 72 hours ± 100 nM insulin. (a) Experimental design schematic. (b) Time course of radiolabeled glucose uptake (n = 4). (c) Quantification of HA secretion into the media (n = 4). Data represent mean ± SEM; two-way ANOVA with Sidak’s multiple-comparison correction (b) and one-way ANOVA with Sidak’s multiple-comparison correction (c).

Figure 4

Cardiac fibroblasts exhibit high metabolic flexibility. To block the mitochondrial oxidation of glucose, glutamine and fatty acids, cultures of primary cardiac fibroblasts and oesophageal cancer cells (KYSE) were given the inhibitors UK5099, BPTES and etomoxir, respectively. Using a Seahorse XFe96 Analyzer, the oxygen consumption rate (OCR) was monitored in real time while injections of single pathway inhibitors were administered (Target inhibitor), followed by an injection of the remaining 2 inhibitors (All inhibitors) to determine each pathway’s contribution to the total mitochondrial respiration. (a) OCR time course of cardiac fibroblasts, values displayed relative to pre-injection OCR baseline (n = 4). (b) Relative cardiac fibroblast substrate dependency (n = 4). (c) OCR time course of KYSE cells, values displayed relative to pre-injection OCR baseline (n = 3). (d) Relative KYSE cell substrate dependency (n = 3). Data represent mean ± SEM; one-way ANOVA with Sidak’s multiple-comparison correction (b,d). **P ≤ 0.005, ****P < 0.0001.

Figure 5

Model of diet-induced obesity and insulin resistance. 8-week-old male C57BL/6 J mice were fed a standard chow (chow) or diabetogenic diet (DD) for 11 weeks. (a) Feeding schematic. (b) Body weight (n = 15). (c) Fasting blood glucose (n = 15). (d) Fixed-dose oral glucose tolerance (n = 11,12) with area under the curve (AUC) quantification. (e) Data represent mean ± SEM; two-way ANOVA with Sidak’s multiple-comparison correction (b) and unpaired t-test (c,e). *P ≤ 0.05, ***P ≤ 0.0005, ****P < 0.0001.

Figure 6

Diabetogenic diet does not result in excessive HA accumulation but does promote cardiac fibroblast activation. 8-week-old male C57BL/6J mice were fed standard chow (chow) or diabetogenic diet (DD) for 11 weeks. Subsequently, cardiac HA content was quantified, and cardiac fibroblasts were isolated and treated with media containing 5.5 or 25 mM glucose in 1% FBS ± 10 ng/mL TGF-β1 for 72 hours. (a) Feeding and experimental schematic. (b) Quantification and representative gel of cardiac HA assessed by FACE (n = 15). (c) Quantification of HA secretion by isolated cardiac fibroblasts (n = 8). (d) Quantification of Acta2 mRNA expression of isolated cardiac fibroblasts, expressed as fold of chow-fed without TGF-β1 (n = 6–8). (e) Quantification of Col1a1 mRNA expression of isolated cardiac fibroblasts, expressed as fold of chow-fed without TGF-β1 (n = 6–8). For analysis of mRNA expression, Rn18S was used as an internal control. Data represent mean ± SEM; unpaired t-test (b) and one-way ANOVA with Sidak’s multiple-comparison correction (c,d,e). *P ≤ 0.05.