Hypoxic cardiac fibroblasts from failing human hearts decrease cardiomyocyte beating frequency in an ALOX15 dependent manner

Abstract

A common denominator for patients with heart failure is the correlation between elevated serum levels of proinflammatory cytokines and adverse clinical outcomes. Furthermore, lipoxygenase-induced inflammation is reportedly involved in the pathology of heart failure. Cardiac fibroblasts, which are abundant in cardiac tissue, are known to be activated by inflammation. We previously showed high expression of the lipoxygenase arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), in ischemic cardiac tissue. The exact roles of ALOX15 and 15-HETE in the pathogenesis of heart failure are however unknown. Biopsies were collected from all chambers of explanted failing human hearts from heart transplantation patients, as well as from the left ventricles from organ donors not suffering from chronic heart failure. Biopsies from the left ventricles underwent quantitative immunohistochemical analysis for ALOX15/B. Gene expression of ALOX enzymes, as well as 15-HETE levels, were examined in cardiac fibroblasts which had been cultured in either hypoxic or normoxic conditions after isolation from failing hearts. After the addition of fibroblast supernatants to human induced pluripotent stem cell-derived cardiomyocytes, intracellular calcium concentrations were measured to examine the effect of paracrine signaling on cardiomyocyte beating frequency. While ALOX15 and ALOX15B were expressed throughout failing hearts as well as in hearts from organ donors, ALOX15 was expressed at significantly higher levels in donor hearts. Hypoxia resulted in a significant increase in gene and protein expression of ALOX15 and ALOX15B in fibroblasts isolated from the different chambers of failing hearts. Finally, preconditioned medium from hypoxic fibroblasts decreased the beating frequency of human cardiomyocytes derived from induced pluripotent stem cells in an ALOX15-dependent manner. In summary, our results demonstrate that ALOX15/B signaling by hypoxic cardiac fibroblasts may play an important role in ischemic cardiomyopathy, by decreasing cardiomyocyte beating frequency.

Fig 1. Expression of ALOX15 and ALOX15B in left ventricular tissue from failing human hearts and donor hearts.

Biopsies were snap frozen with liquid nitrogen and processed for IHC staining. Slides were incubated with primary antibodies for ALOX15 (red) and ALOX15B (yellow), followed by incubation with fluorochrome-conjugated secondary antibodes and mounting with DAPI (blue). (A) Representative images of ALOX15 and ALOX15B expression in the failing human heart. (B) Representative images of ALOX15 and ALOX15B in donor hearts not suffering from chronic heart failure. Corresponding isotype controls are shown for a failing heart (C) as well as for a donor heart (D). (E) Quantification of ALOX15 and ALOX15B expression. Both heart failure patients and donors expressed ALOX15 and ALOX15B, with a significantly higher expression of ALOX15 in donor hearts. Group wise statistical comparisons were performed using two-sided Student’s T-tests.

Fig 2. Expression of ALOX15 and ALOX15B in hypoxic human cardiac fibroblasts.

Cardiac fibroblasts were isolated from left (n = 3) and right (n = 5) atrium as well as left (n = 6) and right (n = 6) ventricle and subjected to 1% or 21% oxygen for 24 h. (A) Harvested cells were subjected to RT-qPCR and paired T-tests was performed using log-transformed RQ values. Paired RQ values are demonstrated as dots combined by a line. *** p < 0.001, **p < 0.01, *p < 0.05. (B) Immunocytochemical staining of human cardiac fibroblasts with primary antibodies against ALOX15 (green), ALOX15B (red) followed by flurochrome-conjugated secondary antibodies and DAPI (blue). Representative images of normoxic and hypoxic fibroblasts are shown. Scale bar = 40 μM.

Fig 3. 15-HETE levels in cardiac fibroblasts under different oxygen tensions and ALOX15 inhibition.

Cardiac fibroblasts isolated from left (LA) and right (RA) atrium as well as left (LV) and right (RV) ventricle were subjected to 1% or 21% oxygen for 24 h. Hypoxic fibroblasts were cultured with or without the specific ALOX15 inhibitor ML351 (10 μmol/L) or the less specific ALOX12 and ALOX15 inhibitor baicalein (10 μmol/L). n = 5 for every chamber in the hypoxic and normoxic groups. n = 3 for every chamber in the inhibitor groups, except for ML351 treated fibroblasts from right atrium (n = 2). Two-way ANOVA was performed with Tukey's multiple comparisons test using log-transformed data. Concentrations were then normalized by subtracting the normoxic controls and means were calculated for demonstration. * p < 0.05, ** p < 0.001.

Fig 4. Preconditioned medium from hypoxic human cardiac fibroblasts affected cardiomyocyte beating frequency.

Cardiomyocytes derived from human induced pluripotent stem cells were cultured with preconditioned medium from human cardiac fibroblasts subjected to 1% or 21% oxygen with or without the addition of ML351 or baicalein, and calcium probe solution. Intracellular calcium concentrations were registered as relative fluorescence units (RFU) for up to 120 minutes and beating frequency was determined. (A) Representative images of changes in RFU after 60 minutes incubation with preconditioned medium. (B) Changes in beating frequency for different time points. One-way ANOVA was performed for each time point with Tukey's multiple comparisons test using log-transformed data. Data is demonstrated as mean ± SEM. * p < 0.05 for hypoxia vs. normoxia, baicalein and ML351 at 60 minutes.