Pharmacological inhibition of Integrin a5b1 improves insulin sensitivity in H9C2 cells and cardiac performance in obese mice

Background and aims: Extracellular matrix (ECM) remodelling has recently emerged as a key contributor to the development of insulin resistance (IR). Inhibition of ECM collagen and hyaluronan deposition by clinical and preclinical antifibrotic drugs attenuated cardiac IR and cardiac dysfunction in diet-induced obese mice. Fibronectin is a multifunctional glycoprotein in the ECM known to communicate with cells mainly via the integrin α5β1 receptor. The present study investigated the effects of CLT-28643, an α5β1 integrin antagonist, on insulin sensitivity in H9C2 cardiomyocytes, and cardiac function in high-fat-high-sucrose (HFHS) fed obese mice.

Materials and methods: In vitro effect of ECM on insulin signalling was investigated by culturing H9C2 cells on fibronectin coated plate for 5 days followed by 15 min treatment with insulin (10nM). Furthermore, insulin resistant H9C2 cells, induced by high glucose (33.3 mM) and palmitic acid incubation (100 μM) for 24-h, were subjected to CLT-28643 treatment for 3 hrs followed by 15 min incubation with insulin (100nM). Insulin sensitivity of treated cells was examined by phosphorylation of Akt using western blot. In vivo, male C57BL/6 mice fed 45% high-fat diet and 30% sucrose water for 10 weeks received twice-daily oral gavage of either vehicle or CLT-28643 (75 mg/kg body weight) for 4 weeks. A separate group of mice fed with chow diet for 14 weeks were used as lean controls (n=8). Cardiac function was measured by Pressure-Volume (PV) loop analysis (Transonic) using PV conductance catheter in closed-chest preparation. Immunohistochemical analysis was performed to assess changes in collagen and alpha-smooth muscle actin (α-SMA) expression in the left ventricle (LV) of the heart.

Results: In H9C2 cells, FB coating decreased insulin-stimulated phosphorylation of Akt (Ser473); CLT28643 prevented high glucose & palmitate-induced IR as indicated by an increased ratio of the phosphorylated Akt (Ser473) to total AKT compared to vehicle-treated cells (2.67±1.55 fold increase, P<0.05). In mice, HFHS diet resulted in significant increases in body weight (29.05 ± 0.82 (chow) vs 40.86 ± 1.34 (HFHS) gram, P<0.0001), %fat mass (15.52 ± 0.80 vs. 51.30 ± 5.89%, P<0.0001) and decreases in %lean mass (78.29 ± 0.98 vs. 58.61 ± 1.01%, P<0.0001) compared to chow-fed mice. CLT28643 displayed a trend of decrease in %fat mass, despite no changes in body weight or %lean mass. HFHS diet impaired cardiac function in mice as evidenced by a decrease in end-systolic elastance (31.06 ± 6.23 (chow) vs 10.88 ± 1.47 (HFHS) mmHg/µL, P<0.01), ejection fraction (86.14 ± 4.24 vs 72.82 ± 3.60%, P<0.05) and an increase in heart rate (554.3 ± 21.41 vs 603.7 ± 9.72bpm, P<0.05) and ventricular arterial coupling (VAC) index (0.19 ± 0.06 vs 0.46 ± 0.07, P<0.05) compared to lean controls. Interestingly, elevated heart rate was reversed by CLT-28643 treatment, suggesting an improved cardiac performance; while the slope of the end-systolic pressure-volume relationship (ESPVR) and end-diastolic pressure-volume relation (EDPVR) were not different among groups. Immunohistochemical analysis revealed no significant changes in LV collagen and α-SMA expression in experimental mice.

Conclusion: Pharmacological inhibition of integrin α5β1 improved insulin signalling in H9C2 cells and cardiac performance in obese mice. These findings suggest that therapies targeting FB-α5β1 interaction may be beneficial to obesity-associated cardiometabolic complications.