INCREASED TISSUE STIFFNESS WITH AGE ALTERS DISTAL COLON SMOOTH MUSCLE CONTRACTILITY

Background: Gastrointestinal (GI) motility disorders, such as chronic constipation, dysphagia, and fecal impaction, are more common in the elderly. In the vascular system, increased tissue stiffness with age is associated with a decline in contractile function of smooth muscle cells (SMCs). Our preliminary studies, using open angle measurements, showed that GI tissue stiffness increases in a region-specific manner with age, with greatest changes in the ileum and distal colon. It is not known whether changes in GI smooth muscle tissue stiffness affect the contractile function of GI SMCs, and, subsequently, impact GI physiology.
Aim: To test the hypothesis that age-induced changes in GI smooth muscle tissue stiffness modulate the contractile function of GI SMCs.
Methods: Mucosa-stripped distal colon smooth muscle tissues from 3- and 24-month-old wild type C57BL/6J mice were used for atomic force microscopy (AFM), muscle bath and RT-qPCR experiments. Expression of contractile SMC markers smoothelin (Smtn), transgelin (Tagln), myosin heavy chain 11 (Myh11) and smooth muscle actin-α2 (Acta2) were analyzed through RT-qPCR. Immortalized human colonic SMCs (HuSMCs) and freshly dissociated SMCs from 3-month-old mouse colons (primary SMCs) were cultured on soft (1kPa) and stiff (50kPa) substrate dishes to model age-dependent shifts in distal colon stiffness for 48 hours and used for RT-qPCR.
Results: AFM measurements showed increased stiffness with age (3mo:8.24±2kPa; 24mo:10.8±2.5kPa, n=10-12, p<0.05). Isometric length-tension measurements of circular muscle confirmed increased stiffness with age (3mo:31.3±4.5kPa, 24mo:43.3±8.4kPa, at 100% strain, n=4, p<0.05). The expression of contractile SMC marker decreased with age (Smtn: 0.58±0.25-fold, Tagln: 0.57±0.17-fold, Myh11: 0.54±0.16-fold, Acta2: 0.6±0.2-fold, n=3, p<0.05). Carbachol-induced active contractions of circular muscle was higher in the young tissues compared to aged tissues (3mo:56.7±10.5kPa, 24mo:33.5±14kPa, at 100% strain, n=4, p<0.05) suggesting loss of smooth muscle contractility with age. HuSMCs and primary SMCs on stiff substrate showed decreased expression of contractile SMC markers (HuSMCs: SMTN:0.6±0.1-fold, MYH11: 0.3±0.1-fold, ACTA2: 0.3±0.03-fold; Primary SMCs: Smtn: 0.4±0.1-fold, Tagln: 0.35±0.18-fold, Myh11: 0.3±0.17-fold, Acta2: 0.37±0.16-fold,, n=3, p<0.05) compared to SMCs on soft substrate suggesting stiffness modulates contractile phenotype of GI SMCs.
Conclusion: Colonic smooth muscle tissue stiffness increases with age, and likely contributes to a loss of SMC contractility. Current studies are focused on determining the nature of age-related gut wall stiffening and the molecular mechanisms connecting tissue stiffening and contractility. Funding: NIH-DK052766, DK123549, HL163168; Fundamental Mechanisms of Aging Award, Robert and Arlene Kogod Center on Aging, Mayo Clinic.