Cell signaling and regulation of smooth muscle contraction from a physiological and a pathophysiological perspective

Abstract

The aim of this thesis was to examine the cell signaling and regulation of smooth muscle in different smooth muscle tissues and under pathophysiological conditions. The thesis is based on 4 papers and the experimental work is based on in vitro studies in mouse.

In Paper I we addressed the question if key characteristics of fast and slow smooth muscle types could be identified, based on contractile, cell signaling and metabolic properties. We examined 4 different smooth muscle mouse tissues (aorta, muscular arteries, intestine, urinary bladder) with a large span in contractile kinetics (Vmax, maximal shortening velocity) based on SM-B expression (”fast” inserted myosin heavy chain). A quantitative PCR (qPCR) and Western blot approach was used to examine expression of key components in the contractile, metabolic and cell signaling pathways. A large variability between different smooth muscle tissues was found regarding contractile, cell signaling and metabolism. The reported main characteristics of fast and slow smooth muscle can serve as a basis for future studies of smooth muscle properties.

In Paper II we addressed the question if the two main Ca2+-sensitizing pathways: RhoARhokinase and protein kinase C (PKC) are altered in response to hypertrophic growth in the urinary bladder. We used a mouse model, partial urinary outlet obstruction, to induce hypertrophic growth of the smooth muscle. It mimics the over active bladder syndrome (OAB) in man, a pathophysiological condition affecting the urinary bladder with sudden and frequent urges to urinate, nocturia and urge incontinence. To examine if active force was altered, we used in vitro force recordings and direct nerve stimulation in open organ baths. Western blot analysis was used to determine if the relative protein expression of components mediating signaling in the RhoA-Rhokinase and PKC pathways. Direct nerve stimulation showed an increased cholinergic response in the hypertrophic smooth muscle, with a lower purinergic and increased nerve independent component. The hypertrophic smooth muscle also had an increased sensitivity to cholinergic stimulation and increased Rho dependent Ca2+ sensitivity that correlated with a lower phosphatase (MYPT1) expression and higher expression of both RhoGDI and RhoA. Based on these results and the profiling of the cell signaling in Paper I, it seems likely that hypertrophic growth of the urinary bladder induces transition from a fast smooth muscle type towards a slow smooth muscle type.

In Paper III we addressed the question if nonmuscle myosin (NMM) can be upregulated in response to hypertrophic growth in the urinary bladder and be involved in a PKC-induced contractile component observed in the hypertrophying urinary bladder. We examined the relative expression of NMM with Western blot and immunohistochemistry. Active force was analyzed using in vitro force recordings in open organ baths. In addition to smooth muscle myosin (SMM), the smooth muscle can express NMM, able to support a contraction with slow kinetics. However, in the urinary bladder NMM is only expressed during fetal life and downregulated shortly after birth. Western blot analysis showed an increased NMM expression in the hypertrophic smooth muscle compared to control. Immunohistochemistry showed an increased expression of NMM in the suburothelium, the smooth muscle layer and the serosa, for the hypertrophic urinary bladder compared to the control bladder. Direct activation of protein kinase C (PKC) with PDBu gave a prominent contraction that was independent of Rhokinase. Blebbistatin is an inhibitor of nonmuscle myosin, with higher affinity for NMM than for SMM. The PKC induced contraction was almost completely abolished by blebbistatin, indicating that NMM is involved in this unique contractile component in hypertrophic urinary bladder. Smooth muscle from the hypertrophying urinary bladder can thus develop a unique PKC activated contractile component based on nonmuscle myosin, mainly localized to the serosa. However, this contractile component is not a major part of the normal muscarinic contraction, instead it may contribute to wall stiffness and be activated by other (unknown) upstream pathways.

In Paper IV we addressed the question if smooth muscle contraction is sensitive to metabolic inhibition and if there is a difference in sensitivity to metabolic block between fast and slow smooth muscle types, due to their different metabolic properties determined in Paper I. The mechanisms of metabolic control of smooth muscle are poorly understood. We approached this question by introducing a partial metabolic blocker (rotenone) of complex I in the mitochondria, resembling e.g. ischemic conditions due to atherosclerotic changes or ageing. To confirm that rotenone slows down the mitochondrial respiration, we measured oxygen consumption in the relaxed smooth muscle tissue and found about 50% inhibition by rotenone. We measured active force using in vitro force recordings in open organ bath, in the presence of blockers and activators to target membrane channels and cellular component that potentially might be affected by the metabolic stress induced by rotenone. Active force of the fast (urinary bladder) was more sensitive to rotenone than that of the slow smooth muscle (aorta), which correlates well with the metabolic profiling in Paper I. AMP-kinase, a metabolic sensor, is activated by metabolic stress (increased ADP:ATP and/or AMP:ATP ratios) and initiates a range of energy-saving processes in the cell. AICAR (AMPK activator) partially attenuated the rotenone effects on contraction, whereas dorsomorphin (AMPK blocker) dramatically increases the inhibitory effect of rotenone. Thus, AMPkinase appears to have a protective action during metabolic stress induced by rotenone in the smooth muscle.

In summary, this thesis demonstrates a large variability between fast and slow smooth muscle tissues regarding contractile properties, signaling and metabolism. Smooth muscle has an impressive ability to adapt during pathophysiological stress, e.g. in the urinary bladder. In the hypertrophic bladder muscle cell signaling is affected, increasing both the nerve induced cholinergic component and Rho-mediated Ca2+ sensitivity. In addition, hypertrophic smooth muscle can also develop a unique contractile component dependent on nonmuscle myosin that is activated by PKC. Partial metabolic block inhibits active force in the smooth muscle and can partially be prevented by AMPkinase. Compared to the slow smooth muscle, the fast smooth muscle is more sensitive to metabolic stress induced by rotenone.