Visceral Myopathy: Clinical syndromes, genetics, pathophysiology, and Fall of the Cytoskeleton

Visceral smooth muscle is a crucial component of the walls of hollow organs like the gut, bladder, and uterus. This specialized smooth muscle has unique properties that distinguish it from other muscle types and that facilitate robust dilation and contraction. Visceral myopathies are diseases where severe visceral smooth muscle dysfunction prevents efficient movement of air and nutrients though the bowel, impairs bladder emptying, and affects normal uterine contraction and relaxation, particularly during pregnancy. Disease severity exists along a spectrum. The most debilitating defects cause highly dysfunctional bowel, reduced intrauterine colon growth (microcolon), and bladder emptying defects requiring catheterization, a condition called Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS). People with MMIHS often die early in childhood. When the bowel is the main organ affected and microcolon is absent, the condition is known as myopathic chronic intestinal pseudo-obstruction (CIPO). Visceral myopathies like MMIHS and myopathic CIPO are most commonly caused by mutations in contractile apparatus cytoskeletal proteins. Here, we review visceral myopathy-causing mutations and normal functions of these disease-associated proteins. We propose molecular, cellular, and tissue-level models that may explain clinical and histopathological features of visceral myopathy and hope these observations prompt new mechanistic studies.

Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca2+-activated K+ channels in bronchial smooth muscle cells

In visceral smooth muscle cells (SMCs), the large-conductance Ca2+-activated K+ (BK) channel is one of the key elements underlying a negative feedback mechanism that is essential for the regulation of intracellular Ca2+ concentration. Although leucine-rich repeat-containing (LRRC) proteins have been identified as novel auxiliary γ-subunits of the BK channel (BKγ) in several cell types, its physiological roles in SMCs are unclear. The BKγ expression patterns in selected SM tissues were examined using real-time PCR analyses and Western blotting. The functional contribution of BKγ1 to BK channel activity was examined by whole cell patch-clamp in SMCs and heterologous expression systems. BKγ1 expression in mouse bronchial SMCs (mBSMCs) was higher than in other several SMC types. Coimmunoprecipitation and total internal reflection fluorescence imaging analyses revealed molecular interaction between BKα and BKγ1 in mBSMCs. Under voltage-clamp, steady-state activation of BK channel currents at pCa 8.0 in mBSMCs occurred in a voltage range comparable to that of reconstituted BKα/BKγ1 complex. However, this range was much more negative than in mouse aortic SMCs (mASMCs) or in HEK293 cells expressing BKα alone and β-subunit (BKβ1). Mallotoxin, a selective activator of BK channel that lacks BKγ1, dose-dependently activated BK currents in mASMCs but not in mBSMCs. The abundant expression of BKγ1 in mBSMCs extensively facilitates BK channel activity to keep the resting membrane potential at negative values and prevents contraction under physiological conditions.

MONOSODIUM GLUTAMATE POTENTIATES THE CONTRACTION OF THE VISCERAL SMOOTH MUSCLE OF DUODENUM BY AUGMENTING THE ACTIVITY OF INTRINSIC CHOLINERGIC EFFERENTS, INDUCING OXIDATIVE STRESS AND PROLIFERATING SMOOTH MUSCLE CELLS

Objective: The objective of the present study was to examine the effects of monosodium glutamate (MSG) on the contraction of visceral smooth muscle (VSM) of the duodenum in a rat model to understand the MSG-induced impairment of the function of the small intestine. Methods: Male albino rats of Charles Foster strain were exposed with MSG at three different dosages (632, 1264, and 2528 mg/kg BW/day) for 30-day duration. The records of the contraction of the duodenum were achieved with isotonic transducer (IT-2245) coupled with RMS-Polyrite D by our standard laboratory protocol. Results: We have observed potentiation of contraction of duodenum ex vivo dose-dependently in MSG exposed groups of rats compared to control. Furthermore, the enzymatic activity of acetylcholinesterase (AChE) in VSM tissue homogenate and expression of AChE protein in fixed duodenal muscle cell layers have been decreased in a dosage response manner comparing to control rats. We have found a significant decrease in the activities of some antioxidant enzymes such as Cu-Zn superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-s-transferase, and increase in the level of malondialdehyde in MSG exposed VSM tissue homogenate of the duodenum. We have also observed thickening of muscularis externa layer and increase in the number of muscle cells in circular and longitudinal muscle layers of the duodenal wall in transverse duodenal wall sections stained with eosin-hematoxylin. Conclusion: MSG potentiates the contraction of VSM of duodenum by augmenting the activity of intrinsic cholinergic efferents predominantly, and inducing oxidative stress and proliferating smooth muscle cells.

Pulmonary Smooth Muscle in Vertebrates: A Comparative Review of Structure and Function

Although the airways of vertebrates are diverse in shape, complexity, and function, they all contain visceral smooth muscle. The morphology, function, and innervation of this tissue in airways is reviewed in actinopterygians, lungfish, amphibians, non-avian reptiles, birds, and mammals. Smooth muscle was likely involved in tension regulation ancestrally, and may serve to assist lung emptying in fishes and aquatic amphibians, as well as maintain internal lung structure. In certain non-avian reptiles and anurans antagonistic smooth muscle fibers may contribute to intrapulmonary gas mixing. In mammals and birds, smooth muscle regulates airway caliber, and may be important in controlling the distribution of ventilation at rest and exercise, or during thermoregulatory and vocal hyperventilation. Airway smooth muscle is controlled by the autonomic nervous system: cranial cholinergic innervation generally causes excitation, cranial non-adrenergic, non-cholinergic innervation causes inhibition, and spinal adrenergic (SA) input causes species-specific, often heterogeneous contractions and relaxations.

A novel human cell culture model to study visceral smooth muscle phenotypic modulation in health and disease

Adaptation of the smooth muscle cell (SMC) phenotype is essential for homeostasis and is often involved in pathologies of visceral organs (e.g., uterus, bladder, gastrointestinal tract). In vitro studies of the behavior of visceral SMCs under (patho)-physiological conditions are hampered by a spontaneous, uncontrolled phenotypic modulation of visceral SMCs under regular tissue culture conditions. We aimed to develop a new visceral SMC culture model that allows controlled phenotypic modulation. Human uterine SMCs [ULTR and telomerase-immortalized human myometrial cells (hTERT-HM)] were grown to confluency and kept for up to 6 days on regular tissue culture surfaces or basement membrane (BM) matrix-coated surfaces in the presence of 0–10% serum. mRNA and protein expression and localization of SMC-specific phenotype markers and their transcriptional regulators were investigated by quantitative PCR, Western blotting, and immunofluorescence. Maintaining visceral SMCs confluent for 6 days increased α-smooth muscle actin (1.9-fold) and smooth muscle protein 22-α (3.1-fold), whereas smooth muscle myosin heavy chain was only slightly upregulated (1.3-fold). Culturing on a BM matrix-coated surface further increased these proteins and also markedly promoted mRNA expression of γ-smooth muscle actin (15.0-fold), smoothelin (3.5-fold), h-caldesmon (5.2-fold), serum response factor (7.6-fold), and myocardin (8.1-fold). Whereas additional serum deprivation only minimally affected contractile markers, platelet-derived growth factor-BB and transforming growth factor β1 consistently reduced versus increased their expression. In conclusion, we present a simple and reproducible visceral SMC culture system that allows controlled phenotypic modulation toward both the synthetic and the contractile phenotype. This may greatly facilitate the identification of factors that drive visceral SMC phenotypic changes in health and disease.

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal–contractile coupling.