The bodies of vertebrates include hundreds of skeletal muscles, each involved in performing different motor tasks at specific anatomical locations. This variety in skeletal muscles and their functions implies genotypic and phenotypic diversity among skeletal muscle fibers. In fact, the preferential expression of different muscular protein isoforms and therefore the existence of distinct muscle fiber phenotypes is one of the main determinants of the muscle performance [1
]. Not only does this diversity manifest in multiple muscle groups but also a single muscle expresses heterogeneous populations of slow and fast-type muscle fibers [1
]. Excitation-contraction coupling properties, the sensitivity of the contractile apparatus to Ca2+
, mechanical power output, shortening velocity, and rate of ATP hydrolysis are also known to vary greatly from fiber to fiber [1
]. The intercostal muscles are an exemplary case of such a heterogeneous muscle population, making them an attractive model for comparative studies.
The mechanical functions of the intercostal muscles during ventilation are highly complex. There are two major division criteria for intercostal fibers: anatomical and functional. Anatomical divisions are based on location on the internal or external side of the ribs; functional divisions are based on participation in expiratory or inspiratory respiration [8
]. The external intercostals and the parasternal intercostals function in inspiratory breathing, whereas the internal intercostals have an expiratory function [9
]. Although the function of the diaphragm appears to dominate breathing at rest [10
], the intercostal muscles may contribute c.a. 40% of the volume shift by movement of the thoracic wall [11
]. During intensified breathing, the contribution of the intercostals becomes more prominent [12
]. The lateral internal intercostals are also recruited for a variety of nonventilatory functions including the cough reflex and speech as well as for postural support [10
Previous work estimates that about 60% of the intercostal fibers are slow-type and 40% fast-type fibers [14
]. As a result of this diversity, the same preparation can be used to address many aspects of muscle fiber-type physiology. Also, due to their involvement in the mechanics of ventilation and the fact that respiratory pathology is common in several diseases (e.g., muscular dystrophy, chronic obstructive pulmonary disease, amyotrophic lateral sclerosis, hereditary polyneuropathies, autoimmune conditions like myasthenia gravis, muscular paralysis, etc.), intercostal muscles represent a model of high clinical relevance.
Indeed, in cases where diagnosis and treatment for muscular dystrophy is available, respiratory arrest has become the critical factor in the majority of fatalities arising from muscular dystrophy [16
]. While the vast majority of studies evaluating the function of the respiratory muscles have used the diaphragm muscle as model of study, information on the intercostal muscles, in particular at cellular level, is more fragmented [8
These muscles are also of relevance in Chronic Obstructive Pulmonary Disorder (COPD), given their critical role in manipulating the configuration of the ribs [9
] and therefore the overall morphology of the thorax. In addition to the mechanical reorganization of the intercostal muscles that results from the inflated thorax common to COPD patients, clear changes in the expression of myosin heavy chain isoforms have been documented [18
Here we describe in detail the isolation and preliminary characterization of isolated adherent intercostals muscle fibers in the common laboratory mouse based on techniques and methods previously developed by our laboratory for use on other muscles [19
]. The loading of fluorescent Ca2+
indicators combined with electrophysiological approaches, including electrical field stimulation of the cultured muscle fibers using any stimulation pattern desired, and the transfection and overexpression of fluorescent fusion proteins permit the study of spatiotemporal aspects of excitation-contraction coupling and biological processes such as excitation-transcription coupling. Due to the difficulty encountered in achieving consistent adhesion of the intercostal fibers to commercial glass-bottom culture dishes, we also include detailed methods for the construction and preparation of glass-bottom dishes optimized for such muscle fiber studies. These dishes have improved the reliability and yield of attached fibers in our preparations several fold. The methods presented here can be modified to allow culture of intercostal fibers from animal at various ages or from animal models with specific genetic background. The protocol described here is intended more generally to provide a flexible new primary cell culture of the as yet poorly characterized, but clinically relevant muscular group, the intercostals.