Adherent Primary Cultures of Mouse Intercostal Muscle Fibers for Isolated Fiber Studies

Abstract

Primary culture models of single adult skeletal muscle fibers dissociated from locomotor muscles adhered to glass coverslips are routine and allow monitoring of functional processes in living cultured fibers. To date, such isolated fiber cultures have not been established for respiratory muscles, despite the fact that dysfunction of core respiratory muscles leading to respiratory arrest is the most common cause of death in many muscular diseases. Here we present the first description of an adherent culture system for single adult intercostal muscle fibers from the adult mouse. This system allows for monitoring functional properties of these living muscle fibers in culture with or without electrical field stimulation to drive muscle fiber contraction at physiological or pathological respiratory firing patterns. We also provide initial characterization of these fibers, demonstrating several common techniques in this new model system in the context of the established Flexor Digitorum Brevis muscle primary culture model.

Atypical behavior of NFATc1 in cultured intercostal myofibers

“…The procedures for isolating single muscle fibers have been previously described [24]. In brief, ItC and FDB muscles were dissected and enzymatically digested, then mechanically triturated with a polished glass pipette.…”

“…Six- to eight-week-old female CD1 mice were euthanized and flexor digitorum brevis (FDB), soleus (Sol) and intercostal (ItC) muscles were removed. The procedures for isolating single muscle fibers have been previously described [ 24 ]. In brief, ItC and FDB muscles were dissected and enzymatically digested, then mechanically triturated with a polished glass pipette.…”

“…The exogenous fusion protein NFATc1-GFP was expressed in isolated muscle fibers by exposure to an adenoviral construct [ 8 ] 24 hours following isolation as previously described [ 8 , 24 ]. After two or three days of expression (three to four days in culture), fibers were transferred to L-15 and imaged on an Olympus Fluoview 500 laser scanning confocal imaging system using an Olympus 60x/1.2 NA water immersion objective (Olympus, Center Valley, PA, USA).…”

Impaired calcium signaling in muscle fibers from intercostal and foot skeletal muscle in a cigarette smoke-induced mouse model of COPD

“…This 6‐month whole‐body cigarette smoke exposure is known to cause muscle dysfunction in different species, including mice . The procedures for isolating single muscle fibers were previously described in detail . In brief, intercostal (combined internal and external) and FDB muscles were dissected and enzymatically digested, then mechanically triturated with a polished glass pipette .…”

“…[31][32][33] The procedures for isolating single muscle fibers were previously described in detail. 34 In brief, intercostal (combined internal and external) and FDB muscles were dissected and enzymatically digested, then mechanically triturated with a polished glass pipette. 34 Resulting fibers were suspended in minimum essential media (MEM) supplemented with 10% fetal bovine serum (FBS) for 12 to 24 h until they were plated on laminincoated dishes or used in releasable Ca 21 experiments.…”