The Intercostal Muscles: Development and Applications of a Novel Primary Muscle Fiber Culture System

Abstract

The intercostal muscles are critical participants in respiration, working continuously throughout life. These core respiratory muscles are distinct from their locomotor counterparts in the limbs in lineage and function. In spite of this they are seldom studied, and muscles from the limbs are often used to model skeletal muscle as a whole. The work presented here has three main parts. The first part is focused on developing methods to culture these understudied muscles. The resulting cultures appear healthy, respond to electrical stimuli and efficiently express exogenous proteins. In the second part, these methods are applied to an animal model of Chronic Obstructive Pulmonary Disorder (COPD). These experiments show that respiratory muscles share in the functional defects observed in other skeletal muscles in an animal model of COPD. Specifically the intercostal and flexor digitorum brevis (FDB) muscle fibers derived from COPD both display markedly reduced Ca2+ transient magnitude, although the kinetics of the transient and amount of Ca2+ available for release from internal stores remain unaltered. Some minor differences were detected in the CaV1.1-Ryanodine receptor complex in FDB fibers but not in the intercostals, suggesting the possibility of divergent mechanisms. Finally, cultures of intercostal muscle fibers are used to examine the NFATc1 excitation-transcription coupling pathway. Isolated intercostal muscle fibers display a complete inversion of this pathway under stimulation patterns identical to their locomotor counterparts in the hindlimb. This effect is mediated by at least two activity regulated kinases, JNK and CaMKII, which oppose the activity of Calcineurin in the canonical NFATc1 activation pathway. We suggest that this is driven by imbalances in the expression levels of CaMKII/JNK relative to CN and demonstrate differences in the levels of these kinases in intercostal muscle relative to FDB and soleus muscle.