© 2016. Published by The Company of Biologists Ltd | Development (2016) 143, 1547-1559 doi:10.1242/dev.129676 RESEARCH ARTICLE Restricting calcium currents is required for correct fiber type specification in skeletal muscle Nasreen Sultana1, Beatrix Dienes2, Ariane Benedetti1, Petronel Tuluc3, Peter Szentesi2, Monika Sztretye2, Johannes Rainer4, Michael W. Hess5, Christoph Schwarzer6, Gerald J. Obermair1, Laszlo Csernoch2 and Bernhard E. Flucher1,* ABSTRACT CaV1.1 can directly activate opening of the RyR1. In mature Skeletal muscle excitation-contraction (EC) coupling is independent muscles, calcium is released from the sarcoplasmic reticulum (SR) of calcium influx. In fact, alternative splicing of the voltage-gated calcium stores, whereas calcium influx is dispensable for skeletal muscle EC coupling (Melzer et al., 1995). Actually, calcium calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development currents through the major CaV1.1a splice variant are small and and function of muscle is not known. However, splicing defects that activate slowly, only at strong membrane depolarizations. cause aberrant expression of the calcium-conducting developmental Interestingly, during maturation of mammalian skeletal muscles activity-dependent calcium influx is actively suppressed by CaV1.1e splice variant correlate with muscle weakness in myotonic alternative splicing of CaV1.1. In fetal muscles, exclusion of exon dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although 29 produces a CaV1.1e channel variant that conducts sizable L-type grip force and voluntary running were reduced. Continued expression calcium currents and activates in parallel to SR calcium release at physiological voltages (Tuluc et al., 2009). However, after birth, the of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium developmental CaV1.1e splice variant is almost completely replaced homeostasis, and spontaneous calcium sparklets in isolated by the adult, poorly conducting CaV1.1a splice variant that includes muscle fibers. Contractile force was reduced and endurance exon 29 (Flucher and Tuluc, 2011; Tang et al., 2012). Why calcium enhanced. Key regulators of fiber type specification were influx is present in developing muscle but is then curtailed in mature dysregulated and the fiber type composition was shifted toward skeletal muscles is not known. Conversely, it remains to be slower fibers. However, oxidative enzyme activity and mitochondrial determined whether continued expression of the calcium- content declined. These findings indicate that limiting calcium influx conducting CaV1.1e splice variant alters contractile properties of during skeletal muscle EC coupling is important for the secondary mature skeletal muscles. function of the calcium signal in the activity-dependent regulation of In addition to their primary role in EC coupling, activity-induced fiber type composition and to prevent muscle disease. calcium signals in skeletal muscle are important for maintaining calcium homeostasis and for the regulation of muscle growth and KEY WORDS: Voltage-gated calcium channel, Skeletal muscle differentiation. For example, calcium signals regulate the excitation-contraction coupling, Muscle fiber type specification, transcription of genes involved in the adaptive response to Mouse exercise (Bassel-Duby and Olson, 2006). Therefore, the tight control of calcium influx by alternative splicing of the CaV1.1 INTRODUCTION channel is probably important for tuning muscle function to varying Calcium is the principal second messenger regulating skeletal activity levels. Conversely, calcium influx through CaV1.1e muscle contraction, growth and differentiation. In excitation- channels in mature muscles might be harmful. Abnormal contraction (EC) coupling, cytoplasmic calcium levels are rapidly expression of the embryonic, calcium-conducting CaV1.1e splice increased in response to action potentials and the magnitude of these variant in myotonic dystrophy type 1 (DM1) patients correlates with calcium signals regulates the force of contraction. In skeletal their degree of muscle weakness (Tang et al., 2012). Moreover, muscle, voltage-gated calcium channels (CaV1.1) and calcium aberrant splicing of calcium channels and transporters in cultured release channels (type 1 ryanodine receptors, RyR1) are physically myotubes from DM1 patients leads to altered intracellular calcium coupled to one another so that voltage-dependent activation of signaling (Santoro et al., 2014), and experimentally induced skipping of exon 29 aggravated the disease phenotype in muscles 1Department of Physiology and Medical Physics, Medical University of a myotonia mouse model (Tang et al., 2012). Innsbruck, Innsbruck 6020, Austria. 2Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen 4032, Hungary. 3Department of To identify the physiological importance of curtailing calcium 4 Pharmacology, University of Innsbruck, Innsbruck 6020, Austria. Division of influx through CaV1.1 channels in adult skeletal muscle and to Molecular Pathophysiology, Biocenter, Medical University Innsbruck, Innsbruck reveal a possible involvement of aberrant calcium signaling in 6020, Austria. 5Division of Histology and Embryology, Medical University Innsbruck, Innsbruck 6020, Austria. 6Department of Pharmacology, Medical DM1, we generated a genetic mouse model in which exon 29 has University Innsbruck, Innsbruck 6020, Austria. been permanently deleted. As expected, skeletal muscles of ΔE29/ΔE29 CaV1.1 knockout mice experienced increased calcium *Author for correspondence ([email protected]) influx during EC coupling and at rest. In addition, their contractile This is an Open Access article distributed under the terms of the Creative Commons Attribution properties were altered, calcium-activated downstream regulators License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. were upregulated, and the fiber type composition was shifted towards slower fiber types. However, mitochondrial content and Received 11 August 2015; Accepted 29 February 2016 oxidative enzyme activity were reduced. Together, these findings DEVELOPMENT 1547 RESEARCH ARTICLE Development (2016) 143, 1547-1559 doi:10.1242/dev.129676 indicate that chronically increased calcium influx through the muscle strength. Endurance was tested by making the mice run on a developmental CaV1.1e isoform has little effect on EC coupling, but treadmill at accelerating speed until exhaustion. Overall motor disturbs the normal regulation of muscle fiber type composition. performance was examined with the Rotarod test. In none of these Furthermore, the increased calcium influx causes mitochondrial tests was a significant difference in the performance of wild-type, +/ΔE29 ΔE29/ΔE29 damage and may thus contribute to muscle wasting in DM1. heterozygous CaV1.1 and homozygous CaV1.1 mice Conversely, these results suggest that, during normal development, observed. However, directly measuring grip strength revealed that limiting L-type calcium currents is important to enable the proper the grip force of the front paws was significantly reduced in ΔE29/ΔE29 specification of fiber type composition and to protect the muscles homozygous CaV1.1 mice (Fig. 2D). Finally, voluntary from calcium-induced damage. running of the mice in a running wheel was recorded over the period of 7 days. Both the distance run and the duration the mice spent RESULTS running per day were significantly reduced in homozygous ΔE29/ΔE29 Selective deletion of CaV1.1 exon 29 prevents the CaV1.1 mice compared with wild-type controls (Fig. 2E). developmental switch from the CaV1.1e to the CaV1.1a These behavioral and functional analyses indicate that aberrant isoform expression of CaV1.1e alters muscle performance without causing In order to study the importance of the isoform switch from the severe motor deficits as assessed in tests that revealed the disease calcium-conducting developmental CaV1.1e splice variant to the phenotype in other DM mouse models (Gomes-Pereira et al., 2011). poorly conducting adult CaV1.1a splice variant we generated a Also, histological staining of muscle sections did not reveal an ΔE29/ΔE29 mouse model with a constitutive knockout of exon 29 of the CaV1.1 increase in centrally located nuclei in CaV1.1 muscles (Cacna1s) gene (Fig. 1A). We reasoned that because the short (Fig. S1B). transcript CaV1.1e is predominant during fetal development, ΔE29 CaV1.1 mice would develop normally up to birth, but that the Aberrant expression of the developmental CaV1.1e isoform aberrant continuing expression of the high-conductance in mature muscles alters the contractile properties of developmental calcium channel splice variant throughout isolated slow and fast muscles postnatal development and adult life would reveal any influence Because the reduced grip force and voluntary running indicated of the extra calcium influx on EC coupling and/or other calcium- altered muscle performance, we next determined the contractile mediated signaling processes regulating muscle growth and properties directly in isolated mouse muscles. The contractile force ΔE29 differentiation. Furthermore, the CaV1.1 mouse will expose of soleus and EDL muscles was