Effect of Dystrophin Deficiency on Selected Intrinsic Laryngeal Muscles of the mdx Mouse

Transcription

1 Effect of Dystrophin Deficiency on Selected Intrinsic Laryngeal Muscles of the Mouse Lisa B. Thomas Marshall University Acknowledgements Co-Authors Joseph Stemple, Ph.D. Anne Harrison, Ph.D. Richard Andreatta, Ph.D. Francisco Andrade, Ph.D. ASHA Convention November 2008 Intrinsic Laryngeal Muscles Highly specialized form of skeletal muscle Similar to extraocular muscle phenotype Areas of divergence from limb skeletal muscle Morphogenesis Myosin heavy chain isoforms present Mitochondrial content Regenerative capacity Innervation patterns (motor, sensory) Sensitivity to disease DMD (2006 study) Duchenne Muscular Dystrophy Most common form of MD 1 in every 3,500 live male births Motor delays, weakness by age 2 Marked reductions in fx and muscle size by ages 6-11 Death by end of third decade Some muscles preferentially spared extraocular Andrade et al., 2003; Goding et al., 1995; McLoon et al., 2004; Noden & Francis- West 2006; Porter et al., 1995; Thomas, Harrison, et al. (in press) Andrade et al., 2000; Kaminski et al., 1992; Menache & Darris, 2001 Duchenne Muscular Dystrophy Dystrophin-Glycoprotein Complex Absence of dystrophin, a protein of the cytoskeleton Dystrophin Integral component of DGC Scaffolds sarcolemma during muscle contraction Possible signaling role / Control of Ca levels Ervasti et al., 1991; Lapidos, Kakkar, & McNally, 2004; Rando,

2 Pathophysiology In the absence of dystrophin Loss of support structure leads to disruption of sarcolemmal (cell membrane) integrity Influx of Ca 2+ triggers destruction of proteins Fiber necrosis Attempts at fiber regeneration (failed) Eventual muscle fibrosis and fatty infiltration Markers of Pathology Evidence of disrupted sarcolemma Fiber degeneration and regeneration Inflammation, necrosis Central nuclei Pleomorphic fibers Eventual widespread fibrosis, necrosis Menache & Darris, 2001; Petrof, 2002 Davies & Nowak, 2006; Menache & Darris, 2001 The Animal Model of DMD mouse dystrophin deficient Genetic and biological similarities to human DMD Differences in disease severity and functional impact across species Introduction 2006 Preliminary Study Examined effects of dystrophin deficiency on the thyroarytenoid (TA) and posterior cricoarytenoid muscles (PCA) of the larynx mouse Bulfield et al., 1984; Menache & Darris, 2001 Thomas, Joseph, et al. (in press) Normal Appearing Leg Muscle (Gastrocnemius) 2

3 Introduction 2006 Preliminary Study Results: While deficient in dystrophin, TA and PCA did not show the effects of the disease process. Conclusions: TA and PCA possess special features which protect them from the effects of this disease Thomas, Joseph, et al. (in press) Laryngeal Muscle Specialization Much of research on ILM specialization conducted on 2 primary laryngeal muscles (TA, PCA) Are these properties true of all laryngeal muscles? Do all laryngeal muscles share a similar level of biological specialization? Implications for function and rehabilitation Laryngeal Muscle Specialization Continued study in Possible diversity among laryngeal muscles Literature review showed evidence of diversity across laryngeal muscles Two laryngeal muscles with features (eg, contractile speeds, proprioceptive mechanisms, etc.) more reflective of limb muscle Interarytenoid Cricothyroid Katto et al., 1987; Okamura et al., 1988; Tellis et al., 2004 Purpose Statement Current Study Effect of Dystrophin Deficiency on Selected Intrinsic Laryngeal Muscles of the Mouse To further define the biological characteristics of two intrinsic laryngeal muscles (the interarytenoid and cricothyroid) and their similarity / dissimilarity to other laryngeal muscles through the use of the mouse model. 3

4 The Interarytenoid Deviates from other laryngeal muscles in: Motor Innervation Transverse fibers are unpaired; receive bilateral innervation from RLN Supplemental innervation from SLN Sensory: Presence of muscle spindles Contractile profile (ie, myosin isoforms) similar to limb muscle The Cricothyroid Deviates from other laryngeal muscles in: Morphogenesis Innervation Contractile profile (ie, myosin isoforms) similar to limb muscle Sensitivity to disease Katto et al., 1987; Maranillo et al. ; 2005; Mu et al., 1994; Tellis et al., 2004 Benninger et al., 2006; Hyodo et al., 2001; Lucas et al., 1995; Marques et al., 2004; Rhee et al. 2004; Sperber, 1981 Methods Primary Investigation Animals mouse (n = 8); C57BL control (n = 8) Dissected out whole larynges and gastrocnemius muscle Serial 10-um thick cryosections H & E Staining Evans blue dye tests Immunocytochemisty (dystrophin, utrophin) Results H & E Gastrocnemius Results H & E - Laryngeal C57BL CT SCA C57BL PCA 4

5 Mean Percent Central Nucleated Fibers Results Central Nuclei Percentage Central Nucleation Across Muscles * C57 GAST GAST C57 PCA PCA C57 CT CT C57 SCA SCA Muscle Control Gastroc* 3.93% (3.08) PCA 7.13% (3.83) SCA 5.33% (3.01) CT 5.83% (4.3) 65.93% (7.99) 4.88% (2.14) 1.60% (1.62) 11.63% (2.76) SCA 5 33% 1 60% *p <.000 Inter Rater Reliability: ICC =.98 Results Evans Blue Results Evans Blue C57BL C57BL CT Gastroc SCA Strap PCA C57BL Results - Utrophin Results - Dystrophin Gastroc CT SCA Background Utrophin Dystrophin homolog Present only at NMJ in mature muscle fibers To study Mark NMJ (alpha-bungarotoxin) ICC staining for utrophin Expect utrophin at NMJ in mature fibers PCA 5

6 Results Utrophin Monoclonal Gastrocnemius Results Utrophin Monoclonal (Laryngeal) Control Control CT SCA PCA Results Utrophin Monoclonal Gastrocnemius Utrophin at NMJ in C57BL Utrophin re-localized to sarcolemma in Laryngeal Utrophin not present at NMJ in mature C57BL fibers No shifts in utrophin expression in Conclusions SCA spared from the effects of dystrophin deficiency CT shows subtle changes indicative of regeneration (central nucleation) Utrophin upregulation not evidenced in spared muscles Hypothesis Muscles will show effects of disease as in limb muscle Neither muscle demonstrated disease effects as seen in limb muscle SCA - full sparing CT subtle regenerative changes Suggest SCA and CT are not comparable to limb muscle Possess specialized features as other laryngeal muscles SCA Has response to disease similar to other laryngeal muscles Has other features similar to limb muscle May be blended form of muscle Varying level of specialization within the larynx 6

7 CT Two-fold increase in central nuclei (not significant) Similar increase shown by Marques et al No widespread evidence of fiber degeneration Comparable to results in other mildly affected muscles Masseter EOM (levator palpebrae superioris, retractor bulbi) Previous study of CT (2007) CT (con t) Implications re: nature of the CT Blended, transitional form of muscle Mechanical differences Different sarcolemmal management? Mechanical requirements of CT? Marques et al. (2007) Mechanisms of Sparing Opens door for use of murine model in laryngeal study Organization of DGC in laryngeal muscle Implications for voice therapy Limitations Muscle sections Utrophin antibodies Use of the animal model Concluding Remarks Thanks to Laryngeal muscles distinctive What does this mean in the clinic? Study of the biology of laryngeal muscles can lay a foundation for physiologic therapies Will require: Communication of clinical needs to the bench Translation of bench findings to clinicians 7

8 Andrade FH, Merriam AP, Guo W, et al. Paradoxical absence of M lines and downregulation of creatine kinase in mouse extraocular muscle. J Appl Physiol. Aug 2003;95(2): Andrade FH, Porter JD, Kaminski HJ. Eye muscle sparing by the muscular dystrophies: lessons to be learned? Microsc Res Tech. Feb ;48(3-4): Benninger MS, Gardner GM, Schwimmer C, Divi i V. Laryngeal neurophysiology. In: Rubin JS, Sataloff RT, Korovin GS, eds. Diagnosis and Treatment of Voice Disorders. 3rd ed. San Diego: Plural Publishing; 2006: Bulfield G, Siller WG, Wight PA, Moore KJ. X chromosome-linked muscular dystrophy () in the mouse. Proc Natl Acad Sci U S A. Feb 1984;81(4): Choi HS, Ye M, Berke GS. Function of the interarytenoid(ia) muscle in phonation: in vivo laryngeal model. Yonsei Med J. Mar 1995;36(1): Davies KE, Nowak KJ. Molecular mechanisms of muscular dystrophies: old and new players. Nat Rev Mol Cell Biol. Oct 2006;7(10): Ervasti JM, Campbell KP. Membrane organization of the dystrophin-glycoprotein complex. Cell. Sep ;66(6): Goding GS, Jr., Al-Sharif KI, McLoon LK. Myonuclear addition to uninjured laryngeal myofibers in adult rabbits. Ann Otol Rhinol Laryngol. Jul 2005;114(7): Hyodo M, Kawakita S, Desaki J. Scanning electron microscopic study of the muscle fiber ends at the myotendinous junction in the posterior cricoarytenoid and cricothyroid id muscles in rats. Acta Otolaryngol. l Dec 2001;121(8): (8) 956 Kaminski HJ, al-hakim M, Leigh RJ, Katirji MB, Ruff RL. Extraocular muscles are spared in advanced Duchenne dystrophy. Ann Neurol. Oct 1992;32(4): Katto Y, Okamura H, Yanagihara N. Electron microscopic study of muscle spindle in human interarytenoid muscle. Acta Otolaryngol. Nov-Dec 1987;104(5-6): Lapidos KA, Kakkar R, McNally EM. The dystrophin glycoprotein complex: signaling strength and integrity for the sarcolemma. Circ Res. Apr ;94(8): Lucas CA, Rughani A, Hoh JF. Expression of extraocular myosin heavy chain in rabbit laryngeal muscle. J Muscle Res Cell Motil. Aug 1995;16(4): Maranillo E, Leon X, Orus C, Quer M, Sanudo JR. Variability in nerve patterns of the adductor muscle group supplied by the recurrent laryngeal nerve. Laryngoscope. Feb 2005;115(2): Marques MJ, Ferretti R, Vomero VU, Minatel E, Neto HS. Intrinsic laryngeal muscles are spared from myonecrosis in the mouse model of Duchenne muscular dystrophy. Muscle Nerve. Mar 2007;35(3): McLoon LK, Rowe J, Wirtschafter J, McCormick KM. Continuous myofiber remodeling in uninjured extraocular myofibers: myonuclear turnover and evidence for apoptosis. Muscle Nerve. May 2004;29(5): Menache CC, Darris BT. Myopathies: Inherited myopathies. In: Katirji B, Kaminski HJ, Preston DC, Ruff RL, Shapiro BE, eds. Neuromuscular Disorders in Clinical Practice. Boston: Butterworth-Heinemann; 2001: Noden DM, Francis-West P. The differentiation and morphogenesis of craniofacial muscles. Dev Dyn. May 2006;235(5): Okamura H, Katto Y. Fine structure of muscle spindle in interarytenoid muscle of human larynx. In: Fujimura O, ed. Vocal Fold Physiology: Voice Production, Mechanisms and Functionss. Vol 2. New York: Raven Press; 1988: Porter JD, Baker RS, Ragusa RJ, Brueckner JK. Extraocular muscles: basic and clinical aspects of structure and function. Surv Ophthalmol. May-Jun 1995;39(6): Rando TA. The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies. Muscle Nerve. Dec 2001;24(12): Rhee HS, Lucas CA, Hoh JF. Fiber types in rat laryngeal muscles and their transformations after denervation and reinnervation. J Histochem Cytochem. May 2004;52(5): Sperber G. Craniofacial Embryology. 3 ed. Boston: Wright PSG; Mu L, Sanders I, Wu BL, Biller HF. The intramuscular innervation of the human interarytenoid muscle. Laryngoscope. Jan 1994;104(1 Pt 1): Tellis CM, Rosen C, Thekdi A, Sciote JJ. Anatomy and fiber type composition of human interarytenoid muscle. Ann Otol Rhinol Laryngol. Feb 2004;113(2): Thomas LB, Harrison AL, Stemple JC. Aging Thyroarytenoid and Limb Skeletal Muscle: Lessons in Contrast. JVoice Voice. Jan Thomas LB, Joseph G, Adkins T, Andrade FH, Stemple JC. Laryngeal muscles are spared in the dystrophin deficient mouse. Journal of Speech, Language, Hearing Research. in press. Zemlin WR. Speech and Hearing Science: Anatomy and Physiology. 3rd ed. Englewood Cliffs, NJ: Prentice-Hall;