Expression of calcium-buffering proteins in rat intrinsic laryngeal muscles

Abstract

Intrinsic laryngeal muscles (ILM) are highly specialized muscles involved in phonation and airway protection, with unique properties that allow them to perform extremely rapid contractions and to escape from damage in muscle dystrophy. Due to that, they may differ from limb muscles in several physiological aspects. Because a better ability to handle intracellular calcium has been suggested to explain ILM unique properties, we hypothesized that the profile of the proteins that regulate calcium levels in ILM is different from that in a limb muscle. Calcium-related proteins were analyzed in the ILM, cricothyroid (CT), and tibialis anterior (TA) muscles from male Sprague-Dawley rats (8 weeks of age) using quantitative PCR and western blotting. Higher expression of key Ca(2+) regulatory proteins was detected in ILM compared to TA, such as the sarcoplasmic reticulum (SR) Ca(2+)-reuptake proteins (Sercas 1 and 2), the Na(+)/Ca(2+) exchanger, phospholamban, and the Ca(2+)-binding protein calsequestrin. Parvalbumin, calmodulin and the ATPase, Ca(2+)-transporting, and plasma membrane 1 were also expressed at higher levels in ILM compared to TA. The store-operated calcium entry channel molecule was decreased in ILM compared to the limb muscle and the voltage-dependent L-type and ryanodine receptor were expressed at similar levels in ILM and TA. These results show that ILM have a calcium regulation system profile suggestive of a better ability to handle calcium changes in comparison to limb muscles, and this may provide a mechanistic insight for their unique pathophysiological properties.

Keywords: Calcium; Ncx; Pmca1; Serca; laryngeal muscles; store‐operated calcium entry.

Figure 1

Relative mRNA levels of genes encoding Ca²⁺-handling proteins in intrinsic laryngeal muscles (ILM), cricothyroid (CT) muscle, extraocular muscle (EOM), and tibialis anterior (TA) muscle. The genes studied were Ca^2+-ATPase 1, plasma membrane (Pmca1), Na⁺/Ca^2+-exchange, plasma membrane (Ncx), Ca²⁺-ATPase slow twitch (Serca2), calsequestrin 2 (Casq2), phospholanbam (Pln), calmodulin (CaM), calsequestrin 1 (Casq1), Ca²⁺-ATPase, fast twitch (Serca1), parvalbumin (Pvalb), regucalcin (Rgn), ryanodine receptor 1 (RyR1), and calcium channel subunit (Cacna1s). The relative mRNA levels of genes encoding Ca²⁺-handling proteins were determined by SYBR Green qPCR in muscle tissue. Quantifications from three independent muscle samples, each sample containing muscles pooled from three or four different rats. Mean fold changes ± SEM of three independent samples each were calculated and statistically analyzed using REST 2005 and *P < 0.05, **P < 0.01 was considered statistically significant.

Figure 2

Protein levels of sarcoplasmic reticulum Ca²⁺-handling proteins in intrinsic laryngeal muscles (ILM), cricothyroid (CT) muscle, extraocular muscles (EOM), and tibialis anterior (TA) muscle. Western blot analysis showing relative abundance of indicated proteins: calsequestrins 1 and 2 (CASQ1 and CASQ2), Sercas 1 and 2 (SERCA1 and SERCA2), calmodulin (CaM), calmodulin kinase II (CaMKII), and Orai1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for protein loading, Western blot transfer and nonspecific changes in protein levels. The molecular weight, expressed in kDa, for each protein is indicated. Quantifications from three independent muscle samples, each sample containing muscles pooled from three or four different rats. Asterisks and different letter combination indicate statistical significance (*P < 0.05 and ab, ac, ad, bc, or bd P < 0.05, respectively). In ILM, CASQ 1 was more abundant than CASQ2 compared with TA. SERCA1 was less than SERCA2 in ILM and higher compared with TA. CT and the other ILM showed similar levels of the proteins studied.

Figure 3

Relative mRNA levels of genes encoding Ca²⁺-handling proteins in control (Ctrl) and dystrophic (mdx) mice in intrinsic laryngeal muscles (ILM), cricothyroid (CT) muscle, extraocular muscle (EOM), and tibialis anterior (TA) muscle. The genes studied were Ca²⁺-ATPase slow twitch (Serca2), fast twitch (Serca1), calsequestrin 2 (Casq2), and calsequestrin 1 (Casq1). The relative mRNA levels of genes encoding Ca²⁺-handling proteins were determined by SYBR Green qPCR in muscle tissue. Mean fold changes ± SEM of three independent samples each were calculated and statistically analyzed using REST 2005. Each independent muscle sample contained a pool of muscles from 3–4 mice. Asterisks indicate statistical significance (*P < 0.05). Letter “a” was considered statistically significant (P < 0.05) compared with Ctrl.