Microtubule-Mediated Misregulation of Junctophilin-2 Underlies T-Tubule Disruptions and Calcium Mishandling in mdx Mice

Summary Cardiac myocytes from the mdx mouse, the mouse model of Duchenne muscular dystrophy, exhibit t-tubule disarray and increased calcium sparks, but a unifying molecular mechanism has not been elucidated. Recently, improper trafficking of junctophilin (JPH)-2 on an altered microtubule network caused t-tubule derangements and calcium mishandling in a pressure-overload heart failure model. Mdx cardiac myocytes have microtubule abnormalities, but how this may affect JPH-2, t-tubules, and calcium handling has not been established. Here, we investigated the hypothesis that an inverse relationship between microtubules and JPH-2 underlies t-tubule disruptions and calcium mishandling in mdx cardiac myocytes. Confocal microscopy revealed t-tubule disorganization in mdx cardiac myocytes. Quantitative Western blot analysis demonstrated JPH-2 was decreased by 75% and showed an inverse hyperbolic relationship with α- and β-tubulin, the individual components of microtubules, in mdx hearts. Colchicine-induced microtubule depolymerization normalized JPH-2 protein levels and localization, corrected t-tubule architecture, and reduced calcium sparks. In summary, these results suggest microtubule-mediated misregulation of JPH-2 causes t-tubule derangements and altered calcium handling in mdx cardiac myocytes.


SUMMARY
Cardiac myocytes from the mdx mouse, the mouse model of Duchenne muscular dystrophy, exhibit t-tubule disarray and increased calcium sparks, but a unifying molecular mechanism has not been elucidated. Recently, improper trafficking of junctophilin (JPH)-2 on an altered microtubule network caused t-tubule derangements and calcium mishandling in a pressure-overload heart failure model. Mdx cardiac myocytes have microtubule abnormalities, but how this may affect JPH-2, t-tubules, and calcium handling has not been established. Here, we investigated the hypothesis that an inverse relationship between microtubules and JPH-2 underlies t-tubule disruptions and calcium mishandling in mdx cardiac myocytes. Confocal microscopy revealed t-tubule disorganization in mdx cardiac myocytes. Quantitative Western blot analysis demonstrated JPH-2 was decreased by 75% and showed an inverse hyperbolic relationship with aand b-tubulin, the individual components of microtubules, in mdx hearts. Colchicine-induced microtubule depolymerization normalized JPH-2 protein levels and localization, corrected t-tubule architecture, and reduced calcium sparks. In summary, these results suggest microtubule-  (1). Heart failure is the cause of death for 20% to 25% of DMD patients (2); therefore research aimed at understanding the molecular and cellular phenotypes underlying the cardiomyopathy of DMD has been conducted. Altered calcium homeostasis marked by increased calcium sparks (3)(4)(5) and t-tubule disarray were documented in cardiac myocytes from the dystrophin-deficient mdx mouse (6,7), but a unifying molecular mechanism has not been identified.
Junctophilin (JPH)-2, the protein that links the plasma membrane of t-tubules to the ryanodine receptor, is essential for proper t-tubule structure and function (8). Recently, it was reported that JPH-2 mislocalization due to abnormalities in microtubule cytoskeleton caused pathological t-tubule remodeling and abnormal calcium handling in the pressure overload-induced heart failure model (9). However, the relationship between microtubules and JPH-2 in other models of heart failure or cardiomyopathy has not been analyzed. Because previous studies documented microtubule derangements in mdx cardiac myocytes (5,10), we tested the hypothesis that microtubule alterations cause JPH-2 misregulation and result in t-tubule disruptions and calcium mishandling in mdx mice.
Finally, to investigate the translational aspects of our hypothesis, we examined the cardiomyopathy of mdx mice via echocardiography and isoproterenol stress tests as previous studies showed mildly reduced systolic function (11)(12)(13)(14) and excessive mortality with isoproterenol administration (10,15,16) Figures 2C and 2D).
When the relationships between aand b-tubulin and JPH-2 in older mice was analyzed, we observed a significant inverse hyperbolic relationships (r 2 ¼ 0.95 Abbreviations as in Figure 1.
Prins et al.
These data suggested increased microtubule content was associated with reduced JPH-2 protein levels.  2 AU) ( Figures 3F and 3G).  Although we were able to document restoration of t-tubule organization with colchicine treatment, we did not observe organ-level cardiac improvements in mdx mice. One explanation is that t-tubule correc- Finally, our results add to several other publications that document a crucial role for JPH-2 in t-tubule integrity. The first link between JPH-2 and t-tubules was shown when knockout of JPH-2 resulted in embryonic lethality due to lack and cardiac contractility and improper t-tubule formation (27). Several other studies document that alterations of JPH-2 via miR-24 (28,29), RNA interference (21,22,30), or calpain-mediated protein cleavage (23) cause t-tubule derangements and rescue of JPH-2 protein levels or localization via transgenic overexpression (17), inhibition of miR-24 (28)