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

Q: What is the clinical evidence from this study?

Population: Duchenne muscular dystrophy cardiomyopathy (mdx mouse model). Intervention: Colchicine vs. Phosphate-buffered saline (PBS) or vehicle (ddH2O). Primary outcome: Junctophilin-2 (JPH-2) protein levels in older mdx hearts following 2 weeks of colchicine treatment (p=0.0007).

Q: What does this research mean for the field?

Microtubule-mediated misregulation of junctophilin-2 causes t-tubule derangements and altered calcium handling in mdx cardiac myocytes, which can be rescued by colchicine-induced microtubule depolymerization. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

Kurt W. Prins; L. Michelle; Huiliang Zhang; Wang Wang; Joseph M. Metzger

doi:10.1016/j.jacbts.2016.02.002

April 1, 2016JACC Basic to Translational ScienceOpen Access

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

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Key result

Colchicine increases JPH-2 protein levels ~7.6-fold and improves cardiomyocyte calcium handling in mdx mice.

P=0.0007

Why the study?

Does colchicine-induced microtubule depolymerization normalize junctophilin-2 levels, restore t-tubule organization, and improve calcium handling in dystrophin-deficient mdx mice?

Key Points

To determine whether an inverse relationship between microtubules and junctophilin-2 drives t-tubule disarray and calcium mishandling in mdx mouse cardiac myocytes.
Evaluated t-tubule organization, junctophilin-2 localization, and tubulin expression via confocal microscopy and Western blot in young (2 to 3 months) and older (8 to 10 months) WT and mdx mice.
Treated older mdx mice with colchicine for 2 weeks in vivo to depolymerize microtubules and assess junctophilin-2 restoration and t-tubule architecture.
Measured calcium transient kinetics and calcium spark frequency in isolated mdx cardiac myocytes treated acutely with colchicine.
Older mdx myocytes showed disrupted t-tubule structural integrity (39.2 ± 2.2 AU vs 57.3 ± 1.3 AU in WT, p ≤ 0.05), a 75% reduction in junctophilin-2 (p = 0.0003), and a 2.6-fold increase in α-tubulin (p = 0.0008) and β-tubulin (p = 0.0007), showing an inverse hyperbolic relationship (r2 = 0.95).
Colchicine depolymerized microtubules, increased junctophilin-2 protein 7.6-fold (p = 0.0007), and improved t-tubule organization (44.5 ± 2.0 AU to 52.5 ± 2.2 AU, p = 0.02).
Colchicine treatment accelerated calcium transient decay to 25% of baseline (59.4 ± 2.5 ms vs 51.1 ± 3.0 ms, p = 0.03) and reduced calcium sparks by more than 50% (0.84 ± 0.2 to 0.31 ± 0.08 sparks/s/100 μm, p = 0.04).

Structured PICO

Does colchicine-induced microtubule depolymerization normalize junctophilin-2 levels, restore t-tubule organization, and improve calcium handling in dystrophin-deficient mdx mice?

P

Population

Young (2-3 months) and older (8-10 months) dystrophin-deficient mdx mice and wild-type C57BL/10 control mice evaluated in vivo over 2 weeks and in isolated ventricular cardiac myocytes.

I

Intervention

Colchicine 0.5 mg/kg via intraperitoneal injection every other day for 2 weeks (in vivo) or 10 μM colchicine in vitro for 2 hours on freshly isolated adult ventricular cardiac myocytes.

C

Comparator

Filter-sterilized phosphate-buffered saline (PBS) via intraperitoneal injection every other day for 2 weeks (in vivo) or vehicle (double-distilled water) for 2 hours (in vitro), as well as untreated age-matched wild-type C57BL/10 mice.

O

Outcome

Junctophilin-2 (JPH-2) protein expression (by quantitative Western blot), JPH-2 localization and t-tubule organization (by confocal microscopy and TT Power analysis in arbitrary units), and intracellular calcium handling/spark frequency (by Fluo 8-AM line-scan confocal imaging and Fura-2AM transients).surrogate

Main Result

Absolute Event Rate: 7.6% vs 1%

p-value: p=0.0007

Limitations

Preclinical mouse model (mdx mice) with relatively mild baseline cardiomyopathy
T-tubule and cellular corrections with 2 weeks of colchicine did not translate to whole-organ echocardiographic or survival improvements
Need for validation in large animal models with more severe Duchenne cardiomyopathy phenotypes
Colchicine treatment for 2 weeks did not lead to whole-organ echocardiographic improvements in cardiac geometry or systolic function
Colchicine did not confer a statistically significant survival benefit during isoproterenol stress testing
The mdx mouse model exhibits only mild baseline cardiomyopathy (mildly reduced fractional shortening), limiting assessment of organ-level rescue

Implication

Identifies microtubule-mediated downregulation and mislocalization of junctophilin-2 as a unifying molecular mechanism driving t-tubule disruption and ryanodine receptor calcium leak in Duchenne muscular dystrophy cardiomyopathy.

Abstract

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.

Discussion

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Cite This Study

Prins et al. (2016) studied Duchenne muscular dystrophy cardiomyopathy (mdx mouse model). Colchicine vs. Phosphate-buffered saline (PBS) or vehicle (ddH2O) was evaluated on Junctophilin-2 (JPH-2) protein levels in older mdx hearts following 2 weeks of colchicine treatment (p=0.0007). Colchicine-induced microtubule depolymerization increased JPH-2 protein levels 7.6-fold (p=0.0007), improved t-tubule organization (p=0.02), and reduced calcium sparks by >50% (p=0.04) in mdx mice.

synapsesocial.com/papers/6a7736580c6965f1a01ea391 https://doi.org/10.1016/j.jacbts.2016.02.002

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

Key Points

Structured PICO

Main Result

Limitations

Implication

Abstract

Discussion

Cite This Study

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

Key Points

Structured PICO

Main Result

Limitations

Implication

Abstract

Discussion

Cite This Study

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