The N-Terminal Fragment of Urine Titin Is Not a Product of Degradation by Calpain 3

Abstract

Introduction: A 20 kDa fragment at the N-terminus of titin is highly excreted in the urine of patients with Duchenne muscular dystrophy (DMD), making urine titin a prominent biomarker for muscle breakdown. This N-terminal fragment is presumed to be a product of degradation by a protein-degrading enzyme, calpain 3; however, whether calpain 3 is required remains unclear. We aimed to determine whether urine titin elevation occurs in the absence of calpain 3.

Methods: We measured urine titin by ELISA in two genetically confirmed limb-girdle muscular dystrophy type R1(LGMDR1) patients, 11 other LGMD patients, and five healthy controls. Five Capn3-/- and nine wild-type mice were also examined.

Results: Urine titin in LGMDR1 patients was ~100-fold higher than in controls (median 112.3 vs. 1.3 pmol/mg Cr, p < 0.0001), with no difference between LGMDR1 and other LGMD subtypes. Similarly, urine titin levels in Capn3-/- mice were more than four times higher than normal (p < 0.01).

Discussion: These results suggest the involvement of other protein-degrading enzymes leading to the production of the N-terminal fragment.

Keywords: Calpain3 KO mouse; limb‐girdle muscular dystrophy; urine titin.

FIGURE 1

Urine titin of LGMD R1 patients and Capn3 KO mouse. The values of urine titin were expressed as the amount of titin per milligram of creatinine (pmol/mg Cr). (a) The urine titin levels of LGMD R1 patients and those of other LGMD patients and the values for normal adults. Dunn's multiple comparisons test **< 0.01; ****< 0.0001. (b) The urine titin levels of heterozygous (Capn3+/−) and homozygous (Capn3−/−) Capn3 gene knockout mice. Dunn's multiple comparisons test **< 0.01.