Decline in muscle strength and running endurance in klotho deficient C57BL/6 mice

Abstract

Alpha klotho (known as klotho) is a multifunctional protein that may be linked to age-associated decline in tissue homeostasis. The original klotho hypomorphic (klotho (hm) ) mouse, produced on a mixed C57BL/6 and C3H background, is short lived and exhibits extensive aging-like deterioration of several body systems. Differently, klotho (hm) mice on a pure C57BL/6 background do not appear sickly nor die young, which has permitted us to gain insight into the effect of klotho deficiency in adult life. First, analyzing klotho transcript levels in the kidney, the main site of klotho production, we demonstrated a 71-fold decline in klotho (hm) females compared to wildtype females versus only a 4-fold decline in mutant males. We then examined the effect of klotho deficiency on muscle-related attributes in adult mice, focusing on 7-11 month old females. Body weight and forelimb grip strength were significantly reduced in klotho (hm) mice compared to wildtype and klotho overexpressing mice. The female mice were also subjected to voluntary wheel running for a period of 6 days. Running endurance was markedly reduced in klotho (hm) mice, which exhibited a sporadic running pattern that may be characteristic of repeated bouts of exhaustions. When actually running, klotho (hm) females ran at the same speed as wildtype and klotho overexpressing mice, but spent about 65 % less time running compared to the other two groups. Our novel results suggest an important link between klotho deficiency and muscle performance. This study provides a foundation for further research on klotho involvement as a potential inhibitor of age-associated muscle deterioration.

Fig. 1

Genotyping of the klotho^hm mouse. PCR analysis of a wildtype and b mutant alleles in klotho^hm mice (klotho^hm/hm) compared to a heterozygous control mouse (klotho^hm/+). PCR no template control samples (NTC) are included for each primer set

Fig. 2

Characterization of the klotho^hm mouse. a Klotho expression level (ΔCt, mean ± SEM) in the kidney of wild type (male n = 3, female n = 6) and klotho^hm (male n = 3, female n = 3) mice. All samples were independently normalized to the expression of the Eef2 reference gene. b Body weight (mean ± SEM, grams) of wildtype (male n = 9, female n = 6) and klotho^hm (male n = 4, female n = 7) mice between the ages of 7 and 11 months. Brackets represents significant differences with the indicated p value

Fig. 3

Analysis of forelimb grip strength in adult (7–11 months old) female mice. a Maximum grip strength force (mean ± SEM, grams-force) and b normalized force to body weight ratio (mean ± SEM) of wild type (n = 7), klotho^hm (n = 7), and klotho transgenic, EFmKL46 mice (n = 8). Significant differences identified by brackets with respective p values

Fig. 4

Mouse activity over 6 days of voluntary wheel running. Studies were performed with the adult female groups described in Fig. 3. a Klotho^hm mice ran significantly less than wildtype and EFmKL46 mice as shown by the total revolutions ran over the 6 days analyzed. b While klotho^hm mice ran at the same rate as wildtype and EFmKL46 mice (i.e., speed in revolutions/min, per actual time spent running), as shown in c the percentage of time spent running was significantly less compared to the other two groups. d Typical running frequency actograms (showing 2 independent examples per each mouse group), demonstrating a unique sporadic running pattern in klotho^hm mice characterized by frequent gaps in the nightly running routine. A schematic representation of the light and dark cycle (12/12-h), over the 6 experimental days, is shown below the actograms. a–c Significant differences are noted by brackets and respective p values