Age-Dependent Effects of Muscle Resting Calcium on Fasting Blood Glucose: Implications for Prediabetes Risk

Abstract

Background and aims: Skeletal muscle is the primary site for insulin-mediated glucose uptake and is critical in maintaining whole-body glucose homeostasis. Muscle cells from malignant hyperthermia-susceptible (MHS) individuals exhibit elevated resting cytosolic calcium concentration ([Ca²⁺]_cyto), and MHS subjects have a higher incidence of hyperglycaemia. This study investigates the association between elevated resting [Ca²⁺]_cyto and fasting blood sugar (FBS) levels while accounting for subject demographics and clinical variables.

Methods: We measured resting [Ca²⁺]_cyto in myotubes derived from muscle biopsies of control and MHS subjects. We analysed the impact of [Ca²⁺]_cyto on FBS levels based on age, sex, and MH status through correlation and comparative analyses. Data were stratified by FBS, [Ca²⁺]_cyto, and age, and heat map and 3D mesh plot analyses were performed to assess the risk of prediabetes in subjects with varying [Ca²⁺]_cyto and age.

Results: Between 2013 and 2024, muscle biopsies from 152 subjects (90 MHS, 62 controls) were used to establish primary myotube culture. MHS myotubes exhibited significantly higher resting [Ca²⁺]_cyto than controls and [Ca²⁺]_cyto positively correlated with fasting blood sugar (FBS) in MHS subjects (r = 0.227; p = 0.031) and across the entire cohort (r = 0.176; p = 0.034). This correlation was high in prediabetic individuals (r = 0.43; p = 0.005) but absent in those with normal FBS. Subjects over 40 years with [Ca²⁺]_cyto > 150 nM showed a higher risk of prediabetes. While aging is a significant risk factor for type 2 diabetes, increased age did not impact FBS levels in individuals with [Ca²⁺]_cyto below 150 nM. However, in those with [Ca²⁺]_cyto exceeding 150 nM, increasing age significantly influenced FBS.

Conclusions: Elevated resting [Ca²⁺]_cyto in skeletal muscle amplifies the risk of hyperglycemia and may contribute to the onset of type 2 diabetes.

Keywords: FBS levels; resting cytosolic calcium; skeletal muscle; type 2 diabetes.

FIGURE 1

Resting [Ca²⁺]_cyto and its association with FBS in Control and MHS subjects. (A) Resting [Ca²⁺]_cyto is significantly elevated in myotubes derived from MHS subjects compared to control (MH‐negative) myotubes (p < 0.001; 62 MHN, 90 MHS). Each data point represents the average [Ca²⁺]_cyto measured from 10 to 30 myotubes. (B) A positive correlation is observed between fasting blood sugar (FBS) levels and resting [Ca²⁺]_cyto of primary myotubes (r = 0.176; p = 0.034; n = 145) in entire cohort (blue line) composed of control subjects (open black) and MHS subjects (open red). (C) When the cohort is grouped by FBS levels, individuals with prediabetic FBS (above 5.6 mmol/L, open black triangles) show a strong positive correlation between [Ca²⁺]_cyto and FBS (r = 0.43; p = 0.005; n = 40), whereas those with normal FBS (below 5.6 mmol/L, blue open circles) show no significant correlation (r = 0.047; p = 0.63; n = 105). (D) A significant positive correlation is found between the [Ca²⁺]_cyto of primary myotubes and the age of the subjects in MHS cohort (r = 0.249; p = 0.018; n = 90), but not in control subjects (r = −0.049; p = 0.70; n = 62). *** indicate p < 0.001.

FIGURE 2

Combined effect of aging and elevated resting [Ca²⁺]_cyto on FBS levels. (A) Heat map showing FBS levels in relation to age and resting [Ca²⁺]_cyto, with prediabetic FBS range (≥ 5.6 mmol/L) shaded in red and normal FBS levels (< 5.6 mmol/L) shaded in blue. Subjects over 40 years with [Ca²⁺]_cyto above 150 nM appear to have an increased risk of prediabetes. (B) 3D mesh plot illustrating the relationship between FBS, age, and [Ca²⁺]_cyto for each subject with a red headed lane. Best subset regression analysis reveals that the combined effects of increasing age and elevated [Ca²⁺]_cyto significantly increase FBS levels (r = 0.220), compared with the individual influence of elevated [Ca²⁺]_cyto alone (r = 0.176; p = 0.034) or increasing age alone (r = 0.156; p = 0.060).

FIGURE 3

Influence of Age and resting [Ca²⁺]_cyto on FBS Levels. (A) Violin‐box plot comparison of FBS levels in older subjects (≥ 40 years) with [Ca²⁺]_cyto below and above 150 nM, showing a significant difference (p = 0.013; n = 20 for > 150 nM and n = 39 for < 150 nM). (B) Box plot comparison of FBS levels in younger subjects (< 40 years) with [Ca²⁺]_cyto below and above 150 nM, showing no significant difference (p = 0.61; n = 26 for > 150 nM and n = 60 for < 150 nM). (C) Box plots comparing FBS levels between younger (< 40 years) and older (≥ 40 years) subjects with [Ca²⁺]_cyto below 150 nM, showing no significant difference (p = 0.32; n = 60 for < 40 years and n = 39 for ≥ 40 years). (D) Box plot comparing FBS levels between younger (< 40 years) and older (≥ 40 years) subjects with [Ca²⁺]_cyto above 150 nM, revealing a significant difference (p = 0.046; n = 20 for < 40 years and n = 26 for ≥ 40 years). * indicate p < 0.05; ns‐non significant.