Kinetic properties of individual contractions from EDL and soleus muscles.

<p>A) Rate of force generation of individual contractions from EDL muscle at the stimulatory frequencies ranging from 1–130 Hz (^<b>a</b> denotes significant difference in <i>β</i>-alanine group compared to control diet. The bracket indicates significance at all stimulatory frequencies tested). B) The time to reach peak contractile force in contractions from EDL muscle stimulated with the frequencies of 1–130 Hz (* denotes significant difference in HMB diet compared to control diet). C) The time constant (tau) in the decaying exponential fit to the tail of the contractions of EDL muscle stimulated at 1–130 Hz. D) Rate of force generation of individual contractions from soleus muscle at the stimulatory frequencies ranging from 1–130 Hz (^<b>a</b> denotes significant difference in <i>β</i>-alanine group compared to control. E) The time to reach peak contractile force in contractions from soleus muscle stimulated with the frequencies of 1–130 Hz (* denotes significant difference in HMB diet compared to control diet, ^<b>a</b> denotes significant difference in <i>β</i>-alanine group compared to control, ^<b>b</b> denotes significant difference in HMB + <i>β</i>-alanine group compared to control). F) The time constant (tau) in the decaying exponential fit to the tail of the contractions of soleus muscle stimulated at 1–130 Hz. (EDL: Control-n = 24 muscles, HMB-n = 23 muscles, <i>β</i>-alanine-n = 24 muscles, HMB + <i>β</i>-alanine-n = 24 muscles. SOL: Control-n = 24 muscles, HMB-n = 24 muscles, <i>β</i>-alanine-n = 24 muscles, HMB + <i>β</i>-alanine-n = 24 muscles).</p

HMB enhances C₂C₁₂ myoblasts cell viability and proliferation.

<p>C₂C₁₂ myoblasts were treated with either 25 μM or 125 μM free acid HMB or vehicle control. Cell viability was assessed at 48 and 72 hours proliferation using the Trypan Blue exclusion assay. Experiments were performed under conditions of both low serum (A) and normal serum (B), (Control, n = 6; 25 μM HMB, n = 3; 125 μM HMB, n = 3, * denotes significant difference compared to control: 3% FBS, P = 0.044; 10% FBS, P = 0.031, One-way ANOVA). C) C₂C₁₂ myoblasts were treated with 25 μM or125 μM HMB or vehicle control for 48 hours in proliferation media (10% FBS) at which point total viable cells were counted while gating out cellular debris with the Scepter™ Automated Cell Counter (Control, n = 6; 25 μM HMB, n = 3; 125μM HMB, n = 3,* denotes significance compared to control: 25 μM HMB, P<0.042; 125 μM HMB, P<0.01, One-way ANOVA).</p

Cellular and Physiological Effects of Dietary Supplementation with <i>β</i>-Hydroxy-<i>β</i>-Methylbutyrate (HMB) and <i>β</i>-Alanine in Late Middle-Aged Mice

<div><p>There is growing evidence that severe decline of skeletal muscle mass and function with age may be mitigated by exercise and dietary supplementation with protein and amino acid ingredient technologies. The purposes of this study were to examine the effects of the leucine catabolite, beta-hydroxy-beta-methylbutyrate (HMB), in C₂C₁₂ myoblasts and myotubes, and to investigate the effects of dietary supplementation with HMB, the amino acid <i>β</i>-alanine and the combination thereof, on muscle contractility in a preclinical model of pre-sarcopenia. In C₂C₁₂ myotubes, HMB enhanced sarcoplasmic reticulum (SR) calcium release beyond vehicle control in the presence of all SR agonists tested (KCl, P<0.01; caffeine, P = 0.03; ionomycin, P = 0.03). HMB also improved C₂C₁₂ myoblast viability (25 μM HMB, P = 0.03) and increased proliferation (25 μM HMB, P = 0.04; 125 μM HMB, P<0.01). Furthermore, an <i>ex vivo</i> muscle contractility study was performed on EDL and soleus muscle from 19 month old, male C57BL/6nTac mice. For 8 weeks, mice were fed control AIN-93M diet, diet with HMB, diet with <i>β</i>-alanine, or diet with HMB and <i>β</i>-alanine. In <i>β</i>-alanine fed mice, EDL muscle showed a 7% increase in maximum absolute force compared to the control diet (202 ± 3vs. 188± 5 mN, P = 0.02). At submaximal frequency of stimulation (20 Hz), EDL from mice fed HMB plus <i>β</i>-alanine showed an 11% increase in absolute force (88.6 ± 2.2 vs. 79.8 ± 2.4 mN, P = 0.025) and a 13% increase in specific force (12.2 ± 0.4 vs. 10.8 ± 0.4 N/cm², P = 0.021). Also in EDL muscle, <i>β</i>-alanine increased the rate of force development at all frequencies tested (P<0.025), while HMB reduced the time to reach peak contractile force (TTP), with a significant effect at 80 Hz (P = 0.0156). In soleus muscle, all experimental diets were associated with a decrease in TTP, compared to control diet. Our findings highlight beneficial effects of HMB and <i>β</i>-alanine supplementation on skeletal muscle function in aging mice.</p></div

EDL and soleus muscle optimal length and mass.

<p>EDL and soleus muscle optimal length and mass.</p

Fatiguing stimulation and recovery from fatigue in EDL and soleus muscles.

<p>Alternating frequencies of 80 Hz and 20 Hz were used to fatigue the muscles intermittently for 5 minutes, with a periodicity of one second. The periodicity, or interval between stimulations, was then extended to one minute to allow force recovery in the absence and presence of 5 mM caffeine. A and B) EDL fatigue and recovery, respectively, using the low stimulatory frequency of 20 Hz. D) EDL fatigue and recovery, respectively using the high frequency of 80 Hz. E and F) Soleus fatigue and recovery, respectively, using the low stimulatory frequency of 20 Hz (<b>@</b> denotes P = 0.049). G and H) Soleus fatigue and recovery, respectively, using the high frequency of 80 Hz. (EDL: Control-n = 22 muscles, HMB-n = 23 muscles, <i>β</i>-alanine-n = 24 muscles, HMB + <i>β</i>-alanine-n = 24 muscles. SOL: Control-n = 22 muscles, HMB-n = 24 muscles, <i>β</i>-alanine-n = 24 muscles, HMB + <i>β</i>-alanine-n = 22 muscles).</p

Summary of <i>in vivo</i> study timeline and muscle contractility assay.

<p>A) Schematic representation of the dietary intervention study performed on 48 C57BL/6nTac male mice. All mice were 19 months old at the time of sacrifice for contraction studies. B) Representative tracing of force data from the <i>ex vivo</i> contractility assay obtained from one muscle (<i>X-axis</i>: Time; <i>Y-axis</i>: Force). Equilibration, fatigue, and recovery from fatigue protocols are performed using alternating high (80 Hz) and low (20 Hz) frequencies of stimulation. <i>Inset</i>: Magnified view of fatiguing stimulations using alternating high (80 Hz) and low (20 Hz) stimulation frequencies.</p

EDL and soleus muscle force vs. frequency relationships.

<p>Muscle contraction was stimulated with increasing frequencies from 1–130 Hz to determine the force-frequency relationship. A) EDL muscle absolute force. <i>Inset</i>: Raw data traces of 1 Hz, 20 Hz and maximal tetanic contractions (* denotes P<0.025, HMB + <i>β</i>-alanine compared to control diet; ^<b>a</b> denotes P<0.025, <i>β</i>-alanine compared to control diet). B) EDL muscle specific force (* denotes P<0.025, HMB + <i>β</i>-alanine compared to control diet). C) Soleus muscle absolute force. D) Soleus muscle specific force. (EDL: Control-n = 24 muscles, HMB-n = 23 muscles, <i>β</i>-alanine-n = 24 muscles, HMB + <i>β</i>-alanine-n = 24 muscles. SOL: Control-n = 23 muscles, HMB-n = 24 muscles, <i>β</i>-alanine-n = 24 muscles, HMB + <i>β</i>-alanine-n = 24 muscles).</p