Lifetime of autofluorescence: A novel method to determine murine skeletal muscle fiber types

This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes derived from a two-exponential fit (τ1 and τ2) in freshly dissected mouse FDB and soleus muscles. While τ1 values did not change between muscles, the distribution of τ2 shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes in cryosections of both muscles and paired them with immunofluorescence images of myosin heavy chain isoforms (MHC), which allow identification of fiber types. In soleus, τ2 was 3.1 ns for type I (SEM = 0.009, n = 49), 3.4 ns for type IIA (SEM = 0.01, n = 30), and 3.3 ns for type IIX (SEM = 0.01, n = 21). In FDB muscle, τ2 was 3.17 ns for type I (SEM = 0.04, n = 18), 3.5 ns for type IIA (SEM = 0.03, n = 27), and 3.62 ns for type IIX (SEM = 0.03, n = 22). From the distribution of measures, it follows that an FDB fiber with τ2 >3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first- and second-class errors estimated at 0.06 and 0.27, respectively. Studies in progress aim at further elucidating the reasons for the different lifetimes, not just among fiber types but between fibers of the same type in the two muscles. Preliminary results point at differences in both the oxidation-reduction and protein-bound versus free states of flavins as causes for the observed divergence of fluorescence lifetimes. Lifetime maps of autofluorescence therefore constitute a tool to identify fiber type that, being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.

Impaired Function and Altered Morphology in the Skeletal Muscles of Adult Men and Women with Type 1 Diabetes

Context Previous investigations on skeletal muscle health in type 1 diabetes (T1D) has generally focused on later stages of disease progression where comorbidities are present and are posited as a primary mechanism of muscle dysfunction. Objective To investigate skeletal muscle function and morphology across the adult lifespan in those with and without T1D. Design Participants underwent maximal contraction (MVC) testing, resting muscle biopsy and venous blood sampling. Setting Procedures in this study were undertaken at the McMaster University Medical Centre. Participants Sixty-five healthy adult (18-78 years old) men/males and women/females [T1D=34; control=31] matched for age/biological sex/body mass index (BMI)/self-reported physical activity levels were included. Main Outcome Measures Our primary measure in this study was MVC, with supporting histological/immunofluorescent measures. Results After 35 years of age (‘older adults’), MVC declined quicker in T1D subjects compared to controls. Loss of strength in T1D was accompanied by morphological changes associated with accelerated aging. Type 1 myofiber grouping was higher in T1D, and the groups were larger and more numerous than in controls. Older T1D females exhibited more myofibers expressing multiple myosin heavy chain isoforms (hybrid fibers) than controls, another feature of accelerated aging. Conversely, T1D males exhibited a shift towards type 2 fibers, with less evidence of myofiber grouping or hybrid fibers. Conclusions These data suggest impairments to skeletal muscle function and morphology exist in T1D. The decline in strength with T1D is accelerated after 35 years of age and may be responsible for the earlier onset of frailty which characterizes those with diabetes.

DHA Supplementation Attenuates MI-Induced LV Matrix Remodeling and Dysfunction in Mice

Objective. Myocardial ischemia and reperfusion (I/R) injury is associated with oxidative stress and inflammation, leading to scar development and malfunction. The marine omega-3 fatty acids (ω-3 FA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are mediating cardioprotection and improving clinical outcomes in patients with heart disease. Therefore, we tested the hypothesis that docosahexaenoic acid (DHA) supplementation prior to LAD occlusion-induced myocardial injury (MI) confers cardioprotection in mice. Methods. C57BL/6N mice were placed on DHA or control diets (CD) beginning 7 d prior to 60 min LAD occlusion-induced MI or sham surgery. The expression of inflammatory mediators was measured via RT-qPCR. Besides FACS analysis for macrophage quantification and subtype evaluation, macrophage accumulation as well as collagen deposition was quantified in histological sections. Cardiac function was assessed using a pressure-volume catheter for up to 14 d. Results. DHA supplementation significantly attenuated the induction of peroxisome proliferator-activated receptor-α (PPAR-α) (2.3±0.4 CD vs. 1.4±0.3 DHA) after LAD occlusion. Furthermore, TNF-α (4.0±0.6 CD vs. 1.5±0.2 DHA), IL-1β (60.7±7.0 CD vs. 11.6±1.9 DHA), and IL-10 (223.8±62.1 CD vs. 135.5±38.5 DHA) mRNA expression increase was diminished in DHA-supplemented mice after 72 h reperfusion. These changes were accompanied by a less prominent switch in α/β myosin heavy chain isoforms. Chemokine mRNA expression was stronger initiated (CCL2 6 h: 32.8±11.5 CD vs. 78.8±13.6 DHA) but terminated earlier (CCL2 72 h: 39.5±7.8 CD vs. 8.2±1.9 DHA; CCL3 72 h: 794.3±270.9 CD vs. 258.2±57.8 DHA) in DHA supplementation compared to CD mice after LAD occlusion. Correspondingly, DHA supplementation was associated with a stronger increase of predominantly alternatively activated Ly6C-positive macrophage phenotype, being associated with less collagen deposition and better LV function (EF 14 d: 17.6±2.6 CD vs. 31.4±1.5 DHA). Conclusion. Our data indicate that DHA supplementation mediates cardioprotection from MI via modulation of the inflammatory response with timely and attenuated remodeling. DHA seems to attenuate MI-induced cardiomyocyte injury partly by transient PPAR-α downregulation, diminishing the need for antioxidant mechanisms including mitochondrial function, or α- to β-MHC isoform switch.

Molecular basis of impaired extraocular muscle function in a mouse model of congenital myopathy due to compound heterozygous Ryr1 mutations

Mutations in the RYR1 gene are the most common cause of human congenital myopathies, and patients with recessive mutations are severely affected and often display ptosis and/or ophthalmoplegia. In order to gain insight into the mechanism leading to extraocular muscle (EOM) involvement, we investigated the biochemical, structural and physiological properties of eye muscles from mouse models we created knocked-in for Ryr1 mutations. Ex vivo force production in EOMs from compound heterozygous RyR1p.Q1970fsX16+p.A4329D mutant mice was significantly reduced compared with that observed in wild-type, single heterozygous mutant carriers or homozygous RyR1p.A4329D mice. The decrease in muscle force was also accompanied by approximately a 40% reduction in RyR1 protein content, a decrease in electrically evoked calcium transients, disorganization of the muscle ultrastructure and a decrease in the number of calcium release units. Unexpectedly, the superfast and ocular-muscle-specific myosin heavy chain-EO isoform was almost undetectable in RyR1p.Q1970fsX16+p.A4329D mutant mice. The results of this study show for the first time that the EOM phenotype caused by the RyR1p.Q1970fsX16+p.A4329D compound heterozygous Ryr1 mutations is complex and due to a combination of modifications including a direct effect on the macromolecular complex involved in calcium release and indirect effects on the expression of myosin heavy chain isoforms.