The hemizygous Kvβ2^+/− null mice were a generous gift from Dr. Geoffrey Murphy (University of Michigan, MI, United States). The Kvβ2^+/− null mice were interbred to generate the age-matched wildtype (WT) and Kvβ2^−/− knockout (KO) mice as described previously (McCormack et al., 2002). All mice were fed with food and water ad libitum, and were maintained at Morsani College of Medicine, University of South Florida, FL, United States. The young (14–20 weeks) and old (1–2 years) WT and KO male mice were used in this study. All animal work was approved before the start of the study by the Institutional Animal Care and Use Committee (IACUC) at the University of South Florida, FL, United States.

The forelimb grip strengths of 14–16 weeks and 50–58 weeks old WT and KO mice were measured with Chatillon force measurement DFE II grip strength meter (Ametek, Columbus Instruments, OH, United States) as described previously (Manickam et al., 2022). Five individual measurements were recorded per mouse and is represented as the average grip strength expressed as KGF/Kg body weight, where n = 6–15 mice/group.

The ex vivo contractility and force-frequency relationship (FFR) were measured as described previously (Manickam et al., 2022). Briefly, the extensor digitorum longus (EDL) muscles from both hindlimbs were collected after euthanizing the mice in compliance with the University of South Florida, IACUC guidelines. Immediately after dissecting the EDL free, it was tied with surgical suture to a force transducer in buffer containing 137 mM NaCl, 0.4 mM NaH₂PO₄, 5.1 mM KCl, 1.05 mM MgCl₂, 2.0 mM CaCl₂, and 10 mM glucose at pH 7.4 in the bath maintained at 22°C and continuously aerated with 95% O₂/5% CO₂. After equilibration the optimal muscle length (l ₀ ) for each muscle was calculated and subjected to stimulation trains (500 ms) of frequencies ranging from 1 to 125 Hz, 1 per minute to determine the force-frequency relationship. The stimulation was delivered with S48 stimulators (Grass Products, RI, United States), and all pulses were 1 ms in duration. The peak forces of each contraction at the different stimulation frequencies were used to plot the FFR. The FFR was expressed in both absolute (mN) and normalized (mN/mg muscle mass) terms, with n = 4–7 mice/group.

The transmission electron microscopy (TEM) images were obtained as described previously (Manickam et al., 2022). Briefly, the hindlimb EDL muscles were dissected and fixed immediately with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer for 2 h at room temperature (RT), and overnight at 4°C. Subsequent washes were carried out in phosphate buffer solution and then fixed with 1% osmium tetroxide in phosphate buffer for 2 h at 4°C. After rinsing in phosphate buffer, the muscles were then dehydrated in ethanol and acetone at RT and infiltrated in acetone: pure resin for 4 h and then embedded in the embedding wax. Polymerization of the embedding mix was carried out at 70°C overnight. 1 µm and 70 nm thin sections were cut with Reichert Ultracut microtome, stained with 2% uranyl acetate, and contrasted with lead citrate as per standard protocol. Images were obtained at x15,000, ×30,000 and ×50,000 magnifications with JOEL1400 transmission electron microscope. The myofibrillar sarcomere lengths were analyzed at ×30,000 magnified images and quantified using ImageJ software, where n = 3 mice/group. 7–14 images were analyzed per mouse with a total of 184 and 191 myofibrils for the young WT and KO mice, respectively. For quantification, the yellow lines were drawn from one Z-line to the next Z-line of the I bands using NIH ImageJ software to measure the length of myofibrils sarcomeres.

The hindlimb tibialis anterior (TA) muscles were dissected out from the young and old WT and KO mice and embedded in optimal cutting temperature (OCT) medium and frozen in isopentane chilled in liquid nitrogen and stored at −80°C. 10 µm thick transverse serial cryosections were cut at the mid-belly region using Leica (CM1860) cryostat machine and stained with Weigert’s iron hematoxylin for 10 min at RT. After washing with tap water, the sections were left in picrofuchsin solution for 20 min at RT. Washes were done with graded ethanol with picric acid, and finally with 100% ethanol until clear. The sections were then cleared with xylene for 5 min twice at RT and mounted with DPX mountant. Montage images were obtained at ×10 magnifications with Olympus IX73 inverted microscope using CellSens Standard software (Olympus Soft Imaging Solution, United States). Quantification of the fibrotic tissues area (red color) was carried out with ImageJ software (NIH, United States), where n = 3 mice/group. The representative images were taken at ×20 magnification.

Total RNA was isolated from the hindlimb gastrocnemius (GAS) muscles of the young and old WT and KO mice using Exiqon miRCURY RNA isolation kit (Exiqon, MA, United States) as per manufacturer’s recommendation. cDNA was synthesized from the isolated total RNA using iScript cDNA synthesis kit (Bio-Rad Laboratories, CA, United States). Il6 expression was measured with iTaq Universal SYBR Green Supermix with CFX96 Real-Time C1000 Touch Thermal Cycler System (Bio-Rad Laboratories, CA, United States). The cDNA synthesis and qPCR procedures were performed as described previously (Chapalamadugu et al., 2018). The mouse GAPDH was used as an internal control reference gene, where n = 5–8 mice/group.

The hindlimb GAS muscles whole transcriptome sequencing was carried out as described previously (Manickam et al., 2022). Total RNA was extracted from the GAS muscles of young WT and KO mice, and the RNA quality and integrity were determined. RNA Seq library was then prepared. The cDNA library quality was determined before sequencing with Illumina PE150 platform. The data generated were analyzed between the WT and KO mice, where n = 3 mice/group for bioinformatics analysis.

RNA-Seq data was analyzed by using two open-source software programs: TopHat and Cufflinks (Trapnell et al., 2012). Raw reads were mapped to mice GRCm38 genome by using TopHat. Cufflinks quantified genes across WT and KO groups to find differentially expressed genes (q value < 0.05) between these two groups. The differentially expressed genes were then entered into the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (Sherman et al., 2022) to explore the KEGG gene pathways that are enriched in these genes.

Statistical analyses were performed with GraphPad Prism 9.5.1 or SigmaPlot software. An unpaired Student’s t-test was utilized when comparing only two groups. A 3-way ANOVA, with 2 between-subjects factors (age and genotype) and one repeated factor (frequency) was used to test the FFR. Data are represented as mean ± SD. Statistical significance was set at p < 0.05 for all data analyzed either with GraphPad Prism 9.5.1 or SigmaPlot software with post hoc tests.

Consistent with our previous publication, the young (20 weeks) Kvβ2 KO mice displayed a significant (p = 0.0215) decrease in body weight compared to littermate WT mice (Chapalamadugu et al., 2018). In the present study, we demonstrate that a similar significant (p = 0.0161) reduction in body weight was noticed in old (1.5–2 years) Kvβ2 KO mice compared with age-matched WT mice (Figures 1A, B). Together this suggests that the deletion of Kvβ2 in mice results in decreased body weight postnatally and during aging.

Previously, we demonstrated that deletion of Kvβ2 in mice results in decreased hindlimb muscles size and mass, and the muscle stem cells marker Pax7 protein levels (Chapalamadugu et al., 2018). However, it was not known if those changes resulted in any alterations in skeletal muscle contractile function and grip strength. Therefore, in the present study we assessed the forelimb grip strength and ex vivo skeletal muscle force-frequency relationship (FFR) of the hindlimb EDL muscles as a measure of function in the age-matched WT and KO mice. Young (14–16 weeks) Kvβ2-KO mice displayed a significant (p = 0.0446) decrease in forelimb grip strength compared to age-matched WT mice (Figure 2A). A similar significant (p = 0.0417) reduction in the forelimb grip strength was also noticed during aging in the 50–58 weeks-old Kvβ2 KO mice compared to age-matched WT mice (Figure 2B). In addition, the young (Y-20 weeks) and old (O-2yr) Kvβ2 KO mice displayed a decrease in the hindlimb EDL muscles absolute and normalized FFR compared with age-matched WT mice. There was a significant effect of genotype (p < 0.001), and an age x genotype (p = 0.006) interaction (Figures 2C, D). Together, these data suggests that deletion of Kvβ2 in mice results in impaired muscle function both in vivo and ex vivo.

Previously, we showed that deletion of Kvβ2 in young mice results in decreased mass of hindlimb muscles: biceps femoris, gastrocnemius, and soleus (Chapalamadugu et al., 2018). Here we show a similar significant decrease not only in the young (20 weeks) Kvβ2 KO mice hindlimb muscles EDL (p = 0.0343), GAS (p = 0.0056), and Quadriceps (QUAD) (p = 0.0001) but also in the old Kvβ2 KO mice EDL (p = 0.0213), GAS (p = 0.0021), and QUAD (p = 0.0003) muscles compared to age-matched WT mice (Figures 3A–F). In addition, the direct comparison of old (2 years) WT and young (20 weeks) Kvβ2 KO mice hindlimb muscles EDL (p = 0.4146), GAS (p = 0.1065), and QUAD (p = 0.1169) did not show any significant difference in their weights (Supplemental Figures S1A–C). This clearly suggests that the reduction in muscle mass in Kvβ2 KO mice leads to decreased hindlimb muscles grip strengths both in the young and old mice.

The transmission electron microscopy image analysis of the longitudinal sections of the hindlimb EDL muscles displayed a significant (p = 0.0086) reduction in myofibrils sarcomere length in the young (20 weeks) Kvβ2 KO mice compared to age-matched WT mice (Figures 4A–C). In addition, a significant reduction in the myofiber cross-sectional area of the medium (2,000–4,000 µm²) (p = 0.0016) and largest (>4,000 µm²) (p = 0.0009) sized myofiber areas was also observed in the young (20 weeks) Kvβ2 KO mice hindlimb TA muscles cryosections stained with hematoxylin and eosin (H&E) compared to age-matched WT mice (Figures 4D, E), These results further confirm that deletion of Kvβ2 in mice results in decreased hindlimb muscle mass.

We then assessed the inflammaging phenotype of the skeletal muscles of the young (20 weeks) and old (2 years) mice through Van Gieson staining (fibrosis) of the hindlimb TA muscles cryosections and expression of the proinflammatory marker Il6 through RT-qPCR. Deletion of Kvβ2 displayed a significant (p = 0.0103) increase in fibrotic tissue area in the young (20 weeks) mice compared to age-matched WT mice, whereas it was found to be non-significant (p = 0.3081) in the old mice between the genotypes (Figures 5A–C). This was accompanied by a significant (p = 0.0008) increase in the proinflammatory marker Il6 expression in the young (20 weeks) Kvβ2 KO mice compared to age-matched WT mice (Figure 5D). However, the expression levels Il6 was found to be non-significant (p = 0.1699) in the 2 years old WT and KO mice (Figure 5E).

To evaluate the differential gene expression of the young Kvβ2 KO and WT mice, RNA Seq analysis was performed in the hindlimb GAS muscles. The volcano plot shows the number of differentially expressed key genes (Figure 6A) that were significantly (p < 0.05) upregulated (384 genes) and downregulated (40 genes) in the young Kvβ2 KO mice compared to age-matched WT mice. Subsequent heat map analysis of the differentially expressed key genes in the young Kvβ2 KO and WT mice displayed a significant (p < 0.05) increase in the expression of genes involved in skeletal muscle development, cell proliferation and determination (Myo1d, Notch1, and Notch4), muscle atrophy (Trim54, Hdac5, Sumo2, Acvr1b, and N4bp3), energy metabolism and muscle plasticity (Irs2, Akt1, Ucp2, Fndc5, Plin3, Plin4, Ppara, and Ppard), and inflammation (Il6ra and Il34), and a decrease in circadian core clock (Clock and Arntl) genes along with an increase in the negative feedback loop clock gene repressors such as Per1, Per2 and Cry2 in young Kvβ2 KO mice compared to age-matched WT mice. However, there was also a decrease in Acvr2a, Cry1 and Il1r1 genes expression in the young Kvβ2 KO mice compared to age-matched WT mice (Figure 6B) suggesting the differential regulation of various signaling pathways involved in muscle atrophy, peripheral circadian rhythm and inflammation between the genotypes (Figures 6C, D).