High-Fat Diet and Vitamin D Effects on Contractile Performance of Isolated Mouse Soleus and EDL

doi:10.21203/rs.3.rs-3114957/v1

Evidence suggests vitamin D (VD) could mitigate adverse effects of obesity on skeletal muscle (SkM) function, however, this is yet to be directly investigated. Therefore, this study used the work-loop technique to examine effects of high dose dietary VD supplementation on contractile performance of isolated SkM. Female mice (N = 37) consumed standard (SLD) or high-fat diet (HFD), with or without VD (20,000 IU/kg^− 1) for 12-weeks. Soleus and EDL (N = 8–10) were isolated and absolute and normalised (relative to muscle size and body mass) isometric force and power output (PO) were measured, and fatigue resistance determined. Absolute and normalised isometric force and PO of the soleus were unaffected by diet (P > 0.087). However, PO normalised to body mass was reduced in HFD groups (P < 0.001). Isometric force of the EDL was unaffected by diet (P > 0.588). HFD evoked reduced EDL isometric stress (P = 0.048) and absolute and normalised PO (P < 0.031), but there was no effect of VD (P > 0.493). Cumulative work during fatiguing contractions was lower in HFD treated SkM (P < 0.043), but the rate of fatigue was unaffected (P > 0.060). This study uniquely demonstrated that high dose dietary VD had limited effects on SkM contractile function and did not offset the demonstrated adverse effects of HFD. However, there was non-significant small and moderate effects suggesting improvement in EDL muscle performance and animal morphology in HFD VD groups. Given trends observed, coupled with the proposed inverted U-shaped dose-effect curve, future investigations are needed to determine dose/duration specific responses to VD, which may culminate in improved function of HFD treated SkM.

High-fat diet

Obesity

Force

Power output

Fatigue

Work loop

Muscle function

The present study is the first to examine the synergistic effects of HFD consumption and vitamin D supplementation on the contractile performance of isolated skeletal muscle. These findings suggest high dose vitamin D has limited effects on force, power or fatigue resistance of isolated mouse soleus and EDL

Obesity has quickly become a public health crisis, due to its links with poor physical (Chu et al., 2018) and mental health (Talen & Mann, 2009), and increased risk of morbidities and mortality (Abdelaal et al., 2017; Chu et al., 2018; Silvestris et al., 2018). Adequate skeletal muscle (SkM) health is key to maintaining whole-body health (Frontera & Ochala, 2015); reductions in SkM health are associated with increased risk of non-communicable disease, mortality and reduced overall quality of life (Murphy & Hartley, 2018; Wu et al., 2017). Excess adipose impairs SkM metabolism, myogenesis and contraction resulting in a negative cycle of lipid accumulation (Tallis et al., 2018). As such reductions in SkM health associated with excess adipose exacerbate the adverse effects of obesity on whole-body function and health.

Understanding of obesity effects on SkM function has been advanced by high fat diet (HFD) and isolated skeletal muscle models (Tallis et al., 2018). Such methods have distinct advantages over whole body in vivo assessments of muscle function, which have been outlined in recent reviews (Tallis et al., 2015, 2018, 2021). In brief, isolated skeletal models allow for examination of direct muscle and fibre type specific effects, and control of dietary composition and duration which are difficult to ascertain and control in human populations. Furthermore, isolated skeletal muscle models allow a more accurate evaluation of the direct impact of HFD on muscle quality (force normalised to cross-sectional area or power normalised to muscle mass) given that the size of the contractile tissue can be more accurately determined, when compared to in vivo assessments. Understanding muscle quality is important as it gives an indication of HFD effects on the intrinsic force producing capacity of muscle (Tallis et al., 2018). Reductions in muscle quality are impactful in obese individuals, where in addition to an elevated cost of tissue failure, larger muscles are often formed, contributing to an already elevated bodily inertia (Tallis et al., 2017). In addition, direct examination of the effects of HFD on muscular fatigue is important for establishing if there is a reduction in the ability to sustain repetitive muscular contractions, opposed to an increase in muscle fatigability solely occurring in obese individuals due the increase in force requirement of moving a larger mass.

Although effects of HFD consumption on isolated muscle function are contractile mode and muscle specific, evidence suggests that HFD consumption can lead to a reduction in force and power normalised to body mass and muscle size (Tallis et al., 2018, 2021) and fatigue resistance (Hill et al., 2019; Hurst et al., 2019; Tallis et al., 2017). The prevalence and magnitude of HFD effects appear affected by HFD dietary composition, feeding duration, test temperature, age and sex (Tallis et al., 2018). Whilst few studies have directly examined both contractility and underpinning mechanisms associated with change in function, HFD induced reductions in contractile function have been attributed to excessive adiposity and intramuscular lipids (Messa et al., 2020) decreased AMPK activity (Tallis et al., 2017), impaired mitochondrial function (Heo et al., 2017), chronic inflammation (Erskine et al. 2017), impaired excitation contractile coupling (Eshima et al., 2020) and impaired myogenesis (D’Souza et al., 2015). Given the detrimental effects of HFD induced obesity on SkM health, and the link between SKM and whole-body health, understanding strategies to ameliorate HFD effects on SKM function are important for the prevention and management of obesity and its associated comorbidities.

Calorific restriction and increased physical activity are well-established lifestyle interventions for obesity management (Ross 2000; Touati et al. 2011; Foster-Schubert et al. 2012; Julian et al. 2018). However, adherence to such strategies is poor, attributed to requiring large lifestyle modification, which appears unsustainable for most. Nutritional supplements, which are low cost and are low demanding, could be an appealing alternative to current lifestyle interventions (Tallis et al., 2021). One such supplement is Vitamin D (VD) (or in its biologically active form 1α,25(OH)2D3, 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]), which has been suggested as a potential nutritional strategy to reduce the impact of HFD consumption on skeletal muscle contractility (Tallis et al., 2021). VD deficiency (typically defined as serum 25-hydroxyvitamin D [25(OH)D] concentrations < 50 nmol/L) (Liu et al., 2018), independent of obesity, is associated with declines in skeletal muscle contractility (Toffanello et al., 2012, Visser et al., 2003, Gerdhem et al., 2005). An increasing body of evidence supports the notion that obesity may be a contributing factor leading to VD deficiency (VDD) (Duan et al., 2020). As such, VDD itself could be one of the primary mechanisms contributing to skeletal muscle dysfunction in obese individuals. Over recent years, chronic supplementation of VD has been increasingly explored in HFD rodent models (Alkharfy et al., 2012; Benetti et al., 2018; Fan et al., 2016; Marcotorchino et al., 2014; Montenegro et al., 2021; Trovato et al., 2018). There is evidence to suggest VD supplementation can reduce and attenuate the increase in both body mass and adiposity associated with HFD consumption (Benetti et al., 2018; Fan et al., 2016; Marcotorchino et al., 2014) through the inhibition of adipogenesis and increase in UCP3 activation (Fan et al., 2016). Furthermore, research indicates VD supplementation may ameliorate mechanisms reported to contribute to obesity induced declines in muscle function, such as modulating inflammation (Farhangi et al., 2017; Szymczak-Pajor & Śliwińska, 2019) and promoting myogenesis (Cipriani et al., 2014), although the latter has not been directly examined in an obese animal model. Despite this mechanistic potential, to date there are no research studies which have examined the potential of VD for alleviating the decline in isolated muscle contractile function associated with HFD consumption.

Using previously established protocols for examining the impact of HFD on contractile performance of skeletal muscle (Tallis et al. 2017; Hill et al. 2019; Hurst et al. 2019), the present study uniquely examined the effects of 12 weeks HFD, with and without high dose VD, on the maximal isometric force, work loop power output (PO), and fatigue resistance of soleus (predominately slow-twitch) and extensor digitorum longus (EDL; predominantly fast-twitch) muscle, isolated from young adult female CD-1 mice.

Animals

The procedures outlined in this study and the use of animals was approved by the ethics committee of host universities and the British Home Office and was completed in accordance with the Animals (scientific procedures) Act 1986. Female CD-1 mice, purchased at ~ 4 weeks old (Charles River, Kent, UK), were housed in groups of five at the University of Warwick and kept in 12:12 hour light:dark cycles at 50% relative humidity, at 22⁰C. Mice were provided with ad libitum access to food and water. At 4 weeks of age, mice were randomly split into four experimental groups (total starting sample: n = 10 per group; final sample n = 10 SLD VD; n = 9 SLD, HFD and HFD VD; see Table ) and following 13 days of habituation, which included gradual transition from standard lab chow (TestDiet 5755; calories provided by protein 18.3%, fat 22.1% and carbohydrate 59.6%; gross energy 4.07 kcal/g; VD 2200IU/g) to new respective custom diets, mice consumed one of the following for 12 weeks: 1) Standard low-fat diet (TestDiet 58Y2; calories provided by protein 18.0%, fat 10.2% and carbohydrate 71.8%; gross energy 3.76 kcal/g; VD 900IU/kg) (SLD), 2) High-fat diet (TestDiet 58V8; calories provided by protein 18.3%, fat 45.7% and carbohydrate 35.5%; gross energy 4.62 kcal/g; VD 1200IU/kg) (HFD), 3) SLD enriched with VD (20,000IU/kg feed) and 4) HFD enriched with VD, as shown in Table 1. Procedures were carried out blinded.

Table 1

Treatment Groups
Group	Abbreviation	12-wks Treatment
Standard Low-fat Diet (control)	SLD	SLD
High Fat Diet	HFD	HFD
Standard Low-fat Diet + Vitamin D	SLD VD	SLD + VD 20,000IU/kg feed
High Fat Diet + Vitamin D	HFD VD	HFD + VD 20,000IU/kg feed
SLD VD n = 10; SLD, HFD, HFD VD n = 9 HFD = High Fat Diet; SLD = Standard Low-fat Diet; VD = Vitamin D

Twelve-weeks feeding duration and or 45% HFD was selected based on previous work that demonstrated large and statistically significant changes in body composition and muscle mechanics over similar durations (Bott et al., 2017; Eshima et al., 2017, 2020; Hurst et al., 2019; Matsakas et al., 2015). Furthermore, typical human western diets may consist of ~ 30–40% fat content, making a tolerable human HFD around 50–60%; fat content in a typical rodent diet is ~ 10%, thus 45% rodent HFD represents similar dietary distortion found in standard and high-fat human diets (Speakman, 2019). The dose of VD used (20,000IU/kg^− 1 diet) was selected based on previous evidence indicating that similar high doses attenuate the increase in overall and segmental adipose tissue associated with HFD consumption over a 10 week feeding duration (15,000IU/kg^− 1 diet) (Marcotorchino et al., 2014), and increase levels of serum 25(OH)D in blood plasma in HFD and SLD treated mice (25,000IU/kg^− 1 diet) (Park et al., 2020). Furthermore, an identical dose (20,000IU/kg^− 1 diet) has previously shown to elicit improvements in isolated skeletal muscle contractility in healthy mice, when compared to an apparently healthy, VD sufficient control group (Debruin et al., 2019; Hayes et al., 2019). Previous work indicates that 20,000IU/kg^− 1 diet of VD equates to a daily intake of approximately 2,700IU/kg^− 1 body mass in mice (Hayes et al., 2019). Following a previously determined calculation for converting dose in mice to dose in humans (daily animal dose in kg^− 1 body mass ÷ metabolic scaling factor for mice; Nair & Jacob, 2016), the human equivalent dose (HED) of vitamin D is ~ 13,000IU^− 1 day for a 60kg individual.

Muscle preparations

Following the 12 weeks treatment period animals were culled via cervical dislocation (in accordance with the British Home Office Animals Scientific Procedures Act 1986, Schedule 1). Animals were then weighed, to determine body mass, and nasoanal length measured using digital callipers (Fisher Scientific$™$ 3417, Fisher Scientific, Loughborough, UK). From this information body mass index (BMI) and Lee index of obesity (Bernardis & Patterson, 1968) were calculated for each mouse.

$$Body Mass Index=\frac{BM\left[g\right]}{\left({NAL\left[cm\right]}^{2}\right)}/100$$

Equation 1 – Rodent Body Mass Index Calculation (Sjögren et al., 2001).

$$Lee Index of Obesity=\frac{\sqrt[3]{BM\left[g\right]}}{NAL\left[cm\right]}x 1000$$

Equation 2 - Lee Index of Obesity calculation (Bernardis and Patterson, 1968).

The subcutaneous fat pad around the top of the hindlimbs and genitals was extracted and weighed as an indication of overall adiposity (Rogers & Webb, 1980) and was later normalised to body mass for a measure of relative adiposity (Hill et al., 2019). In addition, whole extensor digitorum longus (EDL), from the right hind limb, and soleus, from the left hindlimb, were dissected in refrigerated (1–3°C) oxygenated (95% O₂, 5% CO₂) Krebs Henseleit solution (in mM: NaCl 118; KCl 4.75; MgSO₄ 1.18; NaHCO₃ 24.8; KH₂PO₄ 1.18; glucose 10; CaCl₂ 2.54 in each case; pH 7.55 at room temperature before oxygenation). The soleus and EDL represent locomotor muscles which differ in fibre type and function (soleus 53.6% type I, 31.2% type IIA, 15.2% type IIX; EDL 3.9% type I; 9.3% type IIX; 86.8% type IIB) (Agbulut et al., 2003), which allow for a greater understanding of any potential muscle and fibre type-specific effects of HFD and VD. For each muscle preparation, the tendon attachment at the proximal end was left intact with a small piece of bone still attached and aluminium foil T-clips were wrapped around the distal tendon/s as close to the muscle as possible to avoid slippage when the muscle was producing force (Ford et al., 1977; Goldman & Simmons, 1984; James et al., 2005).

Contractility measures

Upon dissection the muscle was placed in a perspex flow through chamber filled with circulated constantly oxygenated (95% O₂, 5% CO₂) Krebs Henseleit solution. The reservoirs of Krebs solution were stored in external heater/cooler baths (Grant LTD6G, Grant Instruments, Shepreth, UK), which were adjusted to maintain a physiologically relevant temperature of 37 ± 0.2°C inside the muscle bath. Temperature in the bath was continuously monitored using a digital thermometer (Traceable, Fisherbrand, Fisher Scientific, Loughborough, UK). Using the bone at the proximal end, the muscle was attached to a crocodile clip connected to a force transducer (UF1, Pioden Controls Ltd, Henwood Ashford, UK) and the T-foil clip at the distal end was attached to a crocodile clip, connected to a motor arm (V201, Ling Dynamic Systems, Royston, UK).

Isometric force

The procedures utilised to measure the contractile properties of isolated mouse EDL and soleus in this study are based on well-established protocols. For the assessment of isometric contractile properties, platinum electrodes running parallel to the muscle within the bath received an electrical stimulation from a tabletop power supply to activate the muscle (PL320, Thurlby Thandar Instruments, Huntington, UK). Stimulation parameters were controlled using custom-written software (Testpoint, CEC), via a D/A board (KPCI3108, Keithley Instruments), on a standard desktop personal computer. Muscle length and stimulation amplitude (typically 12-16V for SOL and 14-18V for EDL) were optimised to elicit a maximal isometric twitch response, the magnitude of which was determined via a digital storage oscilloscope (2211 or 1002, Tektronix, Marlow, UK). Using the optimised muscle length and stimulation amplitude, stimulation frequency was manipulated (100-130Hz [typically 120Hz] and 200-230Hz [typically 230Hz] for the SOL and EDL respectively) to elicit a maximal isometric tetanus, with a 5-minute rest implemented between each tetanus (Askew & Marsh, 1997; Shelley et al., 2022; Vassilakos et al., 2009). In all cases, burst duration was fixed at 350ms for SOL and 250ms for EDL. After maximal force had been achieved a control tetanus was performed at the first stimulation frequency to monitor change in contractile performance over time. From the maximal tetanus response, time to half peak tetanus force (TTHP) and last stimulus to half relaxation (LSHR) were recorded as indicative measures of activation and relaxation kinetics, respectively (Ebashi & Endo, 1968). The muscle length optimal for isometric performance, defined as L₀, was measured using an eyepiece graticule and microscope. Estimated fibre length for the SOL and EDL were calculated as 85% and 75% of L₀, respectively (James et al., 1995).

Work-loop power output

The work-loop technique (WL), which has been used in previous research examining the effects of HFD on mouse skeletal muscle function (Tallis et al. 2017; Hill et al. 2019; Hurst et al. 2019), was utilised to examine the contractile performance of isolated muscle during cyclical length changes, which better replicate in vivo dynamic muscle activity when compared to other in-vitro assessments (James et al., 1995, 1996; Josephson, 1985, 1993). The WL technique involved each muscle being subjected to four sinusoidal length changes around the previously determined L_0. During the length change cycle, muscle was stimulated to produce force primarily during the shortening phase, using the previously determined stimulation amplitude. Stimulation frequency was increased from that which resulted in maximal isometric performance to 160 and 260Hz for the SOL and EDL respectively, as previous work indicates that a greater stimulation frequency is required for maximal WL PO (Shelley et al., 2022; Vassilakos et al., 2009). Length changes were performed via movement of the motor arm (V201, Ling 220 Dynamic Systems, Royston, UK), the position of which was measured using a Linear Variable Displacement Transformer (DFG5.0, Solartron Metrology, Bognor Regis, UK). During the length change cycle, instantaneous force and velocity were sampled at 10kHz and plotted against each other, forming a WL. The instantaneous power (instantaneous velocity * instantaneous force) calculated at each time point during the work loop was averaged over the entire work loop to determine net power output. A range of cycle frequencies (CF; rate at which each muscle undergoes sinusoidal length change cycle) were utilised to examine whether the CF that elicited maximal PO was influenced by a HFD or VD.

Initially, CF’S of 5Hz and 10Hz, burst durations of 65ms and 50ms, phase shift of -10ms and − 2ms (time of initiation of stimulation prior to muscle reaching its greatest length), and a strain of 0.10 (muscle lengthening by 5%, shortening by 10% and elongating by 5% back to L₀.) were used to examine the SOL and EDL respectively, as previous work has established that these muscles typically achieve maximal WL PO using these parameters (James et al., 1995; Tallis et al., 2017). Burst duration, phase and strain were all subject to change based on WL shape to maximise net work, e.g. where WL shape sloped downwards during re-lengthening, as the muscle was still undergoing relaxation, burst duration was reduced to achieve maximal WL PO. Every 5-minutes, a set of four WL’s were performed, and maximal net work was recorded and later used to quantify power production. Once parameters for optimal PO at 5Hz and 10Hz CF for the soleus and EDL respectively were identified, these CF’s were then used as the control WL’s for the remainder of the experiment. Once maximal WL PO had been achieved at the control CF’s, power output was then assessed across a range of CF’s to establish a PO-CF curve for each experimental group (Hill et al. 2019, 2020; Hurst et al. 2019). The range of CF’s used, and typical parameters utilised to elicit maximal net work of each muscle at each CF, are displayed in Table 2, with the exception of phase, which was typically − 10ms for the SOL and − 2ms for the EDL; at higher CF’s phase would occasionally be reduced to ensure stimulation occurred at the onset of shortening, e.g. at 10Hz for the soleus − 6ms phase was often implemented. On rare occasions strain would be increased beyond that provided in Table 2 (0.2 increments) at lower CF’s when WL’s appeared rectangular in shape, again to ensure maximal net work. Except for the control CF’s, the order of CF’s was randomised using Microsoft Excel, Windows v. 2016. After every three CF’s, and after the final CF examined, a control set of WL’s were performed using the initial parameters to examine the deterioration of power over time as the performance of isolated mouse muscle will deteriorate over time due to the build-up of an anoxic core (Barclay, 2005). Utilising this approach allowed for the correction of net-work for other CF’s relative to the control WL’s, as has been common practice in previous research (Vassilakos et al. 2009; Hurst et al. 2019; Hill et al. 2020). Once the final control WL was performed, the muscle rested for 10 minutes prior to assessment of fatigue and recovery.

Table 2

Typical stimulus burst durations and strains that elicited maximal net work, at each cycle frequency, during the WL assessment of Soleus and EDL.
	Cycle Frequency (Hz)
Muscle	Parameter	2	3	4	5	6	7	8	10	12	14	16
Soleus	Burst Duration (ms)	245	150	92	65	52	35	24	11	-	-	-
Soleus	Strain	0.13	0.12	0.11	0.1	0.09	0.08	0.07	0.06	-	-	-
EDL	Burst Duration (ms)	-	-	110	-	75	-	65	50	32	24	16
EDL	Strain	-	-	0.13	-	0.12	-	0.11	0.1	0.09	0.08	0.07

Dash (-) represents CF’s not used for assessment of contractile performance. Values provided were subject to change based on the shape of WL to ensure maximal WL PO.

Fatigue resistance, cumulative work and recovery

For examination of fatigue resistance, each muscle preparation was subjected to 50 consecutive WL cycles, using the parameters that elicited maximal WL PO at 5Hz and 10Hz CF for the Soleus and EDL respectively. However, stimulation frequency was reduced to 100Hz and 200Hz for the Soleus and EDL respectively, to better represent the in vivo mechanism of fatigue as suggested by previous work (Shelley et al., 2022; Vassilakos et al., 2009). The net work of every loop was recorded and plotted relative to maximal PO recorded during the fatigue protocol. The time taken for power to fall below 50% maximal power-output was recorded, as has been used in previous studies examining fatigue resistance and dietary induced obesity using isolated mouse muscle (Tallis et al. 2017; Hill et al. 2019; Hurst et al. 2019). Cumulative work, calculated as the sum of the net work performed in each cycle (Askew et al., 1997; Vassilakos et al., 2009) was also determined to infer absolute differences in the fatigue response, accounting for potential differences in absolute power output between experimental groups, which may promote a faster rate of fatigue. WL shapes were plotted as force against strain (%L₀) for the individual force and length data points for each work loop cycle.

The ability of each muscle preparation to recover from the fatigue protocol was monitored every 10-minutes for 30-minutes post fatigue. Every 10-minutes the muscle was subjected to one set of WL cycles using the same contractile parameters for the control WL’s. Net work during recovery was expressed as a percentage of pre-fatigue maximal power output.

Muscle mass and dimensions calculations

Upon completion of contractile assessments, which from the point of animal sacrifice to final contractile assessment lasted ~ 210 minutes, the muscle was detached from the crocodile clips and removed from the muscle chamber. The tendons, T-foil clip and bone were removed leaving only muscle tissue, which was then blotted on absorbent paper to remove the excess Krebs solution and weighed to determine wet muscle mass (TL-64, Denver Instrument Company, Arvada, CO). Mean muscle cross-sectional area was calculated from L₀, muscle mass, and an assumed density of 1060 kg.m^− 3 (Méndez & Keys, 1960). Isometric stress (kN.m^− 2) was calculated by dividing peak tetanic force by mean muscle cross sectional area (CSA). WL PO normalised to body mass (W.kg^− 1 body mass) was calculated by dividing absolute PO by body mass. WL PO normalised to muscle mass (W.kg^− 1 muscle mass) was calculated as an indicative measure of muscle quality.

Statistical analysis

Statistical analysis was performed using SPSS v.25 (SPSS Statistics for Windows, IBM Corp, Armonk, NY, USA) and GraphPad Prism v.9 (GraphPad Software, California, US). All data are presented as mean ± standard error of mean (SEM). Following appropriate checks of normality (Shapiro-Wilk) and homogeneity of variance (Levenes test), parametric analysis was performed. Mixed model Analysis of Variance (ANOVA), with HFD, treatment (between subjects) and weeks (within subjects) used as the factors, was utilised to determine the effect of HFD and VD consumption on weekly body mass. Two-factor (ANOVA) were conducted with HFD (SLD vs. HFD) and treatment (Control vs. VD) as the fixed factors to examine if differences existed in measures of animal and muscle morphology (body mass, muscle mass, muscle length and muscle CSA), isometric properties (absolute tetanic force, tetanic stress, TTHP and LSHR), time to reach 50% of maximal PO during fatigue protocol and cumulative work produced after 50 consecutive WL’s. Mixed model repeated measures ANOVA, with HFD, treatment (between subjects) and CF (within subjects) used as the factors, was utilised to determine any changes in SOL and EDL absolute PO and PO normalised to body mass and muscle mass. Mixed model repeated measures ANOVA, with HFD, treatment (between subjects) and stimulation frequency (within subjects) used as the factors, was utilised to determine if the reduction in stimulation frequency used for assessment of fatigue resulted in any changes in SOL and EDL PO normalised to muscle mass and if so, were the changes in PO uniform across diet and treatment groups. Mixed model repeated measures ANOVA, with HFD, treatment (between subjects) and time (within subjects) as the factors, was utilised to examine the recovery of WL PO following the fatigue protocol. Significant interactions observed for ANOVA were explored using Bonferroni post hoc for multiple comparisons. Partial eta squared (ηp²) was calculated to estimate effect sizes for all significant main effects. Thresholds for Partial eta squared effect size were classified as small (< 0.05), moderate (0.06–0.137) or large (> 0.138) (Cohen, 1988). Where possible, Cohen’s d was calculated to measure effect size of interactions observed and was then corrected for bias using Hedge’s g due to differences in sample size between groups (Hedges, 1981). Cohen’s d effect size was interpreted as trivial (< 0.2), small (0.2–0.6), moderate (0.6–1.2) or large (> 1.2) (Hopkins et al., 2009). The level of significance was set at P ≤ 0.05.

Morphology

There was a significant TIME*HFD interaction observed for weekly body mass (Fig. 1. P < 0.001, ηp² = 0.412). Bonferroni post hoc analysis multiple comparisons reveal HFD treated animals had significantly greater body mass from week 1 onwards (P < 0.048). No effect of VD was observed (P > 0.999).

Figure 1. Effect of high-fat diet and Vitamin D on body mass over 12 weeks. Data presented as mean ± SEM; SLD, HFD, HFD VD n = 9; SLD VD n = 10; * indicates significant difference between HFD treated and SLD treated at P < 0.05; week − 1 and 0 reflect habituation period where animals were gradually transitioned to their respective custom diet; some error bars are omitted for clarity.

Final whole animal body mass, body length, BMI, Lee Index of Obesity, fat mass and Adiposity:Body mass were significantly greater in HFD groups compared to SLD groups (Table 3. P < 0.005, ηp² > 0.213). No significant differences were observed for EDL or soleus muscle mass, fibre length or CSA (P > 0.053, ηp² < 0.112). See Table 3 for a detailed report of the results of statistical tests for whole body and muscle morphology.

Isometric Performance

There were no significant effects of HFD or VD on soleus isometric properties (Table 4. P > 0.087, ηp² < 0.092) or EDL THPT and tetanus force (Table 4. P > 0.385, ηp² < 0.024). However, EDL from HFD groups produced significantly lower tetanus stress and slower LSHR (Table 4. P < 0.048, ηp² > 0.117) when compared to SLD groups. For detailed report of statistical tests for isometric properties please see Table 4.

Table 3

Whole body and muscle specific animal morphology
	SLD	SLD-VD	HFD	HFD-VD	HFD Effect		VD Effect		Interaction
	n = 9	n = 10	n = 9	n = 9	P value	ηp²	P value	ηp²	P value	ηp²
Whole Body
Body Mass (g)	35.0 ± 1.5	34.4 ± 2.1	48.8 ± 1.4	48.4 ± 2.5	< 0.001	0.566	0.814	0.002	0.991	< 0.001
Body Length (mm)	101 ± 2	102 ± 2	106 ± 1	108 ± 2	0.005	0.213	0.313	0.031	0.964	< 0.001
Fat Mass (g)	1.1 ± 0.2	0.9 ± 0.3	4.6 ± 0.6	3.5 ± 0.4	< 0.001	0.627	0.152	0.061	0.328	0.029
Lee Index of Obesity	325 ± 3	317 ± 4	346 ± 4	338 ± 4	< 0.001	0.458	0.061	0.102	0.985	< 0.001
BMI (g.cm^− 2)	0.34 ± 0.01	0.33 ± 0.01	0.44 ± 0.01	0.42 ± 0.01	< 0.001	0.599	0.149	0.062	0.889	< 0.001
Adiposity:Body Mass	2.8 ± 0.4	2.2 ± 0.6	9.3 ± 1.0	7.1 ± 0.6	< 0.001	0.627	0.063	0.101	0.313	0.031
EDL
Muscle Mass (mg)	10.7 ± 0.4	10.8 ± 0.3	11.3 ± 0.2	11.4 ± 0.3	0.053	0.112	0.65	0.007	0.993	< 0.001
Fibre Length (mm)	9.2 ± 0.1	9.1 ± 0.1	8.9 ± 0.2	9.2 ± 0.2	0.496	0.015	0.306	0.033	0.22	0.047
CSA (m²)	1.10 ± 0.04	1.11 ± 0.04	1.21 ± 0.03	1.18 ± 0.05	0.064	0.103	0.953	< 0.001	0.608	0.008
SOL
Muscle Mass (mg)	8.7 ± 0.5	7.9 ± 0.3	8.4 ± 0.2	8.8 ± 0.3	0.422	0.021	0.457	0.012	0.13	0.072
Fibre Length (mm)	9.4 ± 0.2	9.5 ± 0.2	9.5 ± 0.2	9.4 ± 0.1	0.940	< 0.001	0.696	0.005	0.528	0.013
CSA (m²)	0.88 ± 0.05	0.78 ± 0.03	0.84 ± 0.02	0.88 ± 0.03	0.407	0.022	0.422	0.021	0.066	0.105
Values are presented as means ± SEM; bold values indicate a significant difference at P < 0.05; CSA: Cross-sectional area

Table 4

The effect of 12-week high-fat diet and vitamin D on the isometric properties of isolated mouse EDL and soleus
	SLD	SLD-VD	HFD	HFD-VD	HFD Effect		VD Effect		Interaction
	n = 8	n = 10	n = 9 / 8	n = 9	P value	ηp²	P value	ηp²	P value	ηp²
EDL
Tetanus Force (mN)	364 ± 28	358 ± 16	340 ± 19	354 ± 30	0.588	0.009	0.869	0.001	0.699	0.005
Tetanus Stress (kN.m^− 2)	333 ± 23	320 ± 11	283 ± 17	297 ± 16	0.048	0.117	0.975	< 0.001	0.470	0.016
THPT (ms)	11.4 ± 0.7	10.8 ± 0.7	10.4 ± 0.6	10.5 ± 0.6	0.385	0.024	0.748	0.003	0.614	0.008
LSHR (ms)	13.2 ± 0.4	13.4 ± 0.5	15 ± 0.4	14.5 ± 0.7	0.013	0.178	0.751	0.003	0.505	0.014
Soleus
Tetanus Force (mN)	236 ± 17	208 ± 10	203 ± 12	220 ± 7	0.436	0.02	0.658	0.006	0.084	0.093
Tetanus Stress (kN.m^− 2)	278 ± 17	266 ± 9	243 ± 14	252 ± 11	0.087	0.092	0.927	< 0.001	0.428	0.019
THPT (ms)	34.5 ± 2.4	35.7 ± 1.6	36.4 ± 1.8	33.4 ± 1.3	0.935	< 0.001	0.654	0.007	0.281	0.037
LSHR (ms)	40.1 ± 2.4	47.6 ± 2.6	45.9 ± 2.2	47.8 ± 2.6	0.292	0.036	0.100	0.085	0.321	0.032
Values are presented as means ± SEM; bold values indicate a significant difference at P < 0.05; THPT, Time to half peak tetanus; LSHR, Last stimulus half relaxation time

Maximal work loop power output

For EDL, absolute PO and PO normalised to muscle mass, there was a significant main effect of cycle frequency (CF) (P < 0.001, ηp² > 0.696) indicating peak PO occurred between 8-10Hz cycle frequency (Fig. 2. P > 0.999) with PO from remaining CF’s significantly lower (P < 0.002) except for PO at 12Hz (P > 0.264), but PO produced at 12Hz did not differ to that produced at 6 and 14Hz (P > 0.242). For PO normalised to body mass there was a significant CF*HFD interaction (P = 0.018, ηp² = 0.092). Bonferroni multiple comparisons revealed that in SLD groups PO produced at CF 8, 10 and 12Hz did not differ (P > 0.999), but PO from 10Hz was greater than 4, 14 and 16Hz (P < 0.015), whereas PO at 8 and 12Hz was only greater than 4 and 16Hz (P < 0.001). In HFD treated groups, PO produced at 8, 10, 12 and 14Hz did significantly differ (P > 0.073), but PO produced at 12 and 14Hz was only greater than 4Hz (P < 0.017), whereas PO at 8 and 10Hz was greater than that produced at 4 and 16Hz (P < 0.002).

EDL absolute PO and PO normalised to muscle mass and body mass was significantly lower in the HFD groups when compared to SLD groups (Fig. 2A, B & C P < 0.031, ηp² > 0.137). No main effect of VD (P > 0.493, ηp² < 0.015) or HFD*VD interaction were observed (P > 0.070, ηp² < 0.099). There were no CF*HFD (P > 0.492, ηp² < 0.025), CF*VD (P > 0.409, ηp² < 0.030) or CF*HFD*VD (P > 0.303, ηp² < 0.037) interactions observed.

For soleus, absolute PO and PO normalised to muscle mass, there was a significant main effect of CF (Fig. 2P < 0.001, ηp² > 0.913), indicating maximal PO occurred between 3-4Hz cycle frequency (P > 0.184) with PO from remaining CF’s being significantly lower (P < 0.002) except between 2 and 4Hz (P > 0.498). No CF*HFD (P < 0.372, ηp² < 0.032), CF*VD (P > 0.636, ηp² < 0.016) or CF*HFD*VD (P > 0.510, ηp² < 0.023) interactions were observed. For PO normalised to body mass there was a significant CF*HFD interaction (P = 0.012, ηp² = 0.128). However, Bonferroni multiple comparisons indicated no significant differences in the PO CF curve between groups and indicated that maximal absolute power occurred between 3-4Hz cycle frequency (P > 0.999) with PO from the remaining CF’s being significantly lower (P < 0.002), except between 2 and 4Hz (P > 0.764).

For soleus, PO normalised to body mass, HFD treated groups produced significantly less power than SLD groups (Fig. 2F. P = 0.001, ηp² = 0.312). For absolute PO and PO normalised to muscle mass no main effects of HFD (P > 0.471, ηp² < 0.017) or VD (P > 0.722, ηp² < 0.004) were observed. No HFD*VD interaction observed (P > 0.250, ηp² < 0.042) for absolute or normalised PO.

Fatigue and Recovery

The reduction in stimulation frequency for assessment of fatigue resulted in a significant reduction in maximal WL PO normalised to muscle mass of the soleus and EDL (Fig. 3P < 0.001, ηp² > 0.725), but the magnitude of reduction in PO was not influenced by diet or treatment (P > 0.199, ηp² < 0.053).

For time to fall below 50% of maximum power output during fatigue, there were no significant effects of HFD (Fig. 4A & D P > 0.220, ηp² < 0.048), VD (P > 0.962, ηp² < 0.001) or HFD*VD interactions observed (P > 0.455, ηp² < 0.018) in either the EDL or soleus.

HFD groups produced significantly less cumulative work after 50 consecutive WL’s when compared to SLD groups in both the EDL (SLD, 213.9 ± 9.5; SLD VD, 197.2 ± 9.8; HFD, 175.9 ± 7.5; HFD VD, 173.4 ± 12.8 J kg^− 1) and soleus (SLD, 212.5 ± 13.0; SLD VD, 207.8 ± 12.6; HFD, 178.6 ± 7.7; HFD VD, 185.1 ± 8.2 J kg^− 1; Fig. 4B & E P < 0.043, ηp² > 0.126). There was no significant main effect of VD (P > 0.417, ηp² < 0.021) or HFD*VD interaction observed (P > 0.545, ηp² < 0.012).

For recovery of maximal power output relative to pre fatigue maximum, assessed every 10 minutes for 30 minutes post fatigue, in both the EDL and soleus, there was a significant main effect of time (Fig. 4C & F P < 0.020, ηp² = 0.656), with significant recovery of muscle power output over the 30 minutes. No TIME*HFD (P > 0.203, ηp² < 0.052), TIME*VD (P > 0.202, ηp² < 0.052) or TIME*HFD*VD interactions were observed (P > 0.370, ηp² < 0.028). There were no main effects of HFD (P = 0.228, ηp² = 0.047 for the EDL; P = 0.060, ηp² = 0.110 for the soleus), VD (P = 0.790, ηp² = 0.002 for the EDL; P = 0.078, ηp² = 0.097 for the soleus) or HFD*VD interaction (P > 0.542, ηp² < 0.012) observed.

Figures 5 and 6 illustrate typical WL shape during a standardised time point for mouse soleus and EDL in each respective group. The area within the WL represents the net work done during the length change cycle. The area within the typical EDL WL traces (Fig. 5) is initially smaller in HFD groups (Fig. 5C & D) compared to SLD groups (Fig. 5A & B), but the shape of the loop is consistent across groups. The reduction of the area within the loop, and subsequent change in WL shape exhibited during the fatigue protocol appears uniform irrespective of diet or treatment. However, typical soleus WL shapes (Fig. 6) demonstrate that at the later stages of the fatigue protocol (WL 30 and onward) there is greater force production during muscle re-lengthening in HFD groups (Fig. 6C & D) when compared to SLD groups (Fig. 6A & B). As a result, from WL 30 there is a marked reduction in the size of WL shape relative to the initial WL in HFD groups.

The present study is the first to evaluate the effects of high dose VD on force, power, and fatigue resistance of isolated mouse fast and slow twitch muscle, in both standard and high-fat diets. Using a methodological approach, which incorporated multiple modes of contractility, it was established that a HFD evokes muscle and contractile mode specific reductions in the contractile performance of isolated skeletal muscle. In the soleus, there was a HFD induced decline in PO normalised to body mass and cumulative work production during fatigue. However, in the EDL there was a reduction in isometric stress, absolute PO, PO normalised to muscle mass (muscle quality) and body mass, and cumulative work. Irrespective of diet, mode of contractility or muscle phenotype, a high dose of vitamin D (VD) over a 12-week period had little effect on contractile function of isolated soleus or EDL.

High-fat Diet Effects on Maximal Muscle Force and Power

With respect to the effect of a HFD, the results of the present study add to the evidence demonstrating detrimental effects of HFD consumption on normalised muscle force and power in a contractile mode and muscle specific manner. However, the results extend the understanding of HFD effects by indicating for the first time that a HFD can evoke reductions in absolute power and cumulative work production of fast twitch skeletal muscle.

The isometric function (absolute or stress) of the EDL is commonly diminished in response to longer periods (> 12 weeks) of HFD consumption (Eshima et al., 2017, 2020; Tallis et al., 2017), although this is not always the case (Bott et al., 2017; Hurst et al., 2019). The HFD induced reduction in isolated EDL stress reported in our results supports the idea that HFD can reduce the intrinsic force producing capacity of skeletal muscle. Furthermore, EDL absolute and normalised PO, and muscle quality, were diminished in the HFD groups. A HFD induced reduction in EDL muscle quality is supported by previous work (HFD 16 and 12 weeks respectively; Tallis et al. 2017, Hurst et al. 2019), but the reduction in absolute EDL power output is a unique and novel insight into the impact of HFD consumption on fast twitch muscle performance. Discrepancies in findings of absolute power may be attributed to increased magnitude of intramuscular lipids occurring earlier in the feeding duration in the current study when compared to previous work. Elevated intramuscular lipids occurred in the EDL after ~ 12 weeks of a fixed HFD (Eshima et al., 2017), but not until ~ 16 weeks in a high-fat forage diet (Messa et al., 2020), such as that utilised in previous work using the WL technique (Tallis et al. 2017, Hurst et al. 2019). Intramuscular lipids are not only associated with a reduction in the contractile capacity of whole isolated skeletal muscle (Biltz et al., 2020), but also linked with a reduction in the process of myogenesis (Akhmedov & Berdeaux, 2013), another key factor regulating contractile performance. HFD effects on contractile performance appear more pronounced in fast twitch muscle, attributed to fast glycolytic fibres having a reduced capacity to metabolise elevated lipids levels within muscle, when compared to muscles comprised of slow oxidative fibres (Tallis et al. 2017, Hurst et al. 2019). The present data support the idea that HFD consumption effects on skeletal muscle may be contractile mode and muscle specific (Ciapaite et al., 2015; Hill et al., 2019; Tallis et al., 2018) and could promote a negative obesity cycle (Tallis et al. 2018); where a HFD leads to muscles of poorer quality, which are required to manoeuvre and support a larger mass and require a much greater metabolic cost to maintain them (Seebacher et al. 2017).

Peak isometric force and force normalised to cross-sectional area (CSA; stress) of the soleus was not affected by a HFD, in concordance with the literature using similar feeding durations (Hurst et al., 2019). During longer feeding durations (16 weeks) in female CD-1 mice, literature has identified greater absolute force of soleus from HFD animals (Tallis et al. 2017). The magnitude of increase in body mass was originally suggested as the primary reason for an increase in absolute force observed over longer feeding durations, increasing demand on postural muscles to support and stabilise an elevated mass (Tallis et al., 2017). However, based on the present data, the concomitant effects of magnitude and duration of HFD, and duration of mechanical loading, may account for previously observed greater absolute force, as the final body masses for the HFD group from the present data (48.8g ± 1.4) were comparable to studies utilising longer feeding durations (16 weeks: 52.7g ± 2.3). Greater absolute force of soleus from HFD mice presented by Tallis et al., (2017) could be a result of the greater soleus muscle mass of HFD treated mice compared to controls, which was not found in the present study. Absolute soleus PO and PO normalised to muscle mass (muscle quality) were unaffected by a HFD, supporting previous observations utilising feeding durations of 12–16 weeks (Tallis et al. 2017, Hurst et al. 2019). Thus, despite elevated loading on postural muscles, no muscular adaptations occur to increase absolute power, which is further compounded by a substantial increase in body mass, supported by these data identifying a reduction in PO normalised to body mass. Therefore, despite the preservation of muscle quality in HFD groups, the magnitude of change in body mass evoked by a HFD leads to a reduction in normalised PO, where in vivo this would result in reductions in the ability to be physically active and to perform activities of daily living, thus the potential for a reduction in quality of life and the onset of a negative obesity cycle (Tallis et al., 2018).

High-fat Diet Effects on Fatigue Resistance

With respect to percentage decline in fatigue, these data suggest that a HFD may not influence the rate at which muscle loses power relative to maximum PO during fatiguing contractions. However, there is little consensus regarding HFD effects on isolated skeletal muscle fatigue during dynamic conditions in young adult female CD-1 mice, with no change or a reduction in the ability to maintain PO relative to maximum being reported in both the soleus and EDL (Tallis et al. 2017; Hurst et al. 2019). Differences in fatigue response may be attributed to the use of submaximal stimulation frequency during fatigue, opposed to that which evokes maximal isometric force. Submaximal stimulation parameters provide a better representation of in vivo fatigue mechanics (Shelley et al., 2022); previous reductions in rate of fatigue may have been exacerbated by mechanical fatigue. Furthermore, previous work has only considered HFD effects on percentage decline during fatigue from a standardised point, typically maximum PO or force. Whilst this provides an insight into the performance of the tissue during fatigue, cumulative work is also an important factor when considering in vivo function, as the amount of work, in addition to the rate of fatigue, will play a key part in exercise capacity and in the ability to complete activities of daily living which require repetitive contractions.

Whilst the rate of fatigue was unaffected by HFD, total cumulative work was reduced in the HFD soleus and EDL. The reduction in cumulative work is not a result of the sub maximal stimulation frequency leading to a greater reduction in PO in the HFD group, as the magnitude of change in maximal PO through a reduction in stimulation frequency did not differ between diet and treatment groups. A reduction in cumulative work in HFD EDL is likely a consequence of the reduction in acute PO. Therefore, despite fatiguing at a similar rate as SLD EDL, work per contraction is lower culminating in lower work production throughout the time course of fatigue. The same justification for a reduction in soleus cumulative work is unlikely given there were no differences in acute PO, suggesting other mechanisms account for a reduced capacity to perform work. It is possible that a HFD induced reduction in the function of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) (Funai et al., 2013), which is responsible for the movement of Ca²⁺ from the cytoplasm back into the sarcoplasmic reticulum, impairs calcium reuptake during fatiguing contractions of the soleus. Consecutive fatiguing contractions have been shown to increase relaxation time in some muscles (Allen et al., 2008); if this response is exacerbated by a HFD, it would likely cause a reduction in cumulative work as the magnitude of negative work would increase per WL cycle, i.e., the muscle is active during lengthening, thus decreasing net-work to a greater extent at each subsequent contraction. This is supported by the soleus WL shapes (Fig. 6) - as the fatigue protocol progresses, HFD treated muscle had a larger negative work component during re-lengthening, indicative of increased relaxation time, reducing work production during each cycle. However, independent of changes in SERCA, HFD induced reductions in the efficacy of actin-myosin cross-bridge cycling (Ciapaite et al., 2015; Schilder et al., 2011) may play an important role in a reduction in cumulative work in both the soleus and EDL, and in the case of the soleus, without influencing acute power production.

Vitamin D Effects on Maximal Force, Power and Fatigue Resistance

The administration of high doses of VD has been shown to beneficially alter mechanisms associated with a HFD induced decline in skeletal muscle contractility (Benetti et al., 2018; Manna et al., 2017; Marcotorchino et al., 2014). However, the dose of VD used in the present study did not elicit any changes in whole body or muscle morphology, or any of the contractile parameters assessed, in either SLD or HFD. There are limited comparisons to be made regarding the potential for VD to alleviate obesity induced declines in contractile performance, in fact, only one previous study has directly considered this (Kim et al., 2020). This previous research study reported that a high dose of VD attenuated the reduction of in vivo grip strength and sensorimotor function in 24 week old, male, P-62 deficient mice (genetic obesity model) (Kim et al., 2020). However, it is difficult to make a direct comparison to the present data, given the differences in the methodological approach, such as different model of obesity (dietary vs genetic), gender and age of mouse, different assessment of contractile function (in vivo vs isolated muscle) and different quantity, duration, and mode of administration of VD (75IU every 3 days for 10 weeks via oral gavage), which are all factors that could influence the effect of VD on contractile function.

Previous research has established high dose VD can evoke improvements in isolated skeletal muscle contractility when compared to control VD sufficient mice (Debruin et al., 2019; Hayes et al., 2019). Much like previous work which assessed VD supplementation on isometric properties of isolated muscle utilising the same dose (20,000IU/Kg^− 1 feed) in SLD C57/BL6 mice, no large changes in calcium handling were observed (Debruin et al., 2019, 2020; Hayes et al., 2019) as inferred by lack of difference in measurers of THPT and LSHR. Our data shows that high dose VD had little effect on isometric force, WL PO or fatigue resistance of either the soleus or EDL, in either dietary group. Previous observations support limited VD effects on EDL force output in SLD treated mice (Debruin et al., 2019, 2020; Hayes et al., 2019; Ray et al., 2016). However, we are the first to show minimal effects of high dose (20,000IU/Kg^− 1 feed) VD on soleus stress, as prior work indicates increased soleus stress after 4 weeks (Debruin et al., 2019; Hayes et al., 2019) but diminished after 8 weeks (Debruin et al., 2020). It could be that excess VD supplementation adversely effects muscle function, as has been reported in both human and animal studies, where large single bolus doses have been associated with impaired isolated muscle contractile performance (Hayes et al., 2019) and increased risk of falls in the elderly (Sanders et al., 2010). Although the dose provided in this study, whilst high, was not given in one bolus dose and nor does the data show a reduction in contractile performance irrespective of muscle or mode of contractility. Whilst speculative, based on previous evidence and the data presented in this study, there could well be an “inverted-U” relationship with duration of high dose VD supplementation and isolated skeletal muscle contractility (Bollen et al. 2022). Initially high dose of VD may be beneficial for isolated skeletal muscle contractility until an optimal quantity of circulating VD is achieved, from that point on there is a downward trajectory in muscle performance; we suspect our data may fit in this downward trajectory. However, more data is needed to explore the effect of dose and duration of VD on isolated muscle contractility.

Despite the findings presented in this study, high dose VD could still be beneficial for contractile performance when supplemented long term during HFD feeding. Throughout the data presented there are consistent small (d = 0.2–0.59) and moderate (d = 0.6–1.19) non-significant effects suggesting improved contractile performance of the EDL (absolute and normalised PO and stress) and lower fat mass and fat mass: body mass in HFD VD when compared to HFD only. Whilst these trends should be interpreted with caution, they are important to note as they further contextualise that VD could improve contractile function and morphology in HFD treated rodents when feeding parameters are optimised, opposed to VD having no anti-obesogenic properties. The response to VD appears dose/duration dependent, whereby excess VD may cause VD dysregulation and result in deleterious (Debruin et al., 2020), or in the case of these data, no significant effects on contractile performance. The reduction (~ 50%) in isometric stress observed after 8 weeks high dose VD was attenuated when supplemented with exercise and even enhanced fatigue resistance compared to exercise only (Debruin et al., 2020). The authors suggest that high dose VD alone results in VD dysregulation, through alterations in the activity of key metabolic enzymes 1,25-alpha-hydroxylase and 24-alpha-hydroxylase involved in VD metabolism. However, exercise induced enhancement of mitochondrial function appeared to abate the increase in metabolic stress evoked by high dose VD, ultimately promoting improvements in contractile function (Debruin et al., 2020). Therefore, it may be that high dose VD is beneficial in attenuating a HFD diet induced decline in muscle contractility when combined with an added stimulus which evokes beneficial alterations in mitochondria, be that exercise or in combination with other nutritional strategies.

The effects of surplus VD on isolated muscle function appear dependent upon a number of factors such as dose, duration, sex and physical activity. There is clear evidence demonstrating that chronic supplementation of high dose VD can improve isolated muscle performance (Debruin et al. 2019; Hayes et al. 2019) and evoke mechanisms which directly oppose HFD induced declines in muscle function (Benetti et al., 2018; Manna et al., 2017; Marcotorchino et al., 2014). Given the available evidence, coupled with the need to explore alternative therapeutic strategies to alleviate the adverse effects of obesity, supplementation of VD warrants further investigation; understanding and identification of suitable dosing strategies could contribute to a reduction in the adverse effects of obesity on overall health and specifically skeletal muscle health (Tallis et al. 2021), which are current public health priorities.

Limitations and Future Direction

One limitation of this study is that the exact levels of VD achieved are unknown in each individual, as serum 25 (OH) D concentration in blood plasma was not measured. As such, serum 25 (OH) D concentration in blood plasma could not be quantified in the present study. However, previous research shows that an identical high dose and duration (20,000IU/kg diet for 12 weeks) elicits ~ 4 fold increase in serum 25 (OH) D3 concentration when compared to a control dose (1000IU/kg diet) in C57 mice (Rowling et al., 2007). Thus, we would expect that high dose VD groups would have substantially greater quantities of circulating VD compared to control doses.

Whilst the present study provides a valuable insight into the effects VD on isolated muscle mechanics, these results are only generalisable to the specific dose and duration used, in female mice. One plausible explanation for differences between our data and previous work may well be sex specific responses, given there may be sex specific determinants in the modulation of 25(OH)D levels (Dupuis et al., 2021; Jungert & Neuhäuser-Berthold, 2015) - there is limited available evidence for how this effects the contractile response. Overall previous studies provide evidence that VD supplementation can, in some instances, promote improvement in mechanisms responsible for optimal skeletal muscle performance, including attenuation of mechanisms responsible for HFD induced declines in contractile performance such as reduced AMPK activity and decreased lipid metabolism (Benetti et al., 2018; Manna et al., 2017; Marcotorchino et al., 2014). As such, future work is needed to establish the optimal dose and duration of VD to evoke improvements in isolated skeletal muscle mechanics in both SLD and HFD treated female and male mice.

The present study demonstrates that HFD effects are muscle and contractile mode specific. For force and power performance there are limited effects of HFD on isolated mouse soleus, but HFD evokes reductions in EDL stress and absolute and normalised PO. During fatiguing contractions, whilst percentage decline relative to maximum PO remained unchanged between groups, total cumulative work was reduced in HFD treated soleus and EDL, although it would appear the mechanisms responsible for this decline are not uniform between muscles. Whilst high dose VD did not attenuate any of the HFD induced declines in contractile performance or alter contractile performance in SLD treated mice in this study, there were small and moderate non-significant effects observed between HFD VD and HFD groups to suggest refinement of feeding protocols may evoke positive effects on contractile performance and morphology. As such, future work should consider the impact of various doses and durations to determine if VD can be effective in reducing the impact of obesity of skeletal muscle function.

ANOVA Analysis of variance

EDL Extensor digitorum longus

PO Power output

SkM Skeletal muscle

WL Work loop

VD Vitamin D

Author Contributions: SS, JT and RSJ conceived and designed the study; SS performed data collection; SS and JT analysed the data; SS prepared figures; SS and JT drafted manuscript; all edited and revised the manuscript; all approved the final version of the manuscript.

Funding: No external funding was received for this work.

Competing interest: The authors declare they have no competing or conflict of interest.

Data accessibility: Source data will be available upon request.

Acknowledgements: The authors would like to thank the members of The University of Warwick’s Biological Services Unit for their support and care of the animals throughout the duration of this study.

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No competing interests reported.

High-Fat Diet and Vitamin D Effects on Contractile Performance of Isolated Mouse Soleus and EDL

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Abstract

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Summary Statement

Introduction

Materials and methods