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. 2022 Jan 7;100(2):skac002. doi: 10.1093/jas/skac002

Pulmonary arterial pressure in fattened Angus steers at moderate altitude influences early postmortem mitochondria functionality and meat color during retail display

Chaoyu Zhai 1, Lance C Li Puma 2, Adam J Chicco 2, Asma Omar 2, Robert J Delmore 1, Ifigenia Geornaras 1, Scott E Speidel 1, Tim N Holt 3, Milton G Thomas 1, R Mark Enns 1, Mahesh N Nair 1,✉
PMCID: PMC8846331  PMID: 35015873

Abstract

Pulmonary hypertension is a noninfectious disease of cattle at altitudes > 1524 m (5,000 ft). Mean pulmonary arterial pressures (PAP) are used as an indicator for pulmonary hypertension in cattle. High PAP cattle (≥50 mmHg) entering the feedlot at moderate elevations have lower feed efficiency as compared to low PAP cattle (< 50 mmHg). The impact of pulmonary arterial pressure on mitochondrial function, oxidative phosphorylation (OXPHOS) protein abundance, and meat color was examined using longissimus lumborum (LL) from high (98 ± 13 mmHg; n = 5) and low (41 ± 3 mmHg; n = 6) PAP fattened Angus steers (live weight of 588 ± 38 kg) during early postmortem period (2 and 48 h) and retail display (days 1 to 9), respectively. High PAP muscle had greater (P = 0.013) OXPHOS-linked respiration and proton leak-associated respiration than low PAP muscles at 2 h postmortem but rapidly declined to be similar (P = 0.145) to low PAP muscle by 48 h postmortem. OXPHOS protein expression was higher (P = 0.045) in low PAP than high PAP muscle. During retail display, redness, chroma, hue, ratio of reflectance at 630 and 580 nm, and metmyoglobin reducing activity decreased faster (P < 0.05) in high PAP steaks than low PAP. Lipid oxidation significantly increased (P < 0.05) in high PAP steaks but not (P > 0.05) in low PAP. The results indicated that high PAP caused a lower OXPHOS efficiency and greater fuel oxidation rates under conditions of low ATP demand in premortem beef LL muscle; this could explain the lower feed efficiency in high PAP feedlot cattle compared to low PAP counterparts. Mitochondrial integral function (membrane integrity or/and protein function) declined faster in high PAP than low PAP muscle at early postmortem. LL steaks from high PAP animals had lower color stability than those from the low PAP animals during simulated retail display, which could be partially attributed to the loss of muscle mitochondrial function at early postmortem by ROS damage in high PAP muscle.

Keywords: beef color, longissimus lumborum, mitochondrial function, meat quality, pulmonary hypertension

Lay Summary

The impact of pulmonary arterial pressure (PAP) on mitochondrial function, oxidative phosphorylation protein abundance, and meat color was examined using longissimus lumborum (LL) from high (98 ± 13 mmHg) and low (41 ± 3 mmHg) PAP fattened Angus steers (live weight of 588 ± 38 kg) during early postmortem period (2 and 48 h) and retail display (days 1 to 9), respectively. The results indicated that high PAP caused a lower oxidative phosphorylation efficiency and greater fuel oxidation rates under conditions of positive energy balance in beef LL muscle. This could explain the lower feed efficiency in high PAP feedlot cattle compared to low PAP counterparts. Mitochondrial integral function declined faster in high PAP than low PAP muscle at early postmortem. LL steaks from high PAP animals had lower color stability than those from the low PAP animals during simulated retail display, which could be partially attributed to the loss of muscle mitochondrial function at early postmortem in high PAP muscle

High pulmonary arterial pressure (PAP) caused a lower oxidative phosphorylation efficiency and greater fuel oxidation rates under conditions of low ATP demand in beef longissimus lumborum(LL) muscle. Moreover, LL steaks from high PAP animals had lower color stability than those from the low PAP animals during simulated retail display, which could be partially attributed to the faster decline of mitochondrial integral function in high PAP than low PAP muscle at early postmortem.

Introduction

Pulmonary hypertension is a noninfectious disease of cattle at altitudes > 1524 m (5,000 ft), characterized by distension of the jugular vein, dyspnea, weakness, ascites, and edema of the jaw and brisket region. This condition results from a reduction in the partial pressure of oxygen in inspired air as elevation increases, which can lead to maladaptive changes in the cardiopulmonary system in an attempt to improve oxygen supply to body tissues. In particular, chronic alveolar hypoxia induces pulmonary arterial vasoconstriction and vascular wall remodeling that favors the development of right ventricular hypertrophy, which can progress to right-sided heart failure and death (Veit and Farrell, 1978; Holt and Callan, 2007; Stenmark et al., 2013). Approximately $60 million are lost annually due to complications associated with pulmonary hypertension in beef cattle, making it the leading cause of mortality in the Rocky Mountain region of the United States (Williams et al., 2012; Neary et al., 2013). Mean pulmonary arterial pressures (PAP) ≥50 mmHg are used as an indicator for pulmonary hypertension susceptibility in cattle (Holt and Callan, 2007). The measurement of mean PAP has allowed for genetic selection against cattle susceptible to pulmonary hypertension, resulting in the reduced incidence of high altitude disease (Shirley et al., 2008; Crawford et al., 2016; Cockrum et al., 2019).

Meat color and tenderness significantly influence consumers’ purchase and repurchase decisions (Shackelford et al., 2001; Mancini and Hunt, 2005; Suman and Joseph, 2013; Neethling et al., 2017). These quality attributes have significant economic implications for the meat industry (Smith et al., 2000; Lusk et al., 2001). The conversion of muscle to meat is a complex process that involves metabolic, chemical, and physical changes and can impact meat quality (Matarneh et al., 2017). As the main organelle responsible for energy metabolism in live animals, mitochondria have an important impact on the health of animals and their production efficiency (Bottje, 2019a, 2019b). In postmortem muscle, mitochondria can maintain structural integrity and oxygen consumption activity for up to 60 d (Tang et al., 2005), and their functionality contributes to meat color (Ramanathan and Mancini, 2018; Ramanathan et al., 2019) and tenderness (Wang et al., 2017, 2018).

Recently, a study indicated that high PAP cattle entering the feedlot at moderate elevations (1,557 m) have around 20% higher feed to gain ratio due to excessive energy demands placed on their pulmonary-cardiovascular system (Thomas et al., 2019; Heffernan et al., 2020). However, the impact of pulmonary hypertension on meat quality attributes is still unknown. As noted above, muscle mitochondrial function is likely a key regulator of meat quality, but the effect of PAP on muscle mitochondria in beef cattle has not been previously investigated. Therefore, objectives of this study were to evaluate the effects of PAP on mitochondrial function during the early postmortem period and meat color during retail display.

Materials and Methods

Animal care and use

The Institutional Animal Care and Use Committee at Colorado State University (approval number 17-7179A) approved all animal procedures.

Cattle information

The fattened Angus steers were placed into two groups determined by their mean PAP: high mean PAP (98 ± 13 mmHg; n = 5) and low mean PAP (41 ± 3 mmHg; n = 6). The animals (588 ± 38 kg body weight) were approximately 14 mo old and came from the same farm with the same diet. The cattle were transported to a USDA-inspected meat laboratory in the Global Food Innovation Center of the Department of Animal Sciences at the Colorado State University. This experiment was originally designed as a balanced study, but one animal from the high PAP group died due to heart failure 8 d before slaughter. After resting time in the lairage, the animals were stunned, bled, and dehided according to commercial practice under USDA inspection. Approximately 5 g of longissimus lumborum (LL) was cut from the left side of each carcass at 2 and 48 h post-exsanguination. Half of the sample was immediately placed into ice-cold biopsy preservation medium (BIOPS; pH 7.1) containing 10 mM Ca-EGTA (0.1 µM free calcium), 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 DTT, 6.56 mM MgCl2, 5.77 mM ATP, and 15 mM phosphocreatine, whereas the other half of the sample was snap-frozen in liquid nitrogen.

Muscle sample preparation

Permeabilized muscle fibers from LL were prepared for high-resolution respirometry experiments as described previously (Li Puma et al., 2020). Briefly, muscle fibers were teased in ice-cold BIOPs solution containing 10 mM Ca-EGTA (0.1 μM free calcium), 20 mM imidazole, 20 mM taurine, 50 mM K-MES, 0.5 mM DTT, 6.56 mM MgCl2, 5.77 mM ATP, and 15 mM phosphocreatine (pH 7.1) before incubation with 50 μg/mL saponin on ice for 20 min with gentle rocking to permeabilize cell membranes while leaving mitochondrial membranes intact (Pesta and Gnaiger, 2012). Permeabilized fiber bundles were then transferred to mitochondrial respiration medium (MiR05) containing 0.5 mM EGTA, 3 mM MgCl2 hexahydrate, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, and 0.1% BSA (pH 7.1), and rinsed by rocking for 10 min on ice, followed by another identical 15-min rinse. Fiber bundles were then gently blotted dry for 10 to 15 s on Whatman paper and weighed immediately before adding approximately 6 mg to the 2-mL oxygraph chamber for experiments.

Mitochondrial respiration

Mitochondrial respiratory function was determined with permeabilized muscle fiber bundles collected at 2 and 48 h postmortem by high-resolution respirometry (HRR) using an Oxygraph-2k high-resolution respirometer (Oroboros Instruments, Innsbruck, Austria). Oxygen flux was monitored in real-time by resolving changes in the negative time derivative of the chamber oxygen concentration signal following standardized instrumental and chemical background calibrations using Datlab software (Oroboros Instruments). All respirometry data were collected at 37 °C in a hyperoxygenated environment (275–400 μmol/L) to avoid potential limitations in oxygen diffusion in the permeabilized fiber bundles (Li Puma et al., 2020). A detailed description of the respiration protocols and associated respiratory states generated by the sequential titration of each substrate is provided in Table 1. All the data collected were normalized by the weight of permeabilized fiber bundles.

Table 1.

High-resolution respirometry protocols and associated respiratory flux states assessed in mitochondrial respiration experiment

Protocol titrations (final concentration in chamber) High-resolution respirometry flux state Explanation
Pyruvate (5 mM) + malate (1 mM) NL Proton leak-associated respiration supported by high NADH, but no ADP
ADP (2.5 mM) NP OXPHOS-linked respiration supported by pyruvate + malate oxidation
Glutamate (10 mM) N Maximal OXPHOS-linked respiration supported by NADH
Succinate (20 mM) NS Maximal OXPHOS-linked respiration supported by NADH and succinate
Ascorbate (2 mM) + TMPD(0.5 mM) CIV Maximal oxygen reductase capacity of Complex IV using an artificial electron donor
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Immunoblot analysis for mitochondrial oxidative phosphorylation (OXPHOS) proteins

Protein was isolated from muscle sample by mammalian lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 20 mM sodium fluoride, 50 mL of Mammalian Protein Extraction Reagent [Thermo Scientific, 78501], 500 µL of Protease Inhibitor Cocktail [Sigma, P8340]). The concentration of the protein was detected by using BCA Protein Assay Kit (Thermo Scientific, 23225) according to manufacturer instructions and read by VERSA max microplate reader. Protein (30 μg) was loaded and separated using Invitrogen Bolt 4%–12% Bis-Tris (Invitrogen, NW04122BOX) and XCell SureLock Elecctrophorsis Cell (Invitrogen, EI0001). Then, it was transferred to PVDF membrane after activation with 100% methanol for 10 min. After three washes with TBST (100 mL of 10× TBS, 900 mL of diH2O, 1 mL of Tween [Fisher BioReagents, 125689]) (10× TBS: 24 g of Tris-base, 88 g of NaCl, 1 L of diH2O, pH 7.6 adjusted by 6 N HCl), the membrane was blocked with 5% milk (10 mL of TBST, 0.5 g of Blotting-Grade Blocker [BIO-RAD, 170-6404]) for 1 h at room temperature. After blocking, the membrane was incubated in primary antibodies (1:1000 dilution) overnight at 4 °C. After three washes with TBST, the membrane was incubated in secondary antibodies (1:3000 dilution) for 1 h at room temperature. Then, the membrane was washed three times with TBST, incubated in SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, 34075) for 30 s, imaged by UVP ChemStudio blot imager (Analytik Jena, Jena, Germany). Band density was normalized to AmidoBlack total protein (Sigma, A8181) and analyzed by ImageJ software. Mitochondrial OXPHOS protein abundance of each sample was estimated by the average of density of two protein bands, Complex II (SDHB) and Complex V (ATP5A).

Beef fabrication and meat sample allocation

After 12 d of aging (at 2 °C in the dark), strip loins (LL) were excised from left sides of beef carcasses (n = 5 for high PAP; n = 6 for low PAP), and six 2-cm-thick steaks were cut from each loin. The first steak from the anterior side of each loin was assigned to characterize pH and myoglobin concentration. The following five steaks from the anterior side were cut and assigned randomly to 1, 3, 5, 7, or 9 d of retail display overwrapped in polyvinyl chloride film (O2 transmission = 23,250 mL × m2 × d−1, 72 gauge; Resinite Packaging Films, Borden, Inc., North Andover, MA).

Retail display

After packaging, steaks were placed in a display case maintained at 2 ± 1 °C under continuous lighting (4548 lx, 1810LX4000 LED FIXTURE; Kason, Newnan, GA; color rendering index = 84; color temperature = 4,500 K). All steaks were rotated daily to minimize differences in light intensity or temperature caused by location.

Meat pH and myoglobin concentration

The pH of steaks was determined as described previously (Nair et al., 2016). Duplicate 5 g samples were homogenized in 30 mL diH2O, and the pH was measured using an Accumet AE150 pH-meter (Fisher Scientific, Pittsburgh, PA).

Myoglobin concentration was determined according to the previous method (Faustman and Phillips, 2001) with modification. Duplicate 5 g frozen samples were homogenized in 35 mL ice cold 40 mM sodiumphosphate buffer at pH 6.8. The homogenate was filtered using Whatman No. 1 filter paper. The absorbance of the filtrate at 525 nm (A525) was recorded using a UV-1800 spectrophotometer (Shimadzu Inc., Candy, OR) with sodium phosphate buffer as blank. Myoglobin concentration was calculated using the following equation:

Myoglobin (mg/g)=[A525/(7.6 mM−1cm−1×1 cm)]×[17,000/1000]×8

where 7.6 mM−1 cm−1 = millimolar extinction coefficient of myoglobin at 525 nm; 1 cm = path length of cuvette; 17,000 Da = average molecular mass of myoglobin; 8 = dilution factor.

Instrumental color

CIE lightness (L∗), redness (a∗), hue, and chroma values were measured at three random locations on the light-exposed steak surfaces with a HunterLab MiniScan LabScan EZ4500 colorimeter (Hunter Associates Laboratory, Reston, VA) using 2.54-cm diameter aperture, illuminant A, and 10° standard observer (AMSA, 2012). The instrument was calibrated with standard black and white plates. In addition, the ratio of reflectance at 630 and 580 nm (R630/580) was calculated as an indirect estimate of surface color stability.

Samples for metmyoglobin reducing activity and lipid oxidation

After color measurement, steaks displayed for 1, 3, 5, 7, or 9 d were halved, with one half being used for measuring MRA, and the other half being used to measure lipid oxidation. The interior of the steak half assigned to MRA was dissected parallel to the oxygenated surface to expose the interior (resulting in two interior pieces) and used for measuring MRA. Representative samples from the other half containing both oxygenated surface and interior were collected for measuring lipid oxidation.

Metmyoglobin reducing activity

Metmyoglobin reducing activity (MRA) was evaluated according to the method described previously (Zhai et al., 2019). Samples from the interior of steak halves (5 × 5 × 1.5 cm) were removed from the light-exposed surfaces and submerged in a solution of 0.3% sodium nitrite for 20 min at room temperature to induce metmyoglobin formation. Samples were blotted dry, vacuum packaged, and the reflectance spectra from 700 to 400 nm were recorded immediately on the light-exposed surface using a HunterLab LabSca XE colorimeter. The vacuum-packaged samples were then incubated at 30 °C for 2 h to induce reduction of metmyoglobin, and the reflectance data were taken again. The percentage of surface metmyoglobin (pre-incubation as well as post-incubation) was calculated based on K/S ratios and according to established formulas (AMSA, 2012). MRA was calculated using the following equation: Metmyoglobin reducing activity = 100 × [(% pre-incubation surface metmyoglobin − % post-incubation surface metmyoglobin)/% pre-incubation surface metmyoglobin].

Lipid oxidation

Lipid oxidation was analyzed employing thiobarbituric acid assay described previously (Yin et al., 1993; Nair et al., 2016). Briefly, 5-g representative samples, taken from multiple locations, were mixed with 22.5 mL of 11% trichloroacetic acid, homogenized (PRO250, PRO Scientific Inc, Oxford, CT), and filtered using Whatman No. 1 filter paper. One milliliter of filtrate was mixed with 1 mL of aqueous thiobarbituric acid (20 mM) and incubated at 25 °C for 20 h. The absorbance of samples at 532 nm measured spectrophotometrically (UV-1800 spectrophotometer, Shimadzu Inc., Candy, OR) was reported as thiobarbituric acid reactive substances (TBARS).

Statistical analysis

A split-plot design was used to evaluate 1) the effects of PAP and postmortem hour on mitochondrial functions and 2) the effects of PAP and retail display day on instrumental color, MRA, and lipid oxidation. Samples were assigned to PAP x postmortem hour combinations for mitochondrial function evaluation and PAP x display day combinations for color, MRA, and lipid oxidation measurement. Data analysis was performed by R (version 3.6.1) using the lme4 package as a mixed model, where PAP (high or low), postmortem hour (or display day), and their interactions were fixed effects, and the random effect in the model was an individual animal. The differences between least-square means (P < 0.05) were determined by Tukey’s multiple comparison.

A completely randomized design was used to evaluate the effects of PAP on ultimate pH and myoglobin concentration. A one-way ANOVA was conducted, followed by least-square means test (emmeans function in R) in order to better understand the difference between two PAP groups (P < 0.05).

Results

Muscle mitochondrial respiration and OXPHOS protein abundance

Mitochondrial respiratory capacity was determined in permeabilized LL muscle fiber bundles from high and low PAP cattle under a variety of substrate oxidation states using high-resolution respirometry (Figure 1). Overall, high PAP muscle tended to have greater substrate oxidation (respiratory) capacity than low PAP muscles at 2 h postmortem (Figure 1B) but rapidly declined to be similar to low PAP muscle by 48 h postmortem (P = 0.008 and 0.023 for PAP × postmortem-time interaction in NL and NP states, respectively; Figure 1B). These differences were most robust under conditions of abundant substrate availability in the absence of ADP (NL or “leak” respiration) at 2 h postmortem, indicating a greater capacity of high PAP muscle for endogenous fuel oxidation premortem when ATP demand is absent. In addition, the “leak” respiration, which reflects the extent of proton leak across the inner mitochondrial membrane (Pesta and Gnaiger, 2012; Chicco et al., 2014), may also reflect lower muscle OXPHOS efficiency in high PAP animals premortem.

Figure 1.

Figure 1.

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Muscle respiration under each respiratory flux state at 2 and 48 h postmortem (n = 6 for low pulmonary arterial pressure [PAP]; n = 5 for high PAP). NL: proton leak-associated respiration supported by high NADH, but no ADP; NP: OXPHOS-linked-respiration supported by pyruvate + malate oxidation; N: OXPHOS-linked respiration supported by malate + pyruvate + glutamate oxidation; NS: maximal OXPHOS-linked respiration supported by NADH and succinate; CIV: maximal oxygen reductase capacity of Complex IV using an artificial electron donor;⊗: a significant PAP ×postmortem-hour interaction (NL: P = 0.008; NP: P = 0.028);⊕: a significant postmortem-hour effect (N: P = 0.0007; NS: P = 0.0003; CIV: P = 0.005).

Note: Detailed information for each respiratory flux state is presented in Table 1.

There was a decline in maximal OXPHOS-linked respiration supported by maximal delivery of NADH (N; P = 0.0007) and NADH + succinate (NS; P = 0.0003), as well as the maximal oxygen reductase capacity of complex IV (CIV; P =0.005) in LL muscle from 2 to 48 h postmortem. This was seen in both high and low PAP groups, suggesting that maximal LL respiratory capacity declined after animal slaughter independent of PAP. In addition, nearly identical maximal CIV enzymatic capacities were seen between high and low PAP groups at 2 and 48 h postmortem, suggesting that the “leak” respiration (NL; oxidative capacity under conditions of low ATP demand) accounted for the observed difference in OXPHOS-linked respiration (NP) between groups, not maximal respiratory capacity. Despite these variations in LL respiratory capacity between groups and postmortem timepoints, tissue OXPHOS protein expression was greater in low PAP than high PAP animals (P = 0.046) and remained stable from 2 to 48 h postmortem (Figure 2).

Figure 2.

Figure 2.

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(A) Representative western blots of the abundance of complex II (SDHB) and complex V (ATP5A) and Amido Black stained total protein in beef longissimus steaks from cattle with different pulmonary arterial pressure (PAP) at early postmortem. (B) Mitochondrial OXPHOS protein abundance in beef longissimus steaks from cattle with different PAP at early postmortem (n = 6 for low PAP; n = 5 for high PAP).

⊕: a significant PAP effect (P < 0.05).

pH value, myoglobin concentration, and instrumental color of LL steak

No difference (P = 0.2622) in pH and myoglobin concentration of LL steak was observed between the high PAP group and low PAP group (Table 2). Therefore, these results suggested that PAP did not influence the ultimate pH and myoglobin concentration.

Table 2.

pH and myoglobin concentration of beef longissimus steaks from cattle with different pulmonary arterial pressure (PAP; n = 6 for low PAP; n = 5 for high PAP)

Parameter PAP Value SE1
pH High 5.53 0.02
Low 5.49 0.02
Myoglobin Concentration High 7.67 0.33
Low 7.66 0.30
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SE = standard error.

The PAP did not affect (P > 0.05) the L∗ value (lightness) during retail display (Table 3). However, a PAP × display-day interaction (P = 0.017) was observed for redness (a∗ value) during retail display. There were no difference (P > 0.05) observed in redness between the two PAP groups from days 1 to 7, but the low PAP group had higher (P = 0.001) redness than the high PAP group on day 9. Within treatment, redness decreased (P = 0.032) from days 1 to 7 in the high PAP group, whereas the low PAP group did not exhibit a decrease in redness until day 9 (P = 0.007). Display-day had a main effect on b∗ value regardless of PAP (P = 0.000001). The yellowness decreased (P = 0.0001) from days 1 to 9 for both PAP groups.

Table 3.

Lightness (L∗ value), redness (a∗ value), chroma, hue, R630/580, MRA, and TBARS of beef longissimus steaks from cattle with different pulmonary arterial pressure (PAP) during display (n = 6 for low PAP; n = 5 for high PAP)

Parameter PAP Display d SE
1 3 5 7 9
L∗ value High 41.5 a1 38.9 b 40.1 ab 39.9 ab 40.4 ab 1.09
Low 41.4 41.6 41.5 41.8 42.4 0.99
a∗ value High 18.6 a,x 17.3 ab,x 17.3 ab,x 16.6 b,x 13.5 c,x 0.60
Low 18.7 a,x 18.0 ab,x 18.1 ab,x 17.6 ab,x 16.6 b,y 0.55
b∗ value High 13.9 a 12.7 bc 13.3 ab 12.7 bc 11.7 c 0.45
Low 14.1 a 13.7 ab 13.8 ab 13.4 ab 13.0 b 0.41
Chroma High 23.2 a,x 21.5 ab,x 21.9 ab,x 20.9 b,x 17.9 c,x 0.72
Low 23.4 a,x 22.6 ab,x 22.8 ab,x 22.1 ab,x 21.1 b,y 0.66
Hue High 53.2 a,x 53.8 a,x 52.5 a,x 52.7 a,x 48.8 b,x 0.60
Low 52.9 a,x 52.6 a,x 52.6 a,x 52.6 a,x 51.9 a,y 0.55
R630/580 High 3.37 a,x 3.24 ab,x 3.09 ab,x 2.97 b,x 2.25 c,x 0.10
Low 3.38 a,x 3.20 a,x 3.18 a,x 3.06 ab,x 2.81 b,y 0.09
% MRA High 53.30 a,x 46.54 a,x 26.10 b,x 15.51 bc,x 3.43 c,x 7.43
Low 55.49 a,x 43.39 ab,x 49.24 a,y 27.67 bc,x 21.52 c,x 6.79
TBARS High 0.163 a 0.223 ab 0.305 ab 0.353 b 0.358 b 0.06
Low 0.164 0.164 0.246 0.284 0.286 0.06
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Least square means for the same trait in a column without a common letter (x−y) differ (P < 0.05). Least square means in a row without a common letter (a−c) differ (P < 0.05).

A PAP × display-day interaction was observed for chroma (P = 0.039), hue (P = 0.007), and the ratio of reflectance at 630 and 580 nm (R630/580; P = 0.011). There were no differences in chroma, hue, and R630/580 between high PAP and low PAP groups from days 1 to 7, but the low PAP group had higher chroma (P = 0.004), hue (P = 0.0004), and R630/580 value (P = 0.0002) than the high PAP group on day 9. Within treatment, chroma (P = 0.0152) and R630/580 (P = 0.015) significantly decrease from days 1 to 7 in the high PAP group, whereas the low PAP group did not show decreased chroma (P = 0.004) and R630/580 (P = 0.0003) value until day 9. Similarly, the hue value significantly decreased from days 1 to 9 in the high PAP group (P = 0.0001), whereas the low PAP group did not show any decrease during retail display (P = 0.669). Overall, our results suggested that although LL is a beef muscle with relevantly high color stability and long color shelf-life (McKenna et al., 2005; Seyfert et al., 2006), the LL steaks from the high PAP group discolored faster than those from the low PAP group during simulated retail display.

MRA and lipid oxidation

A PAP × display-day interaction was observed for MRA (P = 0.017; Table 3). On day 5, the low PAP group had greater (P = 0.035) MRA than the high PAP group, but there was no difference (P > 0.05) between treatments on the other display days. Within treatment, MRA decreased (P = 0.001) from days 1 to 5 in the high PAP group, whereas the low PAP group did not show decreased MRA until day 7 (P = 0.0002). Display-day had a main effect on TBARS values regardless of PAP (P = 0.00003; Table 2); however, within the treatment, TBARS increased from days 1 to 7 in the high PAP group (P = 0.005), whereas no change (P > 0.05) was observed in low PAP group during display. Along with our observation of color attributes, these results suggested that the LL steaks from the high PAP group had lower color stability than those from the low PAP group during simulated retail display.

Discussion

Recently, it was reported that high PAP cattle entering the feedlot at moderate elevations have around 20% higher feed to gain ratio compared to low PAP cattle (Thomas et al., 2019; Heffernan et al., 2020). In the present study, muscle from high PAP cattle exhibited greater proton leak-associated respiration (NL; Figure 1B) than low PAP muscle at 2 h postmortem in the present study, which suggested lower OXPHOS efficiency and greater fuel oxidation rates under conditions of low ATP demand in vivo. Consistent with this interpretation, greater mitochondrial proton leak in broilers’ skeletal muscle has been previously found in low feed efficiency than the high feed efficiency group (Bottje et al., 2009). Therefore, the observed lower feed efficiency in high PAP cattle as compared to low PAP cattle could also be attributed to the higher proton leak-associated respiration in skeletal muscle and warrants additional study.

It is also worth noting that the difference in proton leak-associated respiration (NL; Figure 1B) and between two PAP groups disappeared as postmortem time extended, and a much greater postmortem decline happened in the high PAP’s muscle OXPHOS-linked respiration (NP; Figure 1B) than low PAP cattle. These reflect a loss of mitochondrial integral function (membrane integrity or/and protein function) over time, which could be due to the accumulated damage from reactive oxygen species (ROS) known to be produced at high rates during leak respiration states (Li Puma et al., 2020). Previous research has shown that the broilers with pulmonary hypertension had greater ROS production from respiratory complexes I and III compared to the control group (Tang et al., 2002), which are the primary sites of ROS production during leak respiration (Jastroch et al., 2010). Moreover, increased oxidative stress level is often observed during pulmonary hypertension development (Mikhael et al., 2019).

Meat color is determined by biomolecular interaction between myoglobin, mitochondria, metabolites, and lipid oxidation (Ramanathan et al., 2020). Metmyoglobin reducing activity is one of the essential biochemical processes that facilitates the sustained bright-red color of beef during retail display (Ledward, 1985). A greater and stable mitochondrial functionality can maintain meat color by improved MRA (Ramanathan and Mancini, 2018; Ramanathan et al., 2019). Conversely, mitochondria are the major ROS production sites in mammalian cells (Murphy, 2009; Mazat et al., 2020). In postmortem muscle, ROS can increase oxidative stress, damage mitochondrial membrane, and promote mitochondrial degradation, resulting in faster meat tenderization (Wang et al., 2018; Zhang et al., 2019; Chen et al., 2020), increased lipid oxidation, and reduced color stability (Ke et al., 2017; Mancini et al., 2018; Mitacek et al., 2019). The faster decline of MRA observed in the high PAP group steaks is consistent with these interpretations and further supports a link between muscle mitochondrial function and meat quality.

Additionally, there were differences in the mitochondrial OXPHOS protein abundance between the two PAP groups in this study. A previous human clinical study indicated that pulmonary arterial hypertension caused a decreased type I/type II muscle fiber ratio in skeletal muscle (Batt et al., 2013), which could explain the lower OXPHOS protein abundances in the high PAP group. The muscle fiber type was not measured in the current study. However, we hypothesize a similar change of muscle fiber ratio in skeletal muscle from the high PAP cattle.

The same clinical study also reported that increased phosphorylation of ryanodine receptor 1 receptors in skeletal muscle was caused by pulmonary arterial hypertension (Batt et al., 2013), which could result in destabilization and leakiness of the sarcoplasmic reticulum Ca2+ channel, disturbing the control of cytosolic free Ca2+ and impairing skeletal muscle contractility (Bellinger et al., 2008a, 2008b; Andersson and Marks, 2010). A diminished exercise capacity and atrophy of the quadriceps femoris muscle were observed in human with pulmonary arterial hypertension (Batt et al., 2013).

Conclusions

High PAP muscle had greater proton leak-associated respiration than low PAP muscles at 2 h postmortem, suggesting high PAP caused a lower OXPHOS efficiency and greater fuel oxidation rates under conditions of low ATP demand in premortem beef LL muscle. This difference could explain the lower feed efficiency in high PAP feedlot cattle compared to low PAP counterparts. Although maximal LL respiratory capacity declined after animal slaughter regardless of PAP, OXPHOS-linked respiration declined faster in high PAP muscle from 2 to 48 h postmortem, reflecting a faster loss of mitochondrial integral function (membrane integrity or/and protein function). OXPHOS protein expression was also lower in high PAP compared to low PAP muscle, highlighting the need for further investigation into the potentially decreased type I/type II muscle fiber ratio in high PAP muscle. The muscle pH and myoglobin concentration were similar between the two PAP groups. However, the high PAP group steaks had lower a∗, chroma, hue, and R630/580 values than low PAP steers on day 9 and a lower MRA on day 5. High PAP group steaks also showed a significant increase in lipid oxidation on day 7, whereas no change was observed in the low PAP group during display. These results suggested that high PAP group LL had lower color stability than the low PAP group during simulated retail display, which could be partially attributed to the loss of muscle mitochondrial function at early postmortem by ROS damage in high PAP muscle.

Acknowledgments

This work was partially supported by USDA-NIFA grant 2018-67015-28241 and USDA multistate grant W4177.

Glossary

Abbreviations

HRR

high-resolution respirometry

LL

longissimus lumborum

MRA

metmyoglobin reducing activity

OXPHOS

oxidative phosphorylation

PAP

pulmonary arterial pressures

ROS

reactive oxygen species;

TBARS

thiobarbituric acid reactive substances

Conflict of Interest Statement

The authors declare no real or perceived conflicts of interest.

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