animals Article Elbow Extensor Muscles in Humans and Chimpanzees: Adaptations to Different Uses of the Upper Extremity in Hominoid Primates Marina de Diego 1 , Aroa Casado 1,2 , Mónica Gómez 1 , Neus Ciurana 1 , Patrícia Rodríguez 1 , Yasmina Avià 2,3 , Elisabeth Cuesta-Torralvo 2,3 , Natividad García 4 , Isabel San José 4 , Mercedes Barbosa 4 , Félix de Paz 4 , Juan Francisco Pastor 4 and Josep Maria Potau 1,2, * 1 Unit of Human Anatomy and Embryology, University of Barcelona, 08036 Barcelona, Spain 2 Institut d’Arqueologia de la Universitat de Barcelona (IAUB), Faculty of Geography and History, University of Barcelona (UB), 08001 Barcelona, Spain 3 Biological Anthropology Unit, Department of Animal Biology, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain 4 Department of Anatomy and Radiology, University of Valladolid, 47005 Valladolid, Spain * Correspondence: jpotau@ub.edu; Tel.: +34-9-3402-1906 Simple Summary: Chimpanzees and humans are both species of hominoid primates that are closely related phylogenetically. One of the key differences between these two species is their use of their upper extremities. Humans use this limb mainly in manipulative tasks, while chimpanzees also use it during locomotion. In this study, we have analyzed the muscle architecture and the expression of the myosin heavy chain isoforms in the two elbow extensor muscles, the triceps brachii and the anconeus, Citation: de Diego, M.; Casado, A.; in humans and chimpanzees, in order to find differences that could be related to the different uses of Gómez, M.; Ciurana, N.; Rodríguez, the upper extremities in these species. We have found that the triceps brachii of chimpanzees is more P.; Avià, Y.; Cuesta-Torralvo, E.; prepared for strength and power as an adaptation to locomotion, while the same muscle in humans García, N.; San José, I.; Barbosa, M.; is more prepared for speed and resistance to fatigue as an adaptation to manipulative activities. Our et al. Elbow Extensor Muscles in results increase the knowledge we have of the musculoskeletal system of chimpanzees and can be Humans and Chimpanzees: applied in various fields, such as comparative anatomy, evolutionary anatomy or anthropology. Adaptations to Different Uses of the Upper Extremity in Hominoid Primates. Animals 2022, 12, 2987. Abstract: The anatomical and functional characteristics of the elbow extensor muscles (triceps brachii https://doi.org/10.3390/ and anconeus) have not been widely studied in non-human hominoid primates, despite their great ani12212987 functional importance. In the present study, we have analyzed the muscle architecture and the expression of the myosin heavy chain (MHC) isoforms in the elbow extensors in humans and Academic Editor: Efstratios chimpanzees. Our main objective was to identify differences in these muscles that could be related to D. Valakos the different uses of the upper extremity in the two species. In five humans and five chimpanzees, we Received: 30 September 2022 have analyzed muscle mass (MM), muscle fascicle length (MFL), and the physiological cross-sectional Accepted: 27 October 2022 area (PCSA). In addition, we have assessed the expression of the MHC isoforms by RT-PCR. We have Published: 30 October 2022 found high MM and PCSA values and higher expression of the MHC-IIx isoform in the triceps brachii Publisher’s Note: MDPI stays neutral of chimpanzees, while in humans, the triceps brachii has high MFL values and a higher expression with regard to jurisdictional claims in of the MHC-I and MHC-IIa isoforms. In contrast, there were no significant differences between published maps and institutional affil- humans and chimpanzees in any of the values for the anconeus. These findings could be related to iations. the participation of the triceps brachii in the locomotion of chimpanzees and to the use of the upper extremity in manipulative functions in humans. The results obtained in the anconeus support its primary function as a stabilizer of the elbow joint in the two species. Copyright: © 2022 by the authors. Keywords: triceps brachii; anconeus; chimpanzee; elbow joint Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// 1. Introduction creativecommons.org/licenses/by/ The elbow extensor muscles, the triceps brachii and the anconeus, are located in the 4.0/). posterior compartment of the arm and in the lateral region of the elbow, respectively [1] Animals 2022, 12, 2987. https://doi.org/10.3390/ani12212987 https://www.mdpi.com/journal/animals Keywords: triceps brachii; anconeus; chimpanzee; elbow joint Animals 2022, 12, 2987 1. Introduction 2 of 15 The elbow extensor muscles, the triceps brachii and the anconeus, are located in the posterior compartment of the arm and in the lateral region of the elbow, respectively [1] (Figure (Figure1). 1).The Theorigin originandandinsertion insertionsite siteofofthese thesetwo twomuscles muscleshavehaveaasimilar similaranatomical anatomical structure structureininhumans humans(Homo (Homosapiens) sapiens)and andinincommon commonchimpanzees chimpanzees(Pan (Pantroglodytes)—two troglodytes)—two species speciesthatthatare areclosely closelyrelated relatedphylogenetically phylogenetically[2]. [2].The Thetriceps tricepsbrachii brachiiconsists consistsofofaalong long head, which originates in the infraglenoid tubercle of the scapula in head, which originates in the infraglenoid tubercle of the scapula in humans and at leasthumans and at least half halfofofthe thelength lengthofofthe thelateral lateralborder borderofofthe thescapula scapulaininchimpanzees, chimpanzees,aalateral lateralhead, head,which which originates originates on the posterior aspect of the diaphysis of the humerus superior tothe on the posterior aspect of the diaphysis of the humerus superior to theradial radial groove, groove,and andaamedial medialhead,head,which whichoriginates originateson onthe theposterior posteriorfacefaceofofthe thediaphysis diaphysisofofthethe humerus humerusinferior inferiortotothetheradial radialgroove groove[3,4]. [3,4].The Thethree threeheads headsofofthe thetriceps tricepsbrachii brachiiconverge converge and andinsert insertinto intothetheolecranon olecranonprocess processofofthetheulna ulna[1,5–7]. [1,5–7].TheTheanconeus anconeusoriginates originatesininthe the lateral lateral epicondyle of the humerus and inserts into the lateral aspect of theolecranon epicondyle of the humerus and inserts into the lateral aspect of the olecranonand and into intothe theupper upper region region of the posterior of the posterior aspect aspectofofthe theulna ulna[1,3,6,8–10]. [1,3,6,8–10].Functionally, Functionally, thethe tri- triceps brachii is the main extensor of the elbow in humans [3,7,8,11], ceps brachii is the main extensor of the elbow in humans [3,7,8,11], while the anconeus while the anconeus has hasaamore morecomplex complexfunction function[12]. [12].In Inaddition additiontotoacting actingas asananaccessory accessoryextensor extensorofofthethe elbow [9,11,13,14], the anconeus works to stabilize the elbow [14–18] and elbow [9,11,13,14], the anconeus works to stabilize the elbow [14–18] and is also an abduc- is also an abductor of the ulna during pronation-supination movements [19]. tor of the ulna during pronation-supination movements [19]. Figure1.1.Medial Figure Medialview viewofofaatriceps tricepsbrachii brachiimuscle muscledissection dissectioninin(a) (a)Homo Homosapiens sapiensand and(b) (b)Pan Pantroglodytes troglodytes and lateral view of an anconeus muscle dissection in (c) Homo sapiens and (d) Pan troglodytes. Tclo and lateral view of an anconeus muscle dissection in (c) Homo sapiens and (d) Pan troglodytes. Tclo = = Triceps brachii caput longum; Tcla = Triceps brachii caput laterale; Tcm = Triceps brachii caput mediale; A = Anconeus; ED = Extensor digitorum; ECU = Extensor carpi ulnaris. Despite the close phylogenetic relationship between humans and chimpanzees [2], the main functions of the upper extremities—and hence the use of the elbow extensor muscles— differ between the two species: for locomotion in chimpanzees and for manipulative activities in humans [7]. Taxonomically, humans and chimpanzees are both hominoid primates or apes, a group that also includes bonobos, gorillas, orangutans, gibbons and Animals 2022, 12, 2987 3 of 15 siamangs [20]. These hominoid primates share common anatomical features that increase the mobility of large joint complexes in the upper extremity, such as the shoulder, elbow, and wrist [21–23]. These features have allowed the appearance of different forms of arboreal locomotion in non-human hominoid primates, including vertical climbing, arm suspension and brachiation [20], as well as the manipulative abilities characteristic of humans [24]. In addition, non-human African apes (gorillas, chimpanzees and bonobos) use a characteristic form of terrestrial quadrupedal locomotion called knuckle-walking [5], which represents up to 80–90% of the total locomotor repertoire in chimpanzees [25–28]. The triceps brachii of chimpanzees is highly important during the second part of the swing phase of knuckle- walking, when the elbow is extended to bring the hand forward, as well as during the support phase, when it retracts the humerus while keeping the elbow in extension to move the body over it [29]. A moderate activity of the triceps brachii of chimpanzees has also been observed during the elevation of the upper extremity over the head but not during suspensory behavior [29]. The anconeus of chimpanzees is active during the support phase of knuckle-walking—but not during the second part of the swing phase—and during the elevation of the upper extremity over the head [29]. Although the triceps brachii and the anconeus are both elbow extensors, they have clearly differentiated functions both in humans [3,6,8] and in chimpanzees [29]. Moreover, their functions are different between the two species, as they are used mainly in manipu- lative activities in humans [30] and mainly in arboreal locomotion and knuckle-walking in chimpanzees [29]. These functional differences between the two muscles and between the two species can translate into muscle adaptations affecting the muscle architecture and molecular composition. The study of the architecture of a given muscle can provide information on its functional characteristics, based on the calculation of muscle mass (MM), muscle fascicle length (MFL), and the physiological cross-sectional area (PCSA) [31]. MM is directly related to the ability of a given muscle to produce power [31]; MFL reflects the number of sarcomeres arranged in series and is directly proportional to the maximum rate of shortening of muscle fascicles [32]; and the PCSA reflects the number of sarcomeres ar- ranged in parallel and is a good indicator of the ability of a muscle to generate strength [33]. The functional capabilities of a given muscle are also related to its molecular composi- tion, mainly the expression pattern of the myosin heavy chain (MHC) isoforms [34–36]. In the limb muscles of humans and chimpanzees, three MHC isoforms are expressed: MHC-I, MHC-IIa and MHC-IIx [37–39]. The slow isoform MHC-I is mainly expressed in the slow-twitch oxidative type-I muscle fibers [37], which are characterized by a slow contraction rate, a low capacity to generate power and a high resistance to fatigue [40]. The fast isoforms MHC-IIa and MHC-IIx are mainly expressed in fast-twitch oxidative type IIa and fast-twitch glycolytic type IIx muscle fibers, respectively [37]. These fibers are characterized by a fast-twitch speed, a high capacity to generate power and a low resistance to fatigue [34], with type IIx fibers being faster, more powerful and less resistant to fatigue than type IIa fibers [40,41]. To date, few studies have compared the anatomical, structural, functional and molec- ular characteristics of the elbow extensors in humans and chimpanzees. Here we have studied the muscle architecture of the elbow extensors in these two species, and we have analyzed by real-time polymerase chain reaction (RT-PCR) the expression pattern of the MHC isoforms in these muscles. Our main objective was to identify significant differences that could be related to the different uses of the upper extremity in these two primate species [42]. We also sought to identify intra-species differences between the triceps brachii and the anconeus since they have slightly different functions [11]. We hypothesized that since chimpanzees use the triceps brachii mainly in arboreal and terrestrial locomotion [29], this would be reflected in higher values of the architectural parameters related to strength and power and a higher percentage of expression of the rapid MHC isoforms. We further hypothesized that since the anconeus is used in both humans and chimpanzees as a stabi- lizer of the elbow, differences in muscle architecture and expression of the MHC isoforms would not be so evident between the two species. Additionally, since the morphology of Animals 2022, 12, 2987 4 of 15 muscle insertion sites (enthesis) can be related to the size and activity of the muscles [43], we have also performed a quantitative analysis of the insertion sites of the triceps brachii and anconeus in the ulna to determine if structural and molecular differences between humans and chimpanzees would also translate into differences in their insertion sites. Our analysis of muscle architecture, expression of the MHC isoforms, and morphology of the insertion sites of the elbow extensors in humans and chimpanzees can expand the existing knowledge of this muscle group in a species of hominoid primates closely related to humans and help to better understand the functional differences between the two species. Our findings will therefore be of interest in different fields, including comparative anatomy, physical anthropology, and evolutionary anatomy. 2. Materials and Methods 2.1. Muscle and Osteological Samples For the analyses of muscle architecture and expression of the MHC isoforms, we dissected five upper extremities of humans (five males; mean age 85.8 years [range, 81–91 years]) and five upper extremities of adult chimpanzees (four females and one male). The human individuals came from the Body Donation Service of the University of Barcelona, and the chimpanzees were provided by the Anatomy Museum of the University of Valladolid and had died in different Spanish zoos (Zoo of Madrid, Zoo of Santillana, Bioparc of Fuengirola and Primadomus of Alicante) from causes unrelated to our study. All individuals had been cryopreserved at −18 ◦ C without chemical fixation within 24–48 h post-mortem. For the analysis of the triceps brachii and anconeus insertion sites, 18 human ul- nae were provided by the Human Anatomy and Embryology Unit of the University of Barcelona, and 19 ulnae of chimpanzees were provided by the Anatomy Museum of the University of Valladolid. The human ulnae came from ten females and eight males (mean age, 73.1 years [range, 38–97 years]), while the chimpanzee ulnae came from eight females and 11 males (all adults, as determined by epiphyseal fusion). 2.2. Muscle Architecture The upper extremities of humans and chimpanzees were dissected by the same re- searcher (JMP). Once the adipose and connective tissue were removed, the triceps brachii and anconeus muscles were identified and anatomical data were recorded, including their origin and insertion. After the muscles were disinserted, their MM in grams was calculated using a precision scale (model Sartorius PT610 and resolution of 0.1 g). We also calculated the MM of the triceps brachii and the anconeus relative to the total body mass (%MM). For this purpose, we used an estimated body mass of 59,700 g for the male chimpanzees, 45,800 g for the female chimpanzees, and 62,500 g for the humans [44]. Photographs of the two muscles were then taken with a Canon EOS-50 camera, which made it possible to identify the architectural pattern of the muscle fascicles (Figure 2). These photographs were analyzed with the computer software ImageJ [45] to obtain the MFL in centimeters and the pennation angle (θ). These two values were measured in five different muscle fascicles: the central region of the muscle, the proximal end, the distal end, midway between the central region and the proximal end, and midway between the central region and the distal end. The average MFL and the average θ were recorded for each muscle. We then calculated the PCSA in cm2 for each muscle, using the formula PCSA = (MM × cos θ)/(ρ × MFL), where ρ = muscle density (1.06 g/cm3 ) [46]. Since the upper extremity muscles are generally larger in chimpanzees than in humans [47], in order to compare the two species, the absolute MFL and PCSA values were normalized based on the MM. MFL was normalized by dividing this value by MM1/3 (NMFL) and PCSAs were normalized by dividing this value by MM2/3 (NPCSA) [31]. Finally, a 1-cm3 sample of the central region of each muscle was obtained and cryopreserved at −18 ◦ C in physiological saline solution for later molecular analysis. (MM × cos θ)/(ρ × MFL), where ρ = muscle density (1.06 g/cm3) [46]. Since the upper ex- tremity muscles are generally larger in chimpanzees than in humans [47], in order to com- pare the two species, the absolute MFL and PCSA values were normalized based on the MM. MFL was normalized by dividing this value by MM 1/3 (NMFL) and PCSAs were nor- Animals 2022, 12, 2987 malized by dividing this value by MM2/3 (NPCSA) [31]. Finally, a 1-cm3 sample of the cen- 5 of 15 tral region of each muscle was obtained and cryopreserved at −18 °C in physiological sa- line solution for later molecular analysis. Figure2.2.View Figure Viewofofthe the (a)(a) triceps triceps brachii brachii andand (b)(b) anconeus anconeus of Pan of Pan troglodytes troglodytes displaying displaying the architec- the architectural tural arrangement of their muscle fascicles. MFL = muscle fascicle length; θ = pennation angle. arrangement of their muscle fascicles. MFL = muscle fascicle length; θ = pennation angle. 2.3.Expression 2.3. ExpressionofofMHC MHCIsoforms Isoforms RNAwas RNA wasextracted extractedfromfromthe themuscle musclesamples samplesusingusingthe thecommercial commercialRNeasy RNeasymini minikitkit (Qiagen, Valencia, CA, USA). We used a NanoDrop 1000 Spectrophotometer (Qiagen, Valencia, CA, USA). We used a NanoDrop 1000 Spectrophotometer to determine to determine theconcentration, the concentration,puritypurityand andamount amountof ofRNA RNAand andTaqMan TaqManReverse ReverseTranscription TranscriptionReagent Reagent Kit(Applied Kit (AppliedBiosystems, Biosystems, Foster Foster City, City, CA,CA, USA) USA) to synthesize to synthesize cDNA.cDNA. We performed We performed reversere- transcription using 330 verse transcription ng of330 using total ngRNA in 10 of total µL ofinRT RNA 10Buffer, µ L of 22 RTmL of 25 22 Buffer, mM mL magnesium of 25 mM chloride, magnesium 20 µL dNTPs, 20 chloride, 5 µL µ LRandom dNTPs, Hexamers, 5 µ L Random 2 µLHexamers, RNAse Inhibitor, 2.5 µL Inhibitor, 2 µ L RNAse MultiScribe 2.5 Reverse Transcription µ L MultiScribe ReverseandTranscription RNA sample and plusRNA RNAse-free samplewater, for a final volume plus RNAse-free water,of for100 µL, a final involume the following of 100 µthermal L, in thecycler conditions: following thermal10 25 ◦ C, 48 min min conditions: cycler 30 ◦ C 10 min 25and °C, 548minmin9530◦ C. °C Applied and 5 min Biosystems supplied 95 °C. Applied primers and Biosystems probes. supplied Primers primers andwere labeled probes. Primers 50 end at thewere with labeled the reporter at the 5′ enddye molecule with FAM. MYH-I the reporter (Hs00165276_m1), dye molecule FAM. MYH-I MYH-IIa (Hs00430042_m1) (Hs00165276_m1), and MYH-IIa MYH-IIx (Hs00428600_m1) (Hs00430042_m1) and MYH-IIx genes were analyzed. In (Hs00428600_m1) order genes to avoid were any In analyzed. possible order to effects avoid ofanypost-mortem mRNA possible effects degradation,mRNA of post-mortem the mRNA values the degradation, for mRNA each ofvalues the MHC isoforms for each of the were normalized using the reference gene ACTB [48]. The mRNA MHC isoforms were normalized using the reference gene ACTB [48]. The mRNA of ACTB of ACTB is detectable for more thanfor 22 days ◦ C [49], and is detectable more post-mortem than 22 days in skeletal muscle post-mortem fibers muscle in skeletal preserved at 4preserved fibers at 4 it°Cis [49], one ofandthe reference it is one of thegenes that isgenes reference least that affected is leastbyaffected muscular by degeneration [50]. We muscular degeneration performed RT-PCR inRT-PCR [50]. We performed a total volume in a totalof volume 20 µL inof the 20ABI µ L Prism in the 7700 Sequence ABI Prism 7700Detection Sequence System Detection (Applied SystemBiosystems) using the using (Applied Biosystems) following master mix the following conditions: master 10 µL of10the mix conditions: µL TaqMan Universal PCR Master Mix (Thermo Fisher Scientific, of the TaqMan Universal PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA),Waltham, MA, USA), 1 µL of1 µthe L ofprimers and probes, the primers 2 µL2of and probes, µ Lthe cDNA of the cDNA andand 7 µL7 µof thethe L of RNAse-free RNAse-freewater.water.We We ran all samples ran all samples for foreach each gene gene inin duplicate duplicate using using the the following following thermal thermal cycler cycler conditions: conditions: 22min ◦ ◦ and 40× (15 s 95 ◦ C, 1 min 60 ◦ C). We used genomic DNA as min50 50 C,°C,10 10min min9595 C°C and 40× (15 s 95 °C, 1 min 60 °C). We used genomic DNA as negative control in each run. We captured fluorescent emission data and quantified mRNA concentrations by using the critical threshold value and 2-∆∆Ct. Finally, we calculated the percentage of expression of each MHC isoform relative to the total expression of all MHC isoforms (%MHC-I, %MHC-IIa and %MHC-IIx). 2.4. Muscle Insertion Sites To obtain the area of the triceps brachii insertion site (TBIA) and the anconeus insertion site (AIA) at the ulna in mm2 , the 18 human and 19 chimpanzee ulnae were scanned with a Next Engine 2000 Ultra HD laser scanner (NextEngine, Inc., Santa Monica, CA, USA) and the resulting triangle meshes were edited with the MeshLab 2021.05 software [51]. When Animals 2022, 12, 2987 6 of 15 we had identified the insertion sites of the triceps brachii and the anconeus in the olecranon process of the ulna, in each of the 3-D models, we manually eliminated the remaining bone surface with MeshLab to automatically calculate the TBIA and AIA values. Finally, the TBIA and AIA values were normalized by dividing these values by the length of the ulna in each individual (NTBIA and NAIA). 2.5. Statistical Analysis The normality of the sample was tested using the Shapiro-Wilk test. Variables with a normal distribution were compared with the parametric T-test and variables without a normal distribution were compared with the non-parametric Mann-Whitney U test. All variables related to muscle architecture, expression of MHC isoforms, and area of muscle insertion sites were compared between humans and chimpanzees. Variables related to muscle architecture and expression of MHC isoforms were also compared between the triceps brachii and anconeus in each species. 3. Results 3.1. Muscle Architecture The main findings on muscle architecture are summarized in Table 1. The only signifi- cant differences between humans and chimpanzees were observed in the triceps brachii. Chimpanzees had significantly higher MM (377.5 ± 122.4 g vs. 165.4 ± 39.5 g; p = 0.006), %MM (0.78 ± 0.26 vs. 0.26 ± 0.06; p = 0.003) and NPCSA (0.69 ± 0.05 vs. 0.46 ± 0.08; p < 0.001) values, while humans had significantly higher NMFL values (2.04 ± 0.39 vs. 1.18 ± 0.11; p = 0.001). No significant differences between the two species were observed in the anconeus (Figure 3). In humans, there were no significant differences between the triceps brachii and the anconeus in NMFL (2.04 ± 0.39 vs. 1.87 ± 0.27; p = 0.445) or in NPCSA (0.46 ± 0.08 vs. 0.45 ± 0.05; p = 0.754). In contrast, there were significant differences between the two muscles in chimpanzees: the anconeus had higher NMFL values (1.96 ± 0.29 vs. 1.18 ± 0.11; p = 0.001), while the triceps brachii had higher NPCSA values (0.69 ± 0.05 vs. 0.43 ± 0.06; p = 0.009). 3.2. Expression of MHC Isoforms The main findings on the expression of MHC isoforms are summarized in Table 2. In the triceps brachii, the percentage of expression of the MHC-IIx isoform was significantly higher in chimpanzees (43.2 ± 9.8% vs. 29.9 ± 6.0%; p = 0.032), while the percentage of expression of the MHC-I and MHC-IIa isoforms was higher—though not significantly so—in humans (MHC-I: 33.1 ± 3.1% vs. 27.2 ± 8.6%; p = 0.194; MHC-IIa: 37.0 ± 6.0% vs. 29.5 ± 14.4%; p = 0.316). In one human and one chimpanzee, the analysis of the MHC isoforms in the anconeus was not informative, but in the remaining eight samples, the percentage of expression of all three MHC isoforms in the anconeus was similar in humans and chimpanzees (Figure 4). Animals 2022, 12, 2987 7 of 15 Table 1. Main findings on muscle architecture. MM = muscle mass; %MM = percentage of MM relative to total body mass; MFL = muscle fascicle length in cm; NMFL = normalized muscle fascicle length; PCSA = physiological cross-sectional area in cm2 ; NPCSA = normalized physiological cross-sectional area; HS = Homo sapiens; PT = Pan troglodytes; M = male; F = female. SAMPLE SEX AGE (Years) MM %MM MFL NMFL PCSA NPCSA MM %MM MFL NMFL PCSA NPCSA Triceps brachii Anconeus HS01 M 91 138.8 0.22 9.8 1.90 13.0 0.48 5.2 0.008 3.4 1.98 1.4 0.47 HS02 M 85 195.2 0.31 13.4 2.31 12.8 0.38 4.1 0.007 3.5 2.19 1.0 0.38 HS03 M 81 111.8 0.18 8.2 1.70 12.0 0.52 4.7 0.008 2.6 1.57 1.4 0.50 HS04 M 81 174.8 0.28 14.4 2.58 11.5 0.37 5.6 0.009 3.6 2.01 1.3 0.42 HS05 M 91 206.4 0.33 10.1 1.71 18.8 0.54 7.7 0.012 3.2 1.60 1.8 0.45 Mean 165.4 0.26 11.2 2.04 13.6 0.46 5.5 0.009 3.3 1.87 1.4 0.45 SD 39.5 0.06 2.6 0.39 2.9 0.08 1.4 0.002 0.4 0.27 0.3 0.05 PT01 F 26 521.6 1.14 9.5 1.18 45.8 0.71 2.3 0.005 2.4 1.80 0.8 0.48 PT02 F 25 399.9 0.87 8.9 1.21 36.0 0.66 5.2 0.011 3.2 1.82 1.4 0.47 PT03 M 43 426.3 0.71 8.1 1.08 38.4 0.68 8.7 0.015 3.4 1.64 2.0 0.46 PT04 F 40 189.0 0.41 6.1 1.07 25.1 0.76 0.9 0.002 2.2 2.26 0.4 0.38 PT05 F 28 350.6 0.77 9.6 1.35 31.6 0.64 7.6 0.017 4.5 2.27 1.4 0.36 Mean 377.5 0.78 8.4 1.18 35.4 0.69 4.9 0.010 3.1 1.96 1.2 0.43 SD 122.4 0.26 1.4 0.11 7.7 0.05 3.3 0.006 0.9 0.29 0.6 0.06 p value 0.006 * 0.003 * 0.072 0.001 * <0.001 * <0.001 * 0.756 0.704 0.743 0.632 0.56 0.654 * statistical significance. Animals 2022, 12, 2987 8 of 15 Table 2. Main findings on the expression of the MHC isoforms. MHC = myosin heavy chain; HS = Homo sapiens; PT = Pan troglodytes; M = male; F = female. SAMPLE SEX AGE (Years) %MHC-I %MHC-IIa %MHC-IIx %MHC-II %MHC-I %MHC-IIa %MHC-IIx %MHC-II Triceps brachii Anconeus HS01 M 91 36.6 28.2 35.2 63.4 27.7 50.9 21.4 72.3 HS02 M 85 31.6 43.7 24.7 68.4 27.7 52.9 19.4 72.3 HS03 M 81 36.3 41.5 22.2 63.7 NA NA NA NA HS04 M 81 30.7 35.9 33.3 69.3 39.6 31.1 29.4 60.4 HS05 M 91 30.1 35.8 34.0 69.9 42.5 50.7 6.8 57.5 Mean 33.1 37.0 29.9 66.9 34.4 46.4 19.2 65.6 SD 3.1 6.0 6.0 3.1 7.8 10.3 9.4 7.8 PT01 F A 18.3 48.2 33.5 81.7 29.1 41.7 29.1 70.9 PT02 F A 26.9 19.7 53.4 73.1 NA NA NA NA PT03 M A 20.8 29.8 49.4 79.2 35.4 51.4 13.2 64.6 PT04 F A 40.3 11.8 47.9 59.7 28.8 51.0 20.3 71.2 PT05 F A 29.8 38.2 32.0 70.2 36.6 42.7 20.7 63.4 Mean 27.2 29.5 43.2 72.8 32.5 46.7 20.8 67.5 SD 8.6 14.4 9.8 8.6 4.1 5.2 6.5 4.1 p value 0.194 0.316 0.032 * 0.194 0.678 1.000 0.789 0.678 * statistical significance. Mean 377.5 0.78 8.4 1.18 35.4 0.69 4.9 0.010 3.1 1.96 1.2 0.43 SD 122.4 0.26 1.4 0.11 7.7 0.05 3.3 0.006 0.9 0.29 0.6 0.06 p value Animals 2022, 12, 2987 0.006 * 0.003 * 0.072 0.001 * <0.001 * <0.001 * 0.756 0.704 0.743 0.632 0.56 0.654 9 of 15 * statistical significance. Animals 2022, 12, x 8 of 14 Mean 33.1 37.0 29.9 66.9 34.4 46.4 19.2 65.6 SD 3.1 6.0 6.0 3.1 7.8 10.3 9.4 7.8 PT01 F A 18.3 48.2 33.5 81.7 29.1 41.7 29.1 70.9 PT02 F A 26.9 19.7 53.4 73.1 NA NA NA NA PT03 M A 20.8 29.8 49.4 79.2 35.4 51.4 13.2 64.6 PT04 F A 40.3 11.8 47.9 59.7 28.8 51.0 20.3 71.2 PT05 F A 29.8 38.2 32.0 70.2 36.6 42.7 20.7 63.4 Mean 27.2 29.5 43.2 72.8 32.5 46.7 20.8 67.5 SD 8.6 14.4 9.8 8.6 4.1 5.2 6.5 4.1 p value 0.194 3. Boxplots Figure 0.316 of0.032 the differences * 0.678 Homo 0.194 between sapiens (HS) 1.000 0.789and Pan troglodytes 0.678 (PT) in NMFL * statistical significance. and NPCSA values in the (a,c) triceps brachii (TB) and (b,d) anconeus (A). (A). In humans, there were no significant differences between the triceps brachii and the anconeus in NMFL (2.04 ± 0.39 vs. 1.87 ± 0.27; p = 0.445) or in NPCSA (0.46 ± 0.08 vs. 0.45 ± 0.05; p = 0.754). In contrast, there were significant differences between the two muscles in chimpanzees: the anconeus had higher NMFL values (1.96 ± 0.29 vs. 1.18 ± 0.11; p = 0.001), while the triceps brachii had higher NPCSA values (0.69 ± 0.05 vs. 0.43 ± 0.06; p = 0.009). 3.2. Expression of MHC Isoforms The main findings on the expression of MHC isoforms are summarized in Table 2. In the triceps brachii, the percentage of expression of the MHC-IIx isoform was significantly higher in chimpanzees (43.2 ± 9.8% vs. 29.9 ± 6.0%; p = 0.032), while the percentage of expression of the MHC-I and MHC-IIa isoforms was higher—though not significantly so—in humans (MHC-I: 33.1 ± 3.1% vs. 27.2 ± 8.6%; p = 0.194; MHC-IIa: 37.0 ± 6.0% vs. 29.5 ± 14.4%; p = 0.316). In one human and one chimpanzee, the analysis of the MHC isoforms in the anconeus was not informative, but in the remaining eight samples, the percentage of expression of all three MHC isoforms in the anconeus was similar in humans and chim- panzees (Figure 4). Table 2. Main findings on the expression of the MHC isoforms. MHC = myosin heavy chain; HS = Homo sapiens; PT = Pan troglodytes; M = male; F = female. SAMPLE SEX AGE (Years) %MHC-I %MHC-IIa %MHC-IIx %MHC-II %MHC-I %MHC-IIa %MHC-IIx %MHC-II Triceps brachii Anconeus HS01 M 91 36.6 28.2 35.2 63.4 27.7 50.9 21.4 72.3 HS02 M 85 31.6 43.7 24.7 68.4 27.7 52.9 19.4 72.3 HS03 M 81 36.3 41.5 22.2 63.7 NA NA NA NA HS04 M 81 30.7 35.9 33.3 69.3 39.6 31.1 29.4 60.4 HS05 M 91 30.1 35.8 34.0 69.9 42.5 50.7 6.8 57.5 Figure Figure 4. Expression 4. Expression of MHC of MHCinisoforms isoforms in theand the triceps brachii triceps brachii anconeus and in Homo and Pan in Homo sapiens and anconeus sapiens troglodytes. MHC = myosin heavy chain; HS = Homo sapiens; PT = Pan troglodytes. The data bars rep- Pan troglodytes. MHC = myosin heavy chain; HS = Homo sapiens; PT = Pan troglodytes. The data resent mean values, and the error bars represent the standard error. * indicates statistical signifi- bars represent mean values, and the error bars represent the standard error. * indicates statistical cance. significance. Animals 2022, 12, 2987 10 of 15 In humans, the triceps brachii had a higher percentage of expression of the MHC-IIx isoform than the anconeus (29.9 ± 6.0% vs. 19.2 ± 9.4%; p = 0.075), while the anconeus muscle had a higher percentage of expression of the MHC-IIa isoform (46.4 ± 10.3% vs. 37.0 ± 6.0%; P = 0.129), but neither of these differences was statistically significant. In contrast, in chimpanzees, the triceps brachii had a significantly higher percentage of expression of the MHC-IIx isoform than the anconeus (43.2 ± 9.8% vs. 20.8 ± 6.5%; p = 0.006). In addition, in chimpanzees, the percentage of expression of the MHC-I and MHC-IIa isoforms was higher—though not significantly so—in the anconeus than in the triceps brachii (MHC-I: 32.5 ± 4.1% vs. 27.2 ± 8.6%; p = 0.306; MHC-IIa: 46.7 ± 5.2% vs. 29.5 ± 14.4%; p = 0.060). 3.3. Muscle Insertion Sites Chimpanzees had significantly longer ulnae (291.3 ± 27.4 mm vs. 246.8 ± 24.8 mm; p < 0.001) and larger TBIA (531.3 ± 174.1 mm2 vs. 343.3 ± 122.9 mm2 ; p = 0.001) and AIA (615.3 ± 235.9 mm2 vs. 415.1 ± 137.5 mm2 ; p = 0.007). Chimpanzees also had significantly higher NTBIA values (1.84 ± 0.62 vs. 1.38 ± 0.43; p = 0.013) and non-significantly higher NAIA values (2.11 ± 0.80 vs. 1.70 ± 0.60; p = 0.114). 4. Discussion The functional differences between human and chimpanzee elbow extensors and between triceps brachii and anconeus in each of these species have a greater effect on muscle architecture than on the expression of MHC isoforms. The triceps brachii, the largest and strongest arm muscle [7], is the main elbow extensor in both humans and chimpanzees. In chimpanzees, the triceps brachii is involved in the elbow extension that occurs at the end of the swing phase of knuckle-walking [29] and in the elevation of the upper extremity during arboreal locomotion [29,52]. This function explains the higher values for absolute and relative MM (related to power) and PCSA/NPCSA (related to strength) observed in chimpanzees in the present study (Figure 3). The large size of the triceps brachii in chimpanzees [32,33,53,54] mirrors the greater need for generation of strength in the upper extremity of non-human hominoid primates [55] since it is used in arboreal and terrestrial locomotion [32]. In fact, the upper extremity in chimpanzees accounts for 16% of their total body mass, while in humans, who do not generally use the upper limb for locomotion, it accounts for only 9% of total body mass [47]. The larger size of the triceps brachii in chimpanzees may also be a compensation for its relatively shorter lever arm [1]. The olecranon process of the ulna, the only insertion site of the triceps brachii, is shorter in non-human hominoid primates than in humans [5,7,20,56]. This permits the rapid elbow extension that is required during the swing phase of suspen- sory and climbing locomotion [5,7,20]. In addition, in African apes, the short olecranon process allows the hyperextension of the elbow that is necessary to stabilize the elbow dur- ing the support phase of knuckle-walking [1]. In humans, the presence of a relatively short upper limb [5], the lower relative mass of this limb [47], and a relatively long olecranon process are related to the lower participation of this limb in locomotion [32] and its special- ization in manipulating, carrying, or throwing objects [4], including in the manufacture and use of tools [7,55]. This differential use of the upper extremity in humans and chimpanzees can explain the lower values for absolute and relative MM and PCSA/NPCSA (related to power and strength) and the higher values for MFL/NMFL (related to contraction speed) in humans (Figure 3). The functional differences between humans and chimpanzees in the triceps brachii also translate into differences in the expression patterns of the MHC isoforms (Table 2), although these differences are less striking than those in muscle architecture. Our findings on the expression of MHC isoforms at the mRNA level in the human triceps brachii (Table 2) are in line with those of other studies at the protein level using ATPase staining (37.3 ± 15.8% of type-I fibers; 35.9 ± 19.0% of type-IIa fibers; 26.6 ± 13.9% of type-IIx fibers) or SDS-PAGE (32.9 ± 6.1% of MHC-I; 49.6 ± 16.1% MHC-IIa; 17.6 ± 16.5% of MHC- Animals 2022, 12, 2987 11 of 15 IIx) [57]. The expression pattern of MHC isoforms in the triceps brachii of both humans and chimpanzees is characteristic of phasic muscles, with a higher percentage of expression of the rapid MHC-II isoforms (Table 2) [34], which have a large capacity to generate power, a fast contraction speed, and a relatively low resistance to fatigue [40]. However, a higher percentage of expression of the MHC-I and MHC-IIa isoforms (related to resistance to fatigue) was observed in humans than in chimpanzees (Figure 4) although these differences were not significant. The higher percentage of expression of these two isoforms could be related to the prolonged maintenance of flexed elbow postures in humans during object manipulation [58,59]. In contrast, chimpanzees had a significantly higher percentage of expression of the MHC-IIx isoform (related to power [40,41]) (Figure 4), which is consistent with their higher values for MM and PCSA/NPCSA. These differences between humans and chimpanzees in the expression of MHC isoforms in the triceps brachii thus fit well with the different uses that these two species give to their upper extremities [42]. The functional differences between humans and chimpanzees in the triceps brachii, which are related to the larger MM of this muscle in chimpanzees and to its greater participation in arboreal locomotion and terrestrial knuckle-walking—activities involving the generation of force—also translate into osteological differences in its insertion site. A larger muscle insertion site is related to the larger size and the greater activity of the muscle [43,60], which is in line with our finding that the insertion site of the triceps brachii was larger in chimpanzees than in humans, both in terms of the absolute (TBIA) and normalized (NTBIA) values. The function of the anconeus is more complex than that of the triceps brachii. The anconeus has been described as a weak elbow extensor [12,18] as it contributes to only 15% of elbow extension in humans [11], while the triceps brachii is responsible for the remaining 85%. The primary function of the anconeus in humans is to ensure the posterolateral stability of the elbow joint [3,8,15] and to participate in the abduction of the ulna during pronation [9,19]. Although the function of the anconeus in non-human hominoid primates has not been widely studied, considering the anatomical similarity between the human and the chimpanzee anconeus—with a similar origin and insertion, a similar absolute and relative MM (Table 1) and a close relationship between the muscle and the elbow joint capsule—we can postulate that its elbow stabilizing function is also important in chimpanzees. In fact, in contrast with the triceps brachii, the anconeus is not activated during the elbow extension that occurs at the end of the swing phase of knuckle-walking but is activated during the support phase, when it is needed to guarantee the stability of the elbow joint [29]. The lesser participation of the anconeus in the extension of the elbow and its importance as an elbow stabilizing muscle in both humans and chimpanzees could explain the absence of significant differences observed in our study in muscle architecture and in expression of the MHC isoforms (Figures 3 and 4). This functional similarity of the anconeus between humans and chimpanzees can also explain the lack of significant differences in the size of the anconeus insertion site normalized by the length of the ulna. Finally, when the triceps brachii and the anconeus were compared within each species, the different uses of these muscles by chimpanzees in arboreal and terrestrial locomo- tion [29] were reflected in significant differences in muscle architecture, where NPCSA was significantly higher in the triceps brachii and NMFL was significantly higher in the anconeus. The greater involvement of the anconeus in elbow stabilization could also be seen in a higher percentage of expression of the MHC-I and MHC-IIa isoforms, related to resistance to fatigue, while the participation of the triceps brachii in locomotion as an elbow extensor was shown in a significantly higher percentage of expression of the MHC-IIx isoform. In contrast, the greater involvement of the upper limb in manipulative functions in humans [55] was reflected in an absence of significant differences in muscle architecture between the triceps brachii and the anconeus. However, the functional difference between the triceps brachii (mainly as an elbow extensor) and the anconeus (mainly as an elbow stabilizer) was reflected in a higher percentage of expression of the MHC-IIx isoform in the triceps brachii and a higher percentage of expression of the MHC-IIa isoform in the Animals 2022, 12, 2987 12 of 15 anconeus. These differences, while not significant, suggest a greater capacity in the triceps brachii to generate power, related to its extensor function, and a greater resistance to fatigue in the anconeus, related to its stabilizing function [34,40,41]. Our study has several limitations, including the relatively small sample size. Speci- mens of Pan troglodytes are not abundant and a sample of five individuals is a considerable number in comparative anatomy studies of primates. However, our statistical results should be viewed with caution since significance could vary with a larger sample size. Specifically, we believe that some differences between humans and chimpanzees in the expression of the MHC isoforms could well be significant if the sample size were increased. Therefore, we suggest that, if possible, future studies should expand the sample of both humans and chimpanzees and include other species of hominoid primates. Another lim- itation is the advanced age of the human individuals, which is a result of the source of these individuals: body donations to science tend to be older individuals. This could have affected the values of the MM, since humans suffer a loss of MM with age [61], a condition known as sarcopenia. As a result, elderly humans can have 25–35% less MM than younger individuals [62], which may have affected our findings on the differences in the absolute MM of the triceps brachii between humans and chimpanzees. For this reason, we calculated the value of the MM of the triceps brachii and the anconeus relative to the estimated total body mass (Table 1) but found similar results to those obtained with the absolute MM. In addition, the older age of our humans could have affected our results regarding the expression of the MHC isoforms, which is conditioned by age [63]. Although the results we have obtained at the mRNA level are similar to those obtained at the protein level by other authors [57], it would be useful to analyze the expression of MHC isoforms in biopsies obtained from younger patients to see if the results differ from those obtained in our analysis. 5. Conclusions Our analysis of the muscle architecture and the expression of the MHC isoforms have allowed us to explore differences in the elbow extensors between humans and chimpanzees. The greater involvement of the upper extremity in locomotion in chimpanzees is reflected in higher values of the parameters related to generation of power and strength in the triceps brachii (MM, PCSA/NPCSA, %MHC-IIx), while the greater involvement of the upper limb in manipulative functions in humans is reflected in higher values of the parameters related to contraction speed and resistance to fatigue (MFL/NMFL, %MHC-I, %MHC-IIa). In contrast, the main function of the anconeus as an elbow stabilizer in the two species is reflected in similar values in the two species. Despite the limitations regarding sample size and age of our humans, we believe that our findings can help to increase scientific knowledge of the anatomical, molecular and functional characteristics of the elbow extensors in humans and chimpanzees. This muscle group, which has not been widely studied in non-human hominoid primates, is noteworthy due to its participation in the different types of locomotion used by chimpanzees, a species closely related phylogenetically to humans. The fact we have found quantifiable differences between humans and chimpanzees in the insertion site of the triceps brachii which are related to functional differences in this muscle leads us to suggest that similar studies should be carried out in the proximal epiphysis of fossil hominin ulnae [56,64–66]. These analyses could shed light on morphological changes that occurred during the transition from arboreal to terrestrial locomotion and provide information on whether the fossil hominin continued to use arboreal locomotion during the time it transitioned to bipedalism. Furthermore, the fact that we have been able to study muscle architecture, MHC isoforms, and osteological characteristics provides an integrated vision of the anatomy and function of this important muscle group in two highly related species with different functional uses of the upper extremity. Animals 2022, 12, 2987 13 of 15 Author Contributions: Conceptualization, M.d.D. and J.M.P.; methodology, M.d.D., A.C., M.G., N.C., P.R., Y.A., E.C.-T., N.G., I.S.J., M.B., F.d.P., J.F.P. and J.M.P.; software, M.d.D., A.C. and M.G.; validation, M.d.D., A.C., M.G., N.C., P.R., Y.A., E.C.-T., N.G., I.S.J., M.B., F.d.P., J.F.P. and J.M.P.; formal analysis, M.d.D., A.C. and M.G.; investigation, M.d.D., A.C., M.G., N.C., P.R., Y.A., E.C.-T., N.G., I.S.J., M.B., F.d.P., J.F.P. and J.M.P.; resources, J.F.P. and J.M.P.; data curation, M.d.D., J.F.P. and J.M.P.; writing—original draft preparation, M.d.D.; writing—review and editing, M.d.D. and J.M.P.; visualization, M.d.D., A.C., M.G., N.C., P.R., Y.A., E.C.-T., N.G., I.S.J., M.B., F.d.P., J.F.P. and J.M.P.; supervision, J.M.P.; project administration, J.M.P.; funding acquisition, A.C. and J.M.P. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by the Ministerio de Economía y Competitividad of Spain (project CGL2014-52611-C2-2-P to JMP), by the European Union (FEDER project CGL2014-52611-C2-2-P to JMP) and by the Ajudes Predoctorals of the University of Barcelona (APIF-UB 2016/2017 to AC). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of the University of Barcelona (protocol code 00003099, 18 September 2017). Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors thank Manuel Martín (UB) and Manuel J. Taboada (UVa) and Renee Grupp for assistance in drafting the manuscript. In addition, the authors wish to sincerely thank those who donated their bodies to science so that anatomical research could be performed. 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