In this work, employing for the first time a newly generated systemic mitochondrial CB₁ deletion murine model, we explored the multifaceted consequences of the suppression of mtCB₁ expression, primarily impacting muscle force, muscle mitochondrial morphology, respiration, membrane potential, as well as calcium levels.

We replicated the genetic engineering strategy described by Hebert-Chatelaine in 2016 [39], which is aimed at interfering with mitochondrial targeting by specifically disrupting only the mitochondrial pool of CB₁Rs while leaving plasma membrane CB₁Rs intact (Figure 1A). The strategy involves removing 63 nucleotides (21 amino acids) that have been identified as being responsible for the mitochondrial targeting of the receptor. First, to confirm that mtCB₁Rs were indeed absent in our genetically engineered model, we employed high-resolution STED microscopy, a technique that allowed us to precisely pinpoint the subcellular locations of two separate CB₁R populations in skeletal muscles (Figure 1B,C). The noticeable rightward shift in the special antibody-labeled CB₁ receptor fluorescence frequency distribution histogram (Figure 1E) strongly inferred that the mitochondrial CB₁R pool had been successfully suppressed in the mtCB₁^−/− specimens, while expression of pCB₁Rs remained chiefly unaltered.

Motor function and coordination were assessed through various tests, and suppression of mtCB₁ expression did not affect any of the measured parameters, including grip strength, Rota-Rod latency to fall, or wire hang ability (Figure 2). These data suggest that deletion of mtCB₁ does not modify these capabilities. On the other hand, the global CB₁ knockout mice were described to show hypoactivity, reduced exploratory behavior, and increased immobility in motor tests, which was correlated with reduced motivation for goal-directed behaviors [58], arguing that motivation is most likely not dependent on mtCB₁R, but is rather linked to the plasma membrane subpopulation of CB₁R.

To get a deeper insight into the role of mtCB₁ on skeletal muscle physiology, we analyzed the in vitro force production in the EDL and SOL muscles of both genotypes. Assessment of tetani and twitches revealed significantly smaller specific normalized force values in the mtCB₁^−/− specimens (Figure 3A–H, Table 1 and Table 2). Further scrutiny of the measured parameters unveiled that the EDL muscles from the mtCB₁^−/− mice exhibited signs of hypertrophy as they were significantly heavier than those from wild-type mice (Table 1), and their cross-sectional area was also significantly greater. This suggests a compensatory mechanism or a dysregulation of muscle growth pathways in the absence of mtCB₁ (not investigated in this work), even though the specific force is reduced. In the literature, there are clues related to notable enhancements in muscle size and related growth processes following genetic knockout of the CB₁Rs in skeletal muscles [36]. Furthermore, embryonic and postnatal CB₁ knockout mice display a greater number of muscle fibers and an increase in muscle fiber diameter compared to wild-type controls [25]. The possible molecular mechanisms behind this process could be (1) enhanced AKT signaling, which could promote anabolic processes; (2) upregulated mTOR activity, which is crucial for muscle protein synthesis; (3) reduced myostatin expression, which is a negative regulator of muscle growth; (4) increased IL-6 secretion, a myokine associated with muscle regeneration and growth. On the other hand, there were no significant differences in contraction kinetics (TTP, HRT, duration). Nevertheless, the force–frequency analysis revealed faster fatigue at higher frequencies in the mtCB₁^−/− specimens after reaching the maximum force output upon stimulation (80 Hz for EDL and 60 Hz for SOL, Figure 3I,J). Noteworthy, in our earlier work, we showed that the muscle-specific inducible CB₁ knockdown mice (acute and incomplete CB₁ ablation) displayed altered in vivo and in vitro force generation. The latter displayed similar trends to those described here (see Figure 3C from Singlár et al. 2022 [37]). A change in muscle fiber composition was recently documented by Senese et al. [32]; namely, a decrease in MyoFast (MHCIIb) protein levels and a simultaneous increase in MyoSlow (MHCIb) in the CB₁ global knock-out model (chronic deletion) was seen. Gonzalez-Mariscal et al. had a similar observation in a conditional skeletal muscle-specific CB₁^−/− mice [36]. The authors drew the conclusion that the observed changes resulted in enhanced physical performance and an overall improvement in whole-body metabolism in mice. Importantly, our data strongly argue that lack of the mitochondrial (and not the plasma membrane) subpopulation of CB₁R may play a pivotal role in this improvement.

Our in vitro force measurement data suggest that a mitochondria-expressed subset of CB₁Rs plays a crucial role in regulating muscle strength and possibly muscle size, particularly in fast-twitch fibers. The reduced specific force despite increased size in EDL is a particularly interesting finding, indicating a deficit in the quality or efficiency of force production rather than simply a reduction in muscle mass. Our findings seem to correlate well with earlier data done in cells and mice, showing that the cannabinoid system might orchestrate muscle growth [22]. Furthermore, CB₁ is known to influence mTOR and AMPK signaling (both known signaling pathways that regulate autophagy) and muscle growth [59]. Whether the increased muscle mass observed in the case of EDL muscles is a direct effect of the suppression of mtCB₁R or develops secondarily to compensate for the lower normalized force generation of said muscles remains to be elucidated in a follow-up study.

Studies have indicated that the endocannabinoid system can regulate both the integrity and functionality of mitochondria, thereby contributing to the maintenance of cellular energy homeostasis [19,60]. The TEM image analysis we performed here strongly suggests that suppression of mtCB₁ expression leads to significant morphological abnormalities in muscle mitochondria (Figure 4). Thus, we believe our TEM analysis provides a cellular basis for the observed functional deficits during force measurements. The enlarged mitochondria may contribute to the overall increase in muscle cell volume, or they could be a compensatory hypertrophy where the muscle tries to grow larger to compensate for its functional weakness, even if the individual contractile units are less efficient due to mitochondrial issues. Another aspect worth mentioning is that presumably, the enlarged, swollen, or dysfunctional mitochondria may be less efficient at producing ATP. Moreover, it has been shown that mitochondria become elongated and interconnected in neurons of adult CB₁R^−/− animals, most likely due to an impairment in mitophagy, leading to a significant decline in memory function [61]. Because we observed similar phenomena in the muscles of mtCB₁^−/− animals (i.e., increased mitochondrial area and perimeter; Figure 4), our data favor the idea that the mitochondrial (and not the plasma membrane) subset of CB₁ may be responsible for maintaining “mitochondrial health”.

The presence of CB₁ receptors in the mitochondrial outer membrane allows for a direct, fine-tuned regulation of mitochondrial function, including respiration [31,39,43,44]. Since muscle contraction is highly ATP-dependent, the impaired mitochondrial function could directly contribute to reduced force generation. Hence, we set out to assess mitochondrial respiration, which could indirectly provide clues about cellular ATP production. The respirometry data revealed that Complex IV activity is significantly impaired in muscles of adult mtCB₁^−/− animals. Similarly, even with maximal substrate input for both main entry points into the electron transport chain (i.e., Complex I & II), the overall capacity for ATP production is reduced in the mtCB₁^−/− muscles (Figure 5). Moreover, we also observed reduced overall capacity of the electron transport chain itself in mtCB₁^−/− mitochondria, independent of ATP synthesis coupling. This suggests a problem with the respiratory complexes or their efficiency. The reduced respiratory capacity, despite the possibly larger size of mitochondria (seen in TA muscles, Figure 4), suggests that the enlarged mitochondria are dysfunctional, rather than hyper-functional. Their larger size might be a compensatory response to reduced efficiency or a consequence of impaired mitochondrial dynamics (e.g., fusion without proper fission). Earlier studies have found that in global CB₁ knockout mice, there is an increased oxidative capacity in muscle [32] and liver [62], paired with altered mitochondrial dynamics and mitochondrial quality control. Nevertheless, the impact of a CB₁ knockout on mitochondrial respiration can vary depending on the tissue and the specific experimental setup.

Generally, acute activation of mtCB₁ was shown to decrease mitochondrial respiration (e.g., by inhibiting Complex I) [31,39,43,44], implicating that mtCB₁R-dependent endogenous cannabinoid signaling may protect mitochondria from being “overused” and hence subsequently damaged, e.g., due to increased long-term production of reactive oxygen species [63]. The comprehensive mitochondrial dysfunction identified here may serve as the direct explanation for the previously documented decline in specific force (muscle weakness) across both EDL and SOL muscles in mtCB₁^−/− mice. The substantial ATP demand of muscle contraction means that any impairment in mitochondrial ATP synthesis would directly result in a reduction of contractile force.

To further explore the exact nature of the above mitochondrial dysfunction, we also demonstrated that mitochondrial membrane potential stability is impaired in mtCB₁^−/− muscles (Figure 6). Indeed, mitochondria in mtCB₁^−/− muscle fibers depolarize more rapidly and/or are less able to maintain their membrane potential compared to mtCB₁^+/+. A stable and high Δψ_m is the driving force for ATP production via ATP synthase. The faster depolarization and reduced ability to maintain Δψ_m may directly explain the previously observed reduced oxidative phosphorylation (OXPHOS) capacity and reduced specific force (muscle weakness) in mtCB₁^−/− muscles. If the proton gradient cannot be maintained effectively, ATP synthesis will be compromised, leading to insufficient energy for muscle contraction. The respirometry data with ADP and oligomycin (Figure 5) suggest that the observed depolarization in isolated fibers may be related to processes that consume the proton gradient (like ATP synthesis or even reverse ATPase activity if ATP accumulates or there’s an energy deficit). The faster depolarization in the mtCB₁^−/− suggests a greater susceptibility to these processes, or an intrinsic instability of their inner mitochondrial membrane, or perhaps could be linked to the impaired Complex IV activity.

Mitochondrial calcium is a critical regulator of mitochondrial metabolism (e.g., activating enzymes in the Krebs cycle and oxidative phosphorylation). Moreover, mitochondrial calcium contributes to cellular calcium homeostasis and signaling [3,57]. Altered mitochondrial calcium could impact various calcium-dependent cellular processes, including neurotransmission, muscle contraction, and cell death pathways [64,65,66]. Our present findings suggest that mtCB₁Rs play a role in regulating or buffering mitochondrial calcium uptake (Figure 7). Specifically, the suppression of mtCB₁R expression led to an altered basal mitochondrial calcium distribution within muscle fibers, characterized by a higher F_mito value, implying a more spread-out or less aggregated mitochondrial network. However, acute muscle activity (tetanic stimulation) did not appear to significantly alter this mitochondrial distribution of the dye fluorescence, at least in our current experimental conditions. Our present data suggest that the suppression of mitochondrial CB₁R expression may lead to an increase in mitochondrial calcium level by trend under resting conditions. This trend seemed to be maintained throughout the stimulation-induced fluorescence changes. We believe this data could suggest that mtCB₁Rs possibly act to reduce or at least modulate mitochondrial calcium accumulation in skeletal muscle mitochondria, highlighting their potential role in mitochondrial calcium homeostasis.