The bioenergetic “CK Clamp” technique detects substrate-specific changes in mitochondrial respiration and membrane potential during early VML injury pathology

Abstract

Volumetric muscle loss (VML) injuries are characterized by non-recoverable loss of tissue resulting in contractile and metabolic dysfunction. The characterization of metabolic dysfunction in volumetric muscle loss-injured muscle has been interpreted from permeabilized myofiber respiration experiments involving saturating ADP levels and non-physiologic ATP:ADP concentration ratios. The extent to which this testing condition obscures the analysis of mitochondrial (dys) function after volumetric muscle loss injury is unclear. An alternative approach is described that leverages the enzymatic reaction of creatine kinase and phosphocreatine to assess mitochondrial respiration and membrane potential at clamped physiologic ATP:ADP ratios, “CK Clamp.” The objective of this study was to validate the CK Clamp in volumetric muscle loss-injured muscle and to detect differences that may exist between volumetric muscle loss-injured and uninjured muscles at 1, 3, 5, 7, 10, and 14 days post-injury. Volumetric muscle loss-injured muscle maintains bioenergetic features of the CK Clamp approach, i.e., mitochondrial respiration rate (JO2) titters down and mitochondrial membrane potential is more polarized with increasing ATP:ADP ratios. Pyruvate/malate/succinate-supported JO2 was significantly less in volumetric muscle loss-injured muscle at all timepoints compared to uninjured controls (−26% to −84%, p < 0.001) and electron conductance was less at day 1 (−60%), 5 (−52%), 7 (−35%), 10 (−59%), and 14 (−41%) (p < 0.001). Palmitoyl-carnitine/malate-supported JO2 and electron conductance were less affected following volumetric muscle loss injury. volumetric muscle loss-injury also corresponded with a more polarized mitochondrial membrane potential across the clamped ATP:ADP ratios at day 1 and 10 (pyruvate and palmitoyl-carnitine, respectively) (+5%, p < 0.001). This study supports previous characterizations of metabolic dysfunction and validates the CK Clamp as a tool to investigate bioenergetics in traumatically-injured muscle.

Differential effects of Western diet and traumatic muscle injury on skeletal muscle metabolic regulation in male and female mice

“…Mitochondrial oxygen consumption ( J O 2 ) was assessed over a physiological range of ATP re‐synthesis demands ( Figure 3 A , D , G , J ) using the CK energetic clamp technique previously validated in VML‐injured fibre bundles. 24 In general, VML injury negatively affected carbohydrate‐ and fat‐supported mitochondrial J O 2 ( Figure 3 B , E , H , K ) in agreement with a previous acute VML study. 24 This VML‐induced phenotype was present in both males and females; however, in male mice, there was evidence of a combined effect of diet and injury on carbohydrate‐supported mitochondrial function.…”

“… 24 In general, VML injury negatively affected carbohydrate‐ and fat‐supported mitochondrial J O 2 ( Figure 3 B , E , H , K ) in agreement with a previous acute VML study. 24 This VML‐induced phenotype was present in both males and females; however, in male mice, there was evidence of a combined effect of diet and injury on carbohydrate‐supported mitochondrial function. Notably, maximal J O 2 was 48% lower in VML + WD compared to VML + NC ( Figure 3 B ; P = 0.014), and electron conductance was 47% less in VML + WD compared to VML + NC ( Figure 3 C ; P = 0.026).…”

“… 7 This aligns with findings of lower carbohydrate‐supported mitochondrial respiration in permeabilized fibre bundles from VML‐injured limbs. 24 The current study revealed that WD exacerbated the vulnerability to impaired carbohydrate‐supported mitochondrial respiration caused by VML injury, particularly in male mice. Several non‐mutually exclusive mechanisms may contribute to this primary finding.…”

“…High‐resolution oxygen respiration measurement was conducted on gastrocnemius permeabilized fibre bundles using an Oroboros Oxygraph‐2K (Oroboros Instruments, Innsbruck, Austria) as previously described 23 , 24 with minor adjustments. Experiments were carried out at 30°C in buffer Z (105‐mM K‐MES, 30‐mM KCl, 1‐mM EGTA, 10‐mM K 2 HPO 4 , 5‐mM MgCl and 0.5‐mg/mL bovine serum albumin; pH 7.1) with 20‐mM creatine monohydrate.…”

Resistance wheel running improves contractile strength, but not metabolic capacity, in a murine model of volumetric muscle loss injury

“…High‐resolution oxygen respiration measurement was conducted on gastrocnemius samples using an Oroboros Oxygraph‐2K (Oroboros Instruments, Innsbruck, Austria) and the creatine kinase (CK) clamp technique as previously described (McFalin et al., 2023). Briefly, mitochondrial respiration ($${J_{{{\mathrm{O}}_{\mathrm{2}}}}}$$) and electron conductance were tested by using a CK clamp system wherein the enzymatic reaction of CK and phosphocreatine (PCr) is leveraged to manipulate the levels of cellular energy demand (Gibbs free energy; Δ G ATP ) (Fisher‐Wellman et al., 2018).…”