Increased basal metabolic rate in mice susceptible to malignant hyperthermia and heat stroke

Ryanodine receptor type-1 (RYR1) and Calsequestrin-1 (CASQ1) proteins, located in the sarcoplasmic reticulum (SR), are two of the main players in skeletal excitation–contraction (EC) coupling. Mutations in the human RYR1 gene (encoding for the SR Ca2+ release channel) and ablation in mice of CASQ1 (a SR Ca2+ binding protein) cause hypersensitivity to halogenated anesthetics (malignant hyperthermia [MH] susceptibility) and to heat (heat stroke; HS). As both MH and HS are characterized by excessive cytosolic Ca2+ levels and hypermetabolic responses, we studied the metabolism of 4-mo-old mice from two different lines that are MH/HS susceptible: knock-in mice carrying a human MH mutation (RYR1YS) and CASQ1-knockout (ko) mice. RYR1YS and, to a lesser degree, CASQ1-null mice show an increased volume of oxygen consumption (VO2) and a lower respiratory quotient (RQ) compared with WT mice (indicative of a metabolism that relies more on lipids). This finding is accompanied by a reduction in total body fat mass in both Y522S and CASQ1-null mice (again, compared with WT). In addition, we found that RYR1YS and CASQ1-null mice have an increased food consumption (+26.04% and +25.58% grams/day, respectively) and higher basal core temperature (+0.57°C and +0.54°C, respectively) compared with WT mice. Finally, Western blots and electron microscopy indicated that, in hyperthermic mice, (1) SERCA (used to remove myoplasmic Ca2+) and UCP3 (responsible for a thermogenic process that dissipates mitochondrial H+ gradient) are overexpressed, and (2) mitochondrial volume and percentage of damaged mitochondria are both increased. In conclusion, the MH/HS phenotype in RYR1YS and CASQ1-null mice is associated with an intrinsically increased basal metabolism.

241 Skeletal Muscle Mitochondrial Capacities Are Impacted by Breed and Temperament in Young Angus and Brahman Steers

Temperament impacts skeletal muscle mitochondria in Brahman heifers, but this has not been investigated in steers or between cattle breeds. We hypothesized mitochondrial measures would be greater in Angus than Brahman, temperamental than calm steers, and the trapezius (TRAP) than the longissimus thoracis (LT) muscle. Samples from calm (n = 13 per breed), intermediate (n = 12 per breed), and temperamental (n=13 per breed) Angus and Brahman steers (mean±SD 10.0±0.8 mo) were evaluated for mitochondrial enzyme activities via colorimetry. Calm and temperamental LT samples were evaluated for oxidative phosphorylation (P) and electron transfer (E) capacities by high-resolution respirometry. Data were analyzed using linear models with fixed effects of breed, muscle, temperament, and all interactions. Brahman tended to have greater mitochondrial volume density (citrate synthase activity; CS) than Angus (P = 0.08), while intrinsic (relative to CS) mitochondrial function (cytochrome c oxidase activity) was greater in Angus than Brahman (P = 0.001) and greater in TRAP than LT (P = 0.008). Angus exhibited greater integrative (per mg tissue) and intrinsic P with complex I (PCI), P with complexes I+II (PCI+II), maximum noncoupled E, and E with complex II (ECII; P ≤ 0.04) and tended to have greater intrinsic leak (P = 0.1) than Brahman. Contribution of PCI to total E was greater in Angus than Brahman (P = 0.01), while contribution of ECII to total E was greater in Brahman than Angus (P = 0.05). A trend for the interaction of breed and temperament (P = 0.07) indicated calm Angus had the greatest intrinsic ECII (P ≤ 0.03) while intrinsic ECII was similar between temperamental Angus and calm and temperamental Brahman. Integrative PCI+II and ECII, and the contribution of PCI and PCI+II to overall E tended to be greater in temperamental than calm steers (P ≤ 0.09), while intrinsic ECII tended to be greater in calm than temperamental steers (P = 0.07). The impact of these mitochondrial differences on meat quality measures remains to be determined.

507 Late-Breaking: Heat Stress and Mitoq Supplementation Impact Skeletal Muscle Mitochondrial Capacities in Pigs

Heat stress can negatively impact pig health and performance but the effects of heat stress on skeletal muscle mitochondrial function are largely unknown. We hypothesized that mitochondrial function and capacity would be impaired in heat stressed (HS) compared to thermoneutral (TN) pigs but mitochondrially-targeted coenzyme Q (MitoQ) supplementation would rescue the impairment. Oxidative portions of the semitendinosus muscle were evaluated from TN and HS gilts receiving no supplementation (CON) or MitoQ for 2 d prior to and during the 24h environmental heat treatment (n = 8 per group). Mitochondrial oxidative phosphorylation (P) and electron transfer (E) capacities were determined via high resolution respirometry and mitochondrial volume density and function were quantified by citrate synthase (CS) and cytochrome c oxidase activities, respectively. Data were analyzed using linear models in SAS v9.4 with fixed effects of heat, MitoQ treatment (trt), and heat×trt interaction. There were trends for the interaction of trt and heat (P≤0.1) on integrative (per mg tissue) and intrinsic (relative to CS) P with complexes I and II (PCI+II), maximum noncoupled E (ECI+II), and E with complex II only (ECII), in which all measures were greater in HS-MitoQ than TN-MitoQ (P≤0.03), but measures did not differ due to HS in CON pigs. The contribution of leak to total E (flux control ratio, FCRLeak) was lesser in HS-MitoQ than HS-CON, TN-CON, and TN-MitoQ (P≤0.02). The FCRPCI was greater (P≤0.05) while the FCRPCI+II was lesser (P=0.01) in TN compared to HS pigs. Finally, the FCRPCI+II was greater (P=0.02) while the FCRECII tended to be lesser (P=0.09) for CON than MitoQ pigs. Neither mitochondrial volume density nor function were affected by HS or MitoQ supplementation. In total, these data indicate improved mitochondrial capacities following heat stress in pigs receiving MitoQ but no difference in mitochondrial capacities in unsupplemented, HS pigs.

Skeletal muscle bioenergetic health and function in people living with HIV: association with glucose tolerance and alcohol use

At-risk alcohol use is prevalent and increases dysglycemia among people living with human immunodeficiency virus (PLWH). Skeletal muscle (SKM) bioenergetic dysregulation is implicated in dysglycemia and type 2 diabetes. The objective of this study was to determine the relationship between at-risk alcohol, glucose tolerance, and SKM bioenergetic function in PLWH. Thirty-five PLWH (11 females, 24 males, age: 53±9 yrs, body mass index: 29.0±6.6 kg/m2) with elevated fasting glucose enrolled in the ALIVE-Ex study provided medical history and alcohol use information (Alcohol Use Disorders Identification Test, AUDIT), then underwent an oral glucose tolerance test (OGTT) and SKM biopsy. Bioenergetic health and function and mitochondrial volume were measured in isolated myoblasts. Mitochondrial gene expression was measured in SKM. Linear regression adjusting for age, sex, and smoking was performed to examine the relationship between glucose tolerance (2-h glucose post-OGTT), AUDIT, and their interaction with each outcome measure. Negative indicators of bioenergetic health were significantly (p<0.05) greater with higher 2-h glucose (proton leak) and AUDIT (proton leak, non-mitochondrial oxygen consumption, and bioenergetic health index). Mitochondrial volume was increased with the interaction of higher 2-h glucose and AUDIT. Mitochondrial gene expression decreased with higher 2-h glucose (TFAM, PGC1B, PPARG, MFN1), AUDIT (MFN1, DRP1, MFF), and their interaction (PPARG, PPARD, MFF). Decreased expression of mitochondrial genes were coupled with increased mitochondrial volume and decreased bioenergetic health in SKM of PLWH with higher AUDIT and 2-h glucose. We hypothesize these mechanisms reflect poorer mitochondrial health and may precede overt SKM bioenergetic dysregulation observed in type 2 diabetes.

Mitochondrial Fragmentation and Dysfunction in Type IIx/IIb Diaphragm Muscle Fibers in 24-Month Old Fischer 344 Rats

Sarcopenia is characterized by muscle fiber atrophy and weakness, which may be associated with mitochondrial fragmentation and dysfunction. Mitochondrial remodeling and biogenesis in muscle fibers occurs in response to exercise and increased muscle activity. However, the adaptability mitochondria may decrease with age. The diaphragm muscle (DIAm) sustains breathing, via recruitment of fatigue-resistant type I and IIa fibers. More fatigable, type IIx/IIb DIAm fibers are infrequently recruited during airway protective and expulsive behaviors. DIAm sarcopenia is restricted to the atrophy of type IIx/IIb fibers, which impairs higher force airway protective and expulsive behaviors. The aerobic capacity to generate ATP within muscle fibers depends on the volume and intrinsic respiratory capacity of mitochondria. In the present study, mitochondria in type-identified DIAm fibers were labeled using MitoTracker Green and imaged in 3-D using confocal microscopy. Mitochondrial volume density was higher in type I and IIa DIAm fibers compared with type IIx/IIb fibers. Mitochondrial volume density did not change with age in type I and IIa fibers but was reduced in type IIx/IIb fibers in 24-month rats. Furthermore, mitochondria were more fragmented in type IIx/IIb compared with type I and IIa fibers, and worsened in 24-month rats. The maximum respiratory capacity of mitochondria in DIAm fibers was determined using a quantitative histochemical technique to measure the maximum velocity of the succinate dehydrogenase reaction (SDHmax). SDHmax per fiber volume was higher in type I and IIa DIAm fibers and did not change with age. In contrast, SDHmax per fiber volume decreased with age in type IIx/IIb DIAm fibers. There were two distinct clusters for SDHmax per fiber volume and mitochondrial volume density, one comprising type I and IIa fibers and the second comprising type IIx/IIb fibers. The separation of these clusters increased with aging. There was also a clear relation between SDHmax per mitochondrial volume and the extent of mitochondrial fragmentation. The results show that DIAm sarcopenia is restricted to type IIx/IIb DIAm fibers and related to reduced mitochondrial volume, mitochondrial fragmentation and reduced SDHmax per fiber volume.

Presynaptic Mitochondrial Volume and Density Scale with Presynaptic Power Demand

Stable neural function requires an energy supply that can meet the intense episodic power demands of neuronal activity. The bioenergetic machinery of glycolysis and oxidative phosphorylation is highly responsive to such demands, but it must occupy a minimum volume if it is to accommodate these demands. We examined the trade-off between presynaptic power demands and the volume available to the bioenergetic machinery. We quantified the energy demands of six Drosophila motor nerve terminals through direct measurements of neurotransmitter release and Ca2+ entry, and via theoretical estimates of Na+ entry and power demands at rest. Electron microscopy revealed that terminals with the highest power demands contained the greatest volume of mitochondria, indicating that mitochondria are allocated according to presynaptic power demands. In addition, terminals with the greatest power demand-to-volume ratio (~66 nmol/min/uL) harbor the largest mitochondria packed at the greatest density. If we assume sequential and complete oxidation of glucose by glycolysis and oxidative phosphorylation, then these mitochondria are required to produce ATP at a rate of 52 nmol/min/uL at rest, rising to 963 during activity. Glycolysis would contribute ATP at 0.24 nmol/min/uL of cytosol at rest, rising to 4.36. These data provide a quantitative framework for presynaptic bioenergetics in situ, and reveal that, beyond an immediate capacity to accelerate ATP output from glycolysis and oxidative phosphorylation, over longer time periods presynaptic terminals optimize mitochondrial volume and density to meet power demand.

Mitochondrial K+ Transport: Modulation and Functional Consequences

The existence of a K+ cycle in mitochondria has been predicted since the development of the chemiosmotic theory and has been shown to be crucial for several cellular phenomena, including regulation of mitochondrial volume and redox state. One of the pathways known to participate in K+ cycling is the ATP-sensitive K+ channel, MitoKATP. This channel was vastly studied for promoting protection against ischemia reperfusion when pharmacologically activated, although its molecular identity remained unknown for decades. The recent molecular characterization of MitoKATP has opened new possibilities for modulation of this channel as a mechanism to control cellular processes. Here, we discuss different strategies to control MitoKATP activity and consider how these could be used as tools to regulate metabolism and cellular events.