Defining the role of endotoxemia during heat stress in oxidative skeletal muscle

Abstract

Heat stress (HS) is a multifaceted stress, negatively impacting the skeletal muscle, reproductive, and gastrointestinal systems, among others. Endotoxemia occurs during HS and originates from structural changes to the gastrointestinal system, making it permissive for leakage of typically impermeable bacterial lipopolysaccharides (LPS). In this investigation, to better understand the complexities of HS‐mediated changes in skeletal muscle we aimed to identify contributions of endotoxemia to changes in skeletal muscle that occur during HS. Pigs (44 ± 2.6 kg) were assigned to 1 of 2 treatment groups: control (CON; 2mL saline injection; n=6) or LPS (40 µg/ kg BW; Escherichia coli O55:B5; n=6). Injections were given intramuscularly in the mid‐gluteal/ superficial gluteal muscles. All pigs were individually housed with restricted access to food and water 2 h prior to and throughout the 6 h experimental period. Pigs were euthanized 6 h post injection, and the oxidative (STR) portion of the semitendinosus muscle was collected for analysis. Injection with LPS decreased total circulating Ca2+ (30%, p=0.04), but increased skeletal muscle Ca2+ content (55%, p=0.03). Within STR, LPS decreased SERCA2a (28%, p=0.032) and Phospholamban (PLB; 59%, p=0.05) protein abundance but increased PLB bound to SERCA2 (100%, p=0.052), which antagonizes SERCA2a function. These data suggest decreased capacity of Ca2+ clearance, which can cause alterations in mitochondrial membrane potential, as mitochondria serve as secondary Ca2+ storage reservoirs. The change in mitochondrial membrane potential caused by the influx of Ca2+ can alter proteins associated with mitochondrial clearance via mitophagy. Consistent with this notion, LPS treatment decreased Parkin (52%, p=0.0002) however, increased BNIP3L protein abundance (25%, p=0.005), which may support a PINK1/Parkin‐independent mode of mitophagy. As a result, markers of mitochondrial abundance were decreased (NDUFB8 – 43%, p=0.0035; COX IV – 39%, p=0.0358; SOD2 – 44%, p=0.009) by LPS. Notably, these outcomes are distinct from previous reports in skeletal muscle following 6 h of HS indicating endotoxemia, alone, is not sufficient to drive changes associated with HS in skeletal muscle.