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Prolonged exposure to heat can lead to environment‐induced heat stress (EIHS), which may be a threat to human health. How EIHS affects cardiac morphology and the myocardium are unknown. We hypothesized that EIHS would alter cardiac morphology and cause cellular dysfunction. To test this hypothesis, crossbred female pigs were exposed to thermoneutral (TN; 20.6 ± 0.2 ºC; n=8) or EIHS (37.4 ± 0.2 ºC; n=8) conditions for 24 h. Rectal temperature (RT), skin temperature (ST), and respiratory rate (RR) were recorded every 4 h during the environmental challenge. Pigs were euthanized following the environmental challenge and hearts were collected. Hearts were weighed and heart length (apex to base), width (left/right dimension), and left ventricle (LV) and right ventricle (RV) wall thickness were measured with calipers. Portions of LV and RV were lyophilized to measure tissue water content or homogenized for protein extraction and western blotting. Environment‐induced heat stress increased RT by 1.3 ºC (p<0.01), ST by 11 °C (p<0.01) and RR by 72 breaths per minute (p<0.01) compared to TN. Heart weight tended to be decreased (7.6%; p=0.07) and heart length was decreased (8.5%; p=0.01) by EIHS, but heart width was similar between groups. Left ventricle wall thickness was increased (22%; p=0.02) and RV thickness was decreased (26%; p=0.04) in EIHS compared to TN. Water content in the RV was similar between groups, however, in LV it was increased (8.6%; p<0.01) in EIHS compared to TN, suggesting edema contributed to increased LV thickness. Lastly, using a western blot approach we discovered that pathways regulating energy homeostasis were impacted by EIHS and sometimes differed between RV and LV. In RV and LV, phosphorylation (p) of AMP‐activated protein kinase (AMPK) was decreased by EIHS (RV – 73%, p<0.02; LV – 54%, p=0.04), and in the RV was accompanied by increased protein phosphate type 2A (PP2A; 15.5%, p<0.01), which regulates pAMPK, and decreased p‐Acetyl‐CoA carboxylase (ACC; 40%, p<0.01), a client protein of pAMPK, whereas these were similar in LV. In RV, mTOR signaling appeared to be blunted, mitochondrial content increased, and markers of mitophagy increased by EIHS compared to TN, however, LV was resistant to these changes. In total, these data demonstrate that a single bout of EIHS caused cardiac morphological changes and biochemical changes in the myocardium and that EIHS affects the LV and RV differently.

Heat stress (HS) negatively impacts animal health and impairs growth, but little is known about sex‐specific responses of skeletal muscle mitochondria to HS. To test the hypothesis that HS would have more adverse effects on mitochondria in females than males, samples were collected from the oxidative portion of the semitendinosus muscle of 3‐month‐old female and castrated male pigs under thermoneutral (TN) conditions or after 1 or 7 d of HS at 39.4°C (n=8/sex/group). Mitochondrial volume density and function were determined via citrate synthase (CS) and cytochrome c oxidase activities. Mitochondrial oxidative phosphorylation (P) and electron transfer (E) capacities were evaluated by high resolution respirometry. Data were analyzed using mixed linear models in SAS v9.4 with treatment, sex, and treatment × sex as fixed effects. Overall, integrative (relative to tissue weight) maximal P with complex I and II (PCI+II) and E (ECI+II) were greater after 1 and 7 d of HS compared to TN conditions (P≤0.05). The contribution of PCI+II to maximal E (flux control ratio; FCRPCI+II) was greater in TN pigs compared to either HS group (P≤0.03), suggesting decreased efficiency of mitochondrial energy production following HS. However, there appeared to be some adaptation to heat over time as FCRPCI+IIwas greater in pigs that were heat stressed for 7 d compared to 1 d (P=0.05). As hypothesized, some measures differed by sex. Specifically, in females, intrinsic (relative to CS activity) complex I‐supported P (PCI) tended to be greater following 7 d of HS compared to TN (P=0.07); PCI was unaffected by heat in males, resulting in females having greater intrinsic PCI than males following 7 d of HS (P=0.05). The contribution of PCI to total E (FCRPCI) was lesser with 7 d HS than 1 d or TN conditions in male pigs (P≤0.01) but unchanged in females, resulting in females also having greater FCRPCIthan males following 7 d of HS (P=0.008). Conversely, the contribution of electron transfer supported by complex II to total E (FCRECII) was greater in TN compared to 1 d of HS in males (P=0.004) but then increased at 7 d of HS (P=0.02) so that TN and 7 d HS were similar. In females, FCRECII tended to be lesser following 7 d of HS compared to TN, resulting in a trend for males to have greater FCRECII than females after 7 d of HS (P=0.08). Finally, males tended to have greater FCRPCI+IIthan females (P≤0.08). These results suggest HS increased mitochondrial utilization of complex I in females but reliance on complex II in males, resulting in more efficient mitochondria following HS in males than in females. Therefore, skeletal muscle in females may be more susceptible to negative cellular impacts of HS.

With continued trends toward more frequent and more severe heat events there is a greater risk of environment‐induced heat stress (HS). How persistently elevated core temperatures impacts body tissues remains largely unknown. Our preliminary evidence indicates HS may negatively impact cardiac tissue and that biological sex may play a role in HS‐mediated pathology. To further test the hypothesis that cardiac muscle mitochondria are more negatively impacted by HS in females than in males, samples were isolated from the left ventricle (LV) of the heart from 3‐month‐old castrated male and prepubertal female pigs that were exposed to thermoneutral (TN) conditions (n = 4 per sex) or HS conditions for 24 h at 37°C (HS; n = 6 per sex). Samples were analyzed for citrate synthase (CS) and cytochrome c oxidase (COX) activities, and for mitochondrial oxidative phosphorylation (P) and electron transfer (E) capacities via high resolution respirometry. Data were analyzed by mixed linear models in SAS v9.4 with treatment, sex, and treatment × sex as fixed effects. A traditional marker of mitochondrial volume density, CS activity, tended to be greater in TN males compared to HS males (P = 0.08) but was unaffected by HS in female pigs. Conversely, intrinsic (relative to CS activity) mitochondrial function, as measured by COX activity, was greater in TN females than HS females (P = 0.04) and within HS, males maintained greater intrinsic mitochondrial function than females (P = 0.02). Both integrative (relative to tissue wet weight) and intrinsic mitochondrial leak respiration were greater in HS than TN pigs (P = 0.03) but the contribution of leak to total E (flux control ratio; FCRLEAK) tended to be greater in males than females (P = 0.07). Conversely, both integrative and intrinsic maximal E (ECI+II) tended to be greater in female than male pigs (P = 0.08) indicating a greater degree of excess electron transfer capacity in females. The contribution of complex I‐supported P (PCI) to total E (FCRPCI) tended to be greater in TN compared to HS pigs regardless of sex (P = 0.09) and the contribution of maximal P to E (FCRPCI+II) was greater in TN females compared to HS females (P = 0.05). This resulted in the FCRPCI+IIbeing greater in males compared to females in the HS condition (P = 0.01), suggesting greater coupling of oxidative phosphorylation and electron transfer in cardiac mitochondria in males during 24 h of HS. These results suggest that HS may negatively impact cardiac muscle mitochondria in female pigs compared to males. Moreover, these data indicate that persistent HS has deleterious consequences for the myocardium and may represent an additional health concern caused by global climate trends. This project was supported by USDA NIFA Award #2020‐68014‐31954.