Introduction: Chronic psychological stressors and overconsumption of highly palatable foods are major risk factors in the development of metabolic, cardiovascular and mood disorders. The energy imbalance instigated by these disorders result in mitochondrial dysfunction over time, reportedly causing reduced infarct tolerance and responsiveness to cardioprotection. The effects of low-level mood and metabolic disturbances on mitochondrial function are however unclear. It is also unclear how combined mood and metabolic disturbances impact cardiac mitochondrial function. This study aims to investigate the effects of subclinical levels of chronic restraint stress (RS) and a Western diet (WD) on cerebral and cardiac mitochondrial function and the expression of proteins associated with mitochondrial function.

Methods: Sixty-four male C57Bl/6 mice were fed a control diet (CD, n = 16; 19% fat, 59% carbohydrates, 19% protein) or a WD (mild dietary stressor, n = 16; 32% fat, 57% carbohydrates, 11% protein) for 16 weeks, with RS (mild psychological stressor; 2 hr restraint/day) introduced in sub-sets of these dietary groups during the final 2 weeks of the study (CD+RS and WD+RS, n = 16 for each group). A subset of type 2 diabetic mice (T2DM, n = 15) from a separate study was also used to compare their mitochondrial function with the WD fed mice. The T2DM mice were injected with streptozotocin (75 mg/kg) and then fed a high fat diet (strong dietary stressor; 43% fat, 39.7% carbohydrates, 17.1% protein) for 16 weeks. Baseline and post-intervention behavioural tests (week 0 and week 17) were done through sucrose preference test (SPT) and open field test (OFT) to quantify anxiety-like and depressive-like outcomes, respectively (not including T2DM mice). After sacrifice, Langendorff perfusions were performed on hearts from all groups, and they were exposed to an ischaemia-reperfusion protocol (I/R) or subjected to ischaemic preconditioning (IPC) prior to I/R to assess post-ischaemic myocardial function responses. Select brain regions and the left ventricular (LV) myocardium were collected at sacrifice, homogenised and loaded into an Oroboros O2k-oxygraph to measure mitochondrial respiration or the tissue was used for western blotting to assess mitochondrial protein expression in the brain and the heart.

Results: The consumption of the WD significantly increased body weight over the 16 weeks of feeding while the introduction of RS induced weight loss in the RS groups. The WD fed and the WD+RS groups had reduced preference for the sucrose solution, 14 suggestive of depressive-like behaviour. Anxiety-like behaviours such as inactivity and wall-seeking were also present in both WD and RS groups in the open field tests. The WD impaired I/R tolerance and worsened post ischaemic cardiac function, while the introduction of IPC enhanced post-ischaemic recovery. The RS groups had improved cardiac function after I/R, but this beneficial effect of RS was lost with IPC. Coronary effluent LDH levels were unchanged in all intervention groups. Across all intervention groups, there was a trend towards a dysfunctional complex I respiration with preserved outer membrane integrity in hearts subjected to I/R. Mitochondrial respiration was unchanged by IPC in the WD hearts while IPC impaired maximal and spare respiratory capacity in the CD+RS hearts. Despite minor alterations in mitochondrial respiration and capacity, enhanced complex II respiration in hearts exposed to I/R and IPC normalised overall OXPHOS capacity (CI+CII-linked respiration) of the cardiac mitochondria, and this was significant in the CD+RS group. Cerebral mitochondrial respiration across all brain regions was impaired in the CD+RS groups but was unaltered with a WD. Both cardiac and cerebral mitochondria from T2DM mice were compared to WD and showed an improvement in all mitochondrial outcomes. Mitochondrial protein abundance studies revealed that the expression of mitochondrial PGC-1𝛼 was positively correlated with GSK-3β in both hearts exposed to I/R and IPC. Both parameters were also linked to decreased mitochondrial ETC complex protein expression. This observation suggests that PGC-1𝛼 and GSK-3β may act together to regulate OXPHOS capacity and the protein expression of the mitochondrial ETC complexes.

Conclusion: Mild alterations in behavioural outcomes and metabolism elicited compensatory adaptations in mitochondrial respiration and proteomic expression to support effective function of the heart and brain. These changes in the mitochondria, and particularly in the heart are seemingly governed, in part, by upstream proteins such as PGC-1𝛼 and GSK-3β, though this mechanism remains unclear and needs further investigation.