CGRP as a potential mediator for the sexually dimorphic responses to traumatic brain injury.

BACKGROUND: The outcomes of traumatic brain injury (TBI) exhibit variance contingent upon biological sex. Although female sex hormones exert neuroprotective effects, the administration of estrogen and progesterone has not yielded conclusive results. Hence, it is conceivable that additional mediators, distinct from female sex hormones, merit consideration due to their potential differential impact on TBI outcomes. Calcitonin gene-related peptide (CGRP) exhibits sexually dimorphic expression and demonstrates neuroprotective effects in acute brain injuries. In this study, we aimed to examine sex-based variations in TBI structural and functional outcomes with respect to CGRP expression. METHODS: Male and female Sprague Dawley rats were exposed to controlled cortical impact to induce severe TBI, followed by interventions with and without CGRP inhibition. In the acute phase of TBI, the study centered on elucidating the influence of CGRP on oxidative stress, nuclear factor erythroid 2-related factor 2 (Nrf2) and endothelial nitric oxide synthase (eNOS) signaling in the peri-impact tissue. Subsequently, during the chronic phase of TBI, the investigation expanded to evaluate CGRP expression in relation to lesion volume, microvascular dysfunction, and white matter injury, as well as working and spatial memory, anxiety-like, and depression-like behaviors in subjects of both sexes. RESULTS: Female rats exhibited elevated levels of CGRP in the peri-impact brain tissue during both baseline conditions and in the acute and chronic phases of TBI, in comparison to age-matched male counterparts. Enhanced CGRP levels in specific brain sub-regions among female rats correlated with superior structural and functional outcomes following TBI compared to their male counterparts. CGRP inhibition induced heightened oxidative stress and a reduction in the expression of Nrf2 and eNOS in both male and female rats, with the observed alteration being more pronounced in females than in males. CONCLUSIONS: This study marks the inaugural identification of CGRP as a downstream mediator contributing to the sexually dimorphic response observed in TBI outcomes.

Investigating sex disparities in traumatic brain injury (TBI) is crucial for the advancement of precision therapeutics. Despite the neuroprotective effects demonstrated by female sex hormones, the administration of estrogen and progesterone has not produced conclusive results. Therefore, it is conceivable that additional mediators, separate from female sex hormones, warrant consideration due to their potential differential influence on TBI outcomes. In this study, we examined sex-related variations in calcitonin gene-related peptide (CGRP) expression in peri-impact brain tissue and investigated its potential implications on associated TBI outcomes. CGRP exhibits sexually dimorphic expression and exerts a multifaceted influence on diverse physiological processes that contribute to the pathophysiology of TBI. Our findings reveal that female rats exhibit heightened CGRP levels at both baseline and post-TBI within specific brain sub-regions, thereby contributing to superior structural and functional outcomes compared to their age-matched male counterparts. Additionally, we identified substantial sex-based variations in mechanisms modulated by CGRP pertaining to oxidative stress and microvascular dysfunction. The disparities in CGRP levels may be crucial for comprehending the advantageous outcomes noted in female TBI. Therefore, elucidating the sex-related distinctions in CGRP within TBI brains could pave the way for improved management and treatment strategies for TBI in both male and female individuals.

Intrinsic diving reflex induces potent antioxidative response by activation of NRF2 signaling.

Aims: This study aims to elucidate the underlying mechanisms of diving reflex, a powerful endogenous mechanism supporting underwater mammalian survival. Antioxidative responses, observed in marine mammals, may be contributing factors. Using a multi-organ approach, this study assesses whether acute and chronic diving reflex activate nuclear factor-erythroid-2-related factor 2 (NRF2) signaling pathways, which regulate cellular antioxidant responses. Methods: Male Sprague-Dawley rats ( n =38) underwent either a single diving session to elicit acute diving reflex, or daily diving sessions for 4-weeks to produce chronic diving reflex. NRF2 (total, nuclear, phosphorylated), NRF2-downstream genes, and malondialdehyde were assessed via Western blot, immunofluorescence, RT-PCR, and ELISA in brain, lung, kidney, and serum. Results: Diving reflex increased nuclear NRF2, phosphorylated NRF2, and antioxidative gene expression, in an organ-specific and exposure time-specific manner. Comparing organs, the brain had the highest increase of phosphorylated NRF2 expression, while kidney had the highest degree of nuclear NRF2 expression. Comparing acute and chronic sessions, phosphorylated NRF2 increased the most with chronic diving reflex, but acute diving reflex had the highest antioxidative gene expression. Notably, calcitonin gene-related peptide appears to mediate diving reflex' effects on NRF2 activation. Conclusions: Acute and chronic diving reflex activate potent NRF2 signaling in the brain and peripheral organs. Interestingly, acute diving reflex induces higher expression of downstream antioxidative genes compared to chronic diving reflex. This result contradicts previous assumptions requiring chronic exposure to diving for induction of antioxidative effects and implies that the diving reflex has a strong translational potential during preconditioning and postconditioning therapies. Key Points: Diving reflex activates potent NRF2 signaling via multiple mechanisms, including phosphorylation, nuclear translocation, and KEAP1 downregulation with both acute and chronic exposure.Diving reflex activates NRF2 via differential pathways in the brain and other organs; phosphorylated NRF2 increases more in the brain, while nuclear NRF2 increases more in the peripheral organs.Acute diving reflex exposure induces a more pronounced antioxidative effect than chronic diving reflex exposure, indicating that the antioxidative response activated by diving reflex is not dependent upon chronic adaptive responses and supports diving reflex as both a preconditioning and postconditioning treatment.

Trigeminal nerve stimulation: a current state-of-the-art review.

Nearly 5 decades ago, the effect of trigeminal nerve stimulation (TNS) on cerebral blood flow was observed for the first time. This implication directly led to further investigations and TNS' success as a therapeutic intervention. Possessing unique connections with key brain and brainstem regions, TNS has been observed to modulate cerebral vasodilation, brain metabolism, cerebral autoregulation, cerebral and systemic inflammation, and the autonomic nervous system. The unique range of effects make it a prime therapeutic modality and have led to its clinical usage in chronic conditions such as migraine, prolonged disorders of consciousness, and depression. This review aims to present a comprehensive overview of TNS research and its broader therapeutic potentialities. For the purpose of this review, PubMed and Google Scholar were searched from inception to August 28, 2023 to identify a total of 89 relevant studies, both clinical and pre-clinical. TNS harnesses the release of vasoactive neuropeptides, modulation of neurotransmission, and direct action upon the autonomic nervous system to generate a suite of powerful multitarget therapeutic effects. While TNS has been applied clinically to chronic pathological conditions, these powerful effects have recently shown great potential in a number of acute/traumatic pathologies. However, there are still key mechanistic and methodologic knowledge gaps to be solved to make TNS a viable therapeutic option in wider clinical settings. These include bimodal or paradoxical effects and mechanisms, questions regarding its safety in acute/traumatic conditions, the development of more selective stimulation methods to avoid potential maladaptive effects, and its connection to the diving reflex, a trigeminally-mediated protective endogenous reflex. The address of these questions could overcome the current limitations and allow TNS to be applied therapeutically to an innumerable number of pathologies, such that it now stands at the precipice of becoming a ground-breaking therapeutic modality.

Neurobehavioral Impairments Predict Specific Cerebral Damage in Rat Model of Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a severe form of stroke that can cause unpredictable and diffuse cerebral damage, which is difficult to detect until it becomes irreversible. Therefore, there is a need for a reliable method to identify dysfunctional regions and initiate treatment before permanent damage occurs. Neurobehavioral assessments have been suggested as a possible tool to detect and approximately localize dysfunctional cerebral regions. In this study, we hypothesized that a neurobehavioral assessment battery could be a sensitive and specific method for detecting damage in discrete cerebral regions following SAH. To test this hypothesis, a behavioral battery was employed at multiple time points after SAH induced via an endovascular perforation, and brain damage was confirmed via postmortem histopathological analysis. Our results demonstrate that impairment of sensorimotor function accurately predict damage in the cerebral cortex (AUC 0.905; sensitivity 81.8%; specificity 90.9%) and striatum (AUC 0.913; sensitivity 90.1%; specificity 100%), while impaired novel object recognition is a more accurate indicator of damage to the hippocampus (AUC 0.902; sensitivity 74.1%; specificity 83.3%) than impaired reference memory (AUC 0.746; sensitivity 72.2%; specificity 58.0%). Tests for anxiety-like and depression-like behaviors predict damage to the amygdala (AUC 0.900; sensitivity 77.0%; specificity 81.7%) and thalamus (AUC 0.963; sensitivity 86.3%; specificity 87.8%), respectively. This study suggests that recurring behavioral testing can accurately predict damage in specific brain regions, which could be developed into a clinical battery for early detection of SAH damage in humans, potentially improving early treatment and outcomes.

Nonpharmacological modulation of cortical spreading depolarization.

AIMS: Cortical spreading depolarization (CSD) is a wave of pathologic neuronal dysfunction that spreads through cerebral gray matter, causing neurologic disturbance in migraine and promoting lesion development in acute brain injury. Pharmacologic interventions have been found to be effective in migraine with aura, but their efficacy in acutely injured brains may be limited. This necessitates the assessment of possible adjunctive treatments, such as nonpharmacologic methods. This review aims to summarize currently available nonpharmacological techniques for modulating CSDs, present their mechanisms of action, and provide insight and future directions for CSD treatment. MAIN METHODS: A systematic literature review was performed, generating 22 articles across 3 decades. Relevant data is broken down according to method of treatment. KEY FINDINGS: Both pharmacologic and nonpharmacologic interventions can mitigate the pathological impact of CSDs via shared molecular mechanisms, including modulating K+/Ca2+/Na+/Cl- ion channels and NMDA, GABAA, serotonin, and CGRP ligand-based receptors and decreasing microglial activation. Preclinical evidence suggests that nonpharmacologic interventions, including neuromodulation, physical exercise, therapeutic hypothermia, and lifestyle changes can also target unique mechanisms, such as increasing adrenergic tone and myelination and modulating membrane fluidity, which may lend broader modulatory effects. Collectively, these mechanisms increase the electrical initiation threshold, increase CSD latency, slow CSD velocity, and decrease CSD amplitude and duration. SIGNIFICANCE: Given the harmful consequences of CSDs, limitations of current pharmacological interventions to inhibit CSDs in acutely injured brains, and translational potentials of nonpharmacologic interventions to modulate CSDs, further assessment of nonpharmacologic modalities and their mechanisms to mitigate CSD-related neurologic dysfunction is warranted.

Neurobehavioral impairments predict specific cerebral damage in rat model of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a severe form of stroke that can cause unpredictable and diffuse cerebral damage, which is difficult to detect until it becomes irreversible. Therefore, there is a need for a reliable method to identify dysfunctional regions and initiate treatment before permanent damage occurs. Neurobehavioral assessments have been suggested as a possible tool to detect and approximately localize dysfunctional cerebral regions. In this study, we hypothesized that a neurobehavioral assessment battery could be a sensitive and specific early warning for damage in discrete cerebral regions following SAH. To test this hypothesis, a behavioral battery was employed at multiple time points after SAH induced via an endovascular perforation, and brain damage was confirmed via postmortem histopathological analysis. Our results demonstrate that impairment of sensorimotor function accurately predict damage in the cerebral cortex (AUC: 0.905; sensitivity: 81.8%; specificity: 90.9%) and striatum (AUC: 0.913; sensitivity: 90.1%; specificity: 100%), while impaired novel object recognition is a more accurate indicator of damage to the hippocampus (AUC: 0.902; sensitivity: 74.1%; specificity: 83.3%) than impaired reference memory (AUC: 0.746; sensitivity: 72.2%; specificity: 58.0%). Tests for anxiety-like and depression-like behaviors predict damage to the amygdala (AUC: 0.900; sensitivity: 77.0%; specificity: 81.7%) and thalamus (AUC: 0.963; sensitivity: 86.3%; specificity: 87.8%), respectively. This study suggests that recurring behavioral testing can accurately predict damage in specific brain regions, which could be developed into a clinical battery for early detection of SAH damage in humans, potentially improving early treatment and outcomes.

Trigeminal Nerve Stimulation Improves Cerebral Macrocirculation and Microcirculation After Subarachnoid Hemorrhage: An Exploratory Study.

BACKGROUND: Delayed cerebral ischemia (DCI) is the most consequential secondary insult after aneurysmal subarachnoid hemorrhage (SAH). It is a multifactorial process caused by a combination of large artery vasospasm and microcirculatory dysregulation. Despite numerous efforts, no effective therapeutic strategies are available to prevent DCI. The trigeminal nerve richly innervates cerebral blood vessels and releases a host of vasoactive agents upon stimulation. As such, electrical trigeminal nerve stimulation (TNS) has the capability of enhancing cerebral circulation. OBJECTIVE: To determine whether TNS can restore impaired cerebral macrocirculation and microcirculation in an experimental rat model of SAH. METHODS: The animals were randomly assigned to sham-operated, SAH-control, and SAH-TNS groups. SAH was induced by endovascular perforation on Day 0, followed by KCl-induced cortical spreading depolarization on day 1, and sample collection on day 2. TNS was delivered on day 1. Multiple end points were assessed including cerebral vasospasm, microvascular spasm, microthrombosis, calcitonin gene-related peptide and intercellular adhesion molecule-1 concentrations, degree of cerebral ischemia and apoptosis, and neurobehavioral outcomes. RESULTS: SAH resulted in significant vasoconstriction in both major cerebral vessels and cortical pial arterioles. Compared with the SAH-control group, TNS increased lumen diameters of the internal carotid artery, middle cerebral artery, and anterior cerebral artery, and decreased pial arteriolar wall thickness. Additionally, TNS increased cerebrospinal fluid calcitonin gene-related peptide levels, and decreased cortical intercellular adhesion molecule-1 expression, parenchymal microthrombi formation, ischemia-induced hypoxic injury, cellular apoptosis, and neurobehavioral deficits. CONCLUSION: Our results suggest that TNS can enhance cerebral circulation at multiple levels, lessen the impact of cerebral ischemia, and ameliorate the consequences of DCI after SAH.

The Potential Role of Neuromodulation in Subarachnoid Hemorrhage.

OBJECTIVES: Aneurysmal subarachnoid hemorrhage (SAH) continues to be a difficult cerebrovascular disease with limited pharmacologic treatment options. Cerebral vasospasm (CV) and delayed cerebral ischemia (DCI) are leading causes of morbidity and mortality after SAH. Despite the advances in the understanding of its pathophysiology and tremendous efforts to date, nimodipine is currently the sole Food and Drug Administration-approved treatment for patients with SAH, with benefits that are marginal at best. The neuromodulation therapies are promising, especially those that target CV and DCI to improve functional outcomes. The aim of this review is therefore to summarize the available evidence for each type of neuromodulation for CV and DCI, with a special focus on its pathophysiological mechanisms, in addition to their clinical utility and drawbacks, which we hope will lead to future translational therapy options after SAH. MATERIALS AND METHODS: We conducted a comprehensive review of preclinical and clinical studies demonstrating the use of neuromodulation for SAH. The literature search was performed using PubMed, Embase, and ClinicalTrials.gov. A total of 21 articles published from 1992 to 2021 and eight clinical trials were chosen. RESULTS: The studies reviewed provide a compelling demonstration that neuromodulation is a potentially useful strategy to target multiple mechanisms of DCI and thus to potentially improve functional outcomes from SAH. There are several types of neuromodulation that have been tested to treat CV and DCI, including the trigeminal/vagus/facial nerve stimulation, sphenopalatine ganglion and spinal cord stimulation, transcranial direct electrical stimulation, transcutaneous electrical neurostimulation, and electroacupuncture. Most of them are in the preclinical or early phases of clinical application; however, they show promising results. CONCLUSIONS: DCI has a complex pathogenesis, making the unique anatomical distribution and pleiotropic capabilities of various types of neuromodulation a promising field of study. We may be at the cusp of a breakthrough in the use of these techniques for the treatment of this stubbornly difficult disease.

CBF oscillations induced by trigeminal nerve stimulation protect the pericontusional penumbra in traumatic brain injury complicated by hemorrhagic shock.

Traumatic peri-contusional penumbra represents crucial targets for therapeutic interventions after traumatic brain injury (TBI). Current resuscitative approaches may not adequately alleviate impaired cerebral microcirculation and, hence, compromise oxygen delivery to peri-contusional areas. Low-frequency oscillations in cerebral blood flow (CBF) may improve cerebral oxygenation in the setting of oxygen deprivation. However, no method has been reported to induce controllable oscillations in CBF and it hasn't been applied as a therapeutic strategy. Electrical stimulation of the trigeminal nerve (TNS) plays a pivotal role in modulating cerebrovascular tone and cerebral perfusion. We hypothesized that TNS can modulate CBF at the targeted frequency band via the trigemino-cerebrovascular network, and TNS-induced CBF oscillations would improve cerebral oxygenation in peri-contusional areas. In a rat model of TBI complicated by hemorrhagic shock, TNS-induced CBF oscillations conferred significant preservation of peri-contusional tissues leading to reduced lesion volume, attenuated hypoxic injury and neuroinflammation, increased eNOS expression, improved neurological recovery and better 10-day survival rate, despite not significantly increasing CBF as compared with those in immediate and delayed resuscitation animals. Our findings indicate that low-frequency CBF oscillations enhance cerebral oxygenation in peri-contusional areas, and play a more significant protective role than improvements in non-oscillatory cerebral perfusion or volume expansion alone.

Trigeminal Nerve Control of Cerebral Blood Flow: A Brief Review.

The trigeminal nerve, the fifth cranial nerve, is known to innervate much of the cerebral arterial vasculature and significantly contributes to the control of cerebrovascular tone in both healthy and diseased states. Previous studies have demonstrated that stimulation of the trigeminal nerve (TNS) increases cerebral blood flow (CBF) via antidromic, trigemino-parasympathetic, and other central pathways. Despite some previous reports on the role of the trigeminal nerve and its control of CBF, there are only a few studies that investigate the effects of TNS on disorders of cerebral perfusion (i.e., ischemic stroke, subarachnoid hemorrhage, and traumatic brain injury). In this mini review, we present the current knowledge regarding the mechanisms of trigeminal nerve control of CBF, the anatomic underpinnings for targeted treatment, and potential clinical applications of TNS, with a focus on the treatment of impaired cerebral perfusion.

Percutaneous Trigeminal Nerve Stimulation Induces Cerebral Vasodilation in a Dose-Dependent Manner.

BACKGROUND: The trigeminal nerve directly innervates key vascular structures both centrally and peripherally. Centrally, it is known to innervate the brainstem and cavernous sinus, whereas peripherally the trigemino-cerebrovascular network innervates the majority of the cerebral vasculature. Upon stimulation, it permits direct modulation of cerebral blood flow (CBF), making the trigeminal nerve a promising target for the management of cerebral vasospasm. However, trigeminally mediated cerebral vasodilation has not been applied to the treatment of vasospasm. OBJECTIVE: To determine the effect of percutaneous electrical stimulation of the infraorbital branch of the trigeminal nerve (pTNS) on the cerebral vasculature. METHODS: In order to determine the stimulus-response function of pTNS on cerebral vasodilation, CBF, arterial blood pressure, cerebrovascular resistance, intracranial pressure, cerebral perfusion pressure, cerebrospinal fluid calcitonin gene-related peptide (CGRP) concentrations, and the diameter of cerebral vessels were measured in healthy and subarachnoid hemorrhage (SAH) rats. RESULTS: The present study demonstrates, for the first time, that pTNS increases brain CGRP concentrations in a dose-dependent manner, thereby producing controllable cerebral vasodilation. This vasodilatory response appears to be independent of the pressor response induced by pTNS, as it is maintained even after transection of the spinal cord at the C5-C6 level and shown to be confined to the infraorbital nerve by administration of lidocaine or destroying it. Furthermore, such pTNS-induced vasodilatory response of cerebral vessels is retained after SAH-induced vasospasm. CONCLUSION: Our study demonstrates that pTNS is a promising vasodilator and increases CBF, cerebral perfusion, and CGRP concentration both in normal and vasoconstrictive conditions.

Electrical Stimulation of the Infraorbital Nerve Induces Diving Reflex in a Dose-Controlled Manner.

The "diving reflex" (DR) is a very powerful autonomic reflex that facilitates survival in hypoxic/anoxic conditions and could trigger multifaceted physiologic effects for the treatment of various diseases by modulating the cardiovascular, respiratory, and nervous systems. The DR can be induced by cold water or noxious gases applied to the anterior nasal mucosa and paranasal regions, which can stimulate trigeminal thermo- or chemo-receptors to send afferent signals to medullary nuclei which mediate the sympathetic and parasympathetic nervous systems. Although promising, these approaches have yet to be adopted in routine clinical practice due to the inability to precisely control exposure-response relationships, lack of reproducibility, and difficulty implementing in a clinical setting. In this study, we present the ability of electrical Trigeminal (Infraorbital) Nerve Stimulation (eTINS) to induce the DR in a dose-controllable manner. We found that eTINS not only triggered specific physiological changes compatible with the pattern of "classic" DR observed in animals/humans, but also controlled the induced-DR at varying levels. This study demonstrates, for the first time, that the intensity of the DR is controllable by dose and opens possibility to investigate its protective mechanism against various pathologies in well-controlled research settings.

Trigeminal Nerve Stimulation: A Novel Method of Resuscitation for Hemorrhagic Shock.

OBJECTIVES: To determine if trigeminal nerve stimulation can ameliorate the consequences of acute blood loss and improve survival after severe hemorrhagic shock. DESIGN: Animal study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Severe hemorrhagic shock was induced in rats by withdrawing blood until the mean arterial blood pressure reached 27 ± 1 mm Hg for the first 5 minutes and then maintained at 27 ± 2 mm Hg for 30 minutes. The rats were randomly assigned to either control, vehicle, or trigeminal nerve stimulation treatment groups. The effects of trigeminal nerve stimulation on survival rate, autonomic nervous system activity, hemodynamics, brain perfusion, catecholamine release, and systemic inflammation after severe hemorrhagic shock in the absence of fluid resuscitation were analyzed. MEASUREMENTS AND MAIN RESULTS: Trigeminal nerve stimulation significantly increased the short-term survival of rats following severe hemorrhagic shock in the absence of fluid resuscitation. The survival rate at 60 minutes was 90% in trigeminal nerve stimulation treatment group whereas 0% in control group (p < 0.001). Trigeminal nerve stimulation elicited strong synergistic coactivation of the sympathetic and parasympathetic nervous system as measured by heart rate variability. Without volume expansion with fluid resuscitation, trigeminal nerve stimulation significantly attenuated sympathetic hyperactivity paralleled by increase in parasympathetic tone, delayed hemodynamic decompensation, and improved brain perfusion following severe hemorrhagic shock. Furthermore, trigeminal nerve stimulation generated sympathetically mediated low-frequency oscillatory patterns of systemic blood pressure associated with an increased tolerance to central hypovolemia and increased levels of circulating norepinephrine levels. Trigeminal nerve stimulation also decreased systemic inflammation compared with the vehicle. CONCLUSIONS: Trigeminal nerve stimulation was explored as a novel resuscitation strategy in an animal model of hemorrhagic shock. The results of this study showed that the stimulation of trigeminal nerve modulates both sympathetic and parasympathetic nervous system activity to activate an endogenous pressor response, improve cerebral perfusion, and decrease inflammation, thereby improving survival.

Neuromodulation as a new avenue for resuscitation in hemorrhagic shock.

Hemorrhagic shock (HS), a major cause of early death from trauma, accounts for around 40% of mortality, with 33-56% of these deaths occurring before the patient reaches a medical facility. Intravenous fluid therapy and blood transfusions are the cornerstone of treating HS. However, these options may not be available soon after the injury, resulting in death or a poorer quality of survival. Therefore, new strategies are needed to manage HS patients before they can receive definitive care. Recently, various forms of neuromodulation have been investigated as possible supplementary treatments for HS in the prehospital phase of care. Here, we provide an overview of neuromodulation methods that show promise to treat HS, such as vagus nerve stimulation, electroacupuncture, trigeminal nerve stimulation, and phrenic nerve stimulation and outline their possible mechanisms in the treatment of HS. Although all of these approaches are only validated in the preclinical models of HS and are yet to be translated to clinical settings, they clearly represent a paradigm shift in the way that this deadly condition is managed in the future.

Upregulation of proteasome activity rescues cardiomyocytes following pulse treatment with a proteasome inhibitor.

This study examined the hypothesis that cardiomyocyte metabolism is inherently linked to the Ubiquitin Proteasome System. Rat neonatal ventricular cardiomyocytes were pulse-treated with 5 αM lactacystin for 30 min, resulting in 95% loss of proteasome activity, and then maintained in culture for up to 24 h. Pulse-treatment resulted in 36% decrease in cardiomyocyte mitochondrial reductase activity by 8 h which improved to 15% by 24 h. Bax proteins were increased 2.5-fold by 8 h but declined by 16 h. Similar effects were observed for ubiquitinated proteins suggesting recovery of proteasome function. Proteasome activity started to increase by 4 h and was back to baseline by 16 h. Multiple proteasome subunits, including α1, were upregulated with peak 2 to 2.5-fold increased protein levels at 8-16 h post-lactacystin which then declined. Incubating cardiomyocytes with 4 αM morpholino-antisense oligonucleotides to the α1-subunit for up to 24 h post-lactacystin diminished recovery of proteasome activity (45% at 24 h) and prevented the increase in α1 protein levels. Ubiquitinated proteins remained elevated and cardiomyocyte mitochondrial reductase activity was decreased 35% by 16 h. These results show that diminished function of the ubiquitin proteasome system decreases cardiomyocyte metabolism. If proteasome activity recovers, function improves, but preventing recovery diminishes metabolic function supporting the hypothesis that cardiomyocyte metabolism is inherently linked to the ubiquitin proteasome system.