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Cytokine release syndrome, also termed "cytokine storm," is the leading cause of morbidity and mortality among patients with various conditions such as sepsis. While cytokine storm is associated with multiple organ damage, acute cardiac and renal injury represents a hallmark of cytokine storm. Since recent reports have suggested that cannabidiol (CBD) may assist in the treatment of inflammatory diseases, our objective was to examine the effect of CBD on cytokine storm-induced cardiac and renal injury using the lipopolysaccharide (LPS)-induced sepsis mouse model. At 8 weeks of age, mice were randomly assigned to receive CBD (15 mg/kg) or vehicle one hour before a single injection of either phosphate-buffered saline or LPS (10 mg/kg) for an additional 24 h. Our results show that CBD improves cardiac function and reduces renal injury in a mouse model of cytokine storm. Moreover, our data indicate that CBD significantly reduces systemic and renal inflammation to contribute to the improvements observed in a cytokine storm-model of cardiac and renal injury. Overall, the findings of this study suggest that CBD could be repurposed to reduce morbidity in patients with cytokine storm particularly in severe infections such as sepsis.

The small lipid-derived paracrine signaling molecules known as prostaglandins have been recognized for their ability to modulate many facets of cardiovascular physiology since their initial discovery more than 85 years ago. Although the role of prostaglandins in the vasculature has gained significant attention across time, a handful of historical studies have also directly implicated the cardiomyocyte in both prostaglandin synthesis and release. Recently, our understanding of how prostaglandin receptor modulation impacts and contributes to myocardial structure and function has gained attention while leaving most other components of myocardial prostaglandin metabolism and signaling unexplored. This mini-review highlights both the key historical studies that underpin modern prostaglandin research in the heart, while concurrently presenting the latest findings related to how prostaglandin metabolism and signaling impact myocardial injury and repair.

Sepsis is a life-threatening condition of organ dysfunction caused by dysregulated inflammation which predisposes patients to developing cardiovascular disease. The ketone β-hydroxybutyrate is reported to be cardioprotective in cardiovascular disease and this may be due to their signaling properties that contribute to reducing inflammation. While exogenous ketone esters (KE) increase blood ketone levels, it remains unknown whether KEs can reduce the enhanced inflammatory response and multi-organ dysfunction that is observed in sepsis. Thus, this study assesses whether a recently developed and clinically safe KE can effectively improve the inflammatory response and organ dysfunction in sepsis.To assess the anti-inflammatory effects of a KE, we utilized a model of lipopolysaccharide (LPS)-induced sepsis in which an enhanced inflammatory response results in multi-organ dysfunction. Oral administration of KE for three days prior to LPS-injection significantly protected mice against the profound systemic inflammation compared to their vehicle-treated counterparts. In assessing organ dysfunction, KE protected mice from sepsis-induced cardiac dysfunction as well as renal dysfunction and fibrosis. Furthermore, KE administration attenuated the sepsis-induced inflammation in the heart, kidney, and liver. Moreover, these protective effects occurred independent of changes to enzymes involved in ketone metabolism.These data show that the use of an exogenous KE attenuates the dysregulated systemic and organ inflammation as well as organ dysfunction in a model of severe inflammation. We postulate that this exogenous KE is an appealing and promising approach to capitalize on the protective anti-inflammatory effects of ketones in sepsis and/or other inflammatory responses.

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance. Abbreviations: BCL2: B cell leukemia/lymphoma 2; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; DNM1L/DRP1: dynamin 1-like; FUNDC1: FUN14 domain containing 1; IRS1: insulin receptor substrate 1; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; MFN1: mitofusin 1; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; OPA1: OPA1 mitochondrial dynamin like GTPase; PDE4i: phosphodiesterase 4 inhibitor; PLD1: phospholipase D1; PLD6: phospholipase D family member 6; PRKA/PKA: protein kinase, AMP-activated; PRKCD/PKCδ: protein kinase C, delta; PRKCQ/PKCθ: protein kinase C, theta; RHEB: Ras homolog enriched in brain; RPS6KB/p70S6K: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; YWHAB/14-3-3β: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta

Doxorubicin (DOX) is an effective anthracycline used in chemotherapeutic regimens for a variety of haematological and solid tumors. However, its utility remains limited by its well-described, but poorly understood cardiotoxicity. Despite numerous studies describing various forms of regulated cell death and their involvement in DOX-mediated cardiotoxicity, the predominate form of cell death remains unclear. Part of this inconsistency lies in a lack of standardization of in vivo and in vitro model design. To this end, the objective of this study was to characterize acute low- and high-dose DOX exposure on cardiac structure and function in C57BL/6N mice, and evaluate regulated cell death pathways and autophagy both in vivo and in cardiomyocyte culture models. Acute low-dose DOX had little impact on cardiac structure or function; however, acute high-dose DOX elicited substantial cardiac necrosis resulting in diminished cardiac mass and volume, with a corresponding reduced cardiac output, and without impacting ejection fraction or fibrosis. Low-dose DOX consistently activated caspase-signaling with evidence of mitochondrial permeability transition. However, acute high-dose DOX had only modest impact on common necrotic signaling pathways, but instead led to an inhibition in autophagic flux. Intriguingly, when autophagy was inhibited in cultured cardiomyoblasts, DOX-induced necrosis was enhanced. Collectively, these observations implicate inhibition of autophagy flux as an important component of the acute necrotic response to DOX, but also suggest that acute high-dose DOX exposure does not recapitulate the disease phenotype observed in human cardiotoxicity.

Cell death is now understood to be a highly regulated process that contributes to normal development and tissue homeostasis, alongside its role in the etiology of various pathological conditions. Through detailed molecular analysis, we have come to know that all cells do not always die in the same way, and that there are at least 7 processes involved, including: apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, and autophagy-mediated cell death. These processes act as pieces in the mosaic of cardiomyocyte cell death, which come together depending on context and stimulus. This review details each individual process, as well as highlights how they come together to produce various cardiac pathologies. By knowing how the pieces go together we can aim towards the development of efficacious therapeutics, which will enable us to prevent cardiomyocyte loss in the face of stress, both reducing mortality and improving quality of life.

Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analogue misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury.

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3 Δ Exon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNC’s) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNC’s, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, promoting nuclear calcium accumulation leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited a hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, without fibrosis which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor Nix is responsive to lipotoxicity and accumulates in response to diacylglycerols induced by high-fat (HF) feeding. In addition, previous studies have implicated autophagy and mitophagy in muscle insulin sensitivity. To provide a better understanding of these observations, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes including Beclin-1, ATG3, and -5. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the previously reported roles of these phospholipids and Nix in mitochondrial dynamics, we investigated aberrant mitochondrial fission and turn-over as a mechanism of myocyte insulin resistance. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that Nix accumulation triggers mitochondrial depolarization, fragmentation, calcium-dependent activation of DRP1, and mitophagy. In addition, Nix-induced mitochondrial fission leads to myotube insulin resistance through activation of mTOR-p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and Rheb. Finally, we demonstrate that Nix-induced mitophagy and insulin resistance can be reversed by direct phosphorylation of Nix by PKA, leading to the translocation of Nix from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of Nix-induced mitophagy and myocyte insulin resistance during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggests a mechanism by which exercise or pharmacological activation of PKA may overcome myocyte insulin resistance.Graphical Abstract

Bnip3 is a hypoxia-inducible initiator of cardiomyocyte cell death and has also been implicated as a mitochondrial receptor for the cellular mitophagy machinery. Previous work from our group demonstrates that the prostaglandin E1 analogue, misoprostol, prevents hypoxia-induced mitochondrial dysfunction and is a potent activator of PKA. We hypothesize that misoprostol alters the phosphorylation status of Bnip3, inhibiting its ability to induce cardiomyocyte mitophagy, mitochondrial dysfunction and cell death. Using a rodent model of neonatal hypoxia, in combination with rat primary ventricular neonatal cardiomyocytes (PVNC’s) and H9c2 cells, we assessed the effect of hypoxia and misoprostol drug treatments on mitochondrial function, mitophagy, and cell viability. In postnatal day 5 rats, hypoxia caused a 30% increase in the concentration of serum cardiac troponin-I, a clinically relevant marker for cardiomyocyte cell death, which was absent with the addition of misoprostol drug treatments (n=3). Using PVNC’s we further demonstrated that hypoxia reduced measures of mitochondrial function including membrane potential (47%) and maximal respiration (46%), which were restored back to control levels with the addition of misoprostol (p

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3ΔExon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNCs) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNCs, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, accumulation of nuclear calcium leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

The cellular response to hypoxia involves the activation of a conserved pathway for gene expression regulated by the transcription factor complex called hypoxia-inducible factor (HIF). This pathway has been implicated in both the adaptive response to hypoxia and in several hypoxic-ischemic related pathologies. Perinatal hypoxic injury, often associated with prematurity, leads to multi-organ dysfunction resulting in significant morbidity and mortality. Using a rodent model of neonatal hypoxia and several representative cell lines, we observed HIF1α activation and down-stream induction of the cell death gene Bnip3 in brain, large intestine, and heart which was mitigated by administration of the prostaglandin E1 analog misoprostol. Mechanistically, we determined that misoprostol inhibits full-length Bnip3 (Bnip3-FL) expression through PKA-mediated NF-κB (P65) nuclear retention, and the induction of pro-survival splice variants. We observed that the dominant small pro-survival variant of Bnip3 in mouse cells lacks the third exon (Bnip3ΔExon3), whereas human cells produce a pro-survival BNIP3 variant lacking exon 2 (BNIP3ΔExon2). In addition, these small Bnip3 splice variants prevent mitochondrial dysfunction, permeability transition, and necrosis triggered by Bnip3-FL by blocking calcium transfer from the sarco/endoplasmic reticulum to the mitochondria. Furthermore, misoprostol and Bnip3ΔExon3 promote nuclear calcium accumulation, resulting in HDAC5 nuclear export, NFAT activation, and adaptive changes in cell morphology and gene expression. Collectively, our data suggests that misoprostol can mitigate the potential damaging effects of hypoxia on multiple cell types by activating adaptive cell survival pathways through Bnip3 repression and alternative splicing.