The obesity-autophagy-cancer axis: Mechanistic insights and therapeutic perspectives.

Autophagy, a self-degradative process vital for cellular homeostasis, plays a significant role in adipose tissue metabolism and tumorigenesis. This review aims to elucidate the complex interplay between autophagy, obesity, and cancer development, with a specific emphasis on how obesity-driven changes affect the regulation of autophagy and subsequent implications for cancer risk. The burgeoning epidemic of obesity underscores the relevance of this research, particularly given the established links between obesity, autophagy, and various cancers. Our exploration delves into hormonal influence, notably INS (insulin) and LEP (leptin), on obesity and autophagy interactions. Further, we draw attention to the latest findings on molecular factors linking obesity to cancer, including hormonal changes, altered metabolism, and secretory autophagy. We posit that targeting autophagy modulation may offer a potent therapeutic approach for obesity-associated cancer, pointing to promising advancements in nanocarrier-based targeted therapies for autophagy modulation. However, we also recognize the challenges inherent to these approaches, particularly concerning their precision, control, and the dual roles autophagy can play in cancer. Future research directions include identifying novel biomarkers, refining targeted therapies, and harmonizing these approaches with precision medicine principles, thereby contributing to a more personalized, effective treatment paradigm for obesity-mediated cancer.

Transforming Growth Factor Beta and Alveolar Rhabdomyosarcoma: A Challenge of Tumor Differentiation and Chemotherapy Response.

Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-ß). This overexpression of TGF-ß1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-ß also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-ß in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-ß1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.

Differential impact of doxorubicin dose on cell death and autophagy pathways during acute cardiotoxicity.

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/6 N mice, and evaluate regulated cell death pathways and autophagy both in vivo and in cardiomyocyte culture models. Acute low-dose DOX had no significant 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.

Tafazzin deficiency impairs mitochondrial metabolism and function of lipopolysaccharide activated B lymphocytes in mice.

B lymphocytes are responsible for humoral immunity and play a key role in the immune response. Optimal mitochondrial function is required to support B cell activity during activation. We examined how deficiency of tafazzin, a cardiolipin remodeling enzyme required for mitochondrial function, alters the metabolic activity of B cells and their response to activation by lipopolysaccharide in mice. B cells were isolated from 3-month-old wild type or tafazzin knockdown mice and incubated for up to 72 h with lipopolysaccharide and cell proliferation, expression of cell surface markers, secretion of antibodies and chemokines, proteasome and immunoproteasome activities, and metabolic function determined. In addition, proteomic analysis was performed to identify altered levels of proteins involved in survival, immunogenic, proteasomal and mitochondrial processes. Compared to wild type lipopolysaccharide activated B cells, lipopolysaccharide activated tafazzin knockdown B cells exhibited significantly reduced proliferation, lowered expression of cluster of differentiation 86 and cluster of differentiation 69 surface markers, reduced secretion of immunoglobulin M antibody, reduced secretion of keratinocytes-derived chemokine and macrophage-inflammatory protein-2, reduced proteasome and immunoproteasome activities, and reduced mitochondrial respiration and glycolysis. Proteomic analysis revealed significant alterations in key protein targets that regulate cell survival, immunogenicity, proteasomal processing and mitochondrial function consistent with the findings of the above functional studies. The results indicate that the cardiolipin transacylase enzyme tafazzin plays a key role in regulating mouse B cell function and metabolic activity during activation through modulation of mitochondrial function.

A new trick for an old dog? Myocardial-specific roles for prostaglandins as mediators of ischemic injury and repair.

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.

Misoprostol attenuates neonatal cardiomyocyte proliferation through Bnip3, perinuclear calcium signaling, and inhibition of glycolysis.

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.

Intrauterine exposure to diabetes and risk of cardiovascular disease in adolescence and early adulthood: a population-based birth cohort study.

BACKGROUND: It is unclear whether intrauterine exposure to maternal diabetes is associated with risk factors for cardiovascular disease and related end points in adulthood. We examined this potential association in a population-based birth cohort followed up to age 35 years. METHODS: We performed a cohort study of offspring born between 1979 and 2005 (n = 293 546) and followed until March 2015 in Manitoba, Canada, using registry-based administrative data. The primary exposures were intrauterine exposure to gestational diabetes and type 2 diabetes mellitus. The primary outcome was a composite measure of incident cardiovascular disease events, and the secondary outcome was a composite of risk factors for cardiovascular disease in offspring followed up to age 35 years. RESULTS: The cohort provided 3 628 576 person-years of data (mean age at latest follow-up 20.5 [standard deviation 6.4] years, 49.3% female); 2765 (0.9%) of the offspring experienced a cardiovascular disease end point, and 12 673 (4.3%) experienced a cardiovascular disease risk factor. After propensity score matching, the hazard for cardiovascular disease end points was elevated in offspring exposed to gestational diabetes (adjusted hazard ratio [HR] 1.42, 95% confidence interval [CI] 1.12-1.79) but not type 2 diabetes (adjusted HR 1.40, 95% CI 0.98-2.01). A similar association was observed for cardiovascular disease risk factors (gestational diabetes: adjusted HR 1.92, 95% CI 1.75-2.11; type 2 diabetes: adjusted HR 3.40, 95% CI 3.00-3.85). INTERPRETATION: Intrauterine exposure to maternal diabetes was associated with higher morbidity and risk related to cardiovascular disease among offspring up to 35 years of age.

Cardiac structure and function in youth with type 2 diabetes in the iCARE cohort study: Cross-sectional associations with prenatal exposure to diabetes and metabolomic profiles.

OBJECTIVE: This study aimed to determine the degree of left ventricular (LV) dysfunction and its determinants in adolescents with type 2 diabetes (T2D). We hypothesized that adolescents with T2D would display impaired LV diastolic function and that these cardiovascular complications would be exacerbated in youth exposed to maternal diabetes in utero. METHODS: Left ventricular structure and function, carotid artery intima media thickness and strain, and serum metabolomic profiles were compared between adolescents with T2D (n = 121) and controls (n = 34). Sub-group analyses examined the role of exposure to maternal diabetes as a determinant of LV or carotid artery structure and function among adolescents with T2D. RESULTS: Adolescents with T2D were 15.1 ± 2.5 years old, (65% female, 99% Indigenous), had lived with diabetes for 2.7 ± 2.2 years, had suboptimal glycemic control (HbA1c = 9.4 ± 2.6%) and 58% (n = 69) were exposed to diabetes in utero. Compared to controls, adolescents with T2D displayed lower LV diastolic filling (early diastole/atrial filling rate ratio [E/A] = 1.9 ± 0.6 vs 2.2 ± 0.6, P = 0.012), lower LV relaxation and carotid strain (0.12 ± 0.05 vs 0.17 ± 0.05, P = .03) and elevated levels of leucine, isoleucine and valine. Among adolescents with T2D, exposure to diabetes in utero was not associated with differences in LV diastolic filling, LV relaxation, carotid strain or branched chain amino acids. CONCLUSIONS: Adolescents with T2D display LV diastolic dysfunction, carotid artery stiffness, and elevated levels of select branch chain amino acids; differences were not associated with exposure to maternal diabetes in utero.

Regulation of cardiac myocyte cell death and differentiation by myocardin.

Myocardin is a cardiac- and smooth muscle-enriched transcriptional co-activator that was originally described as an interacting partner of the serum response factor. Shortly after myocardin's discovery, a wealth of published literature described the role of myocardin as a regulator of smooth muscle differentiation and phenotype modulation, while gene-targeting studies confirmed the essential role of myocardin in vascular development. More recently, myocardin has been implicated as an important regulator of cardiac myocyte differentiation in studies demonstrating direct programming of fibroblasts towards the cardiac lineage. This function of myocardin has been attributed to its physical interaction with cardiac-enriched transcription factors such as MEF2C, GATA4, and TBX5. Moreover, conditional knockout models have revealed a critical role for myocardin during cardiac chamber maturation, and a surprising function for myocardin in the regulation of cardiomyocyte proliferation, cell death, and possibly mitochondrial function. This review summarizes the literature surrounding the cardiac-specific roles of myocardin during development and post-natal cardiac remodeling. In addition, we take a bioinformatics and computational approach to discuss known and predicted interactions and biological functions of myocardin, which suggests areas for future research.

Targeting skeletal muscle mitochondria to prevent type 2 diabetes in youth.

The prevalence of type 2 diabetes (T2D) has increased dramatically over the past two decades, not only among adults but also among adolescents. T2D is a systemic disorder affecting every organ system and is especially damaging to the cardiovascular system, predisposing individuals to severe cardiac and vascular complications. The precise mechanisms that cause T2D are an area of active research. Most current theories suggest that the process begins with peripheral insulin resistance that precedes failure of the pancreatic ß-cells to secrete sufficient insulin to maintain normoglycemia. A growing body of literature has highlighted multiple aspects of mitochondrial function, including oxidative phosphorylation, lipid homeostasis, and mitochondrial quality control in the regulation of peripheral insulin sensitivity. Whether the cellular mechanisms of insulin resistance in adults are comparable to that in adolescents remains unclear. This review will summarize both clinical and basic studies that shed light on how alterations in skeletal muscle mitochondrial function contribute to whole body insulin resistance and will discuss the evidence supporting high-intensity exercise training as a therapy to circumvent skeletal muscle mitochondrial dysfunction to restore insulin sensitivity in both adults and adolescents.

Human Milk Fortification Increases Bnip3 Expression Associated With Intestinal Cell Death In Vitro.

OBJECTIVES: The aim of the present study was to determine the in vitro effect(s) of a bovine-based human breast milk fortifier (HMF) on human intestinal cells. HMF increases the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein (Bnip3) and cell death; the prostaglandin analogue misoprostol will rescue this effect. METHODS: Cultured intestinal cells were exposed to in vitro-digested human breast milk (BM) ±â€ŠHMF. Intracellular oxidation, cell damage/cell death, and BNIP3 expression were measured after exposure. RESULTS: In vitro-digested BM + HMF significantly increased intracellular oxidation, cell damage, and cell death in enterocyte cell cultures compared with either saline or BM controls, an effect that was rescued by the prostaglandin analogue, misoprostol. Bnip3 transcript and Bnip3 protein levels were significantly increased in vitro after treatment with BM + HMF. We also provide evidence that transfection of enterocytes with Bnip3 increases cell death, an effect that is rescued by a nonfunctional Bnip3 splice variant. CONCLUSIONS: Our data support the hypothesis that HMF increases intestinal Bnip3 in vitro, and that the gene product triggers cell death. We suggest that misoprostol is a promising therapy, which may reduce intestinal cell death.

Assessment of Stiffness-Dependent Autophagosome Formation and Apoptosis in Embryonal Rhabdomyosarcoma Tumor Cells.

Remodeling of the extracellular matrix (ECM) eventually causes the stiffening of tumors and changes to the microenvironment. The stiffening alters the biological processes in cancer cells due to altered signaling through cell surface receptors. Autophagy, a key catabolic process in normal and cancer cells, is thought to be involved in mechano-transduction and the level of autophagy is probably stiffness-dependent. Here, we provide a methodology to study the effect of matrix stiffness on autophagy in embryonal rhabdomyosarcoma cells. To mimic stiffness, we seeded cells on GelMA hydrogel matrices with defined stiffness and evaluated autophagy-related endpoints. We also evaluated autophagy-dependent pathways, apoptosis, and cell viability. Specifically, we utilized immunocytochemistry and confocal microscopy to track autophagosome formation through LC3 lipidation. This approach suggests that the use of GelMA hydrogels with defined stiffness represents a novel method to evaluate the role of autophagy in embryonal rhabdomyosarcoma and other cancer cells.

A novel RhoA/ROCK-CPI-17-MEF2C signaling pathway regulates vascular smooth muscle cell gene expression.

Differentiation of vascular smooth muscle cells (VSMC) is a fundamental aspect of normal development and vascular disease. During contraction, VSMCs modulate calcium sensitivity through RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex (MLCP). Previous studies have demonstrated that this signaling pathway functions in parallel to increase the expression of smooth muscle genes through the myocardin-family of co-activators. MEF2C fulfills a critical role in VSMC differentiation and regulates myocardin expression, leading us to investigate whether the RhoA/ROCK signaling cascade might regulate MEF2 activity. Depolarization-induced calcium signaling increased the expression of myocardin, which was sensitive to ROCK and p38 MAPK inhibition. We previously identified protein phosphatase 1α (PP1α), a known catalytic subunit of the MLCP in VSMCs, as a potent repressor of MEF2 activity. PP1α inhibition resulted in increased expression of myocardin, while ectopic expression of PP1α inhibited the induction of myocardin by MEF2C. Consistent with these data, shRNA-mediated suppression of a PP1α inhibitor, CPI-17, reduced myocardin expression and inhibited VSMC differentiation, suggesting a pivotal role for CPI-17 in regulating MEF2 activity. These data constitute evidence of a novel signaling cascade that links RhoA-mediated calcium sensitivity to MEF2-dependent myocardin expression in VSMCs through a mechanism involving p38 MAPK, PP1α, and CPI-17.

Multiple facets of NF-κB in the heart: to be or not to NF-κB.

The progression from cardiac injury to symptomatic heart failure has been intensely studied over the last decade, and is largely attributable to a loss of functional cardiac myocytes through necrosis, intrinsic and extrinsic apoptosis pathways and autophagy. Therefore, the molecular regulation of these cellular programs has been rigorously investigated in the hopes of identifying a potential cell target that could promote cell survival and/or inhibit cell death to avert, or at least prolong, the degeneration toward symptomatic heart failure. The nuclear factor (NF)-κB super family of transcription factors has been implicated in the regulation of immune cell maturation, cell survival, and inflammation in many cell types, including cardiac myocytes. Recent studies have shown that NF-κB is cardioprotective during acute hypoxia and reperfusion injury. However, prolonged activation of NF-κB appears to be detrimental and promotes heart failure by eliciting signals that trigger chronic inflammation through enhanced elaboration of cytokines including tumor necrosis factor α, interleukin-1, and interleukin-6, leading to endoplasmic reticulum stress responses and cell death. The underlying mechanisms that account for the multifaceted and differential outcomes of NF-κB on cardiac cell fate are presently unknown. Herein, we posit a novel paradigm in which the timing, duration of activation, and cellular context may explain mechanistically the differential outcomes of NF-κB signaling in the heart that may be essential for future development of novel therapeutic interventions designed to target NF-κB responses and heart failure following myocardial injury.

A novel hypoxia-inducible spliced variant of mitochondrial death gene Bnip3 promotes survival of ventricular myocytes.

RATIONALE: Alternative splicing provides a versatile mechanism by which cells generate proteins with different or even antagonistic properties. Previously, we established hypoxia-inducible death factor Bnip3 as a critical component of the intrinsic death pathway. OBJECTIVE: To investigate alternative splicing of Bnip3 pre-mRNA in postnatal ventricular myocytes during hypoxia. METHODS AND RESULTS: We identify a novel previously unrecognized spliced variant of Bnip3 (Bnip3Δex3) generated by alternative splicing of exon3 exclusively in cardiac myocytes subjected to hypoxia. Sequencing of Bnip3Δex3 revealed a frame shift mutation that terminated transcription up-stream of exon5 and exon6 ablating translation of the BH3-like domain and critical carboxyl-terminal transmembrane domain crucial for mitochondrial localization and cell death. Notably, although the 26-kDa Bnip3 protein (Bnip3FL) encoded by full-length mRNA was localized to mitochondria and provoked cell death, the 8.2-kDa Bnip3Δex3 protein encoded by the truncated spliced mRNA was defective for mitochondrial targeting but interacted with Bnip3FL resulting in less association of Bnip3FL with mitochondria and diminished apoptotic and necrotic cell death. Forced expression of Bnip3FL in cardiac myocytes or Bnip3(-/-) mouse embryonic fibroblasts triggered widespread cell death that was inhibited by coexpression of Bnip3Δex3. Conversely, RNA interference targeted against sequences encompassing the unique exon2-exon4 junction of the Bnip3Δex3 sensitized cardiac myocytes to mitochondrial perturbations and cell death induced by Bnip3FL. CONCLUSIONS: Given the otherwise lethal consequences of deregulated Bnip3FL expression in postmitotic cells, our findings reveal a novel intrinsic defense mechanism that opposes the mitochondrial defects and cell death of ventricular myocytes that is obligatorily linked and mutually dependent on alternative splicing of Bnip3FL during hypoxia or ischemic stress.

Rhabdomyosarcoma: Current Therapy, Challenges, and Future Approaches to Treatment Strategies.

Rhabdomyosarcoma is a rare cancer arising in skeletal muscle that typically impacts children and young adults. It is a worldwide challenge in child health as treatment outcomes for metastatic and recurrent disease still pose a major concern for both basic and clinical scientists. The treatment strategies for rhabdomyosarcoma include multi-agent chemotherapies after surgical resection with or without ionization radiotherapy. In this comprehensive review, we first provide a detailed clinical understanding of rhabdomyosarcoma including its classification and subtypes, diagnosis, and treatment strategies. Later, we focus on chemotherapy strategies for this childhood sarcoma and discuss the impact of three mechanisms that are involved in the chemotherapy response including apoptosis, macro-autophagy, and the unfolded protein response. Finally, we discuss in vivo mouse and zebrafish models and in vitro three-dimensional bioengineering models of rhabdomyosarcoma to screen future therapeutic approaches and promote muscle regeneration.

Autophagy in the heart: too much of a good thing?

Autophagy constitutes a catabolic process involving lysosomal degradation of damaged and redundant cytosolic components into biomolecules, via an elaborate lysosomal pathway. Autophagy is a highly regulated and evolutionary conserved process crucial for normal tissue homeostasis and cell life. Certain members of the Bcl-2 gene family, including the BH3 only protein Bnip3 regulate autophagy during cardiac stress during ischemic or hypoxic injury as means of discarding damaged mitochondria and organelles to avert cell death. Defects in the regulation of autophagy have been associated with a number of human pathologies including cancer, neurodegenerative diseases, and heart failure. Here, we discuss the molecular regulation of autophagy in the heart and cellular demise from "too much a good thing."

BNIP3 and Nix: Atypical regulators of cell fate.

Since their discovery nearly 25 years ago, the BCL-2 family members BNIP3 and BNIP3L (aka Nix) have been labelled 'atypical'. Originally, this was because BNIP3 and Nix have divergent BH3 domains compared to other BCL-2 proteins. In addition, this atypical BH3 domain is dispensable for inducing cell death, which is also unusual for a 'death gene'. Instead, BNIP3 and Nix utilize a transmembrane domain, which allows for dimerization and insertion into and through organelle membranes to elicit cell death. Much has been learned regarding the biological function of these two atypical death genes, including their role in metabolic stress, where BNIP3 is responsive to hypoxia, while Nix responds variably to hypoxia and is also down-stream of PKC signaling and lipotoxic stress. Interestingly, both BNIP3 and Nix respond to signals related to cell atrophy. In addition, our current view of regulated cell death has expanded to include forms of necrosis such as necroptosis, pyroptosis, ferroptosis, and permeability transition-mediated cell death where BNIP3 and Nix have been shown to play context- and cell-type specific roles. Perhaps the most intriguing discoveries in recent years are the results demonstrating roles for BNIP3 and Nix outside of the purview of death genes, such as regulation of proliferation, differentiation/maturation, mitochondrial dynamics, macro- and selective-autophagy. We provide a historical and unbiased overview of these 'death genes', including new information related to alternative splicing and post-translational modification. In addition, we propose to redefine these two atypical members of the BCL-2 family as versatile regulators of cell fate.

The molecular mosaic of regulated cell death in the cardiovascular system.

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.

Characterizing Extracellular Vesicles and Particles Derived from Skeletal Muscle Myoblasts and Myotubes and the Effect of Acute Contractile Activity.

Extracellular vesicles (EVs), released from all cells, are essential to cellular communication and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle−derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells at pre-differentiation (myoblasts) and post-differentiation (myotubes) and also from acutely stimulated myotubes (1 h @ 14 V, C-Pace EM, IonOptix, Westwood, MA, USA) using total exosome isolation reagent (TEI, ThermoFisher (Waltham, MA, USA), referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p < 0.001, n = 8−10). Two-way ANOVA showed a significant main effect for the size distribution of myotube vs. myoblast-EPs (p < 0.01, n = 10−13). In comparison, myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400−600, whereas myotube-Eps were largely 50−300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than from the myoblast-EPs, (p < 0.001 n = 6−9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p = 0.07, n = 4−6) and had comparable size distribution profiles to EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p < 0.05, n = 4−6). Myotube-EPs exhibited significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared with myoblast-EPs (p < 0.05, n = 7−12). Conversely, microvesicle marker ARF6 and lipoprotein marker APO-A1 were only found in the myotube-EPs (p < 0.05, n = 4−12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (n = 7) or on the expression of TSG101, ARF6 or CD81 (n = 5−6). Myoblasts treated with control or acute stimulation−derived EPs (13 µg/well) for 48 h and 72 h showed no changes in mitochondrial mass (MitoTracker Red, ThermoFisher, Waltham, MA, USA), cell viability or cell count (n = 3−4). Myoblasts treated with EP-depleted media (72 h) exhibited ~90% lower cell counts (p < 0.01, n = 3). Our data show that EVs differed in size, distribution, protein yield and expression of subtype markers pre vs. post skeletal muscle−differentiation into myotubes. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation−derived EPs did not alter mitochondrial mass or cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle−specific EVs are warranted.