Cellular and Molecular Targets for Non-Invasive, Non-Pharmacological Therapeutic/Rehabilitative Interventions in Acute Ischemic Stroke

BACKGROUND: Cerebral circulation delivers the blood flow to the brain through a dedicated network of sanguine vessels. A healthy human brain can regulate cerebral blood flow (CBF) according to any physiological or pathological challenges. The brain is protected by its self-regulatory mechanisms, which are dependent on neuronal and support cellular populations, including endothelial ones, as well as metabolic, and even myogenic factors. OBJECTIVES: Accumulating data suggest that “non-pharmacological” approaches might provide new opportunities for stroke therapy, such as electro-/acupuncture, hyperbaric oxygen therapy, hypothermia/cooling, photobiomodulation, therapeutic gases, transcranial direct current stimulations, or transcranial magnetic stimulations. We reviewed the recent data on the mechanisms and clinical implications of these non-pharmaceutical treatments. METHODS: To present the state-of-the-art for currently available non-invasive, non-pharmacological-related interventions in acute ischemic stroke, we accomplished this synthetic and systematic literature review based on the Preferred Reporting Items for Systematic Principles Reviews and Meta-Analyses (PRISMA). RESULTS: The initial number of obtained articles was 313. After fulfilling the five steps in the filtering/selection methodology, 54 fully eligible papers were selected for synthetic review. We enhanced our documentation with other bibliographic resources connected to our subject, identified in the literature within a non-standardized search, to fill the knowledge gaps. Fifteen clinical trials were also identified. DISCUSSION: Non-invasive, non-pharmacological therapeutic/rehabilitative interventions for acute ischemic stroke are mainly holistic therapies. Therefore, most of them are not yet routinely used in clinical practice, despite some possible beneficial effects, which have yet to be supplementarily proven in more related studies. Moreover, few of the identified clinical trials are already completed and most do not have final results. CONCLUSIONS: This review synthesizes the current findings on acute ischemic stroke therapeutic/rehabilitative interventions, described as non-invasive and non-pharmacological.

Resolvin-D2 targets myogenic cells and improves muscle regeneration in Duchenne muscular dystrophy

Lack of dystrophin causes muscle degeneration, which is exacerbated by chronic inflammation and reduced regenerative capacity of muscle stem cells in Duchenne Muscular Dystrophy (DMD). To date, glucocorticoids remain the gold standard for the treatment of DMD. These drugs are able to slow down the progression of the disease and increase lifespan by dampening the chronic and excessive inflammatory process; however, they also have numerous harmful side effects that hamper their therapeutic potential. Here, we investigated Resolvin-D2 as a new therapeutic alternative having the potential to target multiple key features contributing to the disease progression. Our in vitro findings showed that Resolvin-D2 promotes the switch of macrophages toward their anti-inflammatory phenotype and increases their secretion of pro-myogenic factors. Moreover, Resolvin-D2 directly targets myogenic cells and promotes their differentiation and the expansion of the pool of myogenic progenitor cells leading to increased myogenesis. These effects are ablated when the receptor Gpr18 is knocked-out, knocked-down, or blocked by the pharmacological antagonist O-1918. Using different mouse models of DMD, we showed that Resolvin-D2 targets both inflammation and myogenesis leading to enhanced muscle function compared to glucocorticoids. Overall, this preclinical study has identified a new therapeutic approach that is more potent than the gold-standard treatment for DMD.

Role of MiR-325-3p in the Regulation of CFL2 and Myogenic Differentiation of C2C12 Myoblasts

Skeletal myogenesis is required to maintain muscle mass and integrity, and impaired myogenesis is causally linked to the etiology of muscle wasting. Recently, it was shown that excessive uptake of saturated fatty acids (SFA) plays a significant role in the pathogenesis of muscle wasting. Although microRNA (miRNA) is implicated in the regulation of myogenesis, the molecular mechanism whereby SFA-induced miRNAs impair myogenic differentiation remains largely unknown. Here, we investigated the regulatory roles of miR-325-3p on CFL2 expression and myogenic differentiation in C2C12 myoblasts. PA impeded myogenic differentiation, concomitantly suppressed CFL2 and induced miR-325-3p. Dual-luciferase analysis revealed that miR-325-3p directly targets the 3′UTR of CFL2, thereby suppressing the expression of CFL2, a crucial factor for actin dynamics. Transfection with miR-325-3p mimic resulted in the accumulation of actin filaments (F-actin) and nuclear Yes-associated protein (YAP) in myoblasts and promoted myoblast proliferation and cell cycle progression. Consequently, miR-325-3p mimic significantly attenuated the expressions of myogenic factors and thereby impaired the myogenic differentiation of myoblasts. The roles of miR-325-3p on CFL2 expression, F-actin modulation, and myogenic differentiation suggest a novel miRNA-mediated regulatory mechanism of myogenesis and PA-inducible miR-325-3p may be a critical mediator between obesity and muscle wasting.

Muscle Regeneration and Function in Sports: A Focus on Vitamin D

Muscle is one of the main targets for the biological effects of vitamin D. This hormone modulates several functions of skeletal muscles, from development to tissue repair after injury, through genomic and non-genomic mechanisms. Vitamin D deficiency and supplementation seem to significantly affect muscle strength in different populations, including athletes, although optimal serum 25(OH)D3 level for sport performance has not been defined so far. Additionally, vitamin D deficiency results in myopathy characterized by fast-twitch fiber atrophy, fatty infiltration, and fibrosis. However, less is known about regenerative effects of vitamin D supplementation after sport-related muscle injuries. Vitamin D receptor (VDR) is particularly expressed in the embryonic mesoderm during intrauterine life and in satellite cells at all stages of life for recovery of the skeletal muscle after injury. Vitamin D supplementation enhances muscle differentiation, growth, and regeneration by increasing the expression of myogenic factors in satellite cells. The objective of this narrative review is to describe the role of vitamin D in sport-related muscle injury and tissue regeneration.

IGF2 Deficiency Causes Mitochondrial Defects in Skeletal Muscle

Exercise training improves muscle fitness in many aspects, including induction of mitochondrial biogenesis and maintenance of mitochondrial dynamics. The insulin-like growth factors were recently proposed as key regulators of myogenic factors to regulate muscle development. This study aimed to investigate the physical exercise impact on insulin-like growth factor 2 (IGF2) and analyzed its functions on skeletal muscle cells in vitro. Using online databases, we stated that IGF2 was relatively highly expressed in skeletal muscle cells and increased after exercise training. Then, IGF2 deficiency in myotubes from C2C12 and primary skeletal muscle cells (PMSCs) led to impaired mitochondrial function, reduced mitochondrial-related protein content, and decreased mitochondrial biogenesis. Furthermore, we explored the possible regulatory pathway and found that mitochondrial regulation in skeletal muscle cells might occur through IGF2-SIRT1-PGC1α signaling pathway. Therefore, this study first demonstrated the relationship between IGF2 and mitochondria in skeletal muscle.

Maternal Undernutrition Alters The Skeletal Muscle Development and Methylation of Myogenic Factors in Progeny: A Randomized Controlled Trial

Maternal malnutrition alters protein synthesis and muscle development in offspring. Epigenetic modification is one of the mechanisms involved in this developmental programming. However, the role of the methylation of myogenic factors remains unclear. Twenty-four pregnant goats were assigned to a control (100% of the nutrients requirement, n = 12) or a restricted group (60% of the nutrients requirement, n = 12) between 0.3 and 0.7 gestation (G). Descendants were harvested at 0.7 G and at 91 days after birth to analyze the fiber area and fiber type of femoris muscle and to examine the effects on the mTOR signal pathway and methylation of the promoter regions of myogenic factors. Maternal undernutrition increased the fiber area of the vastus muscle in the fetuses (P < 0.05). Undernutrition enhanced (P < 0.01) the proportions of MyHCI and MyHCIIA fibers in the vastus muscle of fetuses and semitendinosus (ST) muscle of kids, while the proportion of MyHCIIX fibers were decreased (P < 0.01) in the restricted fetuses and kids. The mRNA expression levels of EYA1 and MYOZ2 in the restricted fetuses were downregulated (P < 0.05), but no difference was observed in the restricted kids (P > 0.05). The percentage of DNA methylation at the + 530 CpG site of the MYF5 gene in the restricted fetuses was increased (P < 0.05), but the methylation percentages of the MYF5 gene at the +274,280 CpG site and of the MYOD gene at the +252 CpG site in the restricted kids were reduced (P < 0.05). The mTOR protein signals were downregulated (P < 0.05) in the restricted fetuses and kids. These results indicated that maternal undernutrition altered the fiber type of femoris muscle in offspring, but its relationship with the methylation in the promoter regions of myogenic genes needs to be further studied.

Cerebrum, liver, and muscle regulatory networks uncover maternal nutrition effects in developmental programming of beef cattle during early pregnancy

The molecular basis underlying fetal programming in response to maternal nutrition remains unclear. Herein, we investigated the regulatory relationships between genes in fetal cerebrum, liver, and muscle tissues to shed light on the putative mechanisms that underlie the effects of early maternal nutrient restriction on bovine developmental programming. To this end, cerebrum, liver, and muscle gene expression were measured with RNA-Seq in 14 fetuses collected on day 50 of gestation from dams fed a diet initiated at breeding to either achieve 60% (RES, n = 7) or 100% (CON, n = 7) of energy requirements. To build a tissue-to-tissue gene network, we prioritized tissue-specific genes, transcription factors, and differentially expressed genes. Furthermore, we built condition-specific networks to identify differentially co-expressed or connected genes. Nutrient restriction led to differential tissue regulation between the treatments. Myogenic factors differentially regulated by ZBTB33 and ZNF131 may negatively affect myogenesis. Additionally, nutrient-sensing pathways, such as mTOR and PI3K/Akt, were affected by gene expression changes in response to nutrient restriction. By unveiling the network properties, we identified major regulators driving gene expression. However, further research is still needed to determine the impact of early maternal nutrition and strategic supplementation on pre- and post-natal performance.

Marine Phytoplankton Improves Exercise Recovery in Humans and Activates Repair Mechanisms in Rats

This study investigated the effects of marine phytoplankton supplementation on 1) perceived recovery and ground reaction forces in humans following a non-functional overreaching resistance-training program and 2) myogenic molecular markers associated with muscle cell recovery in a rat model. In the human trial, a 5-week resistance-training program with intentional overreaching on weeks 2 and 5 was implemented. Results indicate that marine phytoplankton prompted positive changes in perceived recovery at post-testing and, while both marine phytoplankton and placebo conditions demonstrated decreased peak and mean rate of force development following the overreaching weeks, placebo remained decreased at post-testing while marine phytoplankton returned to baseline levels. In the rat model, rats were divided into four conditions: (i) control, (ii) exercise, (iii) exercise + marine phytoplankton 2.55 mg·d-1, or (iv) exercise+marine phytoplankton 5.1 mg·d-1. Rats in exercising conditions performed treadmill exercise 5 d·wk-1 for 6 weeks. Marine phytoplankton in exercising rats increased positive and decrease negative myogenic factors regulating satellite cell proliferation. Taken together, marine phytoplankton improved perceptual and functional indices of exercise recovery in an overreaching human model and, mechanistically, this could be driven through cell cycle regulation and a potential to improve protein turnover.

MiR-96-5p Induced by Palmitic Acid Suppresses the Myogenic Differentiation of C2C12 Myoblasts by Targeting FHL1

Skeletal myogenesis is a multi-stage process that includes the cell cycle exit, myogenic transcriptional activation, and morphological changes to form multinucleated myofibers. Recent studies have shown that saturated fatty acids (SFA) and miRNAs play crucial roles in myogenesis and muscle homeostasis. Nevertheless, the target molecules and myogenic regulatory mechanisms of miRNAs are largely unknown, particularly when myogenesis is dysregulated by SFA deposition. This study investigated the critical role played by miR-96-5p on the myogenic differentiation in C2C12 myoblasts. Long-chain SFA palmitic acid (PA) significantly reduced FHL1 expression and inhibited the myogenic differentiation of C2C12 myoblasts but induced miR-96-5p expression. The knockdown of FHL1 by siRNA stimulated cell proliferation and inhibited myogenic differentiation of myoblasts. Interestingly, miR-96-5p suppressed FHL1 expression by directly targeting the 3’UTR of FHL1 mRNA. The transfection of an miR-96-5p mimic upregulated the expressions of cell cycle-related genes, such as PCNA, CCNB1, and CCND1, and increased myoblast proliferation. Moreover, the miR-96-5p mimic inhibited the expressions of myogenic factors, such as myoblast determination protein (MyoD), myogenin (MyoG), myocyte enhancer factor 2C (MEF2C), and myosin heavy chain (MyHC), and dramatically impeded differentiation and fusion of myoblasts. Overall, this study highlights the role of miR-96-5p in myogenesis via FHL1 suppression and suggests a novel regulatory mechanism for myogenesis mediated by miRNA in a background of obesity.