Fadia Haddad

Cold water immersion (CWI) purportedly reduces inflammation and improves muscle recovery after exercise, yet its effectiveness in specific contexts (ultraendurance) remains unclear. Thus, our aim was to study hematological profiles, systemic inflammation, and muscle damage responses to a specific post-race CWI (vs. control) during recovery after the Ironman World Championship, a culmination of ∼100 000 athletes competing in global qualifying Ironman events each year. Twenty-nine competitors were randomized into either a CWI or control (CON) group. Physiological parameters and blood samples were taken at pre-race, after intervention (POST), and 24 (+1DAY) and 48 hours (+2DAY) following the race. Muscle damage markers (plasma myoglobin, serum creatine kinase) were elevated at POST, +1DAY, and +2DAY, while inflammatory cytokines interleukin (IL)-6, IL-8, and IL-10 and total leukocyte counts were increased only at POST. CWI had no effect on these markers. Numbers of the most abundant circulating cell type, neutrophils, were elevated at POST more so in CWI (p < 0.05, vs. CON). Despite that neutrophil counts may be a sensitive marker to detect subtle effects, CWI does not affect recovery markers 24- and 48-hours post-race (vs. CON). Overall, we determined that our short CWI protocol was not sufficient to improve recovery. Novelty: Ironman World Championship event increased circulating muscle damage markers, inflammatory markers, and hematological parameters, including circulating immune cell sub-populations that recover 24–48 hours after the race. 12-min CWI post-ultraendurance event affects the absolute numbers of neutrophils acutely, post-race (vs. CON), but does not impact recovery 24- and 48-hours post-race.

BACKGROUND: Although several exercise systems have been developed to mitigate the physiological deconditioning that occurs in microgravity, few have the capacity to positively impact multiple physiological systems and still meet the volume/mass requirements needed for missions beyond low Earth orbit. The purpose of this study was to test the gravity-independent Multi-Mode Exercise Device (M-MED) for both resistance (RE) and aerobic (AE) training stimuli.METHODS: Eight men and nine women (mean age 22.0 ± 0.4 yr) completed 5 wk of training on the M-MED: RE 4 × 7 squats 2 d/wk, and AE 4 × 4-min rowing bouts at ∼90% Vo_2max 3 d/wk. Pre- and post-training data collection included an aerobic capacity test, MR imaging, strength testing, and vastus lateralis muscle biopsy.RESULTS: Vo_2max increased 8%, 3RM strength 18%, and quadriceps femoris cross-sectional area (CSA) 10%. Knee extensor strength increased at all isokinetic speeds tested. Subjects also demonstrated improved fatigue resistance in knee extension. At the cellular and molecular level, the biopsy revealed increases in mixed myofiber CSA (13%), citrate synthase activity (26%), total RNA concentration (24%), IGF-I mRNA (77%), and Type IIa myosin heavy chain (MHC) mRNA (8%), and a concomitant decrease in Type IIx MHC mRNA (−23%). None of the changes were gender-specific.DISCUSSION: Both the functional outcomes and biomarker changes indicate that a very low volume of M-MED exercise results in robust adaptation in the cardiovascular and musculoskeletal systems. The M-MED has the potential to provide a wide range of countermeasure exercises and should be considered for testing in ground-based spaceflight simulation.Owerkowicz T, Cotter JA, Haddad F, Yu AM, Camilon ML, Hoang TN, Jimenez DJ, Kreitenberg A, Tesch PA, Caiozzo VJ, Adams GR. Exercise responses to gravity-independent flywheel aerobic and resistance training. Aerosp Med Hum Perform. 2016; 87(2):93–101.

Cotter JA, Yu A, Kreitenberg A, Haddad FH, Baker MJ, Fox JC, Adams GR. Suction-modified needle biopsy technique for the human soleus muscle. Aviat Space Environ Med 2013; 84:1066–73. Introduction: The needle biopsy technique for the soleus muscle is of particular interest because of the muscle's unique fiber type distribution, contractile properties, and sensitivity to unloading. Unlike other commonly biopsied muscles, the soleus is not fully superficial and is in close proximity to neurovascular structures, resulting in a more challenging biopsy. Because of this, a standardized protocol for performing needle biopsies on the human soleus muscle that is safe, reliable, and repeatable is presented. Methods: Ultrasonography was used on an initial set of 12 subjects to determine the optimal biopsy zone, thereby guiding the location of the incision site. There were 45 subjects recruited who attended 2 separate biopsy sessions. Each biopsy session incorporated 3 passes of the biopsy needle proximal, posterior, and distal using suction from a portable vacuum source producing 3 separate muscle specimens. Results: There were 84 soleus muscle biopsy procedures which were successfully conducted yielding 252 total samples without complication. Ultrasonography was used to confirm biopsy needle infiltration of the soleus muscle. Average sample weight obtained per pass was 61.5 ± 15.7 mg. Histochemistry and molecular analyses demonstrated a considerably higher amount of slow type I MHC in comparison to the vastus lateralis, providing verification for the successful sampling of the soleus muscle. Discussion: The procedure presented consists of a detailed protocol to accurately and consistently obtain muscle biopsy samples from the human soleus muscle. We have demonstrated that the human soleus biopsy is a safe, reliable, and repeatable procedure providing ample tissue for multiple types of analyses.

Skeletal muscle is the largest organ system in mammalian organisms providing postural control and movement patterns of varying intensity. Through evolution, skeletal muscle fibers have evolved into three phenotype clusters defined as a muscle unit which consists of all muscle fibers innervated by a single motoneuron linking varying numbers of fibers of similar phenotype. This fundamental organization of the motor unit reflects the fact that there is a remarkable interdependence of gene regulation between the motoneurons and the muscle mainly via activity-dependent mechanisms. These fiber types can be classified via the primary type of myosin heavy chain (MHC) gene expressed in the motor unit. Four MHC gene encoded proteins have been identified in striated muscle: slow type I MHC and three fast MHC types, IIa, IIx, and IIb. These MHCs dictate the intrinsic contraction speed of the myofiber with the type I generating the slowest and IIb the fastest contractile speed. Over the last ~35 years, a large body of knowledge suggests that altered loading state cause both fiber atrophy/wasting and a slow to fast shift in the contractile phenotype in the target muscle(s). Hence, this review will examine findings from three different animal models of unloading: 1) space flight (SF), i.e., microgravity; 2) hindlimb suspension (HS), a procedure that chronically eliminates weight bearing of the lower limbs; and 3) spinal cord isolation (SI), a surgical procedure that eliminates neural activation of the motoneurons and associated muscles while maintaining neurotrophic motoneuron-muscle connectivity. The collective findings demonstrate: 1) all three models show a similar pattern of fiber atrophy with differences mainly in the magnitude and kinetics of alteration; 2) transcriptional/pretranslational processes play a major role in both the atrophy process and phenotype shifts; and 3) signaling pathways impacting these alterations appear to be similar in each of the models investigated.

The antithetical regulation of cardiac α- and β-myosin heavy chain (MHC) genes by thyroid hormone (T₃) is not well understood but appears to involve thyroid hormone interaction with its nuclear receptor and MHC promoters as well as cis-acting noncoding regulatory RNA (ncRNA). Both of these phenomena involve epigenetic regulations. This study investigated the extent that altered thyroid state induces histone modifications in the chromatin associated with the cardiac MHC genes. We hypothesized that specific epigenetic events could be identified and linked to cardiac MHC gene switching in response to a hypothyroid or hyperthyroid state. A hypothyroid state was induced in rats by propylthiouracil treatment (PTU), whereas a hyperthyroid (T₃) was induced by T₃treatment. The left ventricle was analyzed after 7 days for MHC pre-mRNA expression, and the chromatin was assessed for enrichment in specific histone modifications using chromatin immunoprecipitation quantitative PCR assays. At both the α-MHC promoter and the intergenic region, the enrichment in acetyl histone H3 at K9/14 (H3K9/14ac) and trimethyl histone H3 at K4 (H3K4me3) changed in a similar fashion. They were both decreased with PTU treatment but did not change under T₃, except at a location situated 5′ to the antisense intergenic transcription start site. These same marks varied differently on the β-MHC promoter. For example, H3K4me3 enrichment correlated with the β-promoter activity in PTU and T₃groups, whereas H3K9/14ac was repressed in the T₃group but did not change under PTU. Histone H3K9me was enriched in chromatin of both the intergenic and α-MHC promoters in the PTU group, whereas histone H4K20me1 was enriched in chromatin of β-MHC promoter in the normal control and T₃groups. Collectively, these findings provide evidence that specific epigenetic phenomena modulate MHC gene expression in altered thyroid states.

Reverse transcription (RT) is the synthesis of complementary deoxyribonucleic acids (DNA) from single-stranded ribonucleic acid (RNA) templates. This process is catalyzed by the reverse transcriptase enzyme, which is the replicating enzyme of retroviruses. Reverse transcriptase was discovered in 1970, and since then, it has played an instrumental role in the advancement of molecular biology and biotechnology research. In the presence of all four deoxynucleotides (dNTP: dATP, dCTP, dGTP, and dTTP) and under well-defined salt and pH conditions, the reverse transcriptase extends a primer complementary to the RNA to produce a complementary DNA (cDNA) for the RNA template. In this chapter, a simple method of reverse transcription of total cellular RNA into cDNA is described using Superscript II reverse transcriptase (Invitrogen); the resulting cDNA can be used in polymerase chain reaction (PCR).

The role of calcineurin (Cn) in skeletal muscle fiber-type expression has been a subject of great interest because of reports indicating that it controls the slow muscle phenotype. To delineate the role of Cn in phenotype remodeling, particularly its role in driving expression of the type I myosin heavy chain (MHC) gene, we used a novel strategy whereby a profound transition from fast to slow fiber type is induced and examined in the absence and presence of cyclosporin A (CsA), a Cn inhibitor. To induce the fast-to-slow transition, we first subjected rats to 7 days of hindlimb suspension (HS) + thyroid hormone [triiodothyronine (T₃)] to suppress nearly all expression of type I MHC mRNA in the soleus muscle. HS + T₃ was then withdrawn, and rats resumed normal ambulation and thyroid state, during which vehicle or CsA (30 mg·kg⁻¹·day⁻¹) was administered for 7 or 14 days. The findings demonstrate that, despite significant inhibition of Cn, pre-mRNA, mRNA, and protein abundance of type I MHC increased markedly during reloading relative to HS + T₃ ( P < 0.05). Type I MHC expression was, however, attenuated by CsA compared with vehicle treatment. In addition, type IIa and IIx MHC pre-mRNA, mRNA, and relative protein levels were increased in Cn-treated compared with vehicle-treated rats. These findings indicate that Cn has a modulatory role in MHC transcription, rather than a role as a primary regulator of slow MHC gene expression.

Cardiac myosin heavy chain (MHC) gene expression undergoes a rapid transition from β- to α-MHC during early rodent neonatal development (0–21 days of age). Thyroid hormone (3,5,3′-triiodothyronine, T₃) is a major player in this developmental shift; however, the exact mechanism underlying this transition is poorly understood. The goal of this study was to conduct a more thorough analysis of transcriptional activity of the cardiac MHC gene locus during the early postnatal period in the rodent, in order to gain further insight on the regulation of cardiac MHC genes. We analyzed the expression of α- and β-MHC at protein, mRNA, and pre-mRNA levels at birth and 7, 10, 15, and 21 days after birth in euthyroid and hypothyroid rodents. Using novel technology, we also analyzed RNA expression across the cardiac gene locus, and we discovered that the intergenic (IG) region between the two cardiac genes possesses bidirectional transcriptional activity. This IG transcription results in an antisense RNA product as described previously, which is thought to exert an inhibitory effect on β-MHC gene transcription. On the second half of the IG region, sense transcription occurs, resulting in expression of a sense IG RNA that merges with the α-MHC pre-mRNA. This sense IG RNA transcription was detected in the α-MHC gene promoter, approximately −1.8 kb relative to the α-MHC transcription start site. Both sense and antisense IG RNAs were developmentally regulated and responsive to a hypothyroid state ( 11 , 14 ). This novel observation provides more complexity to the cooperative regulation of the two genes, suggesting the involvement of epigenetic processes in the regulation of cardiac MHC gene locus.

The ability to accurately measure patterns of gene expression is essential in studying gene function. The reverse transcription polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. Our previous results show that there is a significant production of primer-independent cDNA synthesis using a popular RNase H- RT enzyme. A PCR product was amplified from RT reactions that were carried out without addition of RT-primer. This finding jeopardizes the accuracy of RT-PCR when analyzing RNA that is expressed in both orientations. Current literature findings suggest that naturally occurring antisense expression is widespread in the mammalian transcriptome and consists of both coding and non-coding regulatory RNA. The primary purpose of this present study was to investigate the occurrence of primer-independent cDNA synthesis and how it may influence the accuracy of detection of sense-antisense RNA pairs.

Hypertension has been shown to cause cardiac hypertrophy and a shift in myosin heavy chain (MHC) gene expression from the faster α- to slower β-MHC isoform. The expression of the β- and α-MHC pre-mRNAs, mRNAs, as well as the newly discovered antisense β-RNA were analyzed in three regions of the normal control (NC) and 12-day pressure-overloaded (AbCon) hearts: the left ventricle apex, left ventricle base, and the septum. The RNA analyses in the AbCon heart targeted both the 5′ and the 3′ ends of each RNA molecule. β-MHC mRNA expression significantly increased relative to control in all three regions, regardless of the target site (5′ or 3′ end). In contrast, β-MHC pre-mRNA expression in the AbCon heart depended on the site of the measurement (5′ vs. 3′ end). For example, whereas the pre-mRNA did not change when targeted at the 3′ end (last intron), it increased significantly in the AbCon heart when measurement targeted the 5′ end (2nd intron) of the 25-kb molecule. Analyses of the antisense β-RNA revealed that its expression in the AbCon heart was significantly decreased relative to control regardless of its measurement site. A negative correlation was observed between the β-mRNA expression and the antisense β-RNA (P < 0.05), suggesting an inhibitory role of antisense RNA on the sense β-MHC gene expression. In contrast, a positive correlation was observed between the antisense β-RNA and the α-MHC pre-mRNA (P < 0.05). This latter observation along with the α-MHC gene position relative to that of the β-antisense suggest that the α-MHC sense and β-antisense transcription are coregulated likely via common intergenic regulatory sequences. Our results suggest that the increased β-MHC expression in the AbCon heart not only is the result of increased β-MHC transcription but also involves an antisense β-RNA regulation scheme. Although the exact mechanism concerning antisense regulation is not clear, it could involve modulation of both transcriptional activity of the β-MHC gene and posttranscriptional processing.