Cardiac myosin binding protein-C phosphorylation as a function of multiple protein kinase and phosphatase activities.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a determinant of cardiac myofilament function. Although cMyBP-C phosphorylation by various protein kinases has been extensively studied, the influence of protein phosphatases on cMyBP-C's multiple phosphorylation sites has remained largely obscure. Here we provide a detailed biochemical characterization of cMyBP-C dephosphorylation by protein phosphatases 1 and 2 A (PP1 and PP2A), and develop an integrated kinetic model for cMyBP-C phosphorylation using data for both PP1, PP2A and various protein kinases known to phosphorylate cMyBP-C. We find strong site-specificity and a hierarchical mechanism for both phosphatases, proceeding in the opposite direction of sequential phosphorylation by potein kinase A. The model is consistent with published data from human patients and predicts complex non-linear cMyBP-C phosphorylation patterns that are validated experimentally. Our results suggest non-redundant roles for PP1 and PP2A under both physiological and heart failure conditions, and emphasize the importance of phosphatases for cMyBP-C regulation.

GELBOX: OPEN-SOURCE SOFTWARE TO IMPROVE RIGOR AND REPRODUCIBILITY WHEN ANALYZING GELS AND IMMUNOBLOTS.

GelBox is open-source software that was developed with the goal of enhancing rigor, reproducibility, and transparency when analyzing gels and immunoblots. It combines image adjustments (cropping, rotation, brightness, and contrast), background correction, and band-fitting in a single application. Users can also associate each lane in an image with metadata (for example, sample type). GelBox data files integrate the raw data, supplied metadata, image adjustments, and band-level analyses in a single file to improve traceability. GelBox has a user-friendly interface and was developed using MATLAB. The software, installation instructions, and tutorials, are available at https://campbell-muscle-lab.github.io/GelBox/.

Postnatal growth restriction alters myocardial mitochondrial energetics in mice.

Postnatal growth restriction (PGR) can increase the risk of cardiovascular disease (CVD) potentially due to impairments in oxidative phosphorylation (OxPhos) within cardiomyocyte mitochondria. The purpose of this investigation was to determine if PGR impairs cardiac metabolism, specifically OxPhos. FVB (Friend Virus B-type) mice were fed a normal-protein (NP: 20% protein), or low-protein (LP: 8% protein) isocaloric diet 2 weeks before mating. LP dams produce ∼20% less milk, and pups nursed by LP dams experience reduced growth into adulthood as compared to pups nursed by NP dams. At birth (PN1), pups born to dams fed the NP diet were transferred to LP dams (PGR group) or a different NP dam (control group: CON). At weaning (PN21), all mice were fed the NP diet. At PN22 and PN80, mitochondria were isolated for respirometry (oxygen consumption rate, J O 2 ${J_{{{\mathrm{O}}_{\mathrm{2}}}}}$ ) and fluorimetry (reactive oxygen species emission, J H 2 O 2 ${J_{{{\mathrm{H}}_{\mathrm{2}}}{{\mathrm{O}}_{\mathrm{2}}}}}$ ) analysis measured as baseline respiration (LEAK) and with saturating ADP (OxPhos). Western blotting at PN22 and PN80 determined protein abundance of uncoupling protein 3, peroxiredoxin-6, voltage-dependent anion channel and adenine nucleotide translocator 1 to provide further insight into mitochondrial function. ANOVAs with the main effects of diet, sex and age with α-level of 0.05 was set a priori. Overall, PGR (7.8 ± 1.1) had significant (P = 0.01) reductions in respiratory control in complex I when compared to CON (8.9 ± 1.0). In general, our results show that PGR led to higher electron leakage in the form of free radical production and reactive oxygen species emission. No significant diet effects were found in protein abundance. The observed reduced respiratory control and increased ROS emission in PGR mice may increase risk for CVD in mice.

The Effect of Downhill Running on Quadriceps Muscle in Growth-Restricted Mice.

INTRODUCTION: Growth restriction (GR) reduces ribosome abundance and skeletal muscle mass in mice. A reduction in skeletal muscle mass increases the risk of frailty and is associated with high morbidity and mortality rates. As eccentric type exercise increases muscle mass, this investigation aimed to determine if eccentric loading of skeletal muscle via downhill running (DHR) increased muscle mass in GR mice. METHODS: Mice were growth-restricted either gestational undernutrition (GUN, n = 8 litters), postnatal undernutrition (PUN, n = 8 litters), or were not restricted (CON, n = 8 litters) via a validated cross-fostering nutritive model. On postnatal day (PN) 21, all mice were weaned to a healthy diet, isolating the period of GR to early life as seen in humans. At PN45, mice were assigned to either a DHR (CON, n = 4 litters; GUN, n = 4 litters; PUN, n = 4 litters) or sedentary (SED: CON, n = 4 litters; GUN, n = 4 litters; PUN, n = 4 litters) group. Downhill running (16% decline: 18 m·min -1 ) was performed in 30-min bouts, three times per week, for 12 wk on a rodent treadmill. At PN129, the quadriceps femoris was dissected and evaluated for mass, myofiber size and type, and molecular markers of growth. RESULTS: Following training, CON-DHR mice having larger cells than CON-SED, GUN-SED, PUN-SED, and PUN-DHR mice ( P < 0.05). The PUN group (as compared with CON) had reduced body mass ( P < 0.001), upstream binding factor abundance ( P = 0.012), phosphor-mTOR ( P < 0.001), and quadriceps mass ( P = 0.02). The GUN and PUN groups had increased MuRF1 abundance ( P < 0.001) compared with CON ( P < 0.001). CONCLUSIONS: The blunted response to training suggests GR mice may have anabolic resistance when exposed to eccentric type exercise.

Longitudinal effects of growth restriction on the murine gut microbiome and metabolome.

Undernutrition-induced growth restriction in the early stages of life increases the risk of chronic disease in adulthood. Although metabolic impairments have been observed, few studies have characterized the gut microbiome and gut-liver metabolome profiles of growth-restricted animals during early-to-mid-life development. To induce growth restriction, mouse offspring were either born to gestational undernutrition (GUN) or suckled from postnatal undernutrition (PUN) dams fed a protein-restricted diet (8% protein) or control diet (CON; 20% protein) until weaning at postnatal age of 21 days (PN21). At PN21, all mice were fed the CON diet until adulthood (PN80). Livers were collected at PN21 and PN80, and fecal samples were collected weekly starting at PN21 (postweaning week 1) until PN80 (postweaning week 5) for gut microbiome and metabolome analyses. PUN mice exhibited the most alterations in gut microbiome and gut and liver metabolome compared with CON mice. These mice had altered fecal microbial ß-diversity (P = 0.001) and exhibited higher proportions of Bifidobacteriales [linear mixed model (LMM) P = 7.1 × 10-6), Clostridiales (P = 1.459 × 10-5), Erysipelotrichales (P = 0.0003), and lower Bacteroidales (P = 4.1 × 10-5)]. PUN liver and fecal metabolome had a reduced total bile acid pool (P < 0.01), as well as lower abundance of riboflavin (P = 0.003), amino acids [i.e., methionine (P = 0.0018), phenylalanine (P = 0.0015), and tyrosine (P = 0.0041)], and higher excreted total peptides (LMM P = 0.0064) compared with CON. Overall, protein restriction during lactation permanently alters the gut microbiome into adulthood. Although the liver bile acids, amino acids, and acyl-carnitines recovered, the fecal peptides and microbiome remained permanently altered into adulthood, indicating that inadequate protein intake in a specific time frame in early life can have an irreversible impact on the microbiome and fecal metabolome.NEW & NOTEWORTHY Undernutrition-induced early-life growth restriction not only leads to increased disease risk but also permanently alters the gut microbiome and gut-liver metabolome during specific windows of early-life development.

Electrify your class with a simple battery: battery demonstration of electrocardiogram vectors.

William Arthur Ward stated, "The mediocre teacher tells. The good teacher explains. The superior teacher demonstrates. The great teacher inspires." Discovery experimentation is an inductive method that demonstrates and inspires by creating an interest in determining the underlining basis of a phenomenon. This experiential approach also fosters motivation and enhances learning. Starting with what the student knows augments this approach. By starting with what the student already knows, the student can consciously and explicitly link the subsequent new information with previous knowledge. Accordingly, we used a simple battery as an analogy for electrocardiogram vectors to introduce the theoretical physics of how the heart produces voltages that are detectable at the body surface. This extraordinarily complex physics was approached in a straightforward and inexpensive way while still providing an understanding of the fundamental concepts developed by Willem Einthoven in 1895.