Myonuclei gained during exercise-induced skeletal muscle hypertrophy may be long-lasting and could facilitate future muscle adaptability after deconditioning, a concept colloquially termed “muscle memory.” The evidence for this is limited, mostly due to the lack of a murine exercise-training paradigm that is nonsurgical and reversible. To address this limitation, we developed a novel progressive weighted-wheel-running (PoWeR) model of murine exercise training to test whether myonuclei gained during exercise persist after detraining. We hypothesized that myonuclei acquired during training-induced hypertrophy would remain following loss of muscle mass with detraining. Singly housed female C57BL/6J mice performed 8 wk of PoWeR, while another group performed 8 wk of PoWeR followed by 12 wk of detraining. Age-matched sedentary cage-dwelling mice served as untrained controls. Eight weeks of PoWeR yielded significant plantaris muscle fiber hypertrophy, a shift to a more oxidative phenotype, and greater myonuclear density than untrained mice. After 12 wk of detraining, the plantaris muscle returned to an untrained phenotype with fewer myonuclei. A finding of fewer myonuclei simultaneously with plantaris deconditioning argues against a muscle memory mechanism mediated by elevated myonuclear density in primarily fast-twitch muscle. PoWeR is a novel, practical, and easy-to-deploy approach for eliciting robust hypertrophy in mice, and our findings can inform future research on the mechanisms underlying skeletal muscle adaptive potential and muscle memory.

Existing therapies to improve heart function target β-adrenergic receptor (β-AR) signaling and Ca2+ handling and often lead to adverse outcomes. This underscores an unmet need for positive inotropes that improve heart function without any adverse effects. The GTPase Ras associated with diabetes (RAD) regulates L-type Ca2+ channel (LTCC) current (ICa,L). Global RAD-knockout mice (gRAD−/−) have elevated Ca2+ handling and increased cardiac hypertrophy, but RAD is expressed also in noncardiac tissues, suggesting the possibility that pathological remodeling is due also to noncardiac effects. Here, we engineered a myocardial-restricted inducible RAD-knockout mouse (RADΔ/Δ). Using an array of methods and techniques, including single-cell electrophysiological and calcium transient recordings, echocardiography, and radiotelemetry monitoring, we found that RAD deficiency results in a sustained increase of inotropy without structural or functional remodeling of the heart. ICa,L was significantly increased, with RAD loss conferring a β-AR–modulated phenotype on basal ICa,L. Cardiomyocytes from RADΔ/Δ hearts exhibited enhanced cytosolic Ca2+ handling, increased contractile function, elevated sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a) expression, and faster lusitropy. These results argue that myocardial RAD ablation promotes a beneficial elevation in Ca2+ dynamics, which would obviate a need for increased β-AR signaling to improve cardiac function.

IntroductionAcute myocardial infarction (MI) is a primary cause of worldwide morbidity and mortality. Macrophages are fundamental components of post-MI inflammation. Pro-inflammatory macrophages can lead to adverse cardiac remodeling and heart failure while anti-inflammatory/reparative macrophages enhance tissue healing. Shifting the balance between pro-inflammatory and reparative macrophages post-MI is a novel therapeutic strategy. Azithromycin (AZM), a commonly used macrolide antibiotic, polarizes macrophages towards the anti-inflammatory phenotype, as shown in animal and human studies. We hypothesized that AZM modulates post-MI inflammation and improves cardiac recovery.Methods and resultsMale WT mice (C57BL/6, 6–8 weeks old) were treated with either oral AZM (160 mg/kg/day) or vehicle (control) starting 3 days prior to MI and continued to day 7 post-MI. We observed a significant reduction in mortality with AZM therapy. AZM-treated mice showed a significant decrease in pro-inflammatory (CD45+/Ly6G-/F4-80+/CD86+) and increase in anti-inflammatory (CD45+/Ly6G-/F4-80+/CD206+) macrophages, decreasing the pro-inflammatory/anti-inflammatory macrophage ratio in the heart and peripheral blood as assessed by flow cytometry and immunohistochemistry. Macrophage changes were associated with a significant decline in pro- and increase in anti-inflammatory cytokines. Mechanistic studies confirmed the ability of AZM to shift macrophage response towards an anti-inflammatory state under hypoxia/reperfusion stress. Additionally, AZM treatment was associated with a distinct decrease in neutrophil count due to apoptosis, a known signal for shifting macrophages towards the anti-inflammatory phenotype. Finally, AZM treatment improved cardiac recovery, scar size, and angiogenesis.ConclusionAzithromycin plays a cardioprotective role in the early phase post-MI through attenuating inflammation and enhancing cardiac recovery. Post-MI treatment and human translational studies are warranted to examine the therapeutic applications of AZM.

Complex tissue regeneration is extremely rare among adult mammals. An exception, however, is the superior tissue healing of multiple organs in spiny mice (Acomys). While Acomys species exhibit the remarkable ability to heal complex tissue with minimal scarring, little is known about their cardiac structure and response to cardiac injury. In this study, we first examined baseline Acomys cardiac anatomy and function in comparison with commonly used inbred and outbred laboratory Mus strains (C57BL6 and CFW). While our results demonstrated comparable cardiac anatomy and function between Acomys and Mus, Acomys exhibited a higher percentage of cardiomyocytes displaying distinct characteristics. In response to myocardial infarction, all animals experienced a comparable level of initial cardiac damage. However, Acomys demonstrated superior ischemic tolerance and cytoprotection in response to injury as evidenced by cardiac functional stabilization, higher survival rate, and smaller scar size 50 days after injury compared to the inbred and outbred mouse strains. This phenomenon correlated with enhanced endothelial cell proliferation, increased angiogenesis, and medium vessel maturation in the peri-infarct and infarct regions. Overall, these findings demonstrate augmented myocardial preservation in spiny mice post-MI and establish Acomys as a new adult mammalian model for cardiac research.

MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GC-MS–based metabolomics workflow that uses insoluble protein–derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow. We applied this workflow to study heart metabolism by first comparing two different methods of heart removal: the Langendorff heart method (reverse aortic perfusion) and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute β-adrenergic receptor stimulation (through isoproterenol (ISO) treatment) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as LC or capillary electrophoresis.