Serum Neurofilament Light is elevated in COVID-19 Positive Adults in the ICU and is associated with Co-Morbid Cardiovascular Disease, Neurological Complications, and Acuity of Illness

In critically ill COVID-19 patients, the risk of long-term neurological consequences is just beginning to be appreciated. While recent studies have identified that there is an increase in structural injury to the nervous system in critically ill COVID-19 patients, there is little known about the relationship of COVID-19 neurological damage to the systemic inflammatory diseases also observed in COVID-19 patients. The purpose of this pilot observational study was to examine the relationships between serum neurofilament light protein (NfL, a measure of neuronal injury) and co-morbid cardiovascular disease (CVD) and neurological complications in COVID-19 positive patients admitted to the intensive care unit (ICU). In this observational study of one-hundred patients who were admitted to the ICU in Tucson, Arizona between April and August 2020, 89 were positive for COVID-19 (COVID-pos) and 11 was COVID-negative (COVID-neg). A healthy control group (n=8) was examined for comparison. The primary outcomes and measures were subject demographics, serum NfL, presence and extent of CVD, diabetes, sequential organ failure assessment score (SOFA), presence of neurological complications, and blood chemistry panel data. COVID-pos patients in the ICU had significantly higher mean levels of Nfl (229.6 ± 163 pg/ml) compared to COVID-neg ICU patients (19.3 ± 5.6 pg/ml), Welch's t-test, p =.01 and healthy controls (12.3 ± 3.1 pg/ml), Welch's t-test p =.005. Levels of Nfl in COVID-pos ICU patients were significantly higher in patients with concomitant CVD and diabetes (n=35, log Nfl 1.6±.09), and correlated with higher SOFA scores (r=.5, p =.001). These findings suggest that in severe COVID-19 disease, the central neuronal and axonal damage in these patients may be driven, in part, by the level of systemic cardiovascular disease and peripheral inflammation. Understanding the contributions of systemic inflammatory disease to central neurological degeneration in these COVID-19 survivors will be important to the design of interventional therapies to prevent long-term neurological and cognitive dysfunction

Sarcomere length-dependent Ca2+ activation in skinned rabbit psoas muscle fibers: coordinated regulation of thin filament cooperative activation and passive force

In skeletal muscle, active force production varies as a function of sarcomere length (SL). It has been considered that this SL dependence results simply from a change in the overlap length between the thick and thin filaments. The purpose of this study was to provide a systematic understanding of the SL-dependent increase in Ca2+ sensitivity in skeletal muscle, by investigating how thin filament “on–off” switching and passive force are involved in the regulation. Rabbit psoas muscles were skinned, and active force measurements were taken at various Ca2+ concentrations with single fibers, in the short (2.0 and 2.4 μm) and long (2.4 and 2.8 μm) SL ranges. Despite the same magnitude of SL elongation, the SL-dependent increase in Ca2+ sensitivity was more pronounced in the long SL range. MgADP (3 mM) increased the rate of rise of active force and attenuated SL-dependent Ca2+ activation in both SL ranges. Conversely, inorganic phosphate (Pi, 20 mM) decreased the rate of rise of active force and enhanced SL-dependent Ca2+ activation in both SL ranges. Our analyses revealed that, in the absence and presence of MgADP or Pi, the magnitude of SL-dependent Ca2+ activation was (1) inversely correlated with the rate of rise of active force, and (2) in proportion to passive force. These findings suggest that the SL dependence of active force in skeletal muscle is regulated via thin filament “on–off” switching and titin (connectin)-based interfilament lattice spacing modulation in a coordinated fashion, in addition to the regulation via the filament overlap

Synergisitic role of ADP and Ca2+ in diastolic myocardial stiffness

Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca2+] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca2+-handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca2+] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca2+ in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca2+ both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathologicallevels of ADP and diastolic [Ca2+] revealed a 76±1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca2+- overload. In isolated Langendorff-perfused rat hearts, CK-inhibition increased ventricular stiffness only in the presence of diastolic [Ca2+]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges even at low Ca2+ and thereby increase myocardial stiffness

Ectopic Catalase Expression in Mitochondria by Adeno-Associated Virus Enhances Exercise Performance in Mice

Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT) was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 1012 vector genome particles per mouse. Three months later, we observed a ∼2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy) and ameliorate muscle disease

Myosin binding protein C: implications for signal-transduction

Myosin binding protein C (MYBPC) is a crucial component of the sarcomere and an important regulator of muscle function. While mutations in different myosin binding protein C (MYBPC) genes are well known causes of various human diseases, such as hypertrophic (HCM) and dilated (DCM) forms of cardiomyopathy as well as skeletal muscular disorders, the underlying molecular mechanisms remain not well understood. A variety of MYBPC3 (cardiac isoform) mutations have been studied in great detail and several corresponding genetically altered mouse models have been generated. Most MYBPC3 mutations may cause haploinsufficiency and with it they may cause a primary increase in calcium sensitivity which is potentially able to explain major features observed in HCM patients such as the hypercontractile phenotype and the well known secondary effects such as myofibrillar disarray, fibrosis, myocardial hypertrophy and remodelling including arrhythmogenesis. However the presence of poison peptides in some cases cannot be fully excluded and most probably other mechanisms are also at play. Here we shall discuss MYBPC interacting proteins and possible pathways linked to cardiomyopathy and heart failure

Estrogen receptors interact with the alpha catalytic subunit of AMP-activated protein kinase Running title: Estrogen receptors interact with AMPK

SYNOPSIS Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signaling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity, and cancer. AMPK is targeted by 17β-estradiol (E2), the main circulating estrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an estrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit binding domain. Finally, both ERs interacted with the upstream LKB1 kinase complex, which is required for E2-dependent activation of AMPK. We conclude that estradiol activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα. Summary statement: We identified a novel interaction between the classical estrogen receptors (ERα and ERβ) and the catalytic subunit of AMPK in several cell types. In addition we demonstrate that estradiol activates AMPK through ERα and requires the upstream kinase complex LKB1

Length-dependent activation in three striated muscle types of the rat

The process whereby sarcomere length modulates the sensitivity of the myofilaments to Ca2+ is termed length-dependent activation. Length-dependent activation is a property of all striated muscles, yet the relative extent of length-dependent activation between skeletal muscle and cardiac muscle is unclear. Although length-dependent activation may be greater in fast skeletal muscle (FSM) than in slow skeletal muscle (SSM), there has not been a well controlled comparison of length-dependent activation between skeletal muscle and cardiac muscle (CM). Accordingly, we measured sarcomere length-dependent properties in skinned soleus (SSM), psoas (FSM) and ventricular trabeculae (CM) of the rat under carefully controlled conditions. The free Ca2+-force relationship was determined at sarcomere lengths (SL) of 1.95 μm, 2.10 μm and 2.25 μm and fitted to a modified Hill equation. FSM and SSM were more sensitive to Ca2+ than CM. Length-dependent activation was ordered as CM > FSM > SSM. Cooperativity as measured by the Hill coefficient of the Ca2+-force relationship was not significantly different between CM and FSM, both of which exhibited greater cooperativity than SSM. SL did not significantly alter this parameter in each muscle type. To establish whether the observed differences can be explained by alterations in interfilament spacing, we measured myofilament lattice spacing (LS) by synchrotron X-ray diffraction in relaxed, skinned muscle preparations. LS was inversely proportional to SL for each muscle type. The slope of the SL-LS relationship, however, was not significantly different between striated muscle types. We conclude that (1) length-dependent activation differs among the three types of striated muscle and (2) these differences in the length-dependent properties among the striated muscle types may not solely be explained by the differences in the response of interfilament spacing to changes in muscle length in relaxed, skinned isolated muscle preparations