Molecular Events Underlying Pregnancy-Induced Cardiomyopathy

The etiology of cardiomyopathy in pregnant women remains unclear. In this issue, Hilfiker-Kleiner et al. (2007) report that a reduction in STAT3 and a concomitant increase in cathepsin D may be a cause of this disease. Cathepsin D generates an antiangiogenic cleavage product of the hormone prolactin. The authors show that an inhibitor of prolactin secretion may be useful in treating this disease

Skeletal myosin heavy chain function in cultured lung myofibroblasts

Myofibroblasts are unique contractile cells with both muscle and nonmuscle properties. Typically myofibroblasts are identified by the expression of α smooth muscle actin (ASMA); however some myofibroblasts also express sarcomeric proteins. In this study, we show that pulmonary myofibroblasts express three of the eight known sarcomeric myosin heavy chains (MyHCs) (IIa, IId, and embryonic) and that skeletal muscle myosin enzymatic activity is required for pulmonary myofibroblast contractility. Furthermore, inhibition of skeletal myosin activity and myofibroblast contraction results in a decrease in both ASMA and skeletal MyHC promoter activity and ASMA protein expression, suggesting a potential coupling of skeletal myosin activity and ASMA expression in myofibroblast differentiation. To understand the molecular mechanisms whereby skeletal muscle genes are regulated in myofibroblasts, we have found that members of the myogenic regulatory factor family of transcription factors and Ca2+-regulated pathways are involved in skeletal MyHC promoter activity. Interestingly, the regulation of skeletal myosin expression in myofibroblasts is distinct from that observed in muscle cells and suggests that cell context is important in its control

Fighting Fat with Muscle: Bulking Up to Slim Down

Akt1 is a well-characterized mediator of muscle hypertrophy. In this issue of Cell Metabolism, Izumiya et al. (2008) reveal a striking link between Akt1 signaling, fast muscle fiber size, and whole-body metabolism. These results provide new insights into the ability of muscle to combat diet-induced obesity and metabolic dysfunction

IIb or not IIb? Regulation of myosin heavy chain gene expression in mice and men

<p>Abstract</p> <p>Background</p> <p>While the myosin heavy chain IIb isoform (MyHC-IIb) is the predominant motor protein in most skeletal muscles of rats and mice, the messenger RNA (mRNA) for this isoform is only expressed in a very small subset of specialized muscles in adult large mammals, including humans.</p> <p>Results</p> <p>We identify the DNA sequences limiting MyHC-IIb expression in humans and explore the activation of this gene in human skeletal muscle. We demonstrate that the transcriptional activity of ~1.0 kb of the human MyHC-IIb promoter is greatly reduced compared to that of the corresponding mouse sequence in both mouse and human myotubes <it>in vitro </it>and show that nucleotide differences that eliminate binding sites for myocyte enhancer factor 2 (MEF2) and serum response factor (SRF) account for this difference. Despite these differences, we show that MyHC-IIb mRNA is expressed in fetal human muscle cells and that MyHC-IIb mRNA is significantly up-regulated in the skeletal muscle of Duchene muscular dystrophy patients.</p> <p>Conclusions</p> <p>These data identify the genetic basis for a key phenotypic difference between the muscles of large and small mammals, and demonstrate that mRNA expression of the MyHC-IIb gene can be re-activated in human limb muscle undergoing profound degeneration/regeneration.</p

A randomized library approach to identifying functional lox site domains for the Cre recombinase

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system

Nonproductive Splicing Prevents Expression of MYH7b Protein in the Mammalian Heart

Background Although the roles of alpha‐myosin heavy chain (&alpha;‐MyHC) and beta‐myosin heavy chain (&beta;‐MyHC) proteins in cardiac contractility have long been appreciated, the biological contribution of another closely related sarcomeric myosin family member, MYH7b (myosin heavy chain 7b), has become a matter of debate. In mammals, MYH7b mRNA is transcribed but undergoes non‐productive alternative splicing that prevents protein expression in a tissue‐specific manner, including in the heart. However, several studies have recently linked MYH7b variants to different cardiomyopathies or have reported MYH7b protein expression in mammalian hearts. &nbsp; Methods and Results By analyzing mammalian cardiac transcriptome and proteome data, we show that the vast majority of MYH7b RNA is subject to exon skipping and cannot be translated into a functional myosin molecule. Notably, we discovered a lag in the removal of introns flanking the alternatively spliced exon, which could retain the non‐coding RNA in the nucleus. This process could play a significant role in controlling MYH7b expression as well as the activity of other cardiac genes. Consistent with the negligible level of full‐length protein coding mRNA, no MYH7b protein expression was detected in adult mouse, rat, and human hearts by Western blot analysis. Furthermore, proteome surveys including quantitative mass spectrometry analyses revealed only traces of cardiac MYH7b protein and even then, only in a subset of individual samples. &nbsp; Conclusions The comprehensive analysis presented here suggests that previous studies showing cardiac MYH7b protein expression were likely attributable to antibody cross‐reactivity. More importantly, our data predict that the MYH7b disease‐associated variants may operate through the alternately spliced RNA itself. &nbsp;</p

Contractility parameters of human -cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function

Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human ?-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human ?-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human ?-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human ?-cardiac myosin contractility as a result of the R403Q mutation

Breath analysis by ultra-sensitive broadband laser spectroscopy detects SARS-CoV-2 infection

Rapid testing is essential to fighting pandemics such as COVID-19, the disease caused by the SARS-CoV-2 virus. Exhaled human breath contains multiple volatile molecules providing powerful potential for non-invasive diagnosis of diverse medical conditions. We investigated breath detection of SARS-CoV-2 infection using cavity-enhanced direct frequency comb spectroscopy (CE-DFCS), a state-of-the-art laser spectroscopic technique capable of a real-time massive collection of broadband molecular absorption features at ro-vibrational quantum state resolution and at parts-per-trillion volume detection sensitivity. Using a total of 170 individual breath samples (83 positive and 87 negative with SARS-CoV-2 based on Reverse Transcription Polymerase Chain Reaction tests), we report excellent discrimination capability for SARS-CoV-2 infection with an area under the Receiver-Operating-Characteristics curve of 0.849(4). Our results support the development of CE-DFCS as an alternative, rapid, non-invasive test for COVID-19 and highlight its remarkable potential for optical diagnoses of diverse biological conditions and disease states