A MicroRNA Linking Human Positive Selection and Metabolic Disorders.

Positive selection in Europeans at the 2q21.3 locus harboring the lactase gene has been attributed to selection for the ability of adults to digest milk to survive famine in ancient times. However, the 2q21.3 locus is also associated with obesity and type 2 diabetes in humans, raising the possibility that additional genetic elements in the locus may have contributed to evolutionary adaptation to famine by promoting energy storage, but which now confer susceptibility to metabolic diseases. We show here that the miR-128-1 microRNA, located at the center of the positively selected locus, represents a crucial metabolic regulator in mammals. Antisense targeting and genetic ablation of miR-128-1 in mouse metabolic disease models result in increased energy expenditure and amelioration of high-fat-diet-induced obesity and markedly improved glucose tolerance. A thrifty phenotype connected to miR-128-1-dependent energy storage may link ancient adaptation to famine and modern metabolic maladaptation associated with nutritional overabundance.

Mechanisms of mechanical overload-induced skeletal muscle hypertrophy: current understanding and future directions.

Mechanisms underlying mechanical overload-induced skeletal muscle hypertrophy have been extensively researched since the landmark report by Morpurgo (1897) of "work-induced hypertrophy" in dogs that were treadmill trained. Much of the preclinical rodent and human resistance training research to date supports that involved mechanisms include enhanced mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, an expansion in translational capacity through ribosome biogenesis, increased satellite cell abundance and myonuclear accretion, and postexercise elevations in muscle protein synthesis rates. However, several lines of past and emerging evidence suggest that additional mechanisms that feed into or are independent of these processes are also involved. This review first provides a historical account of how mechanistic research into skeletal muscle hypertrophy has progressed. A comprehensive list of mechanisms associated with skeletal muscle hypertrophy is then outlined, and areas of disagreement involving these mechanisms are presented. Finally, future research directions involving many of the discussed mechanisms are proposed.

FOXC1 and FOXC2 regulate growth plate chondrocyte maturation towards hypertrophy in the embryonic mouse limb skeleton.

The Forkhead box transcription factors FOXC1 and FOXC2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, whereas the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed, with fewer Indian hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocytes and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that FOXC1 and FOXC2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, FOXC1 and FOXC2 regulate function in hypertrophic chondrocyte remodeling to allow primary ossification center formation and osteoblast recruitment.

A PGC-1α isoform induced by resistance training regulates skeletal muscle hypertrophy.

PGC-1α is a transcriptional coactivator induced by exercise that gives muscle many of the best known adaptations to endurance-type exercise but has no effects on muscle strength or hypertrophy. We have identified a form of PGC-1α (PGC-1α4) that results from alternative promoter usage and splicing of the primary transcript. PGC-1α4 is highly expressed in exercised muscle but does not regulate most known PGC-1α targets such as the mitochondrial OXPHOS genes. Rather, it specifically induces IGF1 and represses myostatin, and expression of PGC-1α4 in vitro and in vivo induces robust skeletal muscle hypertrophy. Importantly, mice with skeletal muscle-specific transgenic expression of PGC-1α4 show increased muscle mass and strength and dramatic resistance to the muscle wasting of cancer cachexia. Expression of PGC-1α4 is preferentially induced in mouse and human muscle during resistance exercise. These studies identify a PGC-1α protein that regulates and coordinates factors involved in skeletal muscle hypertrophy.

Liver Transplantation in a Woman with Mahvash Disease.

Mahvash disease is an exceedingly rare genetic disorder of glucagon signaling characterized by hyperglucagonemia, hyperaminoacidemia, and pancreatic α-cell hyperplasia. Although there is no known definitive treatment, octreotide has been used to decrease systemic glucagon levels. We describe a woman who presented to our medical center after three episodes of small-volume hematemesis. She was found to have hyperglucagonemia and pancreatic hypertrophy with genetically confirmed Mahvash disease and also had evidence of portal hypertension (recurrent portosystemic encephalopathy and variceal hemorrhage) in the absence of cirrhosis. These findings established a diagnosis of portosinusoidal vascular disease, a presinusoidal type of portal hypertension previously known as noncirrhotic portal hypertension. Liver transplantation was followed by normalization of serum glucagon and ammonia levels, reversal of pancreatic hypertrophy, and resolution of recurrent encephalopathy and bleeding varices.

Autism-related KLHL17 and SYNPO act in concert to control activity-dependent dendritic spine enlargement and the spine apparatus.

Dendritic spines, the tiny and actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain that control synaptic activity and plasticity. Dendritic spines contain a specialized form of endoplasmic reticulum (ER), i.e., the spine apparatus, required for local calcium signaling and that is involved in regulating dendritic spine enlargement and synaptic plasticity. Many autism-linked genes have been shown to play critical roles in synaptic formation and plasticity. Among them, KLHL17 is known to control dendritic spine enlargement during development. As a brain-specific disease-associated gene, KLHL17 is expected to play a critical role in the brain, but it has not yet been well characterized. In this study, we report that KLHL17 expression in mice is strongly regulated by neuronal activity and KLHL17 modulates the synaptic distribution of synaptopodin (SYNPO), a marker of the spine apparatus. Both KLHL17 and SYNPO are F-actin-binding proteins linked to autism. SYNPO is known to maintain the structure of the spine apparatus in mature spines and contributes to synaptic plasticity. Our super-resolution imaging using expansion microscopy demonstrates that SYNPO is indeed embedded into the ER network of dendritic spines and that KLHL17 is closely adjacent to the ER/SYNPO complex. Using mouse genetic models, we further show that Klhl17 haploinsufficiency and knockout result in fewer dendritic spines containing ER clusters and an alteration of calcium events at dendritic spines. Accordingly, activity-dependent dendritic spine enlargement and neuronal activation (reflected by extracellular signal-regulated kinase (ERK) phosphorylation and C-FOS expression) are impaired. In addition, we show that the effect of disrupting the KLHL17 and SYNPO association is similar to the results of Klhl17 haploinsufficiency and knockout, further strengthening the evidence that KLHL17 and SYNPO act together to regulate synaptic plasticity. In conclusion, our findings unravel a role for KLHL17 in controlling synaptic plasticity via its regulation of SYNPO and synaptic ER clustering and imply that impaired synaptic plasticity contributes to the etiology of KLHL17-related disorders.

D-galactose might mediate some of the skeletal muscle hypertrophy-promoting effects of milk-A nutrient to consider for sarcopenia?

Sarcopenia is a process of progressive aging-associated loss of skeletal muscle mass (SMM) recognized as a serious global health issue contributing to frailty and increased all-cause mortality. Exercise and nutritional interventions (particularly intake of dairy products and milk) demonstrate good efficacy, safety, and broad applicability. Here, we propose that at least some of the well-documented favorable effects of milk and milk-derived protein supplements on SMM might be mediated by D-galactose, a monosaccharide present in large quantities in milk in the form of disaccharide lactose (milk sugar). We suggest that ingestion of dairy products results in exposure to D-galactose in concentrations metabolized primarily via the Leloir pathway with the potential to (i) promote anabolic signaling via maintenance of growth factor (e.g., insulin-like growth factor 1 [IGF-1]) receptor mature glycosylation patterns; and (ii) provide extracellular (liver glycogen) and intracellular substrates for short (muscle glycolysis) and long-term (muscle glycogen, intramyocellular lipids) energy availability. Additionally, D-galactose might optimize the metabolic function of skeletal muscles by increasing mitochondrial content and stimulating glucose and fatty acid utilization. The proposed potential of D-galactose to promote the accretion of SMM is discussed in the context of its therapeutic potential in sarcopenia.

The MYPT2-regulated striated muscle-specific myosin light chain phosphatase limits cardiac myosin phosphorylation in vivo.

The physiological importance of cardiac myosin regulatory light chain (RLC) phosphorylation by its dedicated cardiac myosin light chain kinase has been established in both humans and mice. Constitutive RLC-phosphorylation, regulated by the balanced activities of cardiac myosin light chain kinase and myosin light chain phosphatase (MLCP), is fundamental to the biochemical and physiological properties of myofilaments. However, limited information is available on cardiac MLCP. In this study, we hypothesized that the striated muscle-specific MLCP regulatory subunit, MYPT2, targets the phosphatase catalytic subunit to cardiac myosin, contributing to the maintenance of cardiac function in vivo through the regulation of RLC-phosphorylation. To test this hypothesis, we generated a floxed-PPP1R12B mouse model crossed with a cardiac-specific Mer-Cre-Mer to conditionally ablate MYPT2 in adult cardiomyocytes. Immunofluorescence microscopy using the gene-ablated tissue as a control confirmed the localization of MYPT2 to regions where it overlaps with a subset of RLC. Biochemical analysis revealed an increase in RLC-phosphorylation in vivo. The loss of MYPT2 demonstrated significant protection against pressure overload-induced hypertrophy, as evidenced by heart weight, qPCR of hypertrophy-associated genes, measurements of myocyte diameters, and expression of ß-MHC protein. Furthermore, mantATP chase assays revealed an increased ratio of myosin heads distributed to the interfilament space in MYPT2-ablated heart muscle fibers, confirming that RLC-phosphorylation regulated by MLCP, enhances cardiac performance in vivo. Our findings establish MYPT2 as the regulatory subunit of cardiac MLCP, distinct from the ubiquitously expressed canonical smooth muscle MLCP. Targeting MYPT2 to increase cardiac RLC-phosphorylation in vivo may improve baseline cardiac performance, thereby attenuating pathological hypertrophy.

Loss-of-function G6PD variant moderated high-fat diet-induced obesity, adipocyte hypertrophy, and fatty liver in male rats.

Obesity is a major risk factor for liver and cardiovascular diseases. However, obesity-driven mechanisms that contribute to the pathogenesis of multiple organ diseases are still obscure and treatment is inadequate. We hypothesized that increased , glucose-6-phosphate dehydrogenase (G6PD), the key rate-limiting enzyme in the pentose shunt, is critical in evoking metabolic reprogramming in multiple organs and is a significant contributor to the pathogenesis of liver and cardiovascular diseases. G6PD is induced by a carbohydrate-rich diet and insulin. Long-term (8 months) high-fat diet (HFD) feeding increased body weight and elicited metabolic reprogramming in visceral fat, liver, and aorta, of the wild-type rats. In addition, HFD increased inflammatory chemokines in visceral fat. Interestingly, CRISPR-edited loss-of-function Mediterranean G6PD variant (G6PDS188F) rats, which mimic human polymorphism, moderated HFD-induced weight gain and metabolic reprogramming in visceral fat, liver, and aorta. The G6PDS188F variant prevented HFD-induced CCL7 and adipocyte hypertrophy. Furthermore, the G6PDS188F variant increased Magel2 - a gene encoding circadian clock-related protein that suppresses obesity associated with Prader-Willi syndrome - and reduced HFD-induced non-alcoholic fatty liver. Additionally, the G6PDS188F variant reduced aging-induced aortic stiffening. Our findings suggest G6PD is a regulator of HFD-induced obesity, adipocyte hypertrophy, and fatty liver.

The Elusive Hypertrophy of the Python Heart.

The Burmese python, one of the world's largest snakes, has reached celebrity status for its dramatic physiological responses associated with digestion of enormous meals. The meals elicit a rapid gain of mass and function of most visceral organs, particularly the small intestine. There is also a manyfold elevation of oxygen consumption that demands the heart to deliver more oxygen. It therefore made intuitive sense when it was reported that the postprandial response entailed a 40% growth of heart mass that could accommodate a rise in stroke volume. Many studies, however, have not been able to reproduce the 40% growth of the heart. We collated published values on postprandial heart mass in pythons, which include several instances of no change in heart mass. On average, the heart mass is only 15% greater. The changes in heart mass did not correlate to the mass gain of the small intestine or peak oxygen consumption. Hemodynamic studies show that the rise in cardiac output does not require increased heart mass but can be fully explained by augmented cardiac filling and postprandial tachycardia. Under the assumption that hypertrophy is a contingent phenomenon, more recent experiments have employed two interventions such as feeding with a concomitant reduction in hematocrit. The results suggest that the postprandial response of the heart can be enhanced, but the 40% hypertrophy of the python heart remains elusive.

GDF5+ chondroprogenitors derived from human pluripotent stem cells preferentially form permanent chondrocytes.

It has been established in the mouse model that during embryogenesis joint cartilage is generated from a specialized progenitor cell type, distinct from that responsible for the formation of growth plate cartilage. We recently found that mesodermal progeny of human pluripotent stem cells gave rise to two types of chondrogenic mesenchymal cells in culture: SOX9+ and GDF5+ cells. The fast-growing SOX9+ cells formed in vitro cartilage that expressed chondrocyte hypertrophy markers and readily underwent mineralization after ectopic transplantation. In contrast, the slowly growing GDF5+ cells derived from SOX9+ cells formed cartilage that tended to express low to undetectable levels of chondrocyte hypertrophy markers, but expressed PRG4, a marker of embryonic articular chondrocytes. The GDF5+-derived cartilage remained largely unmineralized in vivo. Interestingly, chondrocytes derived from the GDF5+ cells seemed to elicit these activities via non-cell-autonomous mechanisms. Genome-wide transcriptomic analyses suggested that GDF5+ cells might contain a teno/ligamento-genic potential, whereas SOX9+ cells resembled neural crest-like progeny-derived chondroprogenitors. Thus, human pluripotent stem cell-derived GDF5+ cells specified to generate permanent-like cartilage seem to emerge coincidentally with the commitment of the SOX9+ progeny to the tendon/ligament lineage.

Leptin-dependent differential remodeling of visceral and pericardial adipose tissue following chronic exercise and psychosocial stress.

Obesity is driven by an imbalance between caloric intake and energy expenditure, causing excessive storage of triglycerides in adipose tissue at different sites around the body. Increased visceral adipose tissue (VAT) is associated with diabetes, while pericardial adipose tissue (PAT) is associated with cardiac pathology. Adipose tissue can expand either through cellular hypertrophy or hyperplasia, with the former correlating with decreased metabolic health in obesity. The aim of this study was to determine how VAT and PAT remodel in response to obesity, stress, and exercise. Here we have used the male obese Zucker rats, which carries two recessive fa alleles that result in the development of hyperphagia with reduced energy expenditure, resulting in morbid obesity and leptin resistance. At 9 weeks of age, a group of lean (Fa/Fa or Fa/fa) Zucker rats (LZR) and obese (fa/fa) Zucker rats (OZR) were treated with unpredictable chronic mild stress or exercise for 8 weeks. To determine the phenotype for PAT and VAT, tissue cellularity and gene expression were analyzed. Finally, leptin signaling was investigated further using cultured 3T3-derived adipocytes. Tissue cellularity was determined following hematoxylin and eosin (H&E) staining, while qPCR was used to examine gene expression. PAT adipocytes were significantly smaller than those from VAT and had a more beige-like appearance in both LZR and OZR. In the OZR group, VAT adipocyte cell size increased significantly compared with LZR, while PAT showed no difference. Exercise and stress resulted in a significant reduction in VAT cellularity in OZR, while PAT showed no change. This suggests that PAT cellularity does not remodel significantly compared with VAT. These data indicate that the extracellular matrix of PAT is able to remodel more readily than in VAT. In the LZR group, exercise increased insulin receptor substrate 1 (IRS1) in PAT but was decreased in the OZR group. In VAT, exercise decreased IRS1 in LZR, while increasing it in OZR. This suggests that in obesity, VAT is more responsive to exercise and subsequently becomes less insulin resistant compared with PAT. Stress increased PPAR-γ expression in the VAT but decreased it in the PAT in the OZR group. This suggests that in obesity, stress increases adipogenesis more significantly in the VAT compared with PAT. To understand the role of leptin signaling in adipose tissue remodeling mechanistically, JAK2 autophosphorylation was inhibited using 5 µM 1,2,3,4,5,6-hexabromocyclohexane (Hex) in cultured 3T3-derived adipocytes. Palmitate treatment was used to induce cellular hypertrophy. Hex blocked adipocyte hypertrophy in response to palmitate treatment but not the increase in lipid droplet size. These data suggest that leptin signaling is necessary for adipocyte cell remodeling, and its absence induces whitening. Taken together, our data suggest that leptin signaling is necessary for adipocyte remodeling in response to obesity, exercise, and psychosocial stress.

Nuclear-localized androgen receptor content following resistance exercise training is associated with hypertrophy in males but not females.

Androgen receptor (AR) content has been implicated in the differential response between high and low responders following resistance exercise training (RET). However, the influence of AR expression on acute skeletal muscle damage and whether it may influence the adaptive response to RET in females is poorly understood. Thus, the purpose of this exploratory examination was to 1) investigate changes in AR content during skeletal muscle repair and 2) characterize AR-mediated sex-based differences following RET. A skeletal muscle biopsy from the vastus lateralis was obtained from 26 healthy young men (n = 13) and women (n = 13) at baseline and following 300 eccentric kicks. Subsequently, participants performed 10 weeks of full-body RET and a final muscle biopsy was collected. In the untrained state, AR mRNA expression was associated with paired box protein-7 (PAX7) mRNA in males. For the first time in human skeletal muscle, we quantified AR content in the myofiber and localized to the nucleus where AR has been shown to trigger cellular outcomes related to growth. Upon eccentric damage, nuclear-associated AR (nAR) content increased (p < .05) in males and not females. Males with the greatest increase in cross-sectional area (CSA) post-RET had more (p < .05) nAR content than females with the greatest gain CSA. Collectively, skeletal muscle damage and RET increased AR protein, and both gene and hypertrophy measures revealed sex differences in relation to AR. These findings suggest that AR content but more importantly, nuclear localization, is a factor that differentiates RET-induced hypertrophy between males and females.

Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte Hypertrophy.

BACKGROUND: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac ß-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. METHODS: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with ß-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. RESULTS: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. CONCLUSIONS: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors.

Systemic Hypoxemia Induces Cardiomyocyte Hypertrophy and Right Ventricular Specific Induction of Proliferation.

BACKGROUND: A recent study suggests that systemic hypoxemia in adult male mice can induce cardiac myocytes to proliferate. The goal of the present experiments was to confirm these results, provide new insights on the mechanisms that induce adult cardiomyocyte cell cycle reentry, and to determine if hypoxemia also induces cardiomyocyte proliferation in female mice. METHODS: EdU-containing mini pumps were implanted in 3-month-old, male and female C57BL/6 mice. Mice were placed in a hypoxia chamber, and the oxygen was lowered by 1% every day for 14 days to reach 7% oxygen. The animals remained in 7% oxygen for 2 weeks before terminal studies. Myocyte proliferation was also studied with a mosaic analysis with double markers mouse model. RESULTS: Hypoxia induced cardiac hypertrophy in both left ventricular (LV) and right ventricular (RV) myocytes, with LV myocytes lengthening and RV myocytes widening and lengthening. Hypoxia induced an increase (0.01±0.01% in normoxia to 0.11±0.09% in hypoxia) in the number of EdU+ RV cardiomyocytes, with no effect on LV myocytes in male C57BL/6 mice. Similar results were observed in female mice. Furthermore, in mosaic analysis with double markers mice, hypoxia induced a significant increase in RV myocyte proliferation (0.03±0.03% in normoxia to 0.32±0.15% in hypoxia of RFP+ myocytes), with no significant change in LV myocyte proliferation. RNA sequencing showed upregulation of mitotic cell cycle genes and a downregulation of Cullin genes, which promote the G1 to S phase transition in hypoxic mice. There was significant proliferation of nonmyocytes and mild cardiac fibrosis in hypoxic mice that did not disrupt cardiac function. Male and female mice exhibited similar gene expression following hypoxia. CONCLUSIONS: Systemic hypoxia induces a global hypertrophic stress response that was associated with increased RV proliferation, and while LV myocytes did not show increased proliferation, our results minimally confirm previous reports that hypoxia can induce cardiomyocyte cell cycle activity in vivo.

STAT5b is a key effector of NRG-1/ERBB4-mediated myocardial growth.

The growth factor Neuregulin-1 (NRG-1) regulates myocardial growth and is currently under clinical investigation as a treatment for heart failure. Here, we demonstrate in several in vitro and in vivo models that STAT5b mediates NRG-1/EBBB4-stimulated cardiomyocyte growth. Genetic and chemical disruption of the NRG-1/ERBB4 pathway reduces STAT5b activation and transcription of STAT5b target genes Igf1, Myc, and Cdkn1a in murine cardiomyocytes. Loss of Stat5b also ablates NRG-1-induced cardiomyocyte hypertrophy. Dynamin-2 is shown to control the cell surface localization of ERBB4 and chemical inhibition of Dynamin-2 downregulates STAT5b activation and cardiomyocyte hypertrophy. In zebrafish embryos, Stat5 is activated during NRG-1-induced hyperplastic myocardial growth, and chemical inhibition of the Nrg-1/Erbb4 pathway or Dynamin-2 leads to loss of myocardial growth and Stat5 activation. Moreover, CRISPR/Cas9-mediated knockdown of stat5b results in reduced myocardial growth and cardiac function. Finally, the NRG-1/ERBB4/STAT5b signaling pathway is differentially regulated at mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to control human subjects, consistent with a role of the NRG-1/ERBB4/STAT5b pathway in myocardial growth.

PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress.

The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis and autophagy1. Its hyperactivation contributes to disease in numerous organs, including the heart1,2, although broad inhibition of mTORC1 risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 that acts through modulation of RHEB (Ras homologue enriched in brain). TSC2 constitutively inhibits mTORC1; however, this activity is modified by phosphorylation from multiple signalling kinases that in turn inhibits (AMPK and GSK-3ß) or stimulates (AKT, ERK and RSK-1) mTORC1 activity3-9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here we show that phosphorylation or gain- or loss-of-function mutations at either of two adjacent serine residues in TSC2 (S1365 and S1366 in mice; S1364 and S1365 in humans) can bidirectionally control mTORC1 activity stimulated by growth factors or haemodynamic stress, and consequently modulate cell growth and autophagy. However, basal mTORC1 activity remains unchanged. In the heart, or in isolated cardiomyocytes or fibroblasts, protein kinase G1 (PKG1) phosphorylates these TSC2 sites. PKG1 is a primary effector of nitric oxide and natriuretic peptide signalling, and protects against heart disease10-13. Suppression of hypertrophy and stimulation of autophagy in cardiomyocytes by PKG1 requires TSC2 phosphorylation. Homozygous knock-in mice that express a phosphorylation-silencing mutation in TSC2 (TSC2(S1365A)) develop worse heart disease and have higher mortality after sustained pressure overload of the heart, owing to mTORC1 hyperactivity that cannot be rescued by PKG1 stimulation. However, cardiac disease is reduced and survival of heterozygote Tsc2S1365A knock-in mice subjected to the same stress is improved by PKG1 activation or expression of a phosphorylation-mimicking mutation (TSC2(S1365E)). Resting mTORC1 activity is not altered in either knock-in model. Therefore, TSC2 phosphorylation is both required and sufficient for PKG1-mediated cardiac protection against pressure overload. The serine residues identified here provide a genetic tool for bidirectional regulation of the amplitude of stress-stimulated mTORC1 activity.

Emerging role of TAK1 in the regulation of skeletal muscle mass.

Maintenance of skeletal muscle mass and strength throughout life is crucial for heathy living and longevity. Several signaling pathways have been implicated in the regulation of skeletal muscle mass in adults. TGF-ß-activated kinase 1 (TAK1) is a key protein, which coordinates the activation of multiple signaling pathways. Recently, it was discovered that TAK1 is essential for the maintenance of skeletal muscle mass and myofiber hypertrophy following mechanical overload. Forced activation of TAK1 in skeletal muscle causes hypertrophy and attenuates denervation-induced muscle atrophy. TAK1-mediated signaling in skeletal muscle promotes protein synthesis, redox homeostasis, mitochondrial health, and integrity of neuromuscular junctions. In this article, we have reviewed the role and potential mechanisms through which TAK1 regulates skeletal muscle mass and growth. We have also proposed future areas of research that could be instrumental in exploring TAK1 as therapeutic target for improving muscle mass in various catabolic conditions and diseases.

Ablation of lysophosphatidic acid receptor 1 attenuates hypertrophic cardiomyopathy in a mouse model.

Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403+/-) with Lpar1-ablated mice to create mice carrying both genetic changes (403+/- LPAR1 -/-) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403+/- LPAR1WT, 403+/- LPAR1 -/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a. Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.

Molecular basis of force-pCa relation in MYL2 cardiomyopathy mice: Role of the super-relaxed state of myosin.

In this study, we investigated the role of the super-relaxed (SRX) state of myosin in the structure-function relationship of sarcomeres in the hearts of mouse models of cardiomyopathy-bearing mutations in the human ventricular regulatory light chain (RLC, MYL2 gene). Skinned papillary muscles from hypertrophic (HCM-D166V) and dilated (DCM-D94A) cardiomyopathy models were subjected to small-angle X-ray diffraction simultaneously with isometric force measurements to obtain the interfilament lattice spacing and equatorial intensity ratios (I11/I10) together with the force-pCa relationship over a full range of [Ca2+] and at a sarcomere length of 2.1 µm. In parallel, we studied the effect of mutations on the ATP-dependent myosin energetic states. Compared with wild-type (WT) and DCM-D94A mice, HCM-D166V significantly increased the Ca2+ sensitivity of force and left shifted the I11/I10-pCa relationship, indicating an apparent movement of HCM-D166V cross-bridges closer to actin-containing thin filaments, thereby allowing for their premature Ca2+ activation. The HCM-D166V model also disrupted the SRX state and promoted an SRX-to-DRX (super-relaxed to disordered relaxed) transition that correlated with an HCM-linked phenotype of hypercontractility. While this dysregulation of SRX ↔ DRX equilibrium was consistent with repositioning of myosin motors closer to the thin filaments and with increased force-pCa dependence for HCM-D166V, the DCM-D94A model favored the energy-conserving SRX state, but the structure/function-pCa data were similar to WT. Our results suggest that the mutation-induced redistribution of myosin energetic states is one of the key mechanisms contributing to the development of complex clinical phenotypes associated with human HCM-D166V and DCM-D94A mutations.