Young at heart.

In this issue of Cell, Loffredo et al. demonstrate that exposing an old mouse to the circulatory system of a young mouse reverses age-related cardiac hypertrophy. The authors demonstrate that this effect can be recapitulated by treating old mice with growth and differentiation factor 11 (GDF11). These data suggest that GDF11 therapy may be a useful tool in combating age-related cardiac hypertrophy.

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.

Utility of the burmese Python as a model for studying plasticity of extreme physiological systems.

Non-traditional animal models present an opportunity to discover novel biology that has evolved to allow such animals to survive in extreme environments. One striking example is the Burmese python (Python molurus bivittatus), which exhibits extreme physiological adaptation in various metabolic organs after consuming a large meal following long periods of fasting. The response to such a large meal in pythons involves a dramatic surge in metabolic rate, lipid overload in plasma, and massive but reversible organ growth through the course of digestion. Multiple studies have reported the physiological responses in post-prandial pythons, while the specific molecular control of these processes is less well-studied. Investigating the mechanisms that coordinate organ growth and adaptive responses offers the opportunity to gain novel insight that may be able to treat various pathologies in humans. Here, we summarize past research on the post-prandial physiological changes in the Burmese python with a focus on the gastrointestinal tract, heart, and liver. Specifically, we address our recent molecular discoveries in the post-prandial python liver which demonstrate transient adaptations that may reveal new therapeutic targets. Lastly, we explore new biology of the aquaporin 7 gene that is potently upregulated in mammalian cardiac myocytes by circulating factors in post-prandial python plasma.

miR-206 enforces a slow muscle phenotype.

Striated muscle is a highly specialized collection of tissues with contractile properties that vary according to functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-dependent adaptation. Functional adaptation requires molecular adaptation, which is partially provided by miRNA-mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-206 expression increases in both physiological (including female sex and endurance exercise) and pathological conditions (muscular dystrophy and adrenergic agonism) that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-206-knockout mice displays a faster phenotype than wild-type mice. Moreover, left ventricles of male miR-206 knockout mice have a faster myosin profile, accompanied by dilation and systolic dysfunction. Thus, miR-206 appears to be necessary to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms.

Signal-dependent repression of DUSP5 by class I HDACs controls nuclear ERK activity and cardiomyocyte hypertrophy.

Cardiac hypertrophy is a strong predictor of morbidity and mortality in patients with heart failure. Small molecule histone deacetylase (HDAC) inhibitors have been shown to suppress cardiac hypertrophy through mechanisms that remain poorly understood. We report that class I HDACs function as signal-dependent repressors of cardiac hypertrophy via inhibition of the gene encoding dual-specificity phosphatase 5 (DUSP5) DUSP5, a nuclear phosphatase that negatively regulates prohypertrophic signaling by ERK1/2. Inhibition of DUSP5 by class I HDACs requires activity of the ERK kinase, mitogen-activated protein kinase kinase (MEK), revealing a self-reinforcing mechanism for promotion of cardiac ERK signaling. In cardiac myocytes treated with highly selective class I HDAC inhibitors, nuclear ERK1/2 signaling is suppressed in a manner that is absolutely dependent on DUSP5. In contrast, cytosolic ERK1/2 activation is maintained under these same conditions. Ectopic expression of DUSP5 in cardiomyocytes results in potent inhibition of agonist-dependent hypertrophy through a mechanism involving suppression of the gene program for hypertrophic growth. These findings define unique roles for class I HDACs and DUSP5 as integral components of a regulatory signaling circuit that controls cardiac hypertrophy.

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.

Longitudinal characterization of functional, morphologic, and biochemical adaptations in mouse skeletal muscle with hindlimb suspension.

INTRODUCTION: Hindlimb unloading-induced muscle atrophy is often assessed after a homeostatic state is established, thus overlooking the early adaptations that are critical to developing this pattern of atrophy. METHODS: Muscle function and physiology were characterized at 0, 1, 3, 7, and 14 days of hindlimb suspension (HS). RESULTS: Reductions in muscle mass were maximal by Day 14 of HS. Functional strength and isolated muscle strength were reduced. MyHC-I and -IIa expressing fibers were reduced in size by Day 7 in the soleus and by Day 14 in the gastrocnemius (MyHC-I fibers only). Atrogin-1 and MuRF1 expression was increased by Day 1 in both the calf and tibialis anterior while IGF-1 expression was significantly reduced on Day 3. Phosphorylation of Akt was reduced on Day 14. CONCLUSIONS: Insight into these early changes in response to HS improves understanding of the molecular and functional changes that lead to muscle atrophy.

Inhibiting myostatin signaling partially mitigates structural and functional adaptations to hindlimb suspension in mice.

Novel treatments for muscle wasting are of significant value to patients with disease states that result in muscle weakness, injury recovery after immobilization and bed rest, and for astronauts participating in long-duration spaceflight. We utilized an anti-myostatin peptibody to evaluate how myostatin signaling contributes to muscle loss in hindlimb suspension. Male C57BL/6 mice were left non-suspended (NS) or were hindlimb suspended (HS) for 14 days and treated with a placebo vehicle (P) or anti-myostatin peptibody (D). Hindlimb suspension (HS-P) resulted in rapid and significantly decreased body mass (-5.6% by day 13) with hindlimb skeletal muscle mass losses between -11.2% and -22.5% and treatment with myostatin inhibitor (HS-D) partially attenuated these losses. Myostatin inhibition increased hindlimb strength with no effect on soleus tetanic strength. Soleus mass and fiber CSA were reduced with suspension and did not increase with myostatin inhibition. In contrast, the gastrocnemius showed histological evidence of wasting with suspension that was partially mitigated with myostatin inhibition. While expression of genes related to protein degradation (Atrogin-1 and Murf-1) in the tibialis anterior increased with suspension, these atrogenes were not significantly reduced by myostatin inhibition despite a modest activation of the Akt/mTOR pathway. Taken together, these findings suggest that myostatin is important in hindlimb suspension but also motivates the study of other factors that contribute to disuse muscle wasting. Myostatin inhibition benefitted skeletal muscle size and function, which suggests therapeutic potential for both spaceflight and terrestrial applications.

Burmese pythons exhibit a transient adaptation to nutrient overload that prevents liver damage.

As an opportunistic predator, the Burmese python (Python molurus bivittatus) consumes large and infrequent meals, fasting for up to a year. Upon consuming a large meal, the Burmese python exhibits extreme metabolic responses. To define the pathways that regulate these postprandial metabolic responses, we performed a comprehensive profile of plasma metabolites throughout the digestive process. Following ingestion of a meal equivalent to 25% of its body mass, plasma lipoproteins and metabolites, such as chylomicra and bile acids, reach levels observed only in mammalian models of extreme dyslipidemia. Here, we provide evidence for an adaptive response to postprandial nutrient overload by the python liver, a critical site of metabolic homeostasis. The python liver undergoes a substantial increase in mass through proliferative processes, exhibits hepatic steatosis, hyperlipidemia-induced insulin resistance indicated by PEPCK activation and pAKT deactivation, and de novo fatty acid synthesis via FASN activation. This postprandial state is completely reversible. We posit that Burmese pythons evade the permanent hepatic damage associated with these metabolic states in mammals using evolved protective measures to inactivate these pathways. These include a transient activation of hepatic nuclear receptors induced by fatty acids and bile acids, including PPAR and FXR, respectively. The stress-induced p38 MAPK pathway is also transiently activated during the early stages of digestion. Taken together, these data identify a reversible metabolic response to hyperlipidemia by the python liver, only achieved in mammals by pharmacologic intervention. The factors involved in these processes may be relevant to or leveraged for remediating human hepatic pathology.

Effects of rifampicin (rifampin) on the pharmacokinetics and safety of ambrisentan in healthy subjects: a single-sequence, open-label study.

BACKGROUND: Ambrisentan is a once-daily, endothelin (ET) type A receptor-selective antagonist approved for the treatment of pulmonary arterial hypertension. Ambrisentan is primarily metabolized by glucuronidation and undergoes cytochrome P450 (CYP)-mediated oxidation to a lesser extent. OBJECTIVE: To assess the effects of rifampicin (rifampin), a potent inducer of CYP3A4 and inhibitor of organic anion transporter polypeptides (OATPs), on the steady-state pharmacokinetics, safety and tolerability of ambrisentan. METHODS: This was a 14-day, single-sequence, open-label study that was conducted in 24 healthy adults. Subjects were administered oral doses of ambrisentan (10 mg) once daily on days 1 through 5 and were then co-administered ambrisentan (10 mg) plus rifampicin (600 mg) once daily on days 6 through 13. The steady-state pharmacokinetics of ambrisentan and its oxidative metabolite 4-hydroxymethyl ambrisentan were determined in the absence and presence of repeated administration of rifampicin. The main outcome measure was the analysis of ambrisentan pharmacokinetics (area under the plasma concentration-time curve during a dosage interval [AUC(τ)], maximum plasma drug concentration [C(max)] and minimum plasma drug concentration [C(min)]) for steady-state ambrisentan alone (day 5) as compared with steady-state ambrisentan plus steady-state rifampicin (day 13). Adverse events (AEs), ECG recordings, vital signs and clinical laboratory parameters were monitored throughout the study and at follow-up. RESULTS: A transient increase (+87% [95% CI 79, 95]) in ambrisentan steady-state systemic exposure (AUC(τ)) was observed during the first 2 days of rifampicin co-administration. However, in the presence of steady-state rifampicin, ambrisentan C(max) and AUC(τ) values were similar (+2% [95% CI -7, 12] and -4% [-9, 2], respectively) to those observed for ambrisentan alone. Relative systemic exposure of 4-hydroxymethyl ambrisentan was unaffected by either acute or steady-state rifampicin. No serious AEs or AEs leading to withdrawal were reported and there were no clinically significant changes in vital signs, ECG recordings or clinical laboratory parameters with co-administration of ambrisentan and rifampicin. CONCLUSION: Steady-state rifampicin had no clinically relevant effects on the steady-state pharmacokinetics of ambrisentan. The overall safety profile of ambrisentan was similar in the presence and absence of rifampicin. No dose adjustment of ambrisentan should be required when it is co-administered with rifampicin, a strong inducer of CYP3A4 activity and inhibitor of OATPs.

Effects of spaceflight on murine skeletal muscle gene expression.

Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts toward faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11-day, 19-h STS-108 shuttle flight and from normal gravity controls. Spaceflight data also were compared with the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 h of reloading to mimic the time between landing and euthanization of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, whereas reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the phosphatidylinositol 3-kinase regulatory subunit p85alpha, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase, whereas mRNA levels of the myostatin inhibitor FSTL3 tended to decrease, in response to spaceflight. In addition, mRNA levels of the slow oxidative fiber-associated transcriptional coactivator peroxisome proliferator-associated receptor (PPAR)-gamma coactivator-1alpha and the transcription factor PPAR-alpha were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type.

Regulation of cardiac stress signaling by protein kinase d1.

In response to pathological stresses such as hypertension or myocardial infarction, the heart undergoes a remodeling process that is associated with myocyte hypertrophy, myocyte death, and fibrosis. Histone deacetylase 5 (HDAC5) is a transcriptional repressor of cardiac remodeling that is subject to phosphorylation-dependent neutralization in response to stress signaling. Recent studies have suggested a role for protein kinase C (PKC) and its downstream effector, protein kinase D1 (PKD1), in the control of HDAC5 phosphorylation. While PKCs are well-documented regulators of cardiac signaling, the function of PKD1 in heart muscle remains unclear. Here, we demonstrate that PKD1 catalytic activity is stimulated in cardiac myocytes by diverse hypertrophic agonists that signal through G protein-coupled receptors (GPCRs) and Rho GTPases. PKD1 activation in cardiomyocytes occurs through PKC-dependent and -independent mechanisms. In vivo, cardiac PKD1 is activated in multiple rodent models of pathological cardiac remodeling. PKD1 activation correlates with phosphorylation-dependent nuclear export of HDAC5, and reduction of endogenous PKD1 expression with small interfering RNA suppresses HDAC5 shuttling and associated cardiomyocyte growth. Conversely, ectopic overexpression of constitutively active PKD1 in mouse heart leads to dilated cardiomyopathy. These findings support a role for PKD1 in the control of pathological remodeling of the heart via its ability to phosphorylate and neutralize HDAC5.

A fatal case of dermatomyositis with underlying metastatic esophageal adenocarcinoma.

Dermatomyositis (DM) is known to be associated with underlying malignancy, though the strength of this relationship and its predisposing factors are not clearly defined. We present a case of a patient who was first diagnosed with DM and, subsequently, metastatic esophageal adenocarcinoma. Despite aggressive immunosuppressive therapy, the patient's cutaneous eruption failed to resolve and his muscle weakness progressed. He had respiratory failure and died less than 2 months after his initial presentation. To our knowledge, this is only the second case of metastatic esophageal adenocarcinoma associated with DM reported in the English language literature.

Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5.

A variety of stress signals stimulate cardiac myocytes to undergo hypertrophy. Persistent cardiac hypertrophy is associated with elevated risk for the development of heart failure. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy and that stress signals neutralize this repressive function by triggering phosphorylation- and CRM1-dependent nuclear export of these chromatin-modifying enzymes. However, the identities of cardiac HDAC kinases have remained unclear. Here, we demonstrate that signaling by protein kinase C (PKC) is sufficient and, in some cases, necessary to drive nuclear export of class II HDAC5 in cardiomyocytes. Inhibition of PKC prevents nucleocytoplasmic shuttling of HDAC5 in response to a subset of hypertrophic agonists. Moreover, a nonphosphorylatable HDAC5 mutant is refractory to PKC signaling and blocks cardiomyocyte hypertrophy mediated by pharmacological activators of PKC. We also demonstrate that protein kinase D (PKD), a downstream effector of PKC, directly phosphorylates HDAC5 and stimulates its nuclear export. These findings reveal a novel function for the PKC/PKD axis in coupling extracellular cues to chromatin modifications that control cellular growth, and they suggest potential utility for small-molecule inhibitors of this pathway in the treatment of pathological cardiac gene expression.

The CRM1 nuclear export receptor controls pathological cardiac gene expression.

Diverse pathological insults trigger a cardiac remodeling process during which myocytes undergo hypertrophy, with consequent decline in cardiac function and eventual heart failure. Multiple transcriptional regulators of pathological cardiac hypertrophy are controlled at the level of subcellular distribution. For example, prohypertrophic transcription factors belonging to the nuclear factor of activated T cells (NFAT) and GATA families are subject to CRM1-dependent nuclear export but are rapidly relocalized to the nucleus in response to cues for hypertrophic growth. Here, we demonstrate that the antihypertrophic chromatin-modifying enzyme histone deacetylase 5 (HDAC5) is shuttled out of the cardiomyocyte nucleus via a CRM1-mediated pathway in response to diverse signals for hypertrophy. CRM1 antagonists block the agonist-mediated nuclear export of HDAC 5 and repress pathological gene expression and associated hypertrophy of cultured cardiomyocytes. Conversely, CRM1 activity is dispensable for nonpathological cardiac gene activation mediated by thyroid hormone and insulin-like growth factor 1, agonists that fail to trigger the nuclear export of HDAC5. These results suggest a selective role for CRM1 in derepression of pathological cardiac genes via its neutralizing effects on antihypertrophic factors such as HDAC5. Pharmacological approaches targeting CRM1-dependent nuclear export in heart muscle may have salutary effects on cardiac function by suppressing maladaptive changes in gene expression evoked by stress signals.

Differential Roles for Inner Membrane Complex Proteins across Toxoplasma gondii and Sarcocystis neurona Development.

The inner membrane complex (IMC) of apicomplexan parasites contains a network of intermediate filament-like proteins. The 14 alveolin domain-containing IMC proteins in Toxoplasma gondii fall into different groups defined by their distinct spatiotemporal dynamics during the internal budding process of tachyzoites. Here, we analyzed representatives of different IMC protein groups across all stages of the Toxoplasma life cycle and during Sarcocystis neurona asexual development. We found that across asexually dividing Toxoplasma stages, IMC7 is present exclusively in the mother's cytoskeleton, whereas IMC1 and IMC3 are both present in mother and daughter cytoskeletons (IMC3 is strongly enriched in daughter buds). In developing macro- and microgametocytes, IMC1 and -3 are absent, whereas IMC7 is lost in early microgametocytes but retained in macrogametocytes until late in their development. We found no roles for IMC proteins during meiosis and sporoblast formation. However, we observed that IMC1 and IMC3, but not IMC7, are present in sporozoites. Although the spatiotemporal pattern of IMC15 and IMC3 suggests orthologous functions in Sarcocystis, IMC7 may have functionally diverged in Sarcocystis merozoites. To functionally characterize IMC proteins, we knocked out IMC7, -12, -14, and -15 in Toxoplasma. IMC14 and -15 appear to be involved in switching between endodyogeny and endopolygeny. In addition, IMC7, -12, and -14, which are all recruited to the cytoskeleton outside cytokinesis, are critical for the structural integrity of extracellular tachyzoites. Altogether, stage- and development-specific roles for IMC proteins can be discerned, suggesting different niches for each IMC protein across the entire life cycle. IMPORTANCE The inner membrane complex (IMC) is a defining feature of apicomplexan parasites key to both their motility and unique cell division. To provide further insights into the IMC, we analyzed the dynamics and functions of representative alveolin domain-containing IMC proteins across developmental stages. Our work shows universal but distinct roles for IMC1, -3, and -7 during Toxoplasma asexual division but more specialized functions for these proteins during gametogenesis. In addition, we find that IMC15 is involved in daughter formation in both Toxoplasma and Sarcocystis. IMC14 and IMC15 function in limiting the number of Toxoplasma offspring per division. Furthermore, IMC7, -12, and -14, which are recruited in the G1 cell cycle stage, are required for stress resistance of extracellular tachyzoites. Thus, although the roles of the different IMC proteins appear to overlap, stage- and development-specific behaviors indicate that their functions are uniquely tailored to each life stage requirement.

Association of Opioids and Sedatives with Increased Risk of In-Hospital Cardiopulmonary Arrest from an Administrative Database.

BACKGROUND: While opioid use confers a known risk for respiratory depression, the incremental risk of in-hospital cardiopulmonary arrest, respiratory arrest, or cardiopulmonary resuscitation (CPRA) has not been studied. Our aim was to investigate the prevalence, outcomes, and risk profile of in-hospital CPRA for patients receiving opioids and medications with central nervous system sedating side effects (sedatives). METHODS: A retrospective analysis of adult inpatient discharges from 2008-2012 reported in the Premier Database. Patients were grouped into four mutually exclusive categories: (1) opioids and sedatives, (2) opioids only, (3) sedatives only, and (4) neither opioids nor sedatives. RESULTS: Among 21,276,691 inpatient discharges, 53% received opioids with or without sedatives. A total of 96,554 patients suffered CPRA (0.92 per 1000 hospital bed-days). Patients who received opioids and sedatives had an adjusted odds ratio for CPRA of 3.47 (95% CI: 3.40-3.54; p<0.0001) compared with patients not receiving opioids or sedatives. Opioids alone and sedatives alone were associated with a 1.81-fold and a 1.82-fold (p<0.0001 for both) increase in the odds of CPRA, respectively. In opioid patients, locations of CPRA were intensive care (54%), general care floor (25%), and stepdown units (15%). Only 42% of patients survived CPRA and only 22% were discharged home. Opioid patients with CPRA had mean increased hospital lengths of stay of 7.57 days and mean increased total hospital costs of $27,569. CONCLUSIONS: Opioids and sedatives are independent and additive risk factors for in-hospital CPRA. The impact of opioid sparing analgesia, reduced sedative use, and better monitoring on CPRA incidence deserves further study.

A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice.

Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle that can be caused by mutations in sarcomere proteins. Clinical diagnosis depends on an abnormal thickening of the heart, but the earliest signs of disease are hyperdynamic contraction and impaired relaxation. Whereas some in vitro studies of power generation by mutant and wild-type sarcomere proteins are consistent with mutant sarcomeres exhibiting enhanced contractile power, others are not. We identified a small molecule, MYK-461, that reduces contractility by decreasing the adenosine triphosphatase activity of the cardiac myosin heavy chain. Here we demonstrate that early, chronic administration of MYK-461 suppresses the development of ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis and attenuates hypertrophic and profibrotic gene expression in mice harboring heterozygous human mutations in the myosin heavy chain. These data indicate that hyperdynamic contraction is essential for HCM pathobiology and that inhibitors of sarcomere contraction may be a valuable therapeutic approach for HCM.