The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop. Bmal1 encodes a core molecular clock transcription factor. Germline Bmal1 knockout mice show a loss of circadian variation in heart rate and blood pressure, and they develop dilated cardiomyopathy. We tested the role of the molecular clock in adult cardiomyocytes by generating mice that allow for the inducible cardiomyocyte-specific deletion of Bmal1 (iCS⌬Bmal1). ECG telemetry showed that cardiomyocyte-specific deletion of Bmal1 (iCS⌬Bmal1 Ϫ/Ϫ ) in adult mice slowed heart rate, prolonged RR and QRS intervals, and increased episodes of arrhythmia. Moreover, isolated iCS⌬Bmal1 Ϫ/Ϫ hearts were more susceptible to arrhythmia during electromechanical stimulation. Examination of candidate cardiac ion channel genes showed that Scn5a, which encodes the principle cardiac voltage-gated Na ϩ channel (NaV1.5), was circadianly expressed in control mouse and rat hearts but not in iCS⌬Bmal1 Ϫ/Ϫ hearts. In vitro studies confirmed circadian expression of a human Scn5a promoter-luciferase reporter construct and determined that overexpression of clock factors transactivated the Scn5a promoter. Loss of Scn5a circadian expression in iCS⌬Bmal1 Ϫ/Ϫ hearts was associated with decreased levels of NaV1.5 and Na ϩ current in ventricular myocytes. We conclude that disruption of the molecular clock in the adult heart slows heart rate, increases arrhythmias, and decreases the functional expression of Scn5a. These findings suggest a potential link between environmental factors that alter the cardiomyocyte molecular clock and factors that influence arrhythmia susceptibility in humans. cardiac excitability; circadian; heart; ion channels; Scn5a; Na ϩ current CIRCADIAN RHYTHMS are approximate 24-h cycles in biology. These rhythms are present at the systems level, the tissue level, the single cell and molecular levels (16,33,34,38). There are several examples of circadian rhythms in the cardiovascular system, with heart rate, blood pressure, and substrate metabolism exhibiting distinct oscillations over time of day (12,13,40,41). The mechanism that underlies circadian function is the molecular clock. The molecular clock is defined, in a simple way, by a transcription-translation feedback mechanism that is composed of the core clock genes Clock, Bmal1, Per1, Per2, Cry1, and Cry2. CLOCK and BMAL1 are transcription factors that heterodimerize and activate transcription of Per1, Per2, Cry1, and Cry2. PER1, PER2, CRY1, and CRY2 form multimers in the cytoplasm of the cell, translocate to the nucleus, and act to inhibit CLOCK:BMAL1 function. This cycle takes ϳ24 h and is the fundamental mechanism underlying circadian rhythms. Components of the core clock have also been shown to regulate the expression of genes outside the clock mechanism, and these genes are designated as clock-controlled genes (CCGs). CCGs often encode transcription factors or proteins that control rate-limiting steps in cell physiology (42).In the last 8 years, resear...

Rationale: The Xin repeat-containing proteins mXin␣ and mXin␤ localize to the intercalated disc of mouse heart and are implicated in cardiac development and function. The mXin␣ directly interacts with ␤-catenin, p120-catenin, and actin filaments. Ablation of mXin␣ results in adult late-onset cardiomyopathy with conduction defects. An upregulation of the mXin␤ in mXin␣-deficient hearts suggests a partial compensation. Objective: The essential roles of mXin␤ in cardiac development and intercalated disc maturation were investigated. Methods and Results: Ablation of mXin␤ led to abnormal heart shape, ventricular septal defects, severe growth retardation, and postnatal lethality with no upregulation of the mXin␣. Postnatal upregulation of mXin␤ in wild-type hearts, as well as altered apoptosis and proliferation in mXin␤-null hearts, suggests that mXin␤ is required for postnatal heart remodeling. The mXin␤-null hearts exhibited a misorganized myocardium as detected by histological and electron microscopic studies and an impaired diastolic function, as suggested by echocardiography and a delay in switching off the slow skeletal troponin I. Loss of mXin␤ resulted in the failure of forming mature intercalated discs and the mislocalization of mXin␣ and N-cadherin. The mXin␤-null hearts showed upregulation of active Stat3 (signal transducer and activator of transcription 3) and downregulation of the activities of Rac1, insulin-like growth factor 1 receptor, protein kinase B, and extracellular signal-regulated kinases 1 and 2. Conclusions: These findings identify not only an essential role of mXin␤ in the intercalated disc maturation but also mechanisms of mXin␤ modulating N-cadherin-mediated adhesion signaling and its crosstalk signaling for postnatal heart growth and animal survival. (Circ Res. 2010;106:1468-1478.)Key Words: N-cadherin-mediated adhesion signaling Ⅲ Xin repeat-containing protein Ⅲ intercalated disc maturation Ⅲ diastolic dysfunction Ⅲ postnatal heart growth A regulatory network of transcription factors is known to control cardiac morphogenesis. Although the core players in this network are highly conserved, from organisms with simple heart-like cells to those with complex four-chambered hearts, it has been theorized and proven that expansion of this regulatory network by adding new transcription factors is a major force for the heart to evolve new structures. 1,2 However, the addition of new transcription factors can only be a part of the mechanism underlying the formation of complex hearts. The transcription factors must act through their downstream targets, which are directly involved in cardiac morphogenesis, growth and function. However, our inventory of such downstream targets remains incomplete.The Xin repeat-containing proteins from chicken and mouse hearts (cXin and mXin␣, respectively) were first identified as a target of the Nkx2.5-Mef2C pathway. 3,4 Another mouse Xin protein, mXin␤ (or myomaxin), has been subsequently identified as a Mef2A downstream target. 5 Evolutionary studies suggest t...

Endorheic basins around the world are suffering from water and ecosystem crisis. To pursue sustainable development, quantifying the hydrological cycle is fundamentally important. However, knowledge gaps exist in how climate change and human activities influence the hydrological cycle in endorheic basins. We used an integrated ecohydrological model, in combination with systematic observations, to analyze the hydrological cycle in the Heihe River Basin, a typical endorheic basin in arid region of China. The water budget was closed for different landscapes, river channel sections, and irrigation districts of the basin from 2001 to 2012. The results showed that climate warming, which has led to greater precipitation, snowmelt, glacier melt, and runoff, is a favorable factor in alleviating water scarcity. Human activities, including ecological water diversion, cropland expansion, and groundwater overexploitation, have both positive and negative effects. The natural oasis ecosystem has been restored considerably, but the overuse of water in midstream and the use of environmental flow for agriculture in downstream have exacerbated the water stress, resulting in unfavorable changes in surface‐ground water interactions and raising concerns regarding how to fairly allocate water resources. Our results suggest that the water resource management in the region should be adjusted to adapt to a changing hydrological cycle, cropland area must be reduced, and the abstraction of groundwater must be controlled. To foster long‐term benefits, water conflicts should be handled from a broad socioeconomic perspective. The findings can provide useful information on endorheic basins to policy makers and stakeholders around the world.

. cIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries. Am J Physiol Heart Circ Physiol 307: H328 -H336, 2014. First published June 6, 2014; doi:10.1152/ajpheart.00132.2014cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3=,5=-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (PO2: 25-30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5=-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr 853 , which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK. soluble guanylyl cyclase; inosine 3=,5=-cyclic monophosphate; hypoxic vasoconstriction; endothelium A PREVIOUS STUDY reported that acute hypoxia caused a rapid further increase in tension of contracted canine saphenous veins (31). Subsequent findings demonstrated that such a phenomenon, termed hypoxic augmentation of vasoconstriction (5, 15), occurs in a number of blood vessel types (5,7,8,15,21,23,25,26) contracted with norepinephrine (7, 8), phenylephrine (23), PGF 2␣ (15,25), and the TP receptor agonist U-46619 (21), indicating that it is not unique for a specific vasoconstrictor. It occurs in isolated coronary arteries with but not without endothelium (5,15,25,26). The phenomenon is not affected by bosentan, a blocker of endothelin receptors (5), but is prevented by inhibitors of nitric oxide (NO) synthase (5,15,25). Hypoxic augmentation also occurs in arteries without endothelium treated with an exogenous NO donor (5) However, hypoxic augmentation is not accompanied by changes in the intracellular leve...

Circadian rhythms are approximate 24-h oscillations in physiology and behavior. Circadian rhythm disruption has been associated with increased incidence of hypertension, coronary artery disease, dyslipidemia, and other cardiovascular pathologies in both humans and animal models. Mice lacking the core circadian clock gene, brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein (Bmal1), are behaviorally arrhythmic, die prematurely, and display a wide range of organ pathologies. However, data are lacking on the role of Bmal1 on the structural and functional integrity of cardiac muscle. In the present study, we demonstrate that Bmal1(-/-) mice develop dilated cardiomyopathy with age, characterized by thinning of the myocardial walls, dilation of the left ventricle, and decreased cardiac performance. Shortly after birth the Bmal1(-/-) mice exhibit a transient increase in myocardial weight, followed by regression and later onset of dilation and failure. Ex vivo working heart preparations revealed systolic ventricular dysfunction at the onset of dilation and failure, preceded by downregulation of both myosin heavy chain isoform mRNAs. We observed structural disorganization at the level of the sarcomere with a shift in titin isoform composition toward the stiffer N2B isoform. However, passive tension generation in single cardiomyocytes was not increased. Collectively, these findings suggest that the loss of the circadian clock gene, Bmal1, gives rise to the development of an age-associated dilated cardiomyopathy, which is associated with shifts in titin isoform composition, altered myosin heavy chain gene expression, and disruption of sarcomere structure.

Key points• Early physical mobilization of mechanically ventilated intensive care unit (ICU) patients can reduce the length of stay in the ICU and hospital and improve muscle strength and functional outcomes.• A unique experimental rat ICU model has been used to study the effects and underlying mechanisms of unilateral passive mechanical loading on skeletal muscle size and function at durations varying between 6 h and 2 weeks.• Passive mechanical loading attenuated the loss of muscle mass and force-generation capacity associated with the ICU intervention.• The maintained muscle mass and function by passive loading is probably due to lower oxidative stress and a reduced loss of the molecular motor protein myosin.• The beneficial effects of passive mechanical loading on muscle size and function strongly support the importance of early and intense physical therapy in immobilized ICU patients.Abstract The response to mechanical stimuli, i.e. tensegrity, plays an important role in regulating cell physiological and pathophysiological function, and the mechanical silencing observed in intensive care unit (ICU) patients leads to a severe and specific muscle wasting condition. This study aims to unravel the underlying mechanisms and the effects of passive mechanical loading on skeletal muscle mass and function at the gene, protein and cellular levels. A unique experimental rat ICU model has been used allowing long-term (weeks) time-resolved analyses of the effects of standardized unilateral passive mechanical loading on skeletal muscle size and function and underlying mechanisms. Results show that passive mechanical loading alleviated the muscle wasting and the loss of force-generation associated with the ICU intervention, resulting in a doubling of the functional capacity of the loaded versus the unloaded muscles after a 2-week ICU intervention. We demonstrate that the improved maintenance of muscle mass and function is probably a consequence of a reduced oxidative stress revealed by lower levels of carbonylated proteins, and a reduced loss of the molecular motor protein myosin. A complex temporal gene expression pattern, delineated by microarray analysis, was observed with loading-induced changes in transcript levels of sarcomeric proteins, muscle developmental processes, stress response, extracellular matrix/cell adhesion proteins and metabolism. Thus, the results from this study show that passive mechanical loading alleviates the severe negative consequences on muscle size * G. Renaud and M. Llano-Diez contributed equally to this paper

Several cardiac troponin I (cTnI) mutations are associated with restrictive cardiomyopathy (RCM) in humans. We have created transgenic mice (cTnI193His mice) that express the corresponding human RCM R192H mutation. Phenotype of this RCM animal model includes restrictive ventricles, biatrial enlargement and sudden cardiac death, which are similar to those observed in RCM patients carrying the same cTnI mutation. In the present study, we modified the overall cTnI in cardiac muscle by crossing cTnI193His mice with transgenic mice expressing an N-terminal truncated cTnI (cTnI-ND) that enhances relaxation. Protein analyses determined that wild type cTnI was replaced by cTnI-ND in the heart of double transgenic mice (Double TG), which express only cTnI-ND and cTnI R193H in cardiac myocytes. The presence of cTnI-ND effectively rescued the lethal phenotype of RCM mice by reducing the mortality rate. Cardiac function was significantly improved in Double TG mice when measured by echocardiography. The hypersensitivity to Ca2+ and the prolonged relaxation of RCM cTnI193His cardiac myocytes were completely reversed by the presence of cTnI-ND in RCM hearts. The results demonstrate that myofibril hypersensitivity to Ca2+ is a key mechanism that causes impaired relaxation in RCM cTnI mutant hearts and Ca2+ desensitization by cTnI-ND can correct diastolic dysfunction and rescue the RCM phenotypes, suggesting that Ca2+ desensitization in myofibrils is a therapeutic option for treatment of diastolic dysfunction without interventions directed at the systemic β-adrenergic-PKA pathways.

Transgenic mice were generated to express a restrictive cardiomyopathy (RCM) human cardiac troponin I (cTnI) R192H mutation in the heart (cTnI(193His) mice). The objective of this study was to assess cardiac function during the development of diastolic dysfunction and to gain insight into the pathophysiological impact of the RCM cTnI mutation. Cardiac function and pathophysiological changes were monitored in cTnI193His mice and wild-type littermates for a period of 12 mo. It progressed gradually from abnormal relaxation to diastolic dysfunction characterized with high-resolution echocardiography by a reversed E-to-A ratio, increased deceleration time, and prolonged isovolumetric relaxation time. At the age of 12 mo, cardiac output in cTnI(193His) mice was significantly declined, and some transgenic mice showed congestive heart failure. The negative impact of cTnI193His on ventricular contraction and relaxation was further demonstrated in isolated mouse working heart preparations. The main morphological change in cTnI193His myocytes was shortened cell length. Dobutamine stimulation increased heart rate in cTnI193His mice but did not improve CO. The cTnI193His mice had a phenotype similar to that in human RCM patients carrying the cTnI mutation characterized morphologically by enlarged atria and restricted ventricles and functionally by diastolic dysfunction and diastolic heart failure. The results demonstrate a critical role of the COOH-terminal domain of cTnI in the diastolic function of cardiac muscle.