Protein temporal dynamics play a critical role in time-dimensional pathophysiological processes, including the gradual cardiac remodeling that occurs in early-stage heart failure. Methods for quantitative assessments of protein kinetics are lacking, and despite knowledge gained from single-protein studies, integrative views of the coordinated behavior of multiple proteins in cardiac remodeling are scarce. Here, we developed a workflow that integrates deuterium oxide (2H2O) labeling, high-resolution mass spectrometry (MS), and custom computational methods to systematically interrogate in vivo protein turnover. Using this workflow, we characterized the in vivo turnover kinetics of 2,964 proteins in a mouse model of β-adrenergic–induced cardiac remodeling. The data provided a quantitative and longitudinal view of cardiac remodeling at the molecular level, revealing widespread kinetic regulations in calcium signaling, metabolism, proteostasis, and mitochondrial dynamics. We translated the workflow to human studies, creating a reference dataset of 496 plasma protein turnover rates from 4 healthy adults. The approach is applicable to short, minimal label enrichment and can be performed on as little as a single biopsy, thereby overcoming critical obstacles to clinical investigations. The protein turnover quantitation experiments and computational workflow described here should be widely applicable to large-scale biomolecular investigations of human disease mechanisms with a temporal perspective.
Common cardiovascular diseases, such as atherosclerosis and congestive heart failure, are exceptionally complex, involving a multitude of environmental and genetic factors that often show nonlinear interactions as well as being highly dependent on sex, age, and even the maternal environment. Although focused, reductionistic approaches have led to progress in elucidating the pathophysiology of cardiovascular diseases, such approaches are poorly powered to address complex interactions. Over the past decade, technological advances have made it possible to interrogate biological systems on a global level, raising hopes that, in combination with computational approaches, it may be possible to more fully address the complexities of cardiovascular diseases. In this Review, we provide an overview of such systems-based approaches to cardiovascular disease and discuss their translational implications.
Cell death is regulated by a myriad of intracellular molecular pathways, with many involving protein phosphorylation and dephosphorylation. In this review, we will focus on Ser/Thr phosphatases-mediated regulation in cell apoptosis as well as on their potential roles in cell necrosis. The emerging functional importance of Ser/Thr protein phosphatases in cell death regulation adds new dimension to the signaling mechanisms of cellular function, physiology, and diseases.
Mitogen-activated protein (MAP) kinases belong to a highly conserved family of Ser-Thr protein kinases in the human kinome and have diverse roles in broad physiological functions. The 4 best-characterized MAP kinase pathways, ERK1/2, JNK, p38, and ERK5, have been implicated in different aspects of cardiac regulation, from development to pathological remodeling. Recent advancements in the development of kinase-specific inhibitors and genetically engineered animal models have revealed significant new insights about MAP kinase pathways in the heart. However, this explosive body of new information also has yielded many controversies about the functional role of specific MAP kinases as either detrimental promoters or critical protectors of the heart during cardiac pathological processes. These uncertainties have raised questions on whether/how MAP kinases can be targeted to develop effective therapies against heart diseases. In this review, recent studies examining the role of MAP kinase subfamilies in cardiac development, hypertrophy, and survival are summarized.
heart failure; molecular biology; signal transduction
Among the myriad of intra-cellular signaling networks that govern the cardiac development and pathogenesis, mitogen-activated protein kinases (MAPKs) are prominent players that have been the focus of extensive investigations in the past decades. The four best characterized MAPK subfamilies, ERK1/2, JNK, p38, and ERK5, are the targets of pharmacological and genetic manipulations to uncover their roles in cardiac development, function, and diseases. However, information reported in the literature from these efforts has not yet resulted in a clear view about the roles of specific MAPK pathways in heart. Rather, controversies from contradictive results have led to a perception that MAPKs are ambiguous characters in heart with both protective and detrimental effects. The primary object of this review is to provide a comprehensive overview of the current progress, in an effort to highlight the areas where consensus is established verses the ones where controversy remains. MAPKs in cardiac development, cardiac hypertrophy, ischemia/reperfusion injury, and pathological remodeling are the main focuses of this review as these represent the most critical issues for evaluating MAPKs as viable targets of therapeutic development. The studies presented in this review will help to reveal the major challenges in the field and the limitations of current approaches and point to a critical need in future studies to gain better understanding of the fundamental mechanisms of MAPK function and regulation in the heart.
Cardiovascular disease is the leading cause of death and disability in the developed world. To design novel therapeutic strategies to treat and prevent this disease, better understanding of cardiac cell function is necessary. In addition to (and, indeed, in combination with) genetics, physiology and molecular biology, proteomics plays a critical role in our understanding of cardiovascular systems at multiple scales. The purpose of this review is to examine recent developments in the field of myocardial injury and protection, examining how proteomics has informed investigations into organelles, signaling complexes, and cardiac phenotype.
Cardiovascular disease; Heart; Mitochondria; Proteasome; Signal transduction
Doxorubicin (DOX) is a commonly used life-saving antineoplastic agent that also causes dose-dependent cardiotoxicity. Because ATP is absolutely required to sustain normal cardiac contractile function and because impaired ATP synthesis through creatine kinase (CK), the primary myocardial energy reserve reaction, may contribute to contractile dysfunction in heart failure, we hypothesized that impaired CK energy metabolism contributes to DOX-induced cardiotoxicity. We therefore overexpressed the myofibrillar isoform of CK (CK-M) in the heart and determined the energetic, contractile and survival effects of CK-M following weekly DOX (5mg/kg) administration using in vivo31P MRS and 1H MRI. In control animals, in vivo cardiac energetics were reduced at 7 weeks of DOX protocol and this was followed by a mild but significant reduction in left ventricular ejection fraction (EF) at 8 weeks of DOX, as compared to baseline. At baseline, CK-M overexpression (CK-M-OE) increased rates of ATP synthesis through cardiac CK (CK flux) but did not affect contractile function. Following DOX however, CK-M-OE hearts had better preservation of creatine phosphate and higher CK flux and higher EF as compared to control DOX hearts. Survival after DOX administration was significantly better in CK-M-OE than in control animals (p<0.02). Thus CK-M-OE attenuates the early decline in myocardial high-energy phosphates and contractile function caused by chronic DOX administration and increases survival. These findings suggest that CK impairment plays an energetic and functional role in this DOX-cardiotoxicity model and suggests that metabolic strategies, particularly those targeting CK, offer an appealing new strategy for limiting DOX-associated cardiotoxicity.
Although the murine late pregnant (LP) heart is speculated to be a better functioning heart during physiological conditions, the susceptibility of LP hearts to I/R injury is still unknown. The aims of this study were to investigate the cardiac vulnerability of LP rodents to ischemia/reperfusion (I/R) injury and to explore its underlying mechanisms. In-vivo female rat hearts (non-pregnant (NP) or LP) or ex-vivo Langendorff-perfused mouse hearts were subjected to I/R.. The infarct size was ~4-fold larger in LP animals compared to NP both in-vivo and ex-vivo. The heart functional recovery was extremely poor in LP mice compared to NP (~10% recovery in LP vs. 80% recovery in NP at the end of reperfusion, P < 0.01). Interestingly, the poor functional recovery and the larger infarct size in LP were partially restored one day post-partum and almost fully restored one week post-partum to their corresponding NP levels. Mitochondrial respiratory function and the threshold for opening of the mitochondrial permeability transition pore were significantly lower in LP compared to NP when they both were subjected to myocardial I/R injury (Respiratory control ratio=1.9±0.1 vs. 4.0±0.5 in NP, P<0.05; calcium retention capacity(CRC)=167±10 vs. 233±18 nmol/mg protein in NP, P<0.01). Cardiac ROS generation, as well mitochondrial superoxide production, were ~2-fold higher in LP compared to NP following I/R. The phosphorylation levels of Akt, ERK1/2 and STAT3, but not GSK3β, were significantly reduced in the hearts from LP subjected to I/R. In conclusion, increased mitochondrial ROS generation, decreased CRC as well as impaired activation of Akt/ERK/STAT3 at reperfusion are the possible underlying mechanisms for higher vulnerability of LP hearts to I/R.
Pregnancy; ischemia/reperfusion; heart hypertrophy; mPTP; ROS
bone marrow mononuclear cells; Editorials; myocardial infarction; prostaglandins
Rationale and Objective
In this Emerging Science Review, we discuss a systems genetics strategy, which we call Gene Module Association Study (GMAS), as a novel approach complementing Genome Wide Association Studies (GWAS), to understand complex diseases by focusing on how genes work together in groups rather than singly.
The first step is to characterize phenotypic differences among a genetically diverse population. The second step is to use gene expression microarray (or other high throughput) data from the population to construct gene co-expression networks. Co-expression analysis typically groups 20,000 genes into 20–30 modules containing 10’s to 100’s of genes, whose aggregate behavior can be represented by the module’s “eigengene.” The third step is to correlate expression patterns with phenotype, as in GWAS, only applied to eigengenes instead of SNPs.
Results and Conclusions
The goal of the GMAS approach is to identify groups of co-regulated genes that explain complex traits from a systems perspective. From an evolutionary standpoint, we hypothesize that variability in eigengene patterns reflects the “good enough solution” concept, that biological systems are sufficiently complex so that many possible combinations of the same elements (in this case eigengenes) can produce an equivalent output, i.e. a “good enough solution” to accomplish normal biological functions. However, when faced with environmental stresses, some “good enough solutions” adapt better than others, explaining individual variability to disease and drug susceptibility. If validated, GMAS may imply that common polygenic diseases are related as much to group interactions between normal genes, as to multiple gene mutations.
systems genetics; genetics of complex diseases; scale-free networks; hybrid mouse diversity panel; computational biology
The protein phosphatase 1-like gene (PPM1l) was identified as causal gene for obesity and metabolic abnormalities in mice. However, the underlying mechanisms were unknown. In this report, we find PPM1l encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (PP2Ce) and has specific activity to basal and ER stress induced auto-phosphorylation of Inositol-REquiring protein-1 (IRE1). PP2Ce inactivation resulted in elevated IRE1 phosphorylation and higher expression of XBP-1, CHOP, and BiP at basal. However, ER stress stimulated XBP-1 and BiP induction was blunted while CHOP induction was further enhanced in PP2Ce null cells. PP2Ce protein levels are significantly induced during adipogenesis in vitro and are necessary for normal adipocyte maturation. Finally, we provide evidence that common genetic variation of PPM11 gene is significantly associated with human lipid profile. Therefore, PPM1l mediated IRE1 regulation and downstream ER stress signaling is a plausible molecular basis for its role in metabolic regulation and disorder.
IRE1; PPM1l; Protein phosphatase; ER stress; Adipogenesis
Protein phosphorylation is a major form of posttranslational modification critical to cell signaling that also occurs in mitochondrial proteome. Yet, only very limited studies have been performed to characterize mitochondrial-targeted protein kinases or phosphatases. Recently, we identified a novel member of PP2C family (PP2Cm) that is a resident mitochondrial protein phosphatase which plays an important role in normal development and cell survival. In this chapter, we will describe the methods applied in the identification of PP2Cm as a resident mitochondrial protein phosphatase based on sequence analysis and biochemical characterization. We will also provide experimental protocols used to establish the intracellular localization of PP2Cm, to achieve loss and gain function of PP2Cm in cultured cells and intact tissue, and to assess the impact of PP2Cm deficiency on cell death, mitochondria oxidative phosphorylation and permeability transition pore opening.
p38 Mitogen-activated protein kinase (MAPK) is one of the most ancient signaling molecules and is involved in multiple cellular processes, including cell proliferation, cell growth, and cell death. In the heart, enhanced activation of p38 MAPK is associated with ischemia/reperfusion injury and the onset of heart failure. In the present study, we investigated the function of p38 MAPK in regulating cardiac contractility and its underlying mechanisms. In cultured adult rat cardiomyocytes, activation of p38 MAPK by adenoviral gene transfer of an activated mutant of its upstream kinase, MKK3bE, led to a significant reduction in baseline contractility, compared with uninfected cells or those infected with a control adenoviral vector (Adv-β-galactosidase). The inhibitory effect of MKK3bE on contractility was largely prevented by coexpressing a dominant-negative mutant of p38 MAPK or treating cells with a p38 MAPK inhibitor, SB203580. Conversely, inhibition of endogenous p38 MAPK activity by SB203580 rapidly and reversibly enhanced cell contractility in a dose-dependent manner, without altering L-type Ca2+ currents or Ca2+i transients. MKK3bE-induced p38 activation had no significant effect on pHi, whereas SB203580 had a minor effect to elevate pHi. Furthermore, activation of p38 MAPK was unable to increase troponin I phosphorylation. Thus, we conclude that the negative inotropic effect of p38 MAPK is mediated by decreasing myofilament response to Ca2+, rather than by altering Ca2+i homeostasis and that the reduced myofilament Ca2+ sensitivity is unlikely attributable to troponin I phosphorylation or alterations in pHi. These findings reveal a novel function of p38 MAPK and shed a new light on our understanding of the coincidence of p38 MAPK activation and the onset of heart failure.
p38 mitogen-activated protein kinase; cardiac contractility; excitation-contraction coupling; troponin I; intracellular pH
anticancer agents; DNA cross-linking; DNA alkylation; H2O2-activation; arylboronates
Diurnal variation in nitrogen homeostasis is observed across phylogeny. But whether these are endogenous rhythms, and if so, molecular mechanisms that link nitrogen homeostasis to the circadian clock remain unknown. Here, we provide evidence that a clock-dependent peripheral oscillator, Krüppel-like factor15 transcriptionally coordinates rhythmic expression of multiple enzymes involved in mammalian nitrogen homeostasis. In particular, Krüppel-like factor15-deficient mice exhibit no discernable amino acid rhythm, and the rhythmicity of ammonia to urea detoxification is impaired. Of the external cues, feeding plays a dominant role in modulating Krüppel-like factor15 rhythm and nitrogen homeostasis. Further, when all behavioral, environmental and dietary cues were controlled in humans, nitrogen homeostasis still expressed endogenous circadian rhythmicity. Thus, in mammals, nitrogen homeostasis exhibits circadian rhythmicity, and is orchestrated by Krüppel-like factor15.
We have developed an association-based approach using classical inbred strains of mice in which we correct for population structure, which is very extensive in mice, using an efficient mixed-model algorithm. Our approach includes inbred parental strains as well as recombinant inbred strains in order to capture loci with effect sizes typical of complex traits in mice (in the range of 5 % of total trait variance). Over the last few years, we have typed the hybrid mouse diversity panel (HMDP) strains for a variety of clinical traits as well as intermediate phenotypes and have shown that the HMDP has sufficient power to map genes for highly complex traits with resolution that is in most cases less than a megabase. In this essay, we review our experience with the HMDP, describe various ongoing projects, and discuss how the HMDP may fit into the larger picture of common diseases and different approaches.
p38γ kinase is highly enriched in skeletal muscle and is implicated in myotube formation. However, the activation status of p38γ in muscle is unclear.
p38γ activity in slow and fast adult mouse skeletal muscle tissue was examined as well as the impact of p38γ deficiency on muscle development and gene expression.
p38γ is preferentially activated in slow muscle, but it is inactive in fast muscle types. Furthermore, the loss of p38γ in mice led to decreased muscle mass associated with a smaller myofiber diameter in slow muscle, but there was no impact on fast muscle in either mass or myofiber diameter. Finally, p38γ deficient muscle showed selective changes in genes related to muscle growth in slow muscle fibers.
Our study provides evidence that p38γ is selectively activated in slow skeletal muscle and is involved in normal growth and development of a subset of skeletal muscle.
p38γ; mice; skeletal muscle; development; phosphorylation
Phosphorylation of β2-adrenergic receptor (β2AR) by a family of serine/threonine kinases known as G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood.
We seek to determine the relative contribution of PKA- and GRK-mediated phosphorylation of β2AR to the receptor coupling to Gi signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload.
Methods and Results
Overexpression of GRK2 led to a Gi-dependent decrease of contractile response to βAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant β2AR lacking PKA phosphorylation sites (PKA- TG), but not the wild type β2AR (WT TG) or a mutant β2AR lacking GRK sites (GRK- TG), led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure, and early mortality. Furthermore, inhibition of Gi signaling with pertussis toxin restores cardiac function in heart failure associated with increased β2AR to Gi coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of β2AR by GRK and resultant increase in Gi-biased β2AR signaling play an important role in the development of heart failure.
Our data show that enhanced β2AR phosphorylation by GRK, in addition to PKA, leads the receptor to Gi-biased signaling which, in turn, contributes to the pathogenesis of heart failure, marking Gi-biased β2AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.
β2-adrenergic receptor; G protein-coupled receptor kinase; Heart failure; hypertrophy
Accurate and comprehensive de novo transcriptome profiling in heart is a central issue to better understand cardiac physiology and diseases. Although significant progress has been made in genome-wide profiling for quantitative changes in cardiac gene expression, current knowledge offers limited insights to the total complexity in cardiac transcriptome at individual exon level.
To develop more robust bioinformatic approaches to analyze high-throughput RNA sequencing (RNA-Seq) data, with the focus on the investigation of transcriptome complexity at individual exon and transcript levels.
Methods and Results
In addition to overall gene expression analysis, the methods developed in this study were used to analyze RNA-Seq data with respect to individual transcript isoforms, novel spliced exons, novel alternative terminal exons, novel transcript clusters (i.e., novel genes) and long non-coding RNA genes. We applied these approaches to RNA-Seq data obtained from mouse hearts following pressure-overload induced by trans-aortic constriction. Based on experimental validations, analyses of the features of the identified exons/transcripts, and expression analyses including previously published RNASeq data, we demonstrate that the methods are highly effective in detecting and quantifying individual exons and transcripts. Novel insights inferred from the examined aspects of the cardiac transcriptome open ways to further experimental investigations.
Our work provided a comprehensive set of methods to analyze mouse cardiac transcriptome complexity at individual exon and transcript levels. Applications of the methods may infer important new insights to gene regulation in normal and disease hearts in terms of exon utilization and potential involvement of novel components of cardiac transcriptome.
RNA-Seq; transcriptome profiling; hypertrophy; heart failure
The p38 mitogen-activated protein kinases (p38s) are stress activated ser/thr kinases. Their activation has been associated with various pathological stressors in the heart. Activated p38 is implicated in a wide spectrum of cardiac pathologies, including hypertrophy, myocardial infarction, as well as systolic and diastolic heart failure. In this review, the specific contribution of different isoforms of p38 kinases to cardiac diseases as well as TAB-1 mediated non-canonical activation pathway are discussed as a rationale for inhibiting p38 activity to treat cardiac hypertrophy, ischemic injury and heart failure. Finally, a summary of current clinical trials targeting p38 kinases in cardiovascular diseases is provided to highlight the potential promise as well as existing challenges of this therapeutic approach.
In the eukaryotic transcriptome, both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity. These RNA species are generated by the utilization of different transcriptional initiation or termination sites, or more commonly, from different messenger RNA (mRNA) splicing events. Among the 30 000+ genes in human genome, it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing. The protein products generated from different RNA splicing variants can have different intracellular localization, activity, or tissue-distribution. Therefore, alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types. In this review, we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach, and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart.
Alternative RNA splicing; Transcriptome; Gene regulation; Heart; RNA-seq
Proteasomal degradation is altered in many disease phenotypes including cardiac hypertrophy, a prevalent condition leading to heart failure. Our recent investigations identified heterogeneous subpopulations of proteasome complexes in the heart and implicated multiple mechanisms for their regulation.
The study aimed at identification of molecular mechanisms changing proteasome function in the hypertrophic heart.
Method and Results
Proteasome function, expression, and assembly were analyzed during the development of cardiac hypertrophy induced by β-adrenergic stimulation. The analysis revealed, for the first time, divergent regulation of proteasome function in cardiac hypertrophy. Proteasome complexes have 3 different proteolytic activities, which are ATP-dependent for 26S complexes (19S assembled with 20S) and ATP-independent for 20S core particles. The 26S activities were enhanced in hypertrophic hearts, partially because of increased expression and assembly of 19S subunits with 20S core complexes. In contrast, caspase- and trypsin-like 20S activities were significantly decreased. Activation of endogenous cAMP-dependent protein kinase (PKA) rescued the depressed 20S functions, supporting the notion that PKA signaling is a positive regulator of protein degradation in the heart. Chymotrypsin-like 20S activity was stably maintained during cardiac remodeling, indicating a switch in proteasome subpopulations, which was supported by altered expression and incorporation of inducible β subunits.
Three novel mechanisms for the regulation of proteasome activities were discovered in the development of cardiac hypertrophy: (1) increased incorporation of inducible subunits in 20S proteasomes; (2) enhanced 20S sensitivity to PKA activation; and (3) increased 26S assembly. PKA modulation of proteasome complexes may provide a novel therapeutic avenue for restoration of cardiac function in the diseased myocardium.
heart disease; protein degradation; cellular homeostasis
Metabolic remodelling is an integral part of the pathogenesis of heart failure. Although much progress has been made in our current understanding of the metabolic impairment involving carbohydrates and fatty acids in failing hearts, relatively little is known about the changes and potential impact of amino acid metabolism in the onset of heart diseases. Although most amino acid catabolic activities are found in the liver, branched-chain amino acid (BCAA) catabolism requires activity in several non-hepatic tissues, including cardiac muscle, diaphragm, brain and kidney. In this review, the new insights into the regulation of cardiac BCAA catabolism and functional impact on cardiac development and physiology will be discussed along with the potential contribution of impairment in BCAA catabolism to heart diseases. A particular focus will be the new information obtained from recently developed genetic models with BCAA catabolic defects and metabolomic studies in human and animal models. These studies have revealed the potential role of BCAA catabolism in cardiac pathophysiology and have helped to distinguish BCAA metabolic defects as an under-appreciated culprit in cardiac diseases rather than an epiphenomenon associated with metabolic remodelling in the failing heart.
Branched-chain amino acid; PP2Cm; Heart failure
Mutation of BLM helicase causes Blooms syndrome, a disorder associated with genome instability, high levels of sister chromatid exchanges, and cancer predisposition. To study the influence of BLM on double-strand break (DSB) repair in human chromosomes, we stably transfected a normal human cell line with a DNA substrate that contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI. The substrate also contained a closely linked functional tk gene to serve as a recombination partner for the tk-neo fusion gene. We derived two cell lines each containing a single integrated copy of the DNA substrate. In these cell lines, a DSB was introduced within the tk-neo fusion gene by expression of I-SceI. DSB repair events that occurred via homologous recombination (HR) or nonhomologous end-joining (NHEJ) were recovered by selection for G418-resistant clones. DSB repair was examined under conditions of either normal BLM expression or reduced BLM expression brought about by RNA interference. We report that BLM knockdown in both cell lines specifically increased the frequency of HR events that produced deletions by crossovers or single-strand annealing while leaving the frequency of gene conversions unchanged or reduced. We observed no change in the accuracy of individual HR events and no substantial alteration of the nature of individual NHEJ events when BLM expression was reduced. Our work provides the first direct evidence that BLM influences DSB repair pathway choice in human chromosomes and suggests that BLM deficiency can engender genomic instability by provoking an increased frequency of HR events of a potentially deleterious nature.
double-strand break repair; homologous recombination; nonhomologous end-joining; Bloom syndrome; human cell culture
cardiac hypertrophy; miRNA; thioredoxin 1; miR-98/let-7; Cyclin D2; Editorial