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
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
Hutchinson–Gilford progeria syndrome (HGPS) is caused by a mutant prelamin A, progerin, that terminates with a farnesylcysteine. HGPS knock-in mice (LmnaHG/+) develop severe progeria-like disease phenotypes. These phenotypes can be ameliorated with a protein farnesyltransferase inhibitor (FTI), suggesting that progerin's farnesyl lipid is important for disease pathogenesis and raising the possibility that FTIs could be useful for treating humans with HGPS. Subsequent studies showed that mice expressing non-farnesylated progerin (LmnanHG/+ mice, in which progerin's carboxyl-terminal –CSIM motif was changed to –SSIM) also develop severe progeria, raising doubts about whether any treatment targeting protein prenylation would be particularly effective. We suspected that those doubts might be premature and hypothesized that the persistent disease in LmnanHG/+ mice could be an unanticipated consequence of the cysteine-to-serine substitution that was used to eliminate farnesylation. To test this hypothesis, we generated a second knock-in allele yielding non-farnesylated progerin (LmnacsmHG) in which the carboxyl-terminal –CSIM motif was changed to –CSM. We then compared disease phenotypes in mice harboring the LmnanHG or LmnacsmHG allele. As expected, LmnanHG/+ and LmnanHG/nHG mice developed severe progeria-like disease phenotypes, including osteolytic lesions and rib fractures, osteoporosis, slow growth and reduced survival. In contrast, LmnacsmHG/+ and LmnacsmHG/csmHG mice exhibited no bone disease and displayed entirely normal body weights and survival. The frequencies of misshapen cell nuclei were lower in LmnacsmHG/+ and LmnacsmHG/csmHG fibroblasts. These studies show that the ability of non-farnesylated progerin to elicit disease depends on the carboxyl-terminal mutation used to eliminate protein prenylation.
ATP is required for normal cardiac contractile function, and it has long been hypothesized that reduced energy delivery contributes to the contractile dysfunction of heart failure (HF). Despite experimental and clinical HF data showing reduced metabolism through cardiac creatine kinase (CK), the major myocardial energy reserve and temporal ATP buffer, a causal relationship between reduced ATP-CK metabolism and contractile dysfunction in HF has never been demonstrated. Here, we generated mice conditionally overexpressing the myofibrillar isoform of CK (CK-M) to test the hypothesis that augmenting impaired CK-related energy metabolism improves contractile function in HF. CK-M overexpression significantly increased ATP flux through CK ex vivo and in vivo but did not alter contractile function in normal mice. It also led to significantly increased contractile function at baseline and during adrenergic stimulation and increased survival after thoracic aortic constriction (TAC) surgery–induced HF. Withdrawal of CK-M overexpression after TAC resulted in a significant decline in contractile function as compared with animals in which CK-M overexpression was maintained. These observations provide direct evidence that the failing heart is “energy starved” as it relates to CK. In addition, these data identify CK as a promising therapeutic target for preventing and treating HF and possibly diseases involving energy-dependent dysfunction in other organs with temporally varying energy demands.
We tested the hypothesis that bi-directional, gene-targeted regulation of cardiomyocyte cGMP-selective phosphodiesterase type 5 (PDE5) influences maladaptive remodeling in hearts subjected to sustained pressure-overload.
PDE5 expression is up-regulated in human hypertrophied and failing hearts, and its inhibition (e.g. by sildenafil) stimulates protein kinase G activity, suppressing and reversing maladaptive hypertrophy, fibrosis, and contractile dysfunction. Sildenafil is currently being clinically tested for the treatment of heart failure. However, new studies have questioned the role of myocyte PDE5 and protein kinase G (PKG) to this process, proposing alternative targets and mechanisms.
Mice with doxycycline controllable myocyte-specific PDE5 gene-expression were generated (medium (me-TG) and high (hi-TG) expression lines), and subjected to sustained pressure-overload.
Rest myocyte and heart function, histology, and molecular profiling were normal in both TG-lines versus controls at 2 months of age. However, upon exposure to pressure-overload (aortic banding), TG hearts developed more eccentric remodeling, maladaptive molecular signaling, depressed function, and amplified fibrosis with up-regulation of tissue growth factor signaling pathway. PKG activation was inhibited in TG myocytes versus control. After establishing a severe cardiomyopathic state, hi-TG mice received doxycycline to suppress PDE5 expression/activity only in myocytes. This in turn enhanced PKG activity, and reversed all of the previously amplified maladaptive responses despite sustained pressure-overload. Sildenafil was also effective in this regard.
These data strongly support a primary role of myocyte PDE5 regulation to myocardial pathobiology and PDE5 targeting therapy in vivo, and reveal a novel mechanism of myocyte-orchestrated extracellular matrix remodeling via PDE5/cGMP-PKG regulatory pathways
phosphodiesterase-5; cyclic GMP; protein kinase G; heart failure; pressure overload; transgenic mouse models
Cyclooxygenase-1 and -2 are rate-limiting enzymes in the formation of a wide array of bioactive lipid mediators collectively known as prostanoids (prostaglandins, prostacyclins, thromboxanes). Evidence from clinical trials shows that selective inhibition of the second isoenzyme (cyclooxygenase-2, or Cox-2) is associated with increased risk for serious cardiovascular events and findings from animal-based studies have suggested protective roles of Cox-2 for the heart. To further characterize the function of Cox-2 in the heart, mice with loxP sites flanking exons 4 and 5 of Cox-2 were rendered knockout specifically in cardiac myocytes (Cox-2 CKO mice) via cre-mediated recombination. Baseline cardiac performance of CKO mice remained unchanged and closely resembled that of control mice. Furthermore, myocardial infarct size induced after in vivo ischemia/reperfusion (I/R) injury was comparable between CKO and control mice. In addition, cardiac hypertrophy and function four weeks after transverse aortic constriction (TAC) was found to be similar between the two groups. Assessment of Cox-2 expression in purified adult cardiac cells isolated after I/R and TAC suggests that the dominant source of Cox-2 is found in the non-myocyte fraction. In conclusion, our animal-based analyses together with the cell-based observations portray a limited role of cardiomyocyte-produced Cox-2 at baseline and in the context of ischemic or hemodynamic challenge.
cyclooxygenase-2; cardiomyocyte; genetic; recombination; echocardiography; infarction; hypertrophy; fibrosis
Lamin A is formed from prelamin A by four post-translational processing steps—farnesylation, release of the last three amino acids of the protein, methylation of the farnesylcysteine and the endoproteolytic release of the C-terminal 15 amino acids (including the farnesylcysteine methyl ester). When the final processing step does not occur, a farnesylated and methylated prelamin A accumulates in cells, causing a severe progeroid disease, restrictive dermopathy (RD). Whether RD is caused by the retention of farnesyl lipid on prelamin A, or by the retention of the last 15 amino acids of the protein, is unknown. To address this issue, we created knock-in mice harboring a mutant Lmna allele (LmnanPLAO) that yields exclusively non-farnesylated prelamin A (and no lamin C). These mice had no evidence of progeria but succumbed to cardiomyopathy. We suspected that the non-farnesylated prelamin A in the tissues of these mice would be strikingly mislocalized to the nucleoplasm, but this was not the case; most was at the nuclear rim (indistinguishable from the lamin A in wild-type mice). The cardiomyopathy could not be ascribed to an absence of lamin C because mice expressing an otherwise identical knock-in allele yielding only wild-type prelamin A appeared normal. We conclude that lamin C synthesis is dispensable in mice and that the failure to convert prelamin A to mature lamin A causes cardiomyopathy (at least in the absence of lamin C). The latter finding is potentially relevant to the long-term use of protein farnesyltransferase inhibitors, which lead to an accumulation of non-farnesylated prelamin A.
Cyclooxygenase-2 (COX-2) is an important mediator of inflammation in stress and disease states. Recent attention has focused on the role of COX-2 in human heart failure and diseases, due to the finding that highly specific COX-2 inhibitors (i.e. Vioxx) increased the risk of myocardial infarction and stroke in chronic users. However, the specific impact of COX-2 expression in the intact heart remains to be determined. We report here the development of a transgenic mouse model, using a loxP-Cre approach, that displays robust COX-2 overexpression and subsequent prostaglandin synthesis specifically in ventricular myocytes. Histological, functional and molecular analyses showed that ventricular myocyte specific COX-2 overexpression led to cardiac hypertrophy and fetal gene marker activation, but with preserved cardiac function. Therefore, specific induction of COX-2 and prostaglandin in vivo is sufficient to induce compensated hypertrophy and molecular remodeling.
cyclooxygenase; COX-2; transgenic; hypertrophy; compensatory