Pressure-Overload Cardiac Hypertrophy Is Associated with Distinct Alternative Splicing Due to Altered Expression of Splicing Factors

Chronic pressure-overload cardiac hypertrophy is associated with an increased risk of morbidity/mortality, largely due to maladaptive remodeling and dilatation that progresses to dilated cardiomyopathy. Alternative splicing is an important biological mechanism that generates proteomic complexity and diversity. The recent development of next-generation RNA sequencing has improved our understanding of the qualitative signatures associated with alternative splicing in various biological conditions. However, the role of alternative splicing in cardiac hypertrophy is yet unknown. The present study employed RNA-Seq and a bioinformatic approach to detect the RNA splicing regulatory elements involved in alternative splicing during pressure-overload cardiac hypertrophy. We found GC-rich exonic motifs that regulate intron retention in 5′ UTRs and AT-rich exonic motifs that are involved in exclusion of the AT-rich elements that cause mRNA instability in 3′ UTRs. We also identified motifs in the intronic regions involved in exon exclusion and inclusion, which predicted splicing factors that bind to these motifs. We found, through Western blotting, that the expression levels of three splicing factors, ESRP1, PTB and SF2/ASF, were significantly altered during cardiac hypertrophy. Collectively, the present results suggest that chronic pressure-overload hypertrophy is closely associated with distinct alternative splicing due to altered expression of splicing factors.     

Does load-induced ventricular hypertrophy progress to systolic heart failure?

Ventricular hypertrophy develops in response to numerous forms of cardiac stress, including pressure or volume overload, loss of contractile mass from prior infarction, neuroendocrine activation, and mutations in genes encoding sarcomeric proteins. Hypertrophic growth is believed to have a compensatory role that diminishes wall stress and oxygen consumption, but Framingham and other studies established ventricular hypertrophy as a marker for increased risk of developing chronic heart failure, suggesting that hypertrophy may have maladaptive features. However, the relative contribution of comorbid disease to hypertrophy-associated systolic failure is unknown. For instance, coronary artery disease is induced by many of the same risk factors that cause hypertrophy and can itself lead to systolic dysfunction. It is uncertain, therefore, whether ventricular hypertrophy commonly progresses to systolic dysfunction without the contribution of intervening ischemia or infarction. In this review, we summarize findings from epidemiologic studies, preclinical experiments in animals, and clinical trials to lay out what is known-and not known-about this important question.     

Morphological and functional characteristics of models of experimental myocardial injury induced by isoproterenol

The animal models of myocardial injury induced by systemic β-adrenergic receptor agonist administration represent an experimental approach of persisting interest. These models were found useful especially for studies of structural and functional adaptation of myocardium during the progression of cardiac adaptive response towards maladaptive hypertrophy and insufficiency. The pathological alterations induced by isoproterenol (ISO) do not develop evenly. The ISO models may contribute effectively to understanding of pathologies in signal transduction, energetics, excitability and contractility that may contribute concomitantly to cardiac dysfunction and heart failure. In this minireview we focused on the alterations in general characteristics and heart function as well as on the morphological changes of cardiomyocytes developed during ISO administration. The morphological alterations within the cellular macro- and microdomains correspond to the electrical remodelling and contractile dysfunction of ventricular myocardium that could be used to identify pathological changes ranging from hypertrophy to failing heart.     

Cardiac hypertrophy and changes in contractile function of cardiomyocyte

To investigate the cellular mechanisms of pressure-overload cardiac hypertrophy transition to heart failure, we observed time course of changes in morphology and contractile function of cardiomyocytes in transverse abdominal aortic constriction (TAC) rats. Since TAC rats suffered higher stress, body weight had a slower growth rate compared with that of synchronous control rats. Therefore, the left ventricular to body weight ratio produced experimental bias to evaluate the degree of cardiac hypertrophy. Length and width of collagenase-isolated cardiomyocyte were directly measured. Length, width and calculated surface area of cardiomyocyte showed a progressive increase in 8-, 16-, and 20-week TAC rats. The increasing rate of surface area in cardiomyocytes was higher at the middle stage of TAC (from the eighth to sixteenth week). Due to the constraint of fibrosis formation, the increasing rate of surface area in cardiomyocytes was slower at the late stage of TAC (from the sixteenth to twentieth week). The sarcomere length of cardiomyocytes was unchanged, whereas sarcomere numbers were significantly increased in 8-, 16-, and 20-week TAC rats. Shortening amplitude of unloaded contraction in single cardiomyocyte was significantly enhanced in 1-week TAC rats, but not altered in 8-week TAC rats compared with that in the synchronous control rats. On the contrary, unloaded shortening amplitude of single cardiomyocyte was significantly reduced in 16- and 20-week TAC rats. The above results suggest that the reduced shortening amplitude may be associated with intrinsic molecular alterations in hypertrophied cardiomyocytes.     

Reversal of systemic hypertension-associated cardiac remodeling in chronic pressure overload myocardium by ciglitazone

Elevated oxidative stress has been characterized in numerous disorders including systemic hypertension, arterial stiffness, left ventricular hypertrophy (LVH) and heart failure. The peroxisome proliferator activated receptor gamma (PPARgamma) ameliorates oxidative stress and LVH. To test the hypothesis that PPARgamma decreased LVH and cardiac fibrosis in chronic pressure overload, in part, by increasing SOD, eNOS and elastin and decreasing NOX4, MMP and collagen synthesis and degradation, chronic pressure overload analogous to systemic hypertension was created in C57BL/6J mice by occluding the abdominal aorta above the kidneys (aortic stenosis-AS). The sham surgery was used as controls. Ciglitazone (CZ, a PPARgamma agonist, 4 microg/ml) was administered in drinking water. LV function was measured by M-Mode Echocardiography. We found that PPARgamma protein levels were increased by CZ. NOX-4 expression was increased by pressure-overload and such an increase was attenuated by CZ. SOD expression was not affected by CZ. Expression of iNOS was induced by pressure-overload, and such an increase was inhibited by CZ. Protein levels for MMP2, MMP-9, MMP-13 were induced and TIMP levels were decreased by pressure-overload. The CZ mitigated these levels. Collagen synthesis was increased and elastin levels were decreased by pressure-overload and CZ ameliorated these changes. Histochemistry showed that CZ inhibited interstitial and perivascular fibrosis. Echocardiography showed that CZ attenuated the systolic and diastolic LV dysfunction induced by pressure-overload. These observations suggested that CZ inhibited pressure-overlaod-induced cardiac remodeling, and inhibition of an induction of NOX4, iNOS, MMP-2/MMP-13 expression and collagen synthesis/degradation may play a role in pressure-overload induced cardiac remodeling.     

Role of microtubules versus myosin heavy chain isoforms in contractile dysfunction of hypertrophied murine cardiocytes

In large mammals there is a correlation between microtubule network densification and contractile dysfunction in severe pressure-overload hypertrophy. In small mammals there is a similar correlation for the shift to beta-myosin heavy chain (MHC), a MHC isoform having a slower ATPase Vmax. In this study, murine left ventricular (LV) pressure overload invoked both mechanisms: microtubule network densification and beta-MHC expression. Cardiac beta-MHC was also augmented without altering tubulin levels by two load-independent means, chemical thyroidectomy and transgenesis. In hypertrophy, contractile function of the LV and its cardiocytes decreased proportionally; microtubule depolymerization restored normal cellular contraction. In hypothyroid mice having a complete shift from alpha-MHC to beta-MHC, contractile function of the LV and its cardiocytes also decreased, but microtubule depolymerization had no effect on cellular contraction. In transgenic mice having a cardiac beta-MHC increase similar to that in hypertrophy, contractile function of the LV and its cardiocytes was normal, and microtubule depolymerization had no effect. Thus, although both mechanisms may cause contractile dysfunction, for the extent of MHC isoform switching seen even in severe murine LV pressure-overload hypertrophy, microtubule network densification appears to have the more important role.     

Increased insulin-like growth factor-I gene expression precedes left ventricular cardiomyocyte hypertrophy in a rapidly-hypertrophying rat model system

Chronic pressure overload leads to an increase in the size, i.e. hypertrophy, of cardiomyocytes in the heart. However, the molecular mechanisms underlying this hypertrophy are not understood. Insulin-like growth factor-I (IGF-I) synthesized locally in the heart is known to be associated with the hypertrophic process. So far, however, cardiac IGF-I gene expression in the widely used rat model system has only been shown to be increased when the hypertrophy induced by pressure-overload was already established. Therefore, the question of whether IGF-I serves as an initiating or early-enhancing factor for the cardiac hypertrophy remains unanswered. Here, cardiac hypertension and hypertrophy were rapidly induced in the rat by complete constriction of the abdominal aorta between the origins of the renal arteries. Carotid arterial systolic blood pressure remained unchanged in sham rats but increased rapidly in the pressure-overloaded constricted rats with a sustained hypertension established by 3 days. Hypertrophy of left ventricular (LV) cardiomyocytes in constricted rats also occurred by 3 days. However, this hypertrophy was preceded by increases in LV IGF-I mRNA and protein which occurred within 1 day. These results support the hypothesis that cardiac-synthesized IGF-I is an initiating or early-enhancing factor for hypertrophy of LV cardiomyocytes.     

Compensated cardiac hypertrophy is characterised by a decline in palmitate oxidation

Cardiac hypertrophy is an independent risk factor in the development of heart failure. However, the cellular mechanisms underlying the transition from compensated hypertrophy to heart failure are incompletely understood. The aim of this study was to investigate changes in myocardial substrate utilisation and function in pressure-overload hypertrophy (using ¹³C NMR spectroscopy) in parallel with alterations in the expression pattern of genes involved in cardiac fatty acid and glucose uptake and oxidation. Left ventricular hypertrophy was induced surgically in Sprague–Dawley rats by inter-renal aortic constriction. Nine weeks later, hearts were perfused in the isovolumic mode with a physiological mixture of substrates including 5 mM 1-¹³C glucose, 1 mM 3-¹³C lactate, 0.1 mM U-¹³C pyruvate and 0.3 mM U-¹³C palmitate and cardiac function monitored simultaneously. Real-time PCR was used to determine mRNA levels of PPARα and PPARα-regulated metabolic enzymes. Results showed that at the stage of compensated hypertrophy, fatty acid oxidation (FAO) and expression of genes involved in FAO were markedly reduced, whilst pyruvate oxidation was enhanced, highlighting the fact that metabolic remodelling is an early event in the development of cardiac hypertrophy.     

新基因C10orf97参与压力超负荷性心肌肥厚

目的检测新基因C10orf97是否参与压力超负荷型心肌肥厚病程。方法通过缩窄大鼠胸主动脉横支构建压力超负荷诱导的心肌肥厚模型,在缩窄手术后的连续时间点应用血流动力学检测评价心室重构和心功能,应用实时荧光定量PCR法检测心肌肥厚标志基因心房利钠肽和C10orf97的mRNA表达。结果主动脉缩窄手术后,大鼠心脏显著肥厚,心脏体重比逐渐增加,心功能先受损后代偿性增强。心房利钠肽表达显著上调,在缩窄后第15天升高为假手术组40倍。C10orf97基因的表达在缩窄后第2天即显著上调为假手术组的2倍,在第4天降低,随后逐渐上升,第15天时表达量升高为假手术组的3倍。结论C10orf97基因参与了压力超负荷引起的心肌肥厚病程。     

Pathological cardiac remodeling seen by the eyes of proteomics

Cardiac remodeling involves cellular and structural changes that occur as consequence of multifactorial events to maintain the homeostasis. The progression of pathological cardiac remodeling involves a transition from adaptive to maladaptive changes that eventually leads to impairment of ventricular function and heart failure. In this scenario, proteins are key elements that orchestrate molecular events as increased expression of fetal genes, neurohormonal and second messengers' activation, contractile dysfunction, rearrangement of the extracellular matrix and alterations in heart geometry. Mass spectrometry based-proteomics has emerged as a sound method to study protein dysregulation and identification of cardiac diseases biomarkers in plasma. In this review, we summarize the main findings related to large-scale proteome modulation of cardiac cells and extracellular matrix occurred during pathological cardiac remodeling. We describe the recent proteomic progresses in the selection of protein targets and introduce the renin-angiotensin system as an interesting target for the treatment of pathological cardiac remodeling.     

Nitric oxide and the enigma of cardiac hypertrophy

In pathological conditions associated with persistent increases in hemodynamic workload (old myocardial infarction, high blood pressure, valvular heart disease), a number of signalling pathways are activated in the heart, all of which promote hypertrophic growth of the heart, characterised at the cellular level by increases in individual cardiac myocyte size. Some of these pathways are required for a successful adaptation to cardiac injury. Other pathways are maladaptive, however, as they lead to progressive contractile dysfunction and heart failure. The free radical gas nitric oxide and natriuretic peptides, both of which are produced in the heart, have emerged as endogenous inhibitors of maladaptive hypertrophy signalling. Overall, it appears that cardiac hypertrophy is controlled by an interplay of pro- and antihypertrophic signalling networks. This delicate balance can tip towards adaptation or heart failure. In the future, patients living with cardiac disease may benefit from therapeutic strategies targeting maladaptive hypertrophy signalling pathways.     

Role of AMP-activated protein kinase in healthy and diseased hearts

The heart is capable of utilizing a variety of substrates to produce the necessary ATP for cardiac function. AMP-activated protein kinase (AMPK) has emerged as a key regulator of cellular energy homeostasis and coordinates multiple catabolic and anabolic pathways in the heart. During times of acute metabolic stresses, cardiac AMPK activation seems to be primarily involved in increasing energy-generating pathways to maintain or restore intracellular ATP levels. In acute situations such as mild ischemia or short durations of severe ischemia, activation of cardiac AMPK appears to be necessary for cardiac myocyte function and survival by stimulating ATP generation via increased glycolysis and accelerated fatty acid oxidation. Whereas AMPK activation may be essential for adaptation of cardiac energy metabolism to acute and/or minor metabolic stresses, it is unknown whether AMPK activation becomes maladaptive in certain chronic disease states and/or extreme energetic stresses. However, alterations in cardiac AMPK activity are associated with a number of cardiovascular-related diseases such as pathological cardiac hypertrophy, myocardial ischemia, glycogen storage cardiomyopathy, and Wolff-Parkinson-White syndrome, suggesting the possibility of a maladaptive role. Although the precise role AMPK plays in the diseased heart is still in question, it is clear that AMPK is a major regulator of cardiac energy metabolism. The consequences of alterations in AMPK activity and subsequent cardiac energy metabolism in the healthy and the diseased heart will be discussed.     

Ablation of telomerase and telomere loss leads to cardiac dilatation and heart failure associated with p53 upregulation

Cardiac failure is a frequent cause of death in the aging human population. Telomere attrition occurs with age, and is proposed to be causal for the aging process. To determine whether telomere shortening leads to a cardiac phenotype, we studied heart function in the telomerase knockout mouse, Terc-/-. We studied Terc-/- mice at the second, G2, and fifth, G5, generation. Telomere shortening in G2 and G5 Terc-/- mice was coupled with attenuation in cardiac myocyte proliferation, increased apoptosis and cardiac myocyte hypertrophy. On a single-cell basis, telomere shortening was coincidental with increased expression of p53, indicating the presence of dysfunctional telomeres in cardiac myocytes from G5 Terc-/- mice. The impairment in cell division, the enhanced cardiac myocyte death and cellular hypertrophy, are concomitant with ventricular dilation, thinning of the wall and cardiac dysfunction. Thus, inhibition of cardiac myocyte replication provoked by telomere shortening, results in de-compensated eccentric hypertrophy and heart failure in mice. Telomere shortening with age could also contribute to cardiac failure in humans, opening the possibility for new therapies.     

Expression profiling reveals differences in metabolic gene expression between exercise-induced cardiac effects and maladaptive cardiac hypertrophy

While cardiac hypertrophy elicited by pathological stimuli eventually leads to cardiac dysfunction, exercise-induced hypertrophy does not. This suggests that a beneficial hypertrophic phenotype exists. In search of an underlying molecular substrate we used microarray technology to identify cardiac gene expression in response to exercise. Rats exercised for seven weeks on a treadmill were characterized by invasive blood pressure measurements and echocardiography. RNA was isolated from the left ventricle and analysed on DNA microarrays containing 8740 genes. Selected genes were analysed by quantitative PCR. The exercise program resulted in cardiac hypertrophy without impaired cardiac function. Principal component analysis identified an exercise-induced change in gene expression that was distinct from the program observed in maladaptive hypertrophy. Statistical analysis identified 267 upregulated genes and 62 downregulated genes in response to exercise. Expression changes in genes encoding extracellular matrix proteins, cytoskeletal elements, signalling factors and ribosomal proteins mimicked changes previously described in maladaptive hypertrophy. Our most striking observation was that expression changes of genes involved in beta-oxidation of fatty acids and glucose metabolism differentiate adaptive from maladaptive hypertrophy. Direct comparison to maladaptive hypertrophy was enabled by quantitative PCR of key metabolic enzymes including uncoupling protein 2 (UCP2) and fatty acid translocase (CD36). DNA microarray analysis of gene expression changes in exercise-induced cardiac hypertrophy suggests that a set of genes involved in fatty acid and glucose metabolism could be fundamental to the beneficial phenotype of exercise-induced hypertrophy, as these changes are absent or reversed in maladaptive hypertrophy.     

Longstanding Hyperthyroidism Is Associated with Normal or Enhanced Intrinsic Cardiomyocyte Function despite Decline in Global Cardiac Function

Thyroid hormones (THs) play a pivotal role in cardiac homeostasis. TH imbalances alter cardiac performance and ultimately cause cardiac dysfunction. Although short-term hyperthyroidism typically leads to heightened left ventricular (LV) contractility and improved hemodynamic parameters, chronic hyperthyroidism is associated with deleterious cardiac consequences including increased risk of arrhythmia, impaired cardiac reserve and exercise capacity, myocardial remodeling, and occasionally heart failure. To evaluate the long-term consequences of chronic hyperthyroidism on LV remodeling and function, we examined LV isolated myocyte function, chamber function, and whole tissue remodeling in a hamster model. Three-month-old F1b hamsters were randomized to control or 10 months TH treatment (0.1% grade I desiccated TH). LV chamber remodeling and function was assessed by echocardiography at 1, 2, 4, 6, 8, and 10 months of treatment. After 10 months, terminal cardiac function was assessed by echocardiography and LV hemodynamics. Hyperthyroid hamsters exhibited significant cardiac hypertrophy and deleterious cardiac remodeling characterized by myocyte lengthening, chamber dilatation, decreased relative wall thickness, increased wall stress, and increased LV interstitial fibrotic deposition. Importantly, hyperthyroid hamsters demonstrated significant LV systolic and diastolic dysfunction. Despite the aforementioned remodeling and global cardiac decline, individual isolated cardiac myocytes from chronically hyperthyroid hamsters had enhanced function when compared with myocytes from untreated age-matched controls. Thus, it appears that long-term hyperthyroidism may impair global LV function, at least in part by increasing interstitial ventricular fibrosis, in spite of normal or enhanced intrinsic cardiomyocyte function.     

Overexpression of microRNA-99a Attenuates Cardiac Hypertrophy

Pathological cardiomyocyte hypertrophy is associated with significantly increased risk of heart failure, one of the leading medical causes of mortality worldwide. MicroRNAs are known to be involved in pathological cardiac remodeling. However, whether miR-99a participates in the signaling cascade leading to cardiac hypertrophy is unknown. To evaluate the role of miR-99a in cardiac hypertrophy, we assessed the expression of miR-99a in hypertrophic cardiomyocytes induced by isoprenaline (ISO)/angiotensin-II (Ang II) and in mice model of cardiac hypertrophy induced by transverse aortic constriction (TAC). Expression of miR-99a was evaluated in these hypertrophic cells and hearts. We also found that miR-99a expression was highly correlated with cardiac function of mice with heart failure (8 weeks after TAC surgery). Overexpression of miR-99a attenuated cardiac hypertrophy in TAC mice and cellular hypertrophy in stimuli treated cardiomyocytes through down-regulation of expression of mammalian target of rapamycin (mTOR). These results indicate that miR-99a negatively regulates physiological hypertrophy through mTOR signaling pathway, which may provide a new therapeutic approach for pressure-overload heart failure.     

Muscle RING Finger-1 Promotes a Maladaptive Phenotype in Chronic Hypoxia-Induced Right Ventricular Remodeling

Exposure to chronic hypoxia (CH) induces elevated pulmonary artery pressure/resistance, leading to an eventual maladaptive right ventricular hypertrophy (RVH). Muscle RING finger-1 (MuRF1) is a muscle-specific ubiquitin ligase that mediates myocyte atrophy and has been shown to play a role in left ventricular hypertrophy and altered cardiac bioenergetics in pressure overloaded hearts. However, little is known about the contribution of MuRF1 impacting RVH in the setting of CH. Therefore, we hypothesized that MuRF1 deletion would enhance RVH compared to their wild-type littermates, while cardiac-specific overexpression would reduce hypertrophy following CH-induced pulmonary hypertension. We assessed right ventricular systolic pressure (RVSP), right ventricle to left ventricle plus septal weight ratio (RV/LV+S) and hematocrit (Hct) following a 3-wk isobaric CH exposure. Additionally, we conducted dual-isotope SPECT/CT imaging with cardiac function agent ²⁰¹Tl-chloride and cell death agent ^99mTc-annexin V. Predictably, CH induced pulmonary hypertension, measured by increased RVSP, RV/LV+S and Hct in WT mice compared to normoxic WT mice. Normoxic WT and MuRF1-null mice exhibited no significant differences in RVSP, RV/LV+S or Hct. CH-induced increases in RVSP were also similar between WT and MuRF1-null mice; however, RV/LV+S and Hct were significantly elevated in CH-exposed MuRF1-null mice compared to WT. In cardiac-specific MuRF1 overexpressing mice, RV/LV+S increased significantly due to CH exposure, even greater than in WT mice. This remodeling appeared eccentric, maladaptive and led to reduced systemic perfusion. In conclusion, these results are consistent with an atrophic role for MuRF1 regulating the magnitude of right ventricular hypertrophy following CH-induction of pulmonary hypertension.