RNA Expression Profiling of Human iPSC-Derived Cardiomyocytes in a Cardiac Hypertrophy Model

Cardiac hypertrophy is an independent risk factor for cardiovascular disease and heart failure. There is increasing evidence that microRNAs (miRNAs) play an important role in the regulation of messenger RNA (mRNA) and the pathogenesis of various cardiovascular diseases. However, the ability to comprehensively study cardiac hypertrophy on a gene regulatory level is impacted by the limited availability of human cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer the opportunity for disease modeling. Here we utilize a previously established in vitro model of cardiac hypertrophy to interrogate the regulatory mechanism associated with the cardiac disease process. We perform miRNA sequencing and mRNA expression analysis on endothelin 1 (ET-1) stimulated hiPSC-CMs to describe associated RNA expression profiles. MicroRNA sequencing revealed over 250 known and 34 predicted novel miRNAs to be differentially expressed between ET-1 stimulated and unstimulated control hiPSC-CMs. Messenger RNA expression analysis identified 731 probe sets with significant differential expression. Computational target prediction on significant differentially expressed miRNAs and mRNAs identified nearly 2000 target pairs. A principal component analysis approach comparing the in vitro data with human myocardial biopsies detected overlapping expression changes between the in vitro samples and myocardial biopsies with Left Ventricular Hypertrophy. These results provide further insights into the complex RNA regulatory mechanism associated with cardiac hypertrophy.     

Ca<Superscript>2+</Superscript> signalling in cardiovascular disease: the role of the plasma membrane calcium pumps

The plasma membrane calcium ATPases (PMCA) are a family of genes which extrude Ca²⁺ from the cell and are involved in the maintenance of intracellular free calcium levels and/or with Ca²⁺ signalling, depending on the cell type. In the cardiovascular system, Ca²⁺ is not only essential for contraction and relaxation but also has a vital role as a second messenger in signal transduction pathways. A complex array of mechanisms regulate intracellular free calcium levels in the heart and vasculature and a failure in these systems to maintain normal Ca²⁺ homeostasis has been linked to both heart failure and hypertension. This article focuses on the functions of PMCA, in particular isoform 4 (PMCA4), in the heart and vasculature and the reported links between PMCAs and contractile function, cardiac hypertrophy, cardiac rhythm and sudden cardiac death, and blood pressure control and hypertension. It is becoming clear that this family of calcium extrusion pumps have essential roles in both cardiovascular health and disease.     

Cardiac overexpression of myotrophin triggers myocardial hypertrophy and heart failure in transgenic mice

Cardiac hypertrophy and heart failure remain leading causes of death in the United States. Many studies have suggested that, under stress, myocardium releases factors triggering protein synthesis and stimulating myocyte growth. We identified and cloned myotrophin, a 12-kDa protein from hypertrophied human and rat hearts. Myotrophin (whose gene is localized on human chromosome 7q33) stimulates myocyte growth and participates in cellular interaction that initiates cardiac hypertrophy in vitro. In this report, we present data on the pathophysiological significance of myotrophin in vivo, showing the effects of overexpression of cardio-specific myotrophin in transgenic mice in which cardiac hypertrophy occurred by 4 weeks of age and progressed to heart failure by 9-12 months. This hypertrophy was associated with increased expression of proto-oncogenes, hypertrophy marker genes, growth factors, and cytokines, with symptoms that mimicked those of human cardiomyopathy, functionally and morphologically. This model provided a unique opportunity to analyze gene clusters that are differentially up-regulated during initiation of hypertrophy versus transition of hypertrophy to heart failure. Importantly, changes in gene expression observed during initiation of hypertrophy were significantly different from those seen during its transition to heart failure. Our data show that overexpression of myotrophin results in initiation of cardiac hypertrophy that progresses to heart failure, similar to changes in human heart failure. Knowledge of the changes that take place as a result of overexpression of myotrophin at both the cellular and molecular levels will suggest novel strategies for treatment to prevent hypertrophy and its progression to heart failure.     

MicroRNA-375-3p inhibitor suppresses angiotensin II-induced cardiomyocyte hypertrophy by promoting lactate dehydrogenase B expression

Cardiac hypertrophy is a myocardial enlargement due to overload pressure, and the primary cause of heart failure. We investigated the function of miR-375-3p in cardiac hypertrophy and its regulating mechanisms. miR-375-3p was upregulated in hearts of the transverse aortic constriction rat model and angiotensin II (Ang II)-induced primary cardiomyocyte hypertrophy model; the opposite was observed for lactate dehydrogenase B (LDHB) protein expression. miR-375-3p knockdown reduced the surface area of primary cardiomyocytes increased by Ang II treatment and decreased the B-natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) messenger RNA (mRNA) and protein levels. miR-375-3p was also observed to directly target LDHB. LDHB knockdown increased the surface area of Ang II-treated primary cardiomyocytes and increased the BNP and β-MHC mRNA and protein levels. LDHB knockdown attenuated the effects of miR-375-3p on the surface area of primary cardiomyocytes and BNP and β-MHC levels. Therefore, miR-375-3p inhibitor suppresses Ang II-induced cardiomyocyte hypertrophy by promoting LDHB expression.     

Prevention of cardiac hypertrophy and heart failure by silencing of NF-kappaB

Activation of the nuclear factor (NF)-κB signaling pathway may be associated with the development of cardiac hypertrophy and its transition to heart failure (HF). The transgenic Myo-Tg mouse develops hypertrophy and HF as a result of overexpression of myotrophin in the heart associated with an elevated level of NF-κB activity. Using this mouse model and an NF-κB-targeted gene array, we first determined the components of NF-κB signaling cascade and the NF-κB-linked genes that are expressed during the progression to cardiac hypertrophy and HF. Second, we explored the effects of inhibition of NF-κB signaling events by using a gene knockdown approach: RNA interference through delivery of a short hairpin RNA against NF-κB p65 using a lentiviral vector (L-sh-p65). When the short hairpin RNA was delivered directly into the hearts of 10-week-old Myo-Tg mice, there was a significant regression of cardiac hypertrophy, associated with a significant reduction in NF-κB activation and atrial natriuretic factor expression. Our data suggest, for the first time, that inhibition of NF-κB using direct gene delivery of sh-p65 RNA results in regression of cardiac hypertrophy. These data validate NF-κB as a therapeutic target to prevent hypertrophy/HF.     

Protocatechuic acid prevents isoproterenol-induced heart failure in mice by downregulating kynurenine-3-monooxygenase

Protocatechuic acid (3,4-dihydroxybenzoic acid) prevents oxidative stress, inflammation and cardiac hypertrophy. This study aimed to investigate the therapeutic effects of protocatechuic acid in an isoproterenol-induced heart failure mouse model and to identify the underlying mechanisms. To establish the heart failure model, C57BL/6NTac mice were given high-dose isoproterenol (80 mg/kg body weight) for 14 days. Echocardiography revealed that protocatechuic acid reversed the isoproterenol-induced downregulation of fractional shortening and ejection fraction. Protocatechuic acid attenuated cardiac hypertrophy as evidenced by the decreased heart-weight-to-body-weight ratio and the expression of Nppb. RNA sequencing analysis identified kynurenine-3-monooxygenase (Kmo) as a potential target of protocatechuic acid. Protocatechuic acid treatment or transfection with short-interfering RNA against Kmo ameliorated transforming growth factor β1–induced upregulation of Kmo, Col1a1, Col1a2 and Fn1 in vivo or in neonatal rat cardiac fibroblasts. Kmo knockdown attenuated the isoproterenol-induced increase in cardiomyocyte size, as well as Nppb and Col1a1 expression in H9c2 cells or primary neonatal rat cardiomyocytes. Moreover, protocatechuic acid attenuated Kmo overexpression–induced increases in Nppb mRNA levels. Protocatechuic acid or Kmo knockdown decreased isoproterenol-induced ROS generation in vivo and in vitro. Thus, protocatechuic acid prevents heart failure by downregulating Kmo. Therefore, protocatechuic acid and Kmo constitute a potential novel therapeutic agent and target, respectively, against heart failure.     

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.     

Loss of a gp130 cardiac muscle cell survival pathway is a critical event in the onset of heart failure during biomechanical stress

Biomechanical stress is a major stimulus for cardiac hypertrophy and the transition to heart failure. By generating mice that harbor a ventricular restricted knockout of the gp130 cytokine receptor via Cre-IoxP-mediated recombination, we demonstrate a critical role for a gp130-dependent myocyte survival pathway in the transition to heart failure. Such conditional mutant mice have normal cardiac structure and function, but during aortic pressure overload, these mice display rapid onset of dilated cardiomyopathy and massive induction of myocyte apoptosis versus the control mice that exhibit compensatory hypertrophy. Thus, cardiac myocyte apoptosis is a critical point in the transition between compensatory cardiac hypertrophy and heart failure. gp130-dependent cytokines may represent a novel therapeutic strategy for preventing in vivo heart failure.     

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a new promising target for prevention and treatment of cardiac hypertrophy and heart failure. There are three δ isoforms of CaMKII in the heart and previous studies focused primarily on δB and δC types. Here we report the δA isoform of CaMKII is also critically involved in cardiac hypertrophy. We found that δA was significantly upregulated in pathological cardiac hypertrophy in both neonatal and adult models. Upregulation of δA was accompanied by cell enlargement, sarcomere reorganization and reactivation of various hypertrophic cardiac genes including atrial natriuretic factor (ANF) and β-myocin heavy chain (β-MHC). Studies further indicated the pathological changes were largely blunted by silencing the δA gene and an underlying mechanism indicated selective interference with the HDAC4-MEF2 signaling pathway. These results provide new evidence for selective interfering cardiac hypertrophy and heart failure when CaMKII is considered as a therapeutic target.     

Increased expression of the gene for alpha-interferon-inducible protein in cardiomyopathic hamster heart

Cardiomyopathic (CM) hamsters have a disruption in the delta-sarcoglycan gene which leads to progressive cardiac necrosis by 30 to 40 days of age, hypertrophy by 120 days, and heart failure by 250 days. We used differential display to detect other changes in mRNA levels in 30-, 60-, and 90-day-old wild-type and CM hamsters. We identified a 400-bp cDNA with sequence similarity to the human alpha-interferon-inducible protein (p27). This cDNA annealed with a 570-base mRNA whose steady-state levels were increased in 30-, 60-, and 90-day-old CM compared to wild-type heart. Increased expression of this hamster homolog of p27 (p27-h) was detected in CM hamster cardiac and skeletal muscle at 60 days of age but not in liver, kidney, or brain. Thus, an inherited defect in CM hamsters leads to increased expression of p27-h in advance of the development of hypertrophy and heart failure.     

Increased expression of poly(ADP-ribose) polymerase-1 contributes to caspase-independent myocyte cell death during heart failure

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a pivotal role in regulating genome stability, cell cycle progression, and cell survival. However, overactivation of PARP has been shown to contribute to cell death and organ failure in various stress-related disease conditions. In this study, we examined the role of PARP in the development and progression of cardiac hypertrophy. We measured the expression of PARP in mouse hearts with physiological (swimming exercise) and pathological (aortic banding) cardiac hypertrophy as well as in human heart samples taken at the time of transplantation. PARP levels were elevated both in swimming and banded mice hearts and demonstrated a linear positive correlation with the degree of cardiac hypertrophy. A dramatic increase (4-fold) of PARP occurred in 6-wk banded mice, accompanied by apparent signs of ventricular dilation and myocyte cell death. PARP levels were also elevated (2- to 3-fold) in human hearts with end-stage heart failure compared with controls. However, we found no evidence of caspase-mediated PARP cleavage in either mouse or human failing hearts. Overexpression of PARP in primary cultures of cardiac myocytes led to suppression of gene expression and robust myocyte cell death. Furthermore, data obtained from the analysis of PARP knockout mice revealed that these hearts produce an attenuated hypertrophic response to aortic banding compared with controls. Together, these results demonstrate a role for PARP in the onset and progression of cardiac hypertrophy and suggest that some events related to cardiac hypertrophy growth and progression to heart failure are mediated by a PARP-dependent mechanism.     

Endothelin antagonism suppresses plasma and cardiac endothelin-1 levels in SHRSPs at the typical hypertensive stage

Endothelin-1 (ET-1) has been implicated in hypertension, heart failure, atherosclerosis, and pulmonary hypertension. In all these conditions, plasma immunoreactive ET-1 levels are elevated, and tissue ET-1 expression is increased. Clinical trials have demonstrated potentially important benefits of ET antagonism among patients with essential hypertension, pulmonary hypertension, and heart failure. It is unknown whether ET antagonism affects the production of ET-1 in stroke-prone spontaneously hypertensive rat (SHRSP) heart at the typical hypertensive stage. The objective of this study was to investigate the effects of ET blockade on the expression levels of plasma and cardiac ET-1 in SHRSPs. SHRSPs were treated for 3 months with SB209670 (ET(A)/ET(B) dual receptor antagonist) or with saline (vehicle) commencing at the prehypertensive stage (age 6 weeks). Plasma and left ventricular ET-1 peptide levels were measured using enzyme-linked immunoabsorbent assay. Compared with age-matched control Wistar-Kyoto rats, peptide levels of ET-1 were significantly upregulated in vehicle-treated SHRSP heart; this upregulation was reversed by long-term ET antagonism. Plasma ET-1 levels were also significantly increased in vehicle-treated SHRSPs and were normalized by ET antagonism. mRNA expression of preproET-1, which is the source of ET-1 peptide production, was significantly increased in vehicle-treated SHRSP heart and was normalized by ET antagonism. Marked cardiac hypertrophy and fibrosis at the histologic level in SHRSPs were ameliorated by ET antagonism, and left ventricular hypertrophy as seen on echocardiography in SHRSPs was suppressed by ET blockade. After ET antagonism, systolic blood pressures were reduced in SHRSPs; diastolic blood pressures were unchanged. The reversal effect of the upregulated ET system in SHRSP heart by ET antagonism might be independent of blood pressure change. By suppressing the upregulated ET system, ET antagonism might be beneficial in arresting cardiac remodeling.     

Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro.

In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.     

Class III PI3K‐mediated prolonged activation of autophagy plays a critical role in the transition of cardiac hypertrophy to heart failure

Pathological cardiac hypertrophy often leads to heart failure. Activation of autophagy has been shown in pathological hypertrophic hearts. Autophagy is regulated positively by Class III phosphoinositide 3‐kinase (PI3K). However, it is unknown whether Class III PI3K plays a role in the transition of cardiac hypertrophy to heart failure. To address this question, we employed a previously established cardiac hypertrophy model in heat shock protein 27 transgenic mice which shares common features with several types of human cardiomyopathy. Age‐matched wild‐type mice served as control. Firstly, a prolonged activation of autophagy, as reflected by autophagosome accumulation, increased LC3 conversion and decreased p62 protein levels, was detected in hypertrophic hearts from adaptive stage to maladaptive stage. Moreover, morphological abnormalities in myofilaments and mitochondria were presented in the areas accumulated with autophagosomes. Secondly, activation of Class III PI3K Vacuolar protein sorting 34 (Vps34), as demonstrated by upregulation of Vps34 expression, increased interaction of Vps34 with Beclin‐1, and deceased Bcl‐2 expression, was demonstrated in hypertrophic hearts from adaptive stage to maladaptive stage. Finally, administration with Wortmaninn, a widely used autophagy inhibitor by suppressing Class III PI3K activity, significantly decreased autophagy activity, improved morphologies of intracellular apartments, and most importantly, prevented progressive cardiac dysfunction in hypertrophic hearts. Collectively, we demonstrated that Class III PI3K plays a central role in the transition of cardiac hypertrophy to heart failure via a prolonged activation of autophagy in current study. Class III PI3K may serve as a potential target for the treatment and management of maladaptive cardiac hypertrophy.