Dongtak Jeong

Inhibition of miR-25 improves cardiac contractility in the failing heart

Heart failure is characterized by a debilitating decline in cardiac function, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy. MicroRNAs (miRNAs) are dysregulated in heart failure but whether they control contractility or constitute therapeutic targets remains speculative. Using high-throughput functional screening of the human microRNAome, here we identify miRNAs that suppress intracellular calcium handling in heart muscle by interacting with messenger RNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a (also known as ATP2A2). Of 875 miRNAs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas adeno-associated virus 9 (AAV9)-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 markedly halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR oligonucleotide. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggest that it might be targeted therapeutically to restore function.

SUMO1-dependent modulation of SERCA2a in heart failure

The calcium-transporting ATPase ATP2A2, also known as SERCA2a, is a critical ATPase responsible for Ca2+ re-uptake during excitation–contraction coupling. Impaired Ca2+ uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure. Accordingly, restoration of SERCA2a expression by gene transfer has proved to be effective in improving cardiac function in heart-failure patients, as well as in animal models. The small ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target proteins, and is involved in many cellular processes. Here we show that SERCA2a is SUMOylated at lysines 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability in mouse and human cells. The levels of SUMO1 and the SUMOylation of SERCA2a itself were greatly reduced in failing hearts. SUMO1 restitution by adeno-associated-virus-mediated gene delivery maintained the protein abundance of SERCA2a and markedly improved cardiac function in mice with heart failure. This effect was comparable to SERCA2A gene delivery. Moreover, SUMO1 overexpression in isolated cardiomyocytes augmented contractility and accelerated Ca2+ decay. Transgene-mediated SUMO1 overexpression rescued cardiac dysfunction induced by pressure overload concomitantly with increased SERCA2a function. By contrast, downregulation of SUMO1 using small hairpin RNA (shRNA) accelerated pressure-overload-induced deterioration of cardiac function and was accompanied by decreased SERCA2a function. However, knockdown of SERCA2a resulted in severe contractile dysfunction both in vitro and in vivo, which was not rescued by overexpression of SUMO1. Taken together, our data show that SUMOylation is a critical post-translational modification that regulates SERCA2a function, and provide a platform for the design of novel therapeutic strategies for heart failure.

Therapeutic cardiac-targeted delivery of miR-1 reverses pressure overload-induced cardiac hypertrophy and attenuates pathological remodeling.

Background MicroRNAs (miRNAs) play a key role in the development of heart failure, and recent studies have shown that the muscle‐specific miR‐1 is a key regulator of cardiac hypertrophy. We tested the hypothesis that chronic restoration of miR‐1 gene expression in vivo will regress hypertrophy and protect against adverse cardiac remodeling induced by pressure overload. Methods and Results Cardiac hypertrophy was induced by left ventricular pressure overload in male Sprague‐Dawley rats subjected to ascending aortic stenosis. When the hypertrophy was established at 2 weeks after surgery, the animals were randomized to receive either an adeno‐associated virus expressing miR‐1 (AAV9.miR‐1) or green fluorescent protein (GFP) as control (AAV9.GFP) via a single‐bolus tail‐vein injection. Administration of miR‐1 regressed cardiac hypertrophy (left ventricular posterior wall thickness,; 2.32±0.08 versus 2.75±0.07 mm, P<0.001) and (left ventricular septum wall thickness, 2.23±0.06 versus 2.54±0.10 mm, P<0.05) and halted the disease progression compared with control‐treated animals, as assessed by echocardiography (fractional shortening, 37.60±5.01% versus 70.68±2.93%, P<0.05) and hemodynamic analyses (end‐systolic pressure volume relationship/effective arterial elastance, 1.87±0.46 versus 0.96±0.38, P<0.05) after 7 weeks of treatment. Additionally, miR‐1 replacement therapy lead to a marked reduction of myocardial fibrosis, an improvement in calcium handling, inhibition of apoptosis, and inactivation of the mitogen‐activated protein kinase signaling pathways, suggesting a favorable effect on preventing the maladaptive ventricular remodeling. We also identified and validated a novel bona fide target of miR‐1, Fibullin‐2 (Fbln2), a secreted protein implicated in extracellular matrix remodeling. Conclusions Taken together, our findings suggest that restoration of miR‐1 gene expression is a potential novel therapeutic strategy to reverse pressure‐induced cardiac hypertrophy and prevent maladaptive cardiac remodeling.

Resident c-kit + cells in the heart are not cardiac stem cells

Identifying a bona fide population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. Previously, cardiac c-kit+ cells were reported to be CSCs with a potential to become myocardial, endothelial and smooth muscle cells in vitro and after cardiac injury. Here we provide further insights into the nature of cardiac c-kit+ cells. By targeting the c-kit locus with multiple reporter genes in mice, we find that c-kit expression rarely co-localizes with the expression of the cardiac progenitor and myogenic marker Nkx2.5, or that of the myocardial marker, cardiac troponin T (cTnT). Instead, c-kit predominantly labels a cardiac endothelial cell population in developing and adult hearts. After acute cardiac injury, c-kit+ cells retain their endothelial identity and do not become myogenic progenitors or cardiomyocytes. Thus, our work strongly suggests that c-kit+ cells in the murine heart are endothelial cells and not CSCs.

Critical Role for Stromal Interaction Molecule 1 in Cardiac Hypertrophy

Background Cardiomyocytes (CM) utilize Ca2+ not only in excitation-contraction coupling (ECC), but also as a signaling molecule promoting for example cardiac hypertrophy. It is largely unclear how Ca2+ triggers signaling in CM in the presence of the rapid and large Ca2+ fluctuations that occur during ECC. A potential route is store-operated Ca2+ entry (SOCE), a drug-inducible mechanism for Ca2+ signaling that requires stromal interaction molecule 1 (STIM1). SOCE can also be induced in cardiomyocytes, which prompted us to study STIM1-dependent Ca2+-entry with respect to cardiac hypertrophy in vitro and in vivo.Background— Cardiomyocytes use Ca2+ not only in excitation-contraction coupling but also as a signaling molecule promoting, for example, cardiac hypertrophy. It is largely unclear how Ca2+ triggers signaling in cardiomyocytes in the presence of the rapid and large Ca2+ fluctuations that occur during excitation-contraction coupling. A potential route is store-operated Ca2+ entry, a drug-inducible mechanism for Ca2+ signaling that requires stromal interaction molecule 1 (STIM1). Store-operated Ca2+ entry can also be induced in cardiomyocytes, which prompted us to study STIM1-dependent Ca2+ entry with respect to cardiac hypertrophy in vitro and in vivo. Methods and Results— Consistent with earlier reports, we found drug-inducible store-operated Ca2+ entry in neonatal rat cardiomyocytes, which was dependent on STIM1. Although this STIM1-dependent, drug-inducible store-operated Ca2+ entry was only marginal in adult cardiomyocytes isolated from control hearts, it increased significantly in cardiomyocytes isolated from adult rats that had developed compensated cardiac hypertrophy after abdominal aortic banding. Moreover, we detected an inwardly rectifying current in hypertrophic cardiomyocytes that occurs under native conditions (ie, in the absence of drug-induced store depletion) and is dependent on STIM1. By manipulating its expression, we found STIM1 to be both sufficient and necessary for cardiomyocyte hypertrophy in vitro and in the adult heart in vivo. Stim1 silencing by adeno-associated viruses of serotype 9–mediated gene transfer protected rats from pressure overload–induced cardiac hypertrophy. Conclusion— By controlling a previously unrecognized sarcolemmal current, STIM1 promotes cardiac hypertrophy.

The opposing effects of CCN2 and CCN5 on the development of cardiac hypertrophy and fibrosis

CCN family members are matricellular proteins with diverse roles in cell function. The differential expression of CCN2 and CCN5 during cardiac remodeling suggests that these two members of the CCN family play opposing roles during the development of cardiac hypertrophy and fibrosis. We aimed to evaluate the role of CCN2 and CCN5 in the development of cardiac hypertrophy and fibrosis. In isolated cardiomyocytes, overexpression of CCN2 induced hypertrophic growth, whereas the overexpression of CCN5 inhibited both phenylephrine (PE)- and CCN2-induced hypertrophic responses. Deletion of the C-terminal (CT) domain of CCN2 transformed CCN2 into a CCN5-like dominant negative molecule. Fusion of the CT domain to the Carboxy-terminus of CCN5 transformed CCN5 into a CCN2-like pro-hypertrophic molecule. CCN2 transgenic (TG) mice did not develop cardiac hypertrophy at baseline but showed significantly increased fibrosis in response to pressure overload. In contrast, hypertrophy and fibrosis were both significantly inhibited in CCN5 TG mice. CCN2 TG mice showed an accelerated deterioration of cardiac function in response to pressure overload, whereas CCN5 TG mice showed conserved cardiac function. TGF-beta-SMAD signaling was elevated in CCN2 TG mice, but was inhibited in CCN5 TG mice. CCN2 is pro-hypertrophic and -fibrotic, whereas CCN5 is anti-hypertrophic and -fibrotic. CCN5 lacking the CT domain acts as a dominant negative molecule. CCN5 may provide a novel therapeutic target for the treatment of cardiac hypertrophy and heart failure.

JNK modulates FOXO3a for the expression of the mitochondrial death and mitophagy marker BNIP3 in pathological hypertrophy and in heart failure

Bcl-2 E1B 19-KDa interacting protein 3 (BNIP3) is a mitochondrial death and mitophagy marker, which is involved in inducing cardiac remodeling post myocardial infarction. In this study, we show that BNIP3 expression increases in stressed cardiomyocytes in vitro and in response to pressure overload in vivo, and that its transcription is directly related to JNK activity. BNIP3 expression gradually increased in the first weeks after pressure overload and peaked at the heart failure stage. Ultrastructurally, the mitochondrial area was inversely proportional to BNIP3 expression. Both JNK and AKT activities increased with pressure overload; however, JNK signaling dominated over AKT signaling for the activation of the transcription factor FOXO3a and for the transcription of its effector, BNIP3. 3-methyladenine attenuated JNK signaling and significantly decreased BNIP3 expression and reversed cardiac remodeling in heart failure. Ultrastructurally, the mitochondrial area was significantly increased in the 3-methyladenine group compared with placebo. Moreover, adenoviral gene delivery of dominant negative JNK in a rat model of pressure overload hypertrophy abolished the increase in BNIP3 expression in response to pressure overload. These results suggest that JNK signaling is a critical modulator of the transcription factor FOXO3a driving the expression of its effector, BNIP3, in heart failure and that JNK, through BNIP3, induces mitochondrial apoptosis and mitophagy.

PICOT Inhibits Cardiac Hypertrophy and Enhances Ventricular Function and Cardiomyocyte Contractility

Multiple signaling pathways involving protein kinase C (PKC) have been implicated in the development of cardiac hypertrophy. We observed that a putative PKC inhibitor, PICOT (PKC-Interacting Cousin Of Thioredoxin) was upregulated in response to hypertrophic stimuli both in vitro and in vivo. This suggested that PICOT may act as an endogenous negative feedback regulator of cardiac hypertrophy through its ability to inhibit PKC activity, which is elevated during cardiac hypertrophy. Adenovirus-mediated gene transfer of PICOT completely blocked the hypertrophic response of neonatal rat cardiomyocytes to enthothelin-1 and phenylephrine, as demonstrated by cell size, sarcomere rearrangement, atrial natriuretic factor expression, and rates of protein synthesis. Transgenic mice with cardiac-specific overexpression of PICOT showed that PICOT is a potent inhibitor of cardiac hypertrophy induced by pressure overload. In addition, PICOT overexpression dramatically increased the ventricular function and cardiomyocyte contractility as measured by ejection fraction and end-systolic pressure of transgenic hearts and peak shortening of isolated cardiomyocytes, respectively. Intracellular Ca2+ handing analysis revealed that increases in myofilament Ca2+ responsiveness, together with increased rate of sarcoplasmic reticulum Ca2+ reuptake, are associated with the enhanced contractility in PICOT-overexpressing cardiomyocytes. The inhibition of cardiac remodeling by of PICOT with a concomitant increase in ventricular function and cardiomyocyte contractility suggests that PICOT may provide an efficient modality for treatment of cardiac hypertrophy and heart failure.

PICOT Attenuates Cardiac Hypertrophy by Disrupting Calcineurin–NFAT Signaling

PICOT (protein kinase C–interacting cousin of thioredoxin) was previously shown to inhibit pressure overload-induced cardiac hypertrophy, concomitant with an increase in ventricular function and cardiomyocyte contractility. The combined analyses of glutathione S-transferase pull-down experiments and mass spectrometry enabled us to determine that PICOT directly interacts with muscle LIM protein (MLP) via its carboxyl-terminal half (PICOT-C). It was also shown that PICOT colocalizes with MLP in the Z-disc. MLP is known to play a role in anchoring calcineurin to the Z-disc in the sarcomere, which is critical for calcineurin–NFAT (nuclear factor of activated T cells) signaling. We, therefore, suggested that PICOT may affect calcineurin–NFAT signaling through its interaction with MLP. Consistent with this hypothesis, PICOT, or more specifically PICOT-C, abrogated phenylephrine-induced increases in calcineurin phosphatase activity, NFAT dephosphorylation/nuclear translocation, and NFAT-dependent transcriptional activation in neonatal cardiomyocytes. In addition, pressure overload–induced upregulation of NFAT target genes was significantly diminished in the hearts of PICOT-overexpressing transgenic mice. PICOT interfered with MLP–calcineurin interactions in a dose-dependent manner. Moreover, calcineurin was displaced from the Z-disc, concomitant with an abrogated interaction between calcineurin and MLP, in the hearts of PICOT transgenic mice. Replenishment of MLP restored the hypertrophic responses and the increase in calcineurin phosphatase activity that was inhibited by PICOT in phenylephrine-treated cardiomyocytes. Finally, PICOT-C inhibited cardiac hypertrophy to an extent that was comparable to that of full-length PICOT. Taken together, these data suggest that PICOT inhibits cardiac hypertrophy largely by negatively regulating calcineurin–NFAT signaling via disruption of the MLP–calcineurin interaction.

AAV9.I-1c Delivered via Direct Coronary Infusion in a Porcine Model of Heart Failure Improves Contractility and Mitigates Adverse Remodeling

Background—Heart failure is characterized by impaired function and disturbed Ca2+ homeostasis. Transgenic increases in inhibitor-1 activity have been shown to improve Ca2 cycling and preserve cardiac performance in the failing heart. The aim of this study was to evaluate the effect of activating the inhibitor (I-1c) of protein phosphatase 1 (I-1) through gene transfer on cardiac function in a porcine model of heart failure induced by myocardial infarction. Methods and Results—Myocardial infarction was created by a percutaneous, permanent left anterior descending artery occlusion in Yorkshire Landrace swine (n=16). One month after myocardial infarction, pigs underwent intracoronary delivery of either recombinant adeno-associated virus type 9 carrying I-1c (n=8) or saline (n=6) as control. One month after myocardial infarction was created, animals exhibited severe heart failure demonstrated by decreased ejection fraction (46.4±7.0% versus sham 69.7±8.5%) and impaired (dP/dt)max and (dP/dt)min. Intracoronary injection of AAV9.I-1c prevented further deterioration of cardiac function and led to a decrease in scar size. Conclusions—In this preclinical model of heart failure, overexpression of I-1c by intracoronary in vivo gene transfer preserved cardiac function and reduced the scar size.