Mikhail Novikov

Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle

The ability to image cardiomyocyte apoptosis in vivo with high‐resolution MRI could facilitate the development of novel cardioprotective therapies. The sensitivity of the novel nanoparticle AnxCLIO‐Cy5.5 for cardiomyocyte apoptosis was thus compared in vitro to that of annexin V‐FITC and showed a high degree of colocalization. MRI was then performed, following transient coronary artery (LAD) occlusion, in five mice given AnxCLIO‐Cy5.5 and in four mice given an identical dose (2 mg Fe/kg) of CLIO‐Cy5.5. MR signal intensity and myocardial T2* were evaluated, in vivo, in hypokinetic regions of myocardium in the LAD distribution. Ex vivo fluorescence imaging was performed to confirm the in vivo findings. Myocardial T2* was significantly lower in the mice given AnxCLIO‐Cy5.5 (8.1 versus 13.2 ms, P < 0.01), and fluorescence target to background ratio was significantly higher (2.1 versus 1.1, P < 0.01). This study thus demonstrates the feasibility of obtaining high‐resolution MR images of cardiomyocyte apoptosis in vivo with the novel nanoparticle, AnxCLIO‐Cy5.5. Magn Reson Med, 2005.

Fluorescence Tomography and Magnetic Resonance Imaging of Myocardial Macrophage Infiltration in Infarcted Myocardium In Vivo

Background— Fluorescence imaging of the heart is currently limited to invasive ex vivo or in vitro applications. We hypothesized that the adaptation of advanced transillumination and tomographic techniques would allow noninvasive fluorescence images of the heart to be acquired in vivo and be coregistered with in vivo cardiac magnetic resonance images. Methods and Results— The uptake of the magnetofluorescent nanoparticle CLIO-Cy5.5 by macrophages in infarcted myocardium was studied. Ligation of the left coronary artery was performed in 12 mice and sham surgery in 7. The mice were injected, 48 hours after surgery, with 3 to 20 mg of iron per kilogram of CLIO-Cy5.5. Magnetic resonance imaging and fluorescence molecular tomography were performed 48 hours later. An increase in magnetic resonance imaging contrast-to-noise ratio, indicative of myocardial probe accumulation, was seen in the anterolateral walls of the infarcted mice but not in the sham-operated mice (23.0±2.7 versus 5.43±2.4; P<0.01). Fluorescence intensity over the heart was also significantly greater in the fluorescence molecular tomography images of the infarcted mice (19.1±5.2 versus 5.3±1.4; P<0.05). The uptake of CLIO-Cy5.5 by macrophages infiltrating the infarcted myocardium was confirmed by fluorescence microscopy and immunohistochemistry. Conclusions— Noninvasive imaging of myocardial macrophage infiltration has been shown to be possible by both fluorescence tomography and magnetic resonance imaging. This could be of significant value in both the research and clinical settings. The techniques developed could also be used to image other existing fluorescent and magnetofluorescent probes and could significantly expand the role of fluorescence imaging in the heart.

Factor XIII Deficiency Causes Cardiac Rupture, Impairs Wound Healing, and Aggravates Cardiac Remodeling in Mice With Myocardial Infarction

Background— Identification of key molecular players in myocardial healing could lead to improved therapies, reduction of scar formation, and heart failure after myocardial infarction (MI). We hypothesized that clotting factor XIII (FXIII), a transglutaminase involved in wound healing, may play an important role in MI given prior clinical and mouse model data. Methods and Results— To determine whether a truly causative relationship existed between FXIII activity and myocardial healing, we prospectively studied myocardial repair in FXIII-deficient mice. All FXIII−/− and FXIII−/+ (FXIII activity <5% and 70%) mice died within 5 days after MI from left ventricular rupture. In contradistinction, FXIII−/− mice that received 5 days of intravenous FXIII replacement therapy had normal survival rates; however, cardiac MRI demonstrated worse left ventricular remodeling in these reconstituted FXIII−/− mice. Using a FXIII-sensitive molecular imaging agent, we found significantly greater FXIII activity in wild-type mice and FXIII−/− mice receiving supplemental FXIII than in FXIII−/− mice (P<0.05). In FXIII−/− but not in reconstituted FXIII−/− mice, histology revealed diminished neutrophil migration into the MI. Reverse transcriptase–polymerase chain reaction studies suggested that the impaired inflammatory response in FXIII−/− mice was independent of intercellular adhesion molecule and lipopolysaccharide-induced CXC chemokine, both important for cell migration. After MI, expression of matrix metalloproteinase-9 was 650% higher and collagen-1 was 53% lower in FXIII−/− mice, establishing an imbalance in extracellular matrix turnover and providing a possible mechanism for the observed cardiac rupture in the FXIII−/− mice. Conclusions— These data suggest that FXIII has an important role in murine myocardial healing after infarction.

Myostatin Regulates Cardiomyocyte Growth Through Modulation of Akt Signaling

Myostatin is a highly conserved, potent negative regulator of skeletal muscle hypertrophy in many species, from rodents to humans, although its mechanisms of action are incompletely understood. Transcript profiling of hearts from a genetic model of cardiac hypertrophy revealed dramatic upregulation of myostatin, not previously recognized to play a role in the heart. Here we show that myostatin abrogates the cardiomyocyte growth response to phenylephrine in vitro through inhibition of p38 and the serine–threonine kinase Akt, a critical determinant of cell size in many species from drosophila to mammals. Evaluation of male myostatin-null mice revealed that their cardiomyocytes and hearts overall were slightly smaller at baseline than littermate controls but exhibited more exuberant growth in response to chronic phenylephrine infusion. The increased cardiac growth in myostatin-null mice corresponded with increased p38 phosphorylation and Akt activation in vivo after phenylephrine treatment. Together, these data demonstrate that myostatin is dynamically regulated in the heart and acts more broadly than previously appreciated to regulate growth of multiple types of striated muscle.

Serum and Glucocorticoid-Responsive Kinase-1 Regulates Cardiomyocyte Survival and Hypertrophic Response

Background—Serum- and glucocorticoid-responsive kinase-1 (SGK1), a serine-threonine kinase that is highly expressed in the heart, has been previously reported to regulate sodium channels. Because SGK1 is a PI 3-kinase–dependent kinase with structural homology to Akt, we examined its regulation in the heart and its effects on cardiomyocyte (CM) apoptosis and hypertrophy in vitro. Methods and Results—Rats were subjected to aortic banding, and expression of total and phosphorylated SGK1 was examined. Both phospho- and total SGK1 increased 2 to 7 days after banding. Phospho-SGK1 was also upregulated in CMs stimulated in vitro with IGF-I or phenylephrine. Infection of CMs with an adenoviral vector encoding constitutively active SGK1 (Ad.SGK1.CA) inhibited apoptosis after serum-deprivation or hypoxia (P<0.05), whereas expression of kinase-dead SGK1 (Ad.SGK1.KD) increased it and partially mitigated the protective effects of IGF-I (P<0.05). SGK1 activation was also sufficient to increase cell size, protein synthesis, sarcomere organization, and ANF expression both at baseline and in response to phenylephrine but was not necessary for the hypertrophic response to phenylephrine. Evaluation of potential downstream signaling pathways demonstrated that SGK1 induces phosphorylation of tuberin, p70s6kinase, and GSK3&bgr; in CMs, which may contribute to its effects. Conclusions—SGK1 is dynamically regulated during acute biomechanical stress in the heart and inhibits CM apoptosis while enhancing the hypertrophic response.

A20 Is Dynamically Regulated in the Heart and Inhibits the Hypertrophic Response

Background—Nuclear factor (NF)–&kgr;B signaling has been implicated in cardiomyocyte hypertrophy. Here, we determine the cardiac regulation and biological activity of A20, an inhibitor of NF-&kgr;B signaling. Methods and Results—Mice were subjected to aortic banding, and A20 expression was examined. A20 mRNA upregulation (4.3±1.5-fold; P <0.05) was detected 3 hours after banding, coinciding with peak NF-&kgr;B activation. A20 was also upregulated in cultured neonatal cardiomyocytes stimulated with phenylephrine or endothelin-1 (2.8±0.6- and 4±1.1-fold, respectively; P <0.05), again paralleling NF-&kgr;B activation. Infection of cardiomyocytes with an adenoviral vector (Ad) encoding A20 inhibited tumor necrosis factor-&agr;–stimulated NF-&kgr;B signaling with an efficacy comparable to dominant negative inhibitor of &kgr;-B kinase &bgr; (dnIKK&bgr;). Ad.dnIKK&bgr;-infected cardiomyocytes exhibited increased apoptosis when they were serum starved or subjected to hypoxia-reoxygenation, whereas Ad.A20-infected cardiomyocytes did not. Expression of Ad.A20 inhibited the hypertrophic response in cardiomyocytes stimulated with phenylephrine or endothelin-1. Conclusions—A20 is dynamically regulated during acute biomechanical stress in the heart and functions to attenuate cardiac hypertrophy through the inhibition of NF-&kgr;B signaling without sensitizing cardiomyocytes to apoptotic cell death.

Strategic advantages of insulin-like growth factor-I expression for cardioprotection

Insulin‐like growth factor‐I (IGF‐I) peptide has beneficial effects on cardiomyocyte function and survival, many of which are mediated through the serine‐threonine kinase, Akt. However, concerns about systemic effects of IGF‐I peptide limit its clinical application. The present study tested whether local IGF‐I expression could mediate cardioprotection without elevating serum [IGF‐I].

50. Transcript Profiling Identifies Novel Role for SGK1 in Promoting Cardiomyocyte Survival

FasL only induces cardiomyocyte (CM) apoptosis when transcription is inhibited. To identify transcription-dependent cardioprotective pathways, we performed microarray analyses on CM that were untreated or stimulated with FasL, in the absence or presence of low-dose Actinomycin D (ActD, to inhibit transcription). 23 transcripts were identified as upregulated by FasL and inhibited by ActD, including Serum- and Glucocorticoid-responsive Kinase-1 (SGK1), a serine-threonine kinase that is expressed in the heart and previously reported to regulate sodium channels. Studies of rats after aortic constriction demonstrated that both phospho- and total SGK1 increased 2 to 7 days after banding. Phospho-SGK1 was also upregulated in CM stimulated in vitro with IGF-I or phenylephrine (PE). To examine the functional effects of SGK1 in CM, we generated recombinant Adenoviral vectors (Ad) carrying HA-tagged, constitutively-active (CA, S422D) and kinase-dead (KD, K127M) mutants of SGK1. In the absence of stimulation, Ad.SGK1.CA increased SGK1 activity 2.45|[plusmn]|0.21-fold (p<0.05), while Ad.SGK1.KD reduced it (0.44|[plusmn]|0.02-fold, p<0.05) compared to control-Ad infected CMs. Stimulation of control-Ad infected CMs with IGF-I induced a comparable, 2.3-fold increase in SGK1 activity. Treatment of Ad.SGK1.CA-infected CM with IGF-I further increased SGK1 activity while Ad. SGK1.KD inhibited it (2.26|[plusmn]|0.07- and 0.40|[plusmn]|0.02-fold, respectively, p<0.05 vs IGF-I-treated control cells). Thus, Ad.SGK1.CA increases SGK1 activity in CMs but remains responsive to further stimulation, and Ad.SGK1.KD acts as a dominant negative to inhibit endogenous SGK1 activity. Neither Ad.SGK1.CA nor Ad.SGK1.KD had significant effects on CM apoptosis in unstimulated CMs. In contrast, Ad.SGK1.CA substantially protected CMs against both serum deprivation (SD)- and hypoxia-induced apoptosis, while inhibition of SGK1 significantly increased the number of apoptotic nuclei compared with control virus-infected CM in both models. These changes in nuclear morphology correlated well with DNA laddering, the biochemical hallmark of apoptosis, and cleavage (activation) of caspase-3, as well as overall cell viability, as indicated by the MTT assay. Of note, the protective effect of IGF-I was significantly but incompletely reversed by expression of SGK1.KD (p<0.05). Evaluation of potential downstream signaling pathways demonstrated that SGK1 induces phosphorylation of Tuberin, p70s6kinase, and GSK3|[beta]| in CM which may contribute to its effects. We conclude that SGK1 is dynamically regulated during acute biomechanical stress in the heart and inhibits CM apoptosis. Thus, SGK1 may represent a novel therapeutic target for conditions such as heart failure or ischemic injury characterized by CM death.