Targeted overexpression of protein kinase C β2 isoform in myocardium causes cardiomyopathy

Increased cardiovascular mortality occurs in diabetic patients with or without coronary artery disease and is attributed to the presence of diabetic cardiomyopathy. One potential mechanism is hyperglycemia that has been reported to activate protein kinase C (PKC), preferentially the β isoform, which has been associated with the development of micro- and macrovascular pathologies in diabetes mellitus. To establish that the activation of the PKCβ isoform can cause cardiac dysfunctions, we have established lines of transgenic mice with the specific overexpression of PKCβ2 isoform in the myocardium. These mice overexpressed the PKCβ2 isoform transgene by 2- to 10-fold as measured by mRNA, and proteins exhibited left ventricular hypertrophy, cardiac myocyte necrosis, multifocal fibrosis, and decreased left ventricular performance without vascular lesions. The severity of the phenotypes exhibited gene dose-dependence. Up-regulation of mRNAs for fetal type myosin heavy chain, atrial natriuretic factor, c-fos, transforming growth factor, and collagens was also observed. Moreover, treatment with a PKCβ-specific inhibitor resulted in functional and histological improvement. These findings have firmly established that the activation of the PKCβ2 isoform can cause specific cardiac cellular and functional changes leading to cardiomyopathy of diabetic or nondiabetic etiology.

Essential role of β-adrenergic receptor kinase 1 in cardiac development and function

The β-adrenergic receptor kinase 1 (βARK1) is a member of the G protein-coupled receptor kinase (GRK) family that mediates the agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. We have cloned and disrupted the βARK1 gene in mice by homologous recombination. No homozygote βARK1−/− embryos survive beyond gestational day 15.5. Prior to gestational day 15.5, βARK1−/− embryos display pronounced hypoplasia of the ventricular myocardium essentially identical to the “thin myocardium syndrome” observed upon gene inactivation of several transcription factors (RXRα, N-myc, TEF-1, WT-1). Lethality in βARK1−/− embryos is likely due to heart failure as they exhibit a >70% decrease in cardiac ejection fraction determined by direct in utero intravital microscopy. These results along with the virtual absence of endogenous GRK activity in βARK1−/− embryos demonstrate that βARK1 appears to be the predominant GRK in early embryogenesis and that it plays a fundamental role in cardiac development.

MEKK1 suppresses oxidative stress-induced apoptosis of embryonic stem cell-derived cardiac myocytes

A combination of in vitro embryonic stem (ES) cell differentiation and targeted gene disruption has defined complex regulatory events underlying oxidative stress-induced cardiac apoptosis, a model of postischemic reperfusion injury of myocardium. ES cell-derived cardiac myocytes (ESCM) having targeted disruption of the MEKK1 gene were extremely sensitive, relative to wild-type ESCM, to hydrogen peroxide-induced apoptosis. In response to oxidative stress, MEKK1−/− ESCM failed to activate c-Jun kinase (JNK) but did activate p38 kinase similar to that observed in wild-type ESCM. The increased apoptosis was mediated through enhanced tumor necrosis factor α production, a response that was positively and negatively regulated by p38 and the MEKK1-JNK pathway, respectively. Thus, MEKK1 functions in the survival of cardiac myocytes by inhibiting the production of a proapoptotic cytokine. MEKK1 regulation of the JNK pathway is a critical response for the protection against oxidative stress-induced apoptosis in cardiac myocytes.

Expression of lipocalin-type prostaglandin D synthase (β-trace) in human heart and its accumulation in the coronary circulation of angina patients

Lipocalin-type prostaglandin D synthase (L-PGDS) is localized in the central nervous system and male genital organs of various mammals and is secreted as β-trace into the closed compartment of these tissues separated from the systemic circulation. In this study, we found that the mRNA for the human enzyme was expressed most intensely in the heart among various tissues examined. In human autopsy specimens, the enzyme was localized immunocytochemically in myocardial cells, atrial endocardial cells, and a synthetic phenotype of smooth muscle cells in the arteriosclerotic intima, and accumulated in the atherosclerotic plaque of coronary arteries with severe stenosis. In patients with stable angina (75–99% stenosis), the plasma level of L-PGDS was significantly (P < 0.05) higher in the great cardiac vein (0.694 ± 0.054 μg/ml, n = 7) than in the coronary artery (0.545 ± 0.034 μg/ml), as determined by a sandwich enzyme immunoassay. However, the veno-arterial difference in the plasma L-PGDS concentration was not observed in normal subjects without stenosis. After a percutaneous transluminal coronary angioplasty was performed to compress the stenotic atherosclerotic plaques, the L-PGDS concentration in the cardiac vein decreased significantly (P < 0.05) to 0.610 ± 0.051 μg/ml at 20 min and reached the arterial level within 1 h. These findings suggest that L-PGDS is present in both endocardium and myocardium of normal subjects and the stenotic site of patients with stable angina and is secreted into the coronary circulation.

Preservation of myocardial β-adrenergic receptor signaling delays the development of heart failure after myocardial infarction

When the heart fails, there is often a constellation of biochemical alterations of the β-adrenergic receptor (βAR) signaling system, leading to the loss of cardiac inotropic reserve. βAR down-regulation and functional uncoupling are mediated through enhanced activity of the βAR kinase (βARK1), the expression of which is increased in ischemic and failing myocardium. These changes are widely viewed as representing an adaptive mechanism, which protects the heart against chronic activation. In this study, we demonstrate, using in vivo intracoronary adenoviral-mediated gene delivery of a peptide inhibitor of βARK1 (βARKct), that the desensitization and down-regulation of βARs seen in the failing heart may actually be maladaptive. In a rabbit model of heart failure induced by myocardial infarction, which recapitulates the biochemical βAR abnormalities seen in human heart failure, delivery of the βARKct transgene at the time of myocardial infarction prevents the rise in βARK1 activity and expression and thereby maintains βAR density and signaling at normal levels. Rather than leading to deleterious effects, cardiac function is improved, and the development of heart failure is delayed. These results appear to challenge the notion that dampening of βAR signaling in the failing heart is protective, and they may lead to novel therapeutic strategies to treat heart disease via inhibition of βARK1 and preservation of myocardial βAR function.

Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibit target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipultation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Transcytosis of α1-acidic glycoprotein in the continuous microvascular endothelium

By using perfusions and bolus administration, coupled with postembedding immunocytochemical procedures, we have identified the structures involved in the transport of derivatized orosomucoid (α1-acidic glycoprotein) across the continuous microvascular endothelium of the murine myocardium. Our findings indicate that: (i) monomeric orosomucoid binds to the luminal surface of the endothelium; (ii) it is restricted to caveolae during its transport across the endothelium; (iii) it is detected in the perivascular spaces at early time points (by 1 min) and in larger quantities at later time points (>5 min) from the beginning of its perfusion or its intravascular administration; (iv) no orosomucoid molecules are found in the intercellular junctions or at the abluminal exits of interendothelial spaces; and (v) the vesicular transport of orosomucoid is strongly inhibited by N-ethylmaleimide (>80%). Because, by size and shape, the orosomucoid qualifies as a preferential probe for the postulated small pore system, our results are discussed in relation to the pore theory of capillary permeability.

Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, δ-sarcoglycan, in hamster: An animal model of disrupted dystrophin-associated glycoprotein complex

Cardiomyopathy (CM) is a primary degenerative disease of myocardium and is traditionally categorized into hypertrophic and dilated CMs (HCM and DCM) according to its gross appearance. Cardiomyopathic hamster (CM hamster), a representative model of human hereditary CM, has HCM and DCM inbred sublines, both of which descend from the same ancestor. Herein we show that both HCM and DCM hamsters share a common defect in a gene for δ-sarcoglycan (δ-SG), the functional role of which is yet to be characterized. A breakpoint causing genomic deletion was found to be located at 6.1 kb 5′ upstream of the second exon of δ-SG gene, and its 5′ upstream region of more than 27.4 kb, including the authentic first exon of δ-SG gene, was deleted. This deletion included the major transcription initiation site, resulting in a deficiency of δ-SG transcripts with the consequent loss of δ-SG protein in all the CM hamsters, despite the fact that the protein coding region of δ-SG starting from the second exon was conserved in all the CM hamsters. We elucidated the molecular interaction of dystrophin-associated glycoproteins including δ-SG, by using an in vitro pull-down study and ligand overlay assay, which indicates the functional role of δ-SG in stabilizing sarcolemma. The present study not only identifies CM hamster as a valuable animal model for studying the function of δ-SG in vivo but also provides a genetic target for diagnosis and treatment of human CM.

Inhibition of caspase 1 reduces human myocardial ischemic dysfunction via inhibition of IL-18 and IL-1β

The proinflammatory cytokine IL-18 was investigated for its role in human myocardial function. An ischemia/reperfusion (I/R) model of suprafused human atrial myocardium was used to assess myocardial contractile force. Addition of IL-18 binding protein (IL-18BP), the constitutive inhibitor of IL-18 activity, to the perifusate during and after I/R resulted in improved contractile function after I/R from 35% of control to 76% with IL-18BP. IL-18BP treatment also preserved intracellular tissue creatine kinase levels (by 420%). Steady-state mRNA levels for IL-18 were elevated after I/R, and the concentration of IL-18 in myocardial homogenates was increased (control, 5.8 pg/mg vs. I/R, 26 pg/mg; P < 0.01). Active IL-18 requires cleavage of its precursor form by the IL-1β-converting enzyme (caspase 1); inhibition of caspase 1 also attenuated the depression in contractile force after I/R (from 35% of control to 75.8% in treated atrial muscle; P < 0.01). Because caspase 1 also cleaves the precursor IL-1β, IL-1 receptor blockade was accomplished by using the IL-1 receptor antagonist. IL-1 receptor antagonist added to the perifusate also resulted in a reduction of ischemia-induced contractile dysfunction. These studies demonstrate that endogenous IL-18 and IL-1β play a significant role in I/R-induced human myocardial injury and that inhibition of caspase 1 reduces the processing of endogenous precursors of IL-18 and IL-1β and thereby prevents ischemia-induced myocardial dysfunction.

Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo

The Ca2+-calmodulin-activated Ser/Thr protein phosphatase calcineurin and the downstream transcriptional effectors of calcineurin, nuclear factor of activated T cells, have been implicated in the hypertrophic response of the myocardium. Recently, the calcineurin inhibitory agents cyclosporine A and FK506 have been extensively used to evaluate the importance of this signaling pathway in rodent models of cardiac hypertrophy. However, pharmacologic approaches have rendered equivocal results necessitating more specific or genetic-based inhibitory strategies. In this regard, we have generated Tg mice expressing the calcineurin inhibitory domains of Cain/Cabin-1 and A-kinase anchoring protein 79 specifically in the heart. ΔCain and ΔA-kinase-anchoring protein Tg mice demonstrated reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second approach, adenoviral-mediated gene transfer of ΔCain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. ΔCain adenoviral gene transfer inhibited cardiac calcineurin activity and reduced hypertrophy in response to pressure overload without reducing aortic pressure. These results provide genetic evidence implicating calcineurin as an important mediator of the cardiac hypertrophic response in vivo.

Distinct gene-specific mechanisms of arrhythmia revealed by cardiac gene transfer of two long QT disease genes, HERG and KCNE1

The long QT syndrome (LQTS) is a heritable disorder that predisposes to sudden cardiac death. LQTS is caused by mutations in ion channel genes including HERG and KCNE1, but the precise mechanisms remain unclear. To clarify this situation we injected adenoviral vectors expressing wild-type or LQT mutants of HERG and KCNE1 into guinea pig myocardium. End points at 48–72 h included electrophysiology in isolated myocytes and electrocardiography in vivo. HERG increased the rapid component, IKr, of the delayed rectifier current, thereby accelerating repolarization, increasing refractoriness, and diminishing beat-to-beat action potential variability. Conversely, HERG-G628S suppressed IKr without significantly delaying repolarization. Nevertheless, HERG-G628S abbreviated refractoriness and increased beat-to-beat variability, leading to early afterdepolarizations (EADs). KCNE1 increased the slow component of the delayed rectifier, IKs, without clear phenotypic sequelae. In contrast, KCNE1-D76N suppressed IKs and markedly slowed repolarization, leading to frequent EADs and electrocardiographic QT prolongation. Thus, the two genes predispose to sudden death by distinct mechanisms: the KCNE1 mutant flagrantly undermines cardiac repolarization, and HERG-G628S subtly facilitates the genesis and propagation of premature beats. Our ability to produce electrocardiographic long QT in vivo with a clinical KCNE1 mutation demonstrates the utility of somatic gene transfer in creating genotype-specific disease models.

Telomerase expression and activity are coupled with myocyte proliferation and preservation of telomeric length in the failing heart

The role and even the existence of myocyte proliferation in the adult heart remain controversial. Documentation of cell cycle regulators, DNA synthesis, and mitotic images has not modified the view that myocardial growth can only occur from hypertrophy of an irreplaceable population of differentiated myocytes. To improve understanding the biology of the heart and obtain supportive evidence of myocyte replication, three indices of cell proliferation were analyzed in dogs affected by a progressive deterioration of cardiac performance and dilated cardiomyopathy. The magnitude of cycling myocytes was evaluated by the expression of Ki67 in nuclei. Ki67 labeling of left ventricular myocytes increased 5-fold, 12-fold, and 17-fold with the onset of moderate and severe ventricular dysfunction and overt failure, respectively. Telomerase activity in vivo is present only in multiplying cells; this enzyme increased 2.4-fold and 3.1-fold in the decompensated heart, preserving telomeric length in myocytes. The contribution of cycling myocytes to telomerase activity was determined by the colocalization of Ki67 and telomerase in myocyte nuclei. More than 50% of Ki67-positive cells expressed telomerase in the overloaded myocardium, suggesting that these myocytes were the morphological counterpart of the biochemical assay of enzyme activity. Moreover, we report that 20–30% of canine myocytes were telomerase competent, and this value was not changed by cardiac failure. In conclusion, the enhanced expression of Ki67 and telomerase activity, in combination with Ki67-telomerase labeling of myocyte nuclei, support the notion that myocyte proliferation contributes to cardiac hypertrophy of the diseased heart.

A new isoleucine substitution of Val-20 in transthyretin tetramers selectively impairs dimer-dimer contacts and causes systemic amyloidosis.

The most frequent form of inherited amyloidoses is associated with mutations in the transthyretin (TTR) gene coding for 127-amino acid residues of four identical, noncovalently linked subunits that form a pair of dimers in the plasma protein complex. Amyloid fibrils containing the variant and to a lesser extent the wild-type form of the TTR molecule are deposited in various organs, including peripheral nerves and the myocardium, with polyneuropathy and cardiomyopathy as major clinical manifestations. So far, more than 40 distinct amino acid substitutions distributed throughout the TTR sequence over 30 positions have been found to be correlated with an increased amyloidogenicity of TTR. Most of these amyloidogenic amino acid substitutions are suspected to alter the conformation and stability of the monomer. Here we identify and characterize by protein and DNA analysis a novel amyloidogenic Val-20 to Ile mutation in a German three-generation family. The index patient suffered from severe amyloid cardiomyopathy at the age of 60. Conformational stability and unfolding behavior of the Ile-20 monomer in urea gradients was found to be almost indistinguishable from that of wild-type TTR. In contrast, tetramer stability was significantly reduced in agreement with the expected change in the interactions between the two opposing dimers via the side chain of Ile-20. Our observations provide strong evidence for the view that amyloidogenic amino acid substitutions in TTR facilitate the conversion of tetrameric TTR complexes into those conformational intermediates of the TTR folding pathway that have an intrinsic amyloidogenic potential.

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

Targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardial and hematological disorders.

gp130 is a ubiquitously expressed signal-transducing receptor component shared by interleukin 6, interleukin 11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1. To investigate physiological roles of gp130 and to examine pathological consequences of a lack of gp130, mice deficient for gp130 have been prepared. Embryos homozygous for the gp130 mutation progressively die between 12.5 days postcoitum and term. On 16.5 days postcoitum and later, they show hypoplastic ventricular myocardium without septal and trabecular defect. The subcellular ultrastructures in gp130-/- cardiomyocytes appear normal. The mutant embryos have greatly reduced numbers of pluripotential and committed hematopoietic progenitors in the liver and differentiated lineages such as T cells in the thymus. Some gp130-/- embryos show anemia due to impaired development of erythroid lineage cells. These results indicate that gp130 plays a crucial role in myocardial development and hematopoiesis during embryogenesis.