Point Shear Wave Elastography by Acoustic Radiation Force Impulse Quantification in Comparison to Transient Elastography for the Noninvasive Assessment of Liver Fibrosis in Chronic Hepatitis C: A Prospective International Multicenter Study.

PURPOSE: The aim of the present prospective European multicenter study was to demonstrate the non-inferiority of point shear wave elastography (pSWE) compared to transient elastography (TE) for the assessment of liver fibrosis in patients with chronic hepatitis C. MATERIALS AND METHODS: 241 patients with chronic hepatitis C were prospectively enrolled at 7 European study sites and received pSWE, TE and blood tests. Liver biopsy was performed with histological staging by a central pathologist. In addition, for inclusion of cirrhotic patients, a maximum of 10 % of patients with overt liver cirrhosis confirmed by imaging methods were allowed by protocol (n = 24). RESULTS: Owing to slower than expected recruitment due to a reduction of liver biopsies, the study was closed after 4 years before the target enrollment of 433 patients with 235 patients in the 'intention to diagnose' analysis and 182 patients in the 'per protocol' analysis. Therefore, the non-inferiority margin was enhanced to 0.075 but non-inferiority of pSWE could not be proven. However, Paired comparison of the diagnostic accuracy of pSWE and TE revealed no significant difference between the two methods in the 'intention to diagnose' and 'per protocol' analysis (0.81 vs. 0.85 for F ≥ 2, p = 0.15; 0.88 vs. 0.92 for F ≥ 3, p = 0.11; 0.89 vs. 0.94 for F = 4, p = 0.19). Measurement failure was significantly higher for TE than for pSWE (p = 0.030). CONCLUSION: Non-inferiority of pSWE compared to TE could not be shown. However, the diagnostic accuracy of pSWE and TE was comparable for the noninvasive staging of liver fibrosis in patients with chronic hepatitis C.

[Treatment of hepatitis C]. / Therapie der Hepatitis C.

Chronic hepatitis C virus (HCV) infection is the major cause of liver cirrhosis, hepatocellular carcinoma and liver transplantation in the western world. The development and approval of nine directly acting antiviral drugs in recent years has led to a dramatic improvement in therapeutic efficacy accompanied by fewer side effects. With current treatment options sustained virologic response in more than 90 % of patients can be achieved depending on HCV genotype, liver cirrhosis and prior therapies. Modern HCV treatment regimens are interferon-free and should be administered for 12-24 weeks. Shorter courses are possible in selected patients. For the treatment of HCV genotype 1 infection combinations of either the nucleotide polymerase inhibitor sofosbuvir with the protease inhibitor simeprevir or with one of the two NS5A inhibitors daclatasvir or ledipasvir on the one hand or triple DAA therapy of paritaprevir, ombitasvir and dasabuvir on the other hand are applicable. Ribavirin has still a role as an add-on in difficult to treat patients.

Acoustic radiation force impulse imaging for non-invasive assessment of liver fibrosis in chronic hepatitis B.

Acoustic radiation force impulse (ARFI) imaging is a novel ultrasound-based elastography method that is integrated in a conventional ultrasound machine. It might provide an alternative method to transient elastography for the noninvasive assessment of liver fibrosis. While previous studies have shown comparable diagnostic accuracy of ARFI to transient elastography in chronic hepatitis C, the aim of the present prospective multicenter study was to evaluate ARFI for the assessment of liver fibrosis in chronic hepatitis B. ARFI imaging involves the mechanical excitation of tissue using short-duration acoustic pulses to generate localized displacements in tissue. The displacements result in shear-wave propagation which is tracked using ultrasonic, correlation-based methods and recorded in m/s. In the present international prospective study, patients infected with chronic hepatitis B received ARFI imaging, blood tests and if available transient elastography. The results were compared to liver biopsy as reference method analysed by a central pathologist. In 92 of 114 patients, a comparison of ARFI with transient elastography was possible. ARFI imaging and transient elastography correlated significantly with histological fibrosis stage. The diagnostic accuracy expressed as areas under ROC curves for ARFI imaging and transient elastography was 0.75 and 0.83 for the diagnosis of significant fibrosis (F ≥ 2), 0.93 and 0.94 for the diagnosis of severe fibrosis (F ≥ 3), and 0.97 and 0.93 for the diagnosis of liver cirrhosis, respectively. No significant difference was found between ARFI and transient elastography. ARFI imaging is a reliable ultrasound-based method for the assessment of advanced stages of liver fibrosis in chronic hepatitis B.

TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice.

The transforming-growth-factor-beta-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor beta. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, 'fetal' gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.

Differentiation of cardiac myocytes after mitogen withdrawal exhibits three sequential states of the ventricular growth response.

During cardiac myogenesis, ventricular muscle cells lose the capacity to proliferate soon after birth. It is unknown whether this developmental block to mitotic division and DNA replication might involve irreversible repression of the cellular oncogene c-myc. Ventricular myocytes from 2 d-old rats continued to differentiate in vitro during 15 d of mitogen withdrawal, as shown by the formation of cross-striations, increased proportion of the muscle isoenzyme of creatine kinase, stable expression of alpha-cardiac actin and myosin heavy chain mRNAs, and appropriate down-regulation of alpha-skeletal actin mRNA. After mitogen withdrawal for 2 d, serum evoked both DNA synthesis and mitotic division; after 7 d, DNA replication was uncoupled from cell division; after 15 d, DNA synthesis itself was markedly attentuated. These three distinct phenotypic states resemble the sequential properties of growth found in the neonatal rat heart in vivo. Despite failure to induce DNA replication or division after 15 d of mitogen withdrawal, serum elicited both c-myc and alpha-skeletal actin as found during hypertrophy of the intact heart. The results agree with previous evidence that one or more functional pathways that transduce the effects of serum factors may persist in older cardiac muscle cells, and indicate that irreversible down-regulation of c-myc cannot be the basis for the loss of growth responses.

Localization and topography of antigenic domains within the heavy chain of smooth muscle myosin.

We have produced and characterized monoclonal antibodies that label antigenic determinants distributed among three distinct, nonoverlapping peptide domains of the 200-kD heavy chain of avian smooth muscle myosin. Mice were immunized with a partially phosphorylated chymotryptic digest of adult turkey gizzard myosin. Hybridoma antibody specificities were determined by solid-phase indirect radioimmunoassay and immunoreplica techniques. Electron microscopy of rotary-shadowed samples was used to directly visualize the topography of individual [antibody.antigen] complexes. Antibody TGM-1 bound to a 50-kD peptide of subfragment-1 (S-1) previously found to be associated with actin binding and was localized by immunoelectron microscopy to the distal aspect of the myosin head. However, there was no antibody-dependent inhibition of the actin-activated heavy meromyosin ATPase, nor was antibody TGM-1 binding to actin-S-1 complexes inhibited. Antibody TGM-2 detected an epitope of the subfragment-2 (S-2) domain of heavy meromyosin but not the S-2 domain of intact myosin or rod, consistent with recognition of a site exposed by chymotryptic cleavage of the S-2:light meromyosin junction. Localization of TGM-2 to the carboxy-terminus of S-2 was substantiated by immunoelectron microscopy. Antibody TGM-3 recognized an epitope found in the light meromyosin portion of myosin. All three antibodies were specific for avian smooth muscle myosin. Of particular interest is that antibody TGM-1, unlike TGM-3, bound poorly to homogenates of 19-d embryonic smooth muscles. This indicates the expression of different myosin heavy chain epitopes during smooth muscle development.

Mitogens and oncogenes can block the induction of specific voltage-gated ion channels.

The mechanisms underlying the ontogeny of voltage-gated ion channels in muscle are unknown. Whether expression of voltage-gated channels is dependent on mitogen withdrawal and growth arrest, as is generally true for the induction of muscle-specific gene products, was investigated in the BC3H1 muscle cell line by patch-clamp techniques. Differentiated BC3H1 myocytes expressed functional Ca2+ and Na+ channels that correspond to those found in T tubules of skeletal muscle. However, Ca2+ and Na+ channels were first detected after about 5 days of mitogen withdrawal. In order to test whether cellular oncogenes, as surrogates for exogenous growth factors, could prevent the expression of ion channels whose induction was contingent on mitogen withdrawal, BC3H1 cells were modified by stable transfection with oncogene expression vectors. Expression vectors containing v-erbB, or c-myc under the control of the SV40 promoter, delayed but did not prevent the appearance of functional Ca2+ and Na+ channels. In contrast, transfection with a Val12 c-H-ras vector, or cotransfection of c-myc together with v-erbB, suppressed the formation of functional Ca2+ and Na+ channels for greater than or equal to 4 weeks. Potassium channels were affected neither by mitogenic medium nor by transfected oncogenes. Thus, the selective effects of certain oncogenes on ion channel induction corresponded to the suppressive effects of mitogenic medium.

Overlapping roles of pocket proteins in the myocardium are unmasked by germ line deletion of p130 plus heart-specific deletion of Rb.

The pocket protein family of tumor suppressors, and Rb specifically, have been implicated as controlling terminal differentiation in many tissues, including the heart. To establish the biological functions of Rb in the heart and overcome the early lethality caused by germ line deletion of Rb, we used a Cre/loxP system to create conditional, heart-specific Rb-deficient mice. Mice that are deficient in Rb exclusively in cardiac myocytes (CRbL/L) are born with the expected Mendelian distribution, and the adult mice displayed no change in heart size, myocyte cell cycle distribution, myocyte apoptosis, or mechanical function. Since both Rb and p130 are expressed in the adult myocardium, we created double-knockout mice (CRbL/L p130-/-) to determine it these proteins have a shared role in regulating cardiac myocyte cell cycle progression. Adult CRbL/L p130-/- mice demonstrated a threefold increase in the heart weight-to-body weight ratio and showed increased numbers of bromodeoxyuridine- and phosphorylated histone H3-positive nuclei, consistent with persistent myocyte cycling. Likewise, the combined deletion of Rb plus p130 up-regulated myocardial expression of Myc, E2F-1, and G1 cyclin-dependent kinase activities, synergistically. Thus, Rb and p130 have overlapping functional roles in vivo to suppress cell cycle activators, including Myc, and maintain quiescence in postnatal cardiac muscle.

Peptide growth factors can provoke "fetal" contractile protein gene expression in rat cardiac myocytes.

Cardiac-specific gene expression is intricately regulated in response to developmental, hormonal, and hemodynamic stimuli. To test whether cardiac muscle might be a target for regulation by peptide growth factors, the effect of three growth factors on the actin and myosin gene families was investigated by Northern blot analysis in cultured neonatal rat cardiac myocytes. Transforming growth factor-beta 1 (TGF beta 1, 1 ng/ml) and basic fibroblast growth factor (FGF, 25 ng/ml) elicited changes corresponding to those induced by hemodynamic load. The "fetal" beta-myosin heavy chain (MHC) was up-regulated about four-fold, whereas the "adult" alpha MHC was inhibited greater than 50-60%; expression of alpha-skeletal actin increased approximately two-fold, with little or no change in alpha-cardiac actin. Thus, peptide growth factors alter the program of differentiated gene expression in cardiac myocytes, and are sufficient to provoke fetal contractile protein gene expression, characteristic of pressure-overload hypertrophy. Acidic FGF (25 ng/ml) produced seven- to eightfold reciprocal changes in MHC expression but, unlike either TGF-beta 1 or basic FGF, inhibited both striated alpha-actin genes by 70-90%. Expression of vascular smooth muscle alpha-actin, the earliest alpha-actin induced during cardiac myogenesis, was increased by all three growth factors. Thus, three alpha-actin genes demonstrate distinct responses to acidic vs. basic FGF.

Dihydropyridine receptor gene expression is regulated by inhibitors of myogenesis and is relatively insensitive to denervation.

To evaluate developmental and physiological signals that may influence expression of the dihydropyridine-sensitive "slow" Ca2+ channel, we analyzed dihydropyridine receptor (DHPR) mRNA abundance in mouse skeletal muscle. Using synthetic oligonucleotide probes corresponding to the rabbit skeletal muscle DHPR, a 6.5 kb DHPR transcript was identified in postnatal skeletal muscle and differentiated C2 or BC3H1 myocytes, but not cardiac muscle or brain. DHPR gene expression was reversibly suppressed by 0.4 nM transforming growth factor beta-1 or by transfection with a mutant c-H-ras allele, nominal inhibitors of myogenesis that block the appearance of slow channels and DHPR. In contrast, both BC3H1 and C2 myocytes containing the activated ras vector expressed the gene encoding the nicotinic acetylcholine receptor delta subunit, demonstrating that not all muscle-specific genes are extinguished by ras. Denervation stimulated DHPR gene expression less than 0.6-fold, despite 8-fold upregulation of delta-subunit mRNA and reciprocal effects on the skeletal and cardiac alpha-actin genes. Thus, DHPR gene induction is prevented by inhibitors of other muscle-specific genes, whereas, at most, relatively small changes in DHPR mRNA abundance occur during adaptation to denervation.

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replication-deficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 x 10(5) plaque-forming units (PFU) and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. Beta-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the exogenous lacZ gene. At 10(8) PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells.

Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo.

Mouse models of human disease can be generated by homologous recombination for germline loss-of-function mutations. However, embryonic-lethal phenotypes and systemic, indirect dysfunction can confound the use of knock-outs to elucidate adult pathophysiology. Site-specific recombination using Cre recombinase can circumvent these pitfalls, in principle, enabling temporal and spatial control of gene recombination. However, direct evidence is lacking for the feasibility of Cre-mediated recombination in postmitotic cells. Here, we exploited transgenic mouse technology plus adenoviral gene transfer to achieve Cre-mediated recombination in cardiac muscle. In vitro, Cre driven by cardiac-specific alpha-myosin heavy chain (alphaMyHC) sequences elicited recombination selectively at loxP sites in purified cardiac myocytes, but not cardiac fibroblasts. In vivo, this alphaMyHC-Cre transgene elicited recombination in cardiac muscle, but not other organs, as ascertained by PCR analysis and localization of a recombination-dependent reporter protein. Adenoviral delivery of Cre in vivo provoked recombination in postmitotic, adult ventricular myocytes. Recombination between loxP sites was not detected in the absence of Cre. These studies demonstrate the feasibility of using Cre-mediated recombination to regulate gene expression in myocardium, with efficient induction of recombination even in terminally differentiated, postmitotic muscle cells. Moreover, delivery of Cre by viral infection provides a simple strategy to control the timing of recombination in myocardium.

Adenoviral delivery of E2F-1 directs cell cycle reentry and p53-independent apoptosis in postmitotic adult myocardium in vivo.

Irreversible exit from the cell cycle precludes the ability of cardiac muscle cells to increase cell number after infarction. Using adenoviral E1A, we previously demonstrated dual pocket protein- and p300-dependent pathways in neonatal rat cardiac myocytes, and have proven that E2F-1, which occupies the Rb pocket, suffices for these actions of E1A. By contrast, the susceptibility of adult ventricular cells to viral delivery of exogenous cell cycle regulators has not been tested, in vitro or in vivo. In cultured adult ventricular myocytes, adenoviral gene transfer of E2F-1 induced expression of proliferating cell nuclear antigen, cyclin-dependent protein kinase 4, cell division cycle 2 kinase, DNA synthesis, and apoptosis. In vivo, adenoviral delivery of E2F-1 by direct injection into myocardium induced DNA synthesis, shown by 5'-bromodeoxyuridine incorporation, and accumulation in G2/M, by image analysis of Feulgen-stained nuclei. In p53(-)/- mice, the prevalence of G1 exit was more than twofold greater; however, E2F-1 evoked apoptosis and rapid mortality comparably in both backgrounds. Thus, the differential effects of E2F-1 on G1 exit in wild-type versus p53-deficient mice illustrate the combinatorial power of viral gene delivery to genetically defined recipients: E2F-1 can override the G1/S checkpoint in postmitotic ventricular myocytes in vitro and in vivo, but leads to apoptosis even in p53(-)/- mice.

The vascular smooth muscle alpha-actin gene is reactivated during cardiac hypertrophy provoked by load.

Cardiac hypertrophy triggered by mechanical load possesses features in common with growth factor signal transduction. A hemodynamic load provokes rapid expression of the growth factor-inducible nuclear oncogene, c-fos, and certain peptide growth factors specifically stimulate the "fetal" cardiac genes associated with hypertrophy, even in the absence of load. These include the gene encoding vascular smooth muscle alpha-actin, the earliest alpha-actin expressed during cardiac myogenesis; however, it is not known whether reactivation of the smooth muscle alpha-actin gene occurs in ventricular hypertrophy. We therefore investigated myocardial expression of the smooth muscle alpha-actin gene after hemodynamic overload. Smooth muscle alpha-actin mRNA was discernible 24 h after coarctation and was persistently expressed for up to 30 d. In hypertrophied hearts, the prevalence of smooth muscle alpha-actin gene induction was 0.909, versus 0.545 for skeletal muscle alpha-actin (P less than 0.05). Ventricular mass after 2 d or more of aortic constriction was more highly correlated with smooth muscle alpha-actin gene activation (r = 0.852; P = 0.0001) than with skeletal muscle alpha-actin (r = 0.532; P = 0.009); P less than 0.0005 for the difference in the correlation coefficients. Thus, smooth muscle alpha-actin is a molecular marker of the presence and extent of pressure-overload hypertrophy, whose correlation with cardiac growth at least equals that of skeletal alpha-actin. Induction of smooth muscle alpha-actin was delayed and sustained after aortic constriction, whereas the nuclear oncogenes c-jun and junB were expressed rapidly and transiently, providing potential dimerization partners for transcriptional control by c-fos.

Dissociated expression of c-myc and a fos-related competence gene during cardiac myogenesis.

Cardiac myocytes irreversibly lose their proliferative capacity soon after birth, and cardiac DNA synthesis becomes uncoupled from mitotic division. Therefore, we examined cardiac muscle for developmental down regulation of inducible proto-oncogenes associated with cell proliferation. c-myc mRNA decreased continuously from day 13 of embryonic development and was dissociated from expression of the fos-related gene r-fos, which decreased precipitously between days 3 and 7 after birth.

A novel Rb- and p300-binding protein inhibits transactivation by MyoD.

The retinoblastoma protein (Rb) regulates both the cell cycle and tissue-specific transcription, by modulating the activity of factors that associate with its A-B and C pockets. In skeletal muscle, Rb has been reported to regulate irreversible cell cycle exit and muscle-specific transcription. To identify factors interacting with Rb in muscle cells, we utilized the yeast two-hybrid system, using the A-B and C pockets of Rb as bait. A novel protein we have designated E1A-like inhibitor of differentiation 1 (EID-1), was the predominant Rb-binding clone isolated. It is preferentially expressed in adult cardiac and skeletal muscle and encodes a 187-amino-acid protein, with a classic Rb-binding motif (LXCXE) in its C terminus. Overexpression of EID-1 in skeletal muscle inhibited tissue-specific transcription. Repression of skeletal muscle-restricted genes was mediated by a block to transactivation by MyoD independent of G(1) exit and, surprisingly, was potentiated by a mutation that prevents EID-1 binding to Rb. Inhibition of MyoD may be explained by EID-1's ability to bind and inhibit p300's histone acetylase activity, an essential MyoD coactivator. Thus, EID-1 binds both Rb and p300 and is a novel repressor of MyoD function.

Autonomous expression of c-myc in BC3H1 cells partially inhibits but does not prevent myogenic differentiation.

Myogenic differentiation is obligatorily coupled to withdrawal of myoblasts from the cell cycle and is inhibited by specific polypeptide growth factors. To investigate the potential involvement of c-myc in the control of myogenesis, the BC3H1 muscle cell line was stably transfected with a simian virus 40 promoter:c-myc chimeric gene. In quiescent cells in 0.5% serum, the exogenous c-myc gene was expressed at a level more than threefold greater than the level of endogenous c-myc in undifferentiated, proliferating cells of the parental line in 20% serum. The transfected myc gene partially inhibited the expression of both muscle creatine kinase and the nicotinic acetylcholine receptor, but was not sufficient to prevent the induction of these muscle differentiation products upon mitogen withdrawal.

A Ras-dependent pathway regulates RNA polymerase II phosphorylation in cardiac myocytes: implications for cardiac hypertrophy.

Despite extensive evidence implicating Ras in cardiac muscle hypertrophy, the mechanisms involved are unclear. We previously reported that Ras, through an effector-like function of Ras GTPase-activating protein (GAP) in neonatal cardiac myocytes (M. Abdellatif et al., J. Biol. Chem. 269:15423-15426, 1994; M. Abdellatif and M. D. Schneider, J. Biol. Chem. 272:527-533, 1997), can up-regulate expression from a comprehensive set of promoters, including both cardiac cell-specific and constitutive ones. To investigate the mechanism(s) underlying these earlier findings, we have used recombinant adenoviruses harboring a dominant negative Ras (17N Ras) allele or the N-terminal domain of GAP (nGAP), responsible for the Ras-like effector function. Inhibition of endogenous Ras reduced basal levels of [3H]uridine and [3H]phenylalanine incorporation into total RNA, mRNA, and protein, with parallel changes in apparent cell size. In addition, 17N Ras markedly inhibited phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (pol II), known to regulate transcript elongation, accompanied by down-regulation of its principal kinase, cyclin-dependent kinase 7 (Cdk7). In contrast, nGAP elicited the opposite effects on each of these parameters. Furthermore, cotransfection of constitutively active Ras (12R Ras) with wild-type pol II, rather than a truncated mutant lacking the CTD, demonstrated that Ras activation of transcription was dependent on the pol II CTD. Consistent with a potential role for this pathway in the development of cardiac myocyte hypertrophy, alpha1-adrenergic stimulation similarly enhanced pol II phosphorylation and Cdk7 expression, where both effects were inhibited by dominant negative Ras, while pressure overload hypertrophy led to an increase in both hyperphosphorylated and hypophosphorylated pol II in addition to Cdk7.

Inducible gene targeting in postnatal myocardium by cardiac-specific expression of a hormone-activated Cre fusion protein.

Cardiac-restricted expression of Cre recombinase can provoke lineage-specific gene excision in the myocardium. However, confounding early lethality may still preclude using loss-of-function models to study the postnatal heart. Here, we have tested whether inducible, heart-specific recombination can be triggered after birth by transgenic expression of a Cre fusion protein that incorporates a mutated progesterone receptor ligand binding domain (PR1) that is activated by the synthetic antiprogestin, RU486, but not by endogenous steroid hormones. CrePR1 driven by the alpha-myosin heavy chain (alphaMHC) promoter was expressed specifically in heart. Translocation of CrePR1 from cytoplasm to nuclei in ventricular myocytes was induced by RU486. To establish whether this approach can mediate cardiac-specific, drug-dependent excision between loxP sites in vivo, we mated alphaMHC-CrePR1 mice with a ubiquitously expressed (ROSA26) Cre reporter line. Offspring harboring alphaMHC-CrePR1 and/or the floxed allele were injected with RU486 versus vehicle, and the prevalence of beta-galactosidase (beta-gal)-positive cells was determined, indicative of Cre-mediated excision. Little or no baseline recombination was seen 1 week after birth. Cardiac-restricted, RU486-inducible recombination was demonstrated in bigenic mice at age 3 and 6 weeks, using each of 3 independent CrePR1 lines. Recombination in the absence of ligand paralleled the levels of CrePR1 protein expression and was more evident at 6 weeks. Thus, conditional, posttranslational activation of a Cre fusion protein can bypass potential embryonic and perinatal effects on the heart and permits inducible recombination in cardiac muscle. High levels of the chimeric Cre protein, in particular, were associated with progressive recombination in the absence of drug.

Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43.

Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1+/-0.13 versus 1.66+/-0.06; P:<0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate.