Atsushi Sanbe

Reengineering Inducible Cardiac-Specific Transgenesis With an Attenuated Myosin Heavy Chain Promoter

Abstract— Despite the advantages of reversibly altering cardiac transgene expression, the number of successful studies with inducible cardiac-specific transgene expression remains limited. The utility of the current system is hampered by the large number of lines needed before a nonleaky inducible line is isolated and by the use of a heterologous virus-based minimal promoter in the responder line. We developed an efficient, experimentally flexible system that enables us to reversibly affect both abundant and nonabundant cardiomyocyte proteins. The use of bacterial-codon–based transactivators led to aberrant splicing, whereas other more efficient transactivators, by themselves, caused disease when expressed in the heart. The redesign of the system focused on developing stable transactivator-expressing lines in which expression was driven by the mouse &agr;-myosin heavy chain promoter. A minimal responder locus was derived from the same promoter, in which the GATA sites and thyroid responsive elements responsible for robust cardiac specific expression were ablated, leading to an attenuated promoter that could be inducibly controlled. In all cases, whether activated or not, expression mimicked that of the parental promoter. By use of this system, an inducible expression of an abundant contractile protein, the atrial isoform of essential myosin light chain 1, and a powerful biological effector, glycogen synthase kinase-3&bgr; (GSK-3&bgr;), were obtained. Subsequently, we tested the hypothesis that GSK-3&bgr; expression could reverse a preexisting hypertrophy. Inducible expression of GSK-3&bgr; could both attenuate a hypertrophic response and partially reverse a pressure-overload–induced hypertrophy. The system appears to be robust and can be used to temporally control high levels of cardiac-specific transgene expression.

Circadian rhythms govern cardiac repolarization and arrhythmogenesis.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease, but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) or pattern (for example, Brugada’s syndrome) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current. Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis.

A mouse model of myosin binding protein C human familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy can be caused by mutations in genes encoding sarcomeric proteins, including the cardiac isoform of myosin binding protein C (MyBP-C), and multiple mutations which cause truncated forms of the protein to be made are linked to the disease. We have created transgenic mice in which varying amounts of a mutated MyBP-C, lacking the myosin and titin binding domains, are expressed in the heart. The transgenically encoded, truncated protein is stable but is not incorporated efficiently into the sarcomere. The transgenic muscle fibers showed a leftward shift in the pCa2+-force curve and, importantly, their power output was reduced. Additionally, expression of the mutant protein leads to decreased levels of endogenous MyBP-C, resulting in a striking pattern of sarcomere disorganization and dysgenesis.

Mitochondrial Dysfunction and Apoptosis Underlie the Pathogenic Process in α-B-Crystallin Desmin-Related Cardiomyopathy

Background— Mitochondria and sarcomeres have a well-defined architectural relation that partially depends on the integrity of the cytoskeletal network. An R120G missense mutation in the small heat shock protein α-B-crystallin (CryAB) causes desmin-related cardiomyopathy. Desmin-related cardiomyopathy is characterized by the formation of intracellular aggregates containing CryAB and desmin that are amyloid positive, and disease can be recapitulated in transgenic mice by cardiac-specific expression of the mutant protein. Methods and Results— To understand the resultant pathology, we explored the acute effects of R120G expression both in vitro and in vivo. In vitro, transfection of adult cardiomyocytes with R120G-expressing adenovirus resulted in altered contractile mechanics. In vivo, as the cytoskeletal network is disturbed but before deficits in organ function can be detected, alterations in mitochondrial organization and architecture occur, leading to a reduction in the maximal rate of oxygen consumption with substrates that utilize complex I activity, alterations in the permeability transition pore, and compromised inner membrane potential. Apoptotic pathways are subsequently activated, which eventually results in cardiomyocyte death, dilation, and heart failure. Conclusions— Cardiac chaperone dysfunction acutely leads to altered cardiomyocyte mechanics, perturbations in mitochondrial-sarcomere architecture, and deficits in mitochondrial function, which can result in activation of apoptosis and heart failure.

Transgenic Modeling of a Cardiac Troponin I Mutation Linked to Familial Hypertrophic Cardiomyopathy

Multiple mutations in cardiac troponin I (cTnI) have been associated with familial hypertrophic cardiomyopathy. Two mutations are located in the cTnI inhibitory domain, a highly negatively charged region that alternately binds to either actin or troponin C, depending on the intracellular concentration of calcium. This region is critical to the inhibition of actin-myosin crossbridge formation when intracellular calcium is low. We modeled one of the inhibitory domain mutations, arginine145-->glycine (TnI(146Gly) in the mouse sequence), by cardiac-specific expression of the mutated protein in transgenic mice. Multiple lines were generated with varying degrees of expression to establish a dose relationship; the severity of phenotype could be correlated directly with transgene expression levels. Transgenic mice overexpressing wild-type cTnI were generated as controls and analyzed in parallel with the TnI(146Gly) animals. The control mice showed no abnormalities, indicating that the phenotype of TnI(146Gly) was not simply an artifact of transgenesis. In contrast, TnI(146Gly) mice showed cardiomyocyte disarray and interstitial fibrosis and suffered premature death. The functional alterations that seem to be responsible for the development of cardiac disease include increased skinned fiber sensitivity to calcium and, at the whole organ level, hypercontractility with diastolic dysfunction. Severely affected lines develop a pathology similar to human familial hypertrophic cardiomyopathy but within a dramatically shortened time frame. These data establish the causality of this mutation for cardiac disease, provide an animal model for understanding the resultant pathogenic structure-function relationships, and highlight the differences in phenotype severity of the troponin mutations between human and mouse hearts.

Enhancement of proteasomal function protects against cardiac proteinopathy and ischemia/reperfusion injury in mice

The ubiquitin-proteasome system degrades most intracellular proteins, including misfolded proteins. Proteasome functional insufficiency (PFI) has been observed in proteinopathies, such as desmin-related cardiomyopathy, and implicated in many common diseases, including dilated cardiomyopathy and ischemic heart disease. However, the pathogenic role of PFI has not been established. Here we created inducible Tg mice with cardiomyocyte-restricted overexpression of proteasome 28 subunit α (CR-PA28αOE) to investigate whether upregulation of the 11S proteasome enhances the proteolytic function of the proteasome in mice and, if so, whether the enhancement can rescue a bona fide proteinopathy and protect against ischemia/reperfusion (I/R) injury. We found that CR-PA28αOE did not alter the homeostasis of normal proteins and cardiac function, but did facilitate the degradation of a surrogate misfolded protein in the heart. By breeding mice with CR-PA28αOE with mice representing a well-established model of desmin-related cardiomyopathy, we demonstrated that CR-PA28αOE markedly reduced aberrant protein aggregation. Cardiac hypertrophy was decreased, and the lifespan of the animals was increased. Furthermore, PA28α knockdown promoted, whereas PA28α overexpression attenuated, accumulation of the mutant protein associated with desmin-related cardiomyopathy in cultured cardiomyocytes. Moreover, CR-PA28αOE limited infarct size and prevented postreperfusion cardiac dysfunction in mice with myocardial I/R injury. We therefore conclude that benign enhancement of cardiac proteasome proteolytic function can be achieved by CR-PA28αOE and that PFI plays a major pathogenic role in cardiac proteinopathy and myocardial I/R injury.

Cardiomyocyte Expression of a Polyglutamine Preamyloid Oligomer Causes Heart Failure

Background— To determine whether soluble preamyloid oligomers (PAOs) are toxic when expressed internally in the cardiomyocyte, we tested the hypothesis that cardiomyocyte-restricted expression and accumulation of a known PAO is cytotoxic and sufficient to cause heart failure. Methods and Results— Intracellular PAOs, the entities believed to cause toxicity in many neurodegenerative diseases, have been observed in cardiomyocytes derived from mouse and human heart failure samples. Long (>50) polyglutamine (PQ) repeats form PAOs and cause neurotoxicity in Huntington disease and other neurodegenerative diseases, whereas shorter PQ peptides are benign. We created transgenic mice in which cardiomyocyte-autonomous expression of an 83 residue–long PQ repeat (PQ83) or a non–amyloid-forming peptide of 19 PQ repeats (PQ19) as a nonpathological control was expressed. A PQ83 line with relatively low levels of expression was generated, along with a PQ19 line that expressed ≈9-fold the levels observed in the PQ83 line. Hearts expressing PQ83 exhibited reduced cardiac function and dilation by 5 months, and all mice died by 8 months, whereas PQ19 mice had normal cardiac function, morphology, and life span. PQ83 protein accumulated within aggresomes with PAO-specific staining. The PQ83 hearts showed increased autophagosomal and lysosomal content but also showed markers of necrotic death, including inflammatory cell infiltration and increased sarcolemmal permeability. Conclusions— The data confirm the hypothesis that expression of an exogenous PAO-forming peptide is toxic to cardiomyocytes and is sufficient to cause cardiomyocyte loss and heart failure in a murine model.

Functional significance of cardiac myosin essential light chain isoform switching in transgenic mice.

The different functions of the ventricular- and atrial-specific essential myosin light chains are unknown. Using transgenesis, cardiac-specific overexpression of proteins can be accomplished. The transgenic paradigm is more useful than originally expected, in that the mammalian heart rigorously controls sarcomeric protein stoichiometries. In a clinical subpopulation suffering from heart disease caused by congenital malformations of the outflow tract, an ELC1v-->ELC1a isoform shift correlated with increases in cross-bridge cycling kinetics as measured in skinned fibers derived from the diseased muscle. We have used transgenesis to replace the ventricular isoform of the essential myosin light chain with the atrial isoform. The ELC1v--> ELC1a shift in the ventricle resulted in similar functional alterations. Unloaded velocities as measured by the ability of the myosin to translocate actin filaments in the in vitro motility assay were significantly increased as a result of the isoform substitution. Unloaded shortening velocity was also increased in skinned muscle fibers, and at the whole organ level, both contractility and relaxation were significantly increased. This increase in cardiac function occurred in the absence of a hypertrophic response. Thus, ELC1a expression in the ventricle appears to be advantageous to the heart, resulting in increased cardiac function.

In Vivo Modeling of Myosin Binding Protein C Familial Hypertrophic Cardiomyopathy

Myosin binding protein C (MyBP-C) is an integral part of the striated muscle sarcomere. As is the case for other sarcomeric genes in human populations, multiple mutations within the gene have been linked to familial hypertrophic cardiomyopathy. Although some MyBP-C lesions are the result of missense mutations, most show truncated polypeptides lacking either the myosin or myosin and titin binding sites. Previously, we generated transgenic (TG) mice with cardiac-specific expression of a MyBP-C mutant lacking the myosin and titin binding domains. Surprisingly, the mutant protein was stable and made up a majority of the MyBP-C species, with concomitant reductions in endogenous MyBP-C such that overall MyBP-C stoichiometry was conserved. In the present study, we created a second series of TG mice that express, in the heart, a mutant MyBP-C lacking only the myosin binding site. In contrast to the previous data for the MyBP-C lacking both titin and myosin binding sites, only very modest levels of protein were found, consistent with data obtained from human biopsies in which mutated MyBP-C could not be detected. Despite normal levels of wild-type MyBP-C, there were significant changes in the structure and ultrastructure of the heart. Fiber mechanics showed decreased unloading shortening velocity, maximum shortening velocity, and relative maximal power output.

Physiological Growth Synergizes With Pathological Genes in Experimental Cardiomyopathy

Hundreds of signaling molecules have been assigned critical roles in the pathogenesis of myocardial hypertrophy and heart failure based on cardiac phenotypes from &agr;-myosin heavy chain–directed overexpression mice. Because permanent ventricular transgene expression in this system begins during a period of rapid physiological neonatal growth, resulting phenotypes are the combined consequences of transgene effects and normal trophic influences. We used temporally-defined forced gene expression to investigate synergy between postnatal physiological cardiac growth and two functionally divergent cardiomyopathic genes. Phenotype development was compared various times after neonatal (age 2 to 3 days) and adult (age 8 weeks) expression. Proapoptotic Nix caused ventricular dilation and severe contractile depression in neonates, but not adults. Myocardial apoptosis was minimal in adults, but was widespread in neonates, until it spontaneously resolved in adulthood. Unlike normal postnatal cardiac growth, concurrent left ventricular pressure overload hypertrophy did not synergize with Nix expression to cause cardiomyopathy or myocardial apoptosis. Prohypertrophic G&agr;q likewise caused eccentric hypertrophy, systolic dysfunction, and pathological gene expression in neonates, but not adults. Thus, normal postnatal cardiac growth can be an essential cofactor in development of genetic cardiomyopathies, and may confound the interpretation of conventional &agr;-MHC transgenic phenotypes.