Genetically encoded sensor proteins provide unique opportunities to advance the understanding of complex cellular interactions in physiologically relevant contexts; however, previously described sensors have proved to be of limited use to report cell signaling in vivo in mammals. Here, we describe an improved Ca 2؉ sensor, GCaMP2, its inducible expression in the mouse heart, and its use to examine signaling in heart cells in vivo. The high brightness and stability of GCaMP2 enable the measurement of myocyte Ca 2؉ transients in all regions of the beating mouse heart and prolonged pacing and mapping studies in isolated, perfused hearts. Transgene expression is efficiently temporally regulated in cardiomyocyte GCaMP2 mice, allowing recording of in vivo signals 4 weeks after transgene induction. High-resolution imaging of Ca 2؉ waves in GCaMP2-expressing embryos revealed key aspects of electrical conduction in the preseptated heart. At embryonic day (e.d.) 10.5, atrial and ventricular conduction occur rapidly, consistent with the early formation of specialized conduction pathways. However, conduction is markedly slowed through the atrioventricular canal in the e.d. 10.5 heart, forming the basis for an effective atrioventricular delay before development of the AV node, as rapid ventricular activation occurs after activation of the distal AV canal tissue. Consistent with the elimination of the inner AV canal muscle layer at e.d. 13.5, atrioventricular conduction through the canal was abolished at this stage. These studies demonstrate that GCaMP2 will have broad utility in the dissection of numerous complex cellular interactions in mammals, in vivo. atrioventricular node ͉ Ca 2ϩ imaging ͉ genetic sensor ͉ heart development ͉ fluorescent Ca 2ϩ sensor T ransient, highly regulated elevations in cytosolic free Ca 2ϩ underlie numerous cellular processes that enable organ function (1-5). In the mammalian heart, for example, efficient function depends upon the coordinated release and reuptake of Ca 2ϩ ions from intracellular organelles in millions of cells, at rates between 0.5 and 15 Hz throughout life, and even subtle dysfunctions of this process can result in cardiac arrythmias and sudden death. Whereas fluorescent imaging using purpose-designed small Ca 2ϩ -binding indicator molecules has enabled important advances in the understanding of the regulatory processes underlying Ca 2ϩ signaling in single cells (6, 7), these approaches have significant limitations in the context of a complex, multicellular organ such as the beating heart. Thus, difficulties in obtaining an adequate and stable concentration of indicator molecules within cells deep in complex tissues, the incompatibility of loading procedures in the in vivo setting, and the inability to selectively load specific cell lineages constitute substantial experimental constraints on the study of multicellular, processive Ca 2ϩ signaling in complex organ function. Genetically encoded sensors of Ca 2ϩ signaling (7-13) hold great promise in this regard and have been used t...

An R120G missense mutation in the small heat shock protein ␣-B-crystallin (CryAB R120G ) causes desmin-related cardiomyopathy (DRM). DRM is characterized by the formation of aggregates containing CryAB and desmin, and it can be recapitulated in transgenic mice by cardiac-specific expression of the mutant protein. In this article, we show that expression of CryAB R120G leads to the formation of electron-dense bodies characteristic of the DRMs and identify these bodies as aggresomes, which are characteristic of the neurodegenerative diseases. Cardiomyocytes transfected with adenovirus containing CryAB R120G establish the necessity and sufficiency of CryAB R120G expression for aggresome formation. The commonality of these aggresomes with oligomeric protein aggregates found in the amyloid-related degenerative diseases was corroborated by the presence of high levels of amyloid oligomers that may represent a primary toxic species in the amyloid diseases. These oligomeric amyloid intermediates are present also in cardiomyocytes derived from many human dilated and hypertrophic cardiomyopathies.H uman heart failure is the leading cause of death in the developed world, and it represents a final common endpoint for several disease entities, including hypertension, coronary artery disease, and the cardiomyopathies (1, 2). A lack of pathogenic commonality is underscored by the large number of mutations in different classes of cardiac proteins that have been linked to dilated and hypertrophic cardiomyopathy (HCM) (3). Mutations in the cytoskeletal and associated proteins can be causative because these proteins function in structural, sensor, and signaling roles in the normal and diseased cardiomyocyte (4). For example, up-regulation of the intermediate filament protein desmin occurs in cardiac disorders such as cardiac hypertrophy and congestive heart failure (CHF) (5), and desmin mutations are associated with desmin-related cardiomyopathy (DRM) and idiopathic dilated cardiomyopathy (6, 7). Mutations in other proteins also have been associated with the DRMs, and genetic evidence linking an R120G mutation in ␣-B-crystallin (CryAB, CryAB R120G ) to human DRM (8) prompted a series of experiments in which we showed that cardiac-restricted transgenic (TG) expression of CryAB R120G was sufficient to cause heart failure in a mouse model (9). Although up-regulation of CryAB is associated with disease states including DRM, its synthesis probably represents a general cellular response to stress because CryAB has chaperone-like activity. Indeed, CryAB, which binds to both desmin and cytoplasmic actin, probably participates normally as a chaperone in intermediate filament formation and maintenance in the heart.The ␣-crystallins (␣-A and ␣-B) were of interest initially as major structural proteins present in the lens of the vertebrate eye. However, the discovery that they were related to the small heat shock proteins (hsps) in Drosophila (10) prompted reevaluation of their broader role(s), and it is now known that CryAB belongs to the sma...

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease 1, 2, but the molecular basis is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (e.g. short or long QT syndromes, heart failure) 3-5 or pattern (e.g. Brugada syndrome) 6 of myocardial repolarization. Here we provide the first 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 novel clock-dependent oscillator, Krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of KChIP2, a critical subunit required for generating the transient outward potassium current (Ito). 7 Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. In sum, these findings identify circadian transcription of ion channels as a novel mechanism for cardiac arrhythmogenesis.

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 ␣-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␤ (GSK-3␤), were obtained. Subsequently, we tested the hypothesis that GSK-3␤ expression could reverse a preexisting hypertrophy. Inducible expression of GSK-3␤ 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.

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.