ERRgamma directs and maintains the transition to oxidative metabolism in the postnatal heart.

At birth, the heart undergoes a critical metabolic switch from a predominant dependence on carbohydrates during fetal life to a greater dependence on postnatal oxidative metabolism. This remains the principle metabolic state throughout life, although pathologic conditions such as heart failure and cardiac hypertrophy reactivate components of the fetal genetic program to increase carbohydrate utilization. Disruption of the ERRgamma gene (Esrrg), which is expressed at high levels in the fetal and postnatal mouse heart, blocks this switch, resulting in lactatemia, electrocardiographic abnormalities, and death during the first week of life. Genomic ChIP-on-chip and expression analysis identifies ERRgamma as both a direct and an indirect regulator of a nuclear-encoded mitochondrial genetic network that coordinates the postnatal metabolic transition. These findings reveal an unexpected and essential molecular genetic component of the oxidative metabolic gene program in the heart and highlight ERRgamma in the study of cardiac hypertrophy and failure.

Computational design of peptides to target NaV1.7 channel with high potency and selectivity for the treatment of pain.

The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.

Spatial distributions of Kv4 channels and KChip2 isoforms in the murine heart based on laser capture microdissection.

OBJECTIVE: Regional differences in repolarizing K(+) current densities and expression levels of their molecular components are important for coordinating the pattern of electrical excitation and repolarization of the heart. The small size of hearts from mice may obscure these interventricular and/or transmural expression differences of K(+) channels. We have examined this possibility in adult mouse ventricle using a technology that provides very high spatial resolution of tissue collection. METHODS: Conventional manual dissection and laser capture microdissection (LCM) were utilized to dissect tissue from distinct ventricular regions. RNA was isolated from epicardial, mid-myocardial and endocardial layers of both the right and left ventricles. Real-time RT-PCR was used to quantify the transcript expression in these different regions. RESULTS: LCM revealed significant interventricular and transmural gradients for both Kv4.2 and the alpha-subunit of KChIP2. The expression profile of a second K(+) channel transcript, Kir2.1, which is responsible for the inwardly rectifying K(+) current I(k1), showed no interventricular or transmural gradients and therefore served as a negative control. CONCLUSIONS: Our findings are in contrast to previous reports of a relatively uniform left ventricular transmural pattern of expression of Kv4.2, Kv4.3 and KChIP2 in adult mouse heart, which appear to be different than that in larger mammals. Specifically, our results demonstrate significant epi- to endocardial differences in the patterns of expression of both Kv4.2 and KChIP2.

Role of myotonic dystrophy protein kinase (DMPK) in glucose homeostasis and muscle insulin action.

Myotonic dystrophy 1 (DM1) is caused by a CTG expansion in the 3'-unstranslated region of the DMPK gene, which encodes a serine/threonine protein kinase. One of the common clinical features of DM1 patients is insulin resistance, which has been associated with a pathogenic effect of the repeat expansions. Here we show that DMPK itself is a positive modulator of insulin action. DMPK-deficient (dmpk-/-) mice exhibit impaired insulin signaling in muscle tissues but not in adipocytes and liver, tissues in which DMPK is not expressed. Dmpk-/- mice display metabolic derangements such as abnormal glucose tolerance, reduced glucose uptake and impaired insulin-dependent GLUT4 trafficking in muscle. Using DMPK mutants, we show that DMPK is required for a correct intracellular trafficking of insulin and IGF-1 receptors, providing a mechanism to explain the molecular and metabolic phenotype of dmpk-/- mice. Taken together, these findings indicate that reduced DMPK expression may directly influence the onset of insulin-resistance in DM1 patients and point to dmpk as a new candidate gene for susceptibility to type 2-diabetes.

Comparison of contraction and calcium handling between right and left ventricular myocytes from adult mouse heart: a role for repolarization waveform.

In the mammalian heart, the right ventricle (RV) has a distinct structural and electrophysiological profile compared to the left ventricle (LV). However, the possibility that myocytes from the RV and LV have different contractile properties has not been established. In this study, sarcomere shortening, [Ca2+]i transients and Ca2+ and K+ currents in unloaded myocytes isolated from the RV, LV epicardium (LVepi) and LV endocardium (LVendo) of adult mice were evaluated. Maximum sarcomere shortening elicited by field stimulation was graded in the order: LVendo > LVepi > RV. Systolic [Ca2+]i was higher in LVendo myocytes than in RV myocytes. Voltage-clamp experiments in which action potential (AP) waveforms from RV and LVendo were used as the command signal, demonstrated that total Ca2+ influx and myocyte shortening were larger in response to the LVendo AP, independent of myocyte subtypes. Evaluation of possible regional differences in myocyte Ca2+ handling was based on: (i) the current-voltage relation of the Ca2+ current; (ii) sarcoplasmic reticulum Ca2+ uptake; and (iii) mRNA expression of important components of the Ca2+ handling system. None of these were significantly different between RV and LVendo. In contrast, the Ca2+-independent K+ current, which modulates AP repolarization, was significantly different between RV, LVepi and LVendo. These results suggest that these differences in K+ currents can alter AP duration and modulate the [Ca2+]i transient and corresponding contraction. In summary, these findings provide an initial description of regional differences in excitation-contraction coupling in the adult mouse heart [corrected]

Myotonic dystrophy protein kinase phosphorylates phospholamban and regulates calcium uptake in cardiomyocyte sarcoplasmic reticulum.

Myotonic dystrophy (DM) is caused by a CTG expansion in the 3'-untranslated region of a protein kinase gene (DMPK). Cardiovascular disease is one of the most prevalent causes of death in DM patients. Electrophysiological studies in cardiac muscles from DM patients and from DMPK(-/-) mice suggested that DMPK is critical to the modulation of cardiac contractility and to the maintenance of proper cardiac conduction activity. However, there are no data regarding the molecular signaling pathways involved in DM heart failure. Here we show that DMPK expression in cardiac myocytes is highly enriched in the sarcoplasmic reticulum (SR) where it colocalizes with the ryanodine receptor and phospholamban (PLN), a muscle-specific SR Ca(2+)-ATPase (SERCA2a) inhibitor. Coimmunoprecipitation studies showed that DMPK and PLN can physically associate. Furthermore, purified wild-type DMPK, but not a kinase-deficient mutant (K110A DMPK), phosphorylates PLN in vitro. Subsequent studies using the DMPK(-/-) mice demonstrated that PLN is hypo-phosphorylated in SR vesicles from DMPK(-/-) mice compared with wild-type mice both in vitro and in vivo. Finally, we show that Ca(2+) uptake in SR is impaired in ventricular homogenates from DMPK(-/-) mice. Together, our data suggest the existence of a novel regulatory DMPK pathway for cardiac contractility and provide a molecular mechanism for DM heart pathology.

Development of the cardiac conduction system as delineated by minK-lacZ.

INTRODUCTION: Due to the lack of good molecular markers, for decades the morphogenetic origin of the cardiac conduction system has been a matter of debate. More recently, the spatial expression of minK-lacZ in the adult mouse heart has been shown, for the larger part, to be coincident with the conduction tissues. METHODS AND RESULTS: To trace the embryonic development of this system, we performed an analysis of the expression of this construct throughout early cardiac development. Expression was first seen at the eighth embryonic day. Subsequently, discrete rings were found at the sinuatrial, atrioventricular, interventricular, and ventriculoarterial junctions. With time, the expression became restricted to boundary regions of the heart, such as the hinges of the leaflets of the pulmonary and aortic valves, the atrioventricular rings, and the venous valves, as well as becoming incorporated into the definitive conduction tissues themselves. In the postnatal heart, the areas retaining minK-lacZ positivity outside of the definitive conduction tissues are known to be the site of origin of abnormal cardiac rhythms, suggesting that ectopic foci may derive from tissues that share a common developmental pathway with the definitive conduction system. CONCLUSION: Our findings suggest that the boundary regions between compartments, along with the atrioventricular conduction axis, share a common developmental pathway.

Nkx2-5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block.

Human mutations in Nkx2-5 lead to progressive cardiomyopathy and conduction defects via unknown mechanisms. To define these pathways, we generated mice with a ventricular-restricted knockout of Nkx2-5, which display no structural defects but have progressive complete heart block, and massive trabecular muscle overgrowth found in some patients with Nkx2-5 mutations. At birth, mutant mice display a hypoplastic atrioventricular (AV) node and then develop selective dropout of these conduction cells. Transcriptional profiling uncovered the aberrant expression of a unique panel of atrial and conduction system-restricted target genes, as well as the ectopic, high level BMP-10 expression in the adult ventricular myocardium. Further, BMP-10 is shown to be necessary and sufficient for a major component of the ventricular muscle defects. Accordingly, loss of ventricular muscle cell lineage specification into trabecular and conduction system myocytes is a new mechanistic pathway for progressive cardiomyopathy and conduction defects in congenital heart disease.