Faculty Bibliography

MicroRNA cluster miR-17-92 has been implicated in cardiovascular development and function, yet its precise mechanisms of action in these contexts are uncertain. This study aimed to investigate the role of miR-17-92 in morphogenesis and function of cardiac and smooth muscle tissues. To do so, a mouse model of conditional overexpression of miR-17-92 in cardiac and smooth muscle tissues was generated. Extensive cardiac functional studies identified a dose-dependent induction of dilated, hypertrophic cardiomyopathy, and arrhythmia inducibility in transgenic animals, which correlated with premature mortality (98.3+/-42.5 d, P<0.0001). Expression analyses revealed the abundance of Pten transcript, a known miR-17-92 target, to be inversely correlated with miR-17-92 expression levels and heart size. In addition, we demonstrated through 3'-UTR luciferase assays and expression analyses that Connexin43 (Cx43) is a novel direct target of miR-19a/b and its expression is suppressed in transgenic hearts. Taken together, these data demonstrate that dysregulated expression of miR-17-92 during cardiovascular morphogenesis results in a lethal cardiomyopathy, possibly in part through direct repression of Pten and Cx43. This study highlights the importance of miR-17-92 in both normal and pathological functions of the heart, and provides a model that may serve as a useful platform to test novel antiarrhythmic therapeutics.-Danielson, L. S., Park, D. S., Rotllan, N., Chamorro-Jorganes, A., Guijarro, M. V., Fernandez-Hernando, C., Fishman, G. I., Phoon, C. K. L., Hernando, E. Cardiovascular dysregulation of miR-17-92 causes a lethal hypertrophic cardiomyopathy and arrhythmogenesis.

Cardiac gap junctions are specialized membrane structures comprised of arrays of intercellular channels responsible for propagation of the cardiac impulse. These channels are formed by oligomerization of individual protein subunits known as connexins. In response to a broad array of pathologic stressors, gap junction expression is disturbed, resulting in aberrant cardiac conduction and increased propensity for rhythm disturbances. In this article, we review some of the recently identified molecular regulators of connexin assembly, membrane targeting, and degradation, focusing on the role of post-translational phosphorylation of connexin 43, the major gap junctional protein expressed in ventricular myocardium. We also describe efforts to engineer "designer" gap junctions that are resistant to pathologic remodeling.

Genetically modified mice mimicking ODDD (oculodentodigital dysplasia), a disease characterized by reduced Cx43 (connexin 43)-mediated gap junctional intercellular communication, represent an in vivo model to assess the role of Cx43 in mammary gland development and function. We previously reported that severely compromised Cx43 function delayed mammary gland development and impaired milk ejection in mice that harboured a G60S Cx43 mutant, yet there are no reports of lactation defects in ODDD patients. To address this further, we obtained a second mouse model of ODDD expressing an I130T Cx43 mutant to assess whether a mutant with partial gap junction channel activity would be sufficient to retain mammary gland development and function. The results of the present study show that virgin Cx43I130T/+ mice exhibited a temporary delay in ductal elongation at 4 weeks. In addition, Cx43I130T/+ mice develop smaller mammary glands at parturition due to reduced cell proliferation despite similar overall gland architecture. Distinct from Cx43G60S/+ mice, Cx43I130T/+ mice adequately produce and deliver milk to pups, suggesting that milk ejection is unaffected. Thus the present study suggests that a loss-of-function mutant of Cx43 with partial gap junction channel coupling conductance results in a less severe mammary gland phenotype, which may partially explain the lack of reported lactation defects associated with ODDD patients.

During development, the ventricular conduction system (VCS) arises from the trabecular or spongy myocardium. VCS and trabecular myocytes proliferate at a significantly slower rate than compact zone myocardial cells, establishing a transmural cell cycle gradient. The molecular determinants of VCS/trabecular myocyte cell cycle arrest are not known. Given the importance of pocket proteins (Rb, p107 and p130) in mediating G0/G1 arrest in many cell types, we examined the role of this gene family in regulating cell cycle exit of the trabecular myocardium and ventricular conduction system. Using a combinatorial knockout strategy, we found that graded loss of pocket proteins results in a spectrum of heart and lung defects. p107/p130 double knockout (dKO) hearts manifest dysregulated proliferation within the compact myocardium and trabecular bases, while the remaining trabecular region cell cycle exits normally. Consequently, dKO hearts exhibit defective cardiac compaction, septal hyperplasia and biventricular outflow tract obstruction, while the VCS appears relatively normal. Loss of all three pocket proteins (3KO) is necessary to completely disrupt the transmural cell cycle gradient. 3KO hearts exhibit massive overgrowth of the trabecular myocardium and ventricular conduction system, which leads to fetal heart failure and death. Hearts carrying a single pocket protein allele are able to maintain the transmural cell cycle gradient. These results demonstrate the exquisite sensitivity of trabecular and conduction myocytes to pocket protein function during ventricular chamber development.

Genetically-modified mice mimicking oculodentodigital dysplasia (ODDD), a disease characterized by reduced Cx43-mediated gap junctional intercellular communication, represent an in vivo model to assess the role of Cx43 in mammary gland development and function. We previously reported that severely compromised-Cx43 function delayed mammary gland development and impaired milk ejection in mice that harboured a G60S Cx43 mutant. Surprisingly, there are no definitive reports of lactation defects in ODDD patients. To address this further, we obtained a second mouse model of ODDD expressing an I130T Cx43 mutant to assess if a Cx43 mutant with residual gap junction channel activity would rescue mammary gland development and function. Our findings show that virgin Cx43^I130T/+ mice, distinct from Cx43^G60S/+ mice, develop with similar body weights compared to control, despite having a reduction in the highly phosphorylated species of Cx43 and reduced Cx43 gap junctional plaques. In addition, virgin Cx43^I130T/+ mice exhibit a delay in ductal elongation at four weeks that is not observed by seven weeks. Cx43^I130T/+ mice develop smaller mammary glands at parturition due to reduced proliferation despite similar overall gland architecture. Distinct from Cx43^G60S/+ mice, Cx43^I130T/+ mice adequately produce and deliver milk to pups suggesting milk ejection is unaffected. Thus, these studies suggest that loss-of-function mutants of Cx43 with residual gap junction channel activity can rescue functional defects in the mammary gland and helps to explain the lack of lactation defects associated with ODDD patients

Background: We have previously demonstrated that the cell adhesion protein contactin2 (Cntn2) is enriched in Purkinje cells of the cardiac conduction system (CCS). Objective: Generation of a mouse embryonic stem cell (mESC) reporter line that allows identification of Purkinje-like cardiomyocytes in vitro. Methods and Results: mESC were generated from transgenic mice carrying a BAC Cntn2-eGFP reporter gene and were subsequently transduced with lentivirus coding for a selectable MHCalpha-mCherry cardiomyocyte reporter gene. Immunostaining analysis confirmed that mESC expressed markers of pluripotency (Oct3/4; Klf4) and spontaneously differentiated into cells of all three germ layers in the absence of LIF (alpha- smooth muscle actin; beta-tubulin; alpha-fetoprotein). Spontaneous or serum-free directed cardiac differentiation resulted in generation of double positive, spontaneously beating cardiomyocytes after three weeks. Yield of double positive cells could be increased by adding endocardialderived factors (Nrg1; ET-1). FACS isolated double positive cells were enriched in transcripts of cardiomyocytes (Tbx5; Nkx2.5) and the CCS (Cntn2; Cx40). Action potential recordings of eGFP positive cardiomyocytes demonstrated distinct plateau phase and elongated action potential duration (APD50=79.9+10.4ms, APD90=170.2+17.5ms; n=11) compared with eGFP negative cardiomyocytes (APD50=53.4+9.4ms, APD90=120.6+17.3ms; n=15). Conclusion: We have established a mESC reporter line for the identification of CCS-like cells. This model should be useful for downstream studies of CCS development and pathology. Cntn2 may also be a useful marker of CCS-like cells derived from human ES and/or iPS cells

BACKGROUND: Notch signaling has previously been shown to play an essential role in regulating cell fate decisions and differentiation during cardiogenesis in many systems including Drosophila, Xenopus, and mammals. We hypothesized that Notch may also be involved in directing the progressive lineage restriction of cardiomyocytes into specialized conduction cells. METHODS AND RESULTS: In hearts where Notch signaling is activated within the myocardium from early development onward, Notch promotes a conduction-like phenotype based on ectopic expression of conduction system-specific genes and cell autonomous changes in electrophysiology. With the use of an in vitro assay to activate Notch in newborn cardiomyocytes, we observed global changes in the transcriptome, and in action potential characteristics, consistent with reprogramming to a conduction-like phenotype. CONCLUSIONS: Notch can instruct the differentiation of chamber cardiac progenitors into specialized conduction-like cells. Plasticity remains in late-stage cardiomyocytes, which has potential implications for engineering of specialized cardiovascular tissues.