Publications

Ischemic heart disease is the leading cause of morbidity and mortality in the world. While pharmacological and surgical interventions developed in the late twentieth century drastically improved patient outcomes, mortality rates over the last two decades have begun to plateau. Following ischemic injury, pathological remodeling leads to cardiomyocyte loss and fibrosis leading to impaired heart function. Cardiomyocyte turnover rate in the adult heart is limited, and no clinical therapies currently exist to regenerate cardiomyocytes lost following ischemic injury. In this review, we summarize the progress of therapeutic strategies including revascularization and cell-based interventions to regenerate the heart: transiently inducing cardiomyocyte proliferation and direct reprogramming of fibroblasts into cardiomyocytes. Moreover, we highlight recent mechanistic insights governing these strategies to promote heart regeneration and identify current challenges in translating these approaches to human patients.

Centriolar satellites are small cytoplasmic granules that play important roles in regulating the formation of centrosomes and primary cilia. Ubiquitylation of satellite proteins, including the core satellite scaffold protein pericentriolar material 1 (PCM1), regulates centriolar satellite integrity. Currently, deubiquitylases that control centriolar satellite integrity have not been identified. In this study, we find that the deubiquitylase USP9X binds PCM1, and antagonizes PCM1 ubiquitylation to protect it from proteasomal degradation. Knockdown of USP9X in human cell lines reduces PCM1 protein levels, disrupts centriolar satellite particles and causes localization of satellite proteins, such as CEP290, to centrosomes. Interestingly, knockdown of mindbomb 1 (MIB1), a ubiquitin ligase that promotes PCM1 ubiquitylation and degradation, in USP9X-depleted cells largely restores PCM1 protein levels and corrects defects caused by the loss of USP9X. Overall, our study reveals that USP9X is a constituent of centriolar satellites and functions to maintain centriolar satellite integrity by stabilizing PCM1.

Mutations in lysosomal-associated membrane protein 2 (LAMP-2) gene are associated with Danon disease, which often leads to cardiomyopathy/heart failure through poorly defined mechanisms. Here, we identify the LAMP-2 isoform B (LAMP-2B) as required for autophagosome-lysosome fusion in human cardiomyocytes (CMs). Remarkably, LAMP-2B functions independently of syntaxin 17 (STX17), a protein that is essential for autophagosome-lysosome fusion in non-CMs. Instead, LAMP-2B interacts with autophagy related 14 (ATG14) and vesicle-associated membrane protein 8 (VAMP8) through its C-terminal coiled coil domain (CCD) to promote autophagic fusion. CMs derived from induced pluripotent stem cells (hiPSC-CMs) from Danon patients exhibit decreased colocalization between ATG14 and VAMP8, profound defects in autophagic fusion, as well as mitochondrial and contractile abnormalities. This phenotype was recapitulated by LAMP-2B knockout in non-Danon hiPSC-CMs. Finally, gene correction of LAMP-2 mutation rescues the Danon phenotype. These findings reveal a STX17-independent autophagic fusion mechanism in human CMs, providing an explanation for cardiomyopathy in Danon patients and a foundation for targeting defective LAMP-2B-mediated autophagy to treat this patient population.

RATIONALE: Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown.

OBJECTIVE: We sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart.

METHODS AND RESULTS: RNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-β (transforming growth factor-β) signaling to stimulate conversion of quiescent cardiac fibroblasts into Periostin (Postn)-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the Sertad4 (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for Sertad4 in TGF-β-mediated cardiac fibroblast activation.

CONCLUSIONS: These findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for Sertad4. The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.

Little is known about the biological function of histone deacetylase 11 (HDAC11), which is the lone class IV HDAC. Here, we demonstrate that deletion of HDAC11 in mice stimulates brown adipose tissue (BAT) formation and beiging of white adipose tissue (WAT). Consequently, HDAC11-deficient mice exhibit enhanced thermogenic potential and, in response to high-fat feeding, attenuated obesity, improved insulin sensitivity, and reduced hepatic steatosis. Ex vivo and cell-based assays revealed that HDAC11 catalytic activity suppresses the BAT transcriptional program, in both the basal state and in response to β-adrenergic receptor signaling, through a mechanism that is dependent on physical association with BRD2, a bromodomain and extraterminal (BET) acetyl-histone-binding protein. These findings define an epigenetic pathway for the regulation of energy homeostasis and suggest the potential for HDAC11-selective inhibitors for the treatment of obesity and diabetes.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs. We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.

RATIONALE: Skeletal muscle wasting with accompanying cachexia is a life threatening complication in congestive heart failure. The molecular mechanisms are imperfectly understood, although an activated renin-angiotensin aldosterone system has been implicated. Angiotensin (Ang) II induces skeletal muscle atrophy in part by increased muscle-enriched E3 ubiquitin ligase muscle RING-finger-1 (MuRF1) expression, which may involve protein kinase D1 (PKD1).

OBJECTIVE: To elucidate the molecular mechanism of Ang II-induced skeletal muscle wasting.

METHODS AND RESULTS: A cDNA expression screen identified the lysosomal hydrolase-coordinating transcription factor EB (TFEB) as novel regulator of the human MuRF1 promoter. TFEB played a key role in regulating Ang II-induced skeletal muscle atrophy by transcriptional control of MuRF1 via conserved E-box elements. Inhibiting TFEB with small interfering RNA prevented Ang II-induced MuRF1 expression and atrophy. The histone deacetylase-5 (HDAC5), which was directly bound to and colocalized with TFEB, inhibited TFEB-induced MuRF1 expression. The inhibition of TFEB by HDAC5 was reversed by PKD1, which was associated with HDAC5 and mediated its nuclear export. Mice lacking PKD1 in skeletal myocytes were resistant to Ang II-induced muscle wasting.

CONCLUSION: We propose that elevated Ang II serum concentrations, as occur in patients with congestive heart failure, could activate the PKD1/HDAC5/TFEB/MuRF1 pathway to induce skeletal muscle wasting.

Direct reprogramming of fibroblasts into cardiomyocytes by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2C, Tbx5) or GHMT (GATA4, Hand2, Mef2C, Tbx5), has recently been demonstrated, suggesting a novel therapeutic strategy for cardiac repair. However, current approaches are inefficient. Here we demonstrate that pro-fibrotic signalling potently antagonizes cardiac reprogramming. Remarkably, inhibition of pro-fibrotic signalling using small molecules that target the transforming growth factor-β or Rho-associated kinase pathways converts embryonic fibroblasts into functional cardiomyocyte-like cells, with the efficiency up to 60%. Conversely, overactivation of these pro-fibrotic signalling networks attenuates cardiac reprogramming. Furthermore, inhibition of pro-fibrotic signalling dramatically enhances the kinetics of cardiac reprogramming, with spontaneously contracting cardiomyocytes emerging in less than 2 weeks, as opposed to 4 weeks with GHMT alone. These findings provide new insights into the molecular mechanisms underlying cardiac conversion of fibroblasts and would enhance efforts to generate cardiomyocytes for clinical applications.

Members of the calmodulin-binding transcription activator (CAMTA) family of proteins function as calcium-sensitive regulators of gene expression in multicellular organisms ranging from plants to humans. Here, we show that global or nervous system deletion of CAMTA1 in mice causes severe ataxia with Purkinje cell degeneration and cerebellar atrophy, partially resembling the consequences of haploinsufficiency of the human CAMTA1 locus. Gene-expression analysis identified a large collection of neuronal genes that were dysregulated in the brains of CAMTA1-mutant mice, and elucidation of a consensus sequence for binding of CAMTA proteins to DNA revealed the association of CAMTA-binding sites with many of these genes. We conclude that CAMTA1 plays an essential role in the control of Purkinje cell function and survival. CAMTA1-mutant mice provide a model to study the molecular mechanisms of neurodegenerative diseases and for screening potential therapeutic interventions for such disorders.