AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

Nonsense Variant PRDM16-Q187X Causes Impaired Myocardial Development and TGF-ß Signaling Resulting in Noncompaction Cardiomyopathy in Humans and Mice.

BACKGROUND: PRDM16 plays a role in myocardial development through TGF-ß (transforming growth factor-beta) signaling. Recent evidence suggests that loss of PRDM16 expression is associated with cardiomyopathy development in mice, although its role in human cardiomyopathy development is unclear. This study aims to determine the impact of PRDM16 loss-of-function variants on cardiomyopathy in humans. METHODS: Individuals with PRDM16 variants were identified and consented. Induced pluripotent stem cell-derived cardiomyocytes were generated from a proband hosting a Q187X nonsense variant as an in vitro model and underwent proliferative and transcriptional analyses. CRISPR (clustered regularly interspaced short palindromic repeats)-mediated knock-in mouse model hosting the Prdm16Q187X allele was generated and subjected to ECG, histological, and transcriptional analysis. RESULTS: We report 2 probands with loss-of-function PRDM16 variants and pediatric left ventricular noncompaction cardiomyopathy. One proband hosts a PRDM16-Q187X variant with left ventricular noncompaction cardiomyopathy and demonstrated infant-onset heart failure, which was selected for further study. Induced pluripotent stem cell-derived cardiomyocytes prepared from the PRDM16-Q187X proband demonstrated a statistically significant impairment in myocyte proliferation and increased apoptosis associated with transcriptional dysregulation of genes implicated in cardiac maturation, including TGF-ß-associated transcripts. Homozygous Prdm16Q187X/Q187X mice demonstrated an underdeveloped compact myocardium and were embryonically lethal. Heterozygous Prdm16Q187X/WT mice demonstrated significantly smaller ventricular dimensions, heightened fibrosis, and age-dependent loss of TGF-ß expression. Mechanistic studies were undertaken in H9c2 cardiomyoblasts to show that PRDM16 binds TGFB3 promoter and represses its transcription. CONCLUSIONS: Novel loss-of-function PRDM16 variant impairs myocardial development resulting in noncompaction cardiomyopathy in humans and mice associated with altered TGF-ß signaling.

Screening of GPCR drugs for repurposing in breast cancer.

Drug repurposing can overcome both substantial costs and the lengthy process of new drug discovery and development in cancer treatment. Some Food and Drug Administration (FDA)-approved drugs have been found to have the potential to be repurposed as anti-cancer drugs. However, the progress is slow due to only a handful of strategies employed to identify drugs with repurposing potential. In this study, we evaluated GPCR-targeting drugs by high throughput screening (HTS) for their repurposing potential in triple-negative breast cancer (TNBC) and drug-resistant human epidermal growth factor receptor-2-positive (HER2+) breast cancer (BC), due to the dire need to discover novel targets and drugs in these subtypes. We assessed the efficacy and potency of drugs/compounds targeting different GPCRs for the growth rate inhibition in the following models: two TNBC cell lines (MDA-MB-231 and MDA-MB-468) and two HER2+ BC cell lines (BT474 and SKBR3), sensitive or resistant to lapatinib + trastuzumab, an effective combination of HER2-targeting therapies. We identified six drugs/compounds as potential hits, of which 4 were FDA-approved drugs. We focused on ß-adrenergic receptor-targeting nebivolol as a candidate, primarily because of the potential role of these receptors in BC and its excellent long-term safety profile. The effects of nebivolol were validated in an independent assay in all the cell line models. The effects of nebivolol were independent of its activation of ß3 receptors and nitric oxide production. Nebivolol reduced invasion and migration potentials which also suggests its inhibitory role in metastasis. Analysis of the Surveillance, Epidemiology and End Results (SEER)-Medicare dataset found numerically but not statistically significant reduced risk of all-cause mortality in the nebivolol group. In-depth future analyses, including detailed in vivo studies and real-world data analysis with more patients, are needed to further investigate the potential of nebivolol as a repurposed therapy for BC.

Left Ventricular Noncompaction Is Associated with Valvular Regurgitation and a Variety of Arrhythmias.

Left ventricular noncompaction (LVNC) is a type of cardiomyopathy characterized anatomically by prominent ventricular trabeculation and deep intertrabecular recesses. The mortality associated with LVNC ranges from 5% to 47%. The etiology of LVNC is yet to be fully understood, although decades have passed since its recognition as a clinical entity globally. Furthermore, critical questions, i.e., whether LVNC represents an acquired pathology or has a congenital origin and whether the reduced contractile function in LVNC patients is a cause or consequence of noncompaction, remain to be addressed. In this study, to answer some of these questions, we analyzed the clinical features of LVNC patients. Out of 9582 subjects screened for abnormal cardiac functions, 45 exhibit the characteristics of LVNC, and 1 presents right ventricular noncompaction (RVNC). We found that 40 patients show valvular regurgitation, 39 manifest reduced systolic contractions, and 46 out of the 46 present different forms of arrhythmias that are not restricted to be caused by the noncompact myocardium. This retrospective examination of LVNC patients reveals some novel findings: LVNC is associated with regurgitation in most patients and arrhythmias in all patients. The thickness ratio of the trabecular layer to compact layer negatively correlates with fractional shortening, and reduced contractility might result from LVNC. This study adds evidence to support a congenital origin of LVNC that might benefit the diagnosis and subsequent characterization of LVNC patients.

The Spatiotemporal Expression of Notch1 and Numb and Their Functional Interaction during Cardiac Morphogenesis.

Numb family proteins (NFPs), including Numb and Numblike (Numbl), are commonly known for their role as cell fate determinants for multiple types of progenitor cells, mainly due to their function as Notch inhibitors. Previous studies have shown that myocardial NFP double knockout (MDKO) hearts display an up-regulated Notch activation and various defects in cardiac progenitor cell differentiation and cardiac morphogenesis. Whether enhanced Notch activation causes these defects in MDKO is not fully clear. To answer the question, we examined the spatiotemporal patterns of Notch1 expression, Notch activation, and Numb expression in the murine embryonic hearts using multiple approaches including RNAScope, and Numb and Notch reporter mouse lines. To further interrogate the interaction between NFPs and Notch signaling activation, we deleted both Notch1 or RBPJk alleles in the MDKO. We examined and compared the phenotypes of Notch1 knockout, NFPs double knockout, Notch1; Numb; Numbl and RBPJk; Numb; Numbl triple knockouts. Our study showed that Notch1 is expressed and activated in the myocardium at several stages, and Numb is enriched in the epicardium and did not show the asymmetric distribution in the myocardium. Cardiac-specific Notch1 deletion causes multiple structural defects and embryonic lethality. Notch1 or RBPJk deletion in MDKO did not rescue the structural defects in the MDKO but partially rescued the defects of cardiac progenitor cell differentiation, cardiomyocyte proliferation, and trabecular morphogenesis. Our study concludes that NFPs regulate progenitor cell differentiation, cardiomyocyte proliferation, and trabecular morphogenesis partially through Notch1 and play more roles than inhibiting Notch1 signaling during cardiac morphogenesis.

An intronic DHCR7 genetic polymorphism associates with vitamin D serum level and incidence of acute coronary syndrome.

INTRODUCTION: Vitamin D deficiency has been linked to cardiovascular pathologies including acute coronary syndrome (ACS). Polymorphisms in vitamin D associated genes have been confounding to vitamin D serum levels and pathological predispositions. 7-hydrocholesterol is a common precursor in cholesterol and vitamin D synthesis. DHCR7/NADSYN1 genetic locus expresses 7-hydrocholesterol reductase (DHCR7), an enzyme that recruits 7-hydrocholesterol in cholesterol biosynthesis, and NAD synthetase 1 (NADSYN1), which participates in the hydroxylation of 25 hydroxyvitamin D. AIM: This study aims to correlate two polymorphisms in the DHCR7/NADSYN1 genetic locus with levels of circulatory vitamin D and the presentation of ACS in an Egyptian population. METHODS: In a case control study, 189 ACS patients and 106 healthy control subjects were genotyped for SNPs rs11606033 of the DHCR7 gene and rs2276360 of the NADSYN1 gene using the amplification-refractory mutation system (ARMS). The levels of 25(OH)D2 and 25(OH)D3 were measured using an in-house developed and validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based protocol. RESULTS: ACS patients have significantly lower levels of circulating vitamin D in comparison to healthy controls. Allele A of the DHCR7 polymorphism was found to correlate with serum vitamin D deficiency and incidence of ACS classes: NSTEMI, STEMI and unstable angina, when compared to allele G. On the other hand, the NADSYN1 polymorphism rs2276360 correlated with serum 25(OH)D3 deficiency. Yet, no significant correlation was found with incidences of ACS. CONCLUSION: We conclude that rs11606033, which is an intronic SNP between exon 4 and exon 5 of the DHCR7 gene, influences vitamin D serum abundance and more importantly ACS incidence.