Maryam Sharifi-Sanjani, PhD

Right ventricle (RV) dysfunction, remodeling, and eventual failure is a consequence of Pulmonary arterial hypertension (PAH) contributing to 44% of deaths in these patients. However, the underlying causes and availability of effective RV targeted therapy stand unresolved. Preliminary data support mitigated expression of Telomeric repeat-binding factor 2 (Trf2), an essential telomere length protecting protein, in RVs of PAH mouse models of hypoxia and pressure-overload model of pulmonary arterial banding (PAB). Interestingly, telomere attrition was not present in Trf2 deficient CMs, supportive of a telomere length maintenance-independent role for Trf2 in this cell type. To identify the role of Trf2 in RV dysfunction associated with PAH, the PI generated unique mice with inducible CM-specific deletion of Trf2 (conTrf2-/-) in adulthood and assessed in vivo RV function. Indeed, PI’s RV hemodynamic analyses revealed premature RV dysfunction in conTrf2-/- mice following both hypoxia and PAB compared with control mice and show that conTrf2-/- mice suffer from contractile dysfunction, which was not associated with elevated RV fibrosis or cardiomyocyte hypertrophy. Interestingly, preliminary data show that Trf2 deficient CMs present relocalized H3K9me3 (a marker of heterochromatin) away from nuclear periphery, a hallmark of gene transcription activation, coupled with ventricular dysfunction marker genes (ß-MHC and ANP) expression changes, suggesting that Trf2 governs RV CM gene transcription changes via regulating nuclear chromatin localization. To test this, we assessed collected RV tissues for aberrant PAH related RV dysfunction-associated gene expression changes, and found that at least ANP/BNP, ß-Actin and GATA4 were altered in conTrf2-/- mice. We will assess H3K9me3-DNA interaction at genomic binding sites of candidate genes (ANP, BNP, ß-Actin and GATA4) by Chromatin Immunoprecipitation (ChIP) coupled with qPCR (we have now optimized the technique specifically for our designed experiments). Further, nuclear relocalization, evaluated as distance from nuclear periphery, of identified genes will be assessed via high-resolution 2D FISH (fluorescence in situ hybridization) combined with immunostaining. We have further identified chromatin interacting nuclear membrane proteins (Syne3 and emerin) affected at transcript level by CM-Trf2 deficiency, providing a possible mechanism through which Trf2 regulates chromatin localization and shedding light on a novel and potential RV targeted therapeutic avenue in the field of PAH. To determine whether Trf2 overexpression has translational therapeutic potential in humans, we will adenovirally overexpress Trf2 in human induced pluripotent stem cell (hiPSC)-derived CMs with and without hypoxia, TGF-ß or TNF-a (PAH-related stimuli) in the presence and absence of heterochromatin protein1 (known to induce chromatin compaction via H3K9me3) siRNA and outcome on PAH-associated RV dysfunction genes’ expression rescue and nuclear localization will be evaluated. The adenovirus is expected to be ready in a month.
 

Update: Right ventricular (RV) failure is the leading cause of death in pulmonary arterial hypertension (PAH), and an RV-targeted therapy remains elusive due to incomplete understanding of mechanisms behind the etiology of RV-failure. We recently identified a protein that shows mitigated expression in RVs of PAH mouse models of hypoxia and pressure-overload model of pulmonary arterial banding (PAB), while its expression does not change in lungs nor cardiac fibroblasts, suggesting its cardiomyocyte (CM)-specific role in current PAH models. Hence, we generated a unique mouse with CM-specific deletion of our protein in adulthood. RV function hemodynamic analyses revealed premature RV dysfunction in our unique mice following both hypoxia and PAB, and found that these mice suffer from contractile dysfunction, which is not associated with RV fibrosis or CM hypertrophy. Molecular analyses identified aberrant PAH related RV dysfunction-associated gene expression changes in RVs of our novel mice, and more importantly, identified possible contribution of nuclear membrane proteins to the observed RV dysfunction. Our finding shed light on a novel and potentially RV-aimed therapy in the field of PAH.