Author ORCID Identifier

https://orcid.org/0000-0003-4219-3798

Semester

Summer

Date of Graduation

2026

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Elizabeth Bowdridge

Committee Co-Chair

Stanley Hileman

Committee Member

Scott P. Levick

Committee Member

Eric Kelley

Committee Member

Giselle Melendez

Abstract

In the United States, heart failure affects approximately 6.7 million individuals. Despite a number of available therapies, heart failure prevalence continues to grow, suggesting the need for a broader range of effective therapies. More specifically, heart failure with preserved ejection fraction comprises approximately 50% of all heart failure cases and, as the name suggests, is a disease of diastolic dysfunction as opposed to systolic dysfunction. A hallmark of heart failure with preserved ejection fraction is myocardial fibrosis, which is a key contributor to diastolic dysfunction, and is the major focus of this dissertation work. In the heart, histamine-3 receptor activation was previously demonstrated to be anti-arrhythmic via inhibition of norepinephrine release from cardiac synaptosomes. Our laboratory further identified the presence of the histamine-3 receptor on mouse cardiac fibroblasts in vitro and demonstrated that histamine-3 receptor activation was anti-fibrotic and inhibited macrophage infiltration in a mouse model of adverse cardiac remodeling. As such, the premise of this present dissertation work was to further investigate the potential of exogenous histamine-3 receptor activation as a treatment for cardiac fibrosis underlying diastolic dysfunction by determining the cellular mechanisms through which histamine-3 receptor activation exerts its anti-fibrotic effects. We hypothesized that activation of the histamine-3 receptor is protective against cardiac fibrosis via direct actions on cardiac fibroblasts. This hypothesis was tested through two aims: 1) Assess the pharmacological properties of the histamine-3 receptor as a G-protein coupled receptor; and 2) Determine the extent to which histamine-3 receptor activation inhibits cardiac fibrosis and diastolic dysfunction through direct actions on cardiac fibroblast function. In a series of pharmacological experiments, activation of the histamine-3 receptor induced canonical Gαi/o G-protein subunit dissociation and subsequent inhibition of cAMP accumulation in HRH3-transfected HEK293T cells, as well as induced β-arrestin recruitment in HRH3-transfected HTLA cells. Furthermore, synthetic histamine-3 receptor agonists (imetit dihydrobromide, immepip dihydrobromide, and (R)-α-Methylhistamine dihydrobromide) were more potent than histamine at inducing Gαi/o G-protein subunit dissociation, inhibiting cAMP accumulation, and inducing β-arrestin recruitment. In a mouse model of adverse cardiac remodeling, angiotensin II infusion (1500 ng/kg) for 7 days led to mild diastolic dysfunction, which was inhibited by exogenous histamine-3 receptor activation with imetit dihydrobromide (40 mg/kg/day subcutaneous injection). At the cardiac structural level, angiotensin II infusion induced both cardiac fibrosis and cardiomyocyte hypertrophy, which were inhibited by treatment with imetit dihydrobromide. After confirming both the anti-fibrotic effects of histamine-3 receptor activation and the presence of the histamine-3 receptor on cardiac fibroblasts in the mouse left ventricle and on primary mouse cardiac fibroblasts in vitro, we then assessed the effects of histamine-3 receptor activation on cardiac fibroblast function using TGF-β1 (30 ng/mL) as a pro-fibrotic stimulus. Upon TGF-β1 treatment, extracellular collagen I was significantly increased, and histamine-3 receptor activation with imetit dihydrobromide (300 nM) inhibited the release of collagen I. Furthermore, TGF-β1 treatment led to a decrease in cardiac fibroblast migration, which was significantly increased following treatment with imetit dihydrobromide. Upon further examination, the inhibition of extracellular collagen by imetit dihydrobromide was not through inhibition of collagen gene transcription. Instead, cardiac fibroblast treatment with imetit dihydrobromide led to retention of collagen I within the cardiac fibroblasts. Next, we assessed the potential mechanisms by which this collagen I retention occurred. Despite the pharmacologic studies indicating that histamine-3 receptor activation induced Gαi/o G-protein subunit dissociation and inhibited cAMP accumulation, studies examining inhibition of both PKA using cAMPs-RP (10 µM) and EPAC using ESI-09 (3.5 µM) suggested this was not the mechanism by which histamine 3-receptor activation was exerting effects on cardiac fibroblast function.  We then reasoned that if collagen is being retained in the cardiac fibroblasts, histamine-3 receptor activation must be interfering with the export of collagen. As such, we found that imetit dihydrobromide treatment led to a reduction in HSP-47, a key endoplasmic reticulum-resident collagen-specific molecular chaperone involved in proper collagen assembly and release. Furthermore, imetit dihydrobromide inhibited phosphorylation of P38 MAPK, and subsequent examination of P38 inhibition with SB203580 (10 µM) revealed significant retention of intracellular collagen in cardiac fibroblasts and a reduction in collagen I release, consistent with our findings with imetit dihydrobromide treatment. Finally, we examined whether the inhibition of P38 phosphorylation downregulated HSP-47, or whether the reduction of HSP-47 inhibited P38 phosphorylation. Through examination of HSP-47 following inhibition of P38, we determined that it was not inhibition of P38 MAPK regulating HSP-47, suggesting it could be inhibition of HSP-47 leading to subsequent inhibition of P38 MAPK phosphorylation. Taken together, the results from this dissertation work demonstrate that histamine-3 receptor activation is protective against cardiac fibrosis, at least in part through direct actions on cardiac fibroblasts by inhibiting collagen I release through a novel mechanism involving downregulation of HSP-47 and subsequent inhibition of P38 MAPK phosphorylation. However, further studies are needed to determine the viability of histamine-3 receptor activation as a potential treatment for adverse cardiac remodeling in humans.

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