Cardiovascular-specific PTH1R activation enhances exercise-induced cardiac remodeling in a mouse model of calcific atherosclerosis

Abstract

Exercise reduces cardiovascular events, yet its interactions with parathyroid hormone (PTH) and atherosclerotic calcification as well as their combined effects on cardiac remodeling remain unclear. Thus, we tested effects of constitutive activation of the PTH type I receptor (PTH1R) with and without exercise on aortic calcification and cardiovascular remodeling in a mouse model of atherosclerotic calcification.

Female Ldlr^{−/−;Tagln-PTH1R} transgenic (Tg) and Ldlr^−/− littermate control (Ctrl) mice were fed a Western diet beginning at 9–10 weeks of age. At age 40 weeks, mice were assigned to sedentary (SED) or treadmill exercise (TM) regimens for 9 weeks. MicroPET/CT and echocardiography were performed at baseline and study completion.

By microCT, aortic calcium content increased in all four groups. However, ¹⁸F-NaF uptake, reflecting mineral surface area, increased only in Ctrl/TM mice. Systolic function improved and left ventricular (LV) mass decreased in Tg/TM and Ctrl/TM groups. However, in Ctrl/TM mice, LV chamber diameter increased while anterior wall thickness decreased, consistent with eccentric remodeling. In contrast, in Tg/TM mice, diastolic wall thickness decreased without chamber enlargement, indicating preserved geometry and enhanced contractile efficiency. In sedentary mice (Tg/SED and Ctrl/SED), diastolic LV diameter increased without changes in wall thickness or systolic function, consistent with ventricular remodeling due to aortic stiffening in hyperlipidemia.

These findings suggest that vascular PTH1R signaling selectively modulates cardiovascular adaptation to exercise. By preserving geometry and improving contractile efficiency, PTH1R activation augments the beneficial effects of exercise on adverse cardiac remodeling in atherosclerotic calcification, supporting endocrine and exercise contributions to ventricular–vascular coupling.

INTRODUCTION

Exercise is recommended to lower risk for cardiovascular events and mortality [1], yet intense exercise is associated with increased coronary artery calcification. Exercise induces transient release of parathyroid hormone (PTH), high levels of which are also associated with cardiovascular calcification.

PTH type 1 receptor (PTH1R) is expressed throughout the cardiovascular system during development, including smooth muscle cells (SMC) and cardiomyocytes [2,3]. Notably, PTH1R signaling in vascular smooth muscle has emerged as a modulator of arterial structure and compliance, potentially influencing both calcific remodeling and the heart’s response to mechanical stress [4,5]. Consistent with this, we previously demonstrated that intermittent elevation of PTH levels via daily administration of PTH(1–34) or treadmill exercise, favorably altered the morphology of aortic calcium mineral deposits, specifically reducing mineral surface area and dynamic calcific activity, evidenced by ¹⁸F-NaF microPET/microCT imaging [6,7].

Clinical studies have shown that high risk plaques tend to have clusters of multiple, small (1–3 mm) calcium deposits [8], which have more surface area than contiguous calcification. Theoretical analysis suggests that the risk of plaque rupture and intramural hemorrhage, which lead to cardiovascular events, increases with the surface area of calcium deposits due to compliance mismatch and debonding [9]. Given the known adsorption and covalent binding of fluoride to hydroxyapatite mineral [10], the surface area of calcium deposits can now be quantified using PET imaging with ¹⁸F-NaF radiotracer, as demonstrated by Irkle and colleagues using calcified human carotid atherosclerotic plaques [11]. Thus, ¹⁸F-NaF tracer uptake may be a more relevant biomarker of plaque rupture risk than total calcium content, such as by Agatston calcium scores. Together, these observations suggest that vascular PTH1R may serve as a key regulator of cardiovascular adaptation to mechanical and metabolic stress, providing a mechanistic link between vascular remodeling, arterial compliance, and cardiac functional reserve.

In this study, to investigate the effects of vascular PTH1R activation on cardiovascular function, we used hyperlipidemic mice with genetic activation of PTH1R specifically in vascular smooth muscle (Ldlr^−/−;^Tagln-PTH1R). Hyperlipidemia is associated with both cardiovascular calcification and increased systemic vascular resistance, which causes increased cardiac afterload, also resulting in adverse cardiac remodeling such as left ventricular hypertrophy. Exercise tends to reduce systemic vascular resistance, thus potentially mitigating the adverse cardiac remodeling. The net effects of these interacting factors are not well established.

MATERIALS AND METHODS

Mice

All procedures were approved by the Institutional Animal Care and Use Committee at the University of California, Los Angeles, and conform to NIH guidelines for animal research (ARC-2006–052, valid through September 2026). Studies are reported in accordance with ARRIVE guidelines. Female Ldlr^−/−;^Tagln-PTH1R (Tagln-PTH1R) hemizygous mice[4] (Jackson Laboratory, stock #022488) and non-carrier Ldlr^−/− littermate controls on a C57BL/6 background were obtained from the Jackson Laboratory (Bar Harbor, ME). The SM22α (Tagln) promoter drives vascular smooth muscle–specific expression of constitutively active PTH1R. Female mice were used to maintain consistency with prior studies in this model and to reduce variability associated with sex-dependent differences in calcification progression.

To induce vascular calcification, mice were placed on a Western diet (TD88137; 21% fat, 0.2% cholesterol; Inotiv) beginning at 9–11 weeks of age. At ~40 weeks of age, mice underwent baseline microPET/CT imaging and echocardiography. To minimize confounding effects of prolonged Western diet exposure, all mice were transitioned to standard chow (#7013, Inotiv) at the initiation of the intervention (Week 0). Sample size was determined a priori based on prior studies using this model demonstrating similar effect sizes [7,12]. Mice were then stratified by genotype and baseline CT-derived aortic calcium burden and assigned to sedentary (SED) or treadmill exercise (TM) groups, ensuring equivalent baseline calcification across groups [n = 13 (Ctrl/SED), 14 (Ctrl/TM), 13 (Tg/SED), 15 (Tg/TM)].

Treadmill exercise protocol

Mice were acclimated to a motorized treadmill (Columbus Instruments Exer-3/6, 6-lane). A single 10-minute run at 12.5 m/min was performed, and the animals unable to tolerate exercise were excluded from the study. The exercise regimen consisted of treadmill running at 12.5 m/min, 30 minutes/session, 5 days/week, 0° incline, without electric shock stimulation, for 9 weeks. “Sedentary” mice were not subjected to the treadmill exercise and were maintained under standard housing conditions with unrestricted cage activity.

microPET/CT imaging and quantification

Fused ¹⁸F-NaF microPET/microCT imaging was performed at week 0 (40 w.o.) and 9 (49 w.o.) at the Crump Preclinical Imaging Facility (California NanoSystems Institute, UCLA). Acquisition and analysis protocols were performed as previously described (11,17). Aortic calcification was quantified by CT using a ≥ 250 Hounsfield Unit (HU) threshold. PET tracer uptake was expressed as percent injected dose per cubic centimeter (%ID/cc). Total CT calcium burden and total PET uptake were calculated as mean density × volume. Lumbar bone mineral density was assessed by geometric region-of-interest (ROI) analysis of the third lumbar vertebra (L3) using a threshold of 1000 HU. Mean HU values were used for comparison.

Echocardiography

Echocardiography was performed at week 0 (40 w.o.) and 9 (49 w.o), using a VisualSonics Vevo 3100 system with a 30-MHz linear-array transducer. Mice were anesthetized with isoflurane (3.0% induction, 1.5–2.0% maintenance via nose cone). M-mode and tissue Doppler images were obtained at baseline (Week 0) and Week 9. Standard measurements included left ventricular (LV) end-diastolic and end-systolic diameters, anterior and posterior wall thickness, LV mass, and ejection fraction. Analyses were performed offline by investigators blinded to group allocation.

Statistical analysis

Data are presented as mean ± SD. Normality of data distribution was assessed using the Shapiro–Wilk test. For echocardiographic parameters demonstrating normal distribution, two-way ANOVA was performed with genotype and treatment (sedentary vs. treadmill) as fixed factors, followed by Sidak post hoc testing for multiple comparisons (GraphPad Prism v9.4.1, GraphPad Software). For PET/CT outcomes, a three-way repeated-measures ANOVA was applied with genotype and treatment as between-subject factors and time (pre- vs. post-intervention) as the within-subject repeated factor. When significant main effects or interactions were detected, Sidak-adjusted pairwise comparisons were performed. All tests were two-sided, and p ≤ 0.05 was considered statistically significant.

RESULTS

Effects of PTH1R signaling on morphology and total content of aortic calcium deposits

Over the 9-week exercise period (Figure 1A), ¹⁸F-NaF PET uptake in the non-exercised Ldlr^−/− mice (either Tg/SED or Ctrl/SED) was not significantly increased (p = 0.9 and 0.2, respectively, Fig. 1B). In non-transgenic mice undergoing treadmill exercise (Ctrl/TM), ¹⁸F-NaF PET tracer uptake increased significantly over the 9-week-study period, indicating increased mineral surface area Fig. 1B). In contrast, in the PTH1R transgenic mice undergoing treadmill exercise (Tg/TM), tracer uptake was not significantly increased (p = 0.06, Fig. 1B), suggesting that vascular PTH1R activation attenuates exercise-associated increases in radiolabeled mineral surface area, indicating morphologic changes of increased porosity or new mineral foci.

Figure 1. Effects of PTH1R signaling on morphology and total content of aortic calcium deposits.

(A) Schematic diagram of experimental design. (B) Aortic ¹⁸F-NaF tracer uptake, assessed by microPET imaging. White arrow indicates PET signal. (C) Total calcium content, assessed by microCT imaging. N = 13 (Ctrl/SED), 14 (Ctrl/TM), 13 (Tg/SED), 15 (Tg/TM).

Despite these differences in tracer uptake, total CT-detectable aortic calcium burden increased significantly in all four groups (Fig. 1C), independent of genotype or exercise. These findings indicate progressive calcification over time, while differential regulation of morphology by PTH1R signaling during exercise.

Effects of PTH1R signaling on cardiac structure and function

As shown in Fig. 2, both sedentary groups (Ldlr^−/− and Ldlr^−/−;^Tagln-PTH1R) exhibited an increase in diastolic LV chamber diameter over the 9-week period without significant changes in LV wall thickness, mass, or systolic function, consistent with mild chamber dilation in the absence of exercise.

Figure 2. Effects PTH1R signaling on cardiac structure and function.

Cardiac structure and function were assessed by echocardiography. (A) Ejection fraction; (B) fractional shortening; (C) LV chamber diameter in systole (left) and in diastole (right); (D) LV anterior wall thickness in systole (left) and in diastole (right); (E) LV posterior wall thickness in systole (left) and in diastole (right); and (F) LV mass. N = 13 (Ctrl/SED), 14 (Ctrl/TM), 13 (Tg/SED), 15 (Tg/TM).

In exercised control Ldlr^−/− mice, ejection fraction, fractional shortening, and diastolic chamber diameter increased significantly over the 9-week exercise period, while LV anterior wall thickness and mass decreased significantly. Together, these changes suggest improved systolic performance, attenuation of hypertrophic remodeling, and a pattern consistent with chamber enlargement during exercise adaptation.

In contrast, in exercised transgenic (Ldlr^−/−;^Tagln-PTH1R) mice, ejection fraction and fractional shortening increased and LV mass decreased over the 9-week exercise period. However, unlike non-transgenics, chamber diameter did not increase. Instead, exercise was associated with reduced posterior wall thickness with preserved overall LV geometry. These findings suggest improved systolic function and prevention of hypertrophy without exercise-induced chamber dilation in the presence of vascular PTH1R activation.

Effects of PTH1R signaling on lumbar bone mineral density

Because skeletal bone mineral density is influenced by hyperlipidemia, exercise, and PTH signaling [13–17], we also assessed bone mineral density at the lumbar L3 site. As shown in Fig. 3, control (non-transgenic) mice exhibited bone loss in response to exercise, whereas transgenic mice showed significant bone loss irrespective of exercise.

Figure 3. Effects PTH1R signaling on lumbar bone mineral density.

(A) Representative CT images of lumbar vertebrae (L2-L4). (B) Mean lumbar bone mineral density at L3, assessed by microCT imaging. N = 13 (Ctrl/SED), 14 (Ctrl/TM), 13 (Tg/SED), 15 (Tg/TM).

DISCUSSION

The present study suggests cardiovascular PTH1R signaling modulates the relationship between aortic calcification and cardiac remodeling in female Ldlr^−/− mice with established calcific atherosclerosis. Over the 9-week-study period, calcification morphology, cardiac function, and most of the structural parameters remained unchanged under sedentary conditions but were selectively altered by exercise.

Although all groups demonstrated progression of CT-detectable aortic calcification, ¹⁸F-NaF tracer uptake increased significantly only in exercised non-transgenic mice, indicating the formation of new microcalcifications or mineral remodeling in this group. In contrast, exercised PTH1R transgenic mice did not show a significant increase in tracer uptake, despite a strong trend (p = 0.06). This difference may reflect PTH1R-mediated reduction in mineral surface area through calcification coalescence [7] and/or attenuation of exercise-induced new microcalcifications. This dissociation between calcium burden and tracer uptake in the exercised groups suggests that exercise reveals active mineral formation in the absence of enhanced PTH1R signaling, which appears to restrain stress-induced increases in mineral surface area, despite comparable overall calcification burden.

Importantly, these vascular differences were paralleled by distinct patterns of cardiac remodeling. In sedentary mice (Ldlr^−/− and Ldlr^−/−;^Tagln-PTH1R), isolated LV chamber dilation occurred in the setting of increased calcification but without evidence of significant change in mineral surface area by PET, suggesting that established calcific burden may be sufficient to increase arterial stiffness and impair ventricular–vascular coupling. Chronic elevation in effective arterial elastance would increase wall stress and promote eccentric remodeling despite preserved systolic function. The absence of compensatory hypertrophy further implies maladaptive load sensing or altered mechano-transduction, potentially downstream of constitutive PTH1R signaling in cardiac and smooth muscle cells.

Strikingly, treadmill exercise completely prevented LV dilation in the PTH1R transgenic mice while reducing LV mass and enhancing systolic performance, despite the persistence of progression in CT-detectable calcification. This cardiac remodeling is consistent with changes seen in human subjects with hypertension following endurance exercise regimens [18,19]. The dissociation between structural calcification and ventricular geometry in these mice suggests that cardiovascular PTH1R mitigates exercise-induced functional consequences of vascular mineralization. These findings suggest that calcification burden alone does not determine ventricular remodeling; rather, its hemodynamic impact depends on the morphologic changes of mineral deposits in the arterial wall.

In contrast, exercised Ldlr^−/− controls lacking the PTH1R transgene demonstrated both increased chamber diameter and elevated ¹⁸F-NaF tracer uptake over the study period, consistent with active mineral remodeling and increased mineral surface area. The concomitant, eccentric cardiac adaptation in this group may therefore reflect a combined effect of exercise-induced preload augmentation and ongoing calcific stiffening of the aorta. Thus, in the absence of constitutive PTH1R signaling, exercise appears to promote physiological chamber enlargement rather than geometric preservation.

Taken together, these findings support a unifying model in which PTH1R activation in cardiovascular cells reshapes the interaction between vascular calcification and cardiac structure. CT-detectable calcification was present across all groups, but exercise promoted evidence of active mineral propagation only in the control Ldlr^−/− mice. The presence of actively growing mineral surface area, rather than total calcium burden alone, appears more closely associated with eccentric ventricular adaptation. Meanwhile, constitutive PTH1R signaling predisposes to dilation under sedentary conditions yet enables exercise-mediated normalization of geometry despite persistent calcification. These data highlight ventricular–vascular coupling as the central mechanistic axis and underscore the importance of distinguishing calcification burden from calcification activity when interpreting cardiac remodeling. Collectively, our results position vascular smooth muscle signaling and dynamic mineral turnover—not simply calcium accumulation—as key determinants of age-associated cardiac structural adaptation in hyperlipidemic females.

In normolipemic mice, exercise transiently increases serum PTH levels and has been shown to enhance skeletal bone mineral density [17]. However, in hyperlipidemic mice, bone loss following treadmill exercise was consistently observed [7,12,20]. In the present study, PTH1R transgenic mice exhibited bone loss even under sedentary conditions over the 9-week study period. Together, these findings support the notion that deleterious skeletal effects of exercise are context-dependent and influenced by hyperlipidemia, potentially due to highly vascularized nature of bone, which may increase its susceptibility to lipid-mediated injury.

Several limitations should be considered when interpreting these findings. Sex-specific effects of vascular PTH1R activation cannot be excluded. The imaging modalities used here did not directly measure arterial stiffness or pulse wave velocity. Future studies incorporating direct measurement of arterial compliance, pressure-volume relationships, and sex-based comparisons will further clarify the potential beneficial effects of vascular PTH1R signaling on cardiovasculopathy.

Highlights.

• Exercise-induced vascular mineral remodeling depends on PTH1R activation: ¹⁸F-NaF uptake increased only in exercised non-transgenic mice despite similar progression of atherosclerotic calcification across all groups.

• Vascular PTH1R activation preserves cardiac geometry under exercise stress: In PTH1R-transgenic mice, exercise reduced diastolic LV wall thickness without chamber dilation, suggesting improved contractile efficiency.

• Sedentary mice exhibit early stiffness-driven remodeling: Mild diastolic LV chamber enlargement occurred without changes in wall thickness or systolic function, independent of PTH1R signaling phenotype.

• Mechanistic implication: Vascular PTH1R is a context-dependent regulator of cardiovascular adaptation, enhancing ventricular–vascular coupling and attenuating stiffness-associated cardiac remodeling under mechanical stress.

Data availability statement

The data that support the findings of this study are available from the corresponding author, [YT], upon a reasonable request.