*3.1. Elevated Levels of Circulating FGF23 Are Associated with Mild Hypertension in Aged Hyp Mice but Do Not Cause LVH*

Several earlier reports have failed to find LV hypertrophy in *Hyp* mice of up to nine months of age [17,18]. However, data about potential changes in cardiovascular function in aged *Hyp* mice as a model of long term FGF23 excess are scarce. Therefore, we examined the cardiovascular phenotype of aged *Hyp* mice by intraarterial and left ventricular catheterization as well as by echocardiography. The HW/BW ratio was distinctly increased in *Hyp* mice relative to WT controls (Figure 2A). However, this increase was mainly driven by lower body weight in *Hyp* mice, and not by a higher heart mass (Figure 2B,C), questioning the relevance of the HW/BW ratio as a read-out for heart hypertrophy in this animal model. Notably, intraarterial catheterization revealed a higher mean arterial pressure (MAP) of about 12 mmHg in aged *Hyp* mice compared to WT controls (Figure 2D). Despite the presence of hypertension, LV function as evidenced by fractional shortening and ejection fraction measured by echocardiography was actually improved in *Hyp* compared with WT mice (Figure 2E,F). In agreement with the echocardiography data, LV contractility as measured by Max dP/dt during LV catheterization was unchanged in 14-month-old *Hyp* mice (Figure 2G). Moreover, there was no difference in mean cardiomyocyte size as measured by wheat germ agglutinin (WGA) staining or in LV collagen content as measured by picrosirius red staining between WT and *Hyp* mice (Figure 2H,I). The absence of LV hypertrophy in *Hyp* mice was further confirmed by qRT-PCR analysis of typical markers of hypertrophy, such as *Anp* and *Bnp,* which remained unaltered between the genotypes (Figure 2J,K). Taken together, these data confirm the presence of mild hypertension in aged *Hyp* mice but strongly argue against LV hypertrophy and functional impairment in these mice.

To further address the question of what drives hypertension in aged *Hyp* mice, we measured the augmentation index in the carotid artery by pulse wave analysis. The latter analysis revealed an increase in augmentation index in aged *Hyp* mice relative to WT controls (Figure 2L). This finding may point to increased peripheral vascular resistance in aged *Hyp* mice. In combination with the increase in aldosterone levels found in *Hyp* mice (Figure 1E), we hypothesized that hypertension in aged *Hyp* mice may be due to crosstalk between FGF23 and RAAS signaling in the kidney and blood vessels, leading to a combination of volume overload through increased aldosterone and FGF23 secretion and increased peripheral vascular resistance by elevated angiotensin II levels.

**Figure 2.** *Hyp* **mice are hypertensive but do not develop LV hypertrophy.** (**A**) Heart/body weight ratio (HW/BW), (**B**) body weight (BW), and (**C**) heart weight (HW) (n = 4–14) in 14-month-old male WT and *Hyp* mice. (**D**) Mean arterial pressure measured by arterial catheterization (n = 12), (**E**,**F**) fractional shortening (FS) and ejection fraction (EF) measured by echocardiography (n = 9–11), and (**G**) LV contractility (Max dP/dt) measured by intracardiac catheterization (n = 12) in 14-monthold male WT and *Hyp* mice. (**H**) Quantification of mean cardiomyocyte size after FITC-WGA staining (n = 6), (**I**) left, representative images of collagen staining using picrosirius red (PSR) in cardiac paraffin sections (bar = 100 μm), right, quantification of fibrosis as measured by PSR-stained area (n = 9–12), (**J**,**K**) relative mRNA expression of markers of hypertrophy, (**J**) atrial natriuretic peptide (Anp) and (**K**) brain natriuretic peptide (Bnp) (n = 5), and (**L**) augmentation index (AI) measured by pulse wave analysis (n = 19) in 14-month-old male WT and *Hyp* mice. Bars in (**A**–**L**) represent mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 by Student's *t*-test. ns, not significant.
