Soluble klotho improves calcium handling and reduces pro-inflammatory signaling in cultured failing human myocardial Slices

Abstract Background Soluble Klotho (sKlotho), a protein primarily produced by the kidney, is one of the key regulators of the heart–kidney axis. sKlotho acts as a co-receptor for fibroblast growth factor 23 (FGF23), and antagonizes the pro-fibrotic, pro-inflammatory and hypertrophic signaling of FGF23. (1). Its age-related decline is linked with increased cardiovascular risk. Purpose To examine the effects of sKlotho on myocardial function and cellular signaling in cultured myocardium from failing (human) and healthy (swine) hearts. Methods Left ventricular tissue was obtained from 8 patients with end-stage heart failure (HF) receiving cardiac transplantation and from 5 healthy swine. Myocardial slices were cultured in biomimetic cultivation chambers (2) and treated with sKlotho (3.4 mM) for two weeks. Contractile function was recorded, and individual contraction peaks were analyzed for force amplitude, contraction duration, time to peak/relaxation, and maximal slopes (+/−dF/dt). Contraction peak parameters were normalized to each slice’s baseline. Additionally, post pause potentiation, an index of calcium handling, was determined. The effect of sKlotho on gene expression profiles were assessed using NanoString analysis in samples from five patients and selected targets were validated by quantitative RT-PCR. Results During culture, human control slices showed worsening of contractile function as evidenced by a prolongation in the time to peak contraction, which was prevented by sKlotho (Fig. A). This was accompanied by enhanced post-pause potentiation, indicating improved calcium handling in sKlotho-treated human myocardial slices (Fig. C). Conversely, this beneficial effect on calcium handling was absent in healthy swine myocardium (Fig. B&D). NanoString analysis revealed differential regulation of pathways related to extracellular matrix (ECM) remodeling, oxidative stress, angiotensin signaling, and apoptosis (Fig. E). RT-PCR analysis confirmed sKlotho induced changes in key genes in ECM remodeling (lower MMP9 and higher α-SMA expression) as well as in angiotensin signaling (reduced expression of type 1 angiotensin receptor (AGTR1), a key mediator of cardiac hypertrophy and fibrosis (Fig. F). Conclusions Our findings show that Klotho exerts beneficial effects on contractile kinetics and calcium-handling dynamics in failing myocardium. These improvements in contractile performance were accompanied by transcriptional changes in pathways related to ECM remodeling, oxidative stress, apoptosis, and angiotensin signaling. Moreover, the downregulation of AGTR1 and coordinated alterations in extracellular matrix gene expression observed following Klotho treatment support a shift towards pro-reparative signaling in the myocardium. These results show that Klotho exerts a multitude of cardioprotective effects, and may therefore provide a therapeutic target to improve cardiac function in heart failure patients.For image description, please refer to the figure legend and surrounding text.