Increased myocardial fibrosis and impaired LV-contractility in a model of uremic cardiomyopathy leads to cardiac mTOR pathway activation with increased ER-stress, inflammation and apoptosis

Aim: Patients with chronic kidney disease (CKD) have an increased risk to develop cardiovascular disease including heart failure but the underlying mechanisms are still incompletely understood. In this study, we used the model of 2,8-dihydroxyadenine (DHA)-induced nephropathy in different mouse stains and with or without transverse aortic constriction (TAC) surgery as an additional hypertrophic and pro-fibrotic stimulus to establish a robust mouse model of uremic cardiomyopathy.

Methods and Results: We first used C57BL/6N mice and induced uremic conditions by feeding an adenine-supplemented diet. Then we performed TAC surgery to induce cardiac hypertrophy and fibrosis (model 1). Then, we used C57BL/6N and 129/Sv mice to induce cardiac hypertrophy by TAC surgery and induced uremia afterwards by adenine supplementation (model 2). In both models, uremic conditions could be detected but we observed no differences in cardiac left ventricular fibrosis and function. Next, we only investigated consequences of uremia in 4 different mouse strains (C57BL/6J, C57BL/6N, 129/Sv and FVB(N) mice) by feeding an adenine-supplemented diet or control diet for a period of 8 weeks (model 3). Expected uremia (C57BL/6J and C57BL/6N: p<0.0001, 129/Sv: p<0.05 and FVB(N): p<0.01), crystal formation (C57BL/6J and 129/Sv: p<0.001, C57BL/6N: p<0.0001 and FVB(N): p<0.05) with tubulointerstitial injury (C57BL/6J, C57BL/6N and 129/Sv: p<0.0001 and FVB(N): p<0.001) and renal collagen deposition (C57BL/6J and FVB(N): p<0.01, C57BL/6N: p<0.0001 and 129/Sv: p=0.05) were found in all strains. Furthermore, renal mitochondrial function (C57BL/6J, C57BL/6N and 129/Sv: p<0.01) was impaired in all strains excluded FVB(N) mice while systolic blood pressure was increased only in C57BL/6J, C57BL/6N mice (after 8 weeks: C57BL/6J and C57BL/6N p<0.01). Analyzing the cardiac phenotype we found uremia to increase cardiac fibrosis only in 129/Sv but not in any of the other strains. No difference in cardiomyocyte hypertrophy, myocardial mitochondrial function nor in left ventricular function in response to uremic conditions was observed. To confirm these results and establish a more robust model of uremic cardiomyopathy, we next extended the time of uremic exposure of 129/Sv mice to 16 weeks. This again caused the expected renal damage with collagen deposition, crystal formation (both p<0.0001) and uremia (p<0.01) and led to a significant increase of myocardial fibrosis (p<0.05) in conjunction with impaired left ventricular contractility under dobutamine stress conditions (p<0.001). Mechanistic, we found significant activation of the mTOR pathway (indicated by its downstream target p70S6K-phosphorylation at Thr389 (p<0.001)) together with downstream endoplasmic reticulum stress (by eIF2α-phosphorylation at Ser51 (p<0.001), ATF4 and CHOP (both p<0.05)), increased apoptosis (via caspase 3 expression (p<0.05)), autophagy (via LC3A/B expression (p<0.001)) and inflammation (via IL-1β expression (p<0.05)).

Conclusions: Our data suggest that 129/Sv mice are the most stable and reproducible mouse model of uremic cardiomyopathy using 2,8-DHA-induced nephropathy for 16 weeks. This resulted in increased myocardial fibrosis, impaired left ventricular function, activation of the mTOR pathway, increased endoplasmic reticulum stress leading to apoptosis, inflammatory signaling and fibrosis.