Optimized protocols for commonly-used murine models of heart failure with preserved ejection fraction

Background HFpEF is a leading cause of death worldwide and clinically relevant preclinical models are required to identify new therapeutic targets. The most clinically representative murine models of heart failure with preserved ejection fraction (HFpEF) in common use include a “2-hit” model combining metabolic stress with hypertension (high-fat diet [HFD] + N(gamma)-nitro-L-arginine methyl ester [L-NAME]) and a “3-hit” model that includes age as an additional “hit” (age + HFD + deoxycorticosterone pivalate [DOCP]). However, both models have reproducibility challenges, and sub-strain and sex dependency. Here we optimize both preclinical models to overcome these challenges. Methods In this study we optimized both models: (1) The 2-hit model was optimised to reproduce HFpEF (defined as the induction and maintenance of obesity, hypertension, diastolic dysfunction, left ventricular hypertrophy, lung congestion, and exercise intolerance) in both C57BL/6N and 6J mice using increasing L-NAME doses (0.5 g/L to 1.75 g/L) and protocol lengths (7 weeks to 13 weeks); and (2) The 3-hit model used 12-week-old C57BL/6N and 6J mice and two aging protocols were compared: HFD for 7 months, or healthy chow for 5 months then high fat diet for 7 months. After HFD, mice received an intraperitoneal injection of DOCP to induce hypertension via sodium retention. To enhance and prolong the effect of DOCP, mice received 1% NaCl drinking water at the time of injection until sacrifice, henceforth called “4-hit”. To ensure the phenotype was maintained, a second bolus of DOCP was administered 8 weeks after the first. Results For the 2-hit protocol, HFpEF was successfully induced in C57BL/6J mice when exposed to a 13-week L-NAME protocol with gradually increasing dosage from 1.0 g/L to 1.75 g/L. C57BL/6N mice showed the desired parameters after 7-weeks of 0.5 g/L L-NAME, which were not augmented by increased dosage or time administered. For the 4-hit mice, after addition of 1% NaCl drinking water following DOCP administration, a clear HFpEF phenotype was observed in C57BL/6N and 6J mice in both male and females, and maintained for up to 12 weeks. Conclusions Our modifications ensure the 2-hit model is equally effective in both commonly used J and N substrains of C57BL/6 mice. Our 4-hit model overcomes the challenges of the 3-hit model, enhances reproducibility and robustness, which we demonstrate across sexes and substrains. Both of these new protocols will enhance clinically relevant mechanistic studies on HFpEF.