Impairment of the adrenergic reserve associated with exercise intolerance in a murine model of heart failure with preserved ejection fraction

This project is funded by grants from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation—SFB 1470—A01 to FRH and PA and A02 to GGS) and from the DZHK (German Centre for Cardiovascular Research to GGS). CUO is additionally funded by the DFG (OE 688/4-1).Aim Exercise intolerance is the central symptom in patients with heart failure with preserved ejection fraction. In the present study, we investigated the adrenergic reserve both in vivo and in cardiomyocytes of a murine cardiometabolic HFpEF model. Methods 12-week-old male C57BL/6J mice were fed regular chow (control) or a high-fat diet and L-NAME (HFpEF) for 15 weeks. At 27 weeks, we performed (stress) echocardiography and exercise testing and measured the adrenergic reserve and its modulation by nitric oxide and reactive oxygen species in left ventricular cardiomyocytes. Results HFpEF mice (preserved left ventricular ejection fraction, increased E/e', pulmonary congestion [wet lung weight/TL]) exhibited reduced exercise capacity and a reduction of stroke volume and cardiac output with adrenergic stress. In ventricular cardiomyocytes isolated from HFpEF mice, sarcomere shortening had a higher amplitude and faster relaxation compared to control animals. Increased shortening was caused by a shift of myofilament calcium sensitivity. With addition of isoproterenol, there were no differences in sarcomere function between HFpEF and control mice. This resulted in a reduced inotropic and lusitropic reserve in HFpEF cardiomyocytes. Preincubation with inhibitors of nitric oxide synthases or glutathione partially restored the adrenergic reserve in cardiomyocytes in HFpEF. Conclusion In this murine HFpEF model, the cardiac output reserve on adrenergic stimulation is impaired. In ventricular cardiomyocytes, we found a congruent loss of the adrenergic inotropic and lusitropic reserve. This was caused by increased contractility and faster relaxation at rest, partially mediated by nitro-oxidative signaling.Peer reviewe

Klotho expression is a prerequisite for proper muscle stem cell function and regeneration of skeletal muscle

Abstract Background Klotho is a well-known anti-aging hormone, which serves as a suppressor of aging through a variety of mechanisms. Aging of skeletal muscle is concomitant with a decrease in muscle stem cell function resulting in impaired regeneration. Methods Here we investigate the functional role of the anti-aging hormone Klotho for muscle stem cell function after cardiotoxin-induced injury of skeletal muscle using a klotho hypomorphic mouse line, which is characterized by a premature aging phenotype. Furthermore, we perform floating single myofiber cultures with their adjacent muscle stem cells to investigate the interplay between canonical Wnt signaling and Klotho function. Results We demonstrate that muscle stem cell numbers are significantly decreased in klotho hypomorphic mice. Furthermore, we show that muscle stem cell function is also severely impaired upon loss of klotho expression, in culture and during regeneration in vivo. Moreover, we demonstrate that addition of recombinant Klotho protein inhibits aberrant excessive Wnt signaling in aged muscle stem cells thereby restoring their functionality. Conclusions The anti-aging hormone Klotho counteracts aberrant canonical Wnt signaling in muscle stem cells and might be one of the naturally occurring inhibitors of canonical Wnt signaling in skeletal muscle

Additional file 1: of Klotho expression is a prerequisite for proper muscle stem cell function and regeneration of skeletal muscle

Figure S1. ΔKlotho mice display signs of sarcopenia. (A) Immunofluorescence of cross-sections of TA muscles from ΔKlotho and control mice stained for DAPI (DNA, blue) and Laminin (green) at p14, p21, and p56. Scale bar = 50 μm. (B) Minimal fiber feret measured on the whole cross-sections from ΔKlotho and control TA muscles at p14, p21, and p56. (p14, p56 n ≥ 3 mice per genotype, p21 n = 3 mice per genotype). (C) Quantification of the cross-section area of the mid-belly region of TA muscles determined from sections from ΔKlotho and control at p14, p21, and p56. (p14, p56 n ≥ 3 mice per genotype, p21 n = 3 mice per genotype). (D) Immunoblot analyses of atrophy associated ubiquitin ligases in TA muscles from ΔKlotho and control littermates at different ages. (E) Fold expression of different ubiquitin ligases as shown in (D), values for ΔKlotho are normalized to control animal of the same age. (F) Muscle weight of tibialis anterior (TA) muscles from adult ΔKlotho (n = 6) and control mice, n = 8 (G) Muscle weight of tibialis anterior (TA) muscles normalized to the length of the tibia bone from adult ΔKlotho (n = 6) and control mice (n = 8). All data are presented as means ± SEM. *p < 0.05, **p < 0.01, *** p < 0.001. (PDF 39024 kb

Additional file 3: of Klotho expression is a prerequisite for proper muscle stem cell function and regeneration of skeletal muscle

Figure S3. The differentiation of myoblasts from ΔKlotho mice is not affected in vitro. (A) Quantification of the average number of nuclei per myotube counted on 6 random regions of interest per condition after 5 days of differentiation (ΔKlotho n = 3 mice, control n = 4 mice). (B) Percentage of myogenin-positive nuclei of all nuclei within myotubes after 5 days of differentiation (ΔKlotho n = 3 mice, control n = 4 mice). (C) Differentiation index (percentage of myotubes with more than three nuclei) after 5 days of differentiation (ΔKlotho n = 3 mice, control n = 4 mice). (D) Distribution of classes of myotubes after 5 days of differentiation (ΔKlotho n = 3 mice, control n = 4 mice). (E) Immunoblot analyses of supernatants from primary myoblasts and lysate from a kidney from wt animals using an antibody directed against klotho showing sKlotho in the supernatant and in whole kidney lysates (as expected). (F) Ponceau stained membrane showing similar loading of concentrated supernatants from primary myoblasts isolated from ΔKlotho and control mice. All data are presented as means ± SEM. (PDF 7378 kb

Impairment of the adrenergic reserve associated with exercise intolerance in a murine model of heart failure with preserved ejection fraction

AimExercise intolerance is the central symptom in patients with heart failure with preserved ejection fraction. In the present study, we investigated the adrenergic reserve both in vivo and in cardiomyocytes of a murine cardiometabolic HFpEF model.Methods12-week-old male C57BL/6J mice were fed regular chow (control) or a high-fat diet and L-NAME (HFpEF) for 15 weeks. At 27 weeks, we performed (stress) echocardiography and exercise testing and measured the adrenergic reserve and its modulation by nitric oxide and reactive oxygen species in left ventricular cardiomyocytes.ResultsHFpEF mice (preserved left ventricular ejection fraction, increased E/e', pulmonary congestion [wet lung weight/TL]) exhibited reduced exercise capacity and a reduction of stroke volume and cardiac output with adrenergic stress. In ventricular cardiomyocytes isolated from HFpEF mice, sarcomere shortening had a higher amplitude and faster relaxation compared to control animals. Increased shortening was caused by a shift of myofilament calcium sensitivity. With addition of isoproterenol, there were no differences in sarcomere function between HFpEF and control mice. This resulted in a reduced inotropic and lusitropic reserve in HFpEF cardiomyocytes. Preincubation with inhibitors of nitric oxide synthases or glutathione partially restored the adrenergic reserve in cardiomyocytes in HFpEF.ConclusionIn this murine HFpEF model, the cardiac output reserve on adrenergic stimulation is impaired. In ventricular cardiomyocytes, we found a congruent loss of the adrenergic inotropic and lusitropic reserve. This was caused by increased contractility and faster relaxation at rest, partially mediated by nitro-oxidative signaling