Introduction. Mitochondrial energy production plays an important role in maintenance of heart function. If it is disturbed, this can lead to heart failure. ATP is the universal form of energy and it is mainly produced at the ATP synthase in mitochondria. To produce ATP, the ATPase relies on a polarized mitochondrial membrane potential (ΔΨ_m), which is also essential for cellular viability. When under pathological conditions, ΔΨ_m dissipates, the ATPase can reverse its direction and consume ATP to maintain ΔΨ_m, but which may come at the cost of compromised cellular function due to ATP-depletion, which may in turn compromise physiological mitophagy. The ATPase inhibiting Factor 1 (ATPIF1) is a pH sensitive mitochondrial protein that is active when ΔΨ_m collapses. ATPIF1 specifically blocks the ATP hydrolysis and is upregulated in human heart failure (HF) and mouse models of HF. Here, we elucidate the role of ATPIF1 in healthy mice under physiological conditions.

Methods and results. Cardiac ventricular myocytes were isolated from ATP IF1-deficient mice (ATPIF1-KO) and WT mice at an age of 13.0+/-0.7 and 28.8+/-0.3 weeks. We determined sarcomere length, cytosolic Ca²⁺ (Indo1,AM) and mitochondrial redox state (autofluorescence of NAD(P)H and FAD), membrane potential (TMRM) and ROS (DCF) in myocytes employing an Ionoptix fluorescence setup. Isolated cardiac myocytes were exposed to a physiological stress protocol by pacing at 0.5 Hz, followed by β-adrenergic stimulation and increased stimulation rate at 5 Hz for 3 minutes. At the age of 13 weeks, [Ca²⁺]_i and Ca²⁺-transient amplitudes were slightly increased in ATPIF1-KO (n=66) vs. CO (n=48), while diastolic and systolic sarcomere length remained unchanged, and fractional sarcomere shortening slightly increased (n=39/49 WT/KO). Mitochondrial redox state (n=39/49 CO/IF1) was unaltered, but the mitochondrial membrane potential (n=38/67 CO/IF1) appeared more stable in ATPIF1-KO vs. CO myocytes during the stress protocol. Furthermore, ROS production was significantly reduced in the ATPIF1-KO (n=38) vs. CO myocytes (n=64). Interestingly, ATPIF1-KO myocytes showed less extra beats after the high pacing phase than CO myocytes. To evaluate whether ATPIF1-KO has a stronger effect in aged mice, we determined mitochondrial redox state and sarcomere length again at an age of 29 weeks. Here, the mitochondrial redox state was also unchanged, but diastolic sarcomere length was significantly extended, while fractional shortening was unaffected. Interestingly, SERCA function, reported by the time to 50% relaxation, was slightly improved in ATPIF1-KO myocytes, which may explain improved diastolic sarcomere function (and length). At this age, we did not observe differences in extra beats.