The single hypertrophic stiffer LV myocytes Figs A and
The single, hypertrophic, stiffer LV myocytes (Figs. 6A and 2D) unveiled functional modifications including stronger and faster contractions with delayed relaxation (Fig. 3C,D,H). This positive, cellular inotropy was determined primarily by an increase in Ca2+ transient amplitude rather than by an amplified Ca2+ sensitivity of the contractile proteins. At the cellular level, our findings of stiffer and hypertrophic LV myocytes were consistent with various reports on HFpEF in human and rodents [17,46,50,51,59,60] and explain in part the increased LV wall stiffness and impaired relaxation of the heart. The force developed by the myocytes depends on both the amount of Ca2+ released by the SR after excitation and the Ca2+ sensitivity of the contractile machinery. Here, single cell contractions were stronger with both a shorter delay for activation and a delayed relaxation, which was consistent with the increased Ca2+ transient amplitude and in line with other studies showing enhanced Ca2+ mobilization, particularly during early stages of pressure overload-induced hypertrophy [17,61]. Delayed Ca2+ extrusion from the cytosol due to impaired NCX activity (Fig. 3J,L) was also a likely contributor. Enhanced Ca2+ cycling between the SR and the cytosol was shown to occur even before hypertrophy development, i.e. during the very first days following AAB . Here, the positive inotropic-like adaptive effect occurred in absence of marked AP plateau prolongation, as confirmed by a lack of modification in the main repolarizing K+ currents (except IK1), which was consistent with unchanged L-type Ca2+ current. It also did not involve enhanced Ca2+ sensitivity of contractile proteins. It remains unclear why the augmented contraction of individual ITF2357 (Givinostat) did not increase global heart function. Possibilities may include a loss of cardiomyocytes, although this is mainly a HFrEF characteristic, or more likely a variety of non-cardiomyocyte factors as recently reviewed . Four weeks after AAB, Ca2+ cycling was characterized by at least four changes occurring in parallel with potentially opposite effects: (i) Ca2+ leakage through RyR2 (Fig. 4C); (ii) impaired Ca2+ extrusion through NCX (Fig. 3J,L); (iii) increased PLN/SERCA ratio (Fig. 5D), and, (iv) increased pPLN/PLN ratio (Fig. 5E), that may compensate the higher PLN/SERCA ratio to rescue SR Ca2+ re-uptake by SERCA2a. Of note, SERCA2a protein abundance was unchanged (Fig. 5A). In normal conditions, Ca2+ removal from the cytosol by the SERCA2a pump prevails over NCX activity. As the decline of the Ca2+ transient evoked by a caffeine challenge is an index of NCX activity, our results were consistent with an impaired Ca2+ extrusion from the cells through the NCX forward mode [62,63]. This was however unrelated to changes in NCX protein expression. Slower decay time of the caffeine-induced Ca2+ transient may reflect changes in Na+ gradient  due to elevated intracellular [Na+] involving possibly factors such as late Na+ currents, electrogenic Na+-K+ ATPase or Na+ co-transport. Impaired NCX-dependent Ca2+ extrusion has been explained by a shift towards an increased reverse mode activity (Ca2+ influx), yet this was excluded in a recent study in human hearts of patients with hypertensive heart disease and HFpEF [60,65]. Leaky RyR2 (late Ca2+ sparks)  associated with both impaired Ca2+ extrusion through NCX and increased PLN/SERCA ratio altogether could contribute to the delay in the decay of the Ca2+ transient (Fig. 3H) and elevate diastolic Ca2+ (Fig. 3I). PLN has a primary role in the regulation of SERCA2a activity and thereby is a major determinant of cardiac contractility and relaxation. Dephosphorylated PLN inhibits SERCA2a activity whereas PLN phosphorylation at either Ser16 by PKA or Thr17 by CaMKII reverses this inhibition . The PLN/SERCA2a ratio in AAB cardiomyocytes was increased, which is expected to inhibit SERCA2a activity and decrease SR Ca2+ reuptake as seen in pathophysiological conditions [68,69]. Remarkably, we detected an increased pPLN/PLN ratio in AAB hearts, i.e. PLN was phosphorylated at the CaMKII-specific site (PLN-Thr17). Activation of this CaMKII-specific site (PLN-Thr17) has complex effects. It has been closely associated with an increase in the relaxant effect of a β-adrenergic response  but also with reduced β-adrenergic responsiveness in a feline model of chronic pressure overload-induced cardiac hypertrophy . This mechanism may also act as a cardiac beat-by-beat frequency-decoder whereas PKA-mediated phosphorylation (PLN-Ser16) instead responds to exercise, stress or “fight and flight” situations . It may reflect a versatile, adaptive role of PLN and its phosphorylated form to counterbalance impaired NCX activity and RyR2-mediated SR Ca2+ leak in an attempt to maintain normal Ca2+ cycling in different physiological demands. The exact implications of the increased phosphorylated state of PLN in our model deserve further explorations.