Hypertrophic cardiomyopathy (HCM) is the most frequent cardiac genetic disease primarily caused by mutations in sarcomeric protein genes (Friedrich and Carrier, 2012; Maron et al., 2014; Ho et al., 2015). The most commonly mutated genes are MYBPC3 (encoding cardiac myosin-binding protein C) and MYH7 (encoding β-myosin-heavy chain) (Walsh et al., 2017). HCM is principally characterized by asymmetric left ventricular hypertrophy, diastolic dysfunction and myocardial disarray (Elliott et al., 2008). Current pharmacological treatment of HCM mainly relies on beta-adrenoceptor (AR) and Ca²⁺ channel blockers, which improve clinical symptoms, partially prevent arrhythmias and improve diastolic dysfunction by prolonging left ventricular (LV) filling time and reducing outflow tract obstruction (Maron et al., 2003; Gersh et al., 2011; Spoladore et al., 2012; Hamada et al., 2014; Tardiff et al., 2015). Increased Ca²⁺ sensitivity seems to be a common factor in HCM as seen in animal HCM models (Tardiff et al., 1999; Cazorla et al., 2006; Pohlmann et al., 2007; Vignier et al., 2009; Fraysse et al., 2012; Barefield et al., 2014; Wijnker et al., 2016), and human HCM samples (Jacques et al., 2008; van Dijk et al., 2009, 2012). The increased Ca²⁺ response may contribute to diastolic dysfunction and arrhythmias (Morimoto et al., 1998; Baudenbacher et al., 2008). Even though the mechanisms accountable for increased myofilament Ca²⁺ sensitivity remain unclear, targeting this pathomechanism by interventions decreasing myofilament Ca²⁺ sensitivity may be an attractive alternative for the treatment of HCM and improvement in symptoms (Jagatheesan et al., 2007; Alves et al., 2014; Tardiff et al., 2015). Among beta-AR blockers that are commonly used in the treatment of cardiovascular diseases, nebivolol has been reported to lower maximal force development and to desensitize rabbit and human cardiac myofilaments to Ca²⁺ (Zeitz et al., 2000; Janssen et al., 2001). However, the effects of nebivolol were never evaluated in HCM models with increased myofilament Ca²⁺ sensitivity. An established HCM mouse model carrying the human c.772G>A MYBPC3 mutation is the Mybpc3 KI mouse model (Vignier et al., 2009). This mutation was frequently found in unrelated HCM patients in Tuscany and is associated with a bad prognosis (Richard et al., 2003; Girolami et al., 2006; Ho et al., 2015). At the homozygous state, this mouse model exhibits HCM-like features such as left ventricular hypertrophy, diastolic dysfunction and increased myofilament Ca²⁺ sensitivity (Vignier et al., 2009; Fraysse et al., 2012). We recently showed that epigallocatechin-3-gallate (EGCg), a major component of green tea, hastened relaxation and Ca²⁺ transient in KI cardiomyocytes and decreased Ca²⁺ sensitivity of KI myofilaments (Friedrich et al., 2016). In this study, we investigated nebivolol effects on myofilament Ca²⁺ sensitivity in Mybpc3 KI cardiac muscle strips. We furthermore assessed nebivolol and EGCg effects in cardiac strips of three HCM patients with MYBPC3 mutations.

HCM patients often present with a normal to supranormal systolic function and diastolic dysfunction. To counteract this hypercontractility, guideline therapies advocate treatment with beta-AR and Ca²⁺ channel blockers. One well established pathomechanism for the hypercontractile phenotype frequently observed in HCM patients and several HCM mouse models is an increased myofilament Ca²⁺ sensitivity (Morimoto et al., 1998; Robinson et al., 2007; Huke and Knollmann, 2010; Kimura, 2010; Fraysse et al., 2012; Moore et al., 2012; van Dijk et al., 2012; Barefield et al., 2014; Elliott et al., 2014; Flenner et al., 2016; Friedrich et al., 2016). Nebivolol, a commonly used beta-AR antagonist, has been reported to lower maximal force development and myofilament Ca²⁺ sensitivity in rabbit and human heart tissues (Zeitz et al., 2000; Janssen et al., 2001). Given the hypercontractile phenotype mentioned above, these pleiotropic actions predestine it for HCM treatment. The aim of this study was to evaluate whether nebivolol would exert similar effects in permeabilized myofilaments of an Mybpc3 HCM mouse model and of HCM patients with mutations in the most frequently mutated gene MYBPC3. The main findings of this study were: (1) At baseline, permeabilized left ventricular trabeculae isolated from Mybpc3 KI mouse hearts showed no difference in maximal force development compared to WT mouse heart strips. (2) Neither 1 nor 10 μM nebivolol had an effect on maximal force development in both genotypes. (3) 10 μM nebivolol induced myofilament Ca²⁺ desensitization in both WT and KI strips and this effect was more pronounced in KI muscle strips, respectively. (4) Nebivolol had no effect on Ca²⁺ sensitivity in cardiac muscle strips of three HCM patients with MYBPC3 mutations, whereas 30 μM of EGCg induced a right shift in the force-Ca²⁺ curve.

In mice, nebivolol did not influence maximal force development. On the other hand, it affected myofilament Ca²⁺ sensitivity in mouse strips. The mechanism behind this is unknown so far. In analogy to the mouse results, 1 and 10 μM nebivolol had no effect on maximal force development in human tissues. In contrast to the observations made in mouse strips, it did not impact on myofilament Ca²⁺ sensitivity.

The reason why nebivolol exerted a myofilament Ca²⁺ desensitizing effect in KI strips at both 1 and 10 μM, whereas in WT strips only 10 μM had an effect and no effect at all in human HCM tissues remains unclear. As mentioned before, discrepancies concerning the effects of nebivolol on maximal force development and myofilament Ca²⁺ sensitivity in cardiac muscle strips have been previously described. Whereas Zeitz et al. reported that 1 μM nebivolol lowered maximal force development and myofilament Ca²⁺ sensitivity in skinned rabbit and human trabeculae (Zeitz et al., 2000), Bundkirchen and colleagues did not observe such an effect at 10 μM in human tissue (Bundkirchen et al., 2001). The findings are contradictory but could be explained by either differences (i) between species (rabbit vs. mouse vs. human), (ii) in experimental setups, (iii) the functional status of the tissues (non-failing vs. failing), or (iv) a combination of it. Interspecies- or setup-dependent differences have been reported in permeabilized strip experiments with other drug interventions (Lues et al., 1988; Edes et al., 1995). Zeitz et al. used non-failing cardiac tissue from rabbit and explanted human tissue from end-stage failing myocardium from patients undergoing heart transplantation and saw an effect in tissues of both species. In another study the same group did not observe any effect on maximal force development in human explanted heart muscle preparations (Janssen et al., 2001). We observed no effect on maximal force development in neither mouse nor human tissue, but on myofilament Ca²⁺ sensitivity in mouse tissue, which was more pronounced in the KI strips. Similar to this study we observed in a previous study with skinned trabeculae that EGCg, another compound with Ca²⁺-desensitizing properties had a more profound effect on strips of the KI than the WT genotype (Friedrich et al., 2016). This is also compatible with results of a study in which the Ca²⁺-desensitizing effect of ranolazine was only present in KI, but not in WT muscle strips (Flenner et al., 2016). The reason for the difference between KI and WT is unclear but could be related to the higher baseline myofilament Ca²⁺ sensitivity in KI or to the proposed antioxidative activity of ranolazine, which might be important in a potentially hyperoxidized KI tissues (Lovelock et al., 2012; Flenner et al., 2016). In analogy to the study of Bundkirchen et al., in which nebivolol had no effect in explanted left ventricular tissue of patients with dilated cardiomyopathy, we did not observe any effect on Ca²⁺ sensitivity at 1 or 10 μM in the HCM samples. In contrast, 30 μM of the positive control compound EGCg, which has been suggested to alter the interaction between cardiac troponin C and I and therefore the sensitivity of the myofilaments to Ca²⁺ (Liou et al., 2008; Robertson et al., 2009), induced a rightward shift in the force-Ca²⁺ curve in human HCM strips. EGCg has been shown to lower the myofilament Ca²⁺ sensitivity in a transgenic HCM mouse model expressing a human cardiac troponin T mutant (Tadano et al., 2010) and in HCM-associated human cardiac troponin I and T mutants (Tadano et al., 2010; Warren et al., 2015; Messer et al., 2016). Similarly, we reported that 30 μM EGCg decreased Ca²⁺ sensitivity in our Mybpc3 KI mouse model that carries a frequent Mybpc3 HCM mutation (Friedrich et al., 2016). EGCg action on myofilament Ca²⁺ sensitivity in cardiac muscle strips of patients carrying a heterozygous MYBPC3 mutation indicates that the human strips can be desensitized for Ca²⁺.

Yet the precise mechanism of Ca²⁺-desensitization of nebivolol in mouse heart tissue remains unaddressed. Nebivolol is a third-generation beta-AR antagonist that exhibits vasodilating properties, most likely due to stimulation of nitric oxide synthase (Cockcroft et al., 1995). Since it was shown to attenuate hydroxyl radical-induced myocardial damage which has been associated with altered intracellular calcium handling and calcium overload of the myocytes (Josephson et al., 1991; Janssen et al., 1999; Piccini et al., 2012), it was proposed that nebivolol has direct free-radical scavenging properties (Janssen et al., 1999). Whether such an indirect effect is the main reason for a decrease in myofilament Ca²⁺ sensitivity or another direct mechanism on the moyfilaments, such as binding to the C-terminal region of cardiac troponin C altering the interaction between cTnC and cTnI as in the case of the positive control compound EGCg (Liou et al., 2008; Robertson et al., 2009), exists, remains to be shown.

Clinically, beta-AR-antagonists are the mainstay of HCM therapy (Elliott et al., 2014). They are thought to potentially improve diastolic filling by a negative chronotropic effect. Some studies support the use of beta-AR-antagonists in HF patients with preserved ejection fraction (EF) but impaired relaxation similar to diastolic dysfunction seen in HCM patients (Lund et al., 2014). Recent data suggest that the effect of nebivolol is similar in HF patients with reduced and preserved EF (van Veldhuisen et al., 2009). This initiated the design of a still ongoing trial (https://clinicaltrials.gov/ct2/show/NCT02619526) investigating the effects of nebivolol and carvedilol on diastolic function of the left ventricle in older HF patients with preserved EF (Park and Park, 2016).

(1) The Mybpc3 KI model shows many HCM characteristics only at the homozygous state. Moreover, Mybpc3 KI mice present a reduced EF. These two points are in contrast to the more common findings in HCM patients who present left ventricular hypertrophy, interstitial fibrosis and diastolic dysfunction with heterozygous mutation states and normal or even supra-normal EF. (2) Our study does not explain the precise mechanism of Ca²⁺-desensitization of nebivolol in mouse heart tissue. (3) The Ca²⁺ desensitizing effect of nebivolol in mouse tissues occurred at concentrations which were above the plasma concentrations (0.8–3.7 nM) observed in humans (Stoschitzky et al., 2004; Prisant, 2008). (4) Even though we did not observe any nebivolol effect in the human strips in this study, this observation cannot be generalized to all HCM patients since the number of tissues was low and they were derived from HCM patients carrying only mutations in MYBPC3.

Nebivolol had no effect on maximal force, but induced a myofilament Ca²⁺ desensitization in both WT and KI mouse cardiac muscle strips, which was more pronounced in KI muscle strips. In human cardiac muscle strips, nebivolol had no effect on force development and myofilament Ca²⁺ sensitivity. Further studies should investigate the exact target and mechanism for Ca²⁺ desensitization in mouse cardiac tissues in order to be able to develop modified compounds with even more potency and specificity for use in human tissue.

SS and NK: Isolation and treatment of cardiac muscle strips and execution of experiments. LC: Analysis and interpretation of data, and correction of the manuscript. FF: Conception and design of research, execution of experiments, analysis and interpretation of data, figure preparation, and drafting of the manuscript. All authors critically discussed the results, and reviewed and approved the manuscript before submission.