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1 J Physiol (217) pp SYMPOSIUM RELATED RESEARCH PAPER Diltiazem prevents stress-induced contractile deficits in cardiomyocytes, but does not reverse the cardiomyopathy phenotype in Mybpc3-knock-in mice Frederik Flenner 1,2,BirgitGeertz 1,2, Silke Reischmann-Düsener 1,2,FlorianWeinberger 1,2, Thomas Eschenhagen 1,2, Lucie Carrier 1,2 and Felix W. Friedrich 1,2 1 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Centre, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Germany The Journal of Physiology Key points Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac illness and can lead to diastolic dysfunction, sudden cardiac death and heart failure. Treatment of HCM patients is empirical and current pharmacological treatments are unable to stop disease progression or reverse hypertrophy. In this study, we tested if the non-dihydropyridine Ca 2+ channel blocker diltiazem, which previously showed potential to stop disease progression, can improve the phenotype of a HCM mouse model (Mybpc3-targeted knock-in), which is based on a mutation commonly found in patients. Diltiazem improved contractile function of isolated ventricular cardiomyocytes acutely, but chronic application did not improve the phenotype of adult mice with a fully developed HCM. Our study shows that diltiazem has beneficial effects in HCM, but long-term treatment success is likely to depend on characteristics and cause of HCM and onset of treatment. Abstract Left ventricular hypertrophy, diastolic dysfunction and fibrosis are the main features of hypertrophic cardiomyopathy (HCM). Guidelines recommend β-adrenoceptor or Ca 2+ channel antagonists as pharmacological treatment. The Ca 2+ channel blocker diltiazem recently showed promising beneficial effects in pre-clinical HCM, particularly in patients carrying MYBPC3 mutations. In the present study we evaluated whether diltiazem could ameliorate or reverse the disease phenotype in cells and in vivo in an Mybpc3-targeted knock-in () mouse model of HCM. Sarcomere shortening and Ca 2+ transients were measured in and wild-type () cardiomyocytes in basal conditions (1-Hz pacing) and under stress conditions (3 nm isoprenaline, 5-Hz pacing) with or without pre-treatment with 1 μm diltiazem. cardiomyocytes exhibited lower diastolic sarcomere length (dsl) at baseline, a tendency to a stronger positive inotropic response to isoprenaline than, a marked reduction of dsl and a tendency towards arrhythmias under stress conditions. Pre-treatment of cardiomyocytes with 1 μm diltiazem reduced the drop in dsl and arrhythmia frequency in, and attenuated the positive inotropic effect of isoprenaline. Furthermore, diltiazem reduced the contraction amplitude at 5 Hz but did not affect diastolic Ca 2+ load and Ca 2+ transient amplitude. Six months of diltiazem treatment of mice did not reverse cardiac hypertrophy and dysfunction, activation of the fetal gene program or fibrosis. L. Carrier and F. W. Friedrich contributed equally to this work. DOI: /JP273769

2 3988 F. Flenner and others J Physiol In conclusion, diltiazem blunted the response to isoprenaline in and cardiomyocytes and improved diastolic relaxation under stress conditions in cardiomyocytes. This beneficial effect of diltiazem in cells did not translate in therapeutic efficacy when applied chronically in mice. (Received 11 November 216; accepted after revision 6 January 217; first published online 15 January 217) Corresponding authors F. W. Friedrich or L. Carrier: Department of Experimental Pharmacology and Toxicology, University Medical Centre Hamburg-Eppendorf, Martinistraße 52, D-2246 Hamburg, Germany. f.friedrich@uke.de or l.carrier@uke.de Abbreviations Ah, anterior wall thickness of the left ventricle; BW, body weight; Ct, cycle threshold; Dil, diltiazem; dsl, diastolic sarcomere length; FAS, fractional area shortening; Gnas, G-protein α-subunit; dp/dt min, minimal rate of pressure change; dp/dt max, maximal rate of pressure change; HCM, hypertrophic cardiomyopathy; ISO, isoprenaline; IVRT, isovolumic relaxation time;, Mybpc3-targeted knock-in; LV, left ventricle; LVM, left ventricular mass; LW, lung weight; MYBPC3 or Mybpc3, human or mouse cardiac myosin-binding protein C gene; MV A, mitral valve velocity in late LV filling; MV E, mitral valve velocity in early LV filling; MYH7, β-myosin heavy chain gene; NFT, non-filling time of the left ventricle; Nppa, atrial natriuretic peptide gene; Nppb, brain natriuretic peptide gene; Ph, posterior wall thickness of the left ventricle; TL, tibia length;, wild type. Introduction Hypertrophic cardiomyopathy (HCM) is the most common inherited myocardial disease (prevalence 1:5). It is considered a monogenic disease with a predominance of sarcomeric gene mutations. Clinically, HCM patients present with cardiac hypertrophy, diastolic dysfunction, increased risk of sudden cardiac death in the young, and sometimes heart failure in elderly patients (Elliott et al. 214). Despite increasing understanding of the genetic causes of HCM, drug treatment remains empirical and no treatment has been shown to prevent or attenuate disease development, to reverse established manifestations or to impact the prognosis. In order to reduce left ventricular (LV) diastolic pressures and improve LV filling, guidelines recommendβ-adrenoceptor antagonists (β-blockers) or Ca 2+ channel blockers in symptomatic patients. β-blockers in particular are effective in patients with outflow tract obstruction under exercise and can relieve them from angina and dyspnoea by lowering the gradient and increasing time for diastolic filling (Spirito et al. 1997; Marian, 29). Ca 2+ channel blockers such as verapamil or diltiazem are primarily applied in patients with non-obstructive HCM and have been shown to improve LV function in early diastole and prolong LV filling time (Hanrath et al. 198; Choudhury et al. 1999). However, they were unable to stop disease progression or even reverse hypertrophy (Frey et al. 212; Hamada et al. 214). Diltiazem is recommended as an alternative for patients who are intolerant of verapamil (Elliott et al. 214). A recent clinical trial showed promising beneficial effects of diltiazem in pre-clinical HCM, particularly in patients carrying MYBPC3 mutations (Ho et al. 215). In addition, chronic diltiazem treatment prevented the development of pathology in αmhc 43/+ HCM mice (Semsarian et al. 22) and heart failure and sudden death induced by acute isoprenaline (ISO) application in TnT-I79N HCM mice (Westermann et al. 26). The goal of this study was to test whether diltiazem would exert beneficial effects in a Mybpc3-targeted knock-in () mouse model that we generated previously (Vignier et al. 29). This model carries a Mybpc3 point mutation (c.772g > A), which is a founder mutation in Tuscany (Girolami et al. 26) and is associated with a severe phenotype and poor prognosis in humans (Richard et al. 23). mice exhibit systolic dysfunction followed by cardiac hypertrophy right after birth (Gedicke-Hornung et al. 213; Mearini et al. 213, 214), increased myofilament Ca 2+ sensitivity and diastolic dysfunction (Fraysse et al. 212). We evaluated the acute effect of diltiazem on the sarcomere function and Ca 2+ transient of and wild-type () cardiomyocytes and the effect of chronic (6 months) diltiazem application on the cardiac phenotype of and mice. Methods Ethical approval. This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the NIH (Publication No , revised 211 published by National Research Council). All experimental procedures were in accordance with German Law for the Protection of Animals, and the protocol was ratified by the Ministry of Science and Public Health of the City State of Hamburg, Germany (No. 13/1). The investigators understand the ethical principles under which The Journal of Physiology operates and state that their work complies with the animal ethics checklist and the principles and regulations, as described in the Editorial by Grundy (Grundy, 215).

3 J Physiol Diltiazem in a Mybpc3 HCM mouse model 3989 Animals. The Mybpc3 cardiomyopathy mouse model was generated by the targeted insertion of a G > A transition on the last nucleotide of exon 6 (c.772g > A) and kept on the Black Swiss background (Vignier et al. 29; Fraysse et al. 212; Schlossarek et al. 212, 214; Gedicke-Hornung et al. 213; Mearini et al. 213, 214; Stohr et al. 213; Friedrich et al. 214; Najafi et al. 214; Thottakara et al. 215; Flenner et al. 216). Mice were kept in the animal facility of the University Medical Centre Hamburg-Eppendorf in conventional cages with sufficient nesting material at room temperature between 2 and 24 C and humidity between 45 and 65%. Mice received feed and water ad libitum. Ventricular myocyte preparation. The isolation of cardiomyocytes from and mouse heart ventricles was performed as formerly described (Pohlmann et al. 27; Flenner et al. 216; Friedrich et al. 216). Mice were sedated with CO 2 and killed by cervical dislocation. Hearts were cut out, cannulated via the aorta and mounted on a temperature-controlled (37 C) perfusion system. After retrograde perfusion with Ca 2+ -free buffer solution (113 mm NaCl, 4.7 mm KCl,.6 mm KH 2 PO 4,.6 mm Na 2 HPO 4, 1.2 mm MgSO 4,12mMNaHCO 3, 1 mm KHCO 3,3mM taurine, 5.55 mm glucose, 1 mm 2,3-butanedione monoxime 1 mm Hepes, ph 7.46) for 6.5 min, hearts were digested with.75 mg ml 1 Liberase TM (Roche Diagnostics, Mannheim, Germany) dissolved in buffer solution containing 12.5 μm CaCl 2 for 7 8 min. Ventricles were separated from the atria and minced with forceps to dissociate single cardiomyocytes. Subsequently Ca 2+ was introduced stepwise up to a concentration of 1mM. Sarcomere shortening and Ca 2+ transient measurements in intact ventricular myocytes. For contractile evaluation of cardiac myocytes the IonOptix system monitoring sarcomere movement and intracellular Ca 2+ levels during contraction was used. Only rod-shaped cells without membrane blebs, hypercontractile zones and spontaneous activity showing a stable contraction amplitude and rhythm at 1-Hz pacing frequency were measured. For details on buffer composition, investigation of sarcomere shortening and Ca 2+ transients, see Flenner et al. (216). Measurements of contraction and Ca 2+ transients were performed with or without diltiazem (1 μm, 5 min incubation, Sigma-Aldrich). Long-term diltiazem treatment, echocardiography and haemodynamic measurements. To test the effects of long-term diltiazem treatment on the HCM phenotype in mice, and mice (n = 1) received diltiazem via their drinking water. Treatment started at 6 8 weeks of age and was maintained for 6 months. Diltiazem (25 mg l 1 ) was dissolved in drinking water, while control groups received normal water. Based on their water consumption, mice received a dose of 25 mg kg 1 day 1 diltiazem (Westermann et al. 26). Transthoracic echocardiography was executed using the Vevo 21 System (VisualSonics, Toronto, Canada) every 6 8 weeks over a period of 6 months as described previously (Flenner et al. 216). Mice were sedated with isoflurane (3.5% for induction, 2% during the recording). B-mode recordings were performed using a MS 4 transducer (18 38 MHz) with a frame rate of 23 4 frames s 1 to assess LV dimensions and fractional area shortening (FAS). Haemodynamic measurements were performed using an open-chest approach in 34-week-old and mice, treated or not for 6 months with diltiazem. Mice were anesthetized with isoflurane (3.5% for induction, 2% during the recording). For analgesia,.5 mg (kg BW) 1 buprenorphine was administered. Animals were fixed to a warming platform in a supine position and abdomen and anterior neck were shaved. Tracheotomy was performed and mice were artificially ventilated with a rodent ventilator (MiniVent Type 845, Hugo Sachs, March-Hugstetten, Germany). The abdomen was opened subxiphoidally. The diaphragm was incised via a transversal subcostal approach and the pericardium was opened. The left ventricle was entered via an apical stab with a 25-gauge needle, followed by a 1.2F transonic catheter. After a stabilization period of 5 min, heart rate, LV end-diastolic and end-systolic pressure and LV contractility (dp/dt max )andrelaxation(dp/dt min )wererecorded with the Scisense ADVANTAGE System (Scisense Inc., London, Ontario, Canada). Expression analysis. At the end of the diltiazem treatment, and mice were killed by cervical dislocation; for further analysis hearts were extracted and cut in three parts (base, middle part and apex). The middle part was used for histological analysis, the base and apex parts were frozen in liquid nitrogen for subsequent molecular analysis. RNA was isolated as reported earlier (Friedrich et al. 215) and 1 ng transcribed into cdna using the SuperScript III Reverse Transcriptase kit (Life Technologies; (Friedrich et al. 212, 214; Thottakara et al. 215). Quantitative determination of atrial natriuretic peptide (Nppa) and brain natriuretic peptide (Nppb) mrna levels was achieved by real-time PCR using the Maxima SYBR Green/Rox qpcr Master Mix (Thermo Scientific), and primers specific for every sequence (Friedrich et al. 215). Cycle threshold (Ct) values were normalized to the stimulatory G-protein α-subunit (Gnas). Ct values were related to control. Histology. For the histological analysis of collagen I and III fibres the middle parts (including left and right ventricle) of extracted hearts were fixed (Histofix, Carl

4 399 F. Flenner and others J Physiol Roth) for 24 h, embedded in paraffin and cut into transverse sections containing both ventricles and the septum. Subsequently, sections were stained with Sirius Red to visualize collagen I and III fibres (7 9 mice per group). Sections were scanned and the extent of Sirius Red-positive area was quantified by ImageJ and related to total cardiac area. Statistical analysis. Data were expressed as means ± SEM. Comparisons were performed by Student s t test, one-way or two-way ANOVA followed by Dunnett s or Bonferroni s post hoc tests, as indicated in the figure legends (GraphPad, Prism 6). A value of P <.5 was considered statistically significant. Results Diltiazem ameliorates contractile deficits under stress conditions in isolated cardiomyocytes from mice. We previously reported that cardiomyocytes demonstrate A ISO Sarcomere length (µm) ISO Dil + ISO Dil + ISO Arrhythmic cardiomyocytes (%) Arrhythmia incidence Dil Dil 2 s B Sarcomere length (µm) Diastolic SL Dil Dil / 5 Hz Sarcomere shortening % Contraction +++ ### + + / 5 Hz Time to peak shortening (s) Contraction time * ** / 5 Hz Time to 5% relengthening (s) Relaxation time + + / 5 Hz C F 34/38 nm ratio Diastolic Ca 2+ level / 5 Hz + + / 5 Hz + + / 5 Hz + + / 5 Hz Ca 2+ peak height (a.u.) Ca 2+ transient amplitude ** ++ Time to Ca 2+ peak (s) ** Ca 2+ transient rise +++ Time to 5% Ca 2+ decay (s) Ca 2+ transient decay Figure 1. Contraction and Ca 2+ transient parameters of and cells with an increased workload protocol and the influence of 1 µm diltiazem A, representative traces of an untreated and a diltiazem-treated and cardiomyocyte paced at 1 and 5 Hz and stimulated with 3 nm ISO. The change of sarcomere length upon electrical field stimulation (marks at the bottom) is recorded over time. The grey line indicates the period of ISO stimulation. Graph to the right shows the number of arrhythmic cardiomyocytes. B, diastolic sarcomere lengths, contraction, contraction time and relaxation time. C, diastolic Ca 2+ level, Ca 2+ transient amplitude, Ca 2+ transient rise and Ca 2+ transient decay. (black) and (grey) cardiomyocytes stimulated at 1 Hz ( ), 1 Hz and 3 nm ISO (+) and 5 Hz and 3 nm ISO (+/5 Hz). Open symbols and dashed lines indicate the presence of 1 µm diltiazem during the measurement: n = 12 for contraction analysis, n = 6 9 for Ca 2+ transient analysis. Two-way ANOVA with Bonferroni s post hoc test; P <.5, P <.1 and P <.1 vs. value in the same condition; + P <.5, ++ P <.1, +++ P <.1 vs. value in the same condition; ### P <.1 vs. untreated.

5 J Physiol Diltiazem in a Mybpc3 HCM mouse model 3991 Table 1. Echocardiographic parameters of the long-term diltiazem study Parameter Age (weeks) control control diltiazem diltiazem BW (g) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.9 LVM (mg) ± ± 7 7 ± ± 9 / ± ± ± ± ± ± 9 79 ± ± ± ± 8 88 ± ± 16 slvid (mm) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.1 dlvid (mm) ± ± ± ± ±.1 5. ± ± ± ± ± ±.1 5. ± ± ± ± ±.1 sah (mm) ±.1.8 ±.1.7 ±.1.8 ± ±.1.8 ±.1.7 ±.1.8 ± ±.1.8 ±.1.7 ±.1.9 ± ±.1.8 ±.1.8 ±.1.9 ±.1 sph (mm) ±.1.7 ±.1.6 ±.1.7 ± ±.1.8 ±.1.6 ±.1.8 ± ±.1.7 ±.1.6 ±.1.8 ± ±.1.8 ±.1.7 ±.1.9 ±.1 Values are expressed as means ± SEM. Statistical analyses were done with the two-way ANOVA with Bonferroni s post hoc test; P <.5, P <.1, P <.1 vs. value in the same condition; P <.5 vs. respective value; n = 8 1 mice. Abbreviations used are: BW, body weight;, Mybpc3-targeted knock-in mice; LVM, left ventricular mass; slvid, left ventricular inner dimension in systole; dlvid, left ventricular inner dimension in diastole; sah, anterior wall thickness in systole; sph, posterior wall thickness in systole;, wild-type mice. a lack of tolerance to stress conditions (3 nm ISO plus 5-Hz pacing; Flenner et al. 216). We therefore applied this stress protocol in the presence or absence of 1 μm diltiazem (Fig. 1). In the absence of diltiazem, diastolic sarcomere length (dsl) was lower in than cardiomyocytes at 1-Hz pacing, and stimulation with 3 nm ISO led to a stronger positive inotropic response in than in (3-fold vs 2-fold increase in contraction amplitude, respectively; Fig. 1). The second phase of the stress condition protocol (= 5-Hz pacing) led to a further shortening of dsl in cardiomyocytes (from 1.8 ±.5 μm to 1.7 ±.11 μm). Additionally, contraction time was longer in than cells under stress conditions. Throughout the whole recording time (up to 5 min), most cells were able to follow the high pacing rate and contracted in a regular manner, whereas almost 4% of cardiomyocytes developed arrhythmias or did not maintain stable contraction amplitudes (Fig. 1A). Neither ISO (3 nm) stimulation nor 5-Hz pacing alone induced this detrimental effect in cardiomyocytes (data not shown). Except for a longer Ca 2+ transient rise, Ca 2+ transient parameters were not different between and cells in all tested conditions. Since diastolic Ca 2+ level did not differ between and in all conditions, the significant drop in dsl in cardiomyocytes under stress conditions waslikelynottheconsequenceofintracellularca 2+ overload. We then investigated whether a 5-min pre-incubation with diltiazem could improve the disease phenotype of cardiomyocytes (Fig. 1). We established concentration response curves for contraction and Ca 2+ transient amplitude with diltiazem concentrations ranging from 1 nm to1mmdiltiazem in Mybpc3 cells. The full (inhibitory) effect of diltiazem was visible within 3 min of application. The IC 5 values of diltiazem for contraction and Ca 2+ transient amplitudes were 7.2 and 246 μm, respectively (data not shown). We therefore used 1 μm, which was the maximal concentration that did not significantly impair sarcomere contraction. At baseline (1-Hz pacing, no ISO stimulation), diltiazem did not influence any parameter in both genotypes (Fig. 1). Diltiazem attenuated the ISO-induced increase in amplitudes of contraction and Ca 2+ transient in

6 3992 F. Flenner and others J Physiol and cardiomyocytes, and prolonged Ca 2+ transient rise in. Furthermore, diltiazem blunted the ISO-induced difference in contraction time between and cells (Fig. 1B) andinfluencedca 2+ transient rise time in, butdidnotaffecttherelaxationandca 2+ transient decay time in both genotypes (Fig. 1C). Further increase in pacing frequency from 1 to 5 Hz induced a reduction in sarcomere shortening in diltiazem-pretreated cells. This effect also appeared in the absence of ISO stimulation (data not shown). Despite the negative inotropic effect, diastolic Ca 2+ levels and Ca 2+ transient amplitude did not decrease in diltiazem-treated cells when pacing was increased. Under stress conditions, cells treated with diltiazem showed no decline in dsl (Fig. 1B). Pretreatment with diltiazem also reduced the occurrence of arrhythmic behaviour under stress conditions in cardiomyocytes (Fig. 1A). Taken together, these data indicate that diltiazem protects cells under the stress condition protocol by stabilizing dsl, by reducing the ISO effect and by decreasing the occurrence of arrhythmias. Long-term diltiazem treatment does not improve the cardiac disease phenotype of mice. Since diltiazem had a protective effect when acutely applied in the stress protocol (Fig. 1), we further evaluated the therapeutic potential of a 6-month diltiazem application (25 mg kg 1 day 1 ) in mice and compared to mice. Cardiac function was measured by echocardiography before, during and at the end of the diltiazem treatment. Echocardiographic recordings (every 8 weeks) of mitral blood flow and movement of the LV were used to determine cardiac function and its changes upon pharmacological treatment. During the study none of the mice died. Body weights (BW) did not significantly differ between and groups and increased from 2 g at the start of the study to up to 3 g at the end of the study (Table 1). The left ventricular mass-to-body weight ratio (LVM/BW)washigherinallthangroupsbeforethe start of the treatment (Fig. 2A). Age-dependent increases in LVM and BW were not influenced by any treatment (Table 1), and, accordingly, LVM/BW did not differ between treated and non-treated and mice, except A mg / g C mm 8 5 # 6 Dil 4 Dil LVM/BW Age (weeks) dah ** Age (weeks) # ** Dil Dil B % D mm Fractional area shortening * Age (weeks) dph ** * ** * ** Age (weeks) Figure 2. Echocardiography of cardiac parameters and function in the long-term treatment study Echocardiographies were performed before the start of treatment at the age of 6 8 weeks, followed by measurements in intervals of 8 weeks (14 17, and weeks of age). A, ratio of left ventricular mass to body weight (LVM/BW) of control and diltiazem-treated (hatched bars) (black) and (grey) mice. B, fractional area shortening of the left ventricle of the same mice. C and D, anterior and posterior wall thicknesses of the left ventricles in diastole (dah and dph, respectively) of the same mice. Two-way ANOVA with Bonferroni s post hoc test; P <.1 vs. respective control group with the same treatment. # P <.5 vs untreated group; n = 7 1. Dil Dil Dil Dil

7 J Physiol Diltiazem in a Mybpc3 HCM mouse model 3993 Table 2. Echocardiographic parameters obtained by tissue Doppler imaging Parameter Age (weeks) control control diltiazem diltiazem NFT (ms) ± ± ± 7 19 ± ± ± 1 99 ± ± ± 9 1 ± ± ± ± ± 7 9 ± 5 13 ± 1 AET (ms) ± 8 35 ± 4 5 ± 8 36 ± ± ± 8 51 ± 6 37 ± ± 4 36 ± 2 52 ± 7 37 ± ± 7 38 ± 1 44 ± 7 34 ± 4 MPI ± ± ± ± ± ± ± ± ± ±.3.9 ±.2 2. ± ± ± ± ±.2 MV E (mm s 1 ) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 95 MV A (mm s 1 ) ± ± ± ± ± ± ± ± ± ± ± ± E/A ± ± ± ± ± ± ± ± ± ± ± ± IVRT (ms) ± 2 47 ± 9 32 ± 4 45 ± ± 3 39 ± 2 29 ± 3 45 ± ± 2 39 ± 2 25 ± 2 42 ± ± 3 33 ± 3 29 ± 2 4 ± 2 Values are expressed as means ± SEM. Statistical analyses were done with the two-way ANOVA with Bonferroni s post hoc test; P <.5. P <.1. P <.1 vs. value in the same condition; n = 8 1 mice. Abbreviations used are:, wild-type (mice);, Mybpc3-targeted knock-in (mice); NFT, non-filling time; AET, aortic ejection time; MPI, myocardial performance index (MPI = (NFT AET)/AET); MV E, early filling of the left ventricle; MV A, late filling of the left ventricle; E/A; ratio of the early (E) to late (A) ventricular filling velocities; IVRT, isovolumic relaxation time. Note that only one mouse treated with diltiazem showed measurable A-waves. in 6- to 8-week-old mice due to a higher LVM (Fig. 2A, Table 1). mice presented larger left anterior (Ah) and posterior wall thicknesses (Ph) in diastole and in systole than mice, and both parameters increased over time (Fig. 2C and D, Table 1). In the course of the study, mice displayed larger left ventricular internal diameters in diastole and systole (dlvid and slvid, respectively) than mice, indicating a dilated phenotype (Table 1). Left ventricular wall thickness and chamber dimensions were not affected by diltiazem treatment in both genotypes (Fig. 2C and D, Table 1). Fractional area shortening (FAS) tended to be lower in than in mice at the beginning of the study (Fig. 2B) and decreased further in controls, leading to significant differences between and from the second echocardiography on. Diltiazem treatment did not influence FAS in or mice. Pulsed-wave Doppler images revealed that the time between diastolic filling phases of the LV (non-filling time, NFT) did not differ between untreated groups during the entire study. On the other hand, the aortic ejection time was shorter in than in mice (Table 2). This resulted in up to 2-fold higher myocardial performance index values, a parameter which calculates the fraction of cardiac output during the NFT. Diltiazem treatment did not influence these parameters in and mice (Table 2). Diastolic function was evaluated by measuring blood flow velocities at the mitral valve in the early (MV E )and late (MV A ) phases of LV filling and isovolumic relaxation time (IVRT). Lower E-wave values were repeatedly found in groups (maximal blood flow velocity 4 mm s 1 vs. 7 mm s 1 in animals; P <.1; Table 2).

8 3994 F. Flenner and others J Physiol Despite the high variability caused by the small number of usable A-wave measurements, this parameter also showed a tendency to be lower in mice (2 3 mm s 1 in and 4 mm s 1 in ; Table 2). IVRT was significantly longer in untreated mice before the start of the study and in the third echocardiography (Table 2). These data suggest impaired diastolic function in, even though the E/A ratios did not differ between and groups. Diltiazem treatment did not influence E-waves in and mice (Table 2). The A-wave peaks were slightly higher in and mice supplied with diltiazem, which led to a tendency to smaller E/A ratios than in untreated mice (Table 2). However, IVRT remained longer in diltiazem-treated than mice throughout the whole study (Table 2). In a subset of mice, cardiac function was further evaluated by performing intraventricular haemodynamic measurements with a catheter (Fig. 3A C). Heart rates were lower in diltiazem-treated mice, but the difference was not significant, while maximal rate of pressure change (dp/dt max ), used as an indicator of LV systolic function, did not differ between the groups in all conditions (Fig. 3A and B). While the minimal rate of pressure change (dp/dt min ), used as another indicator of diastolic function, did not differ between untreated and mice, it was significantly lower in diltiazem-treated than mice (Fig. 3C). At the end of the study we assessed BW, heart and lung weight (HW, LW, respectively) and tibia length (TL). BW, TL, LW/BW, LW/TL did not differ between the groups A 8 Heart rate B 6 dp/dt max C dp/dt min Beats/min mmhg/s 4 2 mmhg/s # * Dil Dil Dil Dil 8 Dil Dil D g BW E mg/g HW/BW F mg/g Lung weight/bw Dil Dil Dil Dil Dil Dil G mm TL H mg/mm HW/TL I mg/mm 5 5 Lung weight/tl Dil Dil Dil Dil Dil Dil Figure 3. Haemodynamic measurements of cardiac function of selected treatment groups and body parameters at the end of the long-term treatment study A, heart rate of (black bars) and (grey bars) control mice and diltiazem-treated animals (hatched bars). B, maximal rate of pressure change (dp/dt max ). C, minimal rate of pressure change (dp/dt min ) of the same animal groups. D, body weight (BW). E, heart weight (HW) to body weight ratio. F, lung weight (LW) to body weight ratio. G, tibia length (TL). H, heart weight to tibia length ratio. I, lung weight to tibia length ratio. One-way ANOVA with Bonferroni s post hoc test; P <.5 and P <.1 vs. in the same condition, or Student s t test # P <.5 vs. control.

9 J Physiol Diltiazem in a Mybpc3 HCM mouse model 3995 (Fig. 3D, F, G and I). In contrast, HW/BW and HW/TL were higher in that in mice and not affected by chronic diltiazem application (Fig. 3E and H). It has been reported that long-term diltiazem application partially prevented the reactivation of the fetal gene program of hypertrophy and the development of fibrosis in αmhc 43/+ HCM mice (Semsarian et al. 22). Despite the absence of beneficial effects of chronic diltiazem treatment on the cardiomyopathy phenotype of mice, we evaluated whether diltiazem impacts on these parameters. The level of Nppa mrna was slightly and the level of Nppb mrna was significantly higher in untreated than mice, but diltiazem treatment did not significantly affect these levels in both genotypes (Fig. 4). Histological analysis of Sirius Red-stained collagen I and III fibres in mouse cardiac sections showed a slight, but not significantly higher percentage of collagen content in untreated than in mice, and a slight, not significant reduction after diltiazem in both genotypes (Fig. 5). Taken together, these in vivo data did not provide evidence for amelioration of the cardiomyopathy phenotype in mice by chronic application of diltiazem. Discussion Guideline-recommended treatment strategies of HCM primarily consist of β-blockers and Ca 2+ channel blockers, which improve clinical symptoms, help to prevent arrhythmias and ameliorate diastolic dysfunction by prolonging LV filling time and reducing outflow tract obstruction (Spoladore et al. 212; Elliott et al. 214; Hamada et al. 214; Tardiff et al. 215). Nevertheless, evidence of a long-term impact on functional capacity or prognosis in HCM patients is missing. A recent clinical trial showed promising beneficial effects of diltiazem in pre-clinical HCM, particularly in patients carrying MYBPC3 mutations (Ho et al. 215). A common feature observed in human HCM and animal models is an increased myofilament Ca 2+ sensitivity. Myofilaments with increased affinity for Ca 2+ may act as Ca 2+ buffers leading to an increase in the Ca 2+ pool at the Z-disc which could also activate hypertrophic signalling cascades such as calcineurin leading to hypertrophy and fibrosis, which are also prominent HCM characteristics (Frank et al. 26; Rohini et al. 21; Shabbir et al. 211). In the present study we evaluated whether the non-dihydropyridine Ca 2+ channel blocker diltiazem would have beneficial effects in an Mybpc3-targeted knock-in mouse model of HCM. The main findings of this study are: (i) diltiazem acutely improved cardiomyocyte performance under stress conditions; (ii) long-term application of diltiazem did not reverse the activation of the fetal gene program, fibrosis, cardiac hypertrophy and dysfunction in mice. Therefore, our study provides additional evidence that acute diltiazem treatment can prevent stress-induced contractile abnormalities, whereas chronic diltiazem treatment does not reverse a pre-existing cardiac disease phenotype. The combination of ISO and high pacing frequency (= stress conditions) worsened the phenotype of cardiomyocytes, showing a drastic decrease in dsl and a trend towards higher frequency of arrhythmic contractions than. This supports previous observations that ISO application worsened diastolic function and increased arrhythmia frequency in the TnT-I79N HCM mouse model (Knollmann et al. 21; Sirenko et al. 26; Baudenbacher et al. 28; Huke et al. 213). Whereas diltiazem did not have major effects at baseline, it reduced Ca 2+ transient amplitude and sarcomere shortening under stress conditions. Interestingly in, diltiazem appeared to diminish the Ca 2+ transient more strongly than sarcomere shortening. This might be explained by high myofilament Ca 2+ sensitivity in (Fraysse et al. 212; Flenner et al. 216; Friedrich et al. 216), a condition in which small changes in cytosolic Ca 2+ can evoke strong changes in contractility. Diltiazem itself neither influenced myofilament Ca 2+ sensitivity directly A Fold change over mrna level of Nppa mrna level of Nppb * Dil Dil B Fold change over Dil Dil Figure 4. Levels of mrna markers for hypertrophy Levels of atrial natriuretic peptide mrna (A) and brain natriuretic peptide mrna (B) in and ventricular samples. One-way-ANOVA, Tukey post hoc test, P <.5 vs. in same condition.

10 3996 F. Flenner and others J Physiol nor indirectly via changing phosphorylation of PKA targets (data not shown). Specifically in cells, diltiazem improved diastolic function and reduced the occurrence of arrhythmias under stress conditions, suggesting that it protects cells under acute stress conditions. The effect could be explained by limiting the ISO-induced increase in Ca 2+ influx via the L-type Ca 2+ channel (LTCC), leading to less Ca 2+ available for Ca 2+ storage in the sarcoplasmic reticulum (SR) than normal after ISO stimulation (Eisner et al. 213), and consequently, less Ca 2+ release from the SR. These data support previous findings that diltiazem prevented acute ISO-induced contractile dysfunction and sudden cardiac death in A Control Diltiazem B Control Diltiazem C % of total tissue area Sirius Red positive area 7 8 Dil 9 9 Dil Figure 5. Sirius-Red positive area in Mybpc3 and hearts A, representative mouse heart images of a Mybpc3 control mouse, a Mybpc3 control mouse, a diltiazem-treated Mybpc3 mouse and a diltiazem-treated Mybpc3 mouse. B and C, quantification of Sirius-Red positive area, 1 section per heart in 7 9 animals as indicated in the bars. Scale bar 1 mm (A) and 1 µm (B). TnT-I79N HCM mice (Westermann et al. 26). Together, the data suggest that the reduction of Ca 2+ influx via blockade of LTCC enabled HCM cells and mice to better tolerate adrenergic stress. On the other hand, long-term treatment of mice with diltiazem did not produce salutary structural or functional effects. All the parameters of LV hypertrophy, dilatation and dysfunction did not differ between diltiazem-treated and untreated mice, and diastolic function (dp/dt min ) obtainedbyhaemodynamicmeasurementsevenworsened in diltiazem-treated mice. Consequently, diltiazem did not reduce the expression of the fetal gene program or the extent of fibrosis. Our findings are in agreement with those obtained with the same dose and treatment duration in the TnT-I79N mice that exhibit hypercontractility and diastolic dysfunction (Westermann et al. 26). On the other hand, our findings differ from the findings obtained in preclinical HCM patients with MYH7 or MYBPC3 mutations (Ho et al. 215) and in the αmhc 43/+ mice with or without cyclosporine treatment (Fatkin et al. 2; Semsarian et al. 22), showing prevention of the development of hypertrophy and hypercontractility, as well as partial prevention of expression of hypertrophic markers and fibrosis in HCM mice (Semsarian et al. 22). The lack of long-term efficacy of diltiazem in both Mybpc3 and TnT-I79N mice (Westermann etal. 26) is likely to be due, at least in part, to the pre-existing cardiac disease phenotype. We previously showed that different pharmacological treatment approaches with ranolazine or the β-blocker metoprolol did not reverse or improve the disease phenotype in the same mice (Friedrich et al. 215; Flenner et al. 216). mice indeed had already developed cardiac dysfunction followed by hypertrophy at the neonatal age (Gedicke-Hornung et al. 213; Mearini et al. 213). Therefore, preventive therapeutic options should be tested at postnatal day 1, before the development of the disease phenotype. In this condition, Mybcp3-gene therapy could prevent the development of cardiac dysfunction and hypertrophy over the long-term in mice (Mearini et al. 214). Although challenging in neonates, further analyses are needed to validate the preventive efficacy of diltiazem, as has been observed in human MYBPC3 mutation carriers (Ho et al. 215). Alternatively, since cardiac function in mice does not further deteriorate, and their overall development, behaviour, food consumption or longevity are not different to mice, it would be advisable to define surrogate endpoints such as exercise performance or adrenergic stress and changes upon treatment. Another limitation is that the Mybpc3 mice show many HCM features only in the homozygous state. Mybpc3 mice also present a lower ejection fraction than. These features are in contrast to the more common characteristics of left ventricular hypertrophy, interstitial fibrosis, diastolic dysfunction and normal or even supra-normal ejection fraction in HCM

11 J Physiol Diltiazem in a Mybpc3 HCM mouse model 3997 patients with heterozygous mutation states. Therefore, it might be valuable to use mice on another genetic background, as the outbred Black Swiss mice of this study seem to be resistant to heart failure-related death, whereas mice on the congenic C57BL/6j background displayed a more severe phenotype and died earlier than mice on the Black Swiss background (Friedrich et al. 215); and authors unpublished data). Finally, we cannot exclude the possibility that the given dose of diltiazem was not sufficient to improve the phenotype of the mice. As we did not evaluate plasma levels of diltiazem, we do not know if the concentration of diltiazem which reached the heart in vivo is comparable with our in vitro experiments. In another study though, the same dose was sufficient to protect mice carrying an HCM mutation from isoprenaline-induced sudden cardiac death (Westermann et al. 26). The results of the clinical trial NCT that evaluated the potential of diltiazem in the prevention of HCM development emphasized the importance of early onset of treatment and showed diltiazem efficacy particularly in MYBPC3,butnotMYH7 mutation carriers (Ho et al. 215). Still, in contrast to homozygous Mybpc3 mice, disease progression in HCM patients often is slow, leaving a larger therapeutic window than in the mouse model used in the present study. The discrepancies between diltiazem effects in different HCM mouse models and patients emphasizes the need for individualized treatment which could be achieved by in vitro models with human cells. By disease modelling in a dish, therapies (drug or gene therapy) could be applied in a mutation- or patient-specific context (Eschenhagen et al. 215). References Baudenbacher F, Schober T, Pinto JR, Sidorov VY, Hilliard F, Solaro RJ, Potter JD & Knollmann BC (28). 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13 J Physiol Diltiazem in a Mybpc3 HCM mouse model 3999 Westermann D, Knollmann BC, Steendijk P, Rutschow S, Riad A, Pauschinger M, Potter JD, Schultheiss HP & Tschope C (26). Diltiazem treatment prevents diastolic heart failure in mice with familial hypertrophic cardiomyopathy. Eur J Heart Fail 8, Additional information Competing interests The authors declare that there are no competing interests or conflict of interest in accordance with the policy of The Journal of Physiology. and analysis of data, contribution to writing the manuscript. T.E.: critical revision of the manuscript and important intellectual contribution to writing the manuscript. L.C.: conception of the study; data interpretation; critical revision of the manuscript and important intellectual contribution. F.W.F.: conception of the study; acquisition, analysis, and interpretation of data; drafting of the manuscript. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Author contributions The experiments were performed in the Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Centre, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany. F.F.: conception of the study; acquisition, analysis, and interpretation of data; critical revision of the manuscript and important intellectual contribution. B.G.: acquisition and analysis of data, contribution to writing the manuscript. S.R.D.: acquisition and analysis of data, contribution to writing the manuscript. FW: acquisition Funding This work was supported by the Deutsche Stiftung für Herzforschung (F/28/12). Acknowledgements We thank the Mouse Pathology Core Facility for histological processing and Giulia Mearini and Marc Hirt for intense discussion.