These experiments adhered to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Pub. Nos. 85–23, Revised 1996). All protocols were approved by the Medical College of Wisconsin’s Institutional Animal Care and Use Committee (IACUC). The IACUC has Animal Welfare Assurance status from the Office of Laboratory Animal Welfare (A3102–01). For all experiments, C57Bl/6 mice were purchased from The Jackson Laboratory.

Echocardiography was performed prior to induction of MI or sham surgery and at subsequent intervals using a VisualSonics 3100 high-frequency ultrasound imaging system. This was performed on mice lightly anesthetized with isoflurane delivered via a nose cone (1.0–1.5%). Parasternal long-axis, short-axis, and apical 4-chamber views were obtained using a transducer (MX550D) operating at 30–40 mHz. Short-axis views in M-mode assessed left ventricular anteroposterior internal diameter (LVID), anterior wall thickness (LVAW), and posterior wall thickness (LVPW) at end-diastole (d) and end-systole (s) at the mid-ventricular level. Long-axis views in B-mode assessed left ventricular internal area (LVA) and length (L) at end-diastole and end-systole. Three short axis views (evenly spaced around a mid-level view at the papillary muscles, one towards the apex or distal portion of the heart and one towards the base or proximal portion of the heart) assessed internal area at end-diastole and end-systole. Left ventricular systolic function was assessed by the following: (i) fractional shortening (FS %), calculated as ([LVIDd – LVIDs]/LVIDd)*100; (ii) fractional area change (FAC %) = ([LVAd – LVAs]/LVA d)* 100, and (iii) ejection fraction (EF %) = (end-diastolic volume – end-systolic volume)/end-diastolic volume, wherein volumes were estimated by B-mode: 4π/3*L/2*(LVA÷π(L/2). In addition, global left ventricular function was assessed by calculating the myocardial performance index: MPI = (isovolumic contraction time + isovolumic relaxation time)/ejection time [24–27]. Time intervals were obtained from pulsed Doppler waveforms of mitral valve inflow and aortic valve outflow obtained from apical 4-chamber views.

MI or sham surgery were induced in 10–12-week-old male mice under general anesthesia with isoflurane (1.5–2.0%) and mechanical respiration (model 845, Harvard Apparatus) via an endotracheal tube with room air supplemented with 100% O₂. Rectal temperature was maintained at 37°C using a servo-controlled heating pad. Following anesthesia onset, mice were subcutaneously injected with sustained release meloxicam (4 mg/kg) to manage post-operative pain. A left-sternal thoracotomy was performed to expose the heart, and the pericardium was opened. To target the MI to the lower half of the ventricle, an 8.0 nylon suture was threaded beneath the left main coronary artery at a level below the tip of the left atrium, with the aid of a microscope. Ischemia was induced by tying a permanent suture with a double knot. Coronary occlusion was verified by visually observing blanching of the myocardium distal to the ligature, and by elevation of the ST segment on the ECG. For sham experiments, the sutures were placed as described above but they were not tightened to occlude the artery. The chest wall was closed using polypropylene suture. Recovery was monitored until mice became fully ambulatory.

ECGs were continuously recorded during surgery and at subsequent intervals post-MI via needle electrodes under general anesthesia with isoflurane (1.5–2.0%) using the lead II configuration. For the analyses, 10 or more beats were averaged to give a precise representation of the ECG waveform; each line marker is manually adjusted to meet the standard waveform as previously described [28–30]. At least 10 averaged recordings (more than 100 total beats) were corrected and analyzed per heart for final average values. The Formula described in Mitchell et al [31] is used for QT correction.

Administration of pentamidine began on the third day post-MI/sham surgery via daily intraperitoneal injections of vehicle (1x PBS; controls) or PBS containing pentamidine (3 mg/kg; C19H24N4O2; MW 340.4). Pentamidine isethionate salt was purchased from Sigma (cat. #P0547). Daily injections were performed for 14 consecutive days, i.e., from the third through the sixteenth day post-surgery. On the day before harvest, mice were intraperitoneally injected with BrdU (1 mg). On the day of harvest (10 or 28 days post-MI), mice were euthanized with CO₂ after which hearts were removed and processed for immunostaining as described below.

TUNEL staining was assessed using the DeadEnd Fluorometric TUNEL System (Promega #G3250) per the manufacturer’s instructions. CM identity was verified by co-immunostaining cytoplasm with cTnT, and by imaging CM boundaries with WGA. Total numbers of TUNEL-positive CMs in the border and remote zones were manually counted at 400x magnification. TUNEL signal was counted only if confined within a DAPI-positive nucleus, and, nuclei were scored as TUNEL-positive only if at least 50% of the nucleus contained fluorescent signal. Apoptosis was also determined by immunostaining cleaved caspase-3 as described below.

Scarring was assessed in Masson trichrome-stained transverse sections (4 μm thick) of hearts removed at 0.3 mm intervals along the axis between the apex and ~1 mm above the ligation site. Trichrome-stained sections were photographed at 10x magnification on a Nikon SMZ800 microscope and MIQuant software was used to quantitate infarct size, as previously described [23]. Results are expressed as the average percentage of area, and midline length, around the left ventricle.

WGA staining was performed using Thermo-Fisher #W21405 Texas Red-X conjugate. Sections were stained with 50 μg/ml WGA in PBS for 10 minutes at room temperature, followed by thorough washing. Images of CMs in transverse orientation were photographed at 400x magnification and processed using ImageJ software to determine numbers of CMs, and numbers of pixels per CM, as respectively indicative of CM density and CM size. Briefly, the Texas Red channel displaying CMs outlined in cross-section was isolated, followed by thresholding to fill-in spaces occupied by CM cytoplasm, then adjusting settings to acquire particle sizes in the 600-infinity range having a circularity of 0.25–1. After results were obtained, particles representing CMs that were non-transversely sectioned, or blood vessels, were removed. Data were analyzed in blind and averaged from >300 CMs per zone in each heart.

On the day before harvest, mice were injected with 1 mg 5’-bromo-2’-deoxyuridine (BrdU; Sigma #B9285). Following removal, hearts were perfused with ice-cold cardioplegic solution and atria were removed. Ventricles were fixed overnight in fresh ice-cold 4% paraformaldehyde/PBS, processed through ethanol series, and embedded in paraffin. Sections (4 μm thick) mounted on microscope slides were de-waxed, subjected to antigen retrieval (100° C in 10 mM trisodium citrate pH6.0/0.05% Tween-20 for 20 minutes) followed by 30 minutes’ cooling at RT, and blocked with 2% goat serum/0.1% Triton-X-100 in PBS. Primary antibodies were diluted in blocking buffer and applied overnight at 4°C; secondary antibodies were applied for one hour in the dark. Combinations and dilutions of primary and secondary antibodies used to immunostain each target antigen are listed in Supplemental Table 1.

Microscopy was performed on a Nikon Eclipse 50i microscope equipped with a Nikon DSU3 digital camera. For counting cell-cycle markers, at least 1,000 CMs were evaluated in 5–6 random 200x photomicrographic fields; to count other markers, at least 300 CMs were evaluated in 5–6 random 400x photomicrographic fields. CM identity was assessed by co-immunostaining cytoplasm with cTnT, and cellular outline with WGA. CMs in the border and remote (the area ~2 mm distal to the infarct boundary) zones relative to the infarction site were separately counted.

Heart tissue, previously disrupted by homogenization with a motorized (Kimble 749,540–0000) Teflon pestle and stored at −80° C in TRIzol reagent, was thawed. RNA was purified using PureLink RNA Mini-Kits (ThermoFisher #12183018A), including a genomic DNA removal step (PureLink DNase kit for on-column protocol, Thermo-Fisher #12185–010), according to the manufacturer’s instructions. RNA yield and quality were determined via 260/280 ratio using an Eppendorf Biophotometer Plus instrument.

For synthesis of cDNA, 1 μg of RNA was suspended in 14 μl of nuclease-free water (NFDW), to which 4.0 μl 5x VILO reaction mixture (ThermoFisher #11754050) were added. To start the reverse transcription reaction, 2.0 μl 10x SuperScript Enzyme Mix were added, followed by transfer to an Applied Biosystems Veriti 96-well Thermocycler programmed as follows: 10 min at 25° C → 60 min at 42° C → 5 min at 85° C. cDNA templates were diluted with NFDW to a concentration of 3.125 ng/μl and stored at −20° C.

qPCR was carried out by subjecting each biological replicate (i.e., sample from each individual heart) to triplicate determinations. Each reaction was performed in a total volume of 10 μl in 96-well arrays, each well containing 1x Taqman Fast-Advanced Master Mix (ThermoFisher #4444557), 1x Tagman Probe Kit (Supp. Table 2), and 12.5 ng cDNA template. The arrayed samples were amplified in a Bio-Rad CFX96 Real Time System (C1000 Touch) programmed as follows: 2 min at 50° C → 20 sec at 95° C → 3 sec at 95° C → 30 sec at 60° C; the last two steps were repeated 39 times. Results were processed using Bio-Rad CFX Manager 3.1 software.

All determinations were performed in blinded fashion and are reported as means ± SEM. Echocardiography and ECG data were analyzed by a two-way repeated measures ANOVA to determine whether there was a main effect of time, treatment, or a time-treatment interaction. If global tests showed an effect, post hoc contrasts between baseline and subsequent timepoints within experimental groups were compared by a Dunnett’s multiple comparison t test; differences between treatment groups at each timepoint were compared by Student’s t testing with the Bonferroni correction for multiple comparisons. All other data were compared by an unpaired, two-tailed Student’s t test with Welch’s correction. P<0.05 was considered statistically significant.

Following its identification as a Tip60 inhibitor in 2010 [32], pentamidine (Fig. 1A) has been applied in molecular modeling studies as a model compound for antagonizing the acetyltransferase domain of Tip60 [22]. Our aim was to assess whether systemic administration of pentamidine would mimic the cardioprotective effects of genetically depleting Tip60 in CMs [20]. As shown in the experimental timeline depicted in Figure 1B, which is similar to the strategy previously employed to pharmacologically inhibit Tip60 using TH1834 [20], we assessed the effect of daily intraperitoneal injection of 3 mg/kg pentamidine to wild-type adult mice, beginning on day 3 after MI/sham surgery, for 14 consecutive days, ending on day 16 post-MI/sham. MI was induced by permanent ligation of the left main coronary artery to generate uniform infarctions in the distal half of the ventricle. In order to specifically assess pentamidine impact during post-MI remodeling, administration was not initiated until 3 days after induction of MI. Mice receiving PBS served as vehicle-treated controls. Electrical conduction of the heart was monitored by electrocardiography and echocardiography was performed to assess cardiac function at the intervals indicated in Figure 1B. On days 10 and 28 post-MI, hearts were removed for histological assessment. Quantitative RT-PCR was performed to assess the expression of genes we previously demonstrated to be dysregulated in the infarcted heart by genetic depletion of Tip60 [15, 19, 20]. Among these targets, Mapt, Osmr, Runx1, Tnni1, and Trp53 (p53) were significantly altered in pentamidine-treated hearts at day 10 post-MI (Fig. 1C), indicating that administration of pentamidine at a dose of 3 mg/kg/day effectively antagonized Tip60 activity. Data were obtained from multiple experiments consisting of small cohorts of 4–5 mice randomly assigned to treatment with pentamidine or vehicle to achieve a minimum sample size of four for histological analyses at the two different timepoints.

As expected, at the onset of treatment on day 3 post-MI, control and pentamidine-treated groups exhibited left ventricular dysfunction across all echocardiographic parameters monitored (Fig. 2A–E). By day 10 post-MI, fractional shortening (FS; Fig. 2B) and stroke volume (SV; Fig. 2E) were preserved in pentamidine-treated mice, persisting until hearts were collected for histology at day 28. By day 19 post-MI, ejection fraction (EF), myocardial performance index (MPI), and fractional area change (FAC) were also significantly improved by pentamidine administration (Fig. 2A, C, D), which persisted at day 28. Preservation of function was consistent with improved post-MI survival of pentamidine-treated mice during the 28-day timeline (Fig. 2F). In a separate subset of mice followed for ~1.5 years (n= 4/group), functional improvement persisted for at least 40 weeks- post-MI resulting in improved long-term survival (Fig. 3). Importantly, pentamidine had no effect on any echocardiographic indices of heart function in sham-operated mice (Supp. Fig. S1). Compared to treatment with vehicle, pentamidine had no effects on electromyographic parameters of cardiac conduction in infarcted mice, including standard time intervals (PR, QRS, QTc, RR) and ST segment height, with the exception that the cQT interval was reduced in infarcted animals at the 28-day timepoint (Table 1). Pentamidine had no effect on heart and body weight measurements taken at days 10 and 28 post-MI (Supp. Fig. S2).

To assess myocardial damage directly, the extent of scarring was quantified by trichrome staining of cross-sections taken at intervals of 0.3 mm from the area between the ventricular apex and the ligation site (Fig. 4A, left). Consistent with enhanced function observed by echocardiography, pentamidine-treated hearts exhibited diminished scarring at 28 days post-MI, as indicated by significant reductions in both left ventricular area and midline length (Fig. 4A, right). To examine whether reduced scarring might be attributed to prevention of cell death, apoptosis was assessed by TUNEL (Fig. 4B, left) and cleaved caspase-3 (Fig. 4C). Although enumeration of TUNEL-positive nuclei in pentamidine-treated mice at day 28 post-MI revealed a trend toward reduced numbers of apoptotic cells in the infarct border zone (Fig. 4B, right), no discernable effects on percentages of cleaved caspase-3-positive CMs (Fig. 4C) were observed.

Immunostaining of tissue sections for phosphorylated histone H3 (pH3, Fig. 5A), 5’-bromo-2’-deoxyuridine (BrdU, Fig. 5B), and Ki67 (Fig. 5C) was performed to assess cell-cycle activation in CMs. BrdU incorporates into nuclear DNA as a thymidine analog during S-phase and pH3 identifies cells in early M-phase. At days 10 and 28 post-MI, percentages of CMs exhibiting both markers were significantly increased in the infarct border zone of hearts in pentamidine-treated mice (Fig. 5A & 5B). Also, trends toward increased numbers of CMs expressing Ki67, a marker that monitors all cell-cycle phases, were observed in the infarct border of hearts in pentamidine-treated mice (Fig. 5C).

To assess the possibility that cell-cycle activation in pentamidine-treated mice promoted CM hypertrophy, CM size was evaluated in transversely sectioned CMs outlined by WGA staining. As shown in Figure 6, CM size was reduced in both the infarct border and remote zones of hearts in pentamidine-treated mice, most prominently at the 10-day timepoint. These findings suggest that pentamidine treatment may induce CM renewal, a notion supported by the increased density of CMs observed in pentamidine-treated hearts (Fig. 6).

Tip60-mediated site-specific acetylation of lysines 4 and 7 in the minor histone variant H2A.Z – i.e., H2A.Zac^K4/K7 - has been associated with maintenance of the differentiated state in hematopoietic stem cells (HSCs; [11, 12], neurons [13, 14], skeletal myoblasts [13], and likely CMs [15]. Because it is now accepted that adult CMs must undergo dedifferentiation prior to resuming cell-cycling and regeneration after MI [33–40], we assessed whether CMs containing H2A.Zac^K4/K7 were reduced in pentamidine-treated mice. While modest in comparison with the reduction of H2A.Zac^K4/K7-positive CMs we observe after genetic depletion of Tip60 [15], pentamidine-treated mice displayed significantly decreased numbers of H2A.Zac^K4/K7-positive CMs within the infarct border zone at 10-days and 28-days post-MI (Fig. 7A), consistent with the dysregulation of genes associated with CM dedifferentiation and proliferation (Fig. 1C). As expected, no effects of pentamidine treatment were observed on the numbers of CMs containing bulk (i.e., acetylated plus non-acetylated) H2A.Z Fig. 7B).