Mice were maintained in accordance with the Guide for Care and Use of Laboratory Animals; animal experiments were approved by the local institutional animal care committees (IACUC) of New York Medical College. When needed, isoflurane (1–1.5%, inhalation) was employed as a methodology of anesthesia. Euthanasia was attained under anesthesia by bilateral thoracotomy and removal of the heart.

For this investigation, data collected from single transgenic mice homozygous or heterozygous for the alpha-MHC-MerCreMer (αMHC-MCM) transgene were used. In these animals, the mouse cardiac-specific alpha-myosin heavy chain promoter (αMHC) directs expression of a tamoxifen-inducible Cre recombinase (MerCreMer), allowing for the temporally regulated modulation of loxP-flanked targeted genes in cardiomyocytes of bi-transgenic mice (17). Mice homozygous or heterozygous for the αMHC-MCM transgene and presenting LoxP-flanked sequences have been widely employed for inducible and conditional gene targeting. Currently, according to the Web of Science database, there are >400 published research articles citing the original investigation reporting the development of the αMHC-MCM mouse line (17). Therefore, based on the relevance of this engineered mouse line, we employed homozygous and heterozygous αMHC-MCM animals, which are typically used as control mice in studies with conditional and temporal regulation of the expressions of genes of interest.

For this purpose, B6.FVB (129)-A1cf^{Tg (Myh6–cre/Esr1*)1Jmk}/J mice (αMHC-MCM, Jackson Labs, Stock No. 005657) (15, 17), backcrossed onto C57BL/6J inbred mice by the vendor to obtain a congenic strain, were utilized for breeding. Homozygous αMHC-MerCreMer mice (Homo) were obtained by inbreed crossing. In contrast, heterozygous αMHC-MerCreMer mice (Het) were generated by selecting the F1 hybrid progeny of mice originated from the crossing of αMHC-MCM homozygous animals with hemizygous ZEG-NICD mice (Jackson Labs, Stock No. 6850), originally derived on 129/Sv strain (15, 18). The F1 hybrid progeny consisted of single transgenic heterozygous αMHC-MCM mice and double transgenic heterozygous αMHC-MCM and ZEG-NICD mice born with normal Mendelian ratio of ∼1:1. From the progeny of the latter breeding scheme, only heterozygous αMHC-MCM mice and lacking ZEG-NICD transgene were used for this study. Therefore, no genes other than the αMHC-MCM were deleted or overexpressed in mice studied in this investigation. Moreover, C57Bl/6 mice and non-carrier mice used as control for the ZEG-NICD strain (wild-type) were introduced to provide information on the influence of genetic background on parameters studies in this investigation. C57Bl/6 mice were obtained from Charles River.

Cre recombinase expression in homozygous and heterozygous αMHC-MerCreMer mice was induced by administration of tamoxifen (Sigma-Aldrich) dissolved in 10% ethanol and 90% peanut oil (Sigma-Aldrich) for 4 days (30 mg/Kg of body weight/day, i.p.) over a period of 4–8 days (15, 19). The dose of tamoxifen was optimized to minimize off-target and confounding effects of drug administration and Cre recombinase expression in the heart, including the development of transient cardiomyopathy and DNA-damage response (19, 20). Mice were enrolled in the study at 3 or more weeks after induction of gene expression. Animals in the naïve state were studied before and after tamoxifen administration and Cre recombinase expression.

Unless otherwise specified, collected data were disaggregated by sex. Electrocardiographic analysis was performed in mice with age ranging from 2.4 to 6.6 months. For tests addressing the influence of the autonomic nervous system on heart rate dynamics of ex vivo perfused hearts, organs were obtained from homozygous αMHC-MerCreMer male and female mice at 4.3–8.5 months of age.

Myocardial infarction was performed under sterile conditions via thoracotomy and coronary artery ligation (21, 22). Under isoflurane anesthesia (∼1.5%), the animal was intubated and ventilated continuously during the surgical procedure. A local anesthetic (lidocaine, ∼4 mg/kg) was injected subcutaneously in the incision site before and after the surgical procedure. The thorax was opened via the third intercostal space, the atrial appendage elevated. The left coronary artery was located, and suture (6–0 Silk DR12 Black 18” Braid, Henry Schein) was inserted around the vessel near the origin and the artery occluded. The chest was closed with suture and pneumothorax reduced by negative pressure. Skin incision was closed using a 9 mm wound clip (Fine Science Tool). To reduce postoperative pain following the procedure, buprenorphine hydrochloride (Buprenex), 0.5–1 mg/kg body weight, was injected i.p. every 12 h for a period of 48 h after surgery.

To record electrocardiograms (ECGs) in conscious animals an ECG-tunnel device (Emka Technologies) was employed (23–25). Animals were placed in a tunnel and ECGs recorded for a period of 10 min. Electrical signals were amplified with a 12 Lead ECG Amplifier (DSI, Ponemah), digitized using a 160 kHz A/D converter (DI-1120 HS, Dataq) and recorded with WinDaq software (Dataq). The bipolar lead I, II, and III and the unipolar lead aVL were collected. Electrical signals were evaluated offline with LabChart 8 for the analysis of heart rate variability and occurrence of rhythm disturbances (24).

Echocardiography was performed in conscious mice, with singlehanded manual restraint method, using an Acuson Sequoia c512 equipped with a 13 MHz (15L8) linear transducer (26–29). By this approach, m-mode images in the parasternal short axis view of the left ventricle (LV) were employed to evaluate chamber diameter and wall thickness in diastole and systole, for computation of LV volume, mass, and ejection fraction (EF) by the Teichholz formula (26–29).

Effects of pharmacological compounds on heart rate and heart rate variability (HRV) were tested by comparing 10 min ECG recordings obtained before and after drug administration. In between acquisitions, mice were returned to their cages.

To interfere with autonomic nervous system, mice were administered with the combination of atropine (0.5 mg/kg body weight, i.p.) plus propranolol (1 mg/kg body weight, i.p.) for combined block of sympathetic and parasympathetic branches of the autonomic nervous system (combined autonomic block) (24, 28). Drugs were dissolved in USP saline solution. Effects of combined autonomic block were evaluated ∼10 min after drug(s) administration.

With the animal under deep anesthesia (isoflurane) and following administration of heparin (∼200 unit, i.p.), bilateral thoracotomy was performed, the ascending aorta was cannulated with PE50 tubing connected to a 23G 3/4 needle, and the heart was excised. Subsequently, hearts were perfused in a Langendorff apparatus (Radnoti). Perfusion was accomplished at a constant pressure of ∼80 mmHg with pre-warmed Krebs–Henseleit buffer (KHB; Sigma-Aldrich) containing, in mmol/L: 118 NaCl, 4.7 KCl, 11 glucose, 1.2 MgSO₄, 1.2 KH₂PO₄, 1.8 CaCl₂, and 25 NaHCO₃, gassed with 95% O₂ and 5% CO₂ (pH 7.4) at 37^°C (27–31). The temperature was maintained by immersing the heart in a water-heated glassware reservoir (Radnoti), containing preheated KHB.

To assess electrical activity of perfused hearts in sinus rhythm, two-lead mini ECG electrodes (Harvard Apparatus) were placed on the right atrium and apex of the left ventricle, respectively, to obtain pseudo-ECG (28–30) for 5–10 min. Electrical signals were amplified (6,600 Amplifier, Gould Instruments), digitized using a 160 kHz A/D converter (DI-1120 HS, Dataq) and recorded with WinDaq software (Dataq). Approximately 5 continuous minutes of ECGs collected in sinus rhythm were employed to assess heart rate dynamics of perfused hearts. At the completion of the procedure, the four chambers of the heart were dissected. A transverse section of the left ventricle was fixed in neutral buffered, 10% formalin solution (Sigma-Aldrich) for histological analysis. Electrophysiological data were analyzed offline with LabChart 8 software.

To assess the influence of intrinsic cardiac ganglia and neural terminations on heart rate dynamics of the excised, isolated heart, ECGs were obtained from organs perfused in the Langendorff system with KHB alone or in combination with 100 nM atropine or 1 μM propranolol. Concentration of these compounds was based on previous studies utilizing isolated or innervated (32) Langendorff perfused rodent hearts (33, 34).

Analysis of heart rate and heart rate variability (HRV) was conducted on electrocardiographic recordings using LabChart 8 (ADinstruments) and the HRV module, as reported previously (24). Lead I was used to obtain HRV parameters and other leads were employed as alternative source of analysis and/or for validation of obtained results. The entire 10 min of recording was analyzed. Company presets of LabChart HRV module for the mouse were adopted, with minor modifications of parameters for beat classification to include valid RR intervals detected by the QRS complex. Ectopic beats were excluded from the computation. A report, providing parameters of HRV, was generated by the software and values exported to Microsoft Excel for quantification.

The RR interval duration was quantified by the average of RR intervals (average RR) obtained during the 10 min of acquisition. Heart rate variability (HRV) was quantified by using time-domain and frequency domain variables, together with non-linear parameters obtained from the Poincaré plots (5, 6, 24, 35, 36). Specifically, time-domain variables included standard deviation of RR intervals (SDRR), coefficient of variation of RR intervals (CVRR, obtained by dividing SDRR by the average RR interval), square root of the mean of the squared differences between adjacent RR intervals (RMSSD, an index of short-term variability). Frequency-domain variables included total power (ms²), corresponding to energy in the entire power spectrum analyzed (0–5 Hz). The spectrum of oscillations of RR interval duration was separated in very low-frequency power (VLF, between 0 and 0.15 Hz), low-frequency power (LF, between 0.15 and 1.5 Hz), and high-frequency power (HF, between 1.5 and 5 Hz). Data for VLF, LF, and HF were computed as percentage of the total power (%). Moreover, low-frequency/high-frequency ratio (LF/HF, ms²/ms²) was computed. High-frequency components of RR interval variations are attributed to respiratory-mediated modulation of heart rate by the parasympathetic system, whereas low-frequency components are attributed to baroreflex-mediated modulation of heart rate (5, 24, 37). Very low-frequency components of RR interval variation are attributed to the modulatory action of the renin-angiotensin system, thermoregulation, and partly, parasympathetic activity (24, 37). It is generally accepted that low-frequency components are influenced by both the sympathetic and parasympathetic system (5) and LF/HF ratio is an index of sympathovagal balance, estimating the ratio of sympathetic to vagus nerve tone (5, 6). However, caution has to be exercised when considering LF/HF ratio as indicator of sympathovagal balance based on the fact that low-frequency components of RR interval variability are affected by sympathetic, parasympathetic, and other unidentified factors (10, 24). Non-linear parameters were obtained from the Poincaré plot, a graphical representation of RR interval (RR_n) plotted against the next one (RR_n+1) (38, 39). Standard deviation of instantaneous beat-to-beat interval variability (SD1) and standard deviation of continuous long-term RR interval variability (SD2) were obtained by an ellipse-fitting technique of plotted data. The SD1 index correlates with the short-term variability of RR interval variation and is mainly influenced by parasympathetic modulation whereas SD2 is a measure of long-term variability and reflects sympathetic activation (24, 39). The SD1/SD2 ratio is an index of autonomic balance and it inversely correlates with the LF/HF ratio (37).

Macroscopic images of formalin-fixed transverse sections of the left ventricle (LV) at the mid-ventricular level were obtained with a stereo microscope (SW-3T13X, Amscope) equipped with a digital camera (MU1003, Amscope). Images were employed to evaluate infarct size using the midline length approach (40). Briefly, using ImageJ software and collected images, a line was manually drawn at the center of the surviving myocardium between the epicardial and endocardial surfaces and at the level of the thin transmural scar tissue. The two lines related to surviving myocardium and scar tissue formed a circumference. Infarct size expressed in percentage was derived by dividing the length of midline at the level of the scar tissue by the total length of the circumference, and multiplying by 100.

For a subgroup of hearts, transverse sections of the LV at the mid-ventricular level were embedded in paraffin and sliced to obtain thin sections (∼4 mm thickness) (21, 28). For detection of fibrotic tissue, slides were trichrome-stained (Masson’s Trichrome Stain Kit, Mastertech, StatLab) following manufacturer’s instruction (28, 41). Images were acquired using a stereo microscope (SW-3T13X, Amscope) equipped with a digital color camera (MU1003, Amscope). Images were employed to evaluate scar size using the area approach (40). Briefly, using collected images, areas of the fibrotic tissue and total area of LV section were traced manually using ImageJ software. Fibrotic tissue size was expressed as percentage of the sum of fibrotic areas by the total LV area or the section.

Data are presented in the text as fold- or percentage- change based on ratio of median values. Graphically, data are presented as scattered plots with indication of median and interquartile ranges, unless otherwise specified. Statistical analysis was performed using GraphPad Prism 9. Data were initially tested for normality (Shapiro-Wilk) and equal variance for assignment to parametric or non-parametric analysis. Parametric tests included Student’s t-test or analysis of variance followed by Bonferroni test for non-paired comparison between two or among multiple groups, respectively. For paired statistical analysis, paired t-test was employed. When normality or equal variance were not met, analysis was performed using Mann–Whitney Rank sum test or Kruskal–Wallis one-way analysis of variance on ranks followed by Dunn’s method, for non-paired comparison between two or among multiple groups, respectively. Wilcoxon signed rank test was employed for paired comparisons (15, 24, 27–29, 31, 41). Fisher’s exact test and survival curve comparison with Gehan-Breslow-Wilcoxon and Log-rank (Mantel-Cox) tests were performed using GraphPad Prism software. P < 0.05 was considered significant. Graphs were prepared using GraphPad Prism.

Heart rate variability was evaluated in naïve mice obtained from a transgenic line routinely employed in studies for temporal regulation of the expression of genes of interest in the heart. Specifically, single transgenic mice homozygous or heterozygous for a genetic construct allowing for expression of a tamoxifen-inducible Cre recombinase (MerCreMer, MCM) under the alpha-myosin heavy chain promoter (αMHC) were employed (αMHC-MCM) (15, 17). Animals were single transgenic and negative for loxP-flanked sequences. Thus, no genes other than Cre recombinase had purposely altered expression.

Homozygous αMHC-MCM^+/+ mice (Homo) were derived by inbreed crossing of animals developed and maintained in a C57Bl/6 background. In contrast, heterozygous αMHC-MCM^± mice (Het) were selected from the single transgenic F1 hybrid progeny of Homo mice crossed with hemizygous ZEG-NICD animals, derived on the 129/Sv strain (15, 18). Double heterozygous mice derived from the latter breeding scheme were not included in the study. Thus Homo and Het mice employed in this investigation differed for the number of MerCreMer alleles and for the hybrid genetic background introduced with the ZEG-NICD strain in Het hybrid mice.

Parameters of heart rate variability were obtained from electrocardiographic recordings in conscious, restrained Homo and Het mice maintained in a tunnel device over a period of 10 min (25). With respect to sex-matched Homo animals, female Het mice presented prolonged average RR interval duration (+7%) (Figures 1A,B) and enhanced RR interval variation, quantified by standard deviation of RR intervals (SDRR, 4.3-fold increase), the coefficient of variance of RR intervals (CVRR, 4-fold increase), and the square root of the mean of the squared differences between adjacent RR intervals (RMSSD, 2.6-fold increase), which reflects short-term variability (Figure 1C).

By frequency-domain analysis, total power and very low-, low-, and high-frequency components of RR interval variations were larger in Het female mice with respect to sex-matched Homo animals (Figure 1D). However, the relative distribution of the three frequency components and the LF/HF ratio, which is generally accepted as an index of sympathovagal balance (5, 6), were similar in the two groups of mice (Figures 1E,F).

By Poincaré plots and non-linear analysis, beat-to-beat (SD1) and long-term (SD2) RR interval variability were, respectively, 2.6- and 4.4-fold larger in Het female mice with respect to sex-matched Homo animals, whereas SD1/SD2 ratio, generally accepted an index of autonomic balance (37), was reduced (Figures 1G,H).

Differences in parameters of RR interval duration and heart rate variability observed between Homo and Het female mice were also detected in cohorts of male animals (Supplementary Figure 1).

Because Homo and Het animals had different genetic background, a group of C57Bl/6 mice and wild-type (non-carrier, WT) mice derived on the 129/Sv strain (18) were studied. With respect to C57Bl/6, female WT mice had prolonged RR interval duration and enhanced RR interval variation, as quantified by SDRR (7.2-fold increase), CVRR (5.3-fold increase), and RMSSD (13-fold increase) (Supplementary Figures 2A,B). Similarly, total power and very low-, low-, and high-frequency components of RR interval variations were larger in female WT mice, with respect to sex-matched C57Bl/6 animals (Supplementary Figure 2C). However, the relative distribution of the three frequency components and the LF/HF ratio, were comparable between the animals with different genetic background (Supplementary Figures 2D,E). By Poincaré plots and non-linear analysis, SD1 and SD2 were, respectively, 13- and 6.7-fold larger in female WT mice with respect to sex-matched C57Bl/6 animals (Supplementary Figure 2F). Similar results were obtained in male mice (Supplementary Figure 3).

Subsequently, to evaluate the influence of genetic background on heart rate dynamics of Homo and Het mice, parameters of heart rate variability for C57Bl/6, Homo (C57Bl/6 background), Het (hybrid C57Bl/6 and 129/Sv background) and WT (129/Sv background) mice were compared. For this analysis data for male and female animals were combined. Overall, for time-domain, frequency domain, and non-linear parameters, C57Bl/6 and Homo mice behaved similarly, but differed from Het and WT animals. In contrast, Het and WT animals had a comparable behavior in the settings of the multiple comparison test (Supplementary Figure 4). Similar results were obtained when data was disaggregated by sex (data not shown).

Therefore, inbred homozygous mice for the MerCreMer transgene present reduced RR interval duration and attenuated short- and long-term heart rate variability with respect to hybrid heterozygous animals. Similar trends were observed between C57Bl/6 and WT mice, suggesting that mouse genetic background represents a variable when considering heart rate dynamics.

To establish the contribution of autonomic nervous tone and circulating catecholamines to differences of heart rate dynamics in homozygous and heterozygous animals studied here, heart rate variability (HRV) was evaluated in the presence of blockers of muscarinic and beta-adrenergic receptors, to inhibit the effects of sympathetic/parasympathetic branches of the autonomic nervous system and circulating factors. Specifically, electrocardiographic recordings were obtained in naïve mice before (baseline, Base) and after combined autonomic block (CAB), which was achieved by administration of atropine, a muscarinic receptor antagonist, and propranolol, a beta-adrenergic receptor blocker (24, 28). CAB prolonged average RR interval duration (+10%, +12%) and reduced RMSSD (−43%, −51%) in both Homo and Het female mice, but attenuated SDRR only in Het mice (−36%) (Figure 2A). Additionally, in Het mice, CAB reduced low- (−92%) and high-frequency (−89%) oscillations of RR interval duration (Figure 2B), whereas only low-frequency components were reduced in Homo mice (−77%). For non-linear parameters, CAB affected SD1/SD2 ratio by reducing SD1 in both groups of mice, but SD2 only in Het mice (Figure 2C). Importantly, CAB abrogated differences of RR interval duration, time-domain parameters, and non-linear SD1 and SD2 observed between Homo and Het mice with intact autonomic nervous function. Moreover, CAB was equally effective in abrogating differences in heart rate variability observed between C57Bl/6 and WT mice (Supplementary Figure 5).

Thus, inhibition of sympathovagal tone response renders HRV parameters comparable in homozygous and heterozygous mice, as well as in C57Bl/6 and WT animals, suggesting that differences in heart rate dynamics in mice with different genetic background are secondary, at least in part, to altered autonomic nervous inputs.

Studies in humans and experimental models have documented that sex is an important biological variable in the physiology of the heart, in normal and diseased conditions (42–45). Thus, to define whether sex affects heart rate dynamics in the cohort of Homo and Het mice, RR interval parameters obtained from male and female naïve animals were compared. No significant differences were observed between the two sexes for each genotype, with respect to RR interval duration, time- and frequency-domain parameters, and non-linear variables (Supplementary Figures 6, 7).

Moreover, based on the genetic construct of the experimental model employed here, heart rate dynamics in Homo and Het mice was compared in cohorts of naïve mice non-treated or treated with tamoxifen, to induce Cre recombinase expression. When considering male and female animals together, differences observed between Homo and Het non-treated mice for RR interval duration and parameters of HRV were maintained following tamoxifen (Tmxf) treatment (Supplementary Figure 8). Additionally, following tamoxifen administration, male and female mice for each genotype had comparable parameters of HRV (data not shown).

To establish whether differences in heart rate dynamics observed in Homo and Het mice were coupled with alterations of cardiac performance, echocardiography was conducted. Sex- and age-matched Homo and Het mice had comparable left ventricular (LV) end-diastolic volume, ejection fraction, and cardiac output (Supplementary Figures 9A,B). LV mass was larger in male Het mice with respect to their female counterparts and with respect to male Homo animals. However, normalization of LV mass by chamber volume or body weight abrogated these differences (Supplementary Figure 9C).

Therefore, for each genotype, heart rate dynamics are comparable between male and female mice. Moreover, Homo and Het mice maintain different behavior of HRV in the absence and presence of Cre recombinase expression.

To establish the consequences of myocardial infarction (MI) on heart rate and its variability, permanent coronary artery ligation (21) was performed in Homo and Het mice. Electrocardiograms were then collected at regular time intervals up to ∼1 month after induction of the ischemic insult. Male and female mice were used in combination.

With respect to the naïve state, ECG waveforms were substantially affected in mice following myocardial infarction (Figure 3A). Specifically, QRS complex, for the various collected leads, presented abnormal orientation in infarcted animals, a feature consistent with lack of electrical activation of the damaged myocardium. By echocardiography at 2 and 4 weeks after coronary artery ligation, all infarcted animals presented akinetic anterior LV free wall (Supplementary Figure 10A), low ejection fraction, and dilated LV chamber. Importantly, Homo and Het infarcted animals had comparable LV ejection fraction, cardiac output, and LV mass. However, with respect to Homo mice, LV end-diastolic volume was increased, and the posterior wall thickness-to-chamber radius ratio was reduced in Het mice (Supplementary Figure 10B). Infarct size, evaluated by measuring the fraction of the thin, scarred tissue with respect to the entire myocardial transverse sections of the LV at the mid-ventricular level, was ∼39% in Homo and ∼44% in Het mice (Supplementary Figure 11A). For a subgroup of animals, sections were processed histologically and trichrome-stained for the identification of fibrotic tissue (Supplementary Figure 11B). The fibrotic tissue constituted ∼33 and 28% of the total area of the LV section for Homo and Het hearts (Supplementary Figure 11C).

By electrocardiographic analysis, low heart rate was transiently observed at ∼24 h after the surgical procedure, when animals were under buprenorphine treatment. Specifically, at 1 day after MI, RR interval duration increased by 16% in Homo and 19% in Het mice, with respect to naïve animals of the same genotype. This behavior, which is consistent with the reported drop in heart rate secondary to buprenorphine treatment in mice at postoperative day 1 (46), precluded the consideration of this time point for evaluation of heart rate variability.

In Homo mice, with respect to naïve animals of the same genotype, RR interval duration was prolonged at 3 days (+2%), ∼7 days (+6%), and ∼14 days after MI (+3%). In contrast, long- and short-term RR interval variation, quantified by SDRR and RMSSD, respectively, were not affected at after MI (Figure 3B). The relative contribution of low-frequency components of RR interval variation was reduced at 3 days after MI, whereas at ∼7 days, contribution of very low-frequency was attenuated and high-frequency was increased (Supplementary Figure 12). No changes were observed for non-linear parameters. When data for Homo infarcted mice were disaggregated by sex, overall comparable results were seen (data not shown).

In Het mice, with respect to naïve animals of the same genotype, RR interval duration was not altered from 3 to ∼28 days after MI. In contrast, both SDRR and RMSSD were decreased with respect to naïve mice by ∼58–78% at time points comprised between 3 and ∼28 days after MI (Figure 3C). Additionally, the relative contribution of very low-frequency components of RR interval variation was reduced at ∼14 and ∼28 days after MI with respect to naïve animals, whereas high-frequency components were increased, with consequent attenuation of LF/HF ratio (Figure 3D). The non-linear parameter SD1 and SD2 followed the pattern of reduction observed for time-domain indices (Supplementary Figure 13). When data for Het infarcted mice were disaggregated by sex, overall similar results were detected (data not shown).

Differences in RR interval duration, short- and long-term RR variability, and total power of RR interval variation between Homo and Het mice observed in the naïve state (see Figure 1 and Supplementary Figures 1, 8) were abrogated at 3–∼28 days after induction of MI. In contrast, the relative contribution of high-frequency band of RR interval variation and of LF/HF ratio, that were comparable between naïve Homo and naïve Het mice, became different for the two groups of mice at ∼14–28 days after MI (Supplementary Figure 14).

Therefore, myocardial infarction in Het, but not in Homo mice, attenuates long- and short-term RR interval variation, affects the relative contribution of very low- and high-frequency components of RR interval oscillations, and alters sympathovagal inputs in the settings of measurements in the conscious, restrained state.

To better define putative mechanisms underlying differences of heart rate dynamics observed in vivo in Homo and Het mice in the naïve state and with chronic myocardial infarction, hearts obtained from naïve animals and animals at ∼1 month after MI were studied using a Langendorff system. Male and female mice were used in combination.

Ex vivo, isolated hearts from Homo and Het naïve animals had comparable intrinsic RR interval duration, SDRR and RMSSD (Figure 4). For both Homo and Het groups, intrinsic RR interval duration was increased in infarcted hearts (+15 and +13%, respectively) in comparison to corresponding non-infarcted organs. However, RR interval variation, assessed by SDRR and RMSSD, was not affected.

In the attempt to establish the contribution of intrinsic cardiac ganglia (33, 47) on RR interval properties of explanted, perfused hearts, organs were studied in the absence or presence of muscarinic or beta-adrenergic receptor inhibitors. Duration of RR interval, SDRR, and RMSSD were comparable for heart perfused without receptor blockers, with atropine, or with propranolol (Supplementary Figure 15). These results suggest that intrinsic cardiac ganglia have limited influence on the firing rate of the sinoatrial node, under experimental conditions employed here.

Therefore, in the absence of higher centers of autonomic nervous system modulation and extrinsic factors, intrinsic heart rate dynamics is comparable in Homo and Het intact organs. Chronic infarct increases RR interval duration in hearts from Homo and Het mice, suggesting that the diseased condition affects mechanisms of pacemaker discharge.

To establish whether the peculiar behavior of heart rate dynamics in Homo and Het mice was associated with the propensity of animals to develop adverse events following MI, occurrence of premature ventricular complexes (PVCs) and ventricular tachycardia (VT) were evaluated from electrocardiographic recordings. Moreover, post-MI survival was computed for the two groups of animals. Male and female mice were used in combination.

Ventricular ectopic events were not detected in naïve mice (data not shown), but isolated or recurrent PVCs (>10 PVCs/10 min) together with VT were observed following myocardial infarction. Specifically, at 1 day after MI, recurrent PVCs were observed in 48% of Homo and 19% of Het mice, but occurrence of ectopic event progressively disappeared at later time points (Figures 5A,B). Also, VT was only detected at 1 day after MI in 17% of Homo and 11% of Het mice. For both Homo and Het mice at 1 day after MI, HRV parameters were overall comparable in mice without or with recurrent PVCs (Supplementary Figure 16).

Importantly, survival after MI was significantly reduced in Het mice with respect to Homo animals (Figure 5C). Parameter of HRV evaluated at 1 and 3 days after MI for each genotype did not discriminate between animals that survived or did not-survive (data not shown).

Thus, Homo and het mice have a different propensity to develop ectopic events and to survive in the early phase following myocardial infarction.