Two month-old male Wistar rats were acquired from the Central Animal House, Botucatu Medical School, UNESP. Animals were housed in a room under controlled temperature and light/dark cycle. Experimental design was approved by the Animal Experimentation Ethics Committee of Botucatu Medical School, UNESP, SP, Brazil.

The rats were randomly distributed to be fed either every day (ad libitum, AL) or every other day (intermittent fasting, IF) with a standard rat diet. After 12 weeks of these feeding regimens, all rats were subjected to coronary artery ligation and the following three groups were created: fed ad libitum (AL); fed ad libitum before MI and intermittently fasted after MI (AL/IF); and intermittently fasted before and after MI (IF). Cardiac structures and left ventricular (LV) function were assessed by transthoracic echocardiograms before MI induction, and 2 and 12 weeks after surgery. At the end of the experiment, after anesthesia, hearts were removed and mounted in the Langendorff apparatus (AL, n = 8; AL/IF, n = 9; IF, n = 13). All remaining animals were anesthetized with pentobarbital (50 mg/kg, intraperitoneal) and euthanized by thoracotomy and hearts removal. Right and left ventricles were dissected, weighed, frozen in liquid nitrogen, and kept at − 80 °C for molecular analyzes. Since in vitro perfusion can alter myocardium tissue, hearts used in Langendorff preparations were not subjected to additional evaluation.

After anesthesia with xylazine (10 mg/kg) and ketamine (70 mg/kg), MI was induced as described in our laboratory [15, 16].

Rats were lightly anesthetized with an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (1 mg/kg). Echocardiogram was performed by the same blinded examiner (KO) using a echocardiograph (General Electric Medical Systems, Vivid S6, Tirat Carmel, Israel) equipped with a 5.0–11.5 MHz multifrequency probe, according to the previously described method [17‐20]. MI size was estimated by two-dimensional image measuring end-diastolic endocardial perimeter of affected myocardium in relation to the total LV endocardial perimeter.

One day after echocardiographic evaluation, rats were anesthetized with sodium thiopental (50 mg/kg, intraperitoneal) and heparin (2000 IU, intraperitoneal) and euthanized by thoracotomy. After removing hearts, they were mounted in the Langendorff apparatus according to the method previously described [21‐23]. After recording functional data, hearts were detached, the atria and great vessels removed, and the ventricles separated and weighed [23].

Hematoxylin and eosin-stained slides from LV tissue were used to measure approximately 50 cardiomyocyte diameters as the lower distance between myocyte borders drawn across the nucleus [24‐26].

Left ventricular concentration of hydroxyproline was quantified to estimate myocardial collagen content according to a previously described method [27‐29]. In brief, myocardial samples were dried and hydrolyzed overnight at 100 °C with 6 N HCl (1 mL/10 mg dry tissue). Aliquots of 50 μL were dried again in a Speedvac Concentrator. After adding 1.0 mL of deionized water and 1.0 mL of potassium borate buffer (pH 8.7), samples were oxidized with 0.3 mL of chloramine T solution for 20 min. The addition of 1 mL of 3.6 M sodium thiosulfate stopped the oxidative process. The solution was then saturated with 1.5 g KCl. The tubes were heated in boiling water for 20 min. After extracting the aqueous layer with 2.5 mL of toluene, 1.5 mL of toluene extract were added to 0.6 mL of Ehrlich’s reagent and the color was allowed to develop for 30 min. Absorbances were read at 565 nm against a reagent blank. Deionized water and hydroxyproline standard (20 μg/mL) were used as the blank and control, respectively.

To assess the fetal gene program, we analyzed sarcoplasmic reticulum calcium ATPase (Serca 2a), myocardial α- and β-myosin heavy chain, and atrial natriuretic peptide (ANP) expression by real time RT-PCR as previously described [30‐33]. Briefly, total RNA was extracted, solubilized in RNase-free H₂O, incubated in DNase I (Invitrogen Life Technologies), quantified and reverse transcribed. Aliquots of cDNA were submitted to real-time PCR using 10 μL 2X TaqMan® Universal PCR Master Mix (Applied Biosystems) and 1 μL of customized assay (20X) containing sense and antisense primers and Taqman (Applied Biosystems, Foster City, CA, EUA) probe specific to the following genes: Serca2a (Taqman assay Rn00568762; Ref. seq. Genbank NM_017290), α-myosin (myosin heavy polypeptide 6, cardiac muscle, alpha; Taqman assay Rn00568304_m1; Ref. seq. Genbank NM_017239.1), β-myosin (myosin heavy polypeptide 7, cardiac muscle, beta; Taqman assay Rn00568328_m1; Ref. seq. Genbank NM_017240.1), and natriuretic peptide precursor type A (Taqman assay Rn00561661_m1; Ref. seq. Genbank NM_012612.1). Amplification and analysis were then performed; reactions were carried out in triplicate. After normalizing data expression to cyclophilin (Taqman assay Rn00690933_m1; Ref. seq. Genbank NM_017101), results were calculated using the CT method (2^-ΔΔCT).