Male and female 8–12 weeks old mice, wild type (WT) (C57BL/6J), were purchased from the Jackson Laboratory (Bar Harbor, ME, United States). The animal procedures were reviewed and subsequently approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Louisville. Furthermore, the animal care and guidelines of the National Institutes of Health (NIH, United States) were also adhered to. Since aging causes chronic congestive CHF primarily due to a chronic increase in preload volume, we created aorta-vena-cava fistula (AVF) below the kidney in WT mice treated with or without remote hind-limb ischemia (RIC) for a total of 16 weeks. AVF was created in anesthetized WT mice between the aorta and the caudal vena cava, ∼0.5 cm below the kidneys, using a 30-gauge needle. An infrarenal AVF creates an unambiguous model of chronic volume overload. In this model, arterial blood, below the kidneys, rapidly enters the venous circulation and overloads the ventricles without the contribution of the stimulation of circulating factors (Givvimani et al., 2010). This model omits the effects of mechanical injuries, such as mitral valve regurgitation and vasodilatation (Tsoporis and Leenen, 1988; Turcani and Jacob, 1998). We selected C57BL/6J mice since this strain serves as the accepted background for genetically engineered mice procedures. Also, results obtained by others have suggested extensive genetic homology between mice and humans (Qureshi et al., 1999). Since CHF due to AVF causes right ventricle (RV), pulmonary, and LV dysfunction, we analyzed RV, LV, and the lung. Due to the chances of oxidative stress and proteolytic activation happening concurrently and thus, to avoid an early activation of MMP and to prevent AVF-mediated oxidative and proteolytic stresses subsequently, we created RIC 1 day after AVF surgical process. To create RIC, the left femoral artery was ligated at the same time as the AVF for 16 weeks. The ligation was established by measuring the decreased blood flow with the help of laser Doppler system monitoring. The counter lateral leg served as the control limb for the experiments (Majumder et al., 2018a,b, 2019). A minimum of 5–8 animals/group were used in the experiments.

Body weight, lung weight, and heart weight were measured at the time of sacrifice. The blood levels of creatine kinase (CK) activity were also measured. In brief, the tissue-specific injury was determined by measuring the CK isoforms in the serum samples from each group of mice. The CK-MM represents the cardiac- and skeletal-muscle-specific isoform, while the CK-BB is primarily a nerve-specific and kidney-specific isoform, respectively. From each mouse, 10 μl of plasma was mixed with 1 μl of activator and loaded onto the CK gel as instructed by the manufacturer (QuickGel^® CK Vis Isoenzyme Procedure; Helena Laboratories, TX, United States). The gels were run at 400 V for 4:15 min. The standard (ST) amounts of CK isoforms were also loaded in parallel to the samples (Miller et al., 2000; Stanisic et al., 2021). Blood exosomes were isolated as described earlier (Familtseva et al., 2019).

Antibodies and reagent antibodies for disheveled associated activator of morphogenesis 1 (DAAM 1), DAAM 2, and regulated in development DNA damage response 1 (REDD 1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States), dynamin-related protein 1 (Drp1), DNA methyl transferase (DNMT) 3B the mitochondrial transcription factor A (TFAM) from Abcam (Cambridge, MA, United States), phosphatidylethanolamine methyltransferase (PEMT) from Lifespan Biosciences, Seattle, WA, United States, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Millipore Sigma, St. Louis, MO, USA. After post 48-h treatment period, cardiac tissue protein was isolated using protein extraction buffer [radio-immuno precipitation assay (RIPA) lysis buffer, protease inhibitor cocktail, and phenylmethylsulphonyl fluoride (PMSF)]. Lysates were spun in an extraction buffer for 12 h and then centrifuged at 12,000 × g for 15 min. The supernatant was transferred to new tubes, and protein concentrations were analyzed via Bradford protein estimation assay. Samples were run on a 10/12% sodium dodecyl sulfate (SDS)-polyacrylamide gel with Tris-glycine SDS buffer. The gel was transferred electrophoretically overnight onto a polyvinylidene difluoride (PVDF) membrane at 4°C. The membrane was blocked with a 5% milk solution for 1 h. Primary antibodies were diluted at a concentration of 1:1,000 in Tris buffered saline with Tween-20 (TBST) and incubated on the membrane overnight. All membranes were washed in TBST solution four times and then incubated with secondary horseradish peroxidase (HRP) conjugated antibody solution for 1 h at room temperature. Four TBST washing steps followed before membranes were developed using a chemiluminescent substrate in a BioRad ChemiDoc (Hercules, CA, United States). Band intensity was determined using densitometry analysis. Beta-actin was used to normalize protein loading. Equal amounts of total protein (50 μg) were resolved on SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene membranes. The membranes were probed overnight at 4°C with primary antibodies followed by 2 h incubation in secondary antibodies. The signal capturing was performed using the Bio-Rad ChemiDoc XRS + system and Image Lab software (Bio-Rad, Hercules, CA, United States). The relative optical density of protein bands was analyzed using gel software Image Lab 3.0. The membranes were stripped and re-probed with GAPDH as a loading control.

The determination of endothelial function in an isolated papillary muscle preparation does not demonstrate what happens in the entire transmyocardial wall. Therefore, to determine endocardial endothelial (EE) function, acetylcholine was perfused in a Langendorff preparation (Wang and Morgan, 1992; Gattuso et al., 1999; Mujumdar and Tyagi, 1999; Cox et al., 2002). However, this also does not differentiate the specific contribution of the regional ischemia, hypertrophy, stunning, and/or hibernation of myocytes in the myocardial wall. Rather it gives a global contractile response to cardiotonic agents. Furthermore, it does not separate the effects of the LV from the RV. Thus, using cardiac ring preparation, we compared data obtained from cardiac rings with the data obtained by Langendorff preparation in hypertensive rats and found similar results. In addition, the cardiac ring preparation separates the effect of the LV from RV. To determine the specific regional differences in contractile function, the rings will be prepared to include or to exclude the homogenous or non-homogeneous regions of the transmural myocardial wall. The endothelial-dependent cardiomyocyte function will be measured in cardiac rings prepared from the LV and RV separately from intact and endothelial-denuded hearts from sham and AVF mice. The response to acetylcholine (an endothelial-dependent) and nitroprusside (endothelial-independent) relaxation will be measured.

To determine vascular density, barium contrast X-angiography was performed as described earlier. In brief, after pentobarbital anesthesia, mice were infused with barium sulfate (0.1 g/ml) in 50 mmol/L Tris-buffer (pH 5.0) at a constant flow (∼1 ml/min) and pressure with a syringe pump through the common carotid artery. Heparin (20 U/ml) was used along with barium sulfate to visualize the vessels. The angiograms were captured using the Carestream whole animal X-ray imaging system (Carestream Molecular Imaging, Woodbridge, CT, United States) as previously described, and the vessel density was quantified using VesSeg tool (Institute for Signal Processing, University of Luebeck, Lübeck, Germany) (Machens et al., 2006; Givvimani et al., 2011; Majumder et al., 2018b;George et al., 2019).

Echocardiography was used to determine overall myocardial function using the ultrasound with the Vevo 2100 imaging system. The cardiac and aortic data were collected as described previously. Experimental animals were placed supine on a warm platform (37°C) under isoflurane anesthesia and fixed. Using an MS550D (22–25 MHz) transducer, the thoracic cavity was imaged. Aortic arch velocity and cardiographic function were assessed in pulse wave and color Doppler modes. The transducer probe was placed on the left hemithorax of the mice in the partial left decubitus position. Two-dimensionally targeted M-mode echocardiograms were obtained from a short-axis view of the LV at or just below the tip of the mitral valve leaflet and were recorded. LV size and the thickness of the LV wall were also measured. Only the M-mode ECHO with well-defined continuous interfaces of the septum and posterior wall were collected (George et al., 2020; Singh et al., 2020).

Data from sham, AVF, sham + RIC, and AVF + RIC were analyzed statistically using the one-way ANOVA to know the difference between the groups, including a Tukey’s post hoc analysis for comparison of groups, comparing AVF with sham and AVF + RIC with AVF groups. Each group has enough mice for the experiments, and the data were presented as the mean ± SEM (n = 5–8 animals/group), ^∗p < 0.05.

The levels of vascular density were decreased in HFrEF. However, these vessels were bigger in size than controls, indicative of leakage of the vessels and causing dilated cardiomyopathy. This also suggested the breakdown of blood-heart barrier (BHB) function/integrity (Figure 2A). The heart and lung weights were increased in AVF-HF, suggestive of congestive HF. Interestingly, this HF was mitigated by RIC (Figure 2B). The cardiac and skeletal muscle injuries were increased by both AVF and RIC. Interestingly, this injury was mitigated together in RIC with AVF (Figure 2C). We isolated and characterized the exosomes released during RIC (Figure 3A). The exosomes isolated from RIC groups revealed elevated levels of musclin, Beta 2-AR, CD71, and vascular endothelial growth factor (VEGF). These results suggested stimulation of bone-marrow, sympathetic activation, and skeletal muscle myokines by RIC (Figure 3B; Marsee et al., 2010; Kalani et al., 2014, 2016). The signal outside-in axis was activated by RIC in terms of inducing connexin-43, CD147, tissue Inhibitor of metalloproteinase-4 (Timp-4), AKT, TFAM, and VEGF (Figure 3C) along with the remodeling axis as by influence by TGF-b, VEGFR, and alpha-smooth muscle actin (a marker of myofibroblast), intracellular caspase1, and calpain by RIC in CHF-AVF hearts (Figure 4A). The cell-cell connexin and adhesive junctions were stimulated by RIC during CHF (Figure 4B). The regeneration axis was stimulated by RIC in CHF by increasing PGC1-a, HDAC1, SIRT1, DAAM 1, and DAAM 2 (differential expression) (Figure 4C).

The epigenetic cascades were instigated in CHF by RIC, as indicated by the levels of DNMT-1, 2, and 3b, PEMT, and betaine–homocysteine S-methyltransferase (BHMT) (Figure 5A). Since epigenetics is the hallmark of homocysteine (Hcy) generation, we measured the Hcy metabolic axis. The results suggested that RIC increased CTH and S-adenosine homocysteine hydrolase (SAHH) and decreased Hcy in CHF (Figure 5B). Since high Hcy induces oxidative stress, we measured the NF-kB, NOx2, SOD-2, SOD2, and malonaldehyde (MDA, a marker of oxidative stress). The results suggested a decrease in oxidative stress after RIC treatment during CHF (Figure 5C).

The cardiac contraction to CaCl₂ and norepinephrine were attenuated in AVF and was mitigated by RIC. The acetylcholine response and endothelial-dependent cardiac muscle relaxation were abrogated by AVF volume overload. Interestingly, the RIC mitigated this endothelial dysfunction (Figure 6A). The cardiac interstitial and perivascular fibrosis were robust in AVF hearts; however, the fibrosis was attenuated by RIC (Figure 6B). The echocardiography data revealed that AVF chronic volume overload decreased cardiac ejection fraction but treatment with RIC reversed this decrease in the ejection fraction to normal levels (Figure 7A). In summary, the results suggest that RIC releases exosomes that contain the myokine, stimulates sympathetic and bone marrow, and all these together mitigates the CHF (Figure 7B; Azevedo et al., 2001; Marsee et al., 2010; Kalani et al., 2014, 2016; Vicencio et al., 2015).