RNA Seq and Proteomics Analysis: We first compared RNA expression of the TGAC8 LV at 3 mo. of age to WT littermate controls utilizing RNA Sequencing and identified 11810 transcripts, of which 2323 were differentially expressed in TGAC8. 1201 transcripts were significantly upregulated and 1117 downregulated. We then performed Mass Spectrometry analysis and identified a total of 6834 proteins in left ventricle, with 2184 differentially expressed in TGAC8. 6233 transcripts and proteins were identified in combined analyses; of these, 5578 transcripts were identified only in RNASEQ and 601 proteins only in mass spec analysis. 544 transcripts and proteins had significantly coordinated changes in their expression in TGAC8 vs TW LV, and 438 of those transcripts and proteins (80.52%) changed concordantly. Top canonical pathways identified by Ingenuity Pathway Analysis (IPA) in the dataset of 544 concordant and differentially expressed genes and proteins included fatty acid beta-oxidation, aryl hydrocarbon receptor signaling, glutaryl-CoA Degradation, NRF2-mediated oxidative stress response, protein kinase A signaling and Ephrin receptor signaling. Protection against excessive cAMP-PKA signaling: To explore the underlying mechanisms involved in TGAC8 increased heart rate and changes in cardiac function, we assessed the expression of factors that could potentially limit PKA signaling, including -AR-receptor desensitization mechanisms, G-proteins, PDEs, muscarinic parasympathetic system activation and catecholamine synthesis in SAN. RNAseq data, confirmed by qRT-PCR, WB and/or Immunolabeling showed human AdCy8 was highly expressed in TGAC8 SAN tissue and expression of multiple genes coding for proteins that desensitize -AR signaling or hydrolyze cAMP was significantly greater in TGAC8. Expression of several SAN genes regulating catecholamine synthesis were significantly reduced, as was circulating plasma epinephrine. Kcnj3, RGS2, RGS5, RGS6 and IKACh are downregulated. Many pathways in TGAC8 SAN are activated to reduce signaling through -adrenergic receptors, thus desensitizing -adrenergic receptor signaling, reducing circulating catecholamines and protecting against excessive Ca2+-cAMP-PKA signaling. Metabolomics analyses: Sustained workload in the TGAC8 heart will likely be matched by metabolic remodeling leading to enhanced energy generation, redox stress elicited by sustained metabolic activity, and activation of nutrient sensing and signaling pathways to respond to those challenges. To test our hypothesis, we performed untargeted metabolomics in the LV from 3 mo old WT and TGAC8 mice and subsequent joint pathway analysis of LV proteome and metabolome profiling data. We found activation of glucose metabolism and a preferred flux redirection towards glycolysis as validated by direct measurement of enzymatic activities. Other metabolic modification in the TGAC8 include an increase in amino acids, relative depletion of fatty acids, an abundance of ketone bodies, enhanced redox metabolism, and amino acid precursors of glutathione synthesis. Together, the data are consistent with underlying LV metabolic remodeling comprising enhancement of glucose, lipid and amino acids metabolism. Redox balance-keeping pathways appear to be activated to counteract increased ROS generation under intense metabolic activity in response to sustained energy demand. Growth Factor Signaling, Biomass and Protein Quality Control: Transcriptomics, proteomics, and phospho-proteomics were combined with Ingenuity Pathway Analysis and Western blotting analyses in 3 mo old TGAC8 LV, focusing on pathways upregulated in response to chronic dynamic exercise endurance training (IGF, PI3K-AKT), changes in nutrient sensing (AMPK) and energy metabolism, and overall protein quality control and misfolded protein disposal mechanisms (autophagy). We found a significant increase in activation of the IGF and PI3K-AKT signaling, no presence of a pathologic hypertrophy profile, and increased AMPK signaling and protein synthesis (WB-SUnSET). Cardiac proteostasis assessment (unbiased ubiquitome analysis) and aggregates quantification showed a significant accumulation of soluble misfolded proteins, and increased proteasome workload. Finally, autophagy markers LC3 and SQSTM1/p62 (abundance, phosphorylation and lipidation status evaluation) were increased, together with the autophagy flux (chloroquine, CQ). Taken together, the upregulation of these survival pathways suggests an overall higher protection in TGAC8. Cardiac Cell Proliferation in the 3 Mo Old TGAC8 Heart: Echocardiography and telemetry data show transgenic mice gain biomass in left ventricular walls at a significantly greater rate than WT mice from 3 wks to 3 mos of age, but average cross-sectional area of cardiomyocytes in TGAC8 left ventricles, as well as SAN cells, are significantly smaller than WT. Cardiomyocyte progenitor cells have noticeably smaller cross-sectional area compared to mature cardiomyocytes, corroborating the hypothesis TGAC8 mice hearts exhibit enhanced cardiomyocyte proliferation. Decreased average cell size indicates TGAC8 hearts are likely adding biomass through a hyperplastic phenotype rather than through cardiomyocyte hypertrophy. Transcriptomics and proteomics analysis on LV cell lysates showed an upregulation of essential cell cycle regulators and enrichment in key signaling pathways (YAP/TAZ, -Catenin, NOTCH), gene targets and gene programs involved in cardiomyocyte cell cycle reentry, cardiac development regulators, progenitor cell maturation and ventricular developmental gene program (TBX5, HAND2, TBX20), further evidence for a hyperplastic cardiomyocyte population. Mitochondrial fitness and resilience: We evaluated the mitochondrial permeability transition pore (mPTP), a key biomarker of aging and mitochondrial fitness and found the mPTP ROS threshold was not significantly different in cardiomyocytes isolated from young 3 mo TGAC8 mice; however, a significant decline in mPTP-ROS threshold was observed in 12-mo-old animals that was significantly more pronounced in TGAC8 compared to WT. Buildup of the undegradable cellular byproduct lipofuscin, which is age-dependent and correlated with (and may contribute to) perturbed cellular/mitochondrial breakdown, was increased in TGAC8 cardiomyocytes in both age groups. We found appreciably enhanced autophagy (mitophagy) in TGAC8 mice vs. WT in both age groups. The observed decrease in mPTP-ROS threshold together with increased lipofuscin formation suggests the enhanced mitophagy flux observed in TGAC8 mice may not be enough to prevent the accumulation of functionally impaired mitochondria in TGAC8 mice which may contribute to the deterioration in mitochondrial fitness and resilience observed in aged TGAC8 mice. Heart-Brain Signaling: We performed behavioral studies in TGAC8 mice concerning the impact of forced expression of cardiac TGAC8 on central functions. At 12-16 wks, these mice neither show signs of anxiety (Elevated Plus Maze (EPM), Open Field (OF) tests), nor any evidence of spatial memory decay (Y-Maze test). However, in both EPM and in OF, we have observed a substantial difference in locomotor activity (distance traveled), in physical prowess (average speed and maximum speed), and in the degree of time spent in motion, all significantly superior in TGAC8 mice. In-depth analysis of EKG and EEG traces collected simultaneously during 24 hrs. with a Holter recording revealed an impairment of HRV in TGAC8 mice (decrease in low frequencies, decrease in high frequencies, decrease in R-R interval). Analysis of a complexity index (alpha-DFA), show an absence of control of external systems (such as PNS) on heart rate in TGAC8, and primary sleep propensity markers (theta activity; theta wave amplitude) were significantly changed.

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