Background:Recent advances in single cell sequencing have led to an increased focus on the role of cell-type composition in phenotypic presentation and disease progression. Cell-type composition research in the heart is challenging due to large, frequently multinucleated cardiomyocytes that preclude most single cell approaches from obtaining accurate measurements of cell composition. Our in silico studies reveal that ignoring cell type composition when calculating differentially expressed genes (DEGs) can have significant consequences. For example, a relatively small change in cell abundance of only 10% can result in over 25% of DEGs being false positives. Methods:We have implemented an algorithmic approach that uses snRNAseq datasets as a reference to accurately calculate cell type compositions from bulk RNAseq datasets through robust data cleaning, gene selection, and multi-sample cross-subject and cross-cell-type deconvolution. We applied our approach to cardiomyocyte-specific α1A adrenergic receptor (CM-α1A-AR) knockout mice. 8-12 week-old mice (either WT or CM-α1A-KO) were subjected to permanent left coronary artery (LCA) ligation or sham surgery (n=4 per group). Transcriptomes from the infarct border zones were collected 3 days later and analyzed using our algorithm to determine cell-type abundances, corrected differential expression calculations using DESeq2, and validated these findings using RNAscope. Results:Uncorrected DEGs for the CM-α1A-KO X LCA interaction term featured many cell-type specific genes such as Timp4 (fibroblasts) and Aplnr (cardiomyocytes) and overall GO enrichment for terms pertaining to cardiomyocyte differentiation (P=3.1E-4). Using our algorithm, we observe a striking loss of cardiomyocytes and gain in fibroblasts in the α1A-KO + LCA mice that was not recapitulated in WT + LCA animals, although we did observe a similar increase in macrophage abundance in both conditions. This recapitulates prior results that showed a much more severe heart failure phenotype in CM-α1A-KO + LCA mice. Following correction for cell-type, our DEGs now highlight a novel set of genes enriched for GO terms such as cardiac contraction (P=3.7E-5) and actin filament organization (P=6.3E-5). Conclusions:Our algorithm identifies and corrects for cell-type abundance in bulk RNAseq datasets opening new avenues for research on novel genes and pathways as well as an improved understanding of the role of cardiac cell types in cardiovascular disease.

AIMS:The sympathetic nervous system regulates numerous critical aspects of mitochondrial function in the heart through activation of adrenergic receptors (ARs) on cardiomyocytes. Mounting evidence suggests that α1-ARs, particularly the α1A subtype, are cardioprotective and may mitigate the deleterious effects of chronic β-AR activation by shared ligands. The mechanisms underlying these adaptive effects remain unclear. Here, we tested the hypothesis that α1A-ARs adaptively regulate cardiomyocyte oxidative metabolism in both the uninjured and infarcted heart.METHODS:We used high resolution respirometry, fatty acid oxidation (FAO) enzyme assays, substrate-specific electron transport chain (ETC) enzyme assays, transmission electron microscopy (TEM) and proteomics to characterize mitochondrial function comprehensively in the uninjured hearts of wild type and α1A-AR knockout mice and defined the effects of chronic β-AR activation and myocardial infarction on selected mitochondrial functions.RESULTS:We found that isolated cardiac mitochondria from α1A-KO mice had deficits in fatty acid-dependent respiration, FAO, and ETC enzyme activity. TEM revealed abnormalities of mitochondrial morphology characteristic of these functional deficits. The selective α1A-AR agonist A61603 enhanced fatty-acid dependent respiration, fatty acid oxidation, and ETC enzyme activity in isolated cardiac mitochondria. The β-AR agonist isoproterenol enhanced oxidative stress in vitro and this adverse effect was mitigated by A61603. A61603 enhanced ETC Complex I activity and protected contractile function following myocardial infarction.CONCLUSIONS:Collectively, these novel findings position α1A-ARs as critical regulators of cardiomyocyte metabolism in the basal state and suggest that metabolic mechanisms may underlie the protective effects of α1A-AR activation in the failing heart.

Introduction: Trametinib (Trm) is a highly selective inhibitor of MEK1/2, downstream targets of the RAS signaling pathway that has been used widely for the treatment of BRAF V600E/K-mutant melanoma. Multiple clinical trials are also underway investigating its use in colorectal, prostate cancer, leukemia, and triple negative breast cancer. Trm is generally well tolerated but can be associated with potentially serious cardiotoxicity in 5-20% of treated patients. The specific mechanisms driving Trm-induced cardiomyopathy (TIC) are largely unexplored. The goal of this project is to investigate the role that resident and infiltrating inflammatory cells play in TIC. Hypothesis: Our data suggest that Trm provokes cardiomyocyte immune responses including increased production of inflammatory cytokines, NF-KB signaling, and macrophage infiltration. We hypothesize that this activation occurs through DAMP-driven activation of the TLR and cGAS-STING pathway. Methods: C57BL/6 mice were gavaged with Trm 3mg/kg/day for 3 days prior to sacrifice. Heart tissue was analyzed via RNAseq, confocal microscopy, and flow cytometry. In vitro assays were completed using neonatal rat ventricular myocytes (NRVM). Results: RNAseq data of whole heart tissue from Trm treated mice surprisingly demonstrated increased expression of transcripts in inflammatory pathways. In vitro stimulation of NRVMs with Trm demonstrated increased production of inflammatory cytokines including IFNy and increased NF-KB signaling. Confocal microscopy of Trm treated heart tissue demonstrated increased CD68+ macrophage recruitment compared to control. None of these findings would have been predicted by our previous knowledge of cardiomyocyte MEK biology. Conclusions: Trm treatment is associated with cardiomyopathy in up to 20% of patients. Understanding the mechanisms driving this disease pathogenesis may help reduce likelihood of adverse cardiac events associated with future MEK inhibitor therapy used alone or in combination with other agents. Our novel findings suggest that Trm causes immune dysregulation and inflammation in the heart. Future studies will focus on further characterization of immune cell subsets involved and mechanisms of innate immune activation in TIC.

Activation of alpha-1-adrenergic receptors (α1-ARs), particularly the α1A subtype, protects the murine heart against injury, whereas human studies show that α1-AR antagonists (α-blockers) may increase the risk of heart failure. We created a cardiomyocyte-specific α1A-AR knockout mouse (cmAKO) to define the mechanisms underlying these effects and to elucidate whether they arise from cardiomyocyte α1A-ARs or systemic factors. Myocardial infarction (MI) resulted in 70% 7-day mortality in cmAKO compared to 10% in wild type (WT) mice. cmAKO mice exhibited exaggerated ventricular remodeling and increased cell death compared to WT mice 3 days post-MI, coupled to upregulation of canonical mediators of necroptosis: receptor-interacting protein (RIP) kinases RIP1 and RIP3 and mixed lineage kinase domain-like protein. An α1A-AR agonist mitigated ischemia-induced cardiomyocyte death and necroptotic signaling in vitro . A RIP1 antagonist abrogated the protective effects of α1A activation in vivo and in vitro . We found that patients at our center who were taking α-blockers at the time of MI experienced a higher risk of mortality (hazard ratio 1.53, p=0.029) during 5-year follow-up, providing clinical correlation for our experimental data. Collectively our findings indicate that cardiomyocyte α1A-ARs constrain ischemia-induced necroptosis and suggest caution in the use of α-blockers in patients at risk for MI.

Trametinib (Trm) is a highly selective inhibitor of the tyrosine kinases MEK1/2 used to treat patients with melanoma and non-small cell lung cancer featuring BRAF mutations. Although Trm is generally well tolerated, it is associated with adverse cardiovascular effects including heart failure. Furthermore, the mechanisms underlying this cardiotoxicity are unclear. Here, we assessed the hypothesis that Trm decreases oxidative phosphorylation in the mouse heart. Female FVB mice were treated with Trm (3 mg/kg/d) or vehicle (DMSO) via oral gavage (n = 10, each group). We assessed cardiac contractility on Day 0 and Day 7 using echocardiography. We then performed electron transport chain (ETC) enzyme assays in isolated cardiac mitochondria and RNA sequencing of cardiac tissue to assess ETC Complex I-IV enzyme activity and ETC transcript abundance, respectively. Additionally, we measured citrate synthase (CS) activity as a marker for mitochondrial abundance. Mice treated with Trm had decreased fractional shortening on Day 7 compared to Day 0 (Panel A, 47.7 ± 1.6% vs. 55.5 ± 0.8%, p < 0.001) (mean ± SEM), confirming the cardiotoxicity of our treatment model. Additionally, mice treated with Trm had increased lung weight to heart weight (LW/HW) ratios compared to vehicle controls (Panel B, 1.69 ± 0.11 vs. 1.39 ± 0.03, p = 0.01), indicating heart failure. Mice treated with Trm had decreased CS (7,235 ± 586 vs. 8,562 ± 192 nmol/mg/min, p = 0.024), Complex II (1,659 ± 207 vs. 2,239 ± 239, p = 0.04), and Complex IV (3,979 ± 460 vs. 5,576 ± 615, p = 0.03) activities with a trend towards decreased Complex III (2,703 ± 452 vs. 3,554 ± 434, p = 0.09) activity (Panel C). Mice treated with Trm had decreased transcript abundance of Complex I (41 of 43), Complex II (6 of 6), Complex III (8 of 8), and Complex IV (18 of 23) transcripts (Panel D). Gene Ontology cellular compartment analysis of transcriptomic data revealed that Trm treatment primarily alters abundance of transcripts related to the mitochondrion (Panel E, p adj = 3.2 x 10 -113 ). KEGG Pathway analysis revealed that oxidative phosphorylation was the most significantly altered process following Trm treatment (Panel F, p adj = 2.9 x 10 -27 ). Collectively, these findings suggest that Trm causes widespread decreases in ETC activity that may hinder cardiomyocyte oxidative phosphorylation and contractility.

Abstract: Adrenergic receptors (ARs) are G protein–coupled receptors that are stimulated by catecholamines to induce a wide array of physiological effects across tissue types. Both α1- and β-ARs are found on cardiomyocytes and regulate cardiac contractility and hypertrophy through diverse molecular pathways. Acute activation of cardiomyocyte β-ARs increases heart rate and contractility as an adaptive stress response. However, chronic β-AR stimulation contributes to the pathobiology of heart failure. By contrast, mounting evidence suggests that α1-ARs serve protective functions that may mitigate the deleterious effects of chronic β-AR activation. Here, we will review recent studies demonstrating that α1- and β-ARs differentially regulate mitochondrial biogenesis and dynamics, mitochondrial calcium handling, and oxidative phosphorylation in cardiomyocytes. We will identify potential mechanisms of these actions and focus on the implications of these findings for the modulation of contractile function in the uninjured and failing heart. Collectively, we hope to elucidate important physiological processes through which these well-studied and clinically relevant receptors stimulate and fuel cardiac contraction to contribute to myocardial health and disease.

Heart failure features decreased electron transport chain (ETC) activity in cardiomyocyte mitochondria. Previous studies have demonstrated that alpha-1A adrenergic receptor (α1A-AR) activation increases cardiac contractility in the failing heart. However, α1A-AR activation as a method of ETC regulation following cardiac injury has not been studied. Here, we assessed the hypothesis that α1A-AR activation increases ETC activity following myocardial infarction (MI) induced by left coronary artery ligation. We measured cardiac contractility using echocardiography at baseline and 7 days after MI in male CL57Bl/6J mice receiving the selective α1A-AR agonist A61603 (10 ng/kg/d) (n = 8) or vehicle (0.9% saline) (n = 9) via subcutaneous osmotic minipumps (Panel A). We then profiled citrate synthase (CS), a key enzyme in the citric acid cycle and common marker for mitochondrial mass, and ETC Complex I-IV activities in ventricular lysates. ETC enzyme activities were normalized to CS to define ETC capacity on a per mitochondrion basis. On Day 0, there were no differences in fractional shortening between groups (Panel B, 46 ± 3% vs. 49 ± 3%, p = 0.27) (mean ± SEM). On Day 7, fractional shortening was preserved in A6-treated mice compared to vehicle controls (Panel B, 24 ± 3% vs. 14 ± 1%, p = 0.01). A6 decreased CS activity compared to vehicle controls (Panel C, 11,024 ± 1,035 vs. 15,966 ± 1,068 nmol/mg/min, p < 0.01). When normalized to CS activity, A6 increased Complex I (16 ± 2% vs. 7 ± 1%, p < 0.01) (1,584 ± 116 vs. 1,106 ± 154 nmol/min/mg), Complex III (82 ± 8% vs. 56 ± 5%, p = 0.01) (8,435 ± 461 vs. 8,542 ± 327 nmol/min/mg), and Complex IV (121 ± 12% vs. 92 ± 9.%, p = 0.04) (12,523 ± 673 vs. 14,174 ± 1,156) activities (Panel D), suggesting enhanced ETC activity on a per mitochondrion basis. In conclusion, we show that a low dose of a selective α1A-AR agonist enhances cardiac contractility following MI. Increased ETC enzyme activity, particularly Complex I, may contribute to this beneficial effect. Ours is the first study in which selective α1A-AR activation has been linked to enhanced cardiac contractility and mitochondrial metabolism following MI. These findings may outline a novel mechanism through which α1A-AR activation preserves cardiac function in the injured and failing heart.

Our previous work has demonstrated essential protective roles for the endogenous cardiomyocyte alpha-1A adrenergic receptor (α1A-AR) subtype in mouse models of heart failure. However, the underlying mechanism of this protective phenotype is unclear. To address this gap in knowledge, we bred a mouse line lacking α1A-ARs on cardiomyocytes by crossing αMHC-cre mice with floxed α1A mice (CMKO= cre+ fl/fl, CMWT= cre- fl/fl), and subjected males to permanent LAD ligation. CMKO mice had increased serum HMGB1 level, larger infarcts and higher mortality. We found that RIP1/3-mediated programmed necrosis (necroptosis), but not apoptosis was exaggerated in CMKO mice 3 days after ligation. We then tested whether RIP1 inhibition with Nec-1s could mitigate this injury. Mice were given Nec-1s (1.65 mg/kg) or vehicle 10 mins prior to LAD ligation, followed by daily IV injection. Nec-1s treatment diminished post-ligation RIP1 (0.62±0.02 vs. 0.78±0.23 A.U., p=NS) and RIP3 expression (0.33±0.1 vs. 0.26±0.10 A.U., p=NS) in CMWT and CMKO mice respectively. Serum level of HMGB1 on D3 was markedly reduced in both CMWT (45.1%) and CMKO (61.1 %) after Nec-1s treatment. There was no difference between Nec-1s treated CMWT and CMKO mice (147±53 vs. 174±37 pg/mL, p=NS), indicating that blocking the RIP kinase pathway abrogates the exaggerated cell death in CMKO mice after ligation. Likewise, Nec-1s-treated CMKO mice had similar infarct areas to CMWT controls (16.2±4.5 vs. 19.9±4.6%, p=NS), further confirming that targeting necroptosis abrogates pathological damage. Collectively these Nec-1s data suggest that RIP-mediated necroptosis may account for larger infarcts in CMKO mice. Interestingly, expression of the apoptosis markers c-caspase-3 and PARP was similar between CMWT and CMKO mice, suggesting that the α1A-AR specifically regulates necroptosis. In sum, our data demonstrate that RIP kinase-mediated necroptosis contributes to susceptibility to injury in mice lacking cardiomyocyte α1A-ARs.

Decreased electron transport chain (ETC) activity in cardiac mitochondria is a hallmark of heart failure. Gain- and loss-of-function studies define the benefits of alpha-1A adrenergic receptor (α1A-AR) activation in the failing heart, such as increased cardiac contractility. However, the mechanisms behind these effects are unknown, and α1A-AR activation as a method of ETC regulation has not been studied. Here, we assessed the hypotheses that decreased α1A-AR activation reduces ETC enzyme activity, whereas increased α1A-AR activation enhances ETC enzyme activity. We profiled citrate synthase and ETC complex I-IV activities in isolated cardiac mitochondria from (1) wild-type (WT) CL57Bl/6J mice or global α1A-AR knockout mice (10-12 wks) and (2) WT mice (10-12 wks) treated with vehicle (0.9% saline) or the selective α1A-AR agonist A61603 (10 ng/kg/d, 3 d). Citrate synthase, a key enzyme in the citric acid cycle, fuels ETC activity and is a commonly used marker for mitochondrial mass. Global α1A-AR knockout increased citrate synthase activity in male mice compared to WT controls (5,292 ± 275 vs. 4,198 ± 339 nmol/min/mg, n = 5 each group, p = 0.04) (mean ± SEM) (Panel A). When normalized to citrate synthase activity, global α1A-AR knockout decreased complex I (37 ± 10% vs. 64 ± 5%, p = 0.02) (1,786 ± 421 vs. 2,766 ± 422 nmol/min/mg) and complex II (25 ± 9% vs. 50 ± 13%, p = 0.01) (1,332 ± 219 vs. 2,032 ± 213 nmol/min/mg) activities with a trend toward decreased complex IV activity (33 ± 13% vs. 49 ± 17%, p = 0.07) (1,707 ± 201 vs. 2,000 ± 238 nmol/min/mg) (Panel B). A61603 treatment led to a trend towards decreased citrate synthase activity in female mice compared to vehicle controls (6,662 ± 501 vs. 7,701 ± 421 nmol/min/mg, n = 3 each group, p = 0.09) (Panel C). When normalized to citrate synthase activity, A61603 increased complex I (27 ± 3% vs. 17 ± 2%, p = 0.03) (1,736 ± 92 vs. 1,326 ± 156 nmol/min/mg), complex III (61 ± 6% vs. 37 ± 5%, p = 0.02) (3,993 ± 258 vs. 2,894 ± 531 nmol/min/mg), and complex IV (70 ± 6% vs. 48 ± 6%, p = 0.03) (4,631 ± 100 vs. 3,676 ± 533 nmol/min/mg) activities (Panel D). In conclusion, we show that global α1A-AR knockout decreases ETC enzyme activity, while treatment with an α1A-AR agonist increases ETC enzyme activity. These findings may identify a novel mechanism through which α1A-AR activation protects the injured and failing heart.