Transcriptome and Functional Profile of Cardiac Myocytes Is Influenced by Biological SexCLINICAL PERSPECTIVE
Background—Although cardiovascular disease is the primary killer of women in the United States, women and female animals have traditionally been omitted from research studies. In reports that do include both sexes, significant sexual dimorphisms have been demonstrated in development, presentation, and outcome of cardiovascular disease. However, there is little understanding of the mechanisms underlying these observations. A more thorough understanding of sex-specific cardiovascular differences both at baseline and in disease is required to effectively consider and treat all patients with cardiovascular disease.
Methods and Results—We analyzed contractility in the whole rat heart, adult rat ventricular myocytes (ARVMs), and myofibrils from both sexes of rats and observed functional sex differences at all levels. Hearts and ARVMs from female rats displayed greater fractional shortening than males, and female ARVMs and myofibrils took longer to relax. To define factors underlying these functional differences, we performed an RNA sequencing experiment on ARVMs from male and female rats and identified ≈600 genes were expressed in a sexually dimorphic manner. Further analysis revealed sex-specific enrichment of signaling pathways and key regulators. At the protein level, female ARVMs exhibited higher protein kinase A activity, consistent with pathway enrichment identified through RNA sequencing. In addition, activating the protein kinase A pathway diminished the contractile sexual dimorphisms previously observed.
Conclusions—These data support the notion that sex-specific gene expression differences at baseline influence cardiac function, particularly through the protein kinase A pathway, and could potentially be responsible for differences in cardiovascular disease presentation and outcomes.
Cardiovascular disease (CVD) is the leading cause of death of American men and women.1 Even though CVD causes 1/3 of women’s deaths each year, women have traditionally been excluded from clinical trials, and female animals have been used less or sex was not reported in basic research studies.1,2 Until recently, consideration of both sexes was not required in clinical and preclinical studies focusing on CVD.3,4 This research bias has led to the development of CVD therapeutics that are either less effective or have different side effects in women when compared with men.5 Sex-specific differences in baseline cardiac function are observed in healthy adults, with women displaying better diastolic function compared with men, as well as preserved systolic function compared with men over the course of aging.6 Similar to humans, baseline differences in cardiac function have been reported in rodent studies, with females generally having better function compared with their male counterparts.7–9 These sex differences are also observed at the level of the cardiac myocyte with male rodent cardiac myocytes generally contracting more strongly and rapidly than female cells, but this difference diminishes with age.10–12 However, the mechanisms responsible for these functional differences are not well understood.
See Editorial by Coronado et al
In addition, differences between men and women are apparent in a variety of CVDs.13,14 With respect to myocardial infarction and heart failure, women are protected in that they develop the disease later in life than men.1,15 Sexually dimorphic responses to cardiac disease stimuli are also observed in a variety of animal models. For example, females are less likely to progress to heart failure in response to a variety of different pathological stressors, and male hypertensive rats typically develop greater increases in blood pressure.14
Estrogen signaling has been credited for the cardioprotection observed in women because this protection is generally lost after the onset of menopause.13 However, contradictory results from many studies reveal that this issue is still not completely understood and highlight that pathways other than estrogen signaling are undoubtedly playing a role. To completely understand these sex-specific differences, the mechanisms underlying these differences, particularly at baseline, need to be defined.
Baseline Sexual Dimorphisms in Cardiac Gene Expression
Left ventricular gene expression is sexually dimorphic in humans and rodents. In reports analyzing the left ventricle of humans, mice, and rats, cardiac genes are differentially expressed between males and females, many of which are expressed on sex chromosomes.16–18 Expression of autosomal cardiac genes also differ between the sexes, and in mice, these differences are not affected by the estrous cycle, suggesting that these differences are not because of varying circulating estrogen levels.16,18 On further investigation, enrichments of GeneOntology categories, such chemotaxis, mitochondrial function, cell cycle, inflammation, and particularly metabolism, are sexually dimorphic, suggesting that these baseline gene expression differences have functional consequences.16,19–21 Although these sexual dimorphisms are intriguing, these studies analyzed whole ventricles, which are a complex mixture of multiple cell types, including fibroblasts, myocytes, smooth muscle, and endothelial cells. In addition, most of these previous studies used microarrays, which are limited compared with next-generation sequencing methods, which provide a more unbiased and in-depth approach. To address how biological sex impacts the genetic profile of the contractile cells of the heart, we conducted an RNA sequencing experiment to analyze baseline gene expression differences between male and female rat cardiac myocytes. This study defines the basic gene expression profiles in cardiac myocytes from each of the sexes and describes the functional consequences of sex-specific gene expression. We think these findings could be useful in the development of cardiovascular therapeutics for both men and women.
Materials and Methods
All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Colorado at Boulder. Rats were fed ad libitum standard rodent chow and housed in a facility with a 12-hour light, 12-hour dark cycle. Male and female (300–400 g, 3–4 months old) Sprague Dawley, CD, rats were purchased from Charles River Laboratory and acclimated to the facility before the described experiments. The females were not staged by estrous cycle for the following experiments. For the ovariectomy experiment, 3-month-old female Sprague Dawley, CD, rats that underwent either a sham or ovariectomy procedure were purchased from Charles River Laboratory. Cardiac myocytes were isolated from sham and ovariectomy animals 3 weeks after surgery.
One week before cardiac myocyte isolation, all rats were subjected to transthoracic echocardiography. Noninvasive echocardiographic images and measurements were made using the Philips Sonos 5500 system as previously described.22 Heart rate measurements were not different between groups (Table I in the Data Supplement).
Adult Rat Ventricular Myocyte Isolation and Culture
Adult rat ventricular myocytes (ARVMs) were isolated from the left ventricle of adult rats using a Langendorff apparatus with modification of previously published protocols.19 Cells were plated on 60-mm plates coated with 10 µg/mL laminin (Invitrogen, Carlsbad, CA) in Springhorn media19 and allowed to adhere for 45 minutes. For cells used for RNA analysis, the media was removed after 45 minutes of culture, and the cells were flash frozen in liquid nitrogen for downstream experiments.
Cardiac Myocyte Contractility Assay and Analysis
ARVMs were isolated as described above with 30 mmol/L blebbistatin (Cayman Chemical, Ann Arbor, MI) added to the type II collagenase (Worthington Biochemical, Lakewood, NJ) solution, plated on laminin-coated coverslips, and allowed to settle for 2 hours in culture before the beginning of each contractility experiment. The coverslips were then transferred to the microscope (Nikon Diaphot) and superfused in Tyrode’s solution (137 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl2, 1.8 mmol/L CaCl2, 0.2 mmol/L Na2HPO4, 12 mmol/L NaHCO3, and 5.5 mmol/L D-glucose, pH 7.4). Myocytes were electrically paced via field stimulation at room temperature by using the IonOptix MyoPacer with a stimulus duration of 4 ms, voltage of 1.2× stimulation threshold, and frequency of 1 Hz. Transients from at most 5 randomly selected myocytes per coverslip were recorded for at least 30 seconds per cell. Cell length measurements and shortening dynamics were determined by edge detection
IonWizard software (IonOptix, Westwood, MA) in which at least 10 transients per cell were averaged and analyzed. To analyze differences in contractile function in response to alterations in the protein kinase A (PKA) pathway, ARVMs from both sexes were treated with 1 μmol/L bucladesine (Cayman Chemical, Ann Arbor, MI) or dimethyl sulfoxide 2 hours after isolation for 30 minutes. Contractility experiments were then performed as described above with the Tyrode’s solution supplemented with either 1 μmol/L bucladesine or dimethyl sulfoxide for the respective treatments. Cells were not in culture for >6 hours post-isolation for the duration of the contractility experiments.
Left Ventricular Myofibrillar Isolation and Analysis
We used previously published techniques to measure the force and kinetics of isolated myofibrils activated and relaxed by fast solution switching.23–25 Myofibrils were stretched 5% to 10% above slack myofibril length to set the sarcomeres to optimally loaded length of 2.1 to 2.3 μm. Average sarcomere length and myofibril diameter were measured using ImageJ (National Institutes of Health). Mounted myofibrils were activated and relaxed by rapidly translating the interface between 2 flowing streams of solutions of different pCa.23,26 Data were collected and analyzed using a customized LabView software. Measured mechanical and kinetic parameters were defined as follows: rate constant of early slow force decline (Linear kREL)—the slope of the linear regression normalized to the amplitude of relaxation transient and duration of early slow force decline—measured from onset of solution change to the beginning of the exponential force decay.
RNA Extraction, Library Preparation, and Sequencing
Total RNA was isolated using the miRNeasy mini kit (Qiagen, Valencia, CA) with on-column DNase digestion. RNA concentration was determined using the Qubit RNA BR Assay (Invitrogen), and RNA integrity was analyzed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA) with all of the RNA samples used for sequencing having an RNA integrity number of at least 9. Ribo-zero gold depleted paired end sequencing libraries were then constructed from 650 ng total RNA using the TruSeq Stranded Total RNA Sample Kit (Illumina, San Diego, CA). All libraries were sequenced using Illumina’s HiSEQ2500 with 2x125bp v4 chemistry.
RNA Sequencing Mapping, Differential Gene Expression Analysis, and Pathway Analysis
All paired end reads were demultiplexed with the Casava pipeline (v1.8.2) and adapter trimmed with Trimmomatic (v0.32). Reads were aligned to the rat rn6 genome using Tophat (v2.0.12, Table II in the Data Supplement). Both the rn6 fasta reference file and the gene annotation (gtf) file were downloaded from the UCSC genome browser database (http://genome.ucsc.edu). To be able to distinguish between multiple isoforms, the UCSC rn6 gtf file was converted with UCSC genome browser’s genePredToGtf Utility and then used for all mapping and differential expression analysis. Aligned reads were counted with HTseq count (v0.6.1), and differentially expressed genes were identified using DESeq (v1.10.1). Genes identified to be differentially expressed between sexes by at least 1.5-fold with a Padj value of <0.01 by DESeq were then analyzed using Qiagen’s ingenuity pathway analysis (IPA) software (content version: 24718999) to identify enriched networks (for complete list, see Tables III and IV in the Data Supplement). We noticed inconsistencies with the gene annotation of Hsp90aa1 between NCBI, Ensemble, and UCSC, so this gene was excluded from our DESeq and IPA analysis.
Estrogen and Androgen Response Element Analysis
We used the genome-wide position weight matrix scanner available on the Computational Cancer Genomics website (http://ccg.vital it.ch/pwmtools/pwmscan.php) to detect estrogen response elements (EREs) and androgen response elements (AREs) in genes that were enriched in the either sex using the JASPAR core vertebrate motif library searching for the Esr1 MA0112.3 and Ar MA0007.3 motifs with a P value cutoff of 0.00001. We detected the presence of the identified response elements within 10 kb of a transcription start site of genes using the Bedtools (v2.22.0) window option. Gene Ontology enrichments for distinct sets of genes, including the presence or absence of ERE or ARE in genes upregulated in male or female cardiac myocytes, was analyzed using David Bioinformatics Resource 6.8.27
Quantitative Polymerase Chain Reaction
cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and random hexamer primers. Gene expression was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using SYBR Green dye (Invitrogen) and gene-specific primer sets. All genes were normalized to 18S expression. All primer sequences used are listed in Table V in the Data Supplement. Data were collected and analyzed using a Bio-Rad CFX-96 Real-Time PCR system.
PKA Activity Assay
Sexual dimorphisms in PKA activity in ARVMs was determined using the PKA activity kit (Abcam, Cambridge, United Kingdom). After isolation, ARVMs from both sexes were cultured for 45 minutes and then washed with phenol-red free media (ThermoFisher, Rochester, NY) and flash frozen. ARVMs were collected in lysis buffer (20 mmol/L MOPS, 50 mmol/L β-glycerolphosphate, 50 mmol/L sodium fluoride, 1 mmol/mol/L sodium vanadate, 5 mmol/L EGTA, 2 mmol/L EDTA, 1% NP40, 1 mmol/L dithiothreitol, benzamidine, phenylmethane-sulphonylfluoride, and 10 μg/mL leupeptin and aprotinin), and this crude enzyme lysate was used for the remainder of the assay according the manufacturer’s protocol.
The number of animals and cells used for each experiment are denoted in the figure legends. Statistical analysis of differentially expressed genes (17 257 genes in total) was performed with DESeq (v1.10.1). DESeq uses a negative binomial model to test for differential expression. Raw P values were then adjusted (Padj values) for FDR control with the Benjamini–Hochberg procedure.28 We only considered genes for downstream analysis that had a DESeq reported Padj value of at least 0.01 and also exhibited at least a 1.5-fold difference in expression relative to the opposite sex. This adjusted Pvalue of 0.01 assumes an FDR of 1%.28 Differences between 2 groups were evaluated for statistical significance using Student 2-tailed t test using GraphPad PRISM 6 software with data presented as mean±SEM. P<0.05 was considered significant. For the Bucladesine experiment, significance was determined using a 2-way ANOVA, followed by a Tukey multiple comparisons post hoc test. For DAVID gene ontology analysis, the top 10 enriched biological keywords with at least P<0.05 were sorted based on fold-enrichment scores. Enrichment of EREs or AREs was analyzed for significance by using the hypergeometric probability calculator (https://www.geneprof.org/GeneProf/tools/hypergeometric.jsp).
Sex Differences Exist in Baseline Cardiac Function From the Level of the Whole Heart to Cardiac Myofibrils
At the level of the whole heart, in vivo echocardiography demonstrated that cardiac function was enhanced in the female animals with fractional shortening being higher in the female hearts compared with their male counterparts (Figure 1A). Similar functional differences between the sexes were also observed in isolated ARVMs with female cells displaying greater percent shortening than the male myocytes (Figure 1B). Interestingly, female ARVMs also took more time than male cells to reach peak contraction, as well as to relax. This sex-specific difference during the relaxation phase was also observed at the level of the myofibril. Female myofibrils exhibited slower relaxation duration and rate constant of the linear relaxation phase (Figure 1C).
Cardiac Myocyte Gene Expression Is Sexually Dimorphic
We next asked what molecular mechanisms could be responsible for the functional differences we observed in the cardiac myocytes by performing an RNA sequencing experiment with ARVMs from both sexes. All paired end sequencing reads were mapped to the rat rn6 genome using the Tophat pipeline leading to ≈100 million reads mapping per sample with an overall mapping rate of at least 80% (Table II in the Data Supplement). Differential expression analysis performed using DESeq identified ≈600 genes that are differentially expressed between male and female cardiac myocytes at baseline by at least 1.5-fold (Figure 2; Tables III and IV in the Data Supplement). These differences in gene expression seem to be sex specific and not merely differences among biological replicates because there are almost no statistical differences in gene expression when the samples were analyzed with DESeq randomly (Figure I in the Data Supplement). Furthermore, the 2 genes with the largest difference in expression between sexes, Ddx3 and Eif2s3y, are located on the Y chromosome (Figure 2A). In addition, there were ≈300 genes that were differentially expressed by at least 1.5-fold in one sex compared with the other (Figure 2B; Tables III and IV in the Data Supplement). To validate our results, we performed qPCR on a subset of mRNAs that were differentially expressed between the sexes and observed the same expression profile that was shown in the RNA sequencing results (Figure 3). For example, genes such as the transferrin receptor (Tfrc) and cytochrome C oxidase (Cox6c) were more highly expressed in the male cardiac myocytes by both qPCR and RNA sequencing methods (Figure 3A). Similarly, expression of the uncoupling protein 2 (Ucp2) and P450 oxidoreductase (Por) genes was higher in the female myocytes in both the RNA sequencing and qPCR data sets (Figure 3B).
Enriched Pathways and Regulators Are Distinct Between the Sexes
We next wanted to investigate the potential functional relevance of the sex-specific gene expression differences observed in cardiac myocytes by performing pathway analysis on genes that were upregulated in either sex (Tables III and IV in the Data Supplement). IPA, which integrates data across previously published studies, was used to identify different pathways, functions, and upstream regulators enriched in genes that were at least 1.5-fold higher in expression between male or female cardiac myocytes. We observed that canonical pathways predicted to be activated by IPA were distinct between sexes (Figure 4A; Tables VI and VII in the Data Supplement). For example, signaling pathways, such as PKA, Wnt/Ca2+, and inducible nitric oxide synthase, were activated in the female data set, whereas integrin, Rac, and insulin receptor pathways were enriched in genes upregulated in male ARVMs. Furthermore, genes that were more highly expressed in female cardiac myocytes are involved processes, such as gene expression, lipid and energy metabolism, and small molecular biochemistry (Figure 4B). However, the male enriched genes are more likely to function in regulating cell growth and movement, the cell cycle, and cellular death processes. To understand more about the effectors responsible for the observed gene expression and pathway differences observed between male and female ARVMs, we used the upstream regulator tools provided by IPA. We discovered that the types of upstream regulators enriched in either sex were largely distinct, similar to what we observed with the activated pathways and cellular functions (Figure 4C). In total, it seems that the sexually dimorphic gene expression differences in cardiac myocytes are pathway specific, which could have functional contractile consequences.
To understand the role of sex hormones in mediating these differences, we identified EREs or AREs within the regulatory regions of genes that were differentially expressed between male and female ARVMs. Almost half of the genes that were more highly expressed in female cardiac myocytes also harbored an ERE within 10 kb of their start site (Figures II and III in the Data Supplement; Table VIII in the Data Supplement). This was also true for genes enriched in female myocytes that contributed to the activated pathways in our IPA analysis (Figure II in the Data Supplement). We observed similar, but not significant, results for genes harboring an ARE that were enriched in male myocytes (Figures II and IV in the Data Supplement; Table IX in the Data Supplement). Furthermore, we performed gene ontology analysis on genes upregulated in either sex with or without an ERE or ARE and discovered enrichment of distinct biological keywords (Figures III and IV in the Data Supplement; Tables X–XIII in the Data Supplement). However, it is important to note that not all genes that were differentially expressed between the sexes contained these response elements (Figures II–IV in the Data Supplement; Tables VIII and IX in the Data Supplement). In addition, genes not differentially expressed between the sexes also harbored EREs or AREs (Figure V in the Data Supplement). We also analyzed the expression of sex-specific differentially expressed genes we validated by qPCR in Figure 3 in sham control and ovariectomized female animals but did not observe differences in expression of these genes despite estrogen depletion observed as decreased uterine weights in the ovariectomized animals (Figure VI in the Data Supplement).
PKA Activity Exhibits Sex-Specific Profiles
Because PKA signaling is an important mediator in cardiac physiology12 and our IPA analysis indicated this pathway was activated in female myocytes (Figure 4A), we measured PKA activity in male and female cells. Female ARVMs displayed increased PKA activity compared with their male counterparts (Figure 5A). We next perturbed the PKA pathway and performed contractility assays in ARVMs from both sexes to determine the functional relevance of the increased PKA activity in the female cells. We treated male and female ARVMs with the cAMP mimic, bucladesine, for 30 minutes to investigate how activating the PKA pathway in cells of both sexes affects contractile function. Similar to what we observe in nontreated cells (Figure 1B), the female dimethyl sulfoxide–treated cells took longer to reach peak shortening, as well as to fully relax relative to their male counterparts (Figure 5B). However, the time it took the male and female bucladesine-treated myocytes to reach peak shortening and relaxation was not statistically different from each other (Figure 5B). In addition, bucladesine treatment seems to affect myocytes of either sex differently because the 2-way ANOVA performed in this experiment resulted in statistically significant P values for an interaction effect between bucladesine treatment and biological sex (P<0.05) for the time it took the cells to relax after treatment. This suggests that activating the PKA pathway in both sexes is mitigating the functional differences observed in the dimethyl sulfoxide control cells.
Baseline Cardiac Function Is Sexually Dimorphic at Multiple Tissue Levels
Although there are multiple reports analyzing the functional differences between male and female cardiac myocytes at baseline, to our knowledge, this is the first report to demonstrate that functional sex differences exist from the level of the whole heart down to isolated myofibrils. Sex differences in whole heart function have been debated within the literature. However, in our study, we observed higher fractional shortening in the female rats relative to the males, which is consistent with previously published work in humans, mice, and rats.6,8,9 Our data also demonstrate that this functional difference is apparent at the level of the cardiac myocyte with female cells displaying greater percent shortening but taking longer to contract and relax. Although other studies have also observed that female myocytes reach peak shortening and relaxation more slowly that male cells, previous reports have commonly observed male myocytes to exhibit greater contractile function, which contrasts with our findings.29 However, a variety of differences in procedures during the contractility studies, such as load conditions, temperature, or pacing frequency,12 could account for these differences, and it is promising that our in vivo echocardiograph data support the functional differences we observed in the isolated myocytes.
This is also the first report comparing isolated myofibrillar function between sexes, further validating that the duration of the relaxation phase is consistently longer in females. In addition, it is encouraging that we observed similar functional relaxation sex differences in both our myocyte and myofibril data because these analyses used distinct experimental loading conditions. These results are particularly interesting considering that women are more likely to develop heart failure with preserved ejection fraction, which is characterized by left ventricular diastolic dysfunction and stiffness.30 The prolonged relaxation times we observed in female cardiac myocytes and myofibrils at baseline, in combination with aging, may provide the underlying mechanism that predisposes women to developing heart failure with preserved ejection fraction, but more focused research on this observation is required.
Sexually Dimorphic Gene Expression Mediates the Functional Differences Observed at Baseline
Many past studies have focused on how estrogen signaling mediates the sexual dimorphisms in cardiac disease development, and while we acknowledge the importance of estrogen in the heart, we also sought to understand what other mechanisms are involved. In addition, we previously described that estrogen receptor transcript expression is low, restricted to estrogen receptor α and not sexually dimorphic in cardiac myocytes,31 prompting our interest in understanding what other pathways are involved in mediating the functional sex differences we observed at baseline. However, differences in hormone signaling between the sexes could be a source of variation leading to the genetic and functional differences in the myocytes. The ERE and ARE motif analysis we report further implicates a role for sex hormones in mediating gene expression differences we observed (Figures II–V in the Data Supplement). Even so, it is important to keep in mind that sexual dimorphisms are much more complex than just hormone signaling. Estrogen and androgen receptors signal in a variety of genomic and rapid nongenomic mechanisms in a variety of different cell types, including cardiac cells. 13,32,33 It is well understood that estrogen receptors can interact with and tether to other transcription factors, such as Fos, Jun, and SP1, promoting expression of genes that do not contain EREs.32 Although we acknowledge the presence of an ERE/ARE within a promoter region of a particular gene suggests that gene could be regulated by that hormone, it does not necessarily mean it actually is affected by estrogen/androgen signaling. In addition, delineating which pathways are not regulated by sex hormones is complex because estrogen and androgen signaling activate a plethora critical cellular pathways, such as MAPK, PI3K, JNK, calcium, and Akt.13,33 Furthermore, we did not observe any difference in expression of the sexually dimorphic genes we validated with qPCR (Figure 3) between the sham and ovariectomized cardiac myocytes, suggesting that estrogen is not mediating these expression differences and other factors are undoubtedly playing a role. By exploring broad gene expression differences between sexes in myocytes from healthy animals, we aimed to gain further insight into how biological sex, not just sex hormones, influences cardiac function and eventually disease development within the cardiac myocyte.
Sexually dimorphic gene expression differences are apparent in multiple somatic tissues, including, liver, adipose, brain, and muscle.18,34,35 Similar to what we observed in cardiac myocytes, there are many genes that are differentially expressed between the sexes with modest fold changes;, however, the gene changes are highly tissue specific and are enriched for distinct signaling pathways.34 Several previous reports have sought to describe sex differences in cardiac gene expression. Studies analyzing healthy human and mouse hearts reported only a modest number of genes (≈30–125) that were expressed in a sexually dimorphic manner.16,36 However, a previous microarray study in our laboratory analyzing mouse left ventricles reported sex-specific expression of ≈2000 genes with most of the genes being enriched in the male samples.19 Although we detected ≈600 genes that were differentially expressed in ARVMs from either sex, we did not observe a difference in the overall number of genes that were more highly expressed in one sex relative to the other (Figure 2B). These previous studies analyzed whole heart and ventricle samples, which because of the many different cardiac cell types could account for the contrast between our findings. In addition, the previous studies used microarray methods to detect differential expression, which would have missed genes not present on the array, whereas our RNA sequencing data provide an unbiased and large-scale approach to detect differences. Interestingly, a large number of genes (≈1800) were differentially expressed in left ventricle samples from male and female patients with dilated cardiomyopathy.17 Similar to our findings, the majority of the fold changes in gene expression reported in the study were modest (≈1.5–2.0), suggesting that biological sex does not mediate large individual changes, but perhaps the enriched pathways are the most important for mediating functional differences.
Irrespective of tissue type, all studies reporting sex-specific differences in gene expression also observed enrichment for distinct pathways. Sexually dimorphic enrichment of pathways, such as the immune response and lipid metabolism, seem to be strongly conserved among a variety of tissues, such as liver, adipose, skeletal muscle, heart, as well as in our cardiac myocyte samples (Figure 4).16,18,20,34–36 Previous reports in the heart have observed GeneOntology categories, such as metabolism21, signaling transduction, regulation of cell growth, size, and cell death to be enriched in a sex-specific manner.16,19,20,36 Our results agree with these reports because Rho and Rac signaling and genes involved in regulating cell growth and death were enriched in male ARVMs, whereas female myocytes were enriched for processes, such as energy metabolism and gene expression (Figure 4). Our findings suggest that these enriched pathways are myocyte specific, but a more thorough gene expression analysis in the whole heart would need to be performed to definitively understand this observation. In addition, we observed the PKA and inducible nitric oxide synthase pathways to be enriched in genes that were more highly expressed in female cardiac myocytes, but this was not observed in the whole heart studies.16,19 Many of the pathways we observed to be sex specific are important in maintaining cardiac function. Integrin signaling, which was enriched in male cardiac myocytes relative to the females, is an integral pathway within the cardiac myocyte because it is involved in maintaining adhesions with the extracellular matrix but also in mechanotransduction and responding to hypertrophic stimuli.37 In addition, estrogen supplementation of cardiac fibroblasts attenuated the angiotensin-II increases in β1-integrin expression,38 suggesting a potential mechanism by which integrin signaling is lower in female cardiac myocytes.
PKA signaling is extremely important in maintaining proper cardiac function, as well as in responding to increases in metabolic demands that was enriched in a sexually dimorphic manner in cardiac myocytes. We also observed that the female cardiac myocytes exhibited higher PKA activity at baseline relative to their male counterparts, further validating our RNA sequencing results (Figure 5A). In addition, treating ARVMs with the cAMP mimic bucladesine abrogated the sex-specific difference in the time it took the cells to reach peak shortening or relax (Figure 5B). This suggests that altering PKA signaling is affecting the cells from either sex differently because the males experienced a slight, but not statistically significant, increase in the time it took them to reach peak shortening and baseline, but the opposite was observed in the female cells.
Because PKA signaling generally results in decreased calcium sensitivity in addition to increased relaxation rates, the increased PKA activity in the female cells was surprising. However, there are conflicting reports on the effect PKA activation has on myofibril relaxation rates, and it seems, based on our data, that sex mediates differences in this pathway.39,40 In addition, our results could be because of the bucladesine treatment protocol. A common dose of bucladesine or its equivalent dibutyryl cAMP in the literature is 1 mmol/L,41 which is much higher than the dose we used for our experiments. However, when we initially performed our contractility experiments with the 1 mmol/L dose, the cells could not maintain proper contraction on electric pacing, prompting us to use a lower concentration. Our chosen bucladesine concentration led to both an increase in p-troponin I levels, as well as consistent, reliable contractions in male ARVMs (data not shown), suggesting this dose appropriately activated the pathway without negatively impacting the cells.
To our knowledge, this study is the first to observe sex differences at multiple levels of the cardiac structure in a single model, as well as report sexually dimorphic gene expression within cardiac myocytes. In addition, our RNA sequencing analysis was able to identify an important cardiac signaling pathway, the PKA pathway, which seems to be differentially regulated between the sexes. Although understanding the influence biological sex exerts on the cardiovascular system is complex, more studies, particularly during healthy baseline conditions, are needed. By obtaining a more thorough picture of how the cardiovascular system differs between men and women, we will be better prepared to effectively treat all patients with CVD.
All RNA sequencing data are publicly available through GEO accession: GSE95231. We thank the BioFrontiers Computing Core at the University of Colorado at Boulder. We also thank Drs Angela Peter, Pamela Harvey, and Mary Allen for helpful assistance, discussions, and preparation of this article.
Sources of Funding
This work was supported, in part, by the American Heart Association 14PRE20380468 (Dr Trexler), the National Science Foundation NSF DBI 1262410 (Dr Dowell), the National Institutes of Health CCTSI KL2, 5KL2TR001080-02 (Dr Jeong), and The Tom Marsico Chair of Excellence (Dr Leinwand).
The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.117.001770/-/DC1.
- Received March 22, 2017.
- Accepted September 5, 2017.
- © 2017 American Heart Association, Inc.
- Mozaffarian D,
- Benjamin EJ,
- Go AS,
- Arnett DK,
- Blaha MJ,
- Cushman M,
- et al
- 3.↵Consideration of sex as a biological variable in NIH-funded research. 2015.
- Chung AK,
- Das SR,
- Leonard D,
- Peshock RM,
- Kazi F,
- Abdullah SM,
- et al
- Grandy SA,
- Howlett SE
- Howlett SE
- Blenck CL,
- Harvey PA,
- Reckelhoff JF,
- Leinwand LA
- Lenzen MJ,
- Rosengren A,
- Scholte op Reimer WJ,
- Follath F,
- Boersma E,
- Simoons ML,
- et al
- Fermin DR,
- Barac A,
- Lee S,
- Polster SP,
- Hannenhalli S,
- Bergemann TL,
- et al
- Murphy E,
- Amanakis G,
- Fillmore N,
- Parks RJ,
- Sun J
- Demos-Davies KM,
- Ferguson BS,
- Cavasin MA,
- Mahaffey JH,
- Williams SM,
- Spiltoir JI,
- et al
- Pugach EK,
- Blenck CL,
- Dragavon JM,
- Langer SJ,
- Leinwand LA
- Heldring N,
- Pike A,
- Andersson S,
- Matthews J,
- Cheng G,
- Hartman J,
- et al
- Yang X,
- Schadt EE,
- Wang S,
- Wang H,
- Arnold AP,
- Ingram-Drake L,
- et al
- Lindholm ME,
- Huss M,
- Solnestam BW,
- Kjellqvist S,
- Lundeberg J,
- Sundberg CJ
- Tsuji M,
- Kawasaki T,
- Matsuda T,
- Arai T,
- Gojo S,
- Takeuchi JK
- Israeli-Rosenberg S,
- Manso AM,
- Okada H,
- Ross RS
- Walker JS,
- Walker LA,
- Margulies K,
- Buttrick P,
- de Tombe P
- Somekawa S,
- Fukuhara S,
- Nakaoka Y,
- Fujita H,
- Saito Y,
- Mochizuki N
Although cardiovascular disease is the primary killer of women in the United States, women and female animals have traditionally not been included in research studies. In reports that do include both sexes, significant sexual dimorphisms have been demonstrated in the development and presentation and outcome of cardiovascular disease. However, there is little understanding of the mechanisms underlying these observations. A more thorough understanding of sex-specific cardiovascular differences both at baseline and in disease is required to effectively consider and treat all patients with cardiovascular disease. To investigate these differences at multiple levels more thoroughly, we analyzed contractility in the whole rat heart, adult rat ventricular myocytes, and myofibrils from both sexes of rats and observed functional sex differences in all preparations. In addition, we performed an RNA sequencing experiment with isolated adult rat ventricular myocytes of each sex and demonstrated that sex-specific gene expression differences are apparent at the level of the cardiac myocyte under physiological conditions. We were able to use this large-scale gene expression approach to identify the protein kinase A pathway as a potential mediator of the functional differences we observed at the level of the cardiac myocyte.