The study describes biventricular changes in myocardial function and protein composition induced by chronic VO due to ACF. In response to ACF, both ventricles displayed eccentric hypertrophy, reduced contractility, prolonged duration of action potential, upregulation of genes associated with myocardial stress (Nppa, Myh7/6 ratio) and expression changes in substrate metabolic genes consistent with enhanced glycolysis and reduced fatty acid oxidation. By proteomic analysis, we identified several novel cardiac proteins differentially regulated by ACF with qualitatively concordant changes in both ventricles. The principal finding is that in response to identical surplus volume load, the pattern of proteome alterations is almost identical between ventricles, but the magnitude of changes is relatively more pronounced in the RV than in the LV. More pronounced changes on the right side can be explained by incremental RV pressure loading due to ACF-induced pulmonary vascular disease that combines with VO, while LV is exposed to lower systolic pressure due to a shunt in systemic circulation. Therefore, quantitative differences in protein expression between ventricles are explainable by hemodynamics, rather than by an existence of a “chamber-specific” regulation10. Study suggests that the primary approach how to preserve RV function and to prevent adverse RV remodeling in HF is to lower excessive hemodynamic loading.
VO-induced changes in biventricular hemodynamics, structure and function
Echocardiography showed eccentric remodeling, relative wall thinning, increased wall stress and depressed volumetric indices of function of both ventricles exposed to ACF. Indices of regional RV function (TAPSE, RV strain and strain rate) were paradoxically increased in ACF, likely due to confounding influence of chamber geometry, grossly changed by ACF. Therefore, TAPSE or strain deformation analysis overestimate RV contractility in volume overloaded ventricles. Limited utility of regional RV function indexes is also supported by low correlation of TAPSE, RV global strain and strain rate with gene expression of markers of myocardial remodeling (Table S1), in contrast to volumetric parameters.
Hemodynamic results are consistent with previous reports, although no study utilized pressure-volume analysis of both ventricles simultaneously in this model. While LV dP/dtmax showed a trend toward reduction in ACF compared to control19,21, RV dP/dtmax was increased in ACF13,19, reflecting either afterload-dependence of this parameter or heterometric (Frank-Starling) adaptation to increased load. Load-independent measure of chamber contractility - preload-recruitable stroke work (PRSW) was reduced in both ventricles, confirming indeed depressed systolic function15. Diastolic function (dP/dtmin and tau) was impaired in ACF LV, which might contribute to development of precapillary pulmonary hypertension component22.
Despite both ventricles handle the same increase in cardiac output in ACF, the increase in myocardial mass was relatively larger in the RV compared to LV (2.5 vs 1.7-fold, corresponding to LV/RV ratio of 0.7). Larger impact of ACF on RV compared to LV was noticed previously19,16,23. The explanation could be in a different stress response compensation, or due to difference in regulation of cardiac growth between ventricles24, or it can be explained hemodynamically. Our data support the latter mechanism. Pressure-volume data showed that ACF RV has to bear relatively higher increment of hemodynamic burden than LV. RV stroke work is increased 6.4-fold while LV stroke work is increased 3.5-fold in ACF compared to normal. Larger loading of the RV can be explained by pulmonary hypertension that adds to VO of the right heart, likely due to latent pulmonary vascular disease25 that is reflected by increased transpulmonary pressure gradient (RV peak pressure-LVEDP). Pulmonary vascular disease in ACF develops due to chronic elevation of pulmonary venous pressure22 and due to excessive pulmonary blood flow16,23,25.
Isolated ventricular trabeculae showed lower attained contraction of RV compared to LV, but no impact of ACF on developed force or force-frequency relationship. Some21,26 but not all27,28 previous studies demonstrated reduced contractility of isolated cardiomyocytes21 or isolated papillary muscle preparations26,28 from rats with VO due to ACF. Further and more detailed analyses of RV and LV myofilament sensitivity are mandated. Both RV and LV from ACF group showed profound electrophysiological remodeling with almost doubling of action potential duration (APD) compared to controls. Prolongation of APD may be a compensatory mechanism how to maintain contraction strength in VO. APD prolongation is pro-arrhythmogenic and together with other mechanisms can contribute to increased risk of arrhythmic sudden death in volume-overloaded hearts29.
VO-induced changes in biventricular mRNA gene expression
VO led to upregulation of gene for natriuretic peptide A (Nppa) in both ventricles8,13,30. ACF-induced upregulation of ANP mRNA was massive and it was more pronounced in LV than in RV. In most HF animal models, including ACF31, as well as in humans with cardiac overload, the progression of cardiac hypertrophy into HF is associated with reduced expression of Myh6 gene, coding α-myosin heavy chain, either absolutely or in relation to Myh7, gene of β-myosin heavy chain 13, 31, 32. Change of Nppa gene expression and Myh7/6 ratio are therefore the most consistent molecular markers of HF and were upregulated in both VO-exposed ventricles. At a given surplus of mass, expression of Nppa or Myh7/6 were more pronounced in RV than in LV (Fig. 2C). Correlation analysis (Table S1) also showed that changes of Myh7/6 and Nppa expression changes are linked with similar hemodynamic variables, and are likely co-regulated, in contrast to metabolic genes.
Volume overload led to change in genes of myocardial substrate metabolism and bioenergetics, such as increased Glut1/4 ratio, indicative of enhanced insulin-independent glucose uptake, and increased Hk1/Mcad ratio, indicative of enhanced glycolysis with reduced transcription of genes of fatty acid β-oxidation (Mcad). These changes were demonstrated in both ventricles. Similar pattern of metabolic gene transcription program was observed previously in pressure-overloaded ventricles, including RV33. In summary, metabolic response to stress resembles a reactivation of fetal gene expression program and is uniform in terms of chamber (RV vs LV) or overload etiology33.
We did not find altered expression of genes coding Vegfa and Hif1a in failing ventricles, but we observed increased ratio of Angpt2/Angpt1 mRNA (coding antiangiogenic angiopoietin-1 and angipoietin2), mostly in ACF LV, indicative of altered angiogenic signaling, similar to the response to myocardial infarction34. There were no consistent differences in genes of cGMP-dependent signaling pathway, speaking against relevance of this pathway in response to VO. Yet myocardial cGMP concentration was increased in ACF ventricles, probably reflecting stimulation of NP receptor-associated (particulate) guanylate cyclase by elevated natriuretic peptides.
VO in both ventricles led to downregulation of apelin, a small peptide with cardioprotective, inotropic and angiogenic properties that has contra-regulatory effects to renin-angiotensin system and acts via apelin receptor (Aplnr)35. Downregulation of myocardial apelin was previously described in failing pressure-overloaded LV or RV7,35, but this is the first study that links apelin to volume overload–induced remodeling.
VO-induced changes in biventricular proteome
VO-induced changes in protein abundance were mostly similar between RV and LV. All differentially expressed proteins were either concordantly altered (≥ 2-fold change FC) in both ventricles, or significantly altered only in one ventricle with concordant and/or not significant change of expression in the second ventricle. The data thus provide no support for chamber-specific protein expression patterns in response to similar hemodynamic stress, as proposed previously on basis of interventricular differences in physiology and embryonic origin10. Yet, protein deregulation was more pronounced in the right ventricle than in left with ratio of 1:0.67, i.e. regression line was tilted from the line of equivalence towards RV (Fig. 4D). Interestingly, this ratio is numerically close to the ratio of ventricular mass increments (RV/LV mass ratio 1:0.7). Proteome data also agree with hemodynamic data, as ACF led to more pronounced change in stroke work and myocardial mass in RV than in LV.
Most proteins emerged as differentially and correspondingly regulated in both ACF ventricles, compared to sham-operated ventricles, some of them for the first time associated with response to VO. The analysis of these proteins helps to understand VO-induced myocardial remodeling shared by both ventricles and they will be discussed by functional groups.
Proteins related to extracellular matrix (ECM): The most upregulated protein in ACF ventricles was periostin (POSTN) - a non-structural component of ECM, marker of myofibroblasts, cells necessary for cardiac adaptive healing and fibrosis36. Periostin assists in deposition of fibronectin-rich ECM and collagen crosslinking37, cardiomyocyte dedifferentiation38 and is extensively upregulated by TGFβ1, angiotensin II, infarction or hemodynamic overload, including VO39,40. VO-driven upregulation is almost twice in RV than in LV. Another TGFβ1-regulated protein upregulated in ACF hearts is cytoskeleton associated protein 4 (CKAP4), known to positively correlate with activated myofibroblast markers in both mouse and human cardiac tissue and to be negative modulator of fibroblast activation in injured heart41. We report again strong upregulation of tissue-type transglutaminase 2 (TGM2) in both ACF ventricles20, both on protein or mRNA level. TGM2 is responsible for crosslinking and stiffening of ECM and it was implicated in development of HF42. Another upregulated ECM protein is Fibrillin-1, a constituent of ECM microfibrils that is enhanced in ANGII-induced cardiac fibrosis43.
Sarcomeric, cytoskeletal, and cell-cell interaction proteins: Second large group of upregulated proteins in ACF were cytoskeletal proteins, sarcomeric proteins and proteins responsible for cell-cell interaction/force transduction. We report here an upregulation of two Xin actin-binding repeat-containing proteins XIRP1 and XIRP2. These proteins with almost cardiac-specific expression are associated with intercalated disks and play a role in myofibril assembly and repair44. XIRP2 modulates the effects of ANG-II on cardiac hypertrophy, fibrosis and myosin isotype switch45, regulates voltage-gated K changes (KV1.5)46, and was found to be upregulated in RV by experimental volume-overload47. XIRPs may therefore represent potential markers of cardiac injury. Mutations of both XIRPs were associated with arrhythmic sudden cardiac death and prolonged action potential46, a feature present also in VO-ACF hearts (Fig. 3B).
From other sarcomeric proteins, we found downregulated gene for α-myosin heavy chain (MYH6), consistently with our targeted mRNA analysis and previous studies32. Across species and types of overload, the downregulation of MYH6 gene is one of the hallmarks of HF48. ACF ventricles displayed also two-fold upregulation of non-muscle myosin 10 and downregulation of tropomodulin 4. Upregulated cytoskeletal signaling protein four and-a-half LIM domains 1 (FHL-1) which binds to and regulates titin stiffness was already linked to VO-induced cardiac LV remodeling in rats30. Another sarcomeric stress-sensing element upregulated in ACF is cardiac cysteine and glycine-rich protein 3 (CSRP3 aka MLP)49. Mutations in FHL150 and CSRP3 are known to cause cardiomyopathies. VO-ventricles showed an upregulation of Annexin-5, intracellular protein that participates in Ca2+ handling, apoptosis and sarcolemma repair and is upregulated in failing human myocardium51.
One of the most upregulated proteins in ACF ventricles was neural cell adhesion molecule 1 (NCAM1, 4.5-fold), a plasma membrane protein relevant for cardiomyocyte cell-cell interactions. NCAM1 is, similarly to identified ECM proteins, regulated by TGFβ52 and overexpressed in cardiomyocytes of other HF models and failing human hearts, proportionally to severity of HF53. Another VO-upregulated protein involved in cell-cell interactions is nebulin-related anchoring protein (NRAP), an actin-associated protein localized in intercalated disc, implicated in sarcomere assembly and force transduction. NRAP overexpression in the mouse leads to right ventricular cardiomyopathy54. Intermediate filament protein synemin (SYNM) that stabilizes intercalated disc and participates in protein kinase A signaling was also upregulated; its absence leads to severe cardiac abnormalities55. ACF ventricles showed an upregulation of microtubule-associated protein 1A (MAP1A), not previously associated with HF, and upregulation of major vault protein – a member of ribonucleoprotein complex relevant for nucleo-cytoplasmatic transport56.
Metabolic genes, ROS and chaperones: Proteomic analysis confirmed upregulation of glycolytic enzymes and downregulation of FA oxidation seen in targeted PCR analysis. Specifically, ACF ventricles showed upregulation of pyruvate kinase (PKM), the final enzyme of glycolysis. Upregulation of fetal isoform (PKM2) was previously observed in failing RV due to PH57. In parallel, we observed downregulation of α and β subunits of trifunctional enzyme of β-oxidation of fatty acids (HADHA, HADHB), more pronounced in volume-overloaded RV than in LV, consistent with switch in myocardial metabolic preference typical to fetal or failing heart33,39. Downregulation of mitochondrial 2,4-dienoyl-CoA reductase, an enzyme of β-oxidation of unsaturated fatty acids, was also observed in both VO ventricles. We observed an upregulation of B (fetal) isoform of creatine kinase (CK-B) that is typical for failing heart58 and a downregulation of its mitochondrial sarcomeric isoform (CKMT2), suggesting abnormalities in creatine shuttle and energy transfer.
Both ACF ventricles, but more RV, display strong upregulation of monoaminoxidase-A (MAOA), an enzyme that is responsible for degradation of catecholamines20. Upregulation of MAOA in ACF ventricles verifies our previous observation and is confirmed also on mRNA level (Fig. 3A) and by western blot (Fig. 5). MAOA might protect myocardium from untoward effects of increased norepinephrine spillover, but it is also a ROS-producing enzyme59. If MAO-A upregulation is adaptive or maladaptive in failing myocardium is therefore not known.
Both ACF ventricles showed upregulation of heat shock proteins: HSPB7 and HSPB1. HSPB7 is expressed almost exclusively in striated muscle and is critical for cardiac sarcomere assembly and proteostasis60. Genome-wide association study found that variation in HSPB7 locus is associated with reduced LV ejection fraction61.
Failing ACF ventricles (more RV than LV) showed upregulated dimethylarginine dimethylhydrolase1 (DDAH1), an enzyme that degrades asymmetric dimethyl arginine (ADMA), an endogenous inhibitor of nitric synthase. DDAH1-deficient rats have more severe PH and RV failure if exposed to monocrotaline62. In contrast, end-stage HF patients without PH have upregulated myocardial DDAH suggesting a contraregulatory response to putative ADMA elevation and ensuing NO deficit 63.
The study has several limitations. To reduce variance, only male rats were studied. Cardiac tolerance to volume overload is worse in males than in females, i.e. changes were more pronounced64. Hemodynamics was tested only in resting state, without provocation maneuvers that could discern more subtle changes in cardiac function. Preload changing maneuvers (vena cava balloon inflation) were not performed due to technical reasons, i.e. we cannot report arterial or ventricular elastance values. Proteomic analysis did not detect some proteins that are known to be differentially regulated in HF and were even detected at mRNA level, such as collagens, apelin or apelin receptor. Such discrepancy can be explained by very low expression, insufficient solubility or low molecular weight of the protein with insufficient number of peptides generated by trypsin.
In conclusion, the study showed that ACF led to changes of molecular markers of heart failure, increased cardiac stress and altered substrate metabolism in both ventricles. RV reacted to ACF relatively more than LV, likely due to larger incremental stroke work due to pulmonary hypertension. Proteomic analysis identified high interventricular concordance of ACF-induced changes, indicating that the RV vs LV differences are explainable hemodynamically, rather than by a presence of “RV-specific” regulatory pathways. Reduction of PA pressure and RV load is therefore the primary instrument how to preserve RV function and adverse remodeling.