Photobiomodulation therapy mitigates cardiovascular aging and improves survival.

BACKGROUND: Photobiomodulation (PBM) therapy, a form of low-dose light therapy, has been noted to be effective in several age-associated chronic diseases such as hypertension and atherosclerosis. Here, we examined the effects of PBM therapy on age-associated cardiovascular changes in a mouse model of accelerated cardiac aging. METHODS: Fourteen months old Adenylyl cyclase type VIII (AC8) overexpressing transgenic mice (n = 8) and their wild-type (WT) littermates (n = 8) were treated with daily exposure to Near-Infrared Light (850 nm) at 25 mW/cm2 for 2 min each weekday for a total dose of 1 Einstein (4.5 p.J/cm2 or fluence 3 J/cm2 ) and compared to untreated controls over an 8-month period. PBM therapy was administered for 3.5 months (Early Treatment period), paused, due to Covid-19 restrictions for the following 3 months, and restarted again for 1.5 months. Serial echocardiography and gait analyses were performed at monthly intervals, and serum TGF-ß1 levels were assessed following sacrifice. RESULTS: During the Early Treatment period PBM treatments: reduced the age-associated increases in left ventricular (LV) mass in both genotypes (p = 0.0003), reduced the LV end-diastolic volume (EDV) in AC8 (p = 0.04); and reduced the left atrial dimension in both genotypes (p = 0.02). PBM treatments substantially increased the LV ejection fraction (p = 0.03), reduced the aortic wall stiffness (p = 0.001), and improved gait symmetry, an index of neuro-muscular coordination (p = 0.005). The effects of PBM treatments, measured following the pause, persisted. Total TGF-ß1 levels were significantly increased in circulation (serum) in AC8 following PBM treatments (p = 0.01). We observed a striking increase in cumulative survival in PBM-treated AC8 mice (100%; p = 0.01) compared to untreated AC8 mice (43%). CONCLUSION: PBM treatment mitigated age-associated cardiovascular remodeling and reduced cardiac function, improved neuromuscular coordination, and increased longevity in an experimental animal model. These responses correlate with increased TGF-ß1 in circulation. Future mechanistic and dose optimization studies are necessary to assess these anti-aging effects of PBM, and validation in future controlled human studies is required for effective clinical translation.

Circulating MicroRNA-30d Is Associated With Response to Cardiac Resynchronization Therapy in Heart Failure and Regulates Cardiomyocyte Apoptosis: A Translational Pilot Study.

BACKGROUND: Biomarkers that predict response to cardiac resynchronization therapy (CRT) in heart failure patients with dyssynchrony (HFDYS) would be clinically important. Circulating extracellular microRNAs (miRNAs) have emerged as novel biomarkers that may also play important functional roles, but their relevance as markers for CRT response has not been examined. METHODS AND RESULTS: Comprehensive miRNA polymerase chain reaction arrays were used to assess baseline levels of 766 plasma miRNAs in patients undergoing clinically indicated CRT in an initial discovery set (n=12) with and without subsequent echocardiographic improvement at 6 months after CRT. Validation of candidate miRNAs in 61 additional patients confirmed that baseline plasma miR-30d was associated with CRT response (defined as an increase in left ventricular ejection fraction ≥10%). MiR-30d was enriched in coronary sinus blood and increased in late-contracting myocardium in a canine model of HFDYS, indicating cardiac origin with maximal expression in areas of high mechanical stress. We examined the functional effects of miR-30d in cultured cardiomyocytes and determined that miR-30d is expressed in cardiomyocytes and released in vesicles in response to mechanical stress. Overexpression of miR-30d in cultured cardiomyocytes led to cardiomyocyte growth and protected against apoptosis by targeting the mitogen-associated kinase 4, a downstream effector of tumor necrosis factor. In HFDYS patients, miR-30d plasma levels inversely correlated with high-sensitivity troponin T, a marker of myocardial necrosis. CONCLUSIONS: Baseline plasma miR-30d level is associated with response to CRT in HFDYS in this translational pilot study. MiR-30d increase in cardiomyocytes correlates with areas of increased wall stress in HFDYS and is protective against deleterious tumor necrosis factor signaling.

Gi-biased ß2AR signaling links GRK2 upregulation to heart failure.

RATIONALE: Phosphorylation of ß(2)-adrenergic receptor (ß(2)AR) by a family of serine/threonine kinases known as G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood. OBJECTIVE: We sought to determine the relative contribution of PKA- and GRK-mediated phosphorylation of ß(2)AR to the receptor coupling to G(i) signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload. METHODS AND RESULTS: Overexpression of GRK2 led to a G(i)-dependent decrease of contractile response to ßAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant ß(2)AR lacking PKA phosphorylation sites (PKA-TG) but not the wild-type ß(2)AR (WT-TG) or a mutant ß(2)AR lacking GRK sites (GRK-TG) led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure and early mortality. Furthermore, inhibition of G(i) signaling with pertussis toxin restores cardiac function in heart failure associated with increased ß(2)AR to G(i) coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of ß(2)AR by GRK and resultant increase in G(i)-biased ß(2)AR signaling play an important role in the development of heart failure. CONCLUSIONS: Our data show that enhanced ß(2)AR phosphorylation by GRK, in addition to PKA, leads the receptor to G(i)-biased signaling, which, in turn, contributes to the pathogenesis of heart failure, marking G(i)-biased ß(2)AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.

Reprogramming of cardiac phosphoproteome, proteome, and transcriptome confers resilience to chronic adenylyl cyclase-driven stress.

Our prior study (Tarasov et al., 2022) discovered that numerous adaptive mechanisms emerge in response to cardiac-specific overexpression of adenylyl cyclase type 8 (TGAC8) which included overexpression of a large number of proteins. Here, we conducted an unbiased phosphoproteomics analysis in order to determine the role of altered protein phosphorylation in the adaptive heart performance and protection profile of adult TGAC8 left ventricle (LV) at 3-4 months of age, and integrated the phosphoproteome with transcriptome and proteome. Based on differentially regulated phosphoproteins by genotype, numerous stress-response pathways within reprogrammed TGAC8 LV, including PKA, PI3K, and AMPK signaling pathways, predicted upstream regulators (e.g. PDPK1, PAK1, and PTK2B), and downstream functions (e.g. cell viability, protein quality control), and metabolism were enriched. In addition to PKA, numerous other kinases and phosphatases were hyper-phosphorylated in TGAC8 vs. WT. Hyper-phosphorylated transcriptional factors in TGAC8 were associated with increased mRNA transcription, immune responses, and metabolic pathways. Combination of the phosphoproteome with its proteome and with the previously published TGAC8 transcriptome enabled the elucidation of cardiac performance and adaptive protection profiles coordinately regulated at post-translational modification (PTM) (phosphorylation), translational, and transcriptional levels. Many stress-response signaling pathways, i.e., PI3K/AKT, ERK/MAPK, and ubiquitin labeling, were consistently enriched and activated in the TGAC8 LV at transcriptional, translational, and PTM levels. Thus, reprogramming of the cardiac phosphoproteome, proteome, and transcriptome confers resilience to chronic adenylyl cyclase-driven stress. We identified numerous pathways/function predictions via gene sets, phosphopeptides, and phosphoproteins, which may point to potential novel therapeutic targets to enhance heart adaptivity, maintaining heart performance while avoiding cardiac dysfunction.

Transcriptome of Left Ventricle and Sinoatrial Node in Young and Old C57 Mice.

Advancing age is the most important risk factor for cardiovascular diseases (CVDs). Two types of cells, within the heart pacemaker, sinoatrial node (SAN), and within the left ventricle (LV), control two crucial characteristics of heart function, heart beat rate and contraction strength. As age advances, the heart's structure becomes remodeled, and SAN and LV cell functions deteriorate, thus increasing the risk for CVDs. However, the different molecular features of age-associated changes in SAN and LV cells have never been compared in omics scale in the context of aging. We applied deep RNA sequencing to four groups of samples, young LV, old LV, young SAN and old SAN, followed by numerous bioinformatic analyses. In addition to profiling the differences in gene expression patterns between the two heart chambers (LV vs. SAN), we also identified the chamber-specific concordant or discordant age-associated changes in: (1) genes linked to energy production related to cardiomyocyte contraction, (2) genes related to post-transcriptional processing, (3) genes involved in KEGG longevity regulating pathway, (4) prolongevity and antilongevity genes recorded and curated in the GenAge database, and (5) CVD marker genes. Our bioinformatic analysis also predicted the regulation activities and mapped the expression of upstream regulators including transcription regulators and post-transcriptional regulator miRNAs. This comprehensive analysis promotes our understanding of regulation of heart functions and will enable discovery of gene-specific therapeutic targets of CVDs in advanced age.

Proteomic Landscape and Deduced Functions of the Cardiac 14-3-3 Protein Interactome.

Rationale: The 14-3-3 protein family is known to interact with many proteins in non-cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis; and the 14-3-3 network is a therapeutic target of critical metabolic and proteostatic signaling in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14-3-3 protein interactome, nor potential functional roles of 14-3-3 protein-protein interactions (PPIs) in heart has been explored. Objective: To establish the comprehensive landscape and characterize the functional role of cardiac 14-3-3 PPIs. Methods and Results: We evaluated both RNA expression and protein abundance of 14-3-3 isoforms in mouse heart, followed by co-immunoprecipitation of 14-3-3 proteins and mass spectrometry in left ventricle. We identified 52 proteins comprising the cardiac 14-3-3 interactome. Multiple bioinformatic analyses indicated that more than half of the proteins bound to 14-3-3 are related to mitochondria; and the deduced functions of the mitochondrial 14-3-3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I. Binding to ribosomal proteins, 14-3-3 proteins likely coordinate protein synthesis and protein quality control. Localizations of 14-3-3 proteins to mitochondria and ribosome were validated via immunofluorescence assays. The deduced function of cardiac 14-3-3 PPIs is to regulate cardiac metabolic homeostasis and proteostasis. Conclusions: Thus, the cardiac 14-3-3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy.

A remarkable adaptive paradigm of heart performance and protection emerges in response to marked cardiac-specific overexpression of ADCY8.

Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TGAC8, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TGAC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TGAC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TGAC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.

RGS2 is a primary terminator of ß2-adrenergic receptor-mediated G(i) signaling.

Two major ß-adrenergic receptor (ßAR) subtypes, ß(1)AR and ß(2)AR, are expressed in mammalian heart with ß(1)AR coupling to G(s) and ß(2)AR dually coupling to G(s) and G(i) proteins. In many types of chronic heart failure, myocardial contractile response to both ß(1)AR and ß(2)AR stimulation is severely impaired. The dysfunction of ßAR signaling in failing hearts is largely attributable to an increase in G(i) signaling, because disruption of the G(i) signaling restores myocardial contractile response to ß(1)AR as well as ß(2)AR stimulation. However, the mechanism terminating the ß(2)AR-G(i) signaling remains elusive, while it has been shown activation of the G(i) signaling is dependent on agonist stimulation and subsequent PKA-mediated phosphorylation of the receptor. Here we demonstrate that regulator of G protein signaling 2 (RGS2) is a primary terminator of the ß(2)AR-G(i) signaling. Specifically, prolonged absence of agonist stimulation for 24h impairs the ß(2)AR-G(i) signaling, resulting in enhanced ß(2)AR- but not ß(1)AR-mediated contractile response in cultured adult mouse cardiomyocytes. Increased ß(2)AR contractile response is accompanied by a selective upregulation of RGS2 in the absence of alterations in other major cardiac RGS proteins (RGS3-5) or G(s), G(i) or ßAR subtypes. Administration of a ßAR agonist, isoproterenol (ISO, 1.0 nM), prevents RGS2 upregulation and restores the ß(2)AR-G(i) signaling in cultured cells. Furthermore, RGS2 ablation, similar to ßAR agonist stimulation, sustains the ß(2)AR-G(i) signaling in cultured cells, whereas adenoviral overexpression of RGS2 suppresses agonist-activated ß(2)AR-G(i) signaling in cardiomyocytes and HEK293 cells. These findings not only define RGS2 as a novel negative regulator of the ß(2)AR-G(i) signaling but also provide a potential novel target for the treatment of chronic heart failure.

Electrophysiological consequences of dyssynchronous heart failure and its restoration by resynchronization therapy.

BACKGROUND: Cardiac resynchronization therapy (CRT) is widely applied in patients with heart failure and dyssynchronous contraction (DHF), but the electrophysiological consequences of CRT in heart failure remain largely unexplored. METHODS AND RESULTS: Adult dogs underwent left bundle-branch ablation and either right atrial pacing (190 to 200 bpm) for 6 weeks (DHF) or 3 weeks of right atrial pacing followed by 3 weeks of resynchronization by biventricular pacing at the same pacing rate (CRT). Isolated left ventricular anterior and lateral myocytes from nonfailing (control), DHF, and CRT dogs were studied with the whole-cell patch clamp. Quantitative polymerase chain reaction and Western blots were performed to measure steady state mRNA and protein levels. DHF significantly reduced the inward rectifier K(+) current (I(K1)), delayed rectifier K(+) current (I(K)), and transient outward K(+) current (I(to)) in both anterior and lateral cells. CRT partially restored the DHF-induced reduction of I(K1) and I(K) but not I(to), consistent with trends in the changes in steady state K(+) channel mRNA and protein levels. DHF reduced the peak inward Ca(2+) current (I(Ca)) density and slowed I(Ca) decay in lateral compared with anterior cells, whereas CRT restored peak I(Ca) amplitude but did not hasten decay in lateral cells. Calcium transient amplitudes were depressed and the decay was slowed in DHF, especially in lateral myocytes. CRT hastened the decay in both regions and increased the calcium transient amplitude in lateral but not anterior cells. No difference was found in Ca(V)1.2 (alpha1C) mRNA or protein expression, but reduced Ca(V)beta2 mRNA was found in DHF cells. DHF reduced phospholamban, ryanodine receptor, and sarcoplasmic reticulum Ca(2+) ATPase and increased Na(+)-Ca(2+) exchanger mRNA and protein. CRT did not restore the DHF-induced molecular remodeling, except for sarcoplasmic reticulum Ca(2+) ATPase. Action potential durations were significantly prolonged in DHF, especially in lateral cells, and CRT abbreviated action potential duration in lateral but not anterior cells. Early afterdepolarizations were more frequent in DHF than in control cells and were reduced with CRT. CONCLUSIONS: CRT partially restores DHF-induced ion channel remodeling and abnormal Ca(2+) homeostasis and attenuates the regional heterogeneity of action potential duration. The electrophysiological changes induced by CRT may suppress ventricular arrhythmias, contribute to the survival benefit of this therapy, and improve the mechanical performance of the heart.

Mechanisms of enhanced beta-adrenergic reserve from cardiac resynchronization therapy.

BACKGROUND: Cardiac resynchronization therapy (CRT) is the first clinical heart failure treatment that improves chamber systolic function in both the short-term and long-term yet also reduces mortality. The mechanical impact of CRT is immediate and well documented, yet its long-term influences on myocyte function and adrenergic modulation that may contribute to its sustained benefits are largely unknown. METHODS AND RESULTS: We used a canine model of dyssynchronous heart failure (DHF; left bundle ablation, atrial tachypacing for 6 weeks) and CRT (DHF for 3 weeks, biventricular tachypacing for subsequent 3 weeks), contrasting both to nonfailing controls. CRT restored contractile synchrony and improved systolic function compared with DHF. Myocyte sarcomere shortening and calcium transients were markedly depressed at rest and after isoproterenol stimulation in DHF (both anterior and lateral walls), and CRT substantially improved both. In addition, beta(1) and beta(2) stimulation was enhanced, coupled to increased beta(1) receptor abundance but no change in binding affinity. CRT also augmented adenylate cyclase activity over DHF. Inhibitory G-protein (Galpha(i)) suppression of beta-adrenergic stimulation was greater in DHF and reversed by CRT. Galpha(i) expression itself was unaltered; however, expression of negative regulators of Galpha(i) signaling (particularly RGS3) rose uniquely with CRT over DHF and controls. CRT blunted elevated myocardial catecholamines in DHF, restoring levels toward control. CONCLUSIONS: CRT improves rest and beta-adrenergic-stimulated myocyte function and calcium handling, upregulating beta(1) receptors and adenylate cyclase activity and suppressing G(i)-coupled signaling associated with novel RGS upregulation. The result is greater rest and sympathetic reserve despite reduced myocardial neurostimulation as components underlying its net benefit.

Rethinking Resynch: Exploring Mechanisms of Cardiac Resynchroniztion Beyond Wall Motion Control.

Cardiac resynchronization (CRT) is a widely used clinical treatment for heart failure patients with depressed function and discoordinate contraction due to conduction delay. It is unique among heart failure treatments as it both acutely and chronically enhances systolic function yet also prolongs survival. While improved chamber mechano-energetics has been considered a primary mechanism for CRT benefit, new animal model data are revealing novel and in many instances unique cellular and molecular modifications from the treatment. Examples of these changes are the reversal of marked regional heterogeneity of the transcriptome and stress kinase signaling, improved ion channel function involved with electrical repolarization, enhanced sarcomere function and calcium handling and upregulation of beta-adrenergic responses, and improved mitochondrial energetic efficiency associated with targeted changes in the mitochondrial proteome. Exploration of these mechanisms may reveal key insights into how CRT can indeed get the failing heart to contract more and perform more work, yet not worsen long-term failure. These changes may provide a more biological marker for both the appropriate patients for CRT as well as point the way for new therapeutic avenues for heart failure in general.

Reversal of global apoptosis and regional stress kinase activation by cardiac resynchronization.

BACKGROUND: Cardiac dyssynchrony in the failing heart worsens global function and efficiency and generates regional loading disparities that may exacerbate stress-response molecular signaling and worsen cell survival. We hypothesized that cardiac resynchronization (CRT) from biventricular stimulation reverses such molecular abnormalities at the regional and global levels. METHODS AND RESULTS: Adult dogs (n=27) underwent left bundle-branch radiofrequency ablation, prolonging the QRS by 100%. Dogs were first subjected to 3 weeks of atrial tachypacing (200 bpm) to induce dyssynchronous heart failure (DHF) and then randomized to either 3 weeks of additional atrial tachypacing (DHF) or biventricular tachypacing (CRT). At 6 weeks, ejection fraction improved in CRT (2.8+/-1.8%) compared with DHF (-4.4+/-2.7; P=0.02 versus CRT) dogs, although both groups remained in failure with similarly elevated diastolic pressures and reduced dP/dtmax. In DHF, mitogen-activated kinase p38 and calcium-calmodulin-dependent kinase were disproportionally expressed/activated (50% to 150%), and tumor necrosis factor-alpha increased in the late-contracting (higher-stress) lateral versus septal wall. These disparities were absent with CRT. Apoptosis assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining, caspase-3 activity, and nuclear poly ADP-ribose polymerase cleavage was less in CRT than DHF hearts and was accompanied by increased Akt phosphorylation/activity. Bcl-2 and BAD protein diminished with DHF but were restored by CRT, accompanied by marked BAD phosphorylation, enhanced BAD-14-3-3 interaction, and reduced phosphatase PP1alpha, consistent with antiapoptotic effects. Other Akt-coupled modulators of apoptosis (FOXO-3alpha and GSK3beta) were more phosphorylated in DHF than CRT and thus less involved. CONCLUSIONS: CRT reverses regional and global molecular remodeling, generating more homogeneous activation of stress kinases and reducing apoptosis. Such changes are important benefits from CRT that likely improve cardiac performance and outcome.

Reversal of cardiac hypertrophy and fibrosis from pressure overload by tetrahydrobiopterin: efficacy of recoupling nitric oxide synthase as a therapeutic strategy.

BACKGROUND: Sustained pressure overload induces pathological cardiac hypertrophy and dysfunction. Oxidative stress linked to nitric oxide synthase (NOS) uncoupling may play an important role. We tested whether tetrahydrobiopterin (BH4) can recouple NOS and reverse preestablished advanced hypertrophy, fibrosis, and dysfunction. METHODS AND RESULTS: C57/Bl6 mice underwent transverse aortic constriction for 4 weeks, increasing cardiac mass (190%) and diastolic dimension (144%), lowering ejection fraction (-46%), and triggering NOS uncoupling and oxidative stress. Oral BH4 was then administered for 5 more weeks of pressure overload. Without reducing loading, BH4 reversed hypertrophy and fibrosis, recoupled endothelial NOS, lowered oxidant stress, and improved chamber and myocyte function, whereas untreated hearts worsened. If BH4 was started at the onset of pressure overload, it did not suppress hypertrophy over the first week when NOS activity remained preserved even in untreated transverse aortic constriction hearts. However, BH4 stopped subsequent remodeling when NOS activity was otherwise declining. A broad antioxidant, Tempol, also reduced oxidant stress yet did not recouple NOS or reverse worsened hypertrophy/fibrosis from sustained transverse aortic constriction. Microarray analysis revealed very different gene expression profiles for both treatments. BH4 did not enhance net protein kinase G activity. Finally, transgenic mice with enhanced BH4 synthesis confined to endothelial cells were unprotected against pressure overload, indicating that exogenous BH4 targeted myocytes and fibroblasts. CONCLUSIONS: NOS recoupling by exogenous BH4 ameliorates preexisting advanced cardiac hypertrophy/fibrosis and is more effective than a less targeted antioxidant approach (Tempol). These data highlight the importance of myocyte NOS uncoupling in hypertrophic heart disease and support BH4 as a potential new approach to treat this disorder.

Overexpression of a Neuronal Type Adenylyl Cyclase (Type 8) in Sinoatrial Node Markedly Impacts Heart Rate and Rhythm.

Heart rate (HR) and HR variability (HRV), predictors of over-all organism health, are widely believed to be driven by autonomic input to the sinoatrial node (SAN), with sympathetic input increasing HR and reducing HRV. However, variability in spontaneous beating intervals in isolated SAN tissue and single SAN cells, devoid of autonomic neural input, suggests that clocks intrinsic to SAN cells may also contribute to HR and HRV in vivo. We assessed contributions of both intrinsic and autonomic neuronal input mechanisms of SAN cell function on HR and HRV via in vivo, telemetric EKG recordings. This was done in both wild type (WT) mice, and those in which adenylyl cyclase type 8 (ADCY8), a main driver of intrinsic cAMP-PKA-Ca2+ mediated pacemaker function, was overexpressed exclusively in the heart (TGAC8). We hypothesized that TGAC8 mice would: (1) manifest a more coherent pattern of HRV in vivo, i.e., a reduced HRV driven by mechanisms intrinsic to SAN cells, and less so to modulation by autonomic input and (2) utilize unique adaptations to limit sympathetic input to a heart with high levels of intrinsic cAMP-Ca2+ signaling. Increased adenylyl cyclase (AC) activity in TGAC8 SAN tissue was accompanied by a marked increase in HR and a concurrent marked reduction in HRV, both in the absence or presence of dual autonomic blockade. The marked increase in intrinsic HR and coherence of HRV in TGAC8 mice occurred in the context of: (1) reduced HR and HRV responses to ß-adrenergic receptor (ß-AR) stimulation; (2) increased transcription of genes and expression of proteins [ß-Arrestin, G Protein-Coupled Receptor Kinase 5 (GRK5) and Clathrin Adaptor Protein (Dab2)] that desensitize ß-AR signaling within SAN tissue, (3) reduced transcripts or protein levels of enzymes [dopamine beta-hydorxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT)] required for catecholamine production in intrinsic cardiac adrenergic cells, and (4) substantially reduced plasma catecholamine levels. Thus, mechanisms driven by cAMP-PKA-Ca2+ signaling intrinsic to SAN cells underlie the marked coherence of TGAC8 mice HRV. Adaptations to limit additional activation of AC signaling, via decreased neuronal sympathetic input, are utilized to ensure the hearts survival and prevent Ca2+ overload.

Basal Spontaneous Firing of Rabbit Sinoatrial Node Cells Is Regulated by Dual Activation of PDEs (Phosphodiesterases) 3 and 4.

BACKGROUND: Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na+/Ca2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown. METHODS: Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking. RESULTS: PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% (P<0.05) and ≈5% (P>0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% (P<0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca2+ current (ICa,L) by ≈60% (P<0.01) or ≈5% (P>0.05), respectively, and PLB phosphorylation by ≈20% (P>0.05) each, but dual PDE3+PDE4 inhibition increased ICa,L by ≈100% (P<0.01) and PLB phosphorylation by ≈110% (P<0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size (P<0.01) and reduced the SR (sarcoplasmic reticulum) Ca2+ refilling time (P<0.01) and the LCR period (time from action potential-induced Ca2+ transient to subsequent LCR; P<0.01), leading to decrease in spontaneous SANC cycle length (P<0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing. CONCLUSIONS: Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length.

Linkage of beta1-adrenergic stimulation to apoptotic heart cell death through protein kinase A-independent activation of Ca2+/calmodulin kinase II.

beta(1)-adrenergic receptor (beta(1)AR) stimulation activates the classic cAMP/protein kinase A (PKA) pathway to regulate vital cellular processes from the change of gene expression to the control of metabolism, muscle contraction, and cell apoptosis. Here we show that sustained beta(1)AR stimulation promotes cardiac myocyte apoptosis by activation of Ca(2+)/calmodulin kinase II (CaMKII), independently of PKA signaling. beta(1)AR-induced apoptosis is resistant to inhibition of PKA by a specific peptide inhibitor, PKI14-22, or an inactive cAMP analogue, Rp-8-CPT-cAMPS. In contrast, the beta(1)AR proapoptotic effect is associated with non-PKA-dependent increases in intracellular Ca(2+) and CaMKII activity. Blocking the L-type Ca(2+) channel, buffering intracellular Ca(2+), or inhibiting CaMKII activity fully protects cardiac myocytes against beta(1)AR-induced apoptosis, and overexpressing a cardiac CaMKII isoform, CaMKII-deltaC, markedly exaggerates the beta(1)AR apoptotic effect. These findings indicate that CaMKII constitutes a novel PKA-independent linkage of beta(1)AR stimulation to cardiomyocyte apoptosis that has been implicated in the overall process of chronic heart failure.

Heterodimerization of beta1- and beta2-adrenergic receptor subtypes optimizes beta-adrenergic modulation of cardiac contractility.

Intermolecular interactions between members of both similar and divergent G protein-coupled receptor subfamilies have been shown in various experimental systems. Here, we demonstrate heterodimerization of predominant beta-adrenergic receptor (betaAR) subtypes expressed in the heart, beta1AR, and beta2AR, and its physiological relevance. In intact adult-mouse cardiac myocytes lacking native beta1AR and beta2AR, coexpression of both betaAR subtypes led to receptor heterodimerization, as evidenced by their coimmunoprecipitation, colocalization at optical resolution, and markedly increased binding affinity for subtype-selective ligands. As a result, the dose-response curve of myocyte contraction to betaAR agonist stimulation with isoproterenol (ISO) was shifted leftward by approximately 1.5 orders of magnitude, and the response of cellular cAMP formation to ISO was enhanced concomitantly, indicating that intermolecular interactions of betaAR subtypes resulted in sensitization of these receptors in response to agonist stimulation. In contrast, the presence of beta1AR greatly suppressed ligand-independent spontaneous activity of coexisting beta2ARs. Thus, heterodimerization of beta1AR and beta2AR in intact cardiac myocytes creates a novel population of betaARs with distinct functional and pharmacological properties, resulting in enhanced signaling efficiency in response to agonist stimulation while silencing ligand-independent receptor activation, thereby optimizing beta-adrenergic modulation of cardiac contractility.

Neonatal Transplantation Confers Maturation of PSC-Derived Cardiomyocytes Conducive to Modeling Cardiomyopathy.

Pluripotent stem cells (PSCs) offer unprecedented opportunities for disease modeling and personalized medicine. However, PSC-derived cells exhibit fetal-like characteristics and remain immature in a dish. This has emerged as a major obstacle for their application for late-onset diseases. We previously showed that there is a neonatal arrest of long-term cultured PSC-derived cardiomyocytes (PSC-CMs). Here, we demonstrate that PSC-CMs mature into adult CMs when transplanted into neonatal hearts. PSC-CMs became similar to adult CMs in morphology, structure, and function within a month of transplantation into rats. The similarity was further supported by single-cell RNA-sequencing analysis. Moreover, this in vivo maturation allowed patient-derived PSC-CMs to reveal the disease phenotype of arrhythmogenic right ventricular cardiomyopathy, which manifests predominantly in adults. This study lays a foundation for understanding human CM maturation and pathogenesis and can be instrumental in PSC-based modeling of adult heart diseases.

Phosphatidylinositol 3-kinase offsets cAMP-mediated positive inotropic effect via inhibiting Ca2+ influx in cardiomyocytes.

Phosphoinositide 3-kinase (PI3K) has been implicated in beta2-adrenergic receptor (beta2-AR)/G(i)-mediated compartmentation of the concurrent G(s)-cAMP signaling, negating beta2-AR-induced phospholamban phosphorylation and the positive inotropic and lusitropic responses in cardiomyocytes. However, it is unclear whether PI3K crosstalks with the beta1-AR signal transduction, and even more generally, with the cAMP/PKA pathway. In this study, we show that selective beta1-AR stimulation markedly increases PI3K activity in adult rat cardiomyocytes. Inhibition of PI3K by LY294002 significantly enhances beta1-AR-induced increases in L-type Ca2+ currents, intracellular Ca2+ transients, and myocyte contractility, without altering the receptor-mediated phosphorylation of phospholamban. The LY294002 potentiating effects are completely prevented by betaARK-ct, a peptide inhibitor of beta-adrenergic receptor kinase-1 (betaARK1) as well as G(betagamma) signaling, but not by disrupting G(i) function with pertussis toxin. Moreover, forskolin, an adenylyl cyclase activator, also elevates PI3K activity and inhibition of PI3K enhances forskolin-induced contractile response in a betaARK-ct sensitive manner. In contrast, PI3K inhibition affects neither the basal contractility nor high extracellular Ca2+-induced increase in myocyte contraction. These results suggest that beta1-AR stimulation activates PI3K via a PKA-dependent mechanism, and that G(betagamma) and the subsequent activation of betaARK1 are critically involved in the PKA-induced PI3K signaling which, in turn, negates cAMP-induced positive inotropic effect via inhibiting sarcolemmal Ca2+ influx and the subsequent increase in intracellular Ca2+ transients, without altering the receptor-mediated phospholamban phosphorylation, in intact cardiomyocytes.