Cardiac Myocyte Function Research Articles

The NADPH oxidase inhibitor diphenyleneiodonium suppresses Ca2+ signaling and contraction in rat cardiac myocytes.

Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca2+ signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca2+ signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC50 of ≅0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca2+ transient and sarcoplasmic reticulum Ca2+ content by 20%-30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca2+ transients by DPI. The L-type Ca2+ current (ICa) was decreased concentration-dependently by DPI (IC50 of ≅40.3 µM) with ≅13.1%-inhibition at 3 µM. The frequency of Ca2+ sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca2+ and Na+. Mitochondrial superoxide level was reduced by DPI at 3-100 µM. Our data suggest that DPI may suppress L-type Ca2+ channel and RyR, thereby attenuating Ca2+-induced Ca2+ release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression.

Abstract 17015: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

Introduction: Protein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiac myocytes. Mutations in PQC pathway proteins, such as CRYAB (arginine to glycine at position 120, R120G) and BAG3 (proline to lysine at position 209, P209L), induce protein aggregates with cardiomyopathy in humans. Novel observations in yeast and mammalian cells demonstrate mitochondrial uptake of cytosolic protein aggregates. The functional relevance of this pathway in myocardial proteostasis is unknown. Hypothesis: We hypothesize that mitophagy removes cytosolic protein aggregates in cardiac myocytes. Methods: Mice with inducible cardiac myocyte specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assayed for protein aggregates with biochemical fractionation and super-resolution imaging in comparison to floxed controls. Transgenic mice expressing R120G CRYAB (R120G TG) were subjected to both TRAF2 gain-of-function (with AAV9-cardiac Troponin T promoter-driven TRAF2 transduction) and TRAF2 loss-of-function (with tamoxifen-inducible ablation of one Traf2 allele) in cardiac myocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology. Results: Poly-ubiquitinated protein-rich aggregates accumulate in the cytosol and within mitochondria with desmin mislocalization in TRAF2icKO myocardium. CRYAB R120G and BAG3 P209L mutant proteins are taken up by isolated mitochondria. CRYAB R120G protein aggregates co-localize with damaged mitochondria and lysosomes by Coupa imaging, and accumulate in the mitochondrial fraction in R120G TG myocardium with upregulation of TRAF2 and induction of mitophagy. Adult-onset haplo-insufficiency of Traf2 accelerated mortality (P=0.003), impaired left ventricular (LV) fractional shortening (P<0.001) and worsened mitochondrial morphology in R120G TG mice. AAV9-cTnT-TRAF2 transduction delayed mortality (P=0.027), rescued LV fractional shortening (P=0.039), reduced mitochondrial protein aggregates and improved desmin localization in R120G TG mice. Conclusions: Mitophagy facilitates removal of cytosolic protein aggregates and can be stimulated to ameliorate proteotoxic cardiomyopathy.

Regulator Of G Protein Signaling-4 Modulates Short-Chain Fatty Acid-induced Cardiac Inflammation And Norepinephrine Release from Sympathetic Neurons

Free fatty acid receptor (FFAR)-3 (also known as GPR41) is a Gi/o protein-coupled receptor (GPCR) mediating many cellular effects of short-chain fatty acids (SCFAs) like propionate and butyrate. It inhibits cyclic 3’,5’-adenosine monophosphate (cAMP) synthesis and promotes sympathetic nervous system (SNS) activity and norepinephrine (NE) release. It is also blocked by ketone bodies, such as beta-hydroxybutyrate. Regulator of G protein Signaling (RGS)-4 deactivates (terminates) Gi/o- and Gq-protein signaling. Cardiac RGS4 protects the heart against arrhythmogenesis via calcium signaling attenuation and against pressure overload-induced hypertrophy. In the present study, we examined whether RGS4 regulates SCFA signaling and function (through FFAR3) in cardiac myocytes and sympathetic neurons in vitro, as well as in mice post-pressure overload in vivo. We found that siRNA-mediated RGS4 depletion in H9c2 cardiomyocytes enhances propionate-induced cAMP lowering, Giα protein subunit activation, and upregulation of pro-inflammatory mediators [p38 mitogen activated protein kinase (MAPK), interleukin (IL)-6 & IL-1β, transforming growth factor (TGF)-β]. On the other hand, RGS4 overexpression in sympathetic neuron-like Neuro-2A cells significantly reduces propionate/FFAR3-mediated NE release. In vivo, in C57/B6 mice undergoing transverse aortic constriction (TAC) to induce pressure overload, cardiac RGS4 was found upregulated, at both the mRNA and protein levels, in post-TAC mice compared to sham-operated controls. This was accompanied by markedly reduced propionic acid-dependent FFAR3 signaling in isolated membranes from the post-TAC hearts. 3-hydroxybutyrate exerts the same effect in vitro in HEK293T cells overexpressing human FFAR3, i.e., inhibits Gi/o protein signaling by FFAR3. Finally, although initially (24 hours post-TAC) elevated, compared to sham (6.6+0.4 nmol/g of tissue vs. 4.1+0.7 nmol/g of tissue, respectively; n=5), cardiac NE levels were markedly suppressed at 7 days post-TAC (5.4+0.3 nmol/g of heart tissue, n=5). In conclusion, RGS4 inhibits SCFA/FFAR3 pro-inflammatory signaling in the myocardium, and neuronal SCFA/FFAR3 signaling that promotes NE release. Pressure overload upregulates cardiac RGS4 in vivo to protect the heart against FFAR3-mediated inflammation and elevated catecholamine toxicity. Thus, RGS4 actions on FFAR3 or pharmacological blockade of this receptor with a ketone body may protect the heart against pathological stimuli (e.g., hypertension) that precipitate heart disease via increased sympathetic activity and elevated catecholaminergic toxicity. 1) NIH (NHLBI); 2) NSU‘s President‘s Faculty Research & Development Grant (PFRDG). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Post-translational modification-dependent regulation of CaMKII-delta conformation in live cardiac myocytes.

Calcium/calmodulin dependent protein kinase II (CaMKII) is a key regulator of numerous functions in cardiac myocytes including Ca regulation, contraction, and transcriptional control. Overactivation of CaMKII due to post-translational modifications like autophosphorylation at Thr287 can autonomously activate and prolong CaMKII activation, even after Ca/CaM dissociates, and thereby promote cardiac pathologies such as heart failure and arrhythmias. The specific molecular mechanisms by which CaMKII PTMs autonomously activate and prolong kinase activity are not fully understood. We found using a novel intermolecular FRET-based approach in live myocytes in a recent study that PTM-dependent alteration in CaMKII's affinity for CaM is one molecular basis by which CaMKII becomes autonomously activated. Here, we investigate how the conformational state of CaMKII in the autonomously activated state may be affected by PTMs using our FRET-based CaMKIIδ conformational state reporter, Camui (RFP-CaMKIIδ-GFP), and its phosphomimetic (T287D) and phosphoresistant (T287A) mutant variants, expressed via adenoviral infection in intact cardiac myocytes. We also conducted parallel in vitro biochemical experiments using HEK cell lysates. We found that the phosphomimetic T287D Camui exhibited prolonged durations in the active open conformational state following pacing-induced activation relative to WT or phosphoresistant T287A Camui. Moreover, the prolonged autonomous conformational state persisted even after intracellular Ca levels returned to diastolic levels at which CaM dissociates. These data suggest that PTM-dependent regulation of the conformational state of CaMKII may serve as an additional molecular mechanism for autonomous CaMKII activation that may function in tandem with PTM-dependent alterations in CaMKII's affinity for CaM.

PMON181 RGS4 Is Essential For The Sympathetic Regulation of The Short-chain Fatty Acid Receptor FFAR3 In The Heart

Abstract Background Free fatty acids (FFAs) are essential cell nutrients but also important signaling molecules regulating various cellular processes. Short-chain fatty acids (SCFAs), like propionate and butyrate, activate two different types of G protein-coupled receptors (GPCRs), FFAR2 and FFAR3. FFAR3 (also known as GPR41) couples to Gi/o proteins lowering cyclic 3', 5'-adenosine monophosphate (cAMP) levels and activating mitogen-activated protein kinases (MAPKs). In addition to adipose tissue, FFAR3 is highly expressed in peripheral sympathetic ganglia augmenting sympathetic nervous system (SNS) outflow. Regulator of G protein Signaling (RGS)-4 belongs to the RGS superfamily of proteins and inactivates (terminates) both Gi/o- and Gq-protein signaling. Cardiac RGS4 has been implicated in atrial fibrillation (AFib) prevention via calcium signaling attenuation. Finally, β-adrenergic receptors (ARs), the major mediators of catecholaminergic and SNS actions in cardiac myocytes, stimulate cAMP synthesis and activate protein kinase A (PKA), which is known to activate RGS4 via phosphorylation. Objective We examined whether RGS4 regulates cardiac FFAR3 signaling. Methods We used H9c2 cardiomyocytes transfected to express the human FFAR3. After treatment with catecholaminergic agonists and/or propionic acid plus various pharmacological inhibitors and siRNA to deplete RGS4, we performed signaling assays (western blotting, cAMP accumulation, GTP binding, ELISA) to delineate the precise involvement of RGS4 in the cardiac βAR-FFAR3 signaling crosstalk. Results RGS4 is essential for the dampening of propionate/FFAR3-dependent signaling in H9c2 cardiomyocytes since siRNA-mediated RGS4 depletion significantly enhances the effect of propionic acid on cAMP lowering, Gi/o activation, and p38 MAPK activation. Notably, concomitant isoproterenol or salbutamol treatment blocks propionic acid-induced signaling via the FFAR3 in H9c2 cardiomyocytes, indicating negative regulation of FFAR3 by catecholamines via the β2AR. Mechanistically, this is mediated by RGS4, since neither isoproterenol, nor salbutamol can inhibit propionate signaling upon siRNA-mediated RGS4 depletion. Finally, β2AR inhibition of FFAR3 signaling via RGS4 is dependent on β2AR-activated PKA, because pre-treatment with the PKA inhibitor H89 abrogates the catecholaminergic inhibition of FFAR3 signaling in H9c2 cardiomyocytes, as measured by cAMP lowering, downstream p38 MAPK activation, and interleukin (IL)-6 production. Conclusion Catecholamine-activated RGS4 abrogates SCFA/FFAR3 signaling and function in cardiac myocytes. This may represent part of the mechanism via which RGS4 protects against both AFib pathogenesis and FFAR3-promoted SNS hyperactivity and inflammation in the myocardium. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Abstract P233: Cardiac Regulator Of G Protein Signaling-4 Upregulation Protects Against FFAR3-mediated Inflammation And Sympathetic Hyperactivity

Background: Free fatty acid receptor (FFAR)-3 is a Gi/o protein-coupled receptor (GPCR) mediating many cellular effects of short-chain fatty acids like propionate. It inhibits cyclic 3’,5’-adenosine monophosphate (cAMP) synthesis and promotes sympathetic nervous system (SNS) activity and norepinephrine (NE) release. Regulator of G protein Signaling (RGS)-4 deactivates (terminates) Gi/o- and Gq-protein signaling. Cardiac RGS4 protects the heart against arrhythmogenesis via calcium signaling attenuation and against pressure overload-induced hypertrophy. Study objective: We examined whether RGS4 regulates FFAR3 signaling and function in cardiac myocytes and sympathetic neurons in vitro, as well as in mice post-pressure overload in vivo. Methods & Results: siRNA-mediated RGS4 depletion in H9c2 cardiomyocytes enhances propionate-induced cAMP lowering, Giα protein subunit activation, and upregulation of pro-inflammatory mediators [p38 MAPK, interleukin (IL)-6 & IL-1β, transforming growth factor (TGF)-β]. On the other hand, RGS4 overexpression in sympathetic neuron-like Neuro-2A cells significantly reduces propionate/FFAR3-mediated NE release. In vivo, in C57/B6 mice undergoing transverse aortic constriction (TAC) to induce pressure overload (increased afterload), cardiac RGS4 was found upregulated, at both the mRNA and protein levels, in post-TAC mice compared to sham-operated controls. This was accompanied by markedly reduced propionic acid-dependent FFAR3 signaling in isolated membranes from the post-TAC hearts. Finally, although initially (24 hours post-TAC) elevated, compared to sham (6.6 + 0.4 nmol/g of tissue vs. 4.1 + 0.7 nmol/g of tissue, respectively; n=5), cardiac NE levels were markedly suppressed at 7 days post-TAC (5.4 + 0.3 nmol/g of heart tissue, n=5). Conclusions: RGS4 inhibits cardiac propionic acid/FFAR3 pro-inflammatory, and neuronal propionic acid/FFAR3 NE release-stimulating signaling. Pressure overload upregulates cardiac RGS4 in vivo to protect the heart against FFAR3-mediated inflammation and elevated SNS activity/NE release. Thus, cardiac RGS4 upregulation might represent a cardioprotective mechanism against the chronically increased afterload imposed by systemic hypertension.

Subcellular β-Adrenergic Receptor Signaling in Cardiac Physiology and Disease.

Adrenergic receptors are critical regulators of cardiac function with profound effects on cardiac output during sympathetic stimulation. Chronic stimulation of the adrenergic system of the heart under conditions of cardiac stress leads to cardiac dysfunction, hypertrophy, and ultimately failure. Emerging data have revealed that G protein-coupled receptors in intracellular compartments are functionally active and regulate distinct cellular processes from those at the cell surface. β2 adrenergic receptors internalize onto endosomes in various cell types where they have recently been shown to continue to stimulate cAMP production to selectively regulate gene expression. Other studies have identified β1 adrenergic receptors at the nuclear envelope and the Golgi apparatus. Here, we discuss data on signaling by β1 and β2 adrenergic receptors in the heart and the possible influence of their subcellular locations on their divergent physiological functions in cardiac myocytes and in cardiac pathology. Understanding the relative roles of these receptors at these locations could have a significant impact on pharmacological targeting of these receptors for the treatment of heart failure and cardiac diseases.

RGS4 Mediates Catecholaminergic Inhibition of Short‐Chain Fatty Acid Receptor FFAR3 Signaling & Function in Cardiomyocytes

BackgroundFree fatty acids (FFAs) are essential cell nutrients but also important signaling molecules regulating various cellular processes. Short‐chain fatty acids (SCFAs), like propionate and butyrate, activate two different types of G protein‐coupled receptors (GPCRs), FFAR2 and FFAR3. FFAR3 (also known as GPR41) couples to Gi/o proteins lowering cyclic 3’,5’‐adenosine monophosphate (cAMP) levels and activating mitogen‐activated protein kinases (MAPKs). In addition to adipose tissue, FFAR3 is highly expressed in peripheral sympathetic ganglia augmenting sympathetic nervous system (SNS) outflow. Regulator of G protein Signaling (RGS)‐4 belongs to the RGS superfamily of proteins and inactivates (terminates) both Gi/o‐ and Gq‐protein signaling. Cardiac RGS4 has been implicated in atrial fibrillation (AFib) prevention via calcium signaling attenuation. Finally, β‐adrenergic receptors (ARs), the major mediators of catecholaminergic and SNS actions in cardiac myocytes, stimulate cAMP synthesis and activate protein kinase A (PKA), which is known to activate RGS4 via phosphorylation.Study objectiveSince catecholamines/SNS and RGS4 play major roles in AFib pathophysiology and FFAR3 promotes SNS activity, we examined whether RGS4 regulates cardiac FFAR3 signaling.MethodsWe used H9c2 cardiomyocytes transfected to express the human FFAR3. After treatment with catecholaminergic agonists and/or propionic acid plus various pharmacological inhibitors and siRNA to deplete RGS4, we performed signaling assays (western blotting, cAMP accumulation, GTP binding, ELISA) to delineate the precise involvement of RGS4 in the cardiac βAR‐FFAR3 signaling crosstalk.ResultsRGS4 is essential for the dampening of propionate/FFAR3‐dependent signaling in H9c2 cardiomyocytes since siRNA‐mediated RGS4 depletion significantly enhances the effect of propionic acid on cAMP lowering, Gi/o activation, and p38 MAPK activation. Notably, concomitant isoproterenol or salbutamol treatment blocks propionic acid‐induced signaling via the FFAR3 in H9c2 cardiomyocytes, indicating negative regulation of FFAR3 by catecholamines via the β2AR. Mechanistically, this is mediated by RGS4, since neither isoproterenol, nor salbutamol can inhibit propionate signaling upon siRNA‐mediated RGS4 depletion. Finally, this β2AR trans‐inhibition of FFAR3 signaling via RGS4 is dependent on β2AR‐activated PKA, because pre‐treatment with the PKA inhibitor H89 abrogated the catecholaminergic inhibition of FFAR3 signaling (cAMP lowering, downstream p38 MAPK activation, interleukin (IL)‐6 production) in H9c2 cardiomyocytes.Conclusionsβ2AR‐activated RGS4 abrogates SCFA/FFAR3 signaling and function in cardiac myocytes. This may represent part of the mechanism via which RGS4 protects against both AFib pathogenesis and FFAR3‐promoted SNS hyperactivity and inflammation in the myocardium.

CMYA5 is a novel interaction partner of FHL2 in cardiac myocytes.

Four-and-a-half LIM domains protein 2 (FHL2) is an anti-hypertrophic adaptor protein that regulates cardiac myocyte signalling and function. Herein, we identified cardiomyopathy-associated 5 (CMYA5) as a novel FHL2 interaction partner in cardiac myocytes. In vitro pull-down assays demonstrated interaction between FHL2 and the N- and C-terminal regions of CMYA5. The interaction was verified in adult cardiac myocytes by proximity ligation assays. Immunofluorescence and confocal microscopy demonstrated co-localisation in the same subcellular compartment. The binding interface between FHL2 and CMYA5 was mapped by peptide arrays. Exposure of neonatal rat ventricular myocytes to a CMYA5 peptide covering one of the FHL2 interaction sites led to an increase in cell area at baseline, but a blunted response to chronic phenylephrine treatment. In contrast to wild-type hearts, loss or reduced FHL2 expression in Fhl2-targeted knockout mouse hearts or in a humanised mouse model of hypertrophic cardiomyopathy led to redistribution of CMYA5 into the perinuclear and intercalated disc region. Taken together, our results indicate a direct interaction of the two adaptor proteins FHL2 and CMYA5 in cardiac myocytes, which might impact subcellular compartmentation of CMYA5.

TRAF2, an Innate Immune Sensor, Reciprocally Regulates Mitophagy and Inflammation to Maintain Cardiac Myocyte Homeostasis

Mitochondria are essential for cardiac myocyte function, but damaged mitochondria trigger cardiac myocyte death. Although mitophagy, a lysosomal degradative pathway to remove damaged mitochondria, is robustly active in cardiac myocytes in the unstressed heart, its mechanisms and physiological role remain poorly defined. We discovered a critical role for TRAF2, an innate immunity effector protein with E3 ubiquitin ligase activity, in facilitating physiological cardiac myocyte mitophagy in the adult heart, to prevent inflammation and cell death, and maintain myocardial homeostasis.

Cardiac myocyte intrinsic contractility and calcium handling deficits underlie heart organ dysfunction in murine cancer cachexia

Cachexia is a muscle wasting syndrome occurring in many advanced cancer patients. Cachexia significantly increases cancer morbidity and mortality. Cardiac atrophy and contractility deficits have been observed in patients and in animal models with cancer cachexia, which may contribute to cachexia pathophysiology. However, underlying contributors to decreased in vivo cardiac contractility are not well understood. In this study, we sought to distinguish heart-intrinsic changes from systemic factors contributing to cachexia-associated cardiac dysfunction. We hypothesized that isolated heart and cardiac myocyte functional deficits underlie in vivo contractile dysfunction. To test this hypothesis, isolated heart and cardiac myocyte function was measured in the colon-26 adenocarcinoma murine model of cachexia. Ex vivo perfused hearts from cachectic animals exhibited marked contraction and relaxation deficits during basal and pacing conditions. Isolated myocytes displayed significantly decreased peak contraction and relaxation rates, which was accompanied by decreased peak calcium and decay rates. This study uncovers significant organ and cellular-level functional deficits in cachectic hearts outside of the catabolic in vivo environment, which is explained in part by impaired calcium cycling. These data provide insight into physiological mechanisms of cardiomyopathy in cachexia, which is critical for the ultimate development of effective treatments for patients.

Mechanisms of cAMP compartmentation in cardiac myocytes: experimental and computational approaches to understanding.

The small diffusible second messenger 3',5'-cyclic adenosine monophosphate (cAMP) is found in virtually every cell in our bodies, where it mediates responses to a variety of different G protein coupled receptors (GPCRs). In the heart, cAMP plays a critical role in regulating many different aspects of cardiac myocyte function, including gene transcription, cell metabolism, and excitation-contraction coupling. Yet, not all GPCRs that stimulate cAMP production elicit the same responses. Subcellular compartmentation of cAMP is essential to explain how different receptors can utilize the same diffusible second messenger to elicit unique functional responses. However, the mechanisms contributing to this behaviour and its significance in producing physiological and pathological responses are incompletely understood. Mathematical modelling has played an essential role in gaining insight into these questions. This review discusses what we currently know about cAMP compartmentation in cardiac myocytes and questions that are yet to be answered.

Immune Cells and Immunotherapy for Cardiac Injury and Repair.

Cardiac injury remains a major cause of morbidity and mortality worldwide. Despite significant advances, a full understanding of why the heart fails to fully recover function after acute injury, and why progressive heart failure frequently ensues, remains elusive. No therapeutics, short of heart transplantation, have emerged to reliably halt or reverse the inexorable progression of heart failure in the majority of patients once it has become clinically evident. To date, most pharmacological interventions have focused on modifying hemodynamics (reducing afterload, controlling blood pressure and blood volume) or on modifying cardiac myocyte function. However, important contributions of the immune system to normal cardiac function and the response to injury have recently emerged as exciting areas of investigation. Therapeutic interventions aimed at harnessing the power of immune cells hold promise for new treatment avenues for cardiac disease. Here, we review the immune response to heart injury, its contribution to cardiac fibrosis, and the potential of immune modifying therapies to affect cardiac repair.

Deletion of Trpm4 Alters the Function of the Nav1.5 Channel in Murine Cardiac Myocytes.

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca2+-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in myocytes. Hearts from Trpm4−/− mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na+ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na+ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Nav1.5 function in murine cardiac myocytes.

Complex Relationship Between Cardiac Fibroblasts and Cardiomyocytes in Health and Disease.

Cardiac fibroblasts are the primary cell type responsible for deposition of extracellular matrix in the heart, providing support to the contracting myocardium and contributing to a myriad of physiological signaling processes. Despite the importance of fibrosis in processes of wound healing, excessive fibroblast proliferation and activation can lead to pathological remodeling, driving heart failure and the onset of arrhythmias. Our understanding of the mechanisms driving the cardiac fibroblast activation and proliferation is expanding, and evidence for their direct and indirect effects on cardiac myocyte function is accumulating. In this review, we focus on the importance of the fibroblast‐to‐myofibroblast transition and the cross talk of cardiac fibroblasts with cardiac myocytes. We also consider the current use of models used to explore these questions.

Rosuvastatin Reduces L-Type Ca2+ Current and Alters Contractile Function in Cardiac Myocytes via Modulation of β-Adrenergic Receptor Signaling.

Rosuvastatin is one of the most used statins to lower plasma cholesterol levels. Although previous studies have reported remarkable cardiovascular effects of rosuvastatin (RSV), the mechanisms of these effects are largely unknown. In this study, we investigated the acute effects of RSV on L-type Ca2+ currents and contractile function of ventricular myocytes under basal conditions and during β-adrenergic stimulation. The effects of RSV were investigated in freshly isolated adult rat ventricular myocytes. L-type Ca+2 currents and myocyte contractility were recorded using patch-clamp amplifier and sarcomere length detection system. All experimental recordings were performed at 36 ± 1°C. L-type Ca+2 currents were significantly reduced with the administration of 1μM RSV (~ 24%) and this reduction in Ca2+ currents was observed at almost all potential ranges applied. Suppression of L-type Ca2+ current by RSV was prevented by adenylyl cyclase (AC) and protein kinase A (PKA) inhibitors SQ 22536 and KT5720, respectively. However, inhibition of Rho-associated kinases (ROCKs) by Y-27632 or nitric oxide synthase (NOS) by L-NAME failed to circumvent the inhibitory effect of RSV. Finally, we examined the effect of RSV during β-adrenergic receptor stimulation by isoproterenol and observed that RSV significantly suppresses the β-adrenergic responses in both L-type Ca2+ currents and contraction parameters. In conclusion, RSV modulates the β-adrenergic signaling cascade and thereby mimics the impact of β-adrenergic receptor blockers in adult ventricular myocytes through modulation of the AC-cAMP-PKA pathway.

Amplification of Cardiac Motor Functions by Prestin (Slc26a5)

The human heart is a durable biological motor, beating approximately 3.4 billion times in an 80-year lifespan. Actual cardiac performance originates in the function of cardiac myocytes, which are electromotile, changing in length in response to a change in membrane potentials. Groundbreaking work by Hugh Huxley and Jean Hanson et al. in the 1950s introduced the “sliding filament theory” that during muscle contraction, myosin and the filamentous actin protein form cross-bridges, allowing myosin to slide along actin, leading to length changes and force generation. Some muscle contraction mechanisms remain unexplained, such as how the sarcolemma accommodates ∼0.2-μm-displacement length changes per sarcomere per cardiac cycle without experiencing significant distortion. Here we invoke and test for the presence and function of a canonical non-conventional motor protein, prestin (Slc26a5), in cardiomyocytes as an amplifier of actin-myosin force generation. Prestin has been thought to be expressed exclusively in outer hair cells (OHCs) of the inner ear. It is a direct voltage-to-force convertor and mediates electromotility of OHCs as part of the molecular element of cochlear sound amplification. We hypothesize that prestin is expressed in mouse and human heart tissue and serves as the cardiac mechanical amplifier. We investigated whether the genetic deletion of Slc26a5 (Slc26a5-/-) produces in vivo and in vitro cardiac contractility changes. Multimodal Second Harmonic Generation (SHG) two-photon fluorescence microscopy and Stimulated Emission Depletion (STED) microscopy analyses were used to substantiate findings of the expression and functional roles of prestin in ventricular myocytes. We conclude that prestin serves to amplify actin-myosin force generation in cardiomyocytes, accounting for the non-linear properties of muscle contraction. The functional significance of prestin is underpinned by alterations of cardiac contractility in Slc26a5-/-mice. Our results suggest that prestin may serve as a broader cellular motor amplifier.

Multi-Compartment, Early Disruption of cGMP and cAMP Signalling in Cardiac Myocytes from the mdx Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is the most frequent and severe form of muscular dystrophy. The disease presents with progressive body-wide muscle deterioration and, with recent advances in respiratory care, cardiac involvement is an important cause of morbidity and mortality. DMD is caused by mutations in the dystrophin gene resulting in the absence of dystrophin and, consequently, disturbance of other proteins that form the dystrophin-associated protein complex (DAPC), including neuronal nitric oxide synthase (nNOS). The molecular mechanisms that link the absence of dystrophin with the alteration of cardiac function remain poorly understood but disruption of NO-cGMP signalling, mishandling of calcium and mitochondrial disturbances have been hypothesized to play a role. cGMP and cAMP are second messengers that are key in the regulation of cardiac myocyte function and disruption of cyclic nucleotide signalling leads to cardiomyopathy. cGMP and cAMP signals are compartmentalised and local regulation relies on the activity of phosphodiesterases (PDEs). Here, using genetically encoded FRET reporters targeted to distinct subcellular compartments of neonatal cardiac myocytes from the DMD mouse model mdx, we investigate whether lack of dystrophin disrupts local cyclic nucleotide signalling, thus potentially providing an early trigger for the development of cardiomyopathy. Our data show a significant alteration of both basal and stimulated cyclic nucleotide levels in all compartments investigated, as well as a complex reorganization of local PDE activities.

Abstract 410: Transthyretin Amyloid Fibrils Deposited in the Microenvironment Alter Cardiac Myocyte and Fibroblast Function and Cytoskeletal Structure

Age-related wild type transthyretin amyloidosis (ATTRwt) is a systemic condition that is characterized by deposition of amyloidogenic fibrils of misfolded transthyretin (TTR) in various organ systems. This disease manifests as an infiltrative cardiomyopathy hallmarked by extracellular deposition of misfolded TTR fibrils in the cardiac tissue, leading to cardiac dysfunction and heart failure with preserved ejection fraction (HFpEF); this disease is underdiagnosed and may affect 25% of elderly men and women. We hypothesize that deposition of amyloidogenic TTR fibrils alters cardiomyocyte contractility and sarcomere structure via modulation of cell growth due to altered microenvironmental features such as substrate stiffness. Recombinant TTR fibrils were deposited on glass substrates or substrates mimicking the stiffness of the healthy myocardium (10kPa). Neonatal rat ventricular myocytes exhibited decreased rate of linear contractility via line scan kymography and altered cytoskeletal structure when exposed to deposited TTR fibrils, with greater maladaptive effects seen on 10kPa substrates. Isolated adult mouse atrial cardiomyocytes plated on TTR fibril-containing substrates had attenuated calcium transient amplitude and a prolonged decay constant, consistent with lower amplitude. We also hypothesize that deposited TTR fibrils induce cardiac fibroblast activation and promote progression of fibrosis. Isolated cardiac fibroblasts grown on TTR-coated glass showed a reorganization of the F-actin cytoskeleton, with preferential distribution to the cell periphery. Barrier removal tests after confluence resulted in significantly faster migration velocity of fibroblasts cultured on TTR fibrils. Increased reduction of MTS tetrazolium by a colorimetric assay suggests a significant TTR-related increase in the proliferation rate of cells. Together, results suggest that amyloidogenic TTR fibrils may affect cardiac myocyte and fibroblast structure and function through both chemical and mechanical pathways. As recently approved therapies for ATTRwt are cost-prohibitive and are intended to slow disease progression only, better understanding of disease mechanisms may elucidate novel therapeutic targets.

Come Together: Protein Assemblies, Aggregates and the Sarcostat at the Heart of Cardiac Myocyte Homeostasis.

Homeostasis in vertebrate systems is contingent on normal cardiac function. This, in turn, depends on intricate protein-based cellular machinery, both for contractile function, as well as, durability of cardiac myocytes. The cardiac small heat shock protein (csHsp) chaperone system, highlighted by αB-crystallin (CRYAB), a small heat shock protein (sHsp) that forms ∼3–5% of total cardiac mass, plays critical roles in maintaining proteostatic function via formation of self-assembled multimeric chaperones. In this work, we review these ancient proteins, from the evolutionarily preserved role of homologs in protists, fungi and invertebrate systems, as well as, the role of sHsps and chaperones in maintaining cardiac myocyte structure and function. We propose the concept of the “sarcostat” as a protein quality control mechanism in the sarcomere. The roles of the proteasomal and lysosomal proteostatic network, as well as, the roles of the aggresome, self-assembling protein complexes and protein aggregation are discussed in the context of cardiac myocyte homeostasis. Finally, we will review the potential for targeting the csHsp system as a novel therapeutic approach to prevent and treat cardiomyopathy and heart failure.