We previously found that in the hearts of hypertensive Dahl salt-sensitive rats, βIIPKC levels increase during the transition from compensated cardiac hypertrophy to cardiac dysfunction. Here we showed that a six-week treatment of these hypertensive rats with a βIIPKC-specific inhibitor, βIIV5-3, prolonged their survival by at least six weeks, suppressed myocardial fibrosis and inflammation, and delayed the transition from compensated hypertrophy to cardiac dysfunction. In addition, changes in the levels of the Ca2+-handling proteins, SERCA2 and the Na+/Ca2+ exchanger, as well as troponin I phosphorylation, seen in the control-treated hypertensive rats were not observed in the βIIPKC-treated rats, suggesting that βIIPKC contributes to the regulation of calcium levels in the myocardium. In contrast, treatment with the selective inhibitor of βIPKC, an alternative spliced form of βIIPKC, had no beneficial effects in these rats. We also found that βIIV5-3, but not βIV5-3, improved calcium handling in isolated rat cardiomyocytes and enhanced contractility in isolated rat hearts. In conclusion, our data using an in vivo model of cardiac dysfunction (late-phase hypertrophy), suggest that βIIPKC contributes to the pathology associated with heart failure and thus an inhibitor of βIIPKC may be a potential treatment for this disease.
Heart failure (HF) afflicts about 5 million people and causes 300 000 deaths a year in the United States alone. An integral part of the pathogenesis of HF is cardiac remodelling, and the signalling events that regulate it are a subject of intense research. Cardiac remodelling is the sum of responses of the heart to causes of HF, such as ischaemia, myocardial infarction, volume and pressure overload, infection, inflammation, and mechanical injury. These responses, including cardiomyocyte hypertrophy, myocardial fibrosis, and inflammation, involve numerous cellular and structural changes and ultimately result in a progressive decline in cardiac performance. Pharmacological and genetic manipulation of cultured heart cells and animal models of HF and the analysis of cardiac samples from patients with HF are all used to identify the molecular and cellular mechanisms leading to the disease. Protein kinase C (PKC) isozymes, a family of serine–threonine protein kinase enzymes, were found to regulate a number of cardiac responses, including those associated with HF. In this review, we describe the PKC isozymes that play critical roles in specific aspects of cardiac remodelling and dysfunction in HF.
Protein kinase C; Heart failure; Cardiac remodeling; Hypertrophy; Fibrosis and inflammation
Cardiac hypertrophy is a response of the myocardium to increased workload and is characterised by an increase of myocardial mass and an accumulation of extracellular matrix (ECM). As an ECM protein, an integrin ligand, and an angiogenesis inhibitor, all of which are key players in cardiac hypertrophy, mindin is an attractive target for therapeutic intervention to treat or prevent cardiac hypertrophy and heart failure. In this study, we investigated the role of mindin in cardiac hypertrophy using littermate Mindin knockout (Mindin−/−) and wild-type (WT) mice. Cardiac hypertrophy was induced by aortic banding (AB) or angiotensin II (Ang II) infusion in Mindin−/− and WT mice. The extent of cardiac hypertrophy was quantitated by echocardiography and by pathological and molecular analyses of heart samples. Mindin−/− mice were more susceptible to cardiac hypertrophy and fibrosis in response to AB or Ang II stimulation than wild type. Cardiac function was also markedly exacerbated during both systole and diastole in Mindin−/− mice in response to hypertrophic stimuli. Western blot assays further showed that the activation of AKT/glycogen synthase kinase 3β (GSK3β) signalling in response to hypertrophic stimuli was significantly increased in Mindin−/− mice. Moreover, blocking AKT/GSK3β signalling with a pharmacological AKT inhibitor reversed cardiac abnormalities in Mindin−/− mice. Our data show that mindin, as an intrinsic cardioprotective factor, prevents maladaptive remodelling and the transition to heart failure by blocking AKT/GSK3β signalling.
Mindin; Hypertrophy; Remodelling; Signal transduction; AKT
The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, δ and εPKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection.
Methods and results
Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished δPKC translocation by 3.8-fold and increased εPKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of δPKC decreased by 60 ± 2.7% in response to IPC, whereas the levels of εPKC did not significantly change. Prolonged ischaemia induced a 48 ± 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 ± 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of εPKC during IPC restored δPKC levels at the mitochondria while decreasing εPKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a δPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol.
Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, δPKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, εPKC.
Cardioprotection; Ischaemia/reperfusion; Apoptosis; Proteasome; PKC; Ischaemic preconditioning
Cardiac hypertrophy is central to the etiology of heart failure. Understanding the molecular pathways promoting cardiac hypertrophy may identify new targets for therapeutic intervention. Sodium-proton exchanger (NHE1) activity and expression levels in the heart are elevated in many models of hypertrophy through protein kinase C (PKC)/MAPK/ERK/p90RSK pathway stimulation. Sustained NHE1 activity, however, requires an acid-loading pathway. Evidence suggests that the Cl−/HCO3− exchanger, AE3, provides this acid load. Here we explored the role of AE3 in the hypertrophic growth cascade of cardiomyocytes.
) mice were compared to wildtype (WT) littermates to examine the role of AE3 protein in the development of cardiomyocyte hypertrophy. Mouse hearts were assessed by echocardiography. As well, responses of cultured cardiomyocytes to hypertrophic stimuli were measured. pH regulation capacity of ae3
and WT cardiomyocytes was assessed in cultured cells loaded with the pH-sensitive dye, BCECF-AM.
mice were indistinguishable from wild type (WT) mice in terms of cardiovascular performance. Stimulation of ae3
cardiomyocytes with hypertrophic agonists did not increase cardiac growth or reactivate the fetal gene program. ae3
mice are thus protected from pro-hypertrophic stimulation. Steady state intracellular pH (pHi) in ae3
cardiomyocytes was not significantly different from WT, but the rate of recovery of pHi from imposed alkalosis was significantly slower in ae3
These data reveal the importance of AE3-mediated Cl−/HCO3− exchange in cardiovascular pH regulation and the development of cardiomyocyte hypertrophy. Pharmacological antagonism of AE3 is an attractive approach in the treatment of cardiac hypertrophy.
AE3; Bicarbonate transport; Chloride/bicarbonate exchange; pH regulation; Cardiomyocyte hypertrophy; Heart failure
Heart failure (HF) is a chronic syndrome in which pathological cardiac remodeling is an integral part of the disease and mast cell (MC) degranulation-derived mediators have been suggested to play a role in its progression. Protein kinase C (PKC) signaling is a key event in the signal transduction pathway of MC degranulation. We recently found that inhibition of εPKC slows down the progression of hypertension-induced HF in salt-sensitive Dahl rats fed a high-salt diet. We therefore determined whether εPKC inhibition affects MC degranulation in this model. Six week-old male Dahl rats were fed with a high-salt diet to induce systemic hypertension, which resulted in concentric left ventricular hypertrophy at the age of 11 weeks, followed by myocardial dilatation and HF at the age of 17 weeks. We administered εV1-2 an εPKC-selective inhibitor peptide (3 mg/Kg/day), δV1-1, a δPKC-selective inhibitor peptide (3 mg/Kg/day), TAT (negative control; at equimolar concentration; 1.6 mg/Kg/day) or olmesartan (angiotensin receptor blocker [ARB] as a positive control; 3mg/Kg/day) between 11 weeks and 17 weeks. Treatment with εV1-2 attenuated cardiac MC degranulation without affecting MC density, myocardial fibrosis, microvessel patency, vascular thickening and cardiac inflammation in comparison to TAT- or δV1-1-treatment. Treatment with ARB also attenuated MC degranulation and cardiac remodeling, but to a lesser extent when compared to εV1-2. Finally, εV1-2 treatment inhibited MC degranulation in isolated peritoneal MCs. Together, our data suggest that εPKC inhibition attenuates pathological remodeling in hypertension-induced HF, at least in part, by preventing cardiac MC degranulation.
Mast cell degranulation; protein kinase C; PKC-selective inhibitor peptide; cardiac remodeling; heart failure
A variety of stress signals stimulate cardiac myocytes to undergo hypertrophy. Persistent cardiac hypertrophy is associated with elevated risk for the development of heart failure. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy and that stress signals neutralize this repressive function by triggering phosphorylation- and CRM1-dependent nuclear export of these chromatin-modifying enzymes. However, the identities of cardiac HDAC kinases have remained unclear. Here, we demonstrate that signaling by protein kinase C (PKC) is sufficient and, in some cases, necessary to drive nuclear export of class II HDAC5 in cardiomyocytes. Inhibition of PKC prevents nucleocytoplasmic shuttling of HDAC5 in response to a subset of hypertrophic agonists. Moreover, a nonphosphorylatable HDAC5 mutant is refractory to PKC signaling and blocks cardiomyocyte hypertrophy mediated by pharmacological activators of PKC. We also demonstrate that protein kinase D (PKD), a downstream effector of PKC, directly phosphorylates HDAC5 and stimulates its nuclear export. These findings reveal a novel function for the PKC/PKD axis in coupling extracellular cues to chromatin modifications that control cellular growth, and they suggest potential utility for small-molecule inhibitors of this pathway in the treatment of pathological cardiac gene expression.
Heart failure (HF) is often the end-phase of maladaptive cardiac hypertrophy. A contributing factor is activation of a hypertrophic gene expression program controlled by decreased class II histone deacetylase (HDAC) transcriptional repression via HDAC phosphorylation. Cardiac-specific overexpression of G protein-coupled receptor kinase-5 (GRK5), has previously been shown to possess nuclear activity as a HDAC5 kinase, promoting an intolerance to in vivo ventricular pressure-overload, however, its endogenous requirement in adaptive and maladaptive hypertrophy remains unknown.
We used mouse models with global or cardiomyocyte-specific GRK5 gene deletion to determine the absolute requirement of endogenous GRK5 for cardiac hypertrophy and HF development following chronic hypertrophic stimuli.
Methods and Results
Mice with global deletion of GRK5 were subjected to transverse aortic constriction (TAC). At 12 weeks, these mice showed attenuated hypertrophy, remodeling, and hypertrophic gene transcription along with preserved cardiac function. Global GRK5 deletion also diminished hypertrophy and related gene expression due to chronic phenylephrine infusion. We then generated mice with conditional, cardiac-specific deletion of GRK5 that also demonstrated similar protection from pathological cardiac hypertrophy and HF following TAC.
These results define myocyte GRK5 as a critical regulator of pathological cardiac growth following ventricular pressure-overload, supporting its role as an endogenous (patho)-physiological HDAC kinase. Further, these results define GRK5 as a potential therapeutic target to limit HF development after hypertrophic stress.
G protein-coupled receptor kinase; hypertrophy; heart failure
Cardiac hypertrophy is the predominant compensatory response of the heart to a wide variety of biomechanical stressors, including exercise, hypertension, myocardial infarction, intrinsic cardiomyopathy or congenital heart disease. Although cardiac hypertrophy can maintain cardiac output in response to elevated wall stress, sustained cardiac hypertrophy is often accompanied by maladaptive remodeling which can ultimately lead to heart failure. Cultured cardiac myocytes, transgenic and knock-out animal models, and pharmacological studies have not only revealed key molecules involved in hypertrophic signaling, but have also highlighted the redundancy in the hypertrophic signaling cascade. Currently, the majority of existing therapies for inhibition of pathologic cardiac hypertrophy and heart failure target molecules on the surface of cardiac myocytes, such as G-protein coupled receptors (GPCRs) and ion channels. Because these molecules are upstream of multiple intracellular signaling pathways, however, current therapy is often accompanied by significant off-target effects and toxicity. More recently, research has focused on identifying the intracellular effectors of these signaling cascades in the hope that more selective drugs may be rationally designed for therapeutic intervention.
Within the cardiac myocyte, the formation of discrete multimolecular complexes, or ‘signalosomes’, is an important mechanism for increasing the specificity and efficiency of hypertrophic signal transduction. In response to extracellular stimuli, these signalosomes can alter gene and protein expression, cell size, and chamber remodeling, such as in the case of the signalosomes formed by the mAKAPβ and AKAP-lbc scaffold proteins. A better understanding of the basic molecular mechanisms regulating the compartmentation and scaffolding of signaling molecules could lead to the development of new clinical tools that may prevent the development of heart failure and minimize negative impacts on physiological processes.
signalosome; mAKAP; AKAP-lbc; heart; therapeutic
Cardiac excitability and electrical activity are determined by the sum of individual ion channels, gap junctions and exchanger activities. Electrophysiological remodeling during heart disease involves changes in membrane properties of cardiomyocytes and is related to higher prevalence of arrhythmia-associated morbidity and mortality. Pharmacological and genetic manipulation of cardiac cells as well as animal models of cardiovascular diseases are used to identity changes in electrophysiological properties and the molecular mechanisms associated with the disease. Protein kinase C (PKC) and several other kinases play a pivotal role in cardiac electrophysiological remodeling. Therefore, identifying specific therapies that regulate these kinases is the main focus of current research. PKC, a family of serine/threonine kinases, has been implicated as potential signaling nodes associated with biochemical and biophysical stress in cardiovascular diseases. Thus, the role of PKC isozymes in regulating cardiac excitability has been a subject of great attention. In this review, we describe the role of PKC isozymes that are involved in cardiac excitability and discuss both genetic and pharmacological tools that were used, their attributes and limitations. Selective and effective pharmacological interventions to normalize cardiac electrical activities and correct cardiac arrhythmias will be of great clinical benefit.
Following activation by G-protein-coupled receptor agonists, protein kinase C (PKC) modulates cardiac myocyte function by phosphorylation of intracellular targets including myofilament proteins cardiac troponin I (cTnI) and cardiac myosin binding protein C (cMyBP-C). Since PKC phosphorylation has been shown to decrease myofibril ATPase activity, we hypothesized that PKC phosphorylation of cTnI and cMyBP-C will lower myocyte power output and, in addition, attenuate the elevation in power in response to protein kinase A (PKA)-mediated phosphorylation. We compared isometric force and power generating capacity of rat skinned cardiac myocytes before and after treatment with the catalytic subunit of PKC. PKC increased phosphorylation levels of cMyBP-C and cTnI and decreased both maximal Ca2+ activated force and Ca2+ sensitivity of force. Moreover, during submaximal Ca2+ activations PKC decreased power output by 62 %, which arose from both the fall in force and slower loaded shortening velocities since depressed power persisted even when force levels were matched before and after PKC. In addition, PKC blunted the phosphorylation of cTnI by PKA, reduced PKA-induced spontaneous oscillatory contractions, and diminished PKA-mediated elevations in myocyte power. To test whether altered thin filament function plays an essential role in these contractile changes we investigated the effects of chronic cTnI pseudo-phosphorylation on myofilament function using myocyte preparations from transgenic animals in which either only PKA phosphorylation sites (Ser-23/Ser-24) (PP) or both PKA and PKC phosphorylation sites (Ser-23/Ser-24/Ser-43/Ser-45/T-144) (All-P) were replaced with aspartic acid. Cardiac myocytes from All-P transgenic mice exhibited reductions in maximal force, Ca2+ sensitivity of force, and power. Similarly diminished power generating capacity was observed in hearts from All-P mice as determined by in situ pressure–volume measurements. These results imply that PKC-mediated phosphorylation of cTnI plays a dominant role in depressing contractility, and, thus, increased PKC isozyme activity may contribute to maladaptive behavior exhibited during the progression to heart failure.
PKC; PKA; Cardiac myocyte; Cardiac troponin I; Power output
Protein kinase Cs (PKCs) constitute a family of serine/threonine kinases, which has distinguished and specific roles in regulating cardiac responses, including those associated with heart failure. We found that the PKCθ isoform is expressed at considerable levels in the cardiac muscle in mouse, and that it is rapidly activated after pressure overload. To investigate the role of PKCθ in cardiac remodeling, we used PKCθ−/− mice. In vivo analyses of PKCθ−/− hearts showed that the lack of PKCθ expression leads to left ventricular dilation and reduced function. Histological analyses showed a reduction in the number of cardiomyocytes, combined with hypertrophy of the remaining cardiomyocytes, cardiac fibrosis, myofibroblast hyper-proliferation and matrix deposition. We also observed p38 and JunK activation, known to promote cell death in response to stress, combined with upregulation of the fetal pattern of gene expression, considered to be a feature of the hemodynamically or metabolically stressed heart. In keeping with these observations, cultured PKCθ−/− cardiomyocytes were less viable than wild-type cardiomyocytes, and, unlike wild-type cardiomyocytes, underwent programmed cell death upon stimulation with α1-adrenergic agonists and hypoxia. Taken together, these results show that PKCθ maintains the correct structure and function of the heart by preventing cardiomyocyte cell death in response to work demand and to neuro-hormonal signals, to which heart cells are continuously exposed.
protein kinase C theta; dilated cardiomyopathy; cardiomyocyte survival; alpha1-adrenergic agonists; protein kinases C
Deciphering the remote conditioning molecular mechanism may provide targets to develop therapeutics that can broaden the clinical application. To further investigate this, we tested whether two protein kinase C isozymes, the ubiquitously expressed epsilon PKC (εPKC) and the neuronal specific gamma PKC (γPKC), mediate nociceptive-induced remote myocardial conditioning.
Male Sprague-Dawley rats were used for both in vivo and ex vivo myocardial ischemia-reperfusion protocols. For the in vivo studies, using a surgical abdominal incision for comparison, applying only to the abdomen either bradykinin or the εPKC activator (ψεRACK) reduced myocardial infarct size (45±1%, 44±2%, respectively, versus incision: 43±2%, and control: 63±2%, P < 0.001). Western blot showed only εPKC, and not γPKC, is highly expressed in the myocardium. However, applying a selective γPKC inhibitor (γV5-3) to the abdominal skin blocked remote protection by any of these strategies.
Using an ex vivo isolated heart model without an intact nervous system, only selective εPKC activation, unlike a selective classical PKC isozyme activator (activating α, β, βII and γ), reduced myocardial injury. Importantly, the classical PKC isozyme activator given to the abdomen in vivo (with an intact nervous system including γPKC) during myocardial ischemia reduced infarct size as effectively as an abdominal incision or ψεRACK (45±1% versus 45±2% and 47±1%, respectively). The classical PKC activator-induced protection was also blocked by spinal cord surgical transection.
These findings identified potential remote conditioning mimetics, with these strategies effective even during myocardial ischemia. A novel mechanism of nociceptive-induced remote conditioning, involving γPKC, was also identified.
infarct size; remote; incision; protein kinase C; gamma; epsilon
In response to pathological stresses such as hypertension or myocardial infarction, the heart undergoes a remodeling process that is associated with myocyte hypertrophy, myocyte death, and fibrosis. Histone deacetylase 5 (HDAC5) is a transcriptional repressor of cardiac remodeling that is subject to phosphorylation-dependent neutralization in response to stress signaling. Recent studies have suggested a role for protein kinase C (PKC) and its downstream effector, protein kinase D1 (PKD1), in the control of HDAC5 phosphorylation. While PKCs are well-documented regulators of cardiac signaling, the function of PKD1 in heart muscle remains unclear. Here, we demonstrate that PKD1 catalytic activity is stimulated in cardiac myocytes by diverse hypertrophic agonists that signal through G protein-coupled receptors (GPCRs) and Rho GTPases. PKD1 activation in cardiomyocytes occurs through PKC-dependent and -independent mechanisms. In vivo, cardiac PKD1 is activated in multiple rodent models of pathological cardiac remodeling. PKD1 activation correlates with phosphorylation-dependent nuclear export of HDAC5, and reduction of endogenous PKD1 expression with small interfering RNA suppresses HDAC5 shuttling and associated cardiomyocyte growth. Conversely, ectopic overexpression of constitutively active PKD1 in mouse heart leads to dilated cardiomyopathy. These findings support a role for PKD1 in the control of pathological remodeling of the heart via its ability to phosphorylate and neutralize HDAC5.
Protein Kinase C (PKC) is a family of serine/threonine-isozymes that are involved in many signaling events in normal and disease states. Previous studies from our lab have demonstrated that εPKC plays a pivotal role in neuroprotection induced by ischemic preconditioning. However, the role of εPKC during and after brain ischemia is not clearly defined. Therefore, in the present study, we tested the hypothesis that activation of εPKC during an ischemic event is neuroprotective. Furthermore, other studies have demonstrated that εPKC mediates cerebral ischemic tolerance in the rat brain by decreasing vascular tone. Thus, we also tested the effects of εPKC activation during ischemia on cerebral blood flow (CBF). We found that ψε-Receptors for activated C kinase (RACK), a εPKC-selective peptide activator, injected intravenously 30 minutes before induction of global cerebral ischemia conferred neuroprotection in the CA1 region of the rat hippocampus. Moreover, measurements of CBF before, during and after cerebral ischemia revealed a significant reduction in the reperfusion phase of rats pretreated with ψεRACK compared to Tat peptide (vehicle). Our results suggest that εPKC can protect the rat brain against ischemic damage by regulating CBF. Thus, εPKC may be one of the treatment modalities against ischemic injury.
Ischemia; epsilon Protein Kinase C; Cerebral Blood Flow; Neuroprotection
A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy) or detrimental (pathological hypertrophy). This study was undertaken to establish the role of different protein kinase-C (PKC) isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus) models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation.
Mindin is a secreted extracellular matrix protein, an integrin ligand, and an angiogenesis inhibitor, other examples of which are all key players in the progression of cardiac hypertrophy. However, its function during cardiac hypertrophy remains unclear. This study was aimed to identify the effect of mindin on cardiac hypertrophy and the underlying mechanisms.
Methods and results
A significant down-regulation of mindin expression was observed in human failing hearts. To further investigate the role of mindin in cardiac hypertrophy, we used cultured neonatal rat cardiomyocytes with gain and loss of mindin function and cardiac-specific Mindin-overexpressing transgenic (TG) mice. In cultured cardiomyocytes, mindin negatively regulated angiotensin II (Ang II)-mediated hypertrophic growth, as detected by [3H]-Leucine incorporation, cardiac myocyte area, and hypertrophic marker protein levels. Cardiac hypertrophy in vivo was produced by aortic banding (AB) or Ang II infusion in TG mice and their wild-type controls. The extent of cardiac hypertrophy was evaluated by echocardiography as well as by pathological and molecular analyses of heart samples. Mindin overexpression in the heart markedly attenuated cardiac hypertrophy, fibrosis, and left ventricular dysfunction in mice in response to AB or Ang II. Further analysis of the signalling events in vitro and in vivo indicated that these beneficial effects of mindin were associated with the interruption of AKT/glycogen synthase kinase 3β (GSK3β) and transforming growth factor (TGF)-β1–Smad signalling.
The present study demonstrates for the first time that mindin serves as a novel mediator that protects against cardiac hypertrophy and the transition to heart failure by blocking AKT/GSK3β and TGF-β1–Smad signalling.
Mindin; Hypertrophy; Remodelling; Signal transduction; AKT
Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac-MLCK (cMLCK); yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined.
Methods and Results
We found that pressure-overload induced by transaortic constriction in wildtype mice reduced phosphorylated-MLC2v levels by ~40% and cMLCK levels by ~85%. To examine how a reduction in cMLCK and the corresponding reduction in pMLC2v affect function, we generated Mylk3 gene-targeted mice as well as transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure-overload led to severe heart failure in cMLCK knockout mice, but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure-overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems.
Our results suggest the novel idea that accelerated cMLCK-protein turnover by the ubiquitin-proteasome system underlie the transition from compensated hypertrophy to decompensated heart failure due to reduced phosphorylation of MLC2v.
myosin light chain; phosphorylation; heart failure
Despite increased understanding of the fundamental biology regulating cardiomyocyte hypertrophy and heart failure, it has been challenging to find novel chemical or genetic modifiers of these pathways. Traditional cell-based methods do not model the complexity of an intact cardiovascular system and mammalian models are not readily adaptable to chemical or genetic screens. Our objective was to create an in vivo model suitable for chemical and genetic screens for hypertrophy and heart failure modifiers
Methods and results
Using the developing zebrafish, we established that the cardiac natriuretic peptide genes (nppa and nppb), known markers of cardiomyocyte hypertrophy and heart failure, were induced in the embryonic heart by pathological cardiac stimuli. This pathological induction was distinct from the developmental regulation of these genes. We created a luciferase-based transgenic reporter line that accurately modelled the pathological induction patterns of the zebrafish nppb gene. Utilizing this reporter line, we were able to show remarkable conservation of pharmacological responses between the larval zebrafish heart and adult mammalian models.
By performing a focused screen of chemical agents, we were able to show a distinct response of a genetic model of hypertrophic cardiomyopathy to the histone deacetylase inhibitor, Trichostatin A, and the mitogen-activated protein kinase kinase 1/2 inhibitor, U0126. We believe this in vivo reporter line will offer a unique approach to the identification of novel chemical or genetic regulators of myocardial hypertrophy and heart failure.
Natriuretic peptides; Hypertrophy; Heart development; Heart failure; Hypertrophic cardiomyopathy
Fibroblasts, which are the most numerous cell type in the heart, interact with cardiomyocytes in vitro and affect their function; however, they are considered to play a secondary role in cardiac hypertrophy and failure. Here we have shown that cardiac fibroblasts are essential for the protective and hypertrophic myocardial responses to pressure overload in vivo in mice. Haploinsufficiency of the transcription factor–encoding gene Krüppel-like factor 5 (Klf5) suppressed cardiac fibrosis and hypertrophy elicited by moderate-intensity pressure overload, whereas cardiomyocyte-specific Klf5 deletion did not alter the hypertrophic responses. By contrast, cardiac fibroblast–specific Klf5 deletion ameliorated cardiac hypertrophy and fibrosis, indicating that KLF5 in fibroblasts is important for the response to pressure overload and that cardiac fibroblasts are required for cardiomyocyte hypertrophy. High-intensity pressure overload caused severe heart failure and early death in mice with Klf5-null fibroblasts. KLF5 transactivated Igf1 in cardiac fibroblasts, and IGF-1 subsequently acted in a paracrine fashion to induce hypertrophic responses in cardiomyocytes. Igf1 induction was essential for cardioprotective responses, as administration of a peptide inhibitor of IGF-1 severely exacerbated heart failure induced by high-intensity pressure overload. Thus, cardiac fibroblasts play a pivotal role in the myocardial adaptive response to pressure overload, and this role is partly controlled by KLF5. Modulation of cardiac fibroblast function may provide a novel strategy for treating heart failure, with KLF5 serving as an attractive target.
Phenylephrine (PE) induces cardiac hypertrophy through multiple signaling pathways including pathways involving protein kinase C (PKC) activation. Docosahexaenoic acid (DHA), an omega-3 fatty acid, has been shown to reduce the PE-induced hypertrophic responses. However, the effects of DHA on PKC activation and translocation are controversial. The present study investigates the effect of DHA on PE-induced activation of PKC. The results indicate that PE induces PKCα translocation (from cytosol to plasma membranes) and activation in cardiomyocytes during the hypertrophic responses. Although DHA itself has no significant effect on basal PKC translocation and activation, it effectively reduced PE-stimulated PKC translocation and activation. The results of the present study suggest a possible mechanism explaining how dietary fish oil may inhibit development of cardiac hypertrophy and therefore may be an attractive dietary agent for preventing cardiac hypertrophy in patients with heart failure.
Cardiomyocytes; hypertrophy; docosahexaenoic acid; protein kinase C
Pathological growth of cardiomyocytes (hypertrophy) is a major determinant for the development of heart failure, one of the leading medical causes of mortality worldwide. Here we show that the microRNA (miRNA)-212/132 family regulates cardiac hypertrophy and autophagy in cardiomyocytes. Hypertrophic stimuli upregulate cardiomyocyte expression of miR-212 and miR-132, which are both necessary and sufficient to drive the hypertrophic growth of cardiomyocytes. MiR-212/132 null mice are protected from pressure-overload-induced heart failure, whereas cardiomyocyte-specific overexpression of the miR-212/132 family leads to pathological cardiac hypertrophy, heart failure and death in mice. Both miR-212 and miR-132 directly target the anti-hypertrophic and pro-autophagic FoxO3 transcription factor and overexpression of these miRNAs leads to hyperactivation of pro-hypertrophic calcineurin/NFAT signalling and an impaired autophagic response upon starvation. Pharmacological inhibition of miR-132 by antagomir injection rescues cardiac hypertrophy and heart failure in mice, offering a possible therapeutic approach for cardiac failure.
Heart failure is often a consequence of pathological growth of cardiomyocytes or cardiac hypertrophy. Here Ucar and colleagues report that the microRNAs miR-132 and miR-212 promote cardiac hypertrophy and inhibit autophagy in cardiomyocytes by downregulating the transcription factor FoxO3.
The adipokine leptin and its receptor are expressed in the heart, and leptin has been shown to promote cardiomyocyte hypertrophy in vitro. Obesity is associated with hyperleptinemia and hypothalamic leptin resistance as well as an increased risk to develop cardiac hypertrophy and heart failure. However, the role of cardiac leptin signaling in mediating the cardiomyopathy associated with increased body weight is unclear, in particular, whether it develops subsequently to cardiac leptin resistance or overactivation of hypertrophic signaling pathways via elevated leptin levels.
The cardiac phenotype of high-fat diet (HFD)-induced obese wildtype (WT) mice was examined and compared to age-matched genetically obese leptin receptor (LepR)-deficient (LepRdb/db) or lean WT mice. To study the role of leptin-mediated STAT3 activation during obesity-induced cardiac remodeling, mice in which tyrosine residue 1138 within LepR had been replaced with a serine (LepRS1138) were also analyzed.
Obesity was associated with hyperleptinemia and elevated cardiac leptin expression in both diet-induced and genetically obese mice. Enhanced LepR and STAT3 phosphorylation levels were detected in hearts of obese WT mice, but not in those with LepR mutations. Moreover, exogenous leptin continued to induce cardiac STAT3 activation in diet-induced obese mice. Although echocardiography revealed signs of cardiac hypertrophy in all obese mice, the increase in left ventricular (LV) mass and diameter was significantly more pronounced in LepRS1138 animals. LepRS1138 mice also exhibited an increased activation of signaling proteins downstream of LepR, including Jak2 (1.8-fold), Src kinase (1.7-fold), protein kinase B (1.3-fold) or C (1.6-fold). Histological analysis of hearts revealed that the inability of leptin to activate STAT3 in LepRdb/db and LepRS1138 mice was associated with reduced cardiac angiogenesis as well as increased apoptosis and fibrosis.
Our findings suggest that hearts from obese mice continue to respond to elevated circulating or cardiac leptin, which may mediate cardioprotection via LepR-induced STAT3 activation, whereas signals distinct from LepR-Tyr1138 promote cardiac hypertrophy. On the other hand, the presence of cardiac hypertrophy in obese mice with complete LepR signal disruption indicates that additional pathways also play a role.
Heart; Hypertrophy; Leptin; Obesity; Signal transduction; STAT3
Protein kinase plays an essential role in controlling cardiac growth and hypertrophic remodeling. The cardiac troponin I-interacting kinase (TNNI3K), a novel cardiac specific kinase, is associated with cardiomyocyte hypertrophy. However, the precise function of TNNI3K in regulating cardiac remodeling has remained controversial.
Methods and Results
In a rat model of cardiac hypertrophy generated by transverse aortic constriction, myocardial TNNI3K expression was significantly increased by 1.62 folds (P<0.05) after constriction for 15 days. To investigate the role of TNNI3K in cardiac hypertrophy, we generated transgenic mouse lines with overexpression of human TNNI3K specifically in the heart. At the age of 3 months, the high-copy-number TNNI3K transgenic mice demonstrated a phenotype of concentric hypertrophy with increased heart weight normalized to body weight (1.31 fold, P<0.01). Echocardiography and non-invasive hemodynamic assessments showed enhanced cardiac function. No necrosis or myocyte disarray was observed in the heart of TNNI3K transgenic mice. This concentric hypertrophy maintained up to 12 months of age without cardiac dysfunction. The phospho amino acid analysis revealed that TNNI3K is a protein-tyrosine kinase. The yeast two-hybrid screen and co-immunoprecipitation assay identified cTnI as a target for TNNI3K. Moreover, TNNI3K overexpression induced cTnI phosphorylation at Ser22/Ser23 in vivo and in vitro, suggesting that TNNI3K is a novel upstream regulator for cTnI phosphorylation.
TNNI3K promotes a concentric hypertrophy with enhancement of cardiac function via regulating the phosphorylation of cTnI. TNNI3K could be a potential therapeutic target for preventing from heart failure.
Protein kinase Cα (PKCα), PKCβ, and PKCγ comprise the conventional PKC isoform subfamily, which is thought to regulate cardiac disease responsiveness. Indeed, mice lacking the gene for PKCα show enhanced cardiac contractility and reduced susceptibility to heart failure. Recent data also suggest that inhibition of conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 enhances heart function and antagonize failure, although the isoform responsible for these effects is unknown. Here we investigated mice lacking PKCα, PKCβ and PKCγ for effects on cardiac contractility and heart failure susceptibility. PKCα−/− mice, but not PKCβγ−/−, showed increased cardiac contractility, myocyte cellular contractility, Ca2+ transients, and sarcoplasmic reticulum Ca2+ load. PKCα−/− mice were less susceptible to heart failure following long-term pressure overload stimulation or 4 weeks after myocardial infarction injury, while PKCβγ−/− mice showed more severe failure. Infusion of ruboxistaurin (LY333531), an orally available PKCα/β/γ inhibitor, increased cardiac contractility in wildtype and PKCβγ−/− mice, but not in PKCα−/− mice. More importantly, ruboxistaurin prevented death in wildtype mice throughout 10 weeks of pressure overload stimulation, reduced ventricular dilation, enhanced ventricular performance, reduced fibrosis, and reduced pulmonary edema comparable to or better than metoprolol treatment. Ruboxistaurin was also administered to PKCβγ−/− mice subjected to pressure overload, resulting in less death and heart failure, implicating PKCα as the primary target of this drug in mitigating heart disease. As an aside, PKCαβγ triple null mice showed no defect in cardiac hypertrophy following pressure overload stimulation. In conclusion, PKCα functions distinct from PKCβ and PKCγ in regulating cardiac contractility and heart failure, and broad acting PKC inhibitors such as ruboxistaurin, could represent a novel therapeutic approach in treating human heart failure.
Heart failure; contractility; PKC; signaling; cardiomyopathy