Small heat shock protein 20 (Hsp20) facilitates nuclear import of protein kinase D 1 (PKD1) during cardiac hypertrophy.

BACKGROUND: Nuclear import of protein kinase D1 (PKD1) is an important event in the transcriptional regulation of cardiac gene reprogramming leading to the hypertrophic growth response, however, little is known about the molecular events that govern this event. We have identified a novel complex between PKD1 and a heat shock protein (Hsp), Hsp20, which has been implicated as cardioprotective. This study aims to characterize the role of the complex in PKD1-mediated myocardial regulatory mechanisms that depend on PKD1 nuclear translocation. RESULTS: In mapping the Hsp20 binding sites on PKD1 within its catalytic unit using peptide array analysis, we were able to develop a cell-permeable peptide that disrupts the Hsp20-PKD1 complex. We use this peptide to show that formation of the Hsp20-PKD1 complex is essential for PKD1 nuclear translocation, signaling mechanisms leading to hypertrophy, activation of the fetal gene programme and pathological cardiac remodeling leading to cardiac fibrosis. CONCLUSIONS: These results identify a new signaling complex that is pivotal to pathological remodelling of the heart that could be targeted therapeutically.

Adult cardiac fibroblast proliferation is modulated by calcium/calmodulin-dependent protein kinase II in normal and hypertrophied hearts.

Increased adult cardiac fibroblast proliferation results in an increased collagen deposition responsible for the fibrosis accompanying pathological remodelling of the heart. The mechanisms regulating cardiac fibroblast proliferation remain poorly understood. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we have assessed fibrosis and cardiac fibroblast proliferation. We have investigated whether calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) regulates proliferation in fibroblasts isolated from normal and hypertrophied hearts. It is known that CaMKIIδ plays a central role in cardiac myocyte contractility, but nothing is known of its role in adult cardiac fibroblast function. The MTAB model used here produces extensive hypertrophy and fibrosis. CaMKIIδ protein expression and activity is upregulated in MTAB hearts and, specifically, in cardiac fibroblasts isolated from hypertrophied hearts. In response to angiotensin II, cardiac fibroblasts isolated from MTAB hearts show increased proliferation rates. Inhibition of CaMKII with autocamtide inhibitory peptide inhibits proliferation in cells isolated from both sham and MTAB hearts, with a significantly greater effect evident in MTAB cells. These results are the first to show selective upregulation of CaMKIIδ in adult cardiac fibroblasts following cardiac hypertrophy and to assign a previously unrecognised role to CaMKII in regulating adult cardiac fibroblast function in normal and diseased hearts.

The cardioprotective role of small heat-shock protein 20.

The small HSP (heat-shock protein) HSP20 is a molecular chaperone that is transiently up-regulated in response to cellular stress/damage. Although ubiquitously expressed in various tissues, it is most highly expressed in skeletal, cardiac and smooth muscle. Phosphorylation at Ser16 by PKA (cAMP-dependent protein kinase) is essential for HSP20 to confer its protective qualities. HSP20 and its phosphorylation have been implicated in a variety of pathophysiological processes, but most prominently cardiovascular disease. A wealth of knowledge of the importance of HSP20 in contractile function and cardioprotection has been gained over the last decade. The present mini-review highlights more recent findings illustrating the cardioprotective properties of HSP20 and its potential as a therapeutic agent.

A novel ryanodine receptor 2 inhibitor, M201-A, enhances natriuresis, renal function and lusi-inotropic actions: Preclinical and phase I study.

BACKGROUND AND PURPOSE: The ryanodine receptor 2 (RyR2) is present in both the heart and kidneys, and plays a crucial role in maintaining intracellular Ca2+ homeostasis in cells in these organs. This study aimed to investigate the impact of M201-A on RyR2, as well as studying its effects on cardiac and renal functions in preclinical and clinical studies. EXPERIMENTAL APPROACH: Following the administration of M201-A (1,4-benzothiazepine-1-oxide derivative), we monitored diastolic Ca2+ leak via RyR2 and intracellular Ca2+ concentration in isolated rat cardiomyocytes and in cardiac and renal function in animals. In a clinical study, M201-A was administered intravenously at doses of 0.2 and 0.4 mg·kg-1 once daily for 20 min for four consecutive days in healthy males, with the assessment of haemodynamic responses. KEY RESULTS: In rat heart cells, M201-A effectively inhibited spontaneous diastolic Ca2+ leakage through RyR2 and exhibited positive lusi-inotropic effects on the rat heart. Additionally, it enhanced natriuresis and improved renal function in dogs. In human clinical studies, when administered intravenously, M201-A demonstrated an increase in natriuresis, glomerular filtration rate and creatinine clearance, while maintaining acceptable levels of drug safety and tolerability. CONCLUSIONS AND IMPLICATIONS: The novel drug M201-A inhibited diastolic Ca2+ leak via RyR2, improved cardiac lusi-inotropic effects in rats, and enhanced natriuresis and renal function in humans. These findings suggest that this drug may offer a potential new treatment option for chronic kidney disease and heart failure.

Ribonucleicacid interference or small molecule inhibition of Runx1 in the border zone prevents cardiac contractile dysfunction following myocardial infarction.

AIMS: Myocardial infarction (MI) is a major cause of death worldwide. Effective treatments are required to improve recovery of cardiac function following MI, with the aim of improving patient outcomes and preventing progression to heart failure. The perfused but hypocontractile region bordering an infarct is functionally distinct from the remote surviving myocardium and is a determinant of adverse remodelling and cardiac contractility. Expression of the transcription factor RUNX1 is increased in the border zone 1-day after MI, suggesting potential for targeted therapeutic intervention. OBJECTIVE: This study sought to investigate whether an increase in RUNX1 in the border zone can be therapeutically targeted to preserve contractility following MI. METHODS AND RESULTS: In this work we demonstrate that Runx1 drives reductions in cardiomyocyte contractility, calcium handling, mitochondrial density, and expression of genes important for oxidative phosphorylation. Both tamoxifen-inducible Runx1-deficient and essential co-factor common ß subunit (Cbfß)-deficient cardiomyocyte-specific mouse models demonstrated that antagonizing RUNX1 function preserves the expression of genes important for oxidative phosphorylation following MI. Antagonizing RUNX1 expression via short-hairpin RNA interference preserved contractile function following MI. Equivalent effects were obtained with a small molecule inhibitor (Ro5-3335) that reduces RUNX1 function by blocking its interaction with CBFß. CONCLUSIONS: Our results confirm the translational potential of RUNX1 as a novel therapeutic target in MI, with wider opportunities for use across a range of cardiac diseases where RUNX1 drives adverse cardiac remodelling.

Surgical optimization and characterization of a minimally invasive aortic banding procedure to induce cardiac hypertrophy in mice.

Left ventricular pressure overload in response to aortic banding is an invaluable model for studying progression of cardiac hypertrophy and transition to heart failure. Traditional aortic banding has recently been superceded by minimally invasive transverse aortic banding (MTAB), which does not require ventilation so is less technically challenging. Although the MTAB approach is superior, few laboratories have documented success, and minimal information on the model is available. The aim of this study was to optimize conditions for MTAB and to characterize the development and progression of cardiac hypertrophy. Isofluorane proved the most suitable anaesthetic for MTAB surgery in mice, and 1 week after surgery the MTAB animals showed significant increases in systolic blood pressure (MTAB 110 ± 6 mmHg versus sham 78 ± 3 mmHg, n = 7, P < 0.0001) and heart weight to body weight ratio (MTAB 6.2 ± 0.2 versus sham 5.1 ± 0.1, n = 12, P < 0.001), together with systolic (e.g. fractional shortening, MTAB 31.7 ± 1% versus sham 36.6 ± 1.4%, P = 0.01) and diastolic dysfunction (e.g. left ventricular end-diastolic pressure, MTAB 12.7 ± 1.0 mmHg versus sham 6.7 ± 0.8 mmHg, P < 0.001). Leucocyte infiltration to the heart was evident after 1 week in MTAB hearts, signifying an inflammatory response. More pronounced remodelling was observed 4 weeks postsurgery (heart weight to body weight ratio, MTAB 9.1 ± 0.6 versus sham 4.6 ± 0.04, n = 10, P < 0.0001) and fractional shortening was further decreased (MTAB 24.3 ± 2.5% versus sham 43.6 ± 1.7%, n = 10, P = 0.003), together with a significant increase in cardiac fibrosis and further cardiac inflammation. Our findings demonstrate that MTAB is a relevant experimental model for studying development and progression of cardiac hypertrophy, which will be highly valuable for future studies examining potential novel therapeutic interventions in this setting.

Preclinical models of myocardial infarction: from mechanism to translation.

Approximately 7 million people are affected by acute myocardial infarction (MI) each year, and despite significant therapeutic and diagnostic advancements, MI remains a leading cause of mortality worldwide. Preclinical animal models have significantly advanced our understanding of MI and have enabled the development of therapeutic strategies to combat this debilitating disease. Notably, some drugs currently used to treat MI and heart failure (HF) in patients had initially been studied in preclinical animal models. Despite this, preclinical models are limited in their ability to fully reproduce the complexity of MI in humans. The preclinical model must be carefully selected to maximise the translational potential of experimental findings. This review describes current experimental models of MI and considers how they have been used to understand drug mechanisms of action and support translational medicine development. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.

RUNX1: an emerging therapeutic target for cardiovascular disease.

Runt-related transcription factor-1 (RUNX1), also known as acute myeloid leukaemia 1 protein (AML1), is a member of the core-binding factor family of transcription factors which modulate cell proliferation, differentiation, and survival in multiple systems. It is a master-regulator transcription factor, which has been implicated in diverse signalling pathways and cellular mechanisms during normal development and disease. RUNX1 is best characterized for its indispensable role for definitive haematopoiesis and its involvement in haematological malignancies. However, more recently RUNX1 has been identified as a key regulator of adverse cardiac remodelling following myocardial infarction. This review discusses the role RUNX1 plays in the heart and highlights its therapeutic potential as a target to limit the progression of adverse cardiac remodelling and heart failure.

Signalling pathways linking cysteine cathepsins to adverse cardiac remodelling.

Adverse cardiac remodelling clinically manifests as deleterious changes to heart architecture (size, mass and geometry) and function. These changes, which include alterations to ventricular wall thickness, chamber dilation and poor contractility, are important because they progressively drive patients with cardiac disease towards heart failure and are associated with poor prognosis. Cysteine cathepsins contribute to key signalling pathways involved in adverse cardiac remodelling including synthesis and degradation of the cardiac extracellular matrix (ECM), cardiomyocyte hypertrophy, impaired cardiomyocyte contractility and apoptosis. In this review, we highlight the role of cathepsins in these signalling pathways as well as their translational potential as therapeutic targets in cardiac disease.

CaMKIIδ interacts directly with IKKß and modulates NF-κB signalling in adult cardiac fibroblasts.

Calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) acts as a molecular switch regulating cardiovascular Ca2+ handling and contractility in health and disease. Activation of CaMKIIδ is also known to regulate cardiovascular inflammation and is reported to be required for pro-inflammatory NF-κB signalling. In this study the aim was to characterise how CaMKIIδ interacts with and modulates NF-κB signalling and whether this interaction exists in non-contractile cells of the heart. Recombinant or purified CaMKIIδ and the individual inhibitory -κB kinase (IKK) proteins of the NF-κB signalling pathway were used in autoradiography and Surface Plasmon Resonance (SPR) to explore potential interactions between both components. Primary adult rat cardiac fibroblasts were then used to study the effects of selective CaMKII inhibition on pharmacologically-induced NF-κB activation as well as interaction between CaMKII and specific IKK isoforms in a cardiac cellular setting. Autoradiography analysis suggested that CaMKIIδ phosphorylated IKKß but not IKKα. SPR analysis further supported a direct interaction between CaMKIIδ and IKKß but not between CaMKIIδ and IKKα or IKKγ. CaMKIIδ regulation of IκΒα degradation was explored in adult cardiac fibroblasts exposed to pharmacological stimulation. Cells were stimulated with agonist in the presence or absence of a CaMKII inhibitor, autocamtide inhibitory peptide (AIP). Selective inhibition of CaMKII resulted in reduced NF-κB activation, as measured by agonist-stimulated IκBα degradation. Importantly, and in agreement with the recombinant protein work, an interaction between CaMKII and IKKß was evident following Proximity Ligation Assays in adult cardiac fibroblasts. This study provides new evidence supporting direct interaction between CaMKIIδ and IKKß in pro-inflammatory signalling in cardiac fibroblasts and could represent a feature that may be exploited for therapeutic benefit.

Targeted disruption of the heat shock protein 20-phosphodiesterase 4D (PDE4D) interaction protects against pathological cardiac remodelling in a mouse model of hypertrophy.

Phosphorylated heat shock protein 20 (HSP20) is cardioprotective. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and a mouse model of pressure overload mediated hypertrophy, we show that peptide disruption of the HSP20-phosphodiesterase 4D (PDE4D) complex results in attenuation of action potential prolongation and protection against adverse cardiac remodelling. The later was evidenced by improved contractility, decreased heart weight to body weight ratio, and reduced interstitial and perivascular fibrosis. This study demonstrates that disruption of the specific HSP20-PDE4D interaction leads to attenuation of pathological cardiac remodelling.

A novel approach for assessing cardiac fibrosis using label-free second harmonic generation.

To determine whether second harmonic generation (SHG) can be used as a novel and improved label-free technique for detection of collagen deposition in the heart. To verify whether SHG will allow accurate quantification of altered collagen deposition in diseased hearts following hypertrophic remodelling. Minimally invasive transverse aortic banding (MTAB) of mouse hearts was used to generate a reproducible model of cardiac hypertrophy. Physiological and functional assessment of hypertrophic development was performed using echocardiography and post-mortem analysis of remodelled hearts. Cardiac fibroblasts were isolated from sham-operated and hypertrophied hearts and proliferation rates compared. Multi-photon laser scanning microscopy was used to capture both two-photon excited autofluorescence (TPEF) and SHG images simultaneously in two channels. TPEF images were subtracted from SHG images and the resulting signal intensities from ventricular tissue sections were calculated. Traditional picrosirius red staining was used to verify the suitability of the SHG application. MTAB surgery induced significant hypertrophic remodelling and increased cardiac fibroblast proliferation. A significant increase in the density of collagen fibres between hypertrophic and control tissues (p < 0.05) was evident using SHG. Similar increases and patterns of staining were observed using parallel traditional picrosirius red staining of collagen. Label-free SHG microscopy provides a new alternative method for quantifying collagen deposition in fibrotic hearts.