CaMKII-dependent responses to ischemia and reperfusion challenges in the heart

Ischemic heart disease is a leading cause of death, and there is considerable imperative to identify effective therapeutic interventions. Cardiomyocyte Ca2+ overload is a major cause of ischemia and reperfusion injury, initiating a cascade of events culminating in cardiomyocyte death, myocardial dysfunction, and occurrence of lethal arrhythmias. Responsive to fluctuations in intracellular Ca2+, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has emerged as an enticing therapeutic target in the management of ischemic heart injury. CaMKII is activated early in ischemia and to a greater extent in the first few minutes of reperfusion, at a time when reperfusion arrhythmias are particularly prominent. CaMKII phosphorylates and upregulates many of the key proteins involved in intracellular Na+ and Ca2+ loading in ischemia and reperfusion. Experimentally, selective inhibition of CaMKII activity reduces cardiomyocyte death and arrhythmic incidence post-ischemia. New evidence is emerging that CaMKII actions in ischemia and reperfusion involve specific splice variant targeted actions, selective and localized post-translational modifications, and organelle-directed substrate interactions. A more complete mechanistic understanding of CaMKII mode of action in ischemia and reperfusion is required to optimize intervention opportunities. This review summarizes the current experimentally derived understanding of CaMKII participation in mediating the pathophysiology of the heart in ischemia and in reperfusion, and highlights priority future research directions.Centro de Investigaciones Cardiovasculare

CaMKII-dependent responses to ischemia and reperfusion challenges in the heart

Ischemic heart disease is a leading cause of death, and there is considerable imperative to identify effective therapeutic interventions. Cardiomyocyte Ca2+ overload is a major cause of ischemia and reperfusion injury, initiating a cascade of events culminating in cardiomyocyte death, myocardial dysfunction, and occurrence of lethal arrhythmias. Responsive to fluctuations in intracellular Ca2+, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has emerged as an enticing therapeutic target in the management of ischemic heart injury. CaMKII is activated early in ischemia and to a greater extent in the first few minutes of reperfusion, at a time when reperfusion arrhythmias are particularly prominent. CaMKII phosphorylates and upregulates many of the key proteins involved in intracellular Na+ and Ca2+ loading in ischemia and reperfusion. Experimentally, selective inhibition of CaMKII activity reduces cardiomyocyte death and arrhythmic incidence post-ischemia. New evidence is emerging that CaMKII actions in ischemia and reperfusion involve specific splice variant targeted actions, selective and localized post-translational modifications, and organelle-directed substrate interactions. A more complete mechanistic understanding of CaMKII mode of action in ischemia and reperfusion is required to optimize intervention opportunities. This review summarizes the current experimentally derived understanding of CaMKII participation in mediating the pathophysiology of the heart in ischemia and in reperfusion, and highlights priority future research directions.Centro de Investigaciones Cardiovasculare

Transcriptomic effects of adenosine 2A receptor deletion in healthy and endotoxemic murine myocardium

Influences of adenosine 2A receptor (A2AR) activity on the cardiac transcriptome and genesis of endotoxemic myocarditis are unclear. We applied transcriptomic profiling (39 K Affymetrix arrays) to identify A2AR-sensitive molecules, revealed by receptor knockout (KO), in healthy and endotoxemic hearts. Baseline cardiac function was unaltered and only 37 A2AR-sensitive genes modified by A2AR KO (≥1.2-fold change, \u3c5 \u3e% FDR); the five most induced are Mtr, Ppbp, Chac1, Ctsk and Cnpy2 and the five most repressed are Hp, Yipf4, Acta1, Cidec and Map3k2. Few canonical paths were impacted, with altered Gnb1, Prkar2b, Pde3b and Map3k2 (among others) implicating modified G protein/cAMP/PKA and cGMP/NOS signalling. Lipopolysaccharide (LPS; 20 mg/kg) challenge for 24 h modified \u3e4100 transcripts in wild-type (WT) myocardium (≥1.5-fold change, FDR \u3c 1 %); the most induced are Lcn2 (+590); Saa3 (+516); Serpina3n (+122); Cxcl9 (+101) and Cxcl1 (+89) and the most repressed are Car3 (−38); Adipoq (−17); Atgrl1/Aplnr (−14); H19 (−11) and Itga8 (−8). Canonical responses centred on inflammation, immunity, cell death and remodelling, with pronounced amplification of toll-like receptor (TLR) and underlying JAK-STAT, NFκB and MAPK pathways, and a ‘cardio-depressant’ profile encompassing suppressed ß-adrenergic, PKA and Ca2+ signalling, electromechanical and mitochondrial function (and major shifts in transcripts impacting function/injury including Lcn2, S100a8/S100a9, Icam1/Vcam and Nox2 induction, and Adipoq, Igf1 and Aplnr repression). Endotoxemic responses were selectively modified by A2AR KO, supporting inflammatory suppression via A2AR sensitive shifts in regulators of NFκB and JAK-STAT signalling (IκBζ, IκBα, STAT1, CDKN1a and RRAS2) without impacting the cardio-depressant gene profile. Data indicate A2ARs exert minor effects in un-stressed myocardium and selectively suppress NFκB and JAK-STAT signalling and cardiac injury without influencing cardiac depression in endotoxemia

Male and female hypertrophic rat cardiac myocyte functional responses to ischemic stress and β-adrenergic challenge are different

Background: Cardiac hypertrophy is the most potent cardiovascular risk factor after age, and relative mortality risk linked with cardiac hypertrophy is greater in women. Ischemic heart disease is the most common form of cardiovascular pathology for both men and women, yet significant differences in incidence and outcomes exist between the sexes. Cardiac hypertrophy and ischemia are frequently occurring dual pathologies. Whether the cellular (cardiomyocyte) mechanisms underlying myocardial damage differ in women and men remains to be determined. In this study, utilizing an in vitro experimental approach, our goal was to examine the proposition that responses of male/female cardiomyocytes to ischemic (and adrenergic) stress may be differentially modulated by the presence of pre-existing cardiac hypertrophy. Methods: We used a novel normotensive custom-derived hypertrophic heart rat (HHR; vs control strain normal heart rat (NHR)). Cardiomyocyte morphologic and electromechanical functional studies were performed using microfluorimetric techniques involving simulated ischemia/reperfusion protocols. Results: HHR females exhibited pronounced cardiac/cardiomyocyte enlargement, equivalent to males. Under basal conditions, a lower twitch amplitude in female myocytes was prominent in normal but not in hypertrophic myocytes. The cardiomyocyte Ca2+ responses to β-adrenergic challenge differed in hypertrophic male and female cardiomyocytes, with the accentuated response in males abrogated in females - even while contractile responses were similar. In simulated ischemia, a marked and selective elevation of end-ischemia Ca2+ in normal female myocytes was completely suppressed in hypertrophic female myocytes - even though all groups demonstrated similar shifts in myocyte contractile performance. After 30 min of simulated reperfusion, the Ca2+ desensitization characterizing the male response was distinctively absent in female cardiomyocytes. Conclusions: Our data demonstrate that cardiac hypertrophy produces dramatically different basal and stress-induced pathophenotypes in female- and male-origin cardiomyocytes. The lower Ca2+ operational status characteristic of female (vs male) cardiomyocytes comprising normal hearts is not exhibited by myocytes of hypertrophic hearts. After ischemia/reperfusion, availability of activator Ca2+ is suppressed in female hypertrophic myocytes, whereas sensitivity to Ca2+ is blunted in male hypertrophic myocytes. These findings demonstrate that selective intervention strategies should be pursued to optimize post-ischemic electromechanical support for male and female hypertrophic hearts.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

A novel small molecule inhibitor of human Drp1

Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1

Fructose Modulates Cardiomyocyte Excitation-Contraction Coupling and Ca2+ Handling In Vitro

BACKGROUND: High dietary fructose has structural and metabolic cardiac impact, but the potential for fructose to exert direct myocardial action is uncertain. Cardiomyocyte functional responsiveness to fructose, and capacity to transport fructose has not been previously demonstrated. OBJECTIVE: The aim of the present study was to seek evidence of fructose-induced modulation of cardiomyocyte excitation-contraction coupling in an acute, in vitro setting. METHODS AND RESULTS: The functional effects of fructose on isolated adult rat cardiomyocyte contractility and Ca²⁺ handling were evaluated under physiological conditions (37°C, 2 mM Ca²⁺, HEPES buffer, 4 Hz stimulation) using video edge detection and microfluorimetry (Fura2) methods. Compared with control glucose (11 mM) superfusate, 2-deoxyglucose (2 DG, 11 mM) substitution prolonged both the contraction and relaxation phases of the twitch (by 16 and 36% respectively, p<0.05) and this effect was completely abrogated with fructose supplementation (11 mM). Similarly, fructose prevented the Ca²⁺ transient delay induced by exposure to 2 DG (time to peak Ca²⁺ transient: 2 DG: 29.0±2.1 ms vs. glucose: 23.6±1.1 ms vs. fructose +2 DG: 23.7±1.0 ms; p<0.05). The presence of the fructose transporter, GLUT5 (Slc2a5) was demonstrated in ventricular cardiomyocytes using real time RT-PCR and this was confirmed by conventional RT-PCR. CONCLUSION: This is the first demonstration of an acute influence of fructose on cardiomyocyte excitation-contraction coupling. The findings indicate cardiomyocyte capacity to transport and functionally utilize exogenously supplied fructose. This study provides the impetus for future research directed towards characterizing myocardial fructose metabolism and understanding how long term high fructose intake may contribute to modulating cardiac function

Sexual dimorphism in cancer.

The incidence of many types of cancer arising in organs with non-reproductive functions is significantly higher in male populations than in female populations, with associated differences in survival. Occupational and/or behavioural factors are well-known underlying determinants. However, cellular and molecular differences between the two sexes are also likely to be important. In this Opinion article, we focus on the complex interplay that sex hormones and sex chromosomes can have in intrinsic control of cancer-initiating cell populations, the tumour microenvironment and systemic determinants of cancer development, such as the immune system and metabolism. A better appreciation of these differences between the two sexes could be of substantial value for cancer prevention as well as treatment

Cardiomyocytes birefringence imaging: simultaneous phase and retardation 2D measurements

Quantitative Phase Microscopy is a simple imaging methodology for resolving internal morphology of unstained living specimens. It computes the 2D phase images of a specimen from a combination of bright field images. However, when used in conjunction with polarised light can lead to the simultaneous mapping of the phase and the retardation of an optically anisotropic specimen. Experimentally the procedure is implemented without the need of expensive compensators used in traditional methods

Autophagic predisposition in the insulin resistant diabetic heart

Existence of a diabetic cardiopathology, independent of vascular abnormalities, has been well reported. Diffuse interstitial fibrosis throughout the diabetic myocardium (even in the absence of an acute coronary event) suggests widespread cardiomyocyte attrition and cytokine activity. In addition to apoptotic and necrotic events, there is now good evidence that significant cardiomyocyte loss in the diabetic heart is driven by a different, non-apoptotic type of programmed cell death: autophagy. Although considered to be beneficial and pro-survival as a short term strategy to deal with acute stress, when chronically elevated or constitutive, excess autophagic activity has potential to be lethal. The insulin resistant myocardium exhibits various pro-autophagic characteristics: suppression of the PI3K(I)-Akt signaling pathway, oxidative stress and metabolic dysregulation, rendering the diabetic heart vulnerable to autophagic demise. There is compelling new evidence that in the diabetic myocardium cardiomyocyte attrition can be linked to autophagic upregulation