Effects of 6 weeks of treatment with dapagliflozin, a sodium- glucose co-transporter-2 inhibitor, on myocardial function and metabolism in patients with type 2 diabetes: A randomized, placebo-controlled, exploratory study

Aim: To explore the early effects of dapagliflozin on myocardial function and metabolism in patients with type 2 diabetes without heart failure.Materials and methods: Patients with type 2 diabetes on metformin treatment were randomized to double-blind, 6-week placebo or dapagliflozin 10 mg daily treatment. Investigations included cardiac function and structure with myocardial resonance imaging; cardiac oxygen consumption, perfusion and efficiency with [11 C]-acetate positron emission tomography (PET); and cardiac and hepatic fatty acid uptake with [18 F]-6-thia-heptadecanoic acid PET, analysed by ANCOVA as least square means with 95% confidence intervals.Results: Evaluable patients (placebo: n = 24, dapagliflozin: n = 25; 53% males) had a mean age of 64.4 years, a body mass index of 30.2 kg/m2 and an HbA1c of 6.7%. Body weight and HbA1c were significantly decreased by dapagliflozin versus placebo. Dapagliflozin had no effect on myocardial efficiency, but external left ventricular (LV) work (-0.095 [-0.145, -0.043] J/g/min) and LV oxygen consumption were significantly reduced (-0.30 [-0.49, -0.12] J/g/min) by dapagliflozin, although the changes were not statistically significant versus changes in the placebo group. Change in left atrial maximal volume with dapagliflozin versus placebo was -3.19 (-6.32, -0.07) mL/m2 (p = .056). Peak global radial strain decreased with dapagliflozin versus placebo (-3.92% [-7.57%, -0.28%]; p = .035), while peak global longitudinal and circumferential strains were unchanged. Hepatic fatty acid uptake was increased by dapagliflozin versus placebo (0.024 [0.004, 0.044] μmol/g/min; p = .018), while cardiac uptake was unchanged.Conclusions: This exploratory study indicates reduced heart work but limited effects on myocardial function, efficiency and cardiac fatty acid uptake, while hepatic fatty acid uptake increased, after 6 weeks of treatment with dapagliflozin.</p

Fumarate is cardioprotective via activation of the Nrf2 antioxidant pathway

The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents. © 2012 Elsevier Inc

The effects of high fat diet feeding on cardiac function in the C57BL6/J mouse strain

It has been established that placing the C57BL6/J mouse on a high fat diet induces obesity and impaired glucose homeostasis, and produces a model that is pathophysiologically relevant to the human condition of the metabolic syndrome. The cardiovascular changes in this model have been relatively poorly explored. I have focussed my work on understanding the effects of this diet on cardiac function using in vivo cardiac functional assessment in combination with in vitro analysis of intact single cardiomyocyte dynamics and Ca2+ handling, as well as demembranated left ventricle trabeculae for the study of myofilament function. High fat diet caused increased left ventricular end diastolic pressure after 30 weeks as assessed by in vivo pressure catheterisation. This change was associated with, increased passive tension in demembranated trabeculae at 30 w, increased collagen 3 expression on mRNA level and increased total tissue collagen from high fat diet after 40 weeks. Reversion to normal diet for 10 weeks had no discernible effect on whole organ function compared with animals continually fed with the high fat diet, despite body fat content and glucose homeostasis becoming normalised. Isolated cardiomyocytes exhibited increased relaxation rate after 40 weeks which was partly reversed by a return to normal diet. The increased cardiomyocyte relaxation rate occurred without changes to the intracellular Ca2+ transient, thus suggesting it may be caused by altered myofilament Ca2+ sensitivity. There was no established decreased myofilament Ca2+ sensitivity seen in demembranated trabeculae at week 40, even though trends were seen towards increased phosphorylation of serines 23/24 of cardiac troponin I, a modification known to induce Ca2+ desensitisation. It is concluded that high fat diet feeding in the C57BL6/J mouse is a mild but valid model for studying the effects on cardiac function of obesity and impaired glucose handling. It results in impaired diastolic function at the whole organ level after 30 weeks feeding. This effect is likely principally caused by extracellular deposition of collagen (which reduces compliance) rather than by changes at the cardiomyocyte level. This whole organ diastolic alteration appears to become established, not responding to dietary fat reduction. The increased relaxation rate of cardiomyocytes more likely originates from changes at the myofilament level as opposed to Ca2+ handling although this could not be statistically verified in this study. As high fat diet feeding was found to increase myocardial NADPH oxidase activity, myofilament function was also assessed in a mouse model of NADPH oxidase 2 over-expression. NADPH oxidase 2 over-expression was found to cause myofilament sensitisation to Ca2+ and increased actomyosin cross bridge cycling rate at maximum Ca2+ activation. This was associated with an increased total phosphorylation of cardiac troponin I independent of phosphorylation of serines 23/24. This finding may represent a new signalling pathway linking NADPH oxidase 2 activity to contractile activation via the myofilament but appears opposite to myofilament changes induced by high fat diet feeding.</p

The effects of high fat diet feeding on cardiac function in the C57BL6/J mouse strain

It has been established that placing the C57BL6/J mouse on a high fat diet induces obesity and impaired glucose homeostasis, and produces a model that is pathophysiologically relevant to the human condition of the metabolic syndrome. The cardiovascular changes in this model have been relatively poorly explored. I have focussed my work on understanding the effects of this diet on cardiac function using in vivo cardiac functional assessment in combination with in vitro analysis of intact single cardiomyocyte dynamics and Ca2+ handling, as well as demembranated left ventricle trabeculae for the study of myofilament function. High fat diet caused increased left ventricular end diastolic pressure after 30 weeks as assessed by in vivo pressure catheterisation. This change was associated with, increased passive tension in demembranated trabeculae at 30 w, increased collagen 3 expression on mRNA level and increased total tissue collagen from high fat diet after 40 weeks. Reversion to normal diet for 10 weeks had no discernible effect on whole organ function compared with animals continually fed with the high fat diet, despite body fat content and glucose homeostasis becoming normalised. Isolated cardiomyocytes exhibited increased relaxation rate after 40 weeks which was partly reversed by a return to normal diet. The increased cardiomyocyte relaxation rate occurred without changes to the intracellular Ca2+ transient, thus suggesting it may be caused by altered myofilament Ca2+ sensitivity. There was no established decreased myofilament Ca2+ sensitivity seen in demembranated trabeculae at week 40, even though trends were seen towards increased phosphorylation of serines 23/24 of cardiac troponin I, a modification known to induce Ca2+ desensitisation. It is concluded that high fat diet feeding in the C57BL6/J mouse is a mild but valid model for studying the effects on cardiac function of obesity and impaired glucose handling. It results in impaired diastolic function at the whole organ level after 30 weeks feeding. This effect is likely principally caused by extracellular deposition of collagen (which reduces compliance) rather than by changes at the cardiomyocyte level. This whole organ diastolic alteration appears to become established, not responding to dietary fat reduction. The increased relaxation rate of cardiomyocytes more likely originates from changes at the myofilament level as opposed to Ca2+ handling although this could not be statistically verified in this study. As high fat diet feeding was found to increase myocardial NADPH oxidase activity, myofilament function was also assessed in a mouse model of NADPH oxidase 2 over-expression. NADPH oxidase 2 over-expression was found to cause myofilament sensitisation to Ca2+ and increased actomyosin cross bridge cycling rate at maximum Ca2+ activation. This was associated with an increased total phosphorylation of cardiac troponin I independent of phosphorylation of serines 23/24. This finding may represent a new signalling pathway linking NADPH oxidase 2 activity to contractile activation via the myofilament but appears opposite to myofilament changes induced by high fat diet feeding.</p

Phospholipase C and cAMP-dependent positive inotropic effects of ATP in mouse cardiomyocytes via P2Y(11)-like receptors.

ATP is released as a cotransmitter together with catecholamines from sympathetic nerves. In the heart ATP has been shown to cause a pronounced positive inotropic effect and may also act in synergy with β-adrenergic agonists to augment cardiomyocyte contractility. The aim of the present study was to investigate the inotropic effects mediated by purinergic P2 receptors using isolated mouse cardiomyocytes. Stable adenine nucleotide analogs were used and the agonist rank order for adenine nucleotide stimulation of the mouse cardiomyocytes was AR-C67085 > ATPγS > 2-MeSATP >>> 2-MeSADP = 0, that fits the agonist profile of the P2Y11 receptor. ATPγS induced a positive inotropic response in single mouse cardiomyocytes. The response was similar to that for the β1 receptor agonist isoproterenol. The most potent response was obtained using AR-C67085, a P2Y11 receptor agonist. This agonist also potentiated contractions in isolated trabecular preparations. The adenylyl cyclase blocker (SQ22563) and phospholipase C (PLC) blocker (U73122) demonstrated that both pathways were required for the inotropic response of AR-C67085. A cAMP enzyme immunoassay confirmed that AR-C67085 increased cAMP in the cardiomyocytes. These findings are in agreement with the P2Y11 receptor, coupled both to activation of IP3 and cAMP, being a major receptor for ATP induced inotropy. Analyzing cardiomyocytes from desmin deficient mice, Des–/–, with a congenital cardiomyopathy, we found a lower sensitivity to AR-C67085, suggesting a down-regulation of P2Y11 receptor function in heart failure. The prominent action of the P2Y11 receptor in controling cardiomyocyte contractility and possible alterations in its function during cardiomyopathy may suggest this receptor as a potential therapeutic target. It is possible that agonists for the P2Y11 receptor could be used to improve cardiac output in patients with circulatory shock and that P2Y11 receptor antagonist could be beneficial in patients with congestive heart failure (CHF)

AMP-activated protein kinase phosphorylates cardiac troponin I and alters contractility of murine ventricular myocytes

Rationale: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. Objective: The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. Methods and Results: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca 2+ sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca 2+ transient and prolonged relaxation despite shortening the time constant of Ca 2+ transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca 2+ sensitivity. Conclusions: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function. </jats:sec

Glucose-insulin-potassium reduces the incidence of low cardiac output episodes after aortic valve replacement for aortic stenosis in patients with left ventricular hypertrophy:results from the Hypertrophy, Insulin, Glucose, and Electrolytes (HINGE) trial

Patients undergoing aortic valve replacement for critical aortic stenosis often have significant left ventricular hypertrophy. Left ventricular hypertrophy has been identified as an independent predictor of poor outcome after aortic valve replacement as a result of a combination of maladaptive myocardial changes and inadequate myocardial protection at the time of surgery. Glucose-insulin-potassium (GIK) is a potentially useful adjunct to myocardial protection. This study was designed to evaluate the effects of GIK infusion in patients undergoing aortic valve replacement surgery

Functional analysis of a gene-edited mouse model to gain insights into the disease mechanisms of a titin missense variant

Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model