Linke, Wolfgang A.

Abstract Metabolic disorders such as obesity, type 2 diabetes, and dyslipidaemia are major drivers of cardiovascular risk and have reached epidemic levels in Western populations. Recent advances in clinical research have uncovered unexpected and often beneficial effects of pharmacological classes of metabolic drugs on cardiovascular outcomes, revealing complex interactions between metabolism and cardiac pathology. Despite their clinical efficacy, the molecular and cellular mechanisms through which many of these agents act remain only partially understood. This gap in knowledge underscores the urgent need for fundamental research that not only dissects the biology of metabolic disorders and cardiovascular disease but also reveals the mechanistic basis of existing and emerging therapies, thereby enabling rational therapeutic development. This scientific statement, developed by researchers from the European Society of Cardiology (ESC) Council on Basic Cardiovascular Science and several ESC Working Groups, provides a state-of-the-art overview of the emerging molecular and cellular pathways linking metabolic abnormalities with vascular and cardiac disease. By illuminating these critical connections and unresolved questions, this article aims to catalyse innovation in the prevention and management of cardiovascular disease in the context of metabolic dysfunction and to suggest new directions for future research.

Myofiber passive stiffness is lowered by phosphorylation of the giant sarcomeric protein titin, with beneficial effects on diastolic function. Titin can be phosphorylated by cGMP-activated protein kinase (PK)G, a pathway stimulated by B-type natriuretic peptide (BNP) or PDE-5A inhibitor (sildenafil). Whether titin phosphorylation and stiffness are affected by PKG activation in vivo had not been studied. Here we examined how dogs with experimental hypertension and diastolic dysfunction, induced by renal wrapping, respond to treatment with β-blockers, sildenafil, and BNP, in terms of altered titin phosphorylation and passive stiffness. Isolated permeabilized cardiomyocytes from left ventricular (LV) biopsies untreated (n=5), treated with β-blockers (n=7), followed by sildenafil (n=7) and BNP (n=7) were attached to a force transducer and the passive length tension relation (F pas ) was measured between 1.8 and 2.4µm sarcomere length (SL) before and after PKA and PKG administration. We also assessed phosphorylation of myofilament proteins including titin, cMyBPC, desmin, cTnT, cTnI and MLC2 by gel electrophoresis (SYPRO Ruby and ProQ Diamond stain) and expression of titin PEVK, phospho-PEVK, cTnI, phospho-cTnI (at Ser 23/24), PKCα and phospho-PKCα by Western blotting. Compared to untreated cardiomyocytes, F pas was unaltered with β-blocker therapy, but significantly reduced with sildenafil; with BNP F pas did not change further. Ex-vivo administration of PKG lowered F pas of untreated and β-blocker cardiomyocytes to the levels measured in sildenafil and BNP samples, and additional administration of PKA did not further change F pas . Total titin phosphorylation was low in untreated samples and with β-blockade, but significantly increased with sildenafil and BNP. Total cTnI phosphorylation and phospho-cTnI at Ser 23/24 were low in untreated compared to treated samples but were similar between β-blocker, sildenafil and BNP groups. Phospho-PEVK and phospho-PKCα were high in untreated samples and with β-blockade, but reduced with sildenafil and BNP. Acute cGMP enhancing therapy with sildenafil and BNP improves LV diastolic function through correction of a titin phosphorylation deficit, thereby reducing cardiomyocyte passive stiffness.

Background: Reduced left atrial (LA) compliance (LAC) contributes to pulmonary venous hypertension in heart failure with preserved ejection fraction (HFpEF); however the underlying mechanisms are poorly defined. We hypothesized that microvascular inflammation with rarefaction and impaired nitric oxide (NO) signaling, LA titin hypophosphorylation and fibrosis may each contribute to reduced LAC in HFpEF. Methods: Invasive LA pressure-volume (PV) analyses (open-chest; pericardiotomy; admittance catheter) were performed in 9 aged dogs with experimental hypertension (bilateral renal wrap + DOCA; HFpEF) compared to 13 sham-operated young adult dogs (control). LAC was measured at end-LA reservoir during acute preload reduction (cava occlusion). LA tissue and LA microvascular function were analyzed. Results: Compared to controls, the curvilinear end-LA reservoir PV relationship ( Fig A ) was shifted upward/leftward in HFpEF (LA diastolic stiffness constant 0.08 vs 0.16 mmHg/mL in controls, p=0.005). LA microvascular density (MVD; CD31 stain) tended to be lower in HFpEF than controls (2154±578 vs 2650±523 vessels/mm 2 , p=0.07) and LA MVD negatively correlated with the LA diastolic stiffness constant (r=-0.66, p=0.006). LA microvessels from HFpEF dogs showed impaired endothelium-dependent shear stress-mediated dilation compared to controls, consistent with impaired NO signaling ( Fig B ). Titin isoform expression was similar between groups, but isoform N2B (stiffer) was (p=0.03) and N2BA (compliant) tended to be (p=0.09) hypophosphorylated in HFpEF LA appendage (LAA) tissue. Fibrosis (Picrosirius Red) was similar between groups in the LA free wall and LAA, but increased in areas adjacent to the pulmonary veins in HFpEF (27±12% vs controls 15±8%, p=0.01). Conclusion: In experimental HFpEF, LAC was reduced in association with evidence of LA microvascular rarefaction and impaired NO signaling, titin hypophosphorylation and regional increases in LA fibrosis.

Background— Left atrial (LA) compliance and contractility influence left ventricular stroke volume. We hypothesized that diminished LA compliance and contractile function occur early during the development of heart failure with preserved ejection fraction (HFpEF) and impair overall cardiac performance. Methods and Results— Cardiac magnetic resonance imaging, echocardiography, left ventricular and LA pressure-volume studies, and tissue analyses were performed in a model of early HFpEF (elderly dogs, renal wrap-induced hypertension, exogenous aldosterone; n=9) and young control dogs (sham surgery; n=13). Early HFpEF was associated with LA enlargement, cardiomyocyte hypertrophy, and enhanced LA contractile function (median active emptying fraction 16% [95% confidence interval, 13–24]% versus 12 [10–14]%, P =0.008; end-systolic pressure-volume relationship slope 2.4 [1.9–3.2]mm Hg/mL HFpEF versus 1.5 [1.2–2.2]mm Hg/mL controls, P =0.01). However, atrioventricular coupling was impaired and the curvilinear LA end-reservoir pressure-volume relationship was shifted upward/leftward in HFpEF (LA stiffness constant [β LA ] 0.16 [0.11–0.18]mm Hg/mL versus 0.06 [0.04–0.10]mm Hg/mL controls; P =0.002), indicating reduced LA compliance. Impaired atrioventricular coupling and lower LA compliance correlated with lower left ventricular stroke volume. Total fibrosis and titin isoform composition were similar between groups; however, titin was hyperphosphorylated in HFpEF and correlated with β LA . LA microvascular reactivity was diminished in HFpEF versus controls. LA microvascular density tended to be lower in HFpEF and inversely correlated with β LA . Conclusions— In early-stage hypertensive HFpEF, LA cardiomyocyte hypertrophy, titin hyperphosphorylation, and microvascular dysfunction occur in association with increased systolic and diastolic LA chamber stiffness, impaired atrioventricular coupling, and decreased left ventricular stroke volume. These data indicate that maladaptive LA remodeling occurs early during HFpEF development, supporting a concept of global myocardial remodeling.

Background— In vitro studies suggest that phosphorylation of titin reduces myocyte/myofiber stiffness. Titin can be phosphorylated by cGMP-activated protein kinase. Intracellular cGMP production is stimulated by B-type natriuretic peptide (BNP) and degraded by phosphodiesterases, including phosphodiesterase-5A. We hypothesized that a phosphodiesterase-5A inhibitor (sildenafil) alone or in combination with BNP would increase left ventricular diastolic distensibility by phosphorylating titin. Methods and Results— Eight elderly dogs with experimental hypertension and 4 young normal dogs underwent measurement of the end-diastolic pressure-volume relationship during caval occlusion at baseline, after sildenafil, and BNP infusion. To assess diastolic distensibility independently of load/extrinsic forces, the end-diastolic volume at a common end-diastolic pressure on the sequential end-diastolic pressure-volume relationships was measured (left ventricular capacitance). In a separate group of dogs (n=7 old hypertensive and 7 young normal), serial full-thickness left ventricular biopsies were harvested from the beating heart during identical infusions to measure myofilament protein phosphorylation. Plasma cGMP increased with sildenafil and further with BNP (7.31±2.37 to 26.9±10.3 to 70.3±8.1 pmol/mL; P <0.001). Left ventricular diastolic capacitance increased with sildenafil and further with BNP (51.4±16.9 to 53.7±16.8 to 60.0±19.4 mL; P <0.001). Changes were similar in old hypertensive and young normal dogs. There were no effects on phosphorylation of troponin I, troponin T, phospholamban, or myosin light chain-1 or -2. Titin phosphorylation increased with sildenafil and BNP, whereas titin-based cardiomyocyte stiffness decreased. Conclusion— Short-term cGMP-enhancing treatment with sildenafil and BNP improves left ventricular diastolic distensibility in vivo, in part by phosphorylating titin.

Background— Altered titin isoforms may modify cardiac function in heart failure (HF), but the nature of isoform switches and associated functional implications are not well defined. Limited studies have reported an increased compliant isoform (N2BA) expression in human systolic HF. Titin may also modulate stretch-regulated responses such as myocardial natriuretic peptide production. Methods and Results— We characterized titin isoform expression and extracellular matrix in all 4 cardiac chambers and the left ventricular (LV) epicardium and endocardium in normal dogs (n=6) and those with HF (n=6) due to tachypacing and characterized functional implications at the LV myofiber and chamber level. Recognizing the potential for uncoupling of the extracellular matrix and cardiomyocyte in tachypacing, myocardial natriuretic peptide production, a molecular marker of stretch-regulated responses, was also assessed. All chambers were dilated in HF, but the extracellular matrix was not increased. HF dogs had markedly lower N2BA in the atria and right ventricle. In failing LVs, N2BA was decreased only in the epicardium, where myofiber passive stiffness was increased. However, LV chamber mechanics were driven by the marked LV dilatation, with no increase in LV diastolic stiffness. Natriuretic peptide concentrations increased markedly in the endocardium in relation to increases in LV wall stress. Conclusions— Tachypacing HF is characterized by decreases in compliant titin isoform expression in the atria, right ventricle, and LV epicardium. However, LV chamber mechanics are principally determined by geometric and extracellular matrix changes rather than titin-based myofiber stiffness in this model. Stretch-regulated myocardial responses (natriuretic peptide production) appeared intact, suggesting that the mechanotransduction role of titin was not impaired in HF.

Heart failure with normal ejection fraction occurs in elderly patients with hypertensive heart disease. We hypothesized that, in such patients, mineralocorticoid receptor activation accelerates the types of ventricular and vascular remodeling and dysfunction believed important in the transition to heart failure. We tested this hypothesis by administering deoxycorticosterone acetate (DOCA) without salt loading or nephrectomy to elderly dogs with experimental hypertension. Elderly dogs were made hypertensive by renal wrapping. After 5 weeks, dogs were randomly assigned to DOCA (1 mg/kg per day IM; old hypertensive [OH]+DOCA; n=11) or not (OH; n=11) for 3 weeks. At week 8, conscious echocardiography and hemodynamic assessment under anesthesia were performed. DOCA resulted in further increases in conscious blood pressure ( P <0.05) without increases in cardiac output or diastolic volume. In the conscious state, effective arterial elastance ( P <0.05) and systemic vascular resistance ( P =0.06) were increased, and systemic arterial compliance ( P <0.05) was decreased in OH+DOCA animals. After anesthesia, instrumentation, and autonomic blockade, blood pressure was lower, whereas left ventricular (LV) systolic elastance, LV diastolic stiffness, and ex vivo myofiber diastolic stiffness were increased in OH+DOCA animals. LV collagen was increased in OH+DOCA animals ( P <0.05 for all), but LV mass, LV brain natriuretic peptide, and titin isoform profiles were not. Neither aortic stiffness nor aortic structure was altered in OH+DOCA animals. These findings suggest that age and hypertensive heart disease enhance sensitivity to exogenous mineralocorticoid administration and that mineralocorticoid receptor activation could contribute to the transition to heart failure in elderly persons by promoting increases in LV diastolic and systolic stiffness.

Aging and hypertension lead to arterial remodeling and tandem increases in arterial (Ea) and ventricular (LV) systolic stiffness (ventricular-arterial [VA] coupling). Age and hypertension also predispose to heart failure with normal ejection fraction (HFnlEF), where symptoms during hypertensive urgencies or exercise are common. We hypothesized that: (1) chronic VA coupling also occurs in diastole, (2) acute changes in Ea are coupled with shifts in the diastolic and systolic pressure-volume relationships (PVR), and (3) diastolic VA coupling reflects changes in LV diastolic stiffness rather than external forces or relaxation. Old chronically hypertensive (OHT, n=8) and young normal (YNL, n=7) dogs underwent assessment of PVR (caval occlusion) and of aortic pressure, dimension, and flow, at baseline and during changes in afterload and preload. Concomitant changes in the slope/position of PVR were accounted for by calculating systolic (ESV 200 ) and diastolic (EDV 20 ) volumes at common pressures (capacitance). OHT displayed marked vascular remodeling. Indices reflecting the pulsatile component of Ea (aortic stiffness and systemic arterial compliance) were more impaired in OHT at any distending pressure. In both groups, acute increases in Ea were associated with decreases in ESV 200 and EDV 20 . However, at any load, OHT had lower ESV 200 and EDV 20 , associated with LV remodeling and myocardial endothelin activation. Acute changes in EDV 20 were not mediated by changes in relaxation or external forces. These observations provide insight into the mechanisms whereby arterial remodeling and acute and chronic VA coupling in both systole and diastole may predispose to and interact with increases in load to cause HFnlEF.