Ogut, Ozgur

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.

The participation of nonmuscle myosin in force maintenance is controversial. To participate in force maintenance, nonmuscle myosin activity must be regulated in smooth muscle. To investigate this question, phosphorylation of the regulatory light chains of smooth muscle myosin (SMLC) and nonmuscle myosin (NMLC) were examined by two dimensional SDS‐PAGE. Intact mouse aortic smooth muscle strips were stimulated with either KCl depolarization or angiotensin II, and 4 distinct LC spots were resolved by two dimensional SDS‐PAGE. Using mass spectroscopy, these spots were identified as four distinct proteins; phosphorylated NMLC, non‐phosphorylated NMLC, phosphorylated SMLC and non‐phosphorylated SMLC. During KCl depolarization, inhibition of Rho kinase or PKC did not affect the increases in SMLC and NMLC phosphorylation. During angiotensin II stimulation, inhibition of PKC or Rho kinase reduced SMLC phosphorylation, while only inhibition of Rho kinase reduced NMLC phosphorylation. These data suggest that in smooth muscle, nonmuscle myosin is regulated by Ca 2+ ‐calmodulin activated MLCK during depolarization, and a Rho kinase mediated pathway during agonist stimulation.

During nitric oxide (NO) signaling, cGMP activates type Iα protein kinase G (PKGIα), which subsequently activates myosin light chain phosphatase (MLCP). The increase in MLCP activity decreases myosin phosphorylation which leads to smooth muscle relaxation. MLCP is a trimeric enzyme consisting of a myosin targeting subunit (MYPT1), a catalytic subunit and a subunit of unknown function. MYPT1 has two isoforms; alterative mRNA splicing of a 3’ exon results in the presence or absence of a C‐terminal leucine zipper (LZ) domain. Further, the MYPT1 LZ+ is required for PKGIα mediated activation of MLCP. Moreover, recent studies have demonstrated PKGIα phosphorylates MYPT1. This study examined the affect of MYPT1 structure for PKGIα phosphorylation. Two MYPT1 fragments differing only by the LZ domain (aa 1010–1039) were used: MYPT1FL (aa 500–1039, LZ+) and MYPT1TR2 (aa 500–1010, LZ−). Both the rate (0.0216±0.005 min −1 vs 0.0596±0.005 min −1 ) and extent of PKGIα mediated phosphorylation of MYPT1TR2 were lower when compared to MYPT1FL. In addition, two‐dimensional SDS‐PAGE demonstrated that following PKGIα phosphorylation, MYPT1TR2 existed primarily as the non‐phosphorylated protein whereas MYPT1FL was diphosphorylated. These results demonstrate that MYPT1 structure is important for determining the sensitivity of smooth muscle to NO mediated vasodilatation.

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.