Christopher Szeto

Cardiotoxic and Cardioprotective Features of Chronic β-Adrenergic Signaling

Rationale: In the failing heart, persistent &bgr;-adrenergic receptor activation is thought to induce myocyte death by protein kinase A (PKA)-dependent and PKA-independent activation of calcium/calmodulin-dependent kinase II. &bgr;-adrenergic signaling pathways also are capable of activating cardioprotective mechanisms. Objective: This study used a novel PKA inhibitor peptide to inhibit PKA activity to test the hypothesis that &bgr;-adrenergic receptor signaling causes cell death through PKA-dependent pathways and cardioprotection through PKA-independent pathways. Methods and Results: In PKA inhibitor peptide transgenic mice, chronic isoproterenol failed to induce cardiac hypertrophy, fibrosis, and myocyte apoptosis, and decreased cardiac function. In cultured adult feline ventricular myocytes, PKA inhibition protected myocytes from death induced by &bgr;1-adrenergic receptor agonists by preventing cytosolic and sarcoplasmic reticulum Ca2+ overload and calcium/calmodulin-dependent kinase II activation. PKA inhibition revealed a cardioprotective role of &bgr;-adrenergic signaling via cAMP/exchange protein directly activated by cAMP/Rap1/Rac/extracellular signal-regulated kinase pathway. Selective PKA inhibition causes protection in the heart after myocardial infarction that was superior to &bgr;-blocker therapy. Conclusions: These results suggest that selective block of PKA could be a novel heart failure therapy.

Calcium Influx through Cav1.2 Is a Proximal Signal for Pathological Cardiomyocyte Hypertrophy

Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca(2+)) is implicated in hypertrophic signaling pathways, the specific role of Ca(2+) influx through the L-type Ca(2+) channel (I(Ca-L)) has been controversial and is the topic of this study. To determine if and how sustained increases in I(Ca-L) induce PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the β2a subunit of Ca(2+) channels (β2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a β2a-GFP were used. In vivo, β2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume, and cross-sectional area and the expression of PCH markers in a time- and dose-dependent manner. PCH was associated with a hypercontractile phenotype including enhanced I(Ca-L), fractional shortening, peak Ca(2+) transient, at the myocyte level, greater ejection fraction, and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of β2a in cultured AFVMs increased I(Ca-L), cell volume, protein synthesis, NFAT, and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of I(Ca-L), intracellular Ca(2+), calcineurin, CaMKII, and SERCA. In conclusion, increasing I(Ca-L) is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca(2+) pools were shown to be involved.

Enhanced basal contractility but reduced excitation-contraction coupling efficiency and β-adrenergic reserve of hearts with increased Cav1.2 activity

Cardiac remodeling during heart failure development induces a significant increase in the activity of the L-type Ca(2+) channel (Cav1.2). However, the effects of enhanced Cav1.2 activity on myocyte excitation-contraction (E-C) coupling, cardiac contractility, and its regulation by the beta-adrenergic system are not clear. To recapitulate the increased Cav1.2 activity, a double transgenic (DTG) mouse model overexpressing the Cavbeta2a subunit in a cardiac-specific and inducible manner was established. We studied cardiac (in vivo) and myocyte (in vitro) contractility at baseline and upon beta-adrenergic stimulation. E-C coupling efficiency was evaluated in isolated myocytes as well. The following results were found: 1) in DTG myocytes, L-type Ca(2+) current (I(Ca,L)) density, myocyte fractional shortening (FS), peak Ca(2+) transients, and sarcoplasmic reticulum (SR) Ca(2+) content (caffeine-induced Ca(2+) transient peak) were significantly increased (by 100.8%, 48.8%, 49.8%, and 46.8%, respectively); and 2) cardiac contractility evaluated with echocardiography [ejection fraction (EF) and (FS)] and invasive intra-left ventricular pressure (maximum dP/dt and -dP/dt) measurements were significantly greater in DTG mice than in control mice. However, 1) the cardiac contractility (EF, FS, dP/dt, and -dP/dt)-enhancing effect of the beta-adrenergic agonist isoproterenol (2 microg/g body wt ip) was significantly reduced in DTG mice, which could be attributed to the loss of beta-adrenergic stimulation on contraction, Ca(2+) transients, I(Ca,L), and SR Ca(2+) content in DTG myocytes; and 2) E-C couplng efficiency was significantly lower in DTG myocytes. In conclusion, increasing Cav1.2 activity by promoting its high-activity mode enhances cardiac contractility but decreases E-C coupling efficiency and the adrenergic reserve of the heart.

β-Adrenergic stimulation increases Cav3.1 activity in cardiac myocytes through protein kinase A.

The T-type Ca2+ channel (TTCC) plays important roles in cellular excitability and Ca2+ regulation. In the heart, TTCC is found in the sinoatrial nodal (SAN) and conduction cells. Cav3.1 encodes one of the three types of TTCCs. To date, there is no report regarding the regulation of Cav3.1 by β-adrenergic agonists, which is the topic of this study. Ventricular myocytes (VMs) from Cav3.1 double transgenic (TG) mice and SAN cells from wild type, Cav3.1 knockout, or Cav3.2 knockout mice were used to study β-adrenergic regulation of overexpressed or native Cav3.1-mediated T-type Ca2+ current (ICa-T(3.1)). ICa-T(3.1) was not found in control VMs but was robust in all examined TG-VMs. A β-adrenergic agonist (isoproterenol, ISO) and a cyclic AMP analog (dibutyryl-cAMP) significantly increased ICa-T(3.1) as well as ICa-L in TG-VMs at both physiological and room temperatures. The ISO effect on ICa-L and ICa-T in TG myocytes was blocked by H89, a PKA inhibitor. ICa-T was detected in control wildtype SAN cells but not in Cav3.1 knockout SAN cells, indicating the identity of ICa-T in normal SAN cells is mediated by Cav3.1. Real-time PCR confirmed the presence of Cav3.1 mRNA but not mRNAs of Cav3.2 and Cav3.3 in the SAN. ICa-T in SAN cells from wild type or Cav3.2 knockout mice was significantly increased by ISO, suggesting native Cav3.1 channels can be upregulated by the β-adrenergic (β-AR) system. In conclusion, β-adrenergic stimulation increases ICa-T(3.1) in cardiomyocytes, which is mediated by the cAMP/PKA pathway. The upregulation of ICa-T(3.1) by the β-adrenergic system could play important roles in cellular functions involving Cav3.1.

Increasing T‐type calcium channel activity by β‐adrenergic stimulation contributes to β‐adrenergic regulation of heart rates

Cav3.1 T‐type Ca2+ channel current (ICa‐T) contributes to heart rate genesis but is not known to contribute to heart rate regulation by the sympathetic/β‐adrenergic system (SAS). We show that the loss of Cav3.1 makes the beating rates of the heart in vivo and perfused hearts ex vivo, as well as sinoatrial node cells, less sensitive to β‐adrenergic stimulation; it also renders less conduction acceleration through the atrioventricular node by β‐adrenergic stimulation. Increasing Cav3.1 in cardiomyocytes has the opposite effects. ICa‐T in sinoatrial nodal cells can be upregulated by β‐adrenergic stimulation. The results of the present study add a new contribution to heart rate regulation by the SAS system and provide potential new mechanisms for the dysregulation of heart rate and conduction by the SAS in the heart. T‐type Ca2+ channel can be a target for heart disease treatments that aim to slow down the heart rate

Reduced Myocardial Reserve in Young X-Linked Muscular Dystrophy Mice Diagnosed by Two-Dimensional Strain Analysis Combined with Stress Echocardiography

Background: Early, sensitive, and reproducible evaluation of left ventricular function is imperative for the diagnosis of cardiac dysfunction in patients with Duchene muscular dystrophy. The aim of this study was to test the hypothesis that combining two‐dimensional strain analysis with catecholamine stress could be a sensitive method for detecting early cardiac dysfunction. Methods: Mdx (C57BL/10ScSn‐Dmdmdx/J, a mouse model of DMD) and control (C57BL/10ScSn) mice were studied with conventional M‐mode and high‐frequency ultrasound‐based two‐dimensional speckle‐tracking echocardiography using long‐ and short‐axis images of the left ventricle at baseline and after intraperitoneal isoprenaline (ISO) administration (2 &mgr;g/g body weight). Results: Conventional M‐mode analysis showed no differences in left ventricular fractional shortening, wall thickness, or internal diameter at diastole between mdx and control mice before the age of 6 months. ISO increased left ventricular ejection fraction and fractional shortening to the same extent in mdx and control mice at young ages (3, 4, and 5 months). No differences in basal peak systolic strain (PSS) but increased SDs of times to PSS between young mdx and control mice were found. After ISO, PSS and percentile changes of PSS were significantly diminished in mdx mice compared with control mice at young ages. ISO increased the normalized maximum difference of times to PSS in young mdx mice but not in young control mice, suggesting that ISO reduces cardiac contractile synchrony in young mdx mice. Conclusions: This study suggests that catecholamine stress coupled with two‐dimensional strain analysis is a feasible and sensitive approach for detecting early onset of cardiac dysfunction, which is instrumental for early diagnosis of cardiac dysfunction and early treatment. HighlightsCardiac dysfunction becomes an important contributing factor to mortality and morbidity in patients with muscular dystrophy.There is still a lack of reliable approaches to diagnose early cardiac dysfunction in patients with DMD.The combination of 2D strain analysis with ISO stress showed reduced &bgr;‐adrenergic reserve and increased contractile dyssynchrony in mdx mice at a very early stage.Two‐dimensional strain analysis with stress testing is a feasible and sensitive approach to diagnose early cardiac dysfunction and to evaluate treatments in patients with DMD.The treatment of cardiac dysfunction should be started at a young age in patients with DMD.