Troponin I phosphorylation is essential for cardiac reserve

With an increase in the body’s metabolic demand (e.g., exercise), the heart must increase its pumping performance. To achieve this increased performance, the heart relies on its cardiac reserve, which is the ability to increase its systolic and diastolic function. The mechanism responsible for cardiac reserve is poorly understood. The myofilaments are essential for contraction/relaxation, with troponin I (the inhibitory subunit of troponin, TnI) being a key regulatory protein. Studies have shown that TnI serine 23/24 (S23/S24) phosphorylation is a key mechanism for accelerating relaxation by decreasing Ca2+ sensitivity. However, the role of TnI in cardiac reserve is unknown. For this study, we characterized the systolic and diastolic reserve in TnI S23/S24 phosphorylation-null transgenic mice (S23/S24 mutated to alanine [AA] mice). Even with increased Ca2+ sensitivity, the AA mice exhibited normal function at resting heart rate with no difference in cardiac structure compared with wild type. To examine the role TnI S23/S24 phosphorylation in systolic and diastolic reserve, we assessed hemodynamics via left ventricular catheterization on the Bowditch effect (i.e., an increase in contractile function with increasing heart rate) by increasing heart rate (from 240 to 420 beats per minute) and sympathetic stimulation (dobutamine). Our data exhibited a clear loss of diastolic and systolic reserve in the AA mice with increasing heart rate and dobutamine. Since we observed a clear inability to increase systolic and diastolic function in AA mice, we performed speckle tracking echocardiography to quantitatively characterize function at resting heart rate. We observed that AA mice demonstrated normal systolic function (radial strain rate) and impaired directional diastolic function (reverse radial strain rate) at resting heart rate. We conclude that TnI S23/S24 phosphorylation is essential for cardiac reserve by enhancing systolic and diastolic function. A blunted cardiac reserve leads to heart disease making TnI S23/S24 phosphorylation a potential therapeutic strategy.

Altered spatio-specific CaMKII activation in autophagy deficient mice

Background Autophagy is linked to preventing the development of cardiac hypertrophy and failure. While aberrant activation of Ca2+/calmodulin-dependent kinase II (CaMKII) promotes myocardial remodeling, the role of autophagy in maintaining cardiac Ca2+ homeostasis and regulating CaMKII signaling is unknown. Objective To test whether loss of autophagy promotes subcellular alterations in CaMKII activation in early myocardial remodelling, and whether compromised in vivo cardiac function parallels those changes. Methods Young (10–15 weeks) cardiomyocyte-specific autophagy protein 5-deficient mice (Atg5−/−) mice and their littermate controls (Atg5+/+) underwent comprehensive in vivo phenotyping using echocardiography, exercise tolerance and hemodynamic stress testing. In vitro assessment included gravimetry, qPCR of hypertrophy marker genes and cellular and nuclear dimensions of isolated ventricular myocytes. CaMKII activation was studied by immunocytochemistry in cardiomyocytes upon exposure to basal (1Hz) or high (4Hz) pacing frequency. Autophosphorylated CaMKII (pT286) signal was evaluated in different subcellular spaces (i.e. cytoplasm, nucleoplasm and nuclear envelope). Results Before symptomatic cardiac dysfunction occurred, Atg5−/− mice showed compromised cardiac reserve in response to β-adrenergic stimulation (dp/dt max: 9475±126 vs 7364±496 mmHg/s, N=4–5; p=0.041), despite similar maximum heart rate. Consequently, effort intolerance (distance run: 251±22 vs 152±13 m, N=8; p=0.03) and maximal oxygen consumption (2093±66 vs 1763±131 ml/h/kg, N=8; p=0.04) were reduced during treadmill exercise tolerance testing. Increased heart-to-body weight ratio (8.1±0.5 vs 10.2±0.8 N=9; p=0.017) was associated with elevated mRNA expression of hypertrophy marker NppB (278% of Atg5+/+, N=5; p=0.016) in Atg5−/− mice, which showed enlarged cardiomyocytes and nuclei, as width-to-length ratio. Because Atg5−/− cardiomyocytes exhibit elevated nuclear Ca2+ levels at high pacing frequency, we now measured subcellular CaMKII activation under the same experimental conditions. Interestingly, at 1Hz, p-CaMKII was increased specifically at the nuclear envelope (154% of Atg5+/+, N=5 mice, 153–159 cells; p=0.029), but not in the cytoplasm or nucleoplasm. Increasing pacing frequency to 4Hz did not alter p-CaMKII levels in Atg5+/+ cells. However, p-CaMKII was increased by ∼30% and ∼20% in the cytoplasm and nucleoplasm of Atg5−/− cells respectively (N=5 mice, 153–155 cells). Conclusion Loss of ATG5-dependent autophagy causes cardiac hypertrophy and impaired cardiac reserve upon acute stress, which involves CaMKII activation, likely through the imbalance of nuclear Ca2+ load. Although, selective increase in p-CaMKII at the nuclear envelope in Atg5−/− mice may temporarily protect from nuclear Ca2+ overload, excessive CaMKII activation in the cytoplasm and the nucleoplasm upon increased workload, likely drives hypertrophic signalling toward heart failure in autophagy-defective mice. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund (FWF)

Abstract MP209: Troponin I Phosphorylation Is Essential For Cardiac Reserve

In response to increase in metabolic demand (e.g., exercise), the heart must increase its pumping performance to meet this demand. To achieve this increase, the heart relies on its cardiac reserve, which is the ability to increase in its contractile and diastolic function. The mechanism responsible for cardiac reserve is poorly understood. The myofilament is the mechanism responsible for contraction and relaxation. Troponin I (the inhibitory subunit of troponin, TnI) is a key regulatory protein. Studies have shown TnI serine 23/24 (S23/S24) phosphorylation, the most abundant and important TnI phosphorylation, is a key mechanism for accelerating relaxation by decreasing Ca 2+ senstivity. The role of TnI in cardiac reserve is unknown. For this study, we thoroughly characterized the systolic and diastolic reserve in TnI S23/S24 phosphorylation-null transgenic mice (S23/S24 mutated to alanine, AA mice). Even with increased Ca 2+ sensitivity, the AA mice exhibited normal function at resting heart rate and no difference in cardiac structure compared to wildtype. To increase in vivo heart performance, the most important system is the Bowditch effect (i.e., an increase in contractile function with increasing heart rate). To examine the role TnI S23/S24 phosphorylation in systolic and diastolic reserve, we assessed hemodynamics via left ventricular catheterization on the Bowditch effect by increasing heart rate from 240 to 420 beats per minute. Our data exhibited a clear loss of diastolic and systolic reserve in the AA mice. Since we observed a clear inability to increase systolic and diastolic function in AA mice, we performed speckle tracking echocardiography to more quantitatively investigate AA mice function. We observed that AA mice demonstrated normal systolic function (radial strain rate) and impaired directional diastolic function (reverse radial strain rate) at resting heart rate. We conclude that TnI S23/S24 phosphorylation is essential for cardiac reserve by enhancing systolic and diastolic function. A blunted cardiac reserve leads to heart disease making TnI S23/S24 phosphorylation a potential therapeutic strategy.

Hemodynamic Heterogeneity of Reduced Cardiac Reserve Unmasked by Volumetric Exercise Echocardiography

Background: Two-dimensional volumetric exercise stress echocardiography (ESE) provides an integrated view of left ventricular (LV) preload reserve through end-diastolic volume (EDV) and LV contractile reserve (LVCR) through end-systolic volume (ESV) changes. Purpose: To assess the dependence of cardiac reserve upon LVCR, EDV, and heart rate (HR) during ESE. Methods: We prospectively performed semi-supine bicycle or treadmill ESE in 1344 patients (age 59.8 ± 11.4 years; ejection fraction = 63 ± 8%) referred for known or suspected coronary artery disease. All patients had negative ESE by wall motion criteria. EDV and ESV were measured by biplane Simpson rule with 2-dimensional echocardiography. Cardiac index reserve was identified by peak-rest value. LVCR was the stress-rest ratio of force (systolic blood pressure by cuff sphygmomanometer/ESV, abnormal values ≤2.0). Preload reserve was defined by an increase in EDV. Cardiac index was calculated as stroke volume index * HR (by EKG). HR reserve (stress/rest ratio) <1.85 identified chronotropic incompetence. Results: Of the 1344 patients, 448 were in the lowest tertile of cardiac index reserve with stress. Of them, 303 (67.6%) achieved HR reserve <1.85; 252 (56.3%) had an abnormal LVCR and 341 (76.1%) a reduction of preload reserve, with 446 patients (99.6%) showing ≥1 abnormality. At binary logistic regression analysis, reduced preload reserve (odds ratio [OR]: 5.610; 95% confidence intervals [CI]: 4.025 to 7.821), chronotropic incompetence (OR: 3.923, 95% CI: 2.915 to 5.279), and abnormal LVCR (OR: 1.579; 95% CI: 1.105 to 2.259) were independently associated with lowest tertile of cardiac index reserve at peak stress. Conclusions: Heart rate assessment and volumetric echocardiography during ESE identify the heterogeneity of hemodynamic phenotypes of impaired chronotropic, preload or LVCR underlying a reduced cardiac reserve.

Cardiac reserve by 6-minute walk stress echocardiography in systemic sclerosis

ObjectivesThere is a high prevalence of left ventricular diastolic dysfunction (LVDD) in systemic sclerosis (SSc) which is associated with high mortality. Thus, early detection of LVDD could be important in management of SSc. We hypothesised that exercise echocardiography in SSc patients with normal resting haemodynamics may expose early phase LVDD, which could affect its prognosis, defined as cardiovascular death and unplanned hospitalisation for heart failure.MethodsBetween January 2014 and December 2018, we prospectively enrolled 140 patients with SSc who underwent 6-minute walk (6MW) stress echocardiographic studies with normal range of estimated mean pulmonary arterial pressure (mPAP) (<25 mm Hg) and mean pulmonary artery wedge pressure (mPAWP) (<15 mm Hg) at rest. We used ΔmPAP/Δcardiac output (CO) to assess pulmonary vascular reserve and ΔmPAWP/ΔCO to assess LV cardiac reserve between resting and post-6MW.ResultsDuring a median period of 3.6 years (IQR 2.0–5.1 years), 25 patients (18%) reached the composite outcome. Both ΔmPAP/ΔCO and ΔmPAWP/ΔCO in patients with events were significantly greater than in those without events (8.9±3.8 mm Hg/L/min vs 3.0±1.7 mm Hg/L/min; p=0.002, and 2.2±0.9 mm Hg/L/min vs 0.9±0.5 mm Hg/L/min; p<0.001, respectively). Patients with both impaired LV cardiac reserve (ΔmPAWP/ΔCO>1.4 mm Hg/L/min) and impaired pulmonary vascular reserve (ΔmPAP/ΔCO>3.0 mm Hg/L/min) had worse outcomes compared with those without these abnormalities (p<0.001).ConclusionThe 6MW stress echocardiography revealed impaired LV cardiac reserve in SSc patients with normal resting haemodynamics. Furthermore, LV cardiac reserve independently associates with clinical worsening in SSc, providing incremental prognostic utility, in addition to pulmonary vascular parameters.

Exercise cardiac MRI unmasks cardiac dysfunction in childhood and adolescent cancer survivors with reduced cardiopulmonary fitness

Background Young cancer survivors are at increased risk of impaired cardiopulmonary fitness (VO2peak) and heart failure. Assessment of exercise cardiac reserve may reveal sub-clinical abnormalities that better explain impairments in fitness and long term heart failure risk. Purpose To investigate the presence of impaired VO2peak in pediatric cancer survivors with increased risk of heart failure, and to assess its relationship with resting cardiac function and cardiac reserve Methods Twenty pediatric cancer survivors (aged 8–24 years) treated with anthracycline chemotherapy underwent cardiopulmonary exercise testing to quantify VO2peak, with a value &lt;85% of predicted defined as impaired VO2peak. Resting cardiac function was assessed using 3-dimensional echocardiography, with cardiac reserve quantified from resting and peak exercise heart rate (HR), stroke volume index (SVi) and cardiac index (CI) using exercise cardiac magnetic resonance imaging. Results 12 of 20 survivors (60%) had impaired VO2peak (97±14% vs. 70±16% of age and gender predicted). There were no differences in echocardiographic or CMR measurements of resting cardiac function between survivors with normal or impaired VO2peak. However, those with reduced VO2peak had diminished cardiac reserve, with a lesser increase in CI (Fig. 1A) and SVi (Fig. 1B) during exercise (Interaction P=0.001 for both), whilst the HR response was similar (Fig. 1C; P=0.71). Conclusions Resting measures of cardiac function are insensitive to significant cardiac dysfunction amongst pediatric cancer survivors with reduced VO2peak. Measures of cardiopulmonary fitness and cardiac reserve may aid in early identification of survivors with heightened risk of long-term heart failure. Figure 1 Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National Heart Foundation