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New Findings: What is the central question of this study?Are there specific contributions of β1‐ and β2‐adrenoceptor subtypes to the impaired β‐adrenoceptor responsiveness of the type 2 diabetic heart?What is the main finding and its importance?In hearts isolated from the Zucker diabetic fatty rat model of type 2 diabetes, we showed that the β1‐adrenoceptors are the main subtype to regulate heart rate, contraction and relaxation. Notably, the β2‐adrenoceptor subtype actions seem to support function in the diabetic heart indirectly. Impaired β‐adrenoceptor (β‐AR) responsiveness causes cardiac vulnerability in patients with type 2 diabetes, but the independent contributions of β1‐ and β2‐AR subtypes to β‐AR‐associated cardiac dysfunction in diabetes are unknown. Our aim was to determine the specific β1‐ and β2‐AR responsiveness of heart rate (HR), contraction and relaxation in the diabetic heart. Isolated Langendorff‐perfused hearts of Zucker type 2 diabetic fatty (ZDF) rats were stimulated with the β‐AR agonist isoprenaline (1 × 10−11 to 3 × 10−8 mol l−1) with or without the selective β1‐AR antagonist CGP20712A (3 × 10−8 mol l−1) or the β2‐AR antagonist ICI‐118,551 (5 × 10−8 mol l−1), and HR, contraction and relaxation were measured. Diabetic hearts showed lower basal HR (non‐diabetic 216 ± 17 beats min−1versus diabetic 151 ± 23 beats min−1, P < 0.05). However, the β‐AR‐induced increase in HR was similar and was completely blocked by the β1‐AR antagonist, but not by the β2‐AR antagonist. The β‐AR‐induced increase in contraction and acceleration of relaxation was impaired in diabetic hearts, completely blocked by the β1‐AR antagonist and partly impaired by the β2‐AR antagonist. Western blots revealed 41% higher phosphorylation levels of AMP kinase (AMPK), a key regulator of cardiac energy metabolism, in diabetic hearts (non‐diabetic 1.62 ± 0.19 a.u. versus diabetic 2.30 ± 0.25 a.u., P < 0.05). In conclusion, the β1‐AR is the main subtype regulating chronotropic, inotropic and lusitropic β‐AR responses in the healthy heart and the type 2 diabetic heart. The β2‐AR subtype indirectly supports the β1‐AR functional response in the diabetic heart. This suggests that β2‐ARs could be an indirect target to improve the function of the heart in type 2 diabetes. [ABSTRACT FROM AUTHOR]

Coronary arterial inflow is impeded and venous outflow is increased as a result of the decrease in coronary vascular volume due to cardiac contraction. We evaluated whether cardiac contraction is influenced by interfering with the changes of the coronary vascular volume over the heart cycle. Length-tension relationships were determined in Tyrode-perfused rat papillary muscle and when coronary vascular volume changes were partly inhibited by filling it with congealed gelatin or perfusing it with a high viscosity dextran buffer. Also, myocyte thickening during contraction was reduced by placing a silicon tube around the muscle. Increasing perfusion pressure from 8 to 80 cm[H.sub.2]O, increased developed tension by ~40%. When compared with the low perfusion state, developed tension of the gelatin-filled vasculature was reduced to 43 [+ or -] 6% at the muscle length where the muscle generates the largest developed tension (n = 5, means [+ or -] SE). Dextran reduced developed tension to 73 [+ or -] 6% (n = 6). The silicon tube, in low perfusion state, reduced the developed tension to 83 [+ or -] 7% (n = 4) of control. Time-control and oxygen-lowering experiments show that the findings are based on mechanical effects. Thus interventions to prevent myocyte thickening reduce developed tension. We hypothesize that when myocyte thickening is prevented, intracellular pressure increases and counteracts the force produced by the contractile apparatus. We conclude that emptying of the coronary vasculature serves a physiological purpose by facilitating cardiomyocyte thickening thereby augmenting force development. cardiac mechanics; cardiomyocytes; rat; shortening; intracellular pressure

New Findings What is the central question of the study? The sympathetic system regulates heart rate via β-adrenoceptors; this is impaired during diabetes. However, the specific β-adrenoceptor subtype contributions in heart rate regulation in diabetes in vivo are unknown. What is the main finding and its importance? Telemetric recordings in conscious non-diabetic and type 2 diabetic rats demonstrated that the β1-adrenoceptor subtype, and not the β2-adrenoceptor, regulated the lower resting heart rate and increased β-adrenoceptor responsiveness in diabetes in vivo. This provides new physiological insight into the dysregulation of heart rate in type 2 diabetes, which is important for improving therapeutic strategies targeting the diabetic chronotropic incompetence. β-Adrenoceptor blockers are widely used to reduce heart rate, the strongest predictor of mortality in cardiac patients, but are less effective in diabetic patients. This study aimed to determine the specific contributions of β1- and β2-adrenoceptor subtypes to chronotropic responses in type 2 diabetes in vivo, which are currently unknown. Type 2 diabetic and non-diabetic rats were implanted with radiotelemeters to measure arterial blood pressure and derive heart rate in conscious conditions. Vascular access ports were implanted to inject isoprenaline (β1- and β2-adrenoceptor agonist, 0.1–300 μg kg−1) in the presence of atenolol (β1-adrenoceptor antagonist, 2000 μg kg−1) or nadolol (β1- and β2-adrenoceptor agonist, 4000 μg kg−1) to determine the chronotropic contributions of the β-adrenoceptor subtypes. Resting heart rate was reduced in diabetic rats (388 ± 62 versus 290 ± 37 beats min−1 non-diabetic versus diabetic, P < 0.05, mean ± SD), which remained after atenolol or nadolol administration. Overall β-adrenoceptor chronotropic responsiveness was increased in diabetic rats (change in heart rate at highest dose of isoprenaline: 135 ± 66 versus 205 ± 28 beats min−1, non-diabetic versus diabetic, P < 0.05), a difference that diminished after β1-adrenoceptor blockade with atenolol (change in heart rate at highest dose of isoprenaline: 205 ± 37 versus 195 ± 22 beats min−1, non-diabetic versus diabetic, P < 0.05). In conclusion, the β1-adrenoceptor is the main subtype to modulate chronotropic β-adrenoceptor responses in healthy and diabetic rats. This study provides new insights into the pathological basis of dysregulation of heart rate in type 2 diabetes, which could be important for improving the current therapeutic strategies targeting diabetic chronotropic incompetence. [ABSTRACT FROM AUTHOR]

Background Diastolic dysfunction is a key factor in the development and pathology of cardiac dysfunction in diabetes, however the exact underlying mechanism remains unknown, especially in humans. We aimed to measure contraction, relaxation, expression of calcium-handling proteins and fibrosis in myocardium of diabetic patients with preserved systolic function. Methods Right atrial appendages from patients with type 2 diabetes mellitus (DM, n = 20) and nondiabetic patients (non-DM, n = 36), all with preserved ejection fraction and undergoing coronary artery bypass grafting (CABG), were collected. From appendages, small cardiac muscles, trabeculae, were isolated to measure basal and β-adrenergic stimulated myocardial function. Expression levels of calcium-handling proteins, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB), and of β1-adrenoreceptors were determined in tissue samples by Western blot. Collagen deposition was determined by picro-sirius red staining. Results In trabeculae from diabetic samples, contractile function was preserved, but relaxation was prolonged (Tau: 74 ± 13 ms vs. 93 ± 16 ms, non-DM vs. DM, p = 0.03). The expression of SERCA2a was increased in diabetic myocardial tissue (0.75 ± 0.09 vs. 1.23 ± 0.15, non-DM vs. DM, p = 0.007), whereas its endogenous inhibitor PLB was reduced (2.21 ± 0.45 vs. 0.42 ± 0.11, non-DM vs. DM, p = 0.01). Collagen deposition was increased in diabetic samples. Moreover, trabeculae from diabetic patients were unresponsive to β-adrenergic stimulation, despite no change in β1-adrenoreceptor expression levels. Conclusions Human type 2 diabetic atrial myocardium showed increased fibrosis without systolic dysfunction but with impaired relaxation, especially during β-adrenergic challenge. Interestingly, changes in calcium-handling protein expression suggests accelerated active calcium re-uptake, thus improved relaxation, indicating a compensatory calcium-handling mechanism in diabetes in an attempt to maintain diastolic function at rest despite impaired relaxation in the diabetic fibrotic atrial myocardium. Our study addresses important aspects of the underlying mechanisms of diabetes-associated diastolic dysfunction, which is crucial to developing new therapeutic treatments. [ABSTRACT FROM AUTHOR]

The positive force–frequency relation, one of the key factors modulating performance of healthy myocardium, has been attributed to an increased Ca2 + influx per unit of time. In failing hearts, a blunted, flat or negative force–frequency relation has been found. In healthy and failing hearts frequency-dependent alterations in Ca2 + sensitivity of the myofilaments, related to different phosphorylation levels of contractile proteins, could contribute to this process. Therefore, the frequency dependency of force, intracellular free Ca2 + ([Ca2 +]i), Ca2 + sensitivity and contractile protein phosphorylation were determined in control and monocrotaline-treated, failing rat hearts. An increase in frequency from 0.5 to 6 Hz resulted in an increase in force in control (14.3 ± 3.0 mN mm−2) and a decrease in force in failing trabeculae (9.4 ± 3.2 mN mm−2), whereas in both groups the amplitude of [Ca2 +]i transient increased. In permeabilized cardiomyocytes, isolated from control hearts paced at 0 and 9 Hz, Ca2 + sensitivity remained constant with frequency (pCa50: 5.55 ± 0.02 and 5.58 ± 0.01, respectively, P > 0.05), whereas in cardiomyocytes from failing hearts Ca2 + sensitivity decreased with frequency (pCa50: 5.62 ± 0.01 and 5.57 ± 0.01, respectively, P < 0.05). After incubation of the cardiomyocytes with protein kinase A (PKA) this frequency dependency of Ca2 + sensitivity was abolished. Troponin I (TnI) and myosin light chain 2 (MLC2) phosphorylation remained constant in control hearts but both increased with frequency in failing hearts. In conclusion, in heart failure frequency-dependent myofilament Ca2 + desensitization, through increased TnI phosphorylation, contributes to the negative force–frequency relation and is counteracted by a frequency-dependent MLC2 phosphorylation. We propose a novel role for PKC-mediated TnI phosphorylation in modulating the force–frequency relation. [ABSTRACT FROM AUTHOR]

Right ventricular (RV) pressure overload causes right ventricular hypertrophy in several types of pulmonary and congenital heart diseases. The associated cardiac dysfunction has generally been attributed to alterations in RV function. However, due to global neurohormonal adaptations and mechanical ventricular interaction left ventricular (LV) function could be affected as well.Therefore,LV function, RV function and their interaction were studied in rats with monocrotaline (MCT)-induced RV hypertrophy and control rats. MCT (30 mg/kg) was used to induce pulmonary hypertension, which resulted, after 28 days, in marked RV hypertrophy (RV-weight: control 220 ± 15,MCT 437 ± 34mg,p < 0.05). In Langendorff-perfused hearts with balloons inserted in both the LV and the RV, the diastolic pressure-volume relations showed increased stiffness, and relaxation was prolonged in the LV and RV in the MCT group compared to controls. In the MCT group, developed pressures were increased only in the RV. An increase of LV volume increased RV diastolic pressure to a similar extent in both groups. However, an increase in RV volume did not affect LV diastolic pressure in controls, but significantly increased LV diastolic pressure in the MCT group. LV and RV developed pressure-volume relations were not affected. Calculated circumferential end-diastolic wall stresses (σ) were larger in the MCT group (LV-σ: 0.55 ± 0.02, RV-σ: 1.94 ± 0.30 kN/m2, both p< 0.05 to control) compared to controls (LV-σ: 0.34 ± 0.06,RV-σ: 1.23 ± 0.46 kN/m2). In the MCT group, collagen content was increased in the LV, septum and RV compared to controls. In conclusion, structural changes of the RV and LV result in depressed LV diastolic function during RV hypertrophy. [ABSTRACT FROM AUTHOR]

Abstract: Objective: In human heart failure β-adrenergic-mediated protein kinase A (PKA) activity is down-regulated, while protein kinase C (PKC) activity is up-regulated. PKC-mediated myofilament protein phosphorylation might be detrimental for contractile function in cardiomyopathy. This study was designed to reveal the effects of PKC on myofilament function in human myocardium under basal conditions and upon modulation of protein phosphorylation by PKA and phosphatases. Methods: Isometric force was measured at different [Ca2+] in single permeabilized cardiomyocytes from non-failing and failing human left ventricular tissue. Basal phosphorylation of myofilament proteins and the influence of PKC, PKA, and phosphatase treatments were analyzed by one- and two-dimensional gel electrophoresis, Western immunoblotting, and ELISA. Results: Troponin I (TnI) phosphorylation at the PKA sites was decreased in failing compared to non-failing hearts and correlated well with myofilament Ca2+ sensitivity (pCa50). Incubation with the catalytic domain of PKC slightly decreased maximal force under basal conditions, but not following PKA and phosphatase pretreatments. PKC reduced Ca2+ sensitivity to a larger extent in failing (ΔpCa50 =0.19±0.03) than in non-failing (ΔpCa50 =0.08±0.01) cardiomyocytes. This shift was reduced, though still significant, when PKC was preceded by PKA, while PKA following PKC did not further decrease pCa50. Protein analysis indicated that PKC phosphorylated PKA sites in human TnI and increased phosphorylation of troponin T, while myosin light chain phosphorylation remained unaltered. Conclusion: In human myocardium PKC-mediated myofilament protein phosphorylation only has a minor effect on maximal force development. The PKC-mediated decrease in Ca2+ sensitivity may serve to improve diastolic function in failing human myocardium in which PKA-mediated TnI phosphorylation is decreased. [Copyright &y& Elsevier]

Objective: This study investigated whether endocardial endothelial cells contribute to intracellular pH (pHi) regulation of subjacent cardiomyocytes. Methods: Right ventricular rabbit papillary muscles were loaded with the pH-sensitive dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) to measure pHi and HCO3- equivalent influx or efflux in muscles with intact endocardial endothelium (+EE) and in muscles with the endocardial endothelium removed (-EE). Results: In steady-state conditions, pHi was consistently higher in +EE than in -EE muscles (7.38±0.03, n=39 vs. 7.27±0.04, n=20, p<0.05). In +EE muscles, removal of HCO3- from the buffer solution or adding 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of HCO3- transport, reduced pHi from 7.38 to 7.24±0.06 (n=14) and to 7.16±0.10 (n=10), respectively, whereas in -EE muscles pHi decreased slightly from 7.27 to 7.15±0.05 (n=14) and to 7.13±0.04 (n=7). In addition, HCO3- equivalent efflux during alkali loads by NH4Cl pulses was smaller in +EE muscles than in -EE muscles (0.89±0.50 vs. 1.99±0.12 mmol/l/min, n=5, p<0.05) and was inhibited by DIDS. HCO3- equivalent influx during recovery from acid load imposed upon wash out of NH4Cl, was larger in +EE muscles than in -EE muscles (2.15±0.54 mmol/l/min, n=6, vs. 1.06±0.20 mmol/l/min, n=5). 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na+/H+ exchange, decreased HCO3- equivalent influx by 50% in both muscle groups but influx was still significantly higher in +EE than in -EE muscles (1.18±0.23 vs. 0.57±0.07 mmol/l/min, p<0.05). Finally, endocardial endothelial cells cultured on collagen-coated inserts established a DIDS-sensitive transendothelial HCO3-gradient. Conclusion: These data suggest that the endocardial endothelium maintains transendothelial fluxes of HCO3- from luminal (blood) to basal (muscle) side of the cells, which modulate pHi regulation in subjacent cardiomyocytes. [Copyright &y& Elsevier]

Human myocardial function can be investigated in the laboratory using human atrial trabeculae. These multicellular preparations provide a translatable model for assessing the ex vivo contractility, electrophysiology, and ionic and metabolic underpinnings of human cardiac muscle. Here, we detail the materials and methods required to determine the baseline function of a human atrial trabecula, from dissection to stimulation. This method is also adaptable for additional non-human myocardial applications., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)