L. Delbridge

Refractive Index Measurement in Viable Cells Using Quantitative Phase-Amplitude Microscopy and Confocal Microscopy

The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen.

Ca2+-dependent proteolysis of junctophilin-1 and junctophilin-2 in skeletal and cardiac muscle.

If skeletal muscle fibres are subjected to excessive activation, or stretched whilst contracting, they subsequently display long‐term reductions in their force response, apparently due in part to structural or molecular changes at the triad junction, where excitation of the surface membrane triggers Ca2+ release from the internal Ca2+ store. The changes appear to be due to excessive or prolonged increases in intracellular Ca2+ levels, which activate Ca2+‐dependent proteases known as calpains, but their target proteins are currently unknown. This study shows that excessive muscle stimulation, or directly raising intracellular Ca2+ levels, causes calpain activation in tandem with proteolysis of junctophilin, a key protein thought to hold the triad junction together. Proteolysis of junctophilin is also seen in muscle of mice with muscular dystrophy and in cardiac muscle following ischaemic damage. Proteolysis of junctophilin may be a major factor causing muscle weakness and cardiac dysfunction in a range of circumstances.

Effects of combined administration of ACE inhibitor and angiotensin II receptor antagonist are prevented by a high NaCl intake.

Background To prevent the action of angiotensin II by blockade with either an angiotensin converting enzyme inhibitor (ACE I) or an angiotensin receptor antagonist (ARA) is difficult due to the physiological compensations. Combined therapy with both drugs may enable complete blockade, and in rats in high doses this has produced a syndrome that results in death. Objective To determine the effect of combined blockade using losartan (10 mg/kg per day) and perindopril (6 mg/kg per day) on blood pressure, cardiac growth, renal function and behaviour, and to determine how this is influenced by different salt intakes in normotensive Sprague Dawley rats. Methods Rats were fed an 0.2 or 4% NaCl diet and received the above drugs intraperitoneally. Blood pressure was measured by telemetry. Cardiac weight was measured after 10 days of therapy. Renal function was assessed by plasma creatinine and electrolytes, plasma renin and angiotensinogen concentrations were measured. Results On 0.2% NaCl intake, combined blockade lowered blood pressure progressively; at day 7, rats on 0.2% NaCl developed a syndrome of listlessness and failure to eat which led to loss of weight and death. Cardiac size was dramatically reduced. Plasma creatinine was elevated to 50% above normal. There was a polyuria. The syndrome was reversed by adding NaCl to the drinking water or prevented in rats on a 4% NaCl intake. In rats on 0.2% NaCl plasma renin rose dramatically with medication and angiotensinogen became depleted. Haematocrit in all groups of rats did not differ. Conclusion Combined blockade of the renin–angiotensin system can cause death in rats on a reduced NaCl intake. This was prevented by a high salt intake. The syndrome may result from depletion of angiotensinogen and the failure to synthesize sufficient angiotensin II that may be critical for normal cardiac growth and function and critical for survival.

Aromatase Deficiency Confers Paradoxical Postischemic Cardioprotection

The conventional view is that estrogen confers female cardioprotection. Estrogen synthesis depends on androgen availability, with aromatase regulating conversion of testosterone to estradiol. Extragonadal aromatase expression mediates estrogen production in some tissues, but a role for local steroid conversion has not yet been demonstrated in the heart. This studys goal was to investigate how aromatase deficiency influences myocardial function and ischemic resilience. RT-PCR analysis of C57Bl/6 mouse hearts confirmed cardiac-specific aromatase expression in adult females. Functional performance of isolated hearts from female aromatase knockout (ArKO) and aromatase wild-type mice were compared. Left ventricular developed pressures were similar in aerobic perfusion, but the maximal rate of rise of ventricular pressure was modestly reduced in ArKO hearts (3725 ± 144 vs. 4272 ± 154 mm Hg/sec, P < 0.05). After 25 min of ischemia, the recovery of left ventricular developed pressure was substantially improved in ArKO (percentage of basal at 60 min of reperfusion, 62 ± 8 vs. 30 ± 6%; P < 0.05). Hypercontracture was attenuated (end diastolic pressure, 25 ± 5 vs. 51 ± 1 mm Hg; P < 0.05), and lactate dehydrogenase content of coronary effluent was reduced throughout reperfusion in ArKO hearts. This was associated with a hyperphosphorylation of phospholamban and a reduction in phosphorylated Akt. Immediately after reperfusion, ArKO hearts exhibited increased incidence of ventricular premature beats (194 ± 70 vs. 46 ± 6, P < 0.05). These observations indicate more robust functional recovery, reduced cellular injury, and modified cardiomyocyte Ca(2+) handling in aromatase-deficient hearts. Our findings indicate that androgen-to-estrogen conversion may be of pathophysiologic importance to the heart and challenge the notion that estrogen deficiency is deleterious. These studies suggest the possibility that aromatase suppression may offer inotropic benefit in the acute ischemia/reperfusion setting with appropriate arrhythmia management.

Stimulus interval-dependent differences in Ca2+ transients and contractile responses of diabetic rat cardiomyocytes

OBJECTIVE The aim of this study was to gain further insights into the consequences of insulin-dependent diabetes mellitus on cardiomyocyte calcium handling. METHODS The effects of steady state and transient changes in stimulus frequency on the intracellular Ca2+ transient and cell shortening were examined in left ventricular cardiomyocytes isolated from the hearts of control and streptozotocin-induced diabetic rats. RESULTS During steady state stimulation diabetic rat cardiomyocytes displayed a slower decay of the Ca2+ transient and longer times for maximum cell shortening and re-lengthening. At 1.5 mM extracellular [Ca2+], increasing stimulus frequency over the range 0.2-1.0 Hz led to an increase in resting and peak [Ca2+]i as well as the amplitude of the transient in both the control and diabetic groups. At frequencies greater than 0.4 Hz the amplitude of the transient was significantly depressed in diabetic rat cells and this was not normalized by increasing extracellular [Ca2+] to 2.5 mM. Recovery of sarcoplasmic reticulum (SR) Ca2+ release was measured from the time course of restitution of the intracellular Ca2+ transient. In both control and diabetic rat cardiomyocytes recovery of the transient occurred in two phases. In diabetic rat myocytes, the initial rapid phase of restitution at intervals <1 s was markedly slowed. The fraction of Ca2+ recirculating between the SR and the cytosol was estimated from the decline in amplitude of transients following post-rest potentiation. There was no difference in this fraction between control and diabetic rat cells either at 1.5 or 2.5 mM extracellular [Ca2+]. CONCLUSION The blunted frequency response of diabetic rat cardiomyocytes at frequencies greater than 0.4 Hz is consistent with reduced SR Ca2+ uptake leading to reduced SR Ca2+ content and subsequent release. At stimulus intervals greater than 1 Hz this is likely to be exacerbated by slower recovery of SR Ca2+ release. Despite the evidence for depressed SR Ca2+ uptake, the relative amount of Ca2+ recirculating within diabetic rat cardiomyocytes remains unaltered. This is most likely due to an accompanying reduction in Ca2+ efflux from the cell due either to depressed Na+/Ca2+ exchanger activity, or an elevation in intracellular Na+ levels.

The intrinsic resistance of female hearts to an ischemic insult is abrogated in primary cardiac hypertrophy.

Important sex differences in cardiovascular disease outcomes exist, including conditions of hypertrophic cardiomyopathy and cardiac ischemia. Studies of sex differences in the extent to which load-independent (primary) hypertrophy modulates the response to ischemia-reperfusion (I/R) damage have not been characterized. We have previously described a model of primary genetic cardiac hypertrophy, the hypertrophic heart rat (HHR). In this study the sex differences in HHR cardiac function and responses to I/R [compared to control normal heart rat (NHR)] were investigated ex vivo. The ventricular weight index was markedly increased in HHR female (7.82 +/- 0.49 vs. 4.80 +/- 0.10 mg/g; P < 0.05) and male (5.76 +/- 0.22 vs. 4.62 +/- 0.07 mg/g; P < 0.05) hearts. Female hearts of both strains exhibited a reduced basal contractility compared with strain-matched males [maximum first derivative of pressure (dP/dt(max)): NHR, 4,036 +/- 171 vs. 4,258 +/- 152 mmHg/s; and HHR, 3,974 +/- 160 vs. 4,540 +/- 259 mmHg/s; P < 0.05]. HHR hearts were more susceptible to I/R (I = 25 min, and R = 30 min) injury than NHR hearts (decreased functional recovery, and increased lactate dehydrogenase efflux). Female NHR hearts exhibited a significantly greater recovery (dP/dt(max)) post-I/R relative to male NHR (95.0 +/- 12.2% vs. 60.5 +/- 9.4%), a resistance to postischemic dysfunction not evident in female HHR (29.0 +/- 5.6% vs. 25.9 +/- 6.3%). Ventricular fibrillation was suppressed, and expression levels of Akt and ERK1/2 were selectively elevated in female NHR hearts. Thus the occurrence of load-independent primary cardiac hypertrophy undermines the intrinsic resistance of female hearts to I/R insult, with the observed abrogation of endogenous cardioprotective signaling pathways consistent with a potential mechanistic role in this loss of protection.

Aromatase transgenic upregulation modulates basal cardiac performance and the response to ischemic stress in male mice.

Estrogen in females is conventionally considered a cardioprotective influence, but a role for estrogen in male cardioprotection has yet to be defined. Estrogen biosynthesis from testosterone is regulated by aromatase. Aromatase has recently been shown to be expressed in the adult heart, although little is known about its involvement in the regulation of myocardial function and stress responses. The goal of this study was to determine whether upregulation of tissue aromatase expression could improve ischemic resilience in male hearts. Isolated hearts from male transgenic aromatase-overexpressing (AROM(+); high estrogen, low testosterone) mice and wild-type (WT) mice (12 wk) were Langendorff perfused and subjected to ischemia-reperfusion (25 min ischemia and 60 min of reperfusion). Basal systolic function was lower in AROM(+) hearts (dP/dtmax: 4,121 ± 255 vs. 4,992 ± 283 mmHg/s, P < 0.05) and associated with augmented Akt phosphorylation, consistent with a suppressor action of estrogen on contractility. Ischemic contracture was attenuated in AROM(+) hearts (43 ± 3 vs. 55 ± 4 mmHg, P < 0.05), yet AROM(+) hearts were more arrhythmic in early reperfusion. At the end of 60 min of reperfusion, AROM(+) systolic functional recovery was lower (left ventricular developed pressure: 39 ± 6 vs. 56 ± 5 %basal, P < 0.05) and diastolic dysfunction was accentuated (36 ± 4 vs. 24 ± 2 mmHg, P < 0.05). This is the first study to show that in vivo aromatase upregulation modulates basal cardiac performance and the response to ischemic stress. These data suggest that while chronic exposure to enhanced estrogenic influence may have benefits in limiting ischemic contracture severity, acute functional recovery in reperfusion is compromised. A temporally targeted, tissue-specific intervention combining aromatase treatment with inotropic support may offer therapeutic potential for men and women.

Insights into the role of maladaptive hexosamine biosynthesis and O-GlcNAcylation in development of diabetic cardiac complications

ABSTRACT Diabetes mellitus significantly increases the risk of heart failure, independent of coronary artery disease. The mechanisms implicated in the development of diabetic heart disease, commonly termed diabetic cardiomyopathy, are complex, but much of the impact of diabetes on the heart can be attributed to impaired glucose handling. It has been shown that the maladaptive nutrient‐sensing hexosamine biosynthesis pathway (HBP) contributes to diabetic complications in many non‐cardiac tissues. Glucose metabolism by the HBP leads to enzymatically‐regulated, O‐linked attachment of a sugar moiety molecule, &bgr;‐N‐acetylglucosamine (O‐GlcNAc), to proteins, affecting their biological activity (similar to phosphorylation). In normal physiology, transient activation of HBP/O‐GlcNAc mechanisms is an adaptive, protective means to enhance cell survival; interventions that acutely suppress this pathway decrease tolerance to stress. Conversely, chronic dysregulation of HBP/O‐GlcNAc mechanisms has been shown to be detrimental in certain pathological settings, including diabetes and cancer. Most of our understanding of the impact of sustained maladaptive HBP and O‐GlcNAc protein modifications has been derived from adipose tissue, skeletal muscle and other non‐cardiac tissues, as a contributing mechanism to insulin resistance and progression of diabetic complications. However, the long‐term consequences of persistent activation of cardiac HBP and O‐GlcNAc are not well‐understood; therefore, the goal of this timely review is to highlight current understanding of the role of the HBP pathway in development of diabetic cardiomyopathy.

HCN channelopathy and cardiac electrophysiologic dysfunction in genetic and acquired rat epilepsy models.

Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post–status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression.

EFFECTS OF ENDOTHELIN-1 ON THE CONTRACTILITY OF CARDIOMYOCYTES FROM THE SPONTANEOUSLY HYPERTENSIVE RAT

1. Disturbances in cardiovascular responsiveness to endogenous endothelin‐1 (ET‐1) may play a significant role in the pathogenesis of essential hypertension. In this study the inotropic responses of cardiomyocytes derived from normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rat (SHR) strains to ET‐1 (10‐11‐10‐8 mol/L) were characterized. Isotonic contraction cycles of ventricular cardiomyocytes isolated from age‐matched (11 week) WKY and SHR rats were recorded using a rapid digital imaging technique and evaluated by computation of a range of normalized parameters.