SLMAP-3 is downregulated in human dilated ventricles and its overexpression promotes cardiomyocyte response to adrenergic stimuli by increasing intracellular calcium

Structural dilation of cardiomyocytes (CMs) imposes a decline in cardiac performance that precipitates cardiac failure and sudden death. Since membrane proteins are implicated in dilated cardiomyopathy and heart failure, we evaluated the expression of the sarcolemmal membrane-associated protein (SLMAP) in dilated cardiomyopathy and its effect on CM contraction. We found that all 3 SLMAP isoforms (SLMAP-1, -2, and -3) are expressed in CMs and are downregulated in human dilated ventricles. Knockdown of SLMAPs in cultured CMs transduced with recombinant adeno-associated viral particles releasing SLMAP-shRNA precipitated reduced spontaneous contractile rate that was not fully recovered in SLMAP-depleted CMs challenged with isoproterenol (ISO), thus phenotypically mimicking heart failure performance. Interestingly, the overexpression of the SLMAP-3 full-length isoform induced a positive chronotropic effect in CMs that was more pronounced in response to ISO insult (vs. ISO-treated naïve CMs). Confocal live imaging showed that H9c2 cardiac myoblasts overexpressing SLMAP-3 exhibit a higher intracellular calcium transient peak when treated with ISO (vs. ISO-treated cells carrying a control adeno-associated viral particle). Proteomics revealed that SLMAP-3 interacts with the regulator of CM contraction, striatin. Collectively, our data demonstrate that SLMAP-3 is a novel regulator of CM contraction rate and their response to adrenergic stimuli. Loss of SLMAPs phenotypically mimics cardiac failure and crystallizes SLMAPs as predictive of dilated cardiomyopathy and heart failure.

Caffeine attenuates contraction-induced diminutions of the intracellular calcium transient in mouse lumbrical muscle ex vivo

The amount of calcium released from the sarcoplasmic reticulum in skeletal muscle rapidly declines during repeated twitch contractions. In this study, we test the hypothesis that caffeine can mitigate these contraction-induced declines in calcium release. Lumbrical muscles were isolated from male C57BL/6 mice and loaded with the calcium-sensitive indicator, AM-furaptra. Muscles were then stimulated at 8 Hz for 2.0 s in the presence or absence of 0.5 mM caffeine, at either 30 °C or 37 °C. The amplitude and area of the furaptra-based intracellular calcium transients and force produced during twitch contractions were calculated. For each of these measures, the values for twitch 16 relative to twitch 1 were higher in the presence of caffeine than in the absence of caffeine at both temperatures. We conclude that caffeine can attenuate contraction-induced diminutions of calcium release during repeated twitch contractions, thereby contributing to the inotropic effects of caffeine.

Impaired mitochondria and intracellular calcium transients in the salivary glands of obese rats

Long-term consumption of a high-fat diet (HFD) causes not only obese-insulin resistance, but is also associated with mitochondrial dysfunction in several organs. However, the effect of obese-insulin resistance on salivary glands has not been investigated. We hypothesized that obese-insulin resistance induced by HFD impaired salivary gland function by reducing salivation, increasing inflammation, and fibrosis, as well as impairing mitochondrial function and calcium transient signaling. Male Wistar rats (200–220 g) were fed either a ND or an HFD (n = 8/group) for 16 weeks. At the end of week 16, salivary flow rates, metabolic parameters, and plasma oxidative stress were determined. Rats were then sacrificed and submandibular glands were removed to determine inflammation, fibrosis, apoptosis, mitochondrial function and dynamics, and intracellular calcium transient signaling. Long-term consumption of an HFD caused obese-insulin resistance and increased oxidative stress, fibrosis, inflammation, and apoptosis in the salivary glands. In addition, impaired mitochondrial function, as indicated by increased mitochondrial reactive oxygen species, mitochondrial membrane depolarization, and mitochondrial swelling in salivary glands and impaired intracellular calcium regulation, as indicated by a reduced intracellular calcium transient rising rate, decay rates, and amplitude of salivary acinar cells, were observed in HFD-fed rats. However, salivary flow rate and level of aquaporin 5 protein were not different between both groups. Although HFD consumption did not affect salivation, it caused obese-insulin resistance, leading to pathophysiological alteration of salivary glands, including impaired intracellular calcium transients, increased oxidative stress and inflammation, and salivary mitochondrial dysfunction.

Abstract 5403: Protein Kinase C-Induced Troponin I Phosphorylation Causes Changes in Cardiac Contractile Function but not the Intracellular Calcium Transient

Both intracellular calcium transients ([Ca] i ) and myofilament properties determine cardiac muscle contractile force. Transgenic mouse models created to perturb specific myofilament proteins often cause a compensatory change in [Ca] i , which confounds the assessment of myofilament structure-function relationships. We have created a new transgenic mouse that has all three protein kinase C (PKC) phosphorylation sites on cardiac troponin I (cTnI) mutated to glutamic acid, rendering these sites constitutively pseudo-phosphorylated. Our goal was to determine the effects of this mutation on cardiac muscle contractile function and whether these effects would be concurrent with changes in the [Ca] i . Two sets of studies were conducted: skinned muscle fiber experiments to characterize the steady-state force-pCa relationships at sarcomere lengths of 1.9 and 2.3 μm and right ventricular papillary muscle experiments to characterize the peak developed force (F dev )-muscle length (L) relationships and [Ca] i (fura-5F calcium dye, emission: 510 nm, excitation: 340 and 380 nm, R = [emission fluorescence 340 ]/[emission fluorescence 380 ]). In skinned fibers, there was a significant decrease in maximally activated force (i.e., force at pCa 4.33) in transgenic mice (Wild-Type, WT (n = 7): 64.4± 8.0, Transgenic, TG (n = 6): 42.6±6.8 mN•mm −2 , P = 0.004), without any changes in calcium sensitivity or cooperativity (Hill coefficient). In intact papillary muscles, TG mice showed a decrease in F dev and slowed relaxation for all muscle lengths examined (F dev @ 100% L max , WT (n = 5): 9.3±3.5, TG (n = 6): 4.2±1.6 mN•mm −2 , P = 0.005; dF/dt min @ 100% L max , WT: −136±32, TG: −74±38 mN•mm −2 •s −1 , P = 0.002). In contrast, [Ca] i was unaltered in TG mice at all muscle lengths examined ([Ca] i amplitude as quantified by R systole / R diaastole , WT: 1.62±0.07, TG: 1.48±0.22; [Ca] i relaxation rate d R /dt min , WT: −96±37, TG: −64±30 s −1 ). Thus, PKC-induced TnI phosphorylation affects cardiac muscle contraction (reduced force magnitude and slowed relaxation) via changes in the myofilament properties (activation and/or crossbridge dynamics), and these contractile effects are not related to any changes in the intracellular calcium transient.